Drosophila melanogaster Models of Motor Neuron Disease 2013 (edited by Ruben Cauchi)

NEURODEGENERATIVE DISEASES - LABORATORY AND CLINICAL RESEARCH

DROSOPHILA MELANOGASTER MODELS

OF MOTOR NEURON DISEASE

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NEURODEGENERATIVE DISEASES -

LABORATORY AND CLINICAL RESEARCH

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NEURODEGENERATIVE DISEASES - LABORATORY AND CLINICAL RESEARCH

DROSOPHILA MELANOGASTER MODELS

OF MOTOR NEURON DISEASE

RUBEN J. CAUCHI

EDITOR

New York

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CONTENTS

Preface vii

Chapter 1 Genetics of Motor Neuron Disorders: From Gene Diversity to

Common Cellular Conspirators in Selective Neuronal Killing 1 Rebecca K. Sheean and Bradley J. Turner

Chapter 2 A Secreted Ligand for Growth Cone Receptors, VAP Mediates the

Cellular Pathological Defects in ALS 35 Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda

Chapter 3 Flies in Motion: What Drosophila Can Tell Us about Amyotrophic

Lateral Sclerosis 57 Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu

Chapter 4 Maintaining Long Supply Lines: Axon Degeneration and the

Function of Hereditary Spastic Paraplegia Genes in Drosophila 85 Belgin Yalçın and Cahir J. O’Kane

Chapter 5 Drosophila as a Model for CMT Peripheral Neuropathy: Mutations

in tRNA Synthetases as an Example 121 Georg Steffes and Erik Storkebaum

Chapter 6 Lessons from Drosophila in Neurodegeneration: Mechanisms

of Toxicity and Therapeutic Targets in Spinal and Bulbar

Muscular Atrophy 147 Adrienne M. Wang

Chapter 7 Spinal Muscular Atrophy: Insights from the Fruit Fly 171 Stuart J. Grice, Kavita Praveen, A. Gregory Matera and

Ji-Long Liu

Chapter 8 Genetic Screens in Drosophila and Their Application in Motor

Neuron Disease Models 185 Liya E. Jose, Patrik Verstreken and Sven Vilain

Index 211

PREFACE

A RESPONSIBLE CHOICE OF MODEL ORGANISM

Motor neuron diseases (MNDs) are the most catastrophic of neurodegenerative disorders

in that cognitive function is spared yet motor neuron degeneration translates into progressive

muscle weakness and paralysis that propel the afflicted patient to eventual death.

Neurodegenerative disorders constitute one of the major challenges of modern medicine in

view of the current lack of effective therapies.

The fruit fly, Drosophila melanogaster, has a distinguished history as an important model

organism capable of shaping our fundamental understanding of life including neuromuscular

development and physiology. Through the efforts of the Drosophila and human genome

projects, more then a decade ago, we learned that the genetic makeup of the fruit fly is

remarkably similar to humans. In this respect, it’s no surprise that the vast majority of all

known human disease genes have a similar fly counterpart that is devoid of the genetic

complexity so typical of the human gene structure and families.

Compared to vertebrate models, the fly is small, has a rapid lifecycle and gives rise to a

large numbers of offspring. Importantly, at a molecular and physiological level, the basic

principles of neuromuscular function are amazing conserved. Combine this with the presence

of numerous genetic tools developed over the last century allowing genes and the proteins

they encode to be manipulated swiftly to decipher their in vivo function and you have a

superb genetic animal model organism of disease. This publication singles out the past and

recent accomplishments of Drosophila in modelling MNDs with particular emphasis on the

emerging molecular pathways underpinning these diseases.

The volume opens with Chapter One where Rebecca K. Sheean and Bradley J. Turner

(University of Melbourne, Australia) introduce us to the features of the two groups of neurons

that are primarily affected in MND, namely upper motor neurons (also known as giant

pyramidal cells or Betz cells) and lower motor neurons (also known as anterior horn cells in

the spinal cord). MNDs are categorised according to which group of motor neurons is

affected and in this context, the authors give us the core clinical and pathological features of

upper MNDs including primary lateral sclerosis (PLS) and hereditary spastic paraplegia

(HSP); lower MNDs including progressive muscular atrophy (PMA), progressive bulbar

palsy (PBP), spinal muscular atrophy (SMA), spinobulbar muscular atrophy (SBMA), axonal

Charcot-Marie-Tooth disease, which significantly overlaps with distal hereditary motor

Ruben J. Cauchi viii

neuropathy (dHMN) and lethal contracture syndrome (LCCS); and amyotrophic lateral

sclerosis (ALS), in which there is combined upper and lower motor neuron loss.

Chapter One also includes a detailed account of the multitude of genes linked to motor

neuron degeneration. Interestingly, most of these genes encode ubiquitously expressed and

fundamental proteins that, when defective, lead to selective motor neuron injury. Based on

this rich genetic evidence captured in the past decades as well as seminal contributions by

mammalian models, the authors eloquently highlight the key cellular pathways that are

disrupted in both motor neurons and their neighbouring cells including mitochondrial

function, intracellular membrane trafficking, axonal transport, cytoskeletal dynamics, RNA

processing, proteostasis, myelination and lipid metabolism. Importantly, various mentioned

genes and the proteins they encode will be subject to deeper investigation in the chapters that

follow.

In Chapter Two, Hiroshi Tsuda et al. (McGill University, Canada) kick-start a series of

accounts that underline the contribution of Drosophila to our understanding of the

pathophysiology of motor neuron degeneration. The authors focus on ALS (also known as

Lou Gehrig’s disease), a mercilessly fatal neurodegenerative disease, which was first

described by the founder of modern neurology, the French neurologist Jean-Martin Charcot.

ALS occurs in hereditary or sporadic forms and since both these forms share several features,

insights into the mechanisms through which gene mutations have a negative impact on motor

neuron physiology can potentially lead to novel therapeutic approaches that are effective in

both forms of the disease.

Identification of the first ALS-linked gene, superoxide dismutase 1 (SOD1), 20 years ago

spurred a successful hunt for additional causative genes among which was the VAMP

associated protein B (VAPB). VAPB, an endoplasmic reticulum (ER) transmembrane protein,

is conserved in Drosophila and numerous other species, including the N-terminal major

sperm protein (MSP) domain, which harbours the dominantly-inherited missense mutation

(proline 56 to serine) that confers motor neuron dysfunction. Human VAPB and its fly

orthologue are functionally interchangeable with regards to their influence on the architecture

and electrophysiological properties of Drosophila neuromuscular junctions (NMJs).

Furthermore, Drosophila studies were pivotal to reveal the VAPB MSP domain cleaved from

the full length protein is secreted in a cell type-specific fashion and acts as a diffusible

hormone. Importantly, VAPB-MSP was identified as a ligand for Ephrine (Eph), Roundabout

(Robo) and leukocyte-antigen related (LAR) family receptors, which were originally

identified as mediators of axon growth cone guidance cues during nervous system

development.

The downstream effects of VAPB signalling are still a work in progress though studies in

the fly implicate a function in the maintenance of muscle mitochondria. The authors conclude

this compelling narrative by delving into the cellular defects associated with mutant VAPB,

which can potentially shed light on the pathophysiology of ALS. Based on several lines of

evidence, the ALS mutation is thought to cause two different types of defects: failed secretion

of mutant VAPB (loss-of-function) and accumulation of mutant VAPB as ubiquitinated ER

inclusions that lead to ER stress (gain-of-function), a common defect observed in the

pathology of both familial and sporadic forms of ALS.

The subject of Chapter Three remains ALS though Daniela C. Zarnescu and colleagues

(University of Arizona, USA) focus on TAR DNA binding protein (TDP-43) and fused in

sarcoma (FUS), two RNA binding proteins that not only associate with ubiquitinated

Preface ix

intracellular inclusions but also act as causative agents of disease in view of the recent

discovery of MND-linked mutations in the respective gene. The identification of these

proteins in addition to the involvement of additional RNA binding proteins such as Senataxin

and Angiogenin as well as RNA itself (C90RF72 noncoding expanded repeats) in ALS,

prompted an ‘earth-shaking’ shift in our thinking about the pathophysiology of ALS whereby

RNA metabolism is presently seen as a central disrupted pathway in the presence of disease.

The authors discuss the contribution of the fruit fly to our understanding of ALS through

a review of the studies on TDP-43 and FUS. At a sequence level, both these proteins share

several domains including those that enable RNA binding and nucleocytoplasmic shuttling.

They also have a similar job description and in this regard, they have been implicated in

several RNA processing steps including transcriptional regulation, mRNA splicing, miRNA

processing as well as mRNA transport and local translation. Drosophila was vital for

demonstrating a link between FUS and TDP-43, whereby through genetic interaction

approaches it was shown that FUS acts downstream of TDP-43. A flurry of studies have tried

to unravel the function of these two proteins through loss-of-function of the orthologous gene

or overexpression of either wild-type or mutant human protein in an otherwise wild-type

background (the Drosophila orthologue of the disease gene is intact). Interestingly, in the

case of TDP-43, overexpression studies tended to produce similar ALS-like phenotypes to

those observed on loss-of-function which raise the question of whether protein

overexpression mirrors the loss-of-function condition. The authors also discuss the genetic

interactions reported in the fly models of TDP-43 and FUS which highlight the cellular

pathways that are functionally important in ALS including protein folding, proteasome-

mediated degradation, apoptosis and microtubule organisation. Differences do exist between

fly models of ALS and human pathology such as the absence of ubiquitinated inclusions,

which are a hallmark feature of the disease. However, the fly model could be telling us that

cytoplasmic aggregates are not a prerequisite for motor neuron degeneration and most

probably these pathological inclusions are a consequence rather than a cause of motor neuron

injury. Rest assured that you will hear about further twists in this riveting story in the years to

come.

It is worth noting that whereas Drosophila was heavily exploited to make great strides in

deciphering the biology of ALS-linked VAMPB, TDP-43 and FUS, the same cannot be said

of SOD1. In this regard, only a few scattered reports exist in the literature. Particularly,

Watson et al. (J Biol Chem 2008) report that expression of wild-type or disease-linked

mutants of human SOD1 selectively in motor neurons induced progressive climbing defect

which were accompanied by defective neural circuit electrophysiology, focal accumulation of

the human SOD1 protein and stress response in glia surrounding motor neurons. The utility of

Drosophila to model SOD1 pathophysiology might have been overtaken by the mutant SOD1

mouse model of ALS. However, since therapeutic success in the mouse model has not

translated into effective therapy for human ALS in clinical trials (Benatar Neurobiol Dis

2007; Aggarwal & Cudkowicz Neurotherapeutics 2008), the use of Drosophila for high-

throughput screening to identify pharmacological and genetic modifiers of disease phenotype

might eventually come of age.

The length of motor axons, which can be up to 105 times longer than that of cell bodies,

presents great challenges to the subcellular trafficking machinery of motor neurons.

Impairment of the mechanisms that maintain axonal function can lead to axon degeneration

diseases, particularly in the distal regions of the axons that lie furthest from the cell body.

Ruben J. Cauchi x

Chapter Four deals with one such group of diseases, namely hereditary spastic paraplegias

(HSPs), which are characterised by progressive spasticity and weakness in lower extremities,

caused by progressive distal axonopathy mostly in the longest upper corticospinal motor

neurons. The large number of genetic loci identified as causative explains the phenotypic

heterogeneity of HSPs although the gene products point at an unexpectedly limited range of

disease mechanisms, including endoplasmic reticulum organisation and function, axonal

microtubule-based transport and endosomal trafficking and signalling, mitochondrial function

as well as the interactions of axons with the myelin sheath.

Most but not all causative human genes have orthologues in Drosophila. In view of the

powerful genetic tools for generation of specific mutant or transgenic flies, as well as the

myriad of analytic tools for understanding the cellular roles of these gene products in neurons,

particularly in axons and synapses, Drosophila offers a compelling system to study HSP-

linked genes as well as the consequences on mutation. Belgin Yalçın and Cahir J. O’Kane

(University of Cambridge, UK) review the major contributions from flies so far including the

dissection of the roles of several HSP proteins in ER organisation, transport of specific

cargoes in axons and in pathways including bone morphogenetic protein (BMP) signalling.

As additional HSP-linked proteins are identified, the fly model offers a great opportunity to

understand their cellular roles and ultimately provide plausible mechanisms for these

diseases.

Charcot-Marie-Tooth (CMT) disease is characterised by the degeneration of peripheral

motor and sensory neurons, leading to progressive muscle weakness and wasting, and sensory

loss. The disease is clinically heterogeneous although electrophysiological and pathological

criteria allow the distinction between demyelinating, axonal and intermediate forms of CMT.

Since more than 30 genes have been causally associated with CMT to date, the disease is also

genetically heterogeneous. These genes encode proteins with often very different molecular

functions suggesting that peripheral motor and sensory neuropathy can result from

impairment of multiple molecular pathways including myelination and myelin maintenance,

axonal transport, mitochondrial dynamics, endosomal trafficking, axon-Schawann cell

interaction, transcriptional regulation and protein chaperone activity. The exact molecular

underpinnings of the peripheral motor and sensory neuropathy are still poorly understand, and

there is no effective drug treatment available. Chapter Five concentrates on the use of

Drosophila as a genetic organism to model CMT in view of the possibility of studying the

effect of CMT-associated mutant proteins on motor and sensory neurons in their

physiological context as well as the suitability of this model system to perform genetic

screens. The organisational principles of the nervous system as well as basic

neurophysiological principles including but not limited to conduction of action potentials,

signal transmission through release of neurotransmitters and the synaptic vesicle cycle, are

remarkably conserved between flies and humans. The development and anatomy of the

Drosophila neuromuscular system is beautifully described in this section.

Mutations in the genes encoding tyrosyl-tRNA synthetase (YARS), glycyl-tRNA

synthetase (GARS), alanyl-tRNA synthetase (AARS) and possibly lysyl-tRNA synthetase

(KARS) and histidyl-tRNA synthetase (HARS) give rise to axonal and intermediate forms of

CMT. Such enzymes ligate amino acids to their cognate tRNA and therefore catalyse an

important step in protein synthesis. Aimed at illustrating the usefulness of the fly as a model

for CMT, Georg Steffes and Erik Storkebaum (Max Planck Institute for Molecular

Biomedicine, Germany) highlight the features of the Drosophila model of CMT associated

Preface xi

with mutations in YARS, which aminoacylates tyrosyl-tRNA with tyrosine. The authors

underline a series of experiments, the results of which suggest unexpectedly that loss of

aminoacylation activity per se is not necessary to cause peripheral motor and sensory

neuropathy, although the possibility that altered subcellular localization of aminoacylation-

active mutants could lead to defects in local protein synthesis and terminal axonal

degeneration cannot be excluded at the present moment. Current evidence suggests that the

disease may be caused by a gain-of-toxic function mechanism, the molecular nature of which

remains elusive. The future use of Drosophila CMT models in genetic screens for disease-

modifying genes may be of great value to unravel the molecular mechanisms of disease, and

to identify possible therapeutic targets.

Chapter Six focuses on spinal and bulbar muscular atrophy (SBMA) or Kennedy’s

disease, a progressive X-linked motor neuron disorder arising from the build-up of toxic

aggregates due to an abnormal expansion of the glutamine tract in the androgen receptor (AR)

gene as well as loss of the endogenous function of the AR. SBMA forms part of an ensemble

of neurodegenerative disorders referred to as polyglutamine (polyQ) diseases which, although

affecting different neuronal subtypes, share several features including an earlier disease onset

and a more acute disease progression, the longer the glutamine expansion. Interestingly,

polyQ-expanded AR is necessary to induce motor impairment, hence suggesting a gain-of-

function by the pathogenic AR in motor neurons and in this regard, patients with loss-of-

function mutations in the AR gene only show androgen insensitivity.

Fly models of SBMA were key to establish that toxicity is dependent on glutamine length

as well as the ligand-dependent activation of the AR, hence, flies that ectopically express the

human AR with either a non-toxic glutamine tract or with an expanded glutamine tract in the

absence of dihydrotestosterone exhibit no neurodegeneration or motor defects. Activation

gives the mutant AR unrestrained access to the nucleus where it is thought to alter numerous

processes. In this context, restricting the mutant AR to the cytosol by genetic manipulation

strategies in the fly was shown to abolish toxicity. Furthermore, Drosophila studies, including

genetic screens for modifiers of polyQ-induced eye degeneration (‘rough-eye phenotype’),

revealed several cellular pathways such as gene expression, axonal trafficking and

mitochondrial physiology that are affected by the disease. Considering that a myriad of

cellular mechanisms sustain a negative impact from a polyQ pathology and highlighting

evidence from SBMA model organisms, Adrienne M. Wang (University of Washington,

USA) makes the case for the stimulation of the cell’s innate protein quality control pathways

as one of the best therapeutic approaches aimed at clearing the mutant protein upstream of its

toxic effects.

Chapter Seven addresses spinal muscular atrophy (SMA) which is the most common

autosomal recessive disorder in the population following cystic fibrosis. The causative gene is

the survival of motor neuron 1 (SMN1), where its homozygous loss in patients with SMA

leads to a situation of low SMN levels resulting from a partially-functional duplicate gene,

SMN2. SMN2 copy number is inversely correlated with disease severity and in this regard,

SMA is usually classified into three types. SMN forms a multimeric complex that participates

in the cytoplasmic phase of spliceosomal Uridine-rich small nuclear ribonucleoprotein

biogenesis. Stuart J. Grice and colleagues (University of Oxford, UK and University of North

Carolina, USA) review the present fly models of SMA by giving a thorough description of

the Smn mutant and transgenic flies that were generated so far. Importantly, the authors

highlight the developmental defects observed in Drosophila SMA models including alteration

Ruben J. Cauchi xii

in P body organisation and nuclear architecture in the Smn knockout germline; growth

defects, stem cell defects, abnormal neuromuscular junction morphology and reduced motor

function at the larval stage; and, flight defects as well as muscle atrophy in the adult stage.

Importantly, this chapter discusses the recent thrilling findings in Drosophila, which shed

light on the selective vulnerability of motor systems. Interestingly, such studies introduce a

concept that was first observed in ALS, specifically, the possibility that a subpopulation of

neurons might be prone to degeneration as a result of alterations in the function of neuronal

circuits that impinge onto these neurons.

The final chapter (Chapter Eight) of this collection revolves around the application of

Drosophila in the conduction of genetic screens aimed at identifying novel genes that cause

motor neuron degeneration or finding modifiers – enhancement or suppression – of the

phenotype resulting from disruption of MND causative genes. Both approaches hold promise

to decipher the molecular mechanisms underpinning both normal physiology and

pathophysiology. Patrik Verstreken and colleagues (KU Leuven, Belgium) first highlight the

key features of the screen phenotype as well as discussing some of the phenotypes that are

amenable to genetic screens such as lifespan, behaviour (crawling of larvae during the larval

stage and climbing or flight during the adult stage), retinal morphology, electroretinogram

(ERG) recordings and NMJ architecture.

Through the use of engaging diagrams, the authors also discuss different screening

strategies including classic genetic screens using either Ethyl Methane Sulphonate (EMS)-

based or transposable element (TE)-based mutagenesis; clonal genetic screens, where

homozygous tissue is generated in an otherwise heterozygous animal, hence allowing the

investigator to assess the phenotypes of genes required for organismal viability; dominant

genetic screens, which aim at identifying dominant modifiers of a phenotype; and, UAS/Gal4-

based screens, whereby the use of the potent UAS/Gal4 system enables researchers to

manipulate gene expression through either overexpression or RNAi-mediated knockdown in a

spatially- and temporally-controlled manner. This account also includes examples of genetic

screens that were successful in fly models of MND including SMA, SBMA and ALS.

Furthermore, the plethora of genetic tools presently available are adequately described, and

exploiting their use will undoubtedly help us to further understand the molecular mechanisms

giving rise to MNDs. Importantly, the ability to conduct high-throughput genetic screens with

relative ease augurs well for the future application of high-throughput pharmacological

screens aimed at identifying novel therapeutics, which can be considered as the final frontier

in MND research.

Whilst editing this assemblage of stimulating works, I confess that I have learned several

interesting things even though I have been in the ‘business’ for quite some time. In this

regard, I am definitely convinced that this timely collection will be welcomed not only by

Drosophilists and MND aficionados alike but also by newcomers to the field. Whilst thanking

wholeheartedly the authors for their expert contribution, I would like to invite the reader to

enjoy this volume, an inspiring look at the indispensability of the fruit fly, and of model

organisms in general, to neuroscience research.

Ruben J. Cauchi

Dept. of Physiology & Biochemistry,

Faculty of Medicine & Surgery,

University of Malta, MALTA G.C.

January 2013

Preface xiii

The author’s laboratory acknowledges the support of research grants from the University

of Malta and Malta’s National Research & Innovation Programme.

In: Drosophila Melanogaster Models of Motor Neuron Disease ISBN: 978-1-62618-747-4

Editor: Ruben J. Cauchi © 2013 Nova Science Publishers, Inc.

Chapter 1

GENETICS OF MOTOR NEURON DISORDERS:

FROM GENE DIVERSITY

TO COMMON CELLULAR CONSPIRATORS

IN SELECTIVE NEURONAL KILLING

Rebecca K. Sheean and Bradley J. Turner Florey Institute of Neuroscience and Mental Health, University of Melbourne,

Parkville, Victoria, Australia

ABSTRACT

Motor neuron disorders (MNDs) are a spectrum of progressive and incapacitating

neurological diseases leading to motor impairment, disability and in most cases death.

The clinical phenotype of MNDs depends on the relative degeneration of (a) upper and

lower motor neurons and (b) their proximal cell bodies and distal axons. Despite limited

progress in the search for effective therapies, there has been recent and explosive

discovery of dozens of causative genes in MNDs. This arsenal of genetic evidence has

illuminated key cellular pathways disrupted in motor neurons, axons and supporting

ensheathing glial cells, providing fresh insights into determinants of neuronal

susceptibility and loss in MNDs. Strikingly, most of these genes encode ubiquitously

expressed and fundamental proteins that, when defective, provoke selective motor neuron

killing. In this chapter, we provide an introduction to the core clinical and pathological

features of upper motor neuron disorders (primary lateral sclerosis and hereditary spastic

paraplegia); lower motor neuron disorders (progressive muscular atrophy, progressive

bulbar palsy, spinal muscular atrophy, spinal bulbar muscular atrophy, axonal Charcot-

Marie-Tooth disease which significantly overlaps with distal hereditary motor

neuropathy, and lethal congenital contracture syndrome); and finally amyotrophic lateral

sclerosis with combined upper and lower motor neuron loss. We provide a systematic

summary of MND causative genes and functionally group them according to six

dominant pathogenic themes established and emerging in MNDs: (1) oxidative stress and

Correspondence: Bradley Turner, Florey Institute of Neuroscience and Mental Health, Kenneth Myer Building,

University of Melbourne, Parkville, Victoria 3010, Australia. Tel: +61 3 90356521, Fax: +61 3 93470446,

Email: [email protected].

Rebecca K. Sheean and Bradley J. Turner 2

mitochondrial dysfunction, (2) dysregulated intracellular membrane trafficking, (3)

abnormal axonal transport and cytoskeleton, (4) defective RNA processing, (5) impaired

proteostasis, and (6) defective myelination and lipid metabolism. We also highlight the

seminal contribution of transgenic, knockout and mutant mice in elucidating MND gene

function and testing disease hypotheses and therapeutic interventions, which are

complemented by other model systems such as Drosophila.

Keywords: Amyotrophic lateral sclerosis; Charcot-Marie-Tooth disease; hereditary spastic

paraplegia, spinal muscular atrophy; spinal bulbar muscular atrophy

ABBREVIATIONS

AD, autosomal dominant

ALS, amyotrophic lateral sclerosis

AR, autosomal recessive

CMT, Charcot-Marie-Tooth disease

CST, corticospinal tract

dHMN, distal hereditary motor neuropathy

DI, dominant intermediate

FALS, familial amyotrophic lateral sclerosis

FTD, frontotemporal dementia

FTDP, frontotemporal dementia-Parkinsonism

HNA, hereditary neuralgic amyotrophy

HSP, hereditary spastic paraplegia

LCCS, lethal congenital contracture syndrome

LMN, lower motor neuron

PBP, progressive bulbar palsy

PLS, primary lateral sclerosis

PMA, progressive muscular atrophy

RI, recessive intermediate

SALS, sporadic amyotrophic lateral sclerosis

SBMA, spinal bulbar muscular atrophy

SMA, spinal muscular atrophy

SPG, spastic paraplegia

X-linked D, X-linked dominant

X-linked R, X-linked recessive

INTRODUCTION

Motor neuron disorders (MNDs) are a collection of clinically and pathologically

heterogeneous neurological diseases characterised by progressive and selective dysfunction

and degeneration of motor neurons. They strike adults, children and infants, occurring in

sporadic and hereditary forms, causing progressive motor disability and in most cases

premature death. The nature and progression of clinical symptoms depends on the relative

Genetics of Motor Neuron Disorders 3

involvement of two groups of neurons primarily affected, upper motor neurons (UMNs) and

lower motor neurons (LMNs). UMNs, also known as giant pyramidal cells or Betz cells, with

diameters up to 100 m, are located in laminar sheets of layer V of the primary motor cortex

(Brodmann area 4) and project long axons to the brainstem and spinal cord through pathways

such as the corticobulbar and corticospinal tracts (CST) (Ravits and La Spada, 2009). These

descending pathways synapse directly, or in most cases indirectly via interneurons, with

LMNs in humans. LMNs, also called anterior horn cells in the spinal cord, with diameters

ranging 35-100 m, are stacked into columns in motor nuclei in the tegmentum of the

brainstem and ventral horns of the spinal cord and send axons to innervate skeletal muscles

(Ravits and La Spada, 2009).

Table 1. Motor neuron disorders classification and clinical features

MN

involvement

Disease Onset Duration Prevalence Clinical features

UMN HSP Childhood Non-fatal 2-5/100,000 Progressive lower limb

spasticity

PLS 50 years 20 years 1/100,000 Slowly progressive limb

and bulbar spasticity

LMN PMA 45-65 years 5-10 years 1/100,000 Slowly progressive

weakness and atrophy

PBP 50-70 years 1-3 years 5-6/100,000 Progressive bulbar

weakness

SMA 6 months 2 years 1/6,000 Symmetrical weakness

and atrophy

SBMA 30-60 years Non-fatal 1-2/100,000 Limb and bulbar

weakness and atrophy,

gynecomastia

CMT/dHMN

Adolescence Non-fatal 1/2,500 Distal lower limb

weakness and

atrophy

LCCS Foetal 10-weeks 1/25,000 Severe muscle atrophy,

foetal hydrops and

akinesia

UMN and

LMN

ALS 45-65 years 2-3 years 7/100,000 Rapidly progressive

spasticity, weakness,

atrophy and paralysis

Abbreviations: ALS, amyotrophic lateral sclerosis; CMT, Charcot-Marie-Tooth disease; dHMN, distal

hereditary motor neuropathy; HSP, hereditary spastic paraplegia; LCCS, lethal congenital

contracture syndrome; LMN, lower motor neuron; PBP, progressive bulbar palsy; PLS, primary

lateral sclerosis; PMA, progressive muscular atrophy; SBMA, spinal bulbar muscular atrophy;

SMA, spinal muscular atrophy.


LMNs are divided into three functional classes: -motor neurons which innervate skeletal

muscle fibres and drive contraction and voluntary movement, -motor neurons which

innervate muscle spindles and sense motor control, and -motor neurons which innervate

both muscle fibres and spindles (Kanning et al., 2010). -motor neurons which are the most

abundant are further classed into fast-fatiguable, fast fatigue-resistant and slow subtypes,

reflecting the contractile properties of their target muscle fibres (Kanning et al., 2010).

Interestingly, fast-fatiguable -motor neurons with large somas and large diameter fast

conducting axons are most vulnerable in MNDs (Pun et al., 2006). MNDs are classified

according to the nerve cells affected whether UMN, LMN or both and the resulting clinical

signs (Table 1). UMN damage is manifested by spasticity and pathological reflexes such as

Babinski and Hoffman signs (Talbot, 2009).

These symptoms arise primarily from the loss of descending motor control and inhibition

of spinal cord reflexes. In contrast, LMN degeneration results in muscle weakness, atrophy,

paralysis and fasciculation caused by chronic muscle denervation (Talbot, 2009). Pure UMN

disorders are hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS). Pure

LMN involvement occurs in progressive muscular atrophy (PMA), progressive bulbar palsy

(PBP), spinal muscular atrophy (SMA), spinal bulbar muscular atrophy (SBMA), Charcot-

Marie-Tooth (CMT) disease which occurs in demyelinating (CMT1 and CMT4) or axonal

forms (CMT2), the latter significantly overlapping with distal hereditary motor neuropathy

(dHMN), and lethal congenital contracture syndrome (LCCS). Finally, amyotrophic lateral

sclerosis (ALS) is distinguished by both UMN and LMN degeneration. ALS, PLS, PMA and

PBP represent the spectrum of motor neuron diseases which are defined by adult-onset and

progressive symptoms that are uniformly fatal and pathologically distinct from SMA, SBMA,

CMT and LCCS, the key differences being the presence of neuronal ubiquitinated inclusions

commonly containing TAR DNA binding protein 43 (TDP-43) and extensive gliosis. In this

chapter, we will refer to all the diseases listed in Table 1 as MNDs which encompasses motor

neuron diseases.

1. AMYOTROPHIC LATERAL SCLEROSIS

ALS, also known as Lou Gehrig's disease in North America or MND in Commonwealth

nations, is the most common motor neuron disease accounting for 85% of cases (Talbot,

2002). It is characterised by a combination of muscle weakness, wasting and spasticity with

rapid progression to respiratory paralysis and death usually within 2-3 years from diagnosis

(Talbot, 2009). Approximately one-third of patients present with lower-limb onset symptoms,

one-third with upper-limb symptoms, and one-third with bulbar-onset (Talbot, 2009). In cases

of limb-onset ALS, symptoms often appear focal and asymmetrical and spread outwardly,

laterally and ascendingly, suggesting dissemination of pathology (Ravits and La Spada,

2009). Although ALS involves death of both UMNs and LMNs, there is evidence for a dying-

back axonopathy preceding cell body loss, at least for LMNs (Fischer et al., 2004). Motor

neuron loss is also associated with cytoplasmic accumulation of misfolded proteins, notably

TDP-43 in over 90% of ALS cases (Neumann et al., 2006) and superoxide dismutase 1

(SOD1) in approximately 50% of ALS cases examined (Bosco et al., 2010).


Table 2. Familial ALS genes and cellular functions

Type

Inheritance Onset Gene Protein Cellular function Reference

ALS1 AD Adult SOD1 Superoxide

dismutase 1

Oxidative stress (Rosen et

al., 1993)

ALS2 AR Juvenile ALS2 ALS2/alsin Endosomal

trafficking

(Hadano et

al., 2001;

Yang et al.,

2001)

ALS4 AD Juvenile SEXT Senataxin RNA processing (Chen et al.,

2004)

ALS5 AR Juvenile SPG11 Spatacsin Endosomal

trafficking

(Stevanin et

al., 2007)

ALS6 AD Adult FUS Fused in sarcoma RNA processing (Kwiatkows

ki et al.,

2009; Vance

et al., 2009)

ALS8 AD Adult VAPB Vesicle-associated

protein B

ER and Golgi

trafficking

(Nishimura

et al., 2004)

ALS9 AD Adult ANG Angiogenin RNA processing (Greenway

et al., 2006)

ALS10 AD Adult TARDBP TAR DNA

binding protein 43

RNA processing (Sreedharan

et al., 2008)

ALS11 AD Adult FIG4 Factor induced

gene 4

Endosomal

trafficking

(Chow et al.,

2009)

ALS12 AD Adult OPTN Optineurin Golgi and

endosomal

trafficking

(Maruyama

et al., 2010)

ALS13 AD Adult ATXN2 Ataxin-2 RNA processing (Elden et al.,

2010)

ALS14 AD Adult VCP Valosin-

containing protein

Ubiquitin-

proteasome

degradation

(Johnson et

al., 2010)

ALS15 X-linked D Adult UBQLN2 Ubiquilin 2 Ubiquitin-

proteasome

degradation

(Deng et al.,

2011)

ALS16 AD Adult SIGMAR1 non-opioid

intracellular

receptor 1

ER trafficking (Al-Saif et

al., 2011)

ALS-

FTD2

AD Adult C9ORF72 C9ORF72 RNA processing (DeJesus-

Hernandez

et al., 2011;

Renton et

al., 2011)

ALS-

FTD3

AD Adult CHMP2B Charged

multivesicular

body protein 2B

Endosomal

trafficking

(Parkinson

et al., 2006)

ALS-

FTDP

AD Adult MAPT Microtubule-

associated protein

tau

Cytoskeleton (Hutton et

al., 1998)


Table 2. (Continued)

Type

Inheritance Onset Gene Protein Cellular function Reference

ALS AD Adult DCTN1 Dynactin 1 Axonal transport (Puls et al.,

2003)

ALS

AD Adult DAO D-amino acid

oxidase

Translation (Mitchell et

al., 2010)

ALS AD Adult SQSTM1 Sequestosome 1 Ubiquitin-

proteasome

degradation

(Fecto et al.,

2011)

ALS

AD Adult PFN1 Profilin 1 Cytoskeleton (Wu et al.,

2012)

Abbreviations: ALS, amyotrophic lateral sclerosis; AD, autosomal dominant; AR, autosomal recessive;

FTD, frontotemporal dementia; FTDP, frontotemporal dementia-Parkinsonism; X-linked D, X-

linked dominant.

The finding of TDP-43 pathology in affected neurons in frontotemporal dementia (FTD)

and co-occurrence of ALS-FTD strongly suggests that ALS represents one extreme of a

clinical spectrum with a similar underlying pathological process, the other extreme being

FTD (Neumann et al., 2006). PLS and PMA (or Duchenne-Aran disease) are rare variants of

motor neuron disease which account for 1-2% and 4% of cases, respectively (Talbot, 2009).

Both are slowly progressive disorders involving predominantly spastic paresis in PLS and

muscle wasting in PMA. PBP is also regarded as distinct from bulbar-onset ALS due to

weakness that rapidly generalises to limbs (Talbot, 2009). While PLS, PMA and PBP are

considered subtypes of motor neuron disease, they may also be phenotypic manifestations of

ALS. It is well established that 5-10% of ALS cases are inherited. To date, 25 loci and 20

genes have been identified in familial ALS (FALS) involving predominantly autosomal

dominant inheritance (Table 2). Most of these genes encode proteins involved either in

intracellular membrane and organelle trafficking or RNA processing and regulation. They are

summarised here according to function and cellular pathways implicated in ALS.

1.1. Common Pathogenic Mechanisms in ALS

1.1.1. Oxidative Stress and Mitochondrial Dysfunction

Mutations in SOD1 were first identified in FALS (Rosen et al., 1993) and over 160

mutations have been reported to date (Al-Chalabi et al., 2012). SOD1 mutations account for

20% of FALS and some sporadic ALS (SALS) cases. SOD1 is a ubiquitously expressed

enzyme that binds copper and zinc ions and degrades superoxide anions produced by normal

mitochondrial metabolism. There are two main proposals for how mutant SOD1 causes motor

neuron damage in ALS. Firstly, mutations induce misfolding of SOD1 leading to abnormal

catalysis and copper-mediated chemistry, producing reactive oxygen species that damage

motor neurons (Wiedau-Pazos et al., 1996). Secondly, mutant SOD1 misfolding promotes

formation of protein aggregates that are injurious to motor neurons (Bruijn et al., 1998).

Irrespective of the primary mechanism of toxicity, tissue markers of oxidative damage and

mitochondrial dysfunction feature prominently in spinal cords of transgenic mice expressing


ALS-linked SOD1 mutations (Turner and Talbot, 2008), suggesting that oxidative stress

contributes to motor neuron degeneration in ALS.

1.1.2. Dysregulated Intracellular Membrane Trafficking

A number of causative genes have been identified in ALS which regulate endosomal

trafficking. In 2001, two groups identified mutations in ALS2 encoding the protein alsin

(Hadano et al., 2010; Yang et al., 2001). Alsin is a guanine nucleotide exchange factor for

Rab5 which is localised to early endosomes and involved in endocytosis and endocytic

trafficking. Mutations in alsin commonly ablate its vacuolar protein sorting 9 domain required

for Rab5 interaction. ALS2 null mice show only mild subclinical motor dysfunction, however

aberrant endosome and vesicle trafficking was observed, as well as a decrease in Rab5-

dependent endosome fusion and accumulation of early endosomes (Devon et al., 2006;

Hadano et al., 2006). Mutations in FIG4 encoding factor induced gene 4 have also been

identified in ALS (Chow et al., 2009). FIG4 regulates the abundance of phosphatidylinositol

3,5-bisphosphate, a signalling lipid located on late endosome membranes, and retrograde

trafficking of endosomes from the trans-Golgi network. Mutations in charged multivesicular

body protein 2B (CHMP2B) in FTD have been reported (Skibinski et al., 2005) and more

recently have been identified in ALS patients (Parkinson et al., 2006). CHMP2B is a

component of the endosomal sorting complex required for transport (ESCRT) which sorts

cargoes to form multivesicular bodies. Mutant CHMP2B expression in transgenic mice

triggers progressive axonal degeneration and accumulation of neuronal inclusions containing

p62, TDP-43 and CHMP2B (Ghazi-Noori et al., 2012). At least 4 genes linked to ALS are

involved in ER-Golgi trafficking. SIGMAR1 encodes an ER chaperone that is mutated in ALS

(Al-Saif et al., 2011), in addition to mutations in OPTN encoding optineurin, a key regulator

of membrane trafficking, exocytosis and Golgi-lysosome trafficking (Maruyama et al., 2010).

Mutations have also been identified in SPG11, encoding spatacsin, which have been linked to

ALS with juvenile onset (Orlacchio et al., 2010) and are discussed further in the HSP section

of this chapter. Finally, VAPB which encodes vesicle-associated protein B involved in ER-

Golgi trafficking is mutated in ALS (Nishimura et al., 2004).

1.1.3. Abnormal Axonal Transport and Cytoskeleton

Three genes associated with axonal transport are associated with ALS. A mutation in the

gene encoding the axonal transport protein, dynactin (DCTN1), which is important for

retrograde axonal transport, was identified in ALS patients, linking disruption of axonal

transport to ALS pathology (Puls et al., 2003). Mutations in DCTN1 are predicted to induce

its misfolding, reducing interaction of the dynactin complex with microtubules, thus slowing

axonal transport. Microtubule-associated protein tau (MAPT) has been linked to a number of

neurodegenerative diseases such as Alzheimer’s disease. Tau-positive inclusions have also

been reported in frontotemporal dementia and parkinsonism linked to chromosome 17

(FTDP), a disease that closely resembles ALS-parkinsonism-dementia complex, and

mutations in MAPT have been identified in FTDP patients (Hutton et al., 1998). These

mutations result in an imbalance in the ratio of tau isoforms and are hypothesized to

destabilise microtubules, leading to impaired axonal transport. Most recently, mutations in

profilin 1 (PFN1) were identified in ALS (Wu et al., 2012). Profilin 1 promotes actin

polymerisation and formation of microfilaments which are vital for cytoskeletal function.


Mutant PFN1 impairs axonal outgrowth and promotes formation of ubiquitinated aggregates

containing TDP-43 (Wu et al., 2012).

1.1.4. Defective RNA Processing

The discovery that TDP-43 is the major component of ubiquitinated inclusions in ALS

(Neumann et al., 2006) drew attention to the contribution of RNA processing defects in ALS.

Links between RNA metabolism and ALS had been formed prior to this, through discovery of

mutations in genes encoding senataxin (SETX) and angiogenin (ANG) (Chen et al., 2004;

Greenway et al., 2006). Mutations in senataxin cause juvenile onset ALS through a

mechanism that may disrupt its DNA/RNA helicase activity involved in transcription (Chen

et al., 2004). Angiogenin is involved in rRNA transcription and regulates expression of

neurotrophic factors such as vascular endothelial growth factor (VEGF). Mutations in ANG

cause aberrant ribosomal transcription, reduced VEGF expression and subsequently, a

reduction in cell repair and proliferation (Greenway et al., 2006). Spurred on by the discovery

of TDP-43 pathology in ALS (Neumann et al., 2006), mutations in TARDBP were soon

identified in ALS (Sreedharan et al., 2008). TDP-43 contains two RNA recognition sites,

nuclear import and localisation sequences and glycine-rich region, with majority of the

known mutations located within the glycine-rich region. TDP-43 is predicted to have a

number of RNA processing functions including regulation of gene expression, transcription

and splicing and microRNA processing. Mutations can result in both a hyper-phosphorylated,

high molecular weight product (45 kDa) and a C-terminal fragment (25 kDa) and cause the

mislocalisation of TDP-43 from the nucleus to cytoplasm (Neumann et al., 2006). TDP-43

depletion from mouse brain causes widespread and abnormal transcription and RNA splicing

defects affecting multiple gene targets (Polymenidou et al., 2011; Tollervey et al., 2011). A

number of these targets have also been identified and include the RNA/DNA binding protein,

fused in sarcoma (FUS) which is also mutated in ALS (Kwiatkowski et al., 2009; Vance et

al., 2009). FUS is a nucleoprotein containing an RNA binding motif, RGG-rich repeat,

transcriptional activation and zinc-ring finger domains which are important for RNA

processing (Vance et al., 2009). A number of similarities exist between TDP-43 and FUS,

with both proteins involved in transcriptional regulation and processing of mRNA and

microRNA. Mutations in FUS also result in its nuclear exclusion which leads to formation of

cytoplasmic FUS-containing stress granules, likely to cause a number of RNA processing

defects (Kwiatkowski et al., 2009). In addition to these DNA/RNA binding proteins,

mutations in the gene encoding D-amino acid oxidase (DAO) were reported in ALS (Mitchell

et al., 2010). DAO is responsible for catabolism of D-amino acids including D-serine and

mutations promote excess D-serine levels which may result in over-activation of NMDA

receptors leading to excitotoxicity. Non-coding gene expansions in Ataxin 2 (Elden et al.,

2010) and very recently C9ORF72 (DeJesus-Hernandez et al., 2011; Renton et al., 2011) have

also been identified in ALS. Ataxin 2 contains a polyglutamine (polyQ) repeat and expansion

in this region can cause spinocerebellar ataxia type 2 (SCA2) (Lorenzetti et al., 1997).

However, intermediate length expansions (27-33 repeats) are associated with ALS which

promote ataxin 2 interaction with TDP-43 to modify its toxicity (Elden et al., 2010). More

recently, two groups identified an expansion in the non-coding GGGGCC hexanucleotide

repeat within C9ORF72 (DeJesus-Hernandez et al., 2011; Renton et al., 2011). This

expansion was shown to cause both ALS and FTD phenotypes and accounts for ~40% of

FALS, which is the most common genetic cause identified to date (Renton et al., 2011).


Although the function of C9ORF72 is currently unknown, the expansion results in a loss of

one of the splice variants of C9ORF72 which causes the formation of RNA foci in the nucleus

(DeJesus-Hernandez et al., 2011). Like most disease-associated repeat expansions, the

C9ORF72 expansion is considered to create an accumulation of toxic mRNA and lead to

disturbances in transcription and other important RNA processes.

1.1.5. Impaired proteostasis

Proteostasis refers to protein homeostasis which is the sum of biogenesis, folding,

trafficking and degradation of intracellular and extracellular proteins. Mutations in valosin-

containing protein (VCP) which is required for maturation of autophagosomes were reported

in ALS, linking dysfunction of autophagy to motor neuron loss (Johnson et al., 2010).

Sequestosome 1 (SQSTM1), also known as p62, is another key regulator of protein

degradation, playing a dual role in both proteasome and autophagic degradation. SQSTM1 is

a ubiquitin binding protein that is found in neuronal inclusions in ALS patients (Mizuno et al.,

2006), and thus was an obvious candidate gene for ALS. This was confirmed by identification

of SQSTM1 mutations in ALS, which were predicted to cause a toxic gain-of-function,

increasing protein-protein interactions and causing deregulation of autophagic signalling

cascades (Fecto et al., 2011). Finally, mutations in the ubiquitin-like protein, ubiquilin 2

(UBQLN2) cause ALS, with UBQLN2 localised to protein inclusions of spinal cords in both

FALS and SALS patients (Deng et al., 2011). UBQLN2 is implicated in regulation of

ubiquitin-dependent protein degradation, with mutations impairing protein clearance and

enhancing inclusion formation.

2. HEREDITARY SPASTIC PARAPLEGIA

HSP, also known as familial spastic paraplegia or Strumpell-Lorrain disease, refers to a

large group of inherited disorders characterised by progressive muscle stiffness, contraction

and weakness predominantly in lower limbs, while upper limbs are largely spared

(Blackstone, 2012).

Table 3. HSP genes and cellular functions

Type Inheritance Gene Protein Cellular function(s) Reference

SPG1

X-linked R L1CAM L1 cell adhesion molecule Cytoskeleton (Jouet et al.,

1994)

SPG2

X-linked R PLP1 Myelin proteolipid protein 1 Myelination (Saugier-Veber

et al., 1994)

SPG3A AD ATL1 Atlastin 1 ER trafficking (Zhao et al.,

2001)

SPG4

AD SPAST Spastin ER and endosome

trafficking,

cytoskeleton

(Hazan et al.,

1999)

SPG5A AR

CYP7B1 Cytochrome P450 family 7

subfamily b polypeptide 1

Myelination, lipid

metabolism

(Tsaousidou et

al., 2008)

SPG6

AD NIPA1 Non-imprinted in Prader-

Willi/Angleman syndrome

region protein 1

Endosomal trafficking (Rainier et al.,

2003)




SPG7 AR SPG7 Paraplegin Mitochondrial

function

(Casari et al.,

1998)

SPG8 AD KIAA0196 Strumpellin Endosomal

trafficking,

cytoskeleton

(Valdmanis et

al., 2007)

SPG10 AD KIF5A Kinesin family member 5A

Axonal transport,

cytoskeleton

(Reid et al.,

2002)

SPG11 AR SPG11 Spatacsin

Endosomal trafficking (Stevanin et al.,

2007)

SPG12

AD RTN2 Reticulon 2 ER trafficking (Montenegro et

al., 2012)

SPG13

AD HSPD1 Heat shock protein 60 Mitochondrial

function

(Hansen et al.,

2002)

SPG15 AR ZFYVE26 Spastizin Endosomal trafficking (Hanein et al.,

2008)

SPG17

AD BSCL2 Seipin ER trafficking, lipid

metabolism

(Windpassinger

et al., 2004)

SPG18

AR ERLIN2 Erlin 2 ER and membrane

trafficking, ubiquitin-

proteasome system

(Alazami et al.,

2011)

SPG20

AR SPG20 Spartin Endosomal

trafficking, lipid

metabolism

(Patel et al.,

2002)

SPG21 AR SPG21 Maspardin Endosomal trafficking (Simpson et al.,

2003)

SPG22 X-linked R SLC16A2 Solute carrier family 16

member 2

Membrane trafficking

(Bohan et al.,

2004)

SPG31

AD REEP1 Receptor-expression

enhancing protein 1

ER trafficking,

cytoskeleton

(Zuchner et al.,

2006)

SPG39

AR PNPLA6 Neuropathy target esterase Lipid metabolism (Rainier et al.,

2008)

SPG35

AR FA2H Fatty acid 2-hydroxylase Myelination, lipid

metabolism

(Dick et al.,

2010)

SPG42

AD SLC33A1 Acetyl-CoA transporter Lipid metabolism (Lin et al.,

2008)

SPG44

AR GJC2 Connexin 47 Myelination (Orthmann-

Murphy et al.,

2009)

SPG47

AR AP4B1 Adaptor-related protein

complex 4,beta 1 subunit

Endosomal trafficking (Bauer et al.,

2012)

SPG48

AR AP5Z1 Adaptor-related protein

complex 5, zeta 1 subunit

Endosomal trafficking (Słabicki et al.,

2010)

SPG50

AR AP4M1 Adaptor-related protein

complex 4, mu 1 subunit

Endosomal trafficking (Verkerk et al.,

2009)

SPG51

AR AP4E1 Adaptor-related protein

complex 4, epsilon 1 subunit

Endosomal trafficking (Moreno-De-

Luca et al.,

2011)

SPG52

AR AP4S1 Adaptor-related protein

complex 4, sigma 1 subunit

Endosomal trafficking (Abou Jamra et

al., 2011)

Abbreviations: HSP, hereditary spastic paraplegia; AD, autosomal dominant; AR, autosomal recessive;

SPG, spastic paraplegia; X-linked R, X-linked recessive.


HSP occurs in pure spastic or complicated forms, the latter involving neurological

symptoms that may include cerebellar ataxia, dementia, mental retardation and sensory

deficits (Harding, 1983). The core pathological feature of HSP is the selective degeneration of

the longest CST axons which innervate the lower extremities. HSP affects distal axons and

there is little neuronal death, thus conforming to a distal axonopathy, and most patients have

normal lifespans (Blackstone, 2012). HSPs are the most genetically diverse of MNDs with

nearly 50 distinct loci and 30 genes identified to date, involving X-linked recessive,

autosomal dominant or autosomal recessive inheritance (Table 3). Most of these genes encode

proteins involved in intracellular membrane trafficking, cytoskeletal function, organelle

shaping and lipid metabolism in axons. We group and discuss them below according to

function and cellular pathways implicated in HSP pathogenesis.

2.1. Common Pathogenic Mechanisms in HSP


Paraplegin and heat shock protein 60 (HSP60) are mitochondrial matrix proteases and

chaperones, respectively, involved in regulation of mitochondrial protein quality control

(Casari et al., 1998; Hansen et al., 2002). HSP patients with paraplegin mutations show

abnormal mitochondrial morphology and energetic defects in skeletal muscle (Casari et al.,

1998), while paraplegin knockout mice develop gait abnormalities preceded by distal

degeneration of central and peripheral axons, accumulation of mitochondria in axonal

swellings and slowing of retrograde axonal transport (Ferreirinha et al., 2004). The phenotype

of HSP60 deficient mice although masked by embryonic lethality, was attributed to

mitochondrial dysfunction early in development (Christensen et al., 2010).


The majority of HSP genes encode proteins involved in endosomal trafficking, such as

NIPA1, strumpellin, spatacsin, spastizin, spartin and maspardin. NIPA1 is an integral

membrane protein localised to endosomes involved in receptor signalling coupled to axonal

growth and mutations have been implicated in abnormal bone morphogenic protein (BMP)

signaling (Rainier et al., 2003). Strumpellin is an endosomal protein containing spectrin

repeats which interact with ankyrin, suggesting roles in stabilising the cell membrane-

cytoskeletal network and vesicle trafficking (Valdmanis et al., 2007). Spatacsin and spastizin

are found in the ER and endosomes with putative roles in endocytic trafficking, particularly

of inositol 1,4,5-trisphosphate receptors (IP3Rs) required for calcium signalling in neurons

(Hanein et al., 2008; Stevanin et al., 2007). Mutations in spartin which contains microtubule

and ESCRT-III interacting domains were predicted to disrupt neuronal endocytic trafficking

in HSP (Patel et al., 2002). Gene deletion of spartin results in progressive locomotor deficits

in adult mice and abnormal axonal branching of cortical neurons without evidence for LMN

loss (Renvoise et al., 2012). Lastly, maspardin which distributes to endosomes has putative

roles in vesicle trafficking between endocytic and trans-Golgi compartments (Simpson et al.,

2003). Maspardin deficient mice produce a similar phenotype to spartin knockouts, revealing

progressive hindlimb weakness and excessive branching of cortical neuron axons (Soderblom

et al., 2010), collectively implicating these two HSP genes as negative regulators of UMN


axon outgrowth. More recently, mutations in subunits of multiple adaptor protein complexes,

AP4 and AP5, involved in endocytic transport of vesicles, were found in HSP families

(Abou Jamra et al., 2011; Bauer et al., 2012; Moreno-De-Luca et al., 2011; Słabicki et al.,

2010; Verkerk et al., 2009). Interestingly, AP4 trafficks AMPA receptors to the cell surface

and HSP mutations were predicted to perturb glutamate-mediated signalling in neurons,

eliciting excitotoxicity (Verkerk et al., 2009). In mice deficient for AP4B, AMPA receptors

were mistrafficked to autophagosomes which accumulate in neurons and animals show motor

impairment (Matsuda et al., 2008). At least six HSP genes encoding atlastin 1, REEP1,

reticulon 2, seipen and erlin 2, are involved in ER formation, morphogenesis and associated

trafficking pathways. Atlastin 1 is an ER-localised GTPase belonging to the dynamin-related

superfamily of large GTPases and mutations are predicted to disrupt ER morphology and

axon outgrowth (Zhao et al., 2001b). REEP1, which interacts with atlastin 1, is involved in

receptor trafficking through the ER, binds microtubules and may play a role in maintaining

the ER network (Zuchner et al., 2006). Mice deficient for REEP1 develop adult-onset gait

abnormalities consistent with spasticity and axonal degeneration in the spinal cord (Beetz,

2010). Mutations in the prototypical ER shaping protein reticulon 2 were recently identified

in HSP, implicating abnormal ER morphogenesis in axonal degeneration (Montenegro et al.,

2012). BSCL2 or seipin is an ER integral membrane protein and HSP mutations disrupt its N-

linked glycosylation site leading to its aggregation in motor neurons (Windpassinger et al.,

2004). Transgenic mice expressing mutant seipin develop spastic motor deficits with

degeneration of peripheral axons and evidence of seipin-positive inclusions, ER stress and

impaired retrograde axonal transport (Yagi et al., 2011). Erlin 2 associates with lipid rafts and

is involved in ER-associated degradation of IP3Rs, implicating dysregulation of calcium

signalling in this form of HSP (Alazami et al., 2011). Finally, SLC16A2 which encodes a

monocarboxylic acid transporter at the plasma membrane may be linked to HSP (Bohan and

Azizi, 2004).


Spastin, L1CAM and KIF5A are key proteins involved in axon development, dynamics

and maintenance. Mutations in spastin, which account for 40% of autosomal-dominant HSP,

were predicted to disrupt its ATPase and microtubule binding domains required for

microtubule severing and cytoskeletal remodeling (Hazan et al., 1999). This was confirmed in

spastin deficient mice which develop progressive axonopathy with disruption of the

cytoskeleton and motor deficits, in the absence of neuronal loss (Tarrade et al., 2006).

L1CAM is a cell surface glycoprotein and adhesion molecule required for neuronal migration

and axon growth during development. Loss-of-function mutations in L1CAM identified in

HSP were proposed to abolish axonal pathfinding (Jouet et al., 1994). This was supported by

L1CAM knockout mice which show reduced length of CST and locomotor deficits (Dahme et

al., 1997). Lastly, mutations in the anterograde motor protein KIF5A underlie a form of HSP,

providing direct evidence of axonal transport defects leading to neurodegeneration (Reid et

al., 2002). Postnatal deletion of KIF5A in neurons provoked sensory neuron loss,

accumulation of neurofilament subunits in cell bodies and hindlimb paralysis (Xia et al.,

2003).


Table 4. Axonal CMT disease genes and cellular functions


CMT2A1 AD KIF1B Kinesin family member 1B

Axonal transport (Zhao et al.,

2001)

CMT2A2 AD MFN2

Mitofusin 2 Mitochondrial

function

(Zuchner et al.,

2004)

CMT2B AD RAB7A Ras-associated protein 7

Endosomal

trafficking

(Verhoeven et

al., 2003)

CMT2B1

AR LMNA Lamin A Cytoskeleton (De Sandre-

Giovannoli et

al., 2002)

CMT2B2

AR MED25 Mediator complex subunit 25 Transcription,

myelination

(Leal et al.,

2009)

CMT2C AD TRPV4

Transient receptor potential

cation channel, subfamily v,

member 4

Membrane

trafficking

(Auer-

Grumbach et

al., 2010)

CMT2D

AD GARS Glycyl-tRNA synthetase Translation (Antonellis et

al., 2003)

CMT2E

AD NEFL Neurofilament light chain Cytoskeleton (Mersiyanova

et al., 2000)

CMT2F AD HSPB1 Heat shock protein 27

Protein degradation (Evgrafov et

al., 2004)

CMT2H/K AD GDAP1 Ganglioside-induced

differentiation-associated

protein 1

Mitochondrial

function

(Baxter et al.,

2002; Cuesta et

al., 2002)

CMT2I/J AD MPZ Myelin protein zero

Myelination (De Jonghe et

al., 1999)

CMT2L AD HSPB8 Heat shock protein 22

Protein degradation (Irobi et al.,

2004)

CMT2M AD DNM2 Dynamin 2

Membrane

trafficking

(Zuchner et al.,

2005)

CMT2N AD AARS Alanyl-tRNA synthetase

Translation (Latour et al.,

2010)

CMT2O AD DYNC1H1 Dynein cytoplasmic heavy

chain 1

Axonal transport

(Weedon et al.,

2011)

CMT2P AR LRSAM1 Leucine-rich repeat and sterile

alpha motif containing 1

Protein degradation,

endosomal

trafficking

(Guernsey et

al., 2010)

CMT2Q

AD HARS Histidyl-tRNA synthetase Translation (Vester et al.,

2012)

CMTDIB AD DNM2 Dynamin 2

Membrane

trafficking

(Zuchner et al.,

2005)

CMTDIC AD YARS Tyrosyl-tRNA synthetase

Translation (Jordanova et

al., 2006)

CMTDID

AD MPZ Myelin protein zero Myelination (Mastaglia et

al., 1999)

CMTDIE

AD INF2 Inverted formin 2 Cytoskeleton (Boyer et al.,

2011)

CMTRIA

AR GDAP1 Ganglioside-induced

differentiation-associated

protein 1

Mitochondrial

function

(Nelis et al.,

2002)

CMTRIB

AR KARS Lysyl-tRNA synthetase Translation (McLaughlin et

al., 2010)

HNA

AD SEPT9 Septin 9 Cytoskeleton (Kuhlenbaumer

et al., 2005)

Abbreviations: ALS, AD, autosomal dominant; AR, autosomal recessive; CMT, Charcot-Marie-Tooth

disease; DI, dominant intermediate; HNA, hereditary neuralgic amyotrophy; RI, recessive

intermediate.


2.1.4. Defective Myelination and Lipid Metabolism

At least 6 HSP genes are linked to myelination and metabolism of lipids and sterols in

axons. Interestingly, 3 of these encoding proteolipid protein 1 (PLP1), connexin 47 and fatty

acid 2-hydroxylase, are expressed by oligodendrocytes, implicating non-cell autonomous

axonal degeneration in HSP. PLP1 is the major structural protein of CNS myelin and HSP

mutations were proposed to disrupt oligodendrocyte maturation and myelination (Saugier-

Veber et al., 1994). In mice either lacking PLP1 or harbouring spontaneous mutations in

PLP1 called jimpy and rumpshaker, oligodendrocyte development was normal, although there

was evidence for dysmyelination at late age (Klugmann et al., 1997; Schneider et al., 1992).

Connexin 47, together with connexin 43, are the main gap junction proteins expressed by

oligodendrocytes essential for proper maintenance of myelin and connexin 47 mutations

linked to HSP disrupt function of these heterotypic channels (Orthmann-Murphy et al., 2009).

Mice with deficiency of connexin 47 are viable, however combined deletion with connexin 32

triggers premature death resulting from severe demyelination and axonal loss (Menichella et

al., 2003). Lastly, mutations in fatty acid 2-hydroxylase which synthesises myelin

galactolipids cause a form of HSP (Dick et al., 2010) and mice lacking this gene develop CNS

demyelination and axonal degeneration (Potter et al., 2011).

CYP7B1 encodes an enzyme essential for cholesterol catabolism in the brain that is

mutated in HSP, providing direct evidence for defective cholesterol metabolism driving motor

neuron degeneration (Tsaousidou et al., 2008). Although CYP7B1 knockout mice appear

normal probably due to functional compensation by CYP7A1, inactivation of liver X receptor

which regulates CYB7B1 results in lipid accumulation and degeneration of motor neurons

in adult mice (Andersson et al., 2005). Next, neuropathy target esterase is an ER protein that

modifies phospholipids that is mutated in HSP, suggesting that altered neuronal membrane

composition can trigger axonal loss (Rainier et al., 2003). Finally, mutations in the acetyl-

CoA transporter necessary for ganglioside and glycoprotein formation in the Golgi apparatus

underlie one form of HSP, suggesting links between acetyl-CoA metabolism and axon

outgrowth and maintenance (Lin et al., 2008).

3. CHARCOT-MARIE-TOOTH DISEASE

CMT disease, also known as hereditary motor and sensory neuropathy, is one of the most

common inherited neurological diseases and peripheral neuropathy (Bucci et al., 2012). It is

characterised by slowly progressive weakness and wasting in distal muscles, commonly

affecting the foot, leading to the classical CMT phenotype of high arch and claw toe. Patients

may also present with mild distal sensory loss, hyporeflexia and skeletal deformity, although

lifespan is not generally affected (Patzko and Shy, 2011). CMT results from selective and

distal degeneration of peripheral nerves. Clinically, CMT disease is divided into two main

types according to nerve conduction velocities (NCVs) of the motor median nerve. CMT1

with NCVs below 38 m/s account for 30% of patients and CMT2 with NCVs above 38 m/s,

which is considered normal, account for 40% of patients (Bucci et al., 2012). This distinction

is useful when classifying CMT disease according to genetics (Table 4). CMT1, or

demyelinating CMT, is associated with 15 genes primarily expressed in Schwann cells,

causing peripheral demyelination and secondary axonal loss. CMT2, or axonal CMT, is


presently linked to over 20 genes expressed in neurons causing primary defects in axons

without demyelination (Bucci et al., 2012). Furthermore, some forms of CMT2 show genetic

and pathological overlap with dHMNs which are predominantly motor neuropathies with

little sensory deficits (Rossor et al., 2012).

In this chapter, we will discuss only CMT2 genes since the focus model organism of this

volume Drosophila lack clear orthologues of myelin genes and Schwann cells. Most CMT2

genes encode proteins involved in mitochondrial morphogenesis, translation machinery and

organelle, vesicle and protein transport in axons as listed below according to pathogenic

themes.

3.1. Common Pathogenic Mechanisms in CMT2


MFN2 is a mitochondrial GTPase required for mitochondrial fusion which coupled to

fission controls morphology, function and motility of mitochondria in axons (Zuchner et al.,

2004). Mutations in MFN2 linked to CMT2 are predicted to disrupt mitochondrial fusion-

fission balance in peripheral nerves (Zuchner et al., 2004). Postnatal gene deletion of MFN2

in the cerebellum causes mitochondrial pathology, electron transport chain defects and loss of

Purkinje cells (Chen et al., 2007), consistent with a role of MFN2 in neurodegeneration.

GDAP1 is implicated in mitochondrial fission and dynamics in axons and mutant forms are

linked to both autosomal dominant (Baxter et al., 2002; Cuesta et al., 2002) and recessive-

intermediate forms of CMT (Nelis et al., 2002).


At least 4 CMT2 genes are associated with membrane and protein trafficking events in

axons. Rab7A which mediates trafficking between late endosomes and lysosomes is mutated

in CMT2 (Verhoeven et al., 2003). Endocytic Rab proteins are implicated in neurite

outgrowth and polarised sorting of vesicles in neurons which may account for axonal

pathology (Verhoeven et al., 2003). LMNA which encodes lamin A/C proteins essential for

maintaining nuclear envelope architecture and transport is linked to a form of CMT2 (De

Sandre-Giovannoli et al., 2002). Interestingly, mice deficient for LMNA develop locomotor

deficits and peripheral axonopathy, consistent with CMT (De Sandre-Giovannoli et al., 2002;

Sullivan et al., 1999). Dynamin 2 is a large GTPase involved in receptor-mediated

endocytosis and trafficking from the plasma membrane which is linked to CMT (Zuchner et

al., 2005). Depletion of dynamin 2 in neurons impaired clathrin-mediated endocytosis and

myelination in mice (Sidiropoulos et al., 2012), highlighting the role of endocytic dysfunction

in axons as a determinant of CMT2. Finally, mutations in the calcium channel TRPV4 which

affect its cell surface trafficking and assembly occur in CMT2 (Auer-Grumbach et al., 2010),

again linking dysregulation of calcium signalling cascades to motor neuron degeneration.


A primary role for axon transport defects in CMT2 is underscored by discovery of

mutations in 5 genes, KIF1B, DYNC1H1, NEFL, INF2 and SEPT9. K1F1B belongs to the

same superfamily of motor proteins as KIF5A and is involved in synaptic vesicle transport


(Zhao et al., 2001a). Mutations in KIF1B associated with CMT2 were shown to abolish its

ATPase activity necessary for vesicle motility and mice partially deficient for K1F1B develop

progressive weakness, motor deficits and impaired axonal transport (Zhao et al., 2001a).

Conversely, DYN1CH1 which encodes the retrograde motor protein cytoplasmic dynein

heavy chain 1 is also mutated in CMT2 (Weedon et al., 2011). This finding was corroborated

by previous studies in two lines of ENU mutant mice, legs at odd angles and cramping 1,

which develop progressive motor deficits and axonal loss due to independent DYNCH1

mutations (Hafezparast et al., 2003). NEFL which encodes the light chain of neurofilament

protein fundamental to axonal structure, organisation and transport is mutated in CMT2

(Mersiyanova et al., 2000) and transgenic mutant NEFL mice show paralysis with selective

spinal motor neuron loss and abnormal accumulation of neurofilaments (Lee et al., 1994).

INF2 which is involved in remodelling actin filaments and microtubules is also defective in a

form of CMT and expression of INF2 variants disrupts the neuronal cytoskeleton (Boyer et

al., 2011). Lastly, SEPT9, which is implicated in filament formation with actin and tubulin is

mutated in hereditary neuralgic amyotrophy (HNA) which shows clinical overlap with CMT2

(Kuhlenbaumer et al., 2005). SEPT9 knockout mice die in utero, revealing evidence for

abnormal cell morphology, cell adhesion and mitotic spindle formation (Fuchtbauer et al.,

2011).

3.1.4. Defective Myelination and Lipid Metabolism

Interestingly, two genes linked to peripheral myelin and expressed in Schwann cells have

been linked to axonal CMT. MED25 which encodes a transcriptional activator implicated in

regulation of myelin genes is mutant in CMT2 (Leal et al., 2009). Mutations in MPZ

predicted to disrupt adhesion and compaction of myelin were reported in CMT patients with

normal NCVs (De Jonghe et al., 1999; Mastaglia et al., 1999). MPZ mutations are

paradoxically linked to CMT1, suggesting that this gene contributes to both demyelinating

and axonal forms of CMT.

3.1.5. Impaired proteostasis

Eight genes associated with translation and protein degradation have been causally linked

to CMT2 forms. HSPB1 and HSPB8 encode heat shock protein chaperones with roles in cell

stress caused by protein misfolding, stabilisation of cytoskeleton and axonal transport.

Mutations in HSPB1 and HSB8 linked to CMT2 promote their aggregation neurons (Evgrafov

et al., 2004; Irobi et al., 2004). Neuronal expression of mutant HSPB1 in transgenic mice

provokes late-onset motor deficits, slowing of retrograde axonal transport and distal axon loss

(d'Ydewalle et al., 2011). LRSAM1 is a predicted E3 type ubiquitin ligase implicated in

trafficking of polyubiquitinated proteins and endosomal vesicle trafficking that is mutated in

CMT (Guernsey et al., 2010). One of the most intriguing discoveries in CMT2, and indeed

biology, was the identification of mutations in aminoacyl tRNA synthetases which charge

tRNAs with their cognate amino acids. Mutations in at least 5 aminoacyl tRNA synthetases

have been reported to date, involving GARS (Antonellis et al., 2003), YARS (Jordanova et

al., 2006), KARS (McLaughlin et al., 2010), AARS (Latour et al., 2010) and most recently

HARS (Vester et al., 2012). How mutations in such ubiquitously expressed and fundamental

enzymes cause peripheral neuropathy is unclear, however proposals include translation

deficiency, defective aminoacylation leading to mis-incorporation of amino acids promoting

protein misfolding and abnormal distribution of tRNA synthetases in axons and terminals


(Latour et al., 2010). Insights into the pathogenesis of mutant aminoacyl tRNA synthetases in

CMT2 were gained from multiple lines of ENU mutant GARS mice which developed

locomotor deficits with peripheral denervation and neuropathy in the absence of reduced

aminoacylation activity (Achilli et al., 2009; Seburn et al., 2006), suggesting a novel toxic

gain-of-function mechanism.

Table 5. SMA and other MND genes and cellular functions

Disease/

Type

Inheritance Gene Protein Cellular function Reference

PLS

AR ALS2 ALS2/Alsin Endosomal

trafficking

(Yang et al.,

2001)

SMA AR SMN1 Survival motor neuron

RNA processing,

axonal transport

(Lefebvre et

al., 1995)

SBMA X-linked R

NR3C4 Androgen receptor Transcription (La Spada et

al., 1991)

SMAX2

X-linked R UBE1 Ubiquitin-like modifier

activating enzyme 1

Protein degradation (Ramser et al.,

2008)

SMAX3

X-linked R ATP7A Menkes copper ATPase Membrane

trafficking

(Kennerson et

al., 2010)

HMN2A AD HSPB8 Heat shock protein 22 Protein degradation (Irobi et al.,

2004)

HMN2B

AD HSPB1 Heat shock protein 27 Protein degradation (Evgrafov et

al., 2004)

HMN2C

AD HSPB3 Heat shock protein 27-like

protein

Protein degradation (Kolb et al.,

2010)

HMN4 AR PLEKHG5 Pleckstrin homology domain

containing, family G member 5

Membrane

trafficking

(Maystadt et

al., 2007)

HMN5A AD GARS

Glycyl-tRNA synthetase Translation (Antonellis et

al., 2003)

HMN5B AD REEP1 Receptor expression-enhancing

protein 1

ER trafficking,

cytoskeleton

(Beetz et al.,

2012)

HMN5C

AD BSCL2 Seipin ER trafficking, lipid

metabolism

(Windpassinger

et al., 2004)

HMN6 AR IGHMBP2

Immunoglobulin helicase -

binding protein 2

RNA processing (Grohmann et

al., 2001)

HMN7B AD DCTN1 Dynactin subunit 1 Axonal transport

(Puls et al.,

2003)

LCCS1

AR GLE1 GLE1 RNA export mediator

homolog

RNA processing (Nousiainen et

al., 2008)

LCCS2

AR ERBB3 V-ERB-B2 avian erythroblastic

leukaemia viral oncogene

homolog 3

Membrane

trafficking

(Narkis et al.,

2007b)

LCCS3

AR PIP5K1C phosphatidylinositol-4-

phosphate 5-kinase, type I,

Endosomal

trafficking

(Narkis et al.,

2007a)

Abbreviations: AD, autosomal dominant; AR, autosomal recessive; dHMN, distal hereditary motor

neuropathy; LCCS, lethal congenital contracture syndrome; PLS, primary lateral sclerosis; SBMA,

spinal bulbar muscular atrophy; SMA, spinal muscular atrophy; SMAX, X-linked spinal muscular

atrophy; X-linked R, X-linked recessive.


5. SPINAL MUSCULAR ATROPHIES AND OTHER MOTOR

NEURON DISORDERS

Spinal muscular atrophies refer to a group of inherited disorders characterised by

selective anterior horn cell death, leading to progressive muscle wasting. They are classified

according to the type of muscles affected, whether proximal or distal, and mode of inheritance

(Table 5). Autosomal recessive proximal SMA, or simply known as SMA, accounts for 95%

of cases and is the most common genetic cause of infant death (Burghes and Beattie, 2009).

SMA results from inactivating mutations in the SMN1 gene and retention of SMN2, which is

polymorphic in copy number, and determines the clinical severity of SMA, which ranges

from infant mortality by 2 years of age (SMA type 1), intermediate (SMA type 2), juvenile

(SMA type 3) and adult-onset forms (SMA type 4) with normal lifespans and mild weakness

(Lefebvre et al., 1995). X-linked SMA shows clinical overlap with SMA Type I, although it is

linked to mutations in 3 distinct genes (Table 5). Next, distal SMA which clinically and

genetically overlaps with dHMN or CMT2, is characterised by muscle weakness in the

extremities with minor sensory abnormalities (Rossor et al., 2012). Around ten genes are

associated with distal SMA or dHMN at present (Table 5). SBMA, also called Kennedy's

disease, is an X-linked recessive disorder characterised by slowly progressive weakness,

atrophy and fasciculations of bulbar, facial and limb muscles (Katsuno et al., 2012). It

predominantly affects males with onset usually in adolescence or middle adult life and is

generally not fatal (Katsuno et al., 2012). SBMA results from pathogenic expansion of a

trinucleotide repeat CAG encoding polyQ in the first exon of the AR gene and was the first

so-called polyQ disease discovered, followed by Huntington's disease (La Spada et al., 1991).

Patients also present with gynecomastia resulting from androgen insensitivity. Lastly, LCCS

is a rare autosomal recessive condition characterised by severe muscle atrophy, anterior horn

cell loss and total immobility of the foetus detected at 13 weeks pregnancy, leading to

premature death by 32 weeks gestation (Nousiainen et al., 2008). Three genes have been

linked to LCCS (Table 5). Most genes associated with SMA and these other MNDs encode

ubiquitous proteins involved in transcription, RNA processing and translation which is

striking given the selective pattern of anterior horn cell death. There are also familiar themes

such as membrane trafficking and axonal transport linked to genes underlying these MNDs as

discussed below.

5.1. Common Pathogenic Mechanisms in SMA and Other MNDs


ATP7A is a copper transporter ATPase required for copper efflux from cells and more

importantly, copper transport across the blood-brain barrier. Mutations in ATP7A occur in

dHMN, implicating defective copper trafficking and deficiency of copper-dependent enzymes

in motor neuropathy (Kennerson et al., 2010). PLEKHG5 encodes an intracellular protein

with a pleckstrin homology domain involved in cell signalling and cytoskeletal function that

is mutant in dHMN (Maystadt et al., 2007). Impaired NFB signalling is linked to axonal

degeneration in this form of motor neuropathy (Maystadt et al., 2007). Seipin and REEP1 are


also mutated in dHMN (Beetz et al., 2012; Windpassinger et al., 2004), again reinforcing the

importance of ER trafficking abnormalities in triggering axonopathy.


A mutation in the p150 subunit of dynactin, part of the retrograde motor in neurons, was

reported in dHMN and ALS (Puls et al., 2003), and together with dynein mutations in CMT,

provide a strong mechanistic link between abnormal axonal transport and neurodegeneration.

5.1.3. Defective RNA Processing

SMN1 encodes survival motor neuron (SMN) which interacts with many proteins,

including Gemins 2-8, to form the mega-Dalton SMN complex which functions in assembly

of small nuclear ribonucleoproteins involved in pre-mRNA splicing (Burghes and Beattie,

2009). There are two main proposals to account for how SMN deficiency causes SMA.

Firstly, SMN reduction may lead to abnormal pre-mRNA splicing to the detriment of motor

neurons, which is supported by findings of altered small nuclear RNA stoichiometry and

defective mRNA processing in spinal cords of SMN deficient mice (Baumer et al., 2009;

Zhang et al., 2008), although this occurred late in disease. Secondly, SMN may be important

for axonal transport of mRNAs required for distal translation in motor neurons and

maintenance of terminals, as evidenced by peripheral denervation in SMA model mice (Le et

al., 2005). IGHMBP2 encodes a DNA/RNA binding protein with helicase activity that is

mutated in SMA with respiratory distress type 1 or dHMN (Grohmann et al., 2001).

Interestingly, the spontaneous mouse mutant neuromuscular degeneration or nmd, which

develops limb and respiratory weakness, results from an IGHMBP2 mutation (Cox et al.,

1998).

The androgen receptor (AR) is a nuclear receptor which mediates the effects of

androgens, testosterone and dihydrotestosterone, by binding androgen response elements in

target genes (Katsuno et al., 2012). Interestingly, AR is highly expressed by anterior horn

cells where it is also required for trophic factor signalling (Katsuno et al., 2012). Transgenic

mice expressing an expanded AR develop progressive and fatal muscle weakness and nuclear

polyglutamine inclusions characteristic of SBMA (Adachi et al., 2001). Two main

mechanisms have been proposed to link pathogenic expanded AR to motor neuron death.

Firstly, the loss of normal AR signalling contributes to neurodegeneration, presumably by

disrupting transcriptional activities linked to neuronal survival. Secondly, expanded AR

acquires properties that are toxic to motor neurons, such as nuclear accumulation of

inclusions, leading to transcriptional dysregulation (Katsuno et al., 2012). GLE1 linked to

LCCS encodes a protein involved in mRNA export from the nucleus to cytoplasm in cells and

mutations are predicted to disrupt gene expression required for early development and

maturation of anterior horn cells (Nousiainen et al., 2008). Interestingly, LCCS mutations in

ERBB3 and PIP5K1C encoding regulators of the phosphatidyl inositol pathway, PIPK and

HER3, are linked to nuclear mRNA export, again pointing towards defective mRNA transport

in promoting motor neuron degeneration (Narkis et al., 2007a; Narkis et al., 2007b).

5.1.4. Impaired Proteostasis

Five genes associated with intracellular protein homeostasis have been linked to SMA or

dHMN. The first UBE1 encodes an E1 ubiquitin activating enzyme that catalyses the first step


in protein ubiquitination and mutations were found in X-linked SMA, implicating defective

protein degradation (Ramser et al., 2008). HSPB1 and HSPB8 mutations were also reported

in dHMN (Evgrafov et al., 2004; Irobi et al., 2004). A third heat shock protein HSPB3 was

recently implicated in dHMN (Kolb et al., 2010), strengthening the case for defective protein

chaperone activity in causing motor neuropathies. Lastly, GARS mutations were also linked

to dHMN (Antonellis et al., 2003), in addition to CMT.

CONCLUSION

Nearly 100 genes have been linked to susceptibility and degeneration of upper and motor

neurons and their distal axons in MNDs to date. Despite the clinical, pathological and genetic

diversity of MNDs, this accumulation and wealth of genetic evidence has highlighted key

cellular pathways and molecular biology disturbed in all forms of MNDs, leading to proposal

of six dominant and unifying pathogenic themes: (1) oxidative stress and mitochondrial

defects; (2) dysregulation of intracellular vesicle, membrane and organelle traffic; (3)

abnormalities in axonal transport and cytoskeletal architecture; (4) dysmyelination and

impaired lipid metabolism in axons; (5) defective splicing and transport of RNA species; and

(6) abnormal proteostasis. These pathways offer important insights into pathogenesis of both

inherited and sporadic forms of MNDs and selection of urgently needed therapeutic targets.

ACKNOWLEDGMENTS

This work was supported by an Australian NHMRC Project Grant 1008910, Mick

Rodger MND Research Grant from MND Research Institute of Australia and Bethlehem

Griffiths Research Foundation Grant. The Florey Institute of Neuroscience and Mental Health

acknowledges the strong support from the Victorian Government and in particular the funding

from the Operational Infrastructure Scheme.

ABOUT THE AUTHORS

Bradley J. Turner is a Research Fellow at the Florey Institute of Neuroscience and

Mental Health at the University of Melbourne, Australia. His research interests include

functional modelling of genes linked to amyotrophic lateral sclerosis (ALS), spinal muscular

atrophy (SMA) and distal hereditary motor neuropathy (dHMN), and investigating the

molecular pathogenesis of ALS in mouse and rat models. His work is focused on the role of

exosomes in the processing, secretion and propagation of misfolded proteins linked to

neurodegeneration, autophagy dysregulation in neuronal death and therapeutic application of

survival motor neuron protein for broad motor neuron injury and disorders.

Rebecca K. Sheean is a Postdoctoral Researcher at the Florey Institute of Neuroscience

and Mental Health at the University of Melbourne, Australia. Her research interests include

investigating the cellular and molecular mechanisms underlying motor neuron degeneration in


ALS, focusing on the role of the endosome-lysosome system and interactions between glial

cells and the immune system in ALS pathogenesis.

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Chapter 2

A SECRETED LIGAND FOR GROWTH CONE

RECEPTORS, VAP MEDIATES THE CELLULAR

PATHOLOGICAL DEFECTS IN ALS

Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda* Department of Neurology and Neurosurgery,

Montreal Neurological Institute, McGill University, Montreal, Canada

ABSTRACT

Drosophila studies have contributed greatly to our understanding of the biological

pathways that might be relevant to the pathogenesis of amyotrophic lateral sclerosis

(ALS). ALS is a progressive neurodegenerative disorder characterized by selective death

of motor neurons. Human VAMP associated protein B (VAPB) is the causative gene of a

familial form of ALS, ALS8. Human VAPB, Drosophila VAP33, and C. elegans VPR-1

are homologous type II transmembrane proteins containing a highly conserved major

sperm protein (MSP) domain in their N-terminal region. A point mutation (P56S) in the

human VAPB MSP domain causes ALS8. Drosophila studies revealed that VAP is

secreted in a cell type-specific fashion and acts as a diffusible hormone. VAP is a ligand

for Ephrin (Eph) and Roundabout (Robo), two receptors originally identified as mediators

of growth cone guidance cues. The ALS mutation causes two different types of defects:

mutant VAP fails to be secreted (loss-of-function) and accumulates as ubiquitinated

inclusions in the endoplasmic reticulum (ER), resulting in ER stress (gain-of-function),

which is a common defect observed in the pathology of familial and sporadic forms of

ALS. Thus, Drosophila studies have revealed the cellular defects associated with mutant

VAP and have helped us understand key pathological features of ALS.

Keywords: Neurodegeneration, ER stress, Mitochondria, Eph, Robo, LAR

* Correspondence: [email protected].

mailto:[email protected]

Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda 36

ABBREVIATIONS

ALS Amyotrophic lateral sclerosis

ER endoplasmic reticulum

LAR Leukocyte common antigen-related

MSP Major sperm protein

Robo Roundabout

SOD1 superoxide dismutase 1

VAP VAMP associated protein

INTRODUCTION

Neurodegenerative disorders constitute one of the major challenges of modern medicine.

Although these diseases are relatively common and highly debilitating, the mechanisms

responsible for their pathologies are poorly understood and there are currently no effective

therapies. Therefore, it is crucial to reveal genetic factors involved in the various pathways

affected, as well as to provide potential therapeutic drug targets.

Powerful genetic techniques available for use in Drosophila have provided new insights

into various aspects of the disease and guided many research efforts in vertebrate

neuroscience. Importantly, Drosophila studies have successfully uncovered several novel

signalling pathways which may be relevant to the pathology of many malignant neurological

diseases, including ALS. The research activities in Drosophila have enabled us to analyse the

resulting cellular phenotypes and uncover the molecular pathways underlying many

neurological diseases.

STUDIES ON MUTATIONS ASSOCIATED WITH FAMILIAL FORMS OF

ALS ENABLE US TO UNDERSTAND THE CORE PATHOLOGY OF ALS

A French neurologist, Jean-Martin Charcot, first noted the characteristics of ALS in 1874

and named the fatal syndrome based on what he found. Damage to upper motor neurons,

which begin at the top of the brain, results in muscle weakness, stiffness, and augmented

reflexes. Lower motor neurons start at the base of the brain and spinal cord; injury to these

neurons causes muscle atrophy, twitching, weakness, and reduced reflexes. Despite over

135 years of vigorous investigations following the discovery of the disease, ALS, also known

as Lou Gehrig’s disease, remains a mercilessly fatal disorder that affects two to eight per

100,000 persons in the world (Kiernan et al., 2011). Unfortunately, there is no primary

therapy for this disease and its pathogenesis is poorly understood, with most persons affected

dying within 3-5 years of diagnosis of the fatal syndrome.

Both environmental and genetic factors have been attributed to the pathology of ALS;

familial cases account for approximately 10% of all instances of the disease. Since both

sporadic and familial forms affect the same types of neurons with comparable disease

hallmarks, they most likely share a similar pathogenesis (Pasinelli and Brown, 2006). Thus,

understanding the mechanisms by which these mutations cause the pathology in ALS may

A Secreted Ligand for Growth Cone Receptors … 37

lead to novel therapeutic strategies of both familial and sporadic forms of the disease. A

landmark discovery reported in 1993 initiated the molecular era of ALS research: the

identification of mutations in the superoxide dismutase 1 (SOD1) gene in a familial form of

ALS (Rosen et al., 1993). Importantly, mice expressing the mutant SOD1 recapitulated the

motor defects observed in ALS patients (Wong et al., 1995). Use of these mice has been

crucial in characterizing the mechanisms of familial and sporadic ALS. On the basis of these

studies, mutant SOD1 has been postulated to be involved in many key aspects of the disease:

(1) accumulation of ubiquitinated SOD1 contributes to ALS cellular toxicity (Bruijn et al.,

1998), (2) synaptic glutamate receptors are aberrantly clustered and cause excitotoxicity

(Bottino et al., 2002; Boyce et al., 2005), (3) motor neuron aberrations alone (cell-

autonomous defects) are insufficient to cause the disease and other cells such as microglia are

involved (cell non-autonomous defect) (Boyle et al., 2006; Brooks, 1994), (4) mitochondrial

morphology and function is impaired (Brostrom et al., 1995; Vielhaber et al., 1999), and (5)

the ER stress response is triggered (Nishitoh et al., 2008; Saxena et al., 2009). The effects of

mutant SOD1 in ALS pathogenesis do not seem to be associated with its wild-type function

and as of yet, there is no unifying hypothesis to explain these observations.

Mutations were subsequently identified in other familial forms of ALS (Table 1),

including TAR DNA-binding protein (TDP)-43 (Sreedharan et al., 2008), fused in sarcoma

(FUS) (Kwiatkowski et al., 2009), valosin-containing protein (VCP) (Johnson et al., 2010),

C9ORF72 (Kudin et al., 1999; Strempel et al., 1999), and VAPB (Nishimura et al., 2004).

Importantly, the proposed functions of these genes are distinct. For example, TDP-43 and

FUS have been implicated in RNA regulation (Brunner et al., 1994; Doering et al., 1999),

while a variety of roles have been proposed for VCP, notably in ER-associated degradation

(Leon and McKearin, 1999; Patel et al., 1998), autophagy, and the ubiquitin-proteasome

system (Cantor et al., 2009). Given that no obvious link between the functions of these

proteins has been established, it is crucial to identify the pathological defects caused by these

mutant proteins implicated in both sporadic and familial ALS to determine the core defects in

ALS. To this end, VAP (ALS8) has been intensively researched in order to elucidate the core

pathology of ALS. Indeed, studies have shown that defects in pathways associated with VAP

lead to key pathological features implicated in ALS (Chai et al., 2008; Chen et al., 2010;

Ratnaparkhi et al., 2008; Tsuda et al., 2008). Thus, uncovering the functions and pathways in

which VAP is operating will provide clues to develop strategies to delay the course of both

the familial and sporadic forms of this disease.

DOMINANTLY INHERITED MISSENSE MUTATIONS IN VAP

ARE ASSOCIATED WITH A FAMILIAL FORM OF ALS

A dominantly inherited proline 56 to serine (P56S) missense mutation in the VAPB gene

was first identified in a large Brazilian family with a slowly progressive and late-onset,

atypical form of ALS, ALS8 (Nishimura et al., 2004) (Figure 1). Interestingly, individuals

carrying the same mutation have been described with three distinct conditions: a late-onset

slowly progressing form of spinal muscular atrophy, an atypical slowly progressing form of

ALS, and a typical severe and rapidly progressing ALS. In a branch of the same large family,

the P56S mutation has been shown to cause a lower motor neuron disorder accompanied by


autonomic involvement and dyslipidaemia (Marques et al., 2006). The mutated proline is

present in a stretch of amino acids that is very highly conserved in species such as yeast,

worms, flies and humans (Figure 1).

Table 1. Genes identified in familial ALS

Mutations in several genes, including the SOD1, TDP-43, FUS, C9orf72, and VAPB genes, cause

familial ALS (Chow et al., 2009; DeJesus-Hernandez et al., 2011; Greenway et al., 2006;

Maruyama et al., 2010; Renton et al., 2011; Topp et al., 2004; Yang et al., 2001).

VAP consists of three domains, MSP domain (MSP), coiled coil domain (CC) and a transmembrane

domain (TM). MSP domain is named from the high similarity with Major Sperm Protein in C.

elegans. Conserved Proline is mutated to Serine in patients associated with ALS8. Note that VAP

and MSP have no signal sequence.

Figure 1. A graphical overview of VAP and MSP proteins.


The same mutation was identified in Germany (Kirches et al., 1999) as well as in a

patient of Japanese descent (Millecamps et al., 2010). Additionally, a recent study found a

new mutation (T46I) in a British ALS patient that leads to similar molecular effects as the

P56S alteration (Chen et al., 2010). Human genetic research on ALS8 has raised three

important questions: (1) What is the function of the wild-type VAP protein, (2) what are the

effects of pathological mutations of this protein, and (3) how does VAP or its associated

pathways contribute to the pathogenesis of sporadic or other familial forms of ALS.

VAP IS AN ER TRANSMEMBRANE PROTEIN CONTAINING

A MSP DOMAIN

VAPB is closely related to VAPA, which has been shown to associate with the

cytoplasmic face of the ER (Soussan et al. 1999; Skehel et al. 2000; Kaiser, 2005) or to tight

junctions as a type II transmembrane protein (Lapierre et al., 1999). The human VAPB

(hereafter named hVAP) protein is about 30kDa and has homologs in numerous other species,

including C. elegans (F33D11.11 or VPR-1), Drosophila (DVAP33-A, hereafter named

dVAP) (Pennetta et al., 2002) and yeast (Suppressor of Choline Sensitivity, Scs2p)

(Kagiwada and Zen, 2003). VAPs contain an amino (N)-terminal domain of about 125

residues called the major sperm protein (MSP) domain, which is conserved among all VAP

family members (Figure 1) (Weir et al. 1998; Nishimura et al. 1999). The MSP domain was

named for its similarity to nematode MSPs, the most abundant proteins in their sperm

(Bottino et al., 2002). The central region of the protein contains an amphipathic helical

structure and is predicted to form a coiled-coil protein-protein interaction motif. The

hydrophobic carboxyl (C)-terminus acts as a membrane anchor to the ER (Kaiser et al., 2005;

Skehel et al., 2000; Soussan et al., 1999). In studies done with Aplysia, VAP was implicated

in synaptic transmission through its interaction with VAMP (Skehel et al., 2000). However, in

other organisms, VAP is not likely to function in synaptic transmission (Chai et al., 2008).

Indeed, VAP proteins have rather been implicated in other biological functions, including

glucose transport trafficking, expression of phospholipid biosynthetic genes, ER-Golgi and

intra-Golgi transport, neurite extension and calcium homeostasis (Foster et al., 2012; Peretti

et al., 2008) (De Vos et al., 2012; Matsuzaki et al., 2011). Yeast homologs are involved in

phosphatidylinositol-4-phosphate (PIP) synthesis and ceramide transport. Strains lacking

SCS2p, a VAP homolog, are unable to activate the inositol-1-phosphate synthase (INO1)

gene. These strains cannot grow in temperatures above 34C due to the absence of inositol

(Brickner and Walter, 2004; Kagiwada and Zen, 2003). Overexpression of hVAP in HeLa

cells affects the structural integrity of the ER through interaction with Nir (N-terminal

domain-interacting receptor) proteins. These interactions are mediated through FFAT (two

phenylalanines [FF] in an acidic tract) domains, which are present in Nir proteins (Amarilio et

al., 2005). VAPs also interact with a lipid-binding protein, oxysterol-binding protein (OSBP),

ceramide transfer protein (CERT) and phosphatidylinositol-four-phosphate adaptor-protein-2

(FAPP-2), all of which contain a FFAT motif (Amarilio et al., 2005; Kaiser et al., 2005;

Loewen and Levine, 2005; Mikitova and Levine, 2012). Therefore, VAPs are also thought to

play a role in the metabolism and transport of lipids (Jansen et al., 2011; Perry and Ridgway,

2006). Importantly, hVAP and dVAP are functionally interchangeable in fly assays. To


further define the role of VAPs in animals, Pennetta et al. characterized the function of dVAP

in flies (Pennetta et al., 2002). Pennetta and colleagues showed that dVAP regulates the

division of boutons at the synaptic terminals at the neuromuscular junctions (NMJs). Loss of

dVAP causes a severe decrease in the number, as well as an increase in the size, of boutons at

the NMJs while presynaptic overexpression of dVAP induces an increase in the number of

boutons and a decrease in their size. Loss of dVAP also causes a disruption of the presynaptic

microtubule architecture, whereas gain of dVAP enhances it. The loss of function of dVAP

has also been shown to lead to an increase in size of miniature excitatory junction potentials

(mEJPs), as well as an increased clustering of postsynaptic glutamate receptors, whereas

presynaptic overexpression of the wild-type dVAP decreases the size of the mEJPs (Chai et

al., 2008). Of note, overexpression of hVAP shows the similar phenotypes as overexpression

of dVAP, and expression of hVAP can rescue the defects associated with loss of dVAP in

flies (Chai et al., 2008), indicating that the function of VAP might be evolutionarily

conserved in Drosophila and humans. The similarity in their structures also suggests a

common function between MSP and VAP. The C. elegans MSP proteins and VAP MSP

domains both fold into evolutionarily conserved immunoglobulin-type seven-stranded beta

sandwiches (Baker et al., 2002; Kaiser et al., 2005), though MSP does not contain a coiled-

coil motif nor a transmembrane domain (Ward et al., 1988). MSP function is required for

fertilization in C. elegans (Kim et al., 2013). Indeed, MSPs are abundantly expressed in

sperm and have an intracellular cytoskeletal function, which depend on their ability to

polymerize in the absence of actin or myosin (Bottino et al., 2002). MSP also has an

important extracellular signalling function during fertilization (Miller et al., 2001). MSP is

secreted from the sperm cytosol into the reproductive tract by an unconventional process

(Kosinski et al., 2005) and extracellular MSP binds to the VAB-1 Eph (Ephrin) receptor and

other, yet to be identified receptors, on the surfaces of unfertilized oocytes and the

surrounding ovarian sheath cells (Corrigan et al., 2005; Govindan et al., 2006; Miller et al.,

2003). Secreted MSP induces oocyte maturation, an essential process that prepares oocytes

for fertilization and embryogenesis, as well as ovarian sheath cell contraction (Corrigan et al.,

2005; Miller et al., 2003). Somatic cAMP signalling in the gonadal sheath cells functions

downstream of MSP signalling and coordinates oocyte growth and meiotic maturation via

soma-germline gap-junction communication (Govindan et al., 2009). Similar molecular

functions to that of MSP were not characterized for VAP proteins.

VAP FUNCTIONS IN NOVEL SIGNALLING PATHWAYS

The presence of the MSP domain in its sequence suggests that VAP might share

signalling functions with MSP sperm-specific proteins. In agreement with this notion,

microinjection of worm, fly, or human VAP MSP domains to the MSP-deficient gonad of C.

elegans rescues the gonadal phenotype (Tsuda et al., 2008). VAP carrying the P56S ALS

mutation mimics the wild-type protein in this assay, demonstrating that the mutant protein

still retains functional signalling properties. This further suggests that the defects caused by

the ALS mutation are primarily a result of aberrant trafficking or localization. This chapter

strives to explain how studies on VAP in Drosophila have revealed that pathways associated

with MSP may be relevant to the pathology of ALS.


CLEAVAGE OF VAP RELEASES MSP DOMAIN-CONTAINING

FRAGMENTS THAT ARE SECRETED

An important feature associated with ALS is that the disease may have a cell non-

autonomous component (Boyle et al., 2006; Brooks, 1994). Complementary in vivo and in

vitro studies have suggested a possible role for trophic factors in ALS (Acsadi et al., 2002;

Kaspar et al., 2003). Tsuda et al. and Han et al. showed that VAP MSP domain cleaved from

full length protein can be secreted in flies and worms (Corey et al., 2012).

VAP exists as an endoplasmic reticulum (ER)-localized type II membrane protein and modulates

diverse pathways including ER homeostasis and ceramide and sphingolipid metabolism. VAP is

cleaved by an unknown protease, releasing MSP domain-containing fragments that are secreted.

The resulting VAP fragments bind to Eph and Robo/LAR receptors. VAP/Eph signalling could act

in an autocrine way to modulate neuronal activity, for example, via glutamate receptors (GluRs).

VAP/Robo/LAR signalling is required for mitochondria fission. The proline 56 to serine (P56S)

mutation (P58S in Drosophila) in VAPB results in aggregation of the mutant (red) and wild-type

(green) VAP protein in the ER, which leads to an upregulation of the ER stress. The mutation

causes loss of VAP function including Eph and Robo/LAR receptor signalling and probably other

ER-associated activities of VAP.

Figure 2. Model of ALS pathology caused by ALS8 (P56S) mutant VAP.

Consistently, the VAP MSP domain is also present in human blood serum, and its

presence was also confirmed in a large survey of serum proteins identified by mass

spectrometry (Omenn et al., 2005). VAP lacks an N-terminal signal sequence and, while still

uncharacterized, VAP MSP secretion likely involves an unconventional mechanism similar to

that of MSP in C. elegans (Kosinski et al., 2005). Significantly, the secreted VAP MSP acts


as a trophic factor via receptors including Eph, Roundabout (Robo) and leukocyte-antigen

related (LAR) in flies and worms, the details of which will be described in this chapter

(Figure 2).

VAP ACTS AS A LIGAND FOR EPH RECEPTOR

AND MEDIATES EPH SIGNALLING

MSP binds to multiple receptors, including the C. elegans VAB-1 Eph-related receptor

protein tyrosine kinase. (Miller et al., 2003). The Eph are an evolutionarily conserved class of

receptor protein-tyrosine kinases that bind to membrane-attached ligands called ephrins

(Palmer and Klein, 2003; Pasquale, 2005). Ephrins inhibit oocyte maturation in the absence of

sperm, and MSP functions to antagonize this inhibitory circuit (Govindan et al., 2006; Miller

et al., 2003; Whitten and Miller, 2007). MSP induces activation of the MAP kinase and

Ca2+

/calmodulin-dependent protein kinase II cascades (Corrigan et al., 2005; Miller et al.,

2001) as well as reorganization of the oocyte microtubule cytoskeleton (Govindan et al.,

2006). Intriguingly, Tsuda et al. have shown that secreted VAP MSP domains also bind to

Eph on the extracellular surface of cells (Tsuda et al., 2008). The genetic and biochemical

binding data shows that VAP acts as a ligand and modulates Eph receptor signalling

pathways. In flies and worms, mutants lacking VAP show phenotypes that overlap with those

observed in Eph mutants (Tsuda et al., 2008). In addition, loss-of-function of the Eph receptor

suppresses the muscle phenotypes induced by overexpression of wild-type dVAP in flies. The

similarity between the phenotypes of VAP mutants and Eph receptor mutants suggests that

VAP MSP functions as an agonist to activate signalling upon binding Eph. In contrast, VAP

functions to antagonize ephrin signalling in chemosensory neuron migration of the C.

elegans, just as MSP antagonizes ephrin signalling during oocyte maturation (Miller et al.,

2003). Biochemical competition assay results are consistent with MSP domains competing

with ephrin for Eph binding (Tsuda et al., 2008). Taken together, the relationship between

MSP and ephrin ligands in Eph signalling may depend on the developmental and cellular

context, as previously observed for ephrins and Eph in mammals (Mawrin et al., 2004).

The agonist effects of VAP MSP on Eph receptors contribute to glutamate excitotoxicity,

which likely plays a role in the pathogenesis of ALS (Bruijn et al., 2004; Rothstein et al.,

1990). Three lines of evidence suggest that VAP MSP domains might regulate glutamate

receptor signalling.

First, Eph receptors directly associate with NMDA-subtype glutamate receptors and

regulate clustering in cultured neurons (reviewed in Dalva et al., 2000; Palmer and Klein,

2003; Takasu et al., 2002). Triple EphB knock-out mice lacking EphB1-3 exhibit homeostatic

upregulation of NMDA receptor surface expression and loss of proper targeting to synaptic

sites (Nolt et al., 2011).

Second, loss of dVAP function in flies is associated with increased glutamate receptor

clustering and increased mEJPs at the NMJs (Chai et al., 2008).

Finally, MSP and VAB-1 Eph regulate NMDA receptor function during worm oocyte

maturation (Corrigan et al., 2005). These observations suggest that defective VAPB-P56S

signalling in ALS patients might alter the susceptibility of motor neurons to potential

pathogenic mechanisms, such as glutamate excitotoxicity.


Alternatively, the antagonist effects of VAP MSP on Eph receptors contribute to the ALS

pathology. In vertebrates, multiple ephrins and Ephs, including EphA4 and A7, are expressed

throughout the adult nervous system and skeletal muscle (Iwamasa et al., 1999; Kullander et

al., 2003; Lai et al., 2001; Martone et al., 1997).

Genetic as well as pharmacological inhibition of EphA4 signalling rescues the mutant

SOD1 phenotype in zebrafish and increases survival in mouse and rat models of ALS (Van

Hoecke et al., 2012).

In humans with ALS, EPHA4 expression inversely correlates with disease onset and

survival, and loss-of-function mutations in EPHA4 are associated with long survival (Van

Hoecke et al., 2012).

Although the mechanism through which deletion of EphA4 suppresses motor neuron

degeneration is not yet understood, MSP VAP may play a role in motor neuron survival or

muscle function through interactions with Eph in the pathogenesis of ALS. Whether the VAP

ligand/Eph receptor interaction leads to positive or negative regulation of downstream

signalling cascades, and whether the effects of VAP/Eph signalling act in an autocrine or

paracrine fashion, or both, will be addressed in the future.

VAP MSP MEDIATES ROBO AND LAR RECEPTORS AND FUNCTIONS

IN THE MAINTENANCE OF THE MITOCHONDRIA IN MUSCLE

MSP domains bind to Eph and other evolutionarily conserved receptors that function

together to regulate a variety of developmental processes (Miller et al., 2003). Accordingly, it

was shown that the cleaved VAP fragment, VAP-MSP, interacts with LAR and Robo family

receptors on the muscle cell surface, which ultimately modulate actin organization and

changes mitochondrial morphology and function (Han et al., 2012). Mitochondria are

remarkably dynamic organelles that migrate, divide and fuse. Since mitochondria cannot

replicate de novo, fission and fusion allow the mixing of metabolites and mitochondrial DNA,

the proliferation and distribution of mitochondria and cellular adaptation to changing energy

demands (Knott et al., 2008). As individual mitochondria are subject to injury and

dysfunction, it is likely that mitochondrial fusion serves as a protective mechanism.

Significantly, Han et al. showed that neuronal VAP is a critical regulator of muscle

mitochondria in C. elegans and Drosophila. Adult C. elegans vap mutants exhibit an

imbalance of mitochondrial fission and fusion in muscle and these aberrant mitochondrial

networks have low transmembrane potential and respiration. Abnormal mitochondrial shapes

and/or electron transport activity have been reported in motor neurons and skeletal muscle of

ALS patients (Crugnola et al., 2010; Wiedemann et al., 2002). Indeed, ALS patients with a

SOD1 mutation show a muscle mitochondrial oxidative defect (Corti et al., 2009).

Cytochrome c oxidase (COX)-negative fibres is a common histochemical finding in skeletal

muscle from patients with sporadic ALS, suggesting the muscle mitochondrial respiratory

chain dysfunction (Crugnola et al., 2010). Therefore, defects in muscle mitochondria may

contribute as a primary cause in ALS pathogenesis.

Importantly, non-autonomous signalling by VAP and sperm-derived MSPs regulates

mitochondria in striated muscles and oocytes in C. elegans. Han et al. observed that the

defects in muscle mitochondria of the vap null mutant can be rescued by expression of VAP


in neurons, but not in the muscles themselves (Han et al., 2012). In motor neurons, secreted

MSPs signal through Eph (Tsuda et al., 2008), but in muscle, loss of Eph does not alter

mitochondrial phenotypes (Boyle et al., 2006). Thus, Han et al. searched for other receptors

mediating MSP signalling in muscle cells. Using genome-wide microarrays and subsequent

functional analysis, Han et al. identified two additional MSP receptors, the Robo and LAR

receptors. Like Eph receptors, Robo, and LAR-like receptors are called growth cone guidance

receptors because of their established roles in regulating the actin cytoskeleton during nervous

system development. However, these receptors are often expressed after guidance decisions

are made, particularly in the adult central nervous system and muscles (Longo et al., 1993;

Zabolotny et al., 2001; Zhang and Goldstein, 1991).

VAP or MSP signals to mitochondria through Robo and LAR receptor pathways, which

controls actin remodelling (Han et al., 2012). Both LAR and Robo mutants have altered

muscle mitochondrial morphology and function in C. elegans. Interestingly, both VAP and

Robo antagonize LAR signalling in muscle mitochondria. Other groups have demonstrated

genetic interactions and shared signalling pathways between LAR and Robo in neurons. For

example, Robo and LAR have opposing functions during midline axon guidance in

Drosophila, suggesting some type of inhibitory crosstalk between these two receptors (Sun).

The data presented by Han et al. in muscle provides additional evidence of crosstalk between

LAR and Robo. They propose the intriguing hypothesis that Robo facilitates VAP and MSP

binding to LAR or Robo/LAR complexes, down-regulating LAR signalling. Similarly, Robo

functions as the stepwise refinement in growth cone guidance (Stein and Tessier-Lavigne,

2001). Growth cones undergo two mutually reinforcing changes upon midline crossing: loss

of response to a midline attractant, and up-regulation of response to a midline repellent, that

helps to expel them from the midline and move them on to the next leg of their trajectory.

Slit, another Robo ligand is a midline repellent. Upregulation of Slit causes loss of response

to the chemoattractant, netrin. This silencing effect of Slit on netrin attraction is mediated by

a direct physical interaction of Slit receptor Robo with the netrin receptor DCC.

In C. elegans, VAP/Robo/Lar signalling antagonizes Arp2/3 activity to position

mitochondria at I-bands in the muscle (Han et al., 2012), actin-enriched sites containing

structures analogous to focal adhesions (Lecroisey et al., 2007). CLR-1 Lar and Arp2/3

complex activity are required for maintenance or elongation of mitochondrial tubule length

along I-bands, a process that may facilitate mitochondrial fusion. In C. elegans vap mutants,

mitochondria mislocalise to the muscle belly along with ectopic actin filaments. Lar or

Arp2/3 loss suppresses the vap mutant mitochondrial position, morphology, and

transmembrane potential defects. These results reveal a critical role for the Arp2/3 complex in

modulating multiple aspects of mitochondrial biology. The actin cytoskeleton has been shown

to regulate mitochondrial position in cultured neurons and yeast (Boldogh and Pon, 2006;

Pathak et al., 2010). The regulation of actin and microtubule effectors downstream of

VAP/LAR and Robo signalling raises intriguing questions regarding the complexity and

specificity of the regulatory process that supports normal mitochondrial structure and function

in muscle cells. This novel signalling pathway mediated by secreted VAP might be important

for the pathogenesis of ALS. Consistently, analysis of single nucleotide polymorphisms

(SNPs) in genome-wide association studies suggested that common variants in Robo and Eph

are associated with sporadic ALS (Lesnick et al., 2008).


ALS MUTATION CAUSES TWO DIFFERENT TYPES OF DEFECTS:

A FAILURE OF VAP SECRETION (LOSS-OF-FUNCTION) AND

ER STRESS (GAIN-OF-FUNCTION OR TOXIC DEFECTS)

Establishing the relevance of VAP signalling to the pathology of ALS will rest on

defining the cellular activities of VAP that are critical for the normal function and

maintenance of motor neurons, and characterizing how each is affected by the P56S mutation.

Significantly, Tsuda et al. demonstrated that the P56S mutation causes a failure of

secretion of VAP MSP protein, resulting in defects in VAP/receptor signalling. Disrupted

secretion of the VAPB MSP domain could mediate the defects of cellular non-autonomous

signalling from glia, endothelia, or muscle cells to the motor neurons described in ALS

patients and in transgenic animal models expressing mutant SOD1 (Pasinelli and Brown,

2006). Tsuda et al. demonstrated that the P56S VAP accumulates as inclusions in the ER.

These inclusions recruit wild-type VAP protein, which causes a dominant negative defect

(Chai et al., 2008; Ratnaparkhi et al., 2008; Tsuda et al., 2008). Interestingly, Ratnaparki et al.

showed that the mutant VAP interferes with BMP signalling at the synapse, suggesting a

possible link between BMP signalling and the ALS pathology.

Accumulated P56S VAP shows key characteristics associated with ALS. First, P56S

dVAP protein induces ubiquitinated inclusions, as observed in ALS patients and mutant

SOD1 mice (Tu et al., 1996). These intracellular ubiquitinated inclusions are a hallmark of

ALS (Wiedemann et al., 2002). Second, the protein inclusions are associated with the ER and

appear to be electron-dense expansions of the ER (Tsuda et al., 2008). Third, several key ER

proteins were found to be associated with these inclusions, including the chaperones Boca

(Culi and Mann, 2003) and PDI (Wilkinson and Gilbert, 2004). Finally, the P56S dVAP

induces ER stress. This data shows at least three important parallels with ALS and SOD1

mouse models: cytoplasmic inclusions, ubiquitination, and ER stress. Consistently, viral-

mediated expression of the ALS mutant human VAPB leads to an ER stress response that

contributes to the selective death of primarily cultured motor neurons (Langou et al., 2010).

The ER stress is typically induced as a response to ER associated stress to refold proteins

(Schroder and Kaufman, 2005). ER stress is initially defensive by up-regulating specific ER

stress-regulated genes and inhibiting general protein translation. If the ER stress is too severe

or prolonged, it can induce cell death and apoptosis (Schroder and Kaufman, 2005). It is

therefore possible that a slow and protracted accumulation of ER protein aggregates

eventually leads to cellular damage and neuronal death, in agreement with recent observations

that overexpression of P56S dVAP causes neuronal death in flies (Chai et al., 2008).

Recent evidence indicates that ER stress primarily contributes to the ALS pathogenesis

(Walker and Atkin, 2011). Activation of ER stress-regulated genes is one of the earliest

events in affected motor neurons of transgenic rodent models expressing ALS-linked mutant

SOD1 (Atkin et al., 2006; Mori et al., 2011). Genetic manipulation of ER stress in several

different SOD1 mouse models was shown to alter disease onset and progression, implicating

an active role for ER stress in disease mechanisms (Mori et al., 2011). ER stress also occurs

in spinal cord tissues of human sporadic ALS patients (Atkin et al., 2008; Sasaki, 2010), and

recent evidence suggests that the perturbation of the ER could occur in ALS cases associated

with TDP-43 and FUS (Farg et al., 2012; Suzuki and Matsuoka, 2012). Together, these


findings implicate ER stress as a potential upstream mechanism involved in both familial and

sporadic forms of ALS.

Based on our data and the published literature, we would like to propose the following

model for the pathogenesis of ALS8, an autosomal dominant disease (Figure 2). The P56S

hVAP protein accumulates in the ER of cells, while the wild-type protein is functional.

However, with time, the aggregates become more prominent, P56S hVAP protein becomes

ubiquitinated, and functional wild-type proteins become trapped in the inclusions. These

protein inclusions initiate ER stress and defects in lipid metabolism that eventually affect cell

viability and lead to a decrease in secretion of the MSP domain. The MSP domain binds to

receptors including Ephrin, Robo and LAR receptors through which it mediates at least some

of its actions. The mutant protein therefore causes two very different types of defects: first,

accumulation in the ER creates the ER stress, which may be toxic to the cells, and could

account for the cell autonomous component. Second, reduced secretion of VAP MSP, which

may function as an autocrine or paracrine signal, exacerbates the problem. Both defects may

synergize to produce the key features of ALS pathology.

Hence, the collective evidence suggests the model that impaired function of VAP causes

core pathological defects in ALS. This model is also supported by other reports showing that

sporadic ALS patients exhibit reduced VAP protein (Teuling et al., 2007) or mRNA levels

(Anagnostou et al., 2008), suggesting that loss of VAP contributes to the pathology of

sporadic ALS.

CONCLUSION

Over the last century, research activities in Drosophila have had a huge impact on our

understanding of vertebrate neuroscience. In particular, the wealth of powerful research tools

available with this model organism has driven important discoveries into the mechanisms

underlying neurodegenerative diseases. In this chapter, we provide evidence that dissecting

pathways in which a disease gene is operating in fruit flies is a productive approach to

understanding the mechanisms leading to the pathology of ALS. The recent Drosophila

studies into the normal function of VAP have provided invaluable novel insights into the

mechanism of the ALS pathogenesis, while analysis of the cellular defects associated with

mutant VAP has helped us understand key pathological features implicated in this disorder.

Future studies aiming at uncovering the functions and pathways in which VAP is operating

will help develop strategies and identify targets for therapeutic intervention, in order to

prevent or delay the course of familial and sporadic ALS.

ABOUT THE AUTHORS

Amina Moustaqim-Barrette is an undergraduate student studying Neuroscience and

Philosophy at McGill University, Montreal, Canada. Upon joining the Tsuda lab at the

Montreal Neurological Institute in early 2012, her main research initiatives have revolved

around the VAP protein and its implication in ALS.


Mario Maira is a research associate scientist at the Montreal Neurological Institute.

After obtaining is PhD in biochemistry, he studied developmental neurobiology at UCSF and

cell signalling at McGill University as a post-doctoral fellow. Then he joined the private

sector to investigate potential drug candidates for treatment of neurodegenerative diseases. He

is currently working in the Tsuda Lab at McGill University where his research is centered

around the molecular mechanisms relevant to the ALS.

Hiroshi Tsuda is an Assistant Professor in the Department of Neurology and

Neurosurgery at the McGill University. He obtained his MD from Kobe University, Japan

and his board certification in neurology. Then he decided to work on the basic mechanisms of

neurodegeneration and obtained his PhD from Kyoto University. He carried out his Postdoc

studies on learning in fruit flies in the Hugo Bellen lab at the Baylor College of Medicine. His

own laboratory in the Montreal Neurological Institute at the McGill University is focused on

understanding neurodegenerative diseases including ALS through the use of the fly and

mouse model.

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Chapter 3

FLIES IN MOTION: WHAT DROSOPHILA CAN TELL

US ABOUT AMYOTROPHIC LATERAL SCLEROSIS

Andrés A. Morera, Alyssa Coyne

and Daniela C. Zarnescu

Department of Molecular and Cellular Biology,

Department of Neuroscience and Department of Neurology,

University of Arizona, Tucson AZ, US

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with

a prevalence of 1 in 30,000 individuals. Generally diagnosed between 40 and 70 years of

age, ALS is accompanied by progressive loss of motor neuron function and a life

expectancy of 2-5 years. Although age is considered to be the highest risk factor for ALS,

it is clear that the disease has a genetic basis and may also be influenced by

environmental factors. Familial ALS (fALS) affects 10% of patients and has been linked

to several loci, the most common of which is C9ORF72, a gene of unknown function. The remaining 90% of ALS cases are sporadic (sALS) and remain poorly understood,

although some loci have been linked to both fALS and sALS. In recent years a dramatic

shift in our thinking about ALS has been catalysed by findings that the RNA binding

proteins TAR DNA binding protein 43 (TDP-43) and fused in sarcoma/translocated in

liposarcoma (FUS/TLS) constitute markers of pathology and when mutated, neural

degeneration occurs in human patients. Studies in a wide range of model systems

including worms, flies, zebrafish and rodents support the notion that alterations in these

RNA binding proteins and RNA metabolism cause motor neuron disease. Together with

the recent discovery of GGGGCC repeat expansions in C9ORF72, these studies led to the

hypothesis that ALS is a disease of RNA dysregulation. Here we will review the

contributions of the fruit fly Drosophila melanogaster to our understanding of ALS with

a focus on TDP-43, FUS/TLS and RNA dysregulation as a disease mechanism.

Corresponding author: Daniela C Zarnescu, PhD, Associate Professor, Molecular and Cellular Biology.

Neuroscience, Neurology, 1007 E Lowell St, Life Sciences South 552, University of Arizona, Tucson AZ

85721, email: [email protected].

Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu 58

Keywords: Drosophila, ALS, motor neuron disease, TDP-43, FUS, RNA binding proteins

INTRODUCTION

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a

neurodegenerative disease with an incidence of 2 in 100,000 people (Roman, 1996). Without

a clear diagnostic test, patients are identified mainly by ruling out a host of other ALS-like

disorders. Although examples of young-adult onset have been reported, most patients are

diagnosed in their forties or later in life (Cleveland and Rothstein, 2001; Gouveia and de

Carvalho, 2007; Logroscino et al., 2005). Those affected by this devastating disease suffer

progressive muscle atrophy due to degeneration of upper and lower motor neurons, leading to

paralysis and death within 2-5 years of diagnosis. Interestingly, about 20% of all ALS

patients also exhibit FrontoTemporal Lobar Degeneration (FTLD), a related

neurodegenerative disorder with overlap at the pathology level (Banks et al., 2008). The

clinical presentation of ALS is heterogeneous, which is in part, a reflection of the complex

genetic and environmental factors linked to this disease. At this time there is no cure for ALS

and the only available treatment, Riluzole, is at best palliative (Miller et al., 2012).

Familial ALS (fALS) affects approximately 10% of patients and has been linked to

several genes, including the recently discovered C9ORF72, which is thought to be

responsible for ~37.6% of fALS cases (DeJesus-Hernandez et al., 2011; Majounie et al.,

2012; Renton et al., 2011). The remaining 90% of ALS cases are sporadic (sALS) and remain

poorly understood, although some loci have been linked to both fALS and sALS (Alexander

et al., 2002). A plethora of studies have focused on superoxide dismutase 1 (SOD1) the first

locus to be identified as causative of ALS (Rosen et al., 1993). SOD1, an enzyme responsible

for converting superoxide radicals (by-products of oxidative phosphorylation in the

mitochondria) into hydrogen peroxide and molecular oxygen, plays an important role in

preventing cellular damage during times of oxidative stress. SOD1 mutations are found in

~20% of all fALS and ~1% of sALS cases, while altered expression of wild-type SOD1 has

been found in a significant fraction of sporadic cases (reviewed in Andersen, 2006).

Interestingly, ALS has been linked to several different genes encoding proteins of

sometimes seemingly unrelated or even unknown functions, as is the case with the recently

identified C9ORF72 locus. In addition to C9ORF72 and SOD1, these include alsin, senataxin,

VAMP/synaptobrevin-associated protein B (VAPB), P150 dynactin, angiogenin, fused in

sarcoma/translocated in liposarcoma (FUS/TLS, referred herein as FUS), and TAR DNA

binding protein 43 (TDP-43) (Beleza-Meireles and Al-Chalabi, 2009; Lagier-Tourenne and

Cleveland, 2009). Based on the known functions of these loci and extensive pathological

studies in autopsy samples and model organisms, ALS appears to be the result of defects in

several cellular processes including oxidative stress, intracellular transport, RNA metabolism

and apoptosis (reviewed in Beleza-Meireles and Al-Chalabi, 2009; Lagier-Tourenne and

Cleveland, 2009). How these diverse processes converge on a common path to motor neuron

disease remains an open question. A combination of molecular and genetic approaches, both

in vitro and in vivo, using a host of model systems is being employed to elucidate the

mechanisms by which individual gene mutations and other, non-genetic factors lead to ALS

(Joyce et al., 2011; McDonald et al., 2011; Tovar et al., 2009).

Flies in Motion 59

In recent years a dramatic shift in our thinking about ALS has been catalysed by findings

that the RNA binding proteins TDP-43 and FUS constitute markers of pathology and when

mutated, neural degeneration occurs in human patients (Colombrita et al., 2011; Kabashi et

al., 2008; Kwiatkowski et al., 2009; Sreedharan et al., 2008; Vance et al., 2009). Studies in

models including yeast, worms, flies, zebrafish and mouse support the notion that alterations

in these RNA binding proteins and RNA metabolism cause motor neuron disease (Couthouis

et al., 2011; Estes et al., 2011; Kabashi et al., 2011; Lanson et al., 2011; Li et al., 2010;

Liachko et al., 2010; Lu et al., 2009; Ramesh et al., 2010; Wegorzewska et al., 2009).

Together with the recent discovery of GGGGCC repeat expansions in the noncoding region

of C9ORF72, these studies support the emerging hypothesis that ALS is a disease of RNA

dysregulation (DeJesus-Hernandez et al., 2011; Lagier-Tourenne and Cleveland, 2009;

Renton et al., 2011).

A plethora of genetic and clinical studies indicate that ALS is a complex disease with a

clear genetic basis although epigenetics and environmental factors are also thought to play a

role (Ahmed and Wicklund, 2011; Horner et al., 2008). Genome Wide Association Studies

(GWAS) have contributed to the identification of several loci linked to ALS and have

provided insights into the genetic complexity of the disease (reviewed in Valdmanis et al.,

2009). Elegant genetic studies in yeast and flies identified functional interactions between

TDP-43 and ataxin 2, which led the way to the discovery that ataxin 2 is a susceptibility

factor for ALS in human patients (Elden et al., 2010). These findings underscore the

importance of studies in model systems, which can provide critical insights into the

mechanisms by which individual gene mutations and mutation combinations contribute to

ALS. Furthermore, model systems can help elucidate the complexity of gene networks and

cellular pathways involved in disease that could be later validated in patients. It is only

through a concerted collaboration between basic scientists, human geneticists and clinicians

that we can expect major progress in ALS research and therapeutics in the coming years.

ALS IN MODEL ORGANISMS: YEAST TO MICE

Some of the early models of ALS based on SOD1 were developed in mice (Gurney,

1997) and rats (Howland et al., 2002; Nagai et al., 2001). These transgenic rodent models

expressing mutant human SOD1 recapitulate many of the disease features seen in human ALS

patients, including muscle atrophy, upper and lower motor neuron degeneration, disease-

associated cytoplasmic inclusions in neurons, and higher mortality (Gurney et al., 1996).

Other organisms in which SOD1-based ALS has been modelled include dog (Awano et al.,

2009), zebrafish (Ramesh et al., 2010), fruit fly (Watson et al., 2008), worm (Gidalevitz et al.,

2009; Wang et al., 2009) and yeast (Nishida et al., 1994). These different models have distinct

advantages as well as limitations that need to be carefully considered.

Recently, models of ALS based on TDP-43 and FUS have also been generated in model

systems ranging from yeast to worms to flies, fish and rodents (Couthouis et al., 2011; Estes

et al., 2011; Kabashi et al., 2011; Laird et al., 2010; Lanson et al., 2011; Li et al., 2010;

Liachko et al., 2010; Lu et al., 2009; Wegorzewska et al., 2009). Although some

discrepancies exist between these models, overall they recapitulate several aspects of ALS

including neurodegeneration, TDP-43 aggregation, locomotor dysfunction and reduced


survival. Here we will review the contributions of the fruit fly Drosophila melanogaster to

our understanding of ALS with a focus on TDP-43, FUS and RNA dysregulation as a disease

mechanism.

Current Hypotheses for ALS

Similar to other neurodegenerative diseases, including Alzheimer’s disease and

Parkinson’s disease, ALS pathology is accompanied by ubiquitin-positive cytoplasmic

aggregates within neurons, with shapes ranging from round to fusiform (Neumann, 2009).

This led to the hypothesis that ALS is a proteinopathy and suggested that protein misfolding,

possibly as a result of cellular stress, is a potential toxic mechanism.

As with other neurodegenerative disorders, the significance of protein aggregates remains

unclear and evidence exists to support both a toxic as well as a protective role for these

abnormal intracellular structures (Young, 2009). Importantly, age, which is the highest risk

factor for ALS, correlates with reduced proteostasis and increased potential for protein

misfolding and aggregation.

This model is supported by several findings including the propensity of TDP-43 and FUS

to aggregate in vitro (Johnson et al., 2009; Sun et al., 2011) and their association with

cytoplasmic puncta in cell models (Liu-Yesucevitz et al., 2010; McDonald et al., 2011). In

addition, TDP-43 and FUS harbour prion domains that are involved in aggregation and thus

are likely to be relevant to the pathology of ALS (Gitler and Shorter, 2011).

Oxidative stress has offered a particularly attractive model for SOD1 cases where toxicity

has been proposed to stem from free radical accumulation. This mechanism however, remains

uncertain due to lack of consistent evidence for oxidative damage (reviewed in Rothstein,

2009). Defects in axonal transport, which are typically found in neural degeneration were also

linked to ALS via mutations in dynactin, a subunit of the motor protein dynein (Munch et al.,

2004).

Additional hypotheses include an imbalance in trophic factors such as VEGF, which is

thought to be neuroprotective (Dodge et al., 2010; Kulshreshtha et al., 2011; Shimazawa et

al., 2010; Tovar-y-Romo and Tapia, 2012; Wang et al., 2007) and synaptic hyperexcitability,

likely due to an inability of glial cells to buffer excess glutamate within the synaptic cleft

(Foran and Trotti, 2009; Vucic and Kiernan, 2009).

RNA Dysregulation in ALS

Although the proteinopathy hypothesis, primarily driven by the overwhelming

pathological evidence for cytoplasmic aggregates, has dominated the field, recent gene

discovery studies point to the involvement of RNA binding proteins including TDP-43 and

FUS, both of which associate with intracellular inclusions and also act as causative agents of

disease (Kabashi et al., 2008; Kwiatkowski et al., 2009; Sreedharan et al., 2008; Vance et al.,

2009). TDP-43 was found to be a major component of cytoplasmic inclusions in motor

neurons of non-SOD1 fALS and sALS cases (Neumann et al., 2006).

Notably, TDP-43and FUS positive inclusions have been found in a wider spectrum of

neurodegenerative disorders including FrontoTemporal Lobar Degeneration and Alzheimer’s

Flies in Motion 61

disease (reviewed in Baloh, 2012; Blair et al., 2010; Gitler and Shorter, 2011). Extensive

mutation analyses in ALS patients showed that TDP-43 and FUS are linked to 4-5% of fALS

and 2% of sALS cases across ethnicities (reviewed in Colombrita et al., 2011; Lagier-

Tourenne and Cleveland, 2009).

Although at this time we do not fully understand how TDP-43 and FUS function in motor

neurons or the surrounding glial cells and how mutations in these RNA binding proteins lead

to motor neuron disease, new details are emerging on their connection to various aspects of

RNA regulation including RNA splicing, export, stability and translation (reviewed in Lagier-

Tourenne and Cleveland, 2009).

The discovery of TDP-43 and FUS together with the involvement of additional RNA

binding proteins (senataxin, angiogenin) and RNA itself (C9ORF72 noncoding expanded

repeats) in ALS, has led to a repositioning of hypotheses now centered on RNA-based

mechanisms. Notably, this newly emerging hypothesis and the protein aggregation model are

not necessarily mutually exclusive and may be interconnected at the molecular level by

multiprotein/RNA complexes known as RNA granules that have been proposed to act as

precursors of protein aggregates (Dewey et al., 2011; Parker et al., 2012).

TAR DNA Binding Protein (TDP-43)

The RNA binding protein TDP-43 has first captured the attention of the ALS field due to

its identification as a component of cytoplasmic inclusions in neurons and the surrounding

glia (Maekawa et al., 2009; Neumann et al., 2006; Tan et al., 2007). Originally identified as a

transcriptional repressor of HIV-1, TDP-43 consists of two RNA recognition motifs (RRM1,

2) and a glycine-rich domain within the C-terminus (Figure 1, Ou et al., 1995). Its cellular

functions are just beginning to be understood and include in addition to transcriptional

repression, pre-mRNA splicing, miRNA biogenesis and apoptosis (Figure 2, reviewed in

Banks et al., 2008). In vitro assays and RNA sequencing approaches have shown that TDP-43

binds with high affinity UG-rich sequences and regulates splicing of numerous mRNA targets

(Ayala et al., 2011; Buratti and Baralle, 2001; Polymenidou et al., 2011; Sephton et al., 2011;

Tollervey et al., 2011). TDP-43 is ubiquitously expressed and co-localizes with Survival of

Motor Neuron (SMN) and Gemin proteins in the nucleus. In hippocampal neurons, TDP-43

associates with cytoplasmic RNA granules and co-localizes with Fragile X mental retardation

protein (FMRP), Staufen and HuD in an activity-dependent manner, suggesting that TDP-43

may regulate synaptic plasticity in vivo by regulating synaptic mRNAs (Fallini et al., 2012;

Wang et al., 2008). Recently, individual TDP-43 mutations have been shown to differentially

regulate stress granule formation in various cell lines subjected to cellular stress (Dewey et

al., 2011; Liu-Yesucevitz et al., 2010; McDonald et al., 2011). RNA granules play critical

roles in post-transcriptional gene expression and are comprised of large RNA/protein

complexes that can be visualized as cytoplasmic puncta. Among these, stress granules

represent foci of RNA storage that form in response to cellular stress and correlate with

translation inhibition (Anderson and Kedersha, 2009).

The majority of TDP-43 mutations found in ALS patients represent aminoacid

substitutions that are thought to increase TDP-43 phosphorylation and caspase cleavage of the

C-terminus followed by proteasome-mediated degradation (Kabashi et al., 2008; Rutherford

et al., 2008; Sreedharan et al., 2008; Xu et al., 2010). It has been proposed that overwhelming


the proteasome degradation machinery may lead to the accumulation of TDP-43 C-terminal

fragments in the cytoplasm, which have been shown to be toxic through a gain of function

mechanism (Zhang et al., 2009). More work is needed to elucidate the mechanisms by which

these individual alterations in TDP-43 lead to motor neuron disease and degeneration.

Interestingly, proteomic analyses of the TDP-43 complex indicate that its protein partners,

which are enriched in components of the splicing and translational machinery do not differ

between wild-type and A315T or M337V variants (Freibaum et al., 2010). In keeping with

this observation, several different TDP-43 variants (i.e., wild-type, A382T, Q331K and

M337V) exhibit comparable binding affinity for specific mRNA targets (Colombrita et al.,

2012). Using biochemical purification approaches coupled with microarray analyses or RNA

sequencing, several RNAs associated with TDP-43 have been identified (Colombrita et al.,

2012; Polymenidou et al., 2011; Sephton et al., 2011; Tollervey et al., 2011). These include

numerous splicing targets containing (TG)n sequences, with a clear preference for RNAs with

long introns (Polymenidou et al., 2011; Tollervey et al., 2011). Given its presence in

cytoplasmic inclusions and the identification of several mutations linked to ALS, TDP-43 has

emerged as a common denominator for the majority of clinical cases known to date (Banks et

al., 2008; Neumann, 2009) and this suggests that TDP-43’s involvement in motor neuron

disease extends beyond its role in pre-mRNA splicing. The identification of TDP-43-

associated RNA targets linked to disease as well as synaptic function and neuronal

development position TDP-43 as a player not only in specific aspects of RNA regulation but

also in the various cellular processes in which its targets are involved. For example, knock-

out (KO) mice lacking TDP-43 exhibit alterations in fat metabolism (Chiang et al., 2010)

while the Drosophila homolog of TDP-43, TBPH has been identified in a genome wide RNAi

screen for genes involved in neuronal development (Sepp et al., 2008). It will be interesting to

see what specific targets mediate these developmental and physiological roles of TDP-43

outside of its involvement in neural degeneration.

Fused in Sarcoma (FUS)

Analysis of brain and spinal cord tissues from patients with ALS has led to the discovery

of the presence of FUS mutations resulting in cytoplasmic inclusions in neurons and glial

cells (Kwiatkowski et al., 2009; Vance et al., 2009). FUS, initially discovered for its role in

human sarcoma, is part of the TET/FET family of proteins that also includes Ewing’s

Sarcoma protein EWSR1 and TATA-binding protein associated factor TAF15 (reviewed in

Gitler and Shorter, 2011). At the structural level, FUS contains an N-terminal domain with a

QGSY region, a glycine-rich region, and RRM domain, several RGG repeats, and a zinc

finger motif in the C-terminus (Figure 1). The majority of the missense mutations associated

with ALS are located in the nuclear localization signal (NLS) region of the C-terminus

(Kwiatkowski et al., 2009; Vance et al., 2009). Although the full function(s) of FUS are still

not fully understood, several reports demonstrate its involvement in transcription, pre-mRNA

splicing, and local translation at the synapse (Figure 2 and reviewed in Colombrita et al.,

2011; Lagier-Tourenne and Cleveland, 2009). In hippocampal neurons, FUS has been shown

to be involved in mRNA transport to dendritic spines and to control spine morphology,

presumably by regulating local protein synthesis of specific targets (Fujii et al., 2005; Fujii

and Takumi, 2005).

Flies in Motion 63

Figure 1. TDP-43 and FUS are nucleocytoplasmic shuttling proteins containing RNA binding domains.

Mutations in the NLS domain impair the protein’s ability to localize to the nucleus and as

a result, FUS is targeted to the cytosol, consistent with the discovery of cytoplasmic

inclusions observed in post-mortem ALS samples. NLS mutations have also been shown to

promote the co-localization of FUS with stress granules in the cytoplasm of cells including

neurons and glial cells (Bosco et al., 2010; Gal et al., 2011). Recently, biochemical

purification and gene profiling approaches have identified CAGGACAGCCAG motifs as

FUS-specific targets (Colombrita et al., 2012; Lagier-Tourenne et al., 2012).

TDP-43 and FUS – Shared and Distinct Features

At the sequence level, both TDP-43 and FUS contain RNA binding domains and harbour

NLS and NES (nuclear export signal) domains that permit nucleocytoplasmic shuttling

(Figure 1). At the functional level, both TDP-43 and FUS have been implicated in similar

RNA processing steps including transcription, splicing, association with the miRNA

machinery as well as mRNA transport, transcript stabilization and local translation (Figure 2

and reviewed in Colombrita et al., 2011; Lagier-Tourenne and Cleveland, 2009). In addition,

both TDP-43 and FUS have been shown to associate with RNA stress granules. A major

difference between the two proteins is that while both wild-type and mutant TDP-43 variants

associate with stress granules, in the case of FUS, only the mutant forms co-localize with

stress granule markers (Bosco et al., 2010; Liu-Yesucevitz et al., 2010; McDonald et al.,

2011). In addition to differences in direct binding sequences their targets are also mostly

distinct with the exception of 45 transcripts that are down-regulated in the absence of either

TDP-43 or FUS (Lagier-Tourenne et al., 2012). Immunoprecipitation experiments indicate


that TDP-43 and FUS associate in a protein complex in whole brains or HEK cells (Freibaum

et al., 2010; Kim et al., 2010; Sephton et al., 2011) but not in motoneuronal NSC-34 cells

(Colombrita et al., 2012). Given their participation in similar RNA processing steps and their

involvement in ALS, these findings are somewhat surprising and suggest cell-type and

context specific interactions between TDP-43 and FUS.

In addition to an NLS and an NES, TDP-43 contains two RNA Recognition Motifs,

RRM1 and RRM2 and a C-terminus glycine-rich region, which harbours the majority of

disease linked mutations as well as a prion domain (Gitler and Shorter, 2011). FUS contains a

Q/G/S/Y rich region followed by a glycine-rich region, which also harbours a prion domain

(Gitler and Shorter, 2011). The C-terminus half of FUS contains an RNA recognition motif

(RRM), an RGG rich region harbouring a Zinc finger motif as well as a second prion domain

in addition to an NES and NLS (Gitler and Shorter, 2011).

Disease linked mutations in FUS cluster within the NLS. Domains defined according to

http://www.uniprot.org and predicted nuclear export signals defined according to

http://www.cbs.dtu.dk/services/NetNES.

Indeed, with the exception of one report of FUS positive inclusions in a patient

harbouring the G298S mutation within TDP-43, pathological inclusions contain either one or

the other but not both (Davidson et al., 2012; Deng et al., 2010). While it is possible that

interactions between TDP-43 and FUS are context dependent, these findings raise questions

about the functional relationship between the two proteins and about where in the pathway

that leads to neuronal dysfunction they may intersect. Using genetic interaction approaches,

experiments in Drosophila have shown that the FUS homolog cabeza (caz) and the TDP-43

homolog, TBPH may function in a common pathway with caz acting downstream of TBPH

(Wang et al., 2011).

Figure 2. Both TDP-43 and FUS are implicated in similar steps of RNA processing.

Flies in Motion 65

A. Regulation of transcription. TDP-43 has been demonstrated to bind TG-rich promoter

regions of target genes, inhibiting transcription, as in the case of mouse acrv1 and SP-10

genes (Abhyankar et al., 2007). FUS/TLS is also involved in transcriptional regulation,

having been shown to be recruited to the promoter region of cyclin D1 by non-coding RNAs

induced by DNA damage (Wang, 2008; Lalmansingh et al., 2011). Also, evidence of

association of FUS to the TFIID complex implicates it in general transcriptional regulation

(Bertolotti et al., 1998). B. pre-mRNA splicing. Recent studies demonstrated that TDP-43

binds to numerous pre-mRNA splicing targets containing (UG)n sequences, with a clear bias

for RNAs with long introns, and likely participates in splicing of these targets (Ayala et al.,

2011; Buratti and Baralle, 2001; Polymenidou et al., 2011; Sephton et al., 2011; Tollervey et

al., 2011). There is also strong evidence indicating that FUS participates in pre-mRNA

splicing as it has been found to be part of the spliceosome (Hartmuth et al., 2002; Zhou et al.,

2002). C. miRNA processing. TDP-43 and FUS/TLS are thought to be involved in miRNA

processing, as both bind to Drosha (Gregory et al., 2004), and TDP-43 interacts with the

Dicer complex (Kawahara and Mieda-Sato, 2012). D. mRNA transport. Both TDP-43 and

FUS proteins are shown to transport mRNA to dendritic spines in hippocampal neurons,

suggesting that they may also be involved in transporting target mRNAs throughout the cell

(Fujii et al., 2005; Fujii and Takumi, 2005). E. Stress granules. FUS and TDP-43 have also

been shown to localize to stress granules, which are associated with repression of translation

(Bosco et al., 2010; Colombrita et al., 2009; Liu-Yesucevitz et al., 2010; McDonald et al.,

2011). F. Regulation of local translation. Through regulation of mRNA transport and stress

granule formation, FUS and TDP-43 may also regulate local translation (Liu-Yesucevitz et

al., 2011; Wang et al., 2008).

Modelling ALS in Drosophila

In recent years Drosophila melanogaster has proven to be a powerful model system for

studying the basic biology of human disease genes as well as elucidating the mechanisms of

disease. Notably, about 75% of the known human disease genes have homologs in

Drosophila (Reiter et al., 2001). Although obvious differences exist between flies and

humans, the conservation of cellular and developmental signalling pathways lends a clear

advantage for employing Drosophila to elucidate the basic biology of genes and to generate

models of human disease. A recent example of success lies with modelling the most common

form of inherited mental retardation, Fragile X syndrome, which led to the identification of

novel neuroanatomical and functional phenotypes as well as interacting genes and small

molecules with therapeutic potential (Chang et al., 2008; Jin et al., 2004; McBride et al.,

2005; Wan et al., 2000; Zarnescu et al., 2005). The Drosophila model offers an unparalleled

set of genetic tools that allow for tissue specific and temporally controlled expression of

genes of interest; these could be wild-type or mutant alleles linked to human disease, or RNAi

constructs for gene knock-down (Brand and Perrimon, 1993; McGuire et al., 2003; Roman et

al., 2001). In addition, clonal analysis approaches allow for loss- and gain-of-function as well

as lineage studies (Lee and Luo, 1999; Pignoni and Zipursky, 1997; Xu and Rubin, 1993).

The use of these sophisticated genetic tools has helped uncover novel components and

functions of the EGFR/Ras, Notch and other signalling pathways, which in turn has advanced

our knowledge of human biology and mechanisms of disease (Doroquez and Rebay, 2006; Hu


and Li, 2010). Recently, Drosophila has proven its usefulness as a tool for drug discovery

(reviewed in Pandey and Nichols, 2011). Together with the relatively low costs involved and

a short generation time (10-12 days at 25oC), Drosophila offers powerful tools for gene and

drug discovery as well as elucidating the pathophysiological mechanisms underlying various

diseases in humans.

TDP-43 and FUS in Drosophila

There are two TDP-43 orthologues in the fly genome, TBPH and CG7804 (Estes et al.,

2011). Interestingly, CG7804 lacks the entire C-terminus domain that harbours the majority

of missense mutations in TDP-43 and is thought to play a major role in disease (Pesiridis et

al., 2009). Although at this time it is not clear what the precise function or evolutionary

significance of CG7804 is, it shares 41.5% amino acid identity to TBPH and 34.5% to TDP-

43 (Estes et al., 2011). Interestingly, loss-of-function of TBPH is sufficient to cause motor

neuron and ALS-like phenotypes (Feiguin et al., 2009). FUS has a single orthologue in

Drosophila, namely caz, which shares 53% aminoacid identity with its human counterpart


TDP-43 Based Models of ALS in Flies

Initial studies focused on elucidating the requirement of TBPH in motor neurons using

loss-of-function approaches. Hypomorphic and null alleles of TBPH exhibit smaller larval

neuromuscular junctions (NMJs), adult climbing defects and reduced longevity. Importantly,

expressing human TDP-43 in motor neurons only rescues these phenotypes indicating that

TBPH and TDP-43 are functionally conserved (Feiguin et al., 2009). Several groups took a

reverse translational approach, whereby either wild-type or mutant human TDP-43 were

overexpressed in various types of neurons including photoreceptors, motor, dendritic

arborisation (da) or mushroom body neurons (Elden et al., 2010; Estes et al., 2011; Hanson et

al., 2010; Lanson et al., 2011; Li et al., 2010; Lu et al., 2009; Miguel et al., 2011; Ritson et

al., 2010; Voigt et al., 2010; Wang et al., 2011). These experiments showed that TDP-43

overexpression is neurotoxic and leads to neuronal degeneration, locomotor dysfunction and

reduced survival, all of which are remarkably similar to the human disease. Notably,

mutations in TDP-43 that affect the RRM1 domain and its RNA binding ability exhibit milder

phenotypes, suggesting that RNA binding is required for toxicity (Voigt et al., 2010).

An important question in the field concerns the role of the C-terminal domain of TDP-43

in disease. It has been proposed that the missense mutations found in human patients, the

majority of which reside in the C-terminus, promote caspase cleavage, which in turn leads to

accumulation of C-terminal fragments in aggregates. While experiments in mammalian cells

have provided support to this model, with the exception of one report (Gregory et al., 2012),

all other in vivo studies using C-terminus overexpression did not produce any obvious

phenotypes (Estes et al., 2011; Li et al., 2010; Voigt et al., 2010; Yang et al., 2010). It will be

interesting to see whether this is a fly-specific response or perhaps, a reflection of the

increased ability of the whole organism to handle excess C-terminal fragments compared to

cultured cells.

Flies in Motion 67

TBPH/TDP-43 Localization and Toxicity: Loss of Nuclear Function or Gain

of Cytoplasmic Function?

Endogenous TBPH appears to localize primarily to the nucleus, while both nuclear,

perinuclear and some cytoplasmic puncta containing TBPH have been observed upon

overexpression (Estes et al., 2011; Hazelett et al., 2012; Lin et al., 2011). This is not unlike

studies of human TDP-43, which appears restricted to the nucleus when expressed at

endogenous levels but associates with cytoplasmic granules upon overexpression in

mammalian neurons (McDonald et al., 2011; Xu et al., 2010). In keeping with this

observation, upon overexpression in Drosophila motor neurons, TDP-43 is primarily

restricted to the nucleus although some reports of axonal TDP-43 also exist (Estes et al.,

2011; Li et al., 2010; Lu et al., 2009). These discrepancies could, in part, be explained by

differences in levels of expression between various published models. Interestingly, when

expressed in the developing neuroepithelium, TDP-43 associates with cytoplasmic puncta

within axons, which may be due to a differential response of photoreceptor neurons compared

to motor neurons (Estes et al., 2011).

A major question remains whether toxicity is conferred by TDP-43 in the nucleus,

cytoplasm or both. In other words, is toxicity due to a loss of nuclear function or a gain of

cytoplasmic function? This issue has been elegantly addressed in flies by expressing TDP-43

mutants that lack either the NLS or the NES (Miguel et al., 2011; Ritson et al., 2010). These

experiments showed that cytoplasmic TDP-43 is sufficient to generate neurotoxic phenotypes

and that in motor neurons, cytoplasmic TDP-43 (NLS- TDP-43) is more toxic than nuclear

TDP-43 (NES- TDP-43).Together, these data suggest that TDP-43 expression is toxic both

in the nucleus and the cytoplasm and support a gain of cytoplasmic function model (Miguel et

al., 2011; Ritson et al., 2010). Notably, these as well as several studies using disease-linked

variants found that cytoplasmic puncta are not a prerequisite for toxicity (Estes et al., 2011;

Hanson et al., 2010; Miguel et al., 2011). Genetic interaction experiments between human

TDP-43 and Drosophila TBPH result in an enhancement of the ALS-like phenotypes, which

provides further support to the model that TDP-43 overexpression mimics a loss-of-function

(Estes et al., 2011). In contrast, a recent transcriptome analysis using TBPH loss-of-function

and overexpression showed that these two conditions affect gene expression in very different

ways (Hazelett et al., 2012). Surprisingly, although phenotypically these conditions are rather

similar, at the molecular level there were only 57 transcripts commonly changed between

TBPH loss-of-function and overexpression. Collectively, these localization, phenotypic and

genetic interaction studies coupled with bioinformatics suggest that the overexpression of

TDP-43 variants (wild-type or mutant) shares both loss- and gain-of-function features.

Elucidating what aspects of TDP-43 function are impacted in these models that recapitulate

the pathology remarkably well, will provide much needed insights into the pathophysiology

of ALS.

TDP-43 Cytoplasmic Puncta: RNA Stress Granules or Protein Aggregates?

While in mammalian models TDP-43 has been clearly associated with RNA stress

granules, it remains to be seen what types of RNA granules if any, contain TDP-43 in flies

(McDonald et al., 2011). The role of RNA stress granules is to pull specific mRNAs from the

translational pool under less favourable environmental conditions and protect them from

degradation for some period of time (Anderson and Kedersha, 2009). At the same time,


biochemical and pathological studies indicate that TDP-43 associates with insoluble

aggregates (Voigt et al., 2010). Could these two cellular structures be connected? While it is

tempting to speculate that RNA stress granules may be precursors of protein aggregates, it is

possible that these two structures represent distinct entities (Dewey et al., 2012). More work

is needed to address this important aspect of ALS pathophysiology.

Reduced Survival and Locomotor Dysfunction

A hallmark of ALS is a progressive reduction in locomotor function and reduced

survival. Importantly, both loss-of-function and overexpression approaches in the Drosophila

model show that this feature is well recapitulated. Removal of TBPH from motor neurons

specifically using RNAi results in larval and adult locomotor dysfunction (Estes et al., 2011;

Feiguin et al., 2009; Li et al., 2010; Voigt et al., 2010). Interestingly, overexpression of TDP-

43 variants in motor neurons produces similar effects with TBPH loss, which lends further

support to the notion that phenotypically, TDP-43 overexpression resembles a loss-of-

function condition. Notably, some differences between wild-type and mutant TDP-43 variants

have begun to emerge. For example, while one study found that mutant TDP-43 exerts more

severe phenotypes compared to wild-type (Guo et al., 2011), others reported that wild-type

TDP-43 is more toxic than mutant variants when expressed at comparable levels (Estes et al.,

2011; Lu et al., 2009; Voigt et al., 2010). Thus while all TDP-43 variants exhibit various

levels of toxicity in flies, phenotypic nuances among different alleles may provide useful

insights into the mechanisms of disease.

Neuroanatomical Phenotypes

Another major feature of ALS pathology involves neuroanatomical defects accompanied

by denervation at the NMJ and more recently, evidence for axonal growth abnormalities

(Fallini et al., 2012). Loss of TBPH results in under grown NMJs characterized by fewer

synaptic boutons (Feiguin et al., 2009). As with the locomotor function studies,

overexpression of either wild-type or mutant human TDP-43 also leads to smaller NMJs,

consistent with a loss-of-function mechanism (Estes et al., 2011; Godena et al., 2011; Li et

al., 2010). At least one report finds that wild-type TDP-43 overexpression generates bigger

NMJs while mutant TDP-43 has no effect, suggesting a gain of function mechanism (Wang et

al., 2011). Despite some differences in the net effect of TDP-43 on NMJ growth, which could

be explained in part by genetic background effects, these findings indicate that TBPH/TDP-

43 modulate the morphology of the larval NMJ synapse. A notable difference between

Drosophila and mammalian models is the lack of obvious denervation and motor neuron

degeneration at least at the larval stage when these experiments were performed. Future

studies involving adult NMJs as well as physiological recordings from the synaptic terminals

are likely to provide additional insights into the precise role of TDP-43 and FUS in the

morphology and function of neuromuscular synapses.

Drosophila Models of ALS Based on FUS

Several recent studies have sought to determine the role of FUS and the mechanisms

underlying FUS pathology in ALS using flies as a model (Chen et al., 2011; Lanson et al.,

Flies in Motion 69

2011; Miguel et al., 2012; Sasayama et al., 2012; Wang et al., 2011; Xia et al., 2012). Loss-

of-function and overexpression approaches have shown that caz/FUS plays a role in both

locomotion and the growth of the NMJ synaptic terminal (Chen et al., 2011; Lanson et al.,

2011; Sasayama et al., 2012; Wang et al., 2011; Xia et al., 2012). caz is ubiquitously

expressed and loss-of-function approaches using either RNAi knock-down or classical

deletions result in lethality, which supports the notion that Caz, like TBPH is required for the

survival of the organism (Sasayama et al., 2012). Overexpression of wild-type and disease

variants of human FUS in the Drosophila eye leads to a progressive degenerative phenotype

accompanied by a loss of ommatidia organization. In motor neurons, FUS overexpression

leads to architectural defects at the NMJ synapse, locomotor dysfunction and reduced

survival, which resemble essential features of ALS pathology (Lanson et al., 2011; Sasayama

et al., 2012; Wang et al., 2011; Xia et al., 2012).

FUS Subcellular Localization and Toxicity

Recent studies aimed at examining the role of FUS mutations in ALS have shown that

wild-type FUS is primarily nuclear while mutant FUS localizes to the cytoplasm, which is

consistent with the inclusions found in disease pathology (Kwiatkowski et al., 2009; Lanson

et al., 2011; Miguel et al., 2012; Vance et al., 2009). Its cytoplasmic localization is required

for toxicity as disease mutants lacking the NES ( FUS) exhibit reduced ALS-like

phenotypes (Lanson et al., 2011). Evidence also exists that the nuclear localization of FUS is

critical for toxicity (Xia et al., 2012). As with TBPH, caz loss-of-function results in locomotor

impairment, suggesting a loss-of-function mechanism (Sasayama et al., 2012). Thus while the

FUS overexpression phenotypes are consistent with a gain of toxicity, it remains unclear

whether the human disease is caused by a loss of nuclear function, gain of cytoplasmic

toxicity or both.

Locomotor Phenotypes and Lifespan Effects

Neuronal specific knock-down of caz using RNAi results in impaired locomotor activity

albeit it has no apparent effect on lifespan (Sasayama et al., 2012). On the other hand,

complete loss of caz function using a null allele leads to severe locomotor defects affecting

both walking and flying as well as decreased lifespan (Wang et al., 2011). Importantly,

neuronal specific expression of caz in a null mutant background rescued most but not all

locomotor phenotypes, suggesting that control of locomotor function is partly intrinsic to

motor neurons. Furthermore, human wild-type FUS overexpression in caz mutant neurons

rescued locomotion defects but mutant FUS linked to fALS did not (Wang et al., 2011).

When overexpressed in wild-type motor neurons, all FUS variants (wild-type and mutants)

led to severe locomotor dysfunction in adult flies (Xia et al., 2012).

Neuroanatomical Defects

Loss-of-function studies show that Caz is required for proper morphology at the larval

NMJ (Sasayama et al., 2012; Wang et al., 2011). Although the structure of the synaptic

boutons themselves appears unaltered, loss of caz leads to undergrown synapses, containing

fewer synaptic boutons (Sasayama et al., 2012). As with TDP-43, FUS overexpression in

motor neurons is neurotoxic. While some report a disorganization of motor neurons and a

reduced NMJ area accompanied by a decrease in the number of small and large axonal


boutons (Xia et al., 2012), others report an increase in the number of synaptic boutons or no

change when FUS is overexpressed (Lanson et al., 2011; Wang et al., 2011). As with TDP-

43, despite some differences between various studies, it is clear that FUS is required to

regulate the architecture of the NMJ synapse.

Pathways and Networks

One of the strongest assets of Drosophila as a model for human disease is the power of

the genetic toolbox. The ability to sort through biochemically identified candidate targets and

partners to determine functional interactions in vivo represents a major strength of the fly

model. While full genome genetic and drug screens have yet to be published, several genetic

interactions have been reported in the fly models of TDP-43 and FUS. Importantly, TDP-43

and FUS exhibit genetic interactions with each other, suggesting that at least some functional

partners are common (Lanson et al., 2011; Wang et al., 2011).

In keeping with the RNA dysregulation hypothesis, TAF15, an RNA binding protein with

a domain structure similar to that of FUS has recently been reported to co-localize with TDP-

43 in pathological inclusions and to mimic several ALS features with the TDP-43 fly model

(Couthouis et al., 2011). It would not be surprising for several other RNA binding proteins to

exhibit similar interactions with TDP-43 and FUS. Identifying which of the known protein

partners can modify TDP-43 or FUS neurotoxicity may be critical for the development of

future therapeutic strategies.

Additional genetic interactions reported for TDP-43 include the HSP70 chaperone, the

caspase inhibitor P35, which suppress photoreceptor neurodegeneration as well as a dominant

negative construct of the small proteasome subunit, which enhances TDP-43 phenotypes

(Estes et al., 2011). These findings provide further evidence that the TDP-43 Drosophila

model recapitulates not only pathological features but also exhibits in vivo interactions with

well-established pathways including protein folding, apoptosis and proteasome mediated-

degradation. Similar to TDP-43, the neurotoxicity induced by FUS in the retina is alleviated

by overexpression of the HSP70 (HSPA1L) chaperone that has also been shown to mitigate

polyglutamine-mediated neurodegeneration (Miguel et al., 2012; Warrick et al., 1999).

In recent years, the discovery of novel genetic and environmental factors for

neurodegenerative disorders have led to the emergence of multi-hit models for

neurodegeneration, similar to Weinberg’s breakthrough cancer models (Hanahan and

Weinberg, 2000). Thus perhaps it is not surprising to discover that mutations in ataxin 2

accompany TDP-43 mutations in patients, making ataxin 2 a risk factor for ALS (Elden et al.,

2010). Similarly, the Type 1, inositol 1,4,5 Triphosphate (IP3) Receptor, ITPR1, a ER

resident, IP3 gated Ca2+

channel, which has been previously linked to cerebellar ataxia,

modulates TDP-43’s nucleocytoplasmic shuttling and its clearance by autophagy (Kim et al.,

2012; van de Leemput et al., 2007). Another example providing support to the multi-hit

model is Valosin Containing Protein (VCP), an ATP-ase involved in segregating

ubiquitinated substrates from large protein complexes and linked to inclusion body myopathy

associated with Paget’s disease of bone and frontotemporal dementia, which interacts

genetically with TBPH (Ritson et al., 2010). This functional interaction was identified in an

unbiased screen for VCP modifiers and suggests that VCP toxicity is mediated by TDP-43, as

evidenced by the redistribution of TDP-43 from the nucleus to the cytoplasm, which is

Flies in Motion 71

consistent with inclusions containing TDP-43 in inclusion body myopathy associated with

Paget’s disease of bone and frontotemporal dementia patients.

Another emerging theme is that TDP-43 and FUS may regulate the stability of the

microtubule network and possibly affect the transport processes dependent on the microtubule

cytoskeleton, which is one of the current ALS hypotheses. For example, HDAC6, a histone

deacetylase implicated in transcriptional control as well as acetylation of tubulin has been

shown to be a target of TDP-43 (Polymenidou et al., 2011). In human cells, TDP-43 and FUS

compete for binding HDAC6 mRNA, which supports the notion that they act in a common

pathway to regulate HDAC6 controlled processes (Kim et al., 2010). At the larval NMJ, the

microtubule associated protein MAP1B/Futsch, which like HDAC6 acts to stabilize

microtubules, is reduced in TBPH mutants, providing a possible explanation for the observed

undergrowth of the synaptic terminals (Feiguin et al., 2009; Godena et al., 2011).

Recently, RNA sequencing approaches using loss-of-function and overexpression

conditions identified significant changes in transcripts linked to the Wnt and BMP pathways,

suggesting that TBPH/TDP-43 may regulate the output of these pathways in the nervous

system (Hazelett et al., 2012).

CONCLUSION: CHALLENGES AND OPPORTUNITIES

The past years since the first reports of TDP-43 mutations in fALS and sALS patients

(Kabashi et al., 2008; Sreedharan et al., 2008; Van Deerlin et al., 2008) have seen an

avalanche of studies aimed at elucidating the basic biology of TDP-43 and the underlying

mechanisms of disease. The discovery of FUS followed shortly (Kwiatkowski et al., 2009;

Vance et al., 2009), which prompted a repositioning of ideas in the field and led to the

emergence of the RNA dysregulation hypothesis (Lagier-Tourenne and Cleveland, 2009).

Does the fly provide a good model for ALS and for studying these new hypotheses? The

sceptics remain concerned about the gulf between the fly model and humans. Eyebrows are

still raised upon discussions of ALS (or other human disease) modelling in the tiny fruit fly.

The evidence however stands to demonstrate that there are remarkable similarities between

the fly phenotypes and ALS pathology. These include among others, locomotor dysfunction

and reduced lifespan, which are, after all, the main problem with this devastating disease.

There are of course caveats that we need to be mindful of, including the overexpression

paradigms used by most models and the differences in glial biology, especially when

considering that glia seems to play a major role in motor neuron disease (Howland et al.,

2002). However, when one considers the strengths of the fly model, they weigh a lot heavier

than the caveats. For example, the ability to express or knock-down genes in a tissue, cell-

type and even temporal-specific manner, allows us to address why various types of neurons

are differentially affected in neurodegenerative disorders. Furthermore, the genetic toolbox

offers multiple strategies for unbiased genetic and drug screens.

Given the high degree of conservation between flies and humans, these screens provide a

relatively high-throughput means of discovering genes and compounds with therapeutic

promise in humans. Most surely, we will see data coming from such endeavours in the

coming years.


So, what have we learned from the fruit fly models? First, loss-of-function studies

demonstrate the requirement of TDP-43 and FUS during development as well as proper

neuronal function afterwards. Thus, there may be a previously unappreciated developmental

component for this neurodegenerative disease. Second, we learned that fALS and sALS

mutations in TDP-43 and FUS affect motor neurons intrinsically, in ways that mimic their

effects in humans, i.e., their function and survival are compromised. Furthermore, genetic

interactions in the fly confirmed that suspected cellular pathways are functionally important

in ALS. These include protein folding, proteasome-mediated degradation, apoptosis and

microtubule organization.

A major difference between fly models of ALS and human pathology remains,

specifically, according to most studies, the absence of ubiquitinated inclusions, which

represent a hallmark of the disease. While the reason and significance for this apparent

discrepancy remains to be seen, it is worth noting that the fly model unequivocally shows that

cytoplasmic aggregates are not a prerequisite of motor neuron disease. This suggests that

pathological inclusions are more likely to be a consequence rather than a cause for motor

neuron dysfunction and death. In addition, this finding shifts the focus from cytoplasmic

aggregates to other aspects of disease pathophysiology, including neuroanatomical and

synaptic function defects.

How far can the fly model ”fly”? Even the most passionate Drosophila geneticists will

probably agree that while the fly provides a rapid and efficient evaluation of the pathways

relevant to disease, it is just the first step to developing strategies with therapeutic potential in

humans. Perhaps a most reasonable approach is to learn as much as possible about the basic

biology of ALS from the fly and other genetically amenable models ranging from yeast to

zebrafish, then validate these findings in mammalian models, which have been the golden

standard for advancing therapies to humans.

A similar logic could be applied to drug screening: compounds identified as

neuroprotective in the fly should be subjected to further testing and validation in rodent

models. However, given the limited success of translating findings from mouse to humans at

least when it comes to the SOD1 model and taking into the account the lack of therapies for

ALS patients who face a rapid loss of motor function and death within 2-5 years of diagnosis,

one has to ask: should we take the tiny fruit fly a little more seriously?

ACKNOWLEDGMENTS

We are grateful to the Jim Himelic foundation as well as the Himelic family and friends

for providing seed funding and inspiration for modelling ALS in Drosophila. We also thank

the Muscular Dystrophy Association (MDA173230) and NIH (1R21NS078429-01A1) for

financial support and Drs. K. Scherer, H. Horak, B. Coull and D. Labiner (UA, Neurology) as

well as members of the Zarnescu laboratory for helpful discussions.

Flies in Motion 73

ABOUT THE AUTHORS

Andrés Morera earned his BS in Biotechnology at Indiana University. He is presently a

PhD student in Biochemistry and Molecular & Cellular Biology (BMCB) at University of

Arizona. His research is focused on the involvement of the TOR pathway and autophagy in

neurodegeneration.

Alyssa Coyne obtained her BS in Biology at Springfield College, MA. Alyssa is

currently pursing a PhD in Neuroscience at the University of Arizona where her research

interests lie in the area of protein homeostasis during neurodevelopment and

neurodegeneration.

Daniela C Zarnescu obtained her BS in Physics at University of Bucharest, Romania.

Following her PhD degree in Biochemistry and Molecular Biology at Penn State, Daniela

trained as a postdoctoral fellow in Molecular Genetics at Emory University School of

Medicine. She is currently Associate Professor of Molecular and Cellular Biology,

Neuroscience and Neurology at University of Arizona. Daniela’s current research interests lie

in the broad area of gene expression with a focus on the role of RNA binding proteins during

development and in disease

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Chapter 4

MAINTAINING LONG SUPPLY LINES:

AXON DEGENERATION AND THE FUNCTION

OF HEREDITARY SPASTIC PARAPLEGIA GENES

IN DROSOPHILA

Belgin Yalçın and Cahir J. O’Kane* Department of Genetics, University of Cambridge, UK

ABSTRACT

The length of motor system axons presents great challenges to the subcellular

trafficking machinery of neurons. Impairment of the mechanisms that maintain axonal

function can lead to axon degeneration diseases, particularly in the distal regions of axons

that lie furthest from the cell body. Hereditary spastic paraplegias (HSPs) are one such

group of diseases – these share a common feature of degeneration of distal upper motor

axons, sometimes with a spectrum of additional mainly neurological symptoms. Their

phenotypic heterogeneity is reflected in their genetic causes - some 50 genetic loci have

been identified as causative, and over 25 of these have been cloned. These spastic

paraplegia gene (SPG) products do however point at an unexpectedly limited range of

disease mechanisms, including endoplasmic reticulum (ER) organization and function,

axonal microtubule-based transport, and endosomal traffic and signalling. Most but not

all causative human genes have orthologues in Drosophila. Despite the shorter lifespan

and short axons compared to humans, Drosophila offers a powerful system to study both

the cellular functions of many of these genes, and what goes wrong when they are

mutated. This stems both from the powerful genetic tools for generation of specific

mutant or transgenic flies, as well as the powerful analytic tools for understanding the

cellular roles of these gene products in neurons, particularly in axons and synapses.

Major contributions from flies so far have included dissection of the roles of several SPG

proteins in ER organization, transport of specific cargoes in axons, and in pathways

including bone morphogenetic protein (BMP) signalling. As additional SPG proteins are

* Correspondence should be addressed to: Cahir J O’Kane, Department of Genetics, University of Cambridge,

Downing Street, Cambridge, CB2 3EH, United Kingdom. Tel: +44-1223-333177; Fax: +44-1223-333992;

Email: [email protected].

Belgin Yalçın and Cahir J. O’Kane 86

identified, Drosophila offers a great opportunity to understand their cellular roles, and

ultimately providing plausible mechanisms for these diseases.

Keywords: Axon degeneration; axon transport; endoplasmic reticulum

INTRODUCTION

Maintaining the functionality of motor axons, whose length can be up to 105 times longer

than that of cell bodies, presents great challenges for the trafficking machinery of motor

neurons. Impairment of this machinery can therefore lead to degeneration of axons,

particularly in the distal regions that lie furthest from the cell body. Hereditary spastic

paraplegias (HSPs) are a diverse group of genetic disorders that show this effect. They are

characterized by progressive spasticity and weakness in lower extremities, caused by

progressive distal axonopathy mostly in the longest “upper” corticospinal motor neurons.

HSPs are conventionally classed as either pure (uncomplicated) or complex (complicated)

depending on whether there are additional neurological symptoms such as dementia,

intellectual impairment, epilepsy or amyotrophy. However, it is becoming clearer that this

division is simplistic, and that HSPs show a spectrum of additional symptoms. The diversity

of HSP-associated symptoms and causative genes suggests that impairments in a range of

cellular processes can result in axonal degeneration.

Many cases of HSP are clearly inherited. Over 50 causative spastic paraplegia genes

(SPGs) have now been mapped, that can give rise to either dominant or recessive forms of the

disease, and about 25 of these have been cloned (Table 1). This suggests great heterogeneity

in the causes of HSP, and in the cellular processes necessary for integrity of longer axons.

However, a closer look suggests a more limited range of cellular processes that are affected –

in particular endoplasmic reticulum (ER) organization and function, microtubule (MT)

trafficking, endosomal trafficking and signalling, mitochondrial function, and interactions of

axons with the myelin sheath.

The axons affected in HSP can be around a meter long in humans, enormous compared to

their cell body diameters of a few tens of µm. Maintenance of distal axons requires transport

of most organelles and proteins from the cell body, transport of other components such as

lipids, and other communication with the cell body. These are problems both of fundamental

interest, and highly relevant to HSP disease mechanisms. Understanding the cellular roles and

mutant phenotypes of SPG proteins is a route to addressing these problems, and for this the

fruit fly Drosophila is an excellent model. Flies have orthologues of most (but not all) SPGs

(Table 2), they have motor axons that house most of the axon transport and cell signalling

machinery of human neurons (albeit much shorter than human neurons), and sophisticated

genetic tools to generate mutant or transgenic genotypes, more rapidly than in mice. Here we

review the contributions of Drosophila to understanding the cellular roles of SPG proteins,

and the processes that may be affected when their functions are lost or altered. We focus

mainly on the many SPG genes with functions in organization or trafficking of the neuronal

endomembrane system, where Drosophila has been most valuable to understand the roles of

these genes in axonal or synapse function.

Table 1. Cloned HSP genes with predicted domains and protein functions. Predictions were performed using the SMART prediction

program (http://smart.embl-heidelberg.de/)

Category Human

Protein

Predicted Domain Protein

length

(AAs)

Protein Function Main References

Intracellular

trafficking

(ER shaping)

SPG3A

atlastin

558 Member of dynamin GTPase

superfamily, localized to ER three-way

junctions

Zhao et al., 2001; Hu et

al., 2009; Orso et al.,

2009; Lee et al., 2009

Intracellular

trafficking

(ER shaping)

SPG4

spastin

616 AAA protein, predominantly ER

localized, binds and severs

microtubules

Fonknechten et al., 2000;

Evans et al., 2005; Roll-

Mecak and Vale, 2005;

Trotta et al., 2004; Du et

al., 2010

Intracellular

trafficking

(ER shaping)

SPG12

reticulon 2

545 ER shaping protein, generating ER

curvature

Shibata et al., 2008;

Montenegro et al., 2012;

O’Sullivan et al., 2012

Intracellular

trafficking

(ER shaping)

SPG31

REEP 1

208

ER shaping protein, generating ER

curvature, might localize to

mitochondria

Zuchner et al., 2006; Park

et al., 2010


Category Human

Protein


length

(AAs)


Intracellular

trafficking

(axonal

transport)

SPG10

KIF5A

1032 Member of kinesin family, microtubule

motor protein in intracellular transport

Reid et al., 2002; Ebbing

et al., 2008

Intracellular

trafficking

(axonal

transport)

SPG30

KIF1A

1791

Member of kinesin family, microtubule

motor protein in intracellular transport

Klebe et al., 2012

Intracellular

trafficking

(endosomal

trafficking)

SPG6

NIPA1

329 Endosomal trafficking, BMP signalling Wang et al., 2007

Intracellular

trafficking

(endosomal

trafficking)

SPG8

strumpellin

1159 Subunit of WASH complex, involved

in microtubule dynamics

Valdmanis et al., 2007;

Clemen et al., 2010;

Harbour et al., 2010

Intracellular

trafficking

(endosomal

trafficking)

SPG11

spatacsin

2443 Protein might be involved in cell

growth, cell cycle or transcriptional

regulation

Stevanin et al., 2007;

Murmu et al., 2011

Intracellular

trafficking

(endosomal

trafficking)

ALS2

alsin

1657 Localized to early endosomes, mediates

endosome fusion via Rab5 GEF activity

Kunita et al., 2004; Otomo

et al., 2011

Category Human

Protein


length

(AAs)


Intracellular

trafficking

(endosomal

trafficking)

SPG15

spastizin

2539 Protein might be involved in axonal

transport and membrane trafficking

Hanein et al., 2008;

Murmu et al., 2011

Intracellular

trafficking

(endosomal

trafficking)

SPG21

maspardin

308 Protein might function in protein

transport and sorting of endosomes

Simpson et al., 2003

Intracellular

trafficking

(endosomal

trafficking)

SPG33

Protrudin

(ZFYVE27)

416 Interacts with spastin, localizes to

endosomes

Mannan et al., 2006;

Zhang et al., 2012

Intracellular

trafficking

(endosomal

trafficking)

SPG42

SLC33A1

549 Localizes to ER membrane, acetyl-CoA

transporter

Lin et al., 2008; Jonas et

al., 2010

mitochondrial SPG7

paraplegin

795 Mitochondrial AAA protease, protein

folding, and proteolysis

Nolden et al., 2005;

Mancuso et al., 2012

mitochondrial SPG13

HSP60

573 Mitochondrial chaperonin Hansen et al., 2002, 2008

Lipid

metabolism

SPG5A

CYP7B1

506 Localizes to ER, hydroxylation

reactions of some steroids and

oxysterols

Tsaousidou et al., 2008;

Rose et al., 2001

Lipid

metabolism

SPG17

seipin

462 Integral ER membrane protein, lipid

droplet biogenesis

Patel et al., 2001;

Windpassinger et al.,

2004; Ito et al., 2008


Category Human

Protein


length

(AAs)


Lipid

metabolism

SPG18

ERLIN2

339 Mediates ER degradation (ERAD)

pathway

Alazami et al., 2011

Lipid

metabolism

SPG20

spartin

666 Multifunctional protein, binds ESCRT-

III complex involved in endosomal

transport. Binds to Eps15, involved in

lipid droplet maintenance

Ciccarelli et al., 2003;

Bakowska et al., 2005; Lu

et al., 2006

Lipid

metabolism

SPG35

FA2H

372 Hydroxylation of myelin lipids

Dick et al., 2010; Eckhardt

et al., 2005

Lipid

metabolism

SPG39 NTE

1375 Membrane lipid homeostasis Kienesberger et al., 2008;

Rainier et al., 2008

Cell adhesion SPG1

L1CAM

1257 Immunoglobulin-related neuronal cell

adhesion molecule

Jouet et al., 1994; De

Angelis et al., 2002

Category Human

Protein


length

(AAs)


Myelination SPG2 PLP1

277 Predominant myelin protein present in

the central nervous system

Grossi et al., 2011; Zappia

et al., 2011

Abbreviations:

AAA: ATPases associated with a variety of cellular activities

Abhydrolase_1: alpha/beta hydrolase fold

cNMP: Cyclic nucleotide-monophosphate binding domain

FHA: Forkhead associated domain

FN3: Fibronectin type 3 domain

FYVE: zinc finger domain

GBP: Guanylate-binding proteins (a family of GTPases)

IG: Immunoglobulin

IGc2: Immunoglobulin C-2 Type

KISc: kinesin motor catalytic domain, ATPase

MIT: Microtubule Interacting and Trafficking molecule domain

MORN: Possible plasma membrane-binding motif in junctophilins, PIP-5-kinases and protein kinases.

PH: Pleckstrin homology domain.

PHB: Prohibitin homologues

PLP: Myelin proteolipid protein (PLP or lipophilin)

RhoGEF: Rho guanine exchange factor (GEF)

TCP1/cpn60: chaperonin containing domain

TM: transmembrane domain; note that SMART usually scores intramembrane loops as TM domains.

VPS9: vacuolar sorting protein 9-like GEF

Table 2. Cloned HSP genes and their Drosophila homologues. Homology search was performed using BLAST

(Basic Local Alignment Search Tool)

Category Gene Inheritance Pure/

complex

Gene product Drosophila

homologue

AA identity of longest

homology, BLASTP

E value

Intracellular trafficking

(ER shaping)

SPG3A Autosomal

Dominant

Pure atlastin CG6668

atlastin

57%, 0.0


(ER shaping)

SPG4 Autosomal

Dominant

Pure spastin CG5977

spastin

52%, 5e-160


(ER shaping)

SPG12 Autosomal

Dominant

Pure reticulon 2 CG33113

Reticulon-like 1

40%, 2e-48


(ER shaping)

SPG31 Autosomal

Dominant

Pure REEP 1 CG42678

REEP A

63%, 2e-57


(axonal transport)

SPG10 Autosomal

Dominant

Complex KIF5A CG7765

Khc

59%, 0.0


(axonal transport)

SPG30 Autosomal

Recessive

Complex KIF1A CG8566

unc-104

55%, 0.0


(endosomal trafficking)

SPG6 Autosomal

Dominant

Pure NIPA1 CG12292

spichthyin

40%, 4e-67



SPG8 Autosomal

Dominant

Pure strumpellin CG12272

strumpellin

44%, 0.0



SPG11 Autosomal

Recessive

Complex spatacsin CG13531

spatacsin

27%, 3e-33



ALS2 Autosomal

Recessive

Complex alsin CG7158

alsin

25%, 5e-53



SPG15 Autosomal

Recessive

Complex spastizin CG5270

spastizin

36%, 4e-39

Category Gene Inheritance Pure/

complex

Gene product Drosophila

homologue

AA identity of longest

homology, BLASTP

E value



SPG21 Autosomal

Recessive

Complex maspardin No



SPG33 Autosomal

Dominant

Pure Protrudin

(ZFYVE27)

No



SPG42 Autosomal

Dominant

Pure SLC33A1 CG9706

SLC33A1

52%, 4e-172

mitochondrial SPG7 Autosomal

Recessive

Complex paraplegin CG2658

paraplegin

58%, 0.0

mitochondrial SPG13 Autosomal

Dominant

Pure HSP60 CG12101

HSP60

74%, 0.0

Lipid metabolism SPG5A Autosomal

Recessive

Pure CYP7B1 many homologues

Lipid metabolism SPG17 Autosomal

Dominant

Complex seipin CG9904

seipin

40%, 7e-59


Recessive

Complex ERLIN2 No


Recessive

Complex spartin CG12001

spartin

28%, 7e-36


Recessive

Complex FA2H CG30502

FA2H

35%, 7e-66


Recessive

Complex NTE CG2212

Swisscheese

44%, 0.0

Cell adhesion SPG1 X-linked Complex L1CAM CG1634

neuroglian

29%, 2e-146

Myelination SPG2 X-linked Complex PLP1 CG7540

PLP1

24%, 8e-10


A ROLE FOR MIS-SHAPING OF ENDOPLASMIC RETICULUM IN HSP?

Most cases of autosomal dominant HSP are caused by mutations affecting four proteins

that shape ER morphology – spastin, atlastin-1, REEP1 and reticulon-2 – suggesting that

defects in ER morphology could be one cellular cause of HSPs. These proteins share a

common feature of one or more intramembrane hairpin-loop domains that insert into the

cytosolic face of the ER membrane (Voeltz et al., 2006; Shibata et al., 2008; Zürek et al.,

2011), increasing its surface area and thus inducing membrane curvature, as in ER tubules or

the edges of sheet ER (Hu et al., 2009; Shibata et al., 2008). Before considering how this

process might relate to axonal function, let us first look at the relevant SPG products.

Spastin (SPG4)

Mutations in spastin are the most common cause of autosomal dominant HSP, normally

pure (Hazan et al., 1999). Spastin is a MT-severing AAA (ATPase associated with various

cellular activities) protein. Many missense, nonsense and splice site point mutations, deletions

and insertions have been observed in patients (Fonknechten et al., 2000), suggesting either a

haploinsufficient and/or dominant-negative disease mechanism, although there is a suggestion

of dominant gain-of-function toxicity caused by mutations in the intramembrane hairpin

domain (Solowska et al., 2010).

The most abundant spastin isoforms are a longer M1 isoform of 616 amino acid residues,

and a shorter M87 isoform translated from a downstream start codon, that lacks the N-

terminal 86 residues of M1 (Claudiani et al., 2005; Mancuso et al., 2008). Both forms contain

a MIT domain that interacts with the late endosomal ESCRT-III component CHMP1B (Reid

et al., 2005), an MT-binding domain, and a C-terminal AAA domain responsible for hexamer

formation and MT-severing activity (Beetz et al., 2004; Claudiani et al., 2005; Roll-Mecak et

al., 2008). The M1 variant contains a single intramembrane hairpin, which inserts in ER

membrane, and mediates interactions with atlastin-1 and reticulon-1 (Sanderson et al., 2006;

Mannan et al., 2006; Evans et al., 2006; Connell et al., 2009). In contrast, the M87 isoform is

abundant in endosomes, probably due to interaction of its MIT domain with the ESCRT-III

component CHMP1B.

It is not yet clear which molecular function(s) and population(s) of spastin are most

relevant in HSP. Both M1 and M87 are found in spinal cord (Claudiani et al., 2005; Salinas et

al., 2005; Solowska et al., 2008); and missense mutations are found throughout the coding

region, including the M1-specific region (Shoukier et al., 2009; McCorquodale et al., 2011).

While the MT-severing activity of spastin suggests a link to MT-based axonal transport, this

could be required on the ER, endosomes, or elsewhere. Other functions of spastin could also

be relevant to SPG4 pathology. First, spastin (like three other HSP proteins spartin, NIPA1,

and atlastin-1) appears to antagonize BMP signalling (Tsang et al., 2009), although a

mechanism for this is lacking. Second, spastin is also found in the cytokinetic midbody

during cell division due to its interaction with centrosome protein NA14 (Errico et al., 2004)

and ESCRT-III (Yang et al., 2008), where it is involved in MT abscission (Connell et al.,

2009).

Maintaining Long Supply Lines 95

Drosophila has one spastin orthologue (CG5977), with one known transcript that encodes

an M1-like protein with a likely intramembrane hairpin. Homozygous spastin mutant flies are

predominantly lethal, and adult escapers have severely disrupted motor function, as do human

patients (Sherwood et al., 2004). Spastin is abundant in axons and synaptic areas (Trotta et al.,

2004). RNAi knockdown results in smaller synaptic area and increased MTs at the

neuromuscular junction (NMJ), whereas overexpression lowers synaptic strength and synaptic

MTs (Trotta et al., 2004). In contrast to knockdown larvae, spastin null mutant larvae exhibit

fewer MTs at the NMJ, and additional satellite boutons (Sherwood et al., 2004). Exogenous

expression of full-length human spastin rescues spastin null phenotypes (Du et al., 2010),

showing phylogenetic conservation of at least some spastin functions, but it is not known

whether rescue is mediated by the M1 and/or M87 variants. Taken together, analysis of

spastin mutant phenotypes in flies suggest possible mechanisms of pathology involving

dysfunction of the neuronal MT cytoskeleton, although it would be simplistic to regard these

Drosophila genotypes as replicas of the human disease.

Drosophila phenotypes also illuminate mutant spastin functions. For example, expression

of a pathogenic ATPase domain mutant has dominant-negative effects and is not simply non-

functional (Orso et al., 2005). Co-expression of different spastin variants shows that as in

human patients, amino acid substitutions in the N-terminal M1-specific domain exacerbate

phenotypes caused by mutations in the ATPase domain (Du et al., 2010), arguing that

impairment of ER-specific spastin function is important for spastin mutant phenotypes in

both flies and humans.

Drosophila genetic screens have also revealed cellular processes that interact with

spastin. Spastin loss-of-function phenotypes are strongly suppressed by loss of the actin

regulatory kinase Pak3 (Ozdowski et al., 2011), suggesting that many effects of spastin loss

act via Pak3-dependent processes. Pak family kinases might therefore be potential therapeutic

targets in SPG4 patients, although this will require further investigation in mammals. A link

with another neurological condition has also been found in Drosophila; heterozygous loss of

spastin dominantly suppresses phenotypes of Drosophila FMRP (Fragile X mental retardation

protein) overexpression (Yao et al., 2011), and so FMRP may act upstream of or in parallel to

spastin.

Atlastin (SPG3A)

Mutations in atlastin-1 cause autosomal dominant usually pure HSP (Zhao et al., 2001). It

is the second most common HSP, generally caused by missense mutations (Zhao et al., 2001).

Humans have three atlastin paralogues: atlastin-1 is predominantly expressed in the CNS

(Zhu et al., 2003) and atlastin-2 and atlastin-3 are widely expressed (Rismanchi et al., 2008).

Atlastins belong to the dynamin GTPase superfamily, and contain a C-terminal

intramembrane loop (Zhu et al., 2003; Rismanchi et al., 2008). Knockdown, or expression of

mutant forms, suggests that atlastin-1 is important for the reticular network of tubular ER,

whereas atlastin-2 and atlastin-3 regulate Golgi morphology (Namekawa et al., 2007;

Rismanchi et al., 2008). Atlastin-1 interacts with spastin, REEP and reticulon family proteins

via their intramembrane loop regions (Evans et al., 2006; Sanderson et al., 2006; Hu et al.,

2009; Park et al., 2010), suggesting that these proteins participate in a common

pathomechanism of HSP.


Drosophila has a single atlastin orthologue (CG6668), which has been central to our

understanding of atlastin function. It localizes to ER membranes, and overexpression causes

ER expansion, while loss of its function causes ER network fragmentation. Moreover atlastin

enhances the in vitro fusion of liposomes. The GTPase domain is required to form trans-

oligomeric complexes, promote membrane fusion in vitro, and form a normal reticular

network in vivo (Orso et al., 2009). The fusion function of the yeast atlastin, Sey1p, is

antagonized by another ER hairpin protein lunapark/lnp-1 (Chen et al., 2012), hinting at a

machinery that regulates ER fusion. Drosophila has a lunapark homolog (CG8735), but there

is no information on its function.

Drosophila atlastin loss-of-function and overexpression phenotypes also show many

similarities to those of spastin, perhaps resulting from their interaction. Loss of atlastin causes

accumulation of stable MTs in muscles whereas its overexpression results in the opposite;

loss of atlastin also causes additional satellite boutons at the NMJ (Lee et al., 2009).

Reticulon 2 (SPG12)

Mutations in reticulon 2 (RTN2) cause autosomal dominant pure HSP (Montenegro et al.,

2012). Mammals have four reticulons, RTN1-4 (Shibata et al., 2008). They are predominantly

localized in the ER (van de Velde et al., 1994; Grandpre et al., 2000; Di Sano et al., 2003).

RTN4 has also been characterized as Nogo, an inhibitor of axon regeneration (reviewed by

Zörner and Schwab, 2010), although its in vivo role in regeneration appears subtle (Lee et al.,

2009).

Reticulons contain two hydrophobic hairpins inserted in the ER membrane that mediate

formation of reticulon oligomers and heteromeric complexes with other hairpin loop proteins

(Shibata et al., 2008). They are localized mainly in curved regions of ER (Voeltz et al., 2006;

Kiseleva et al., 2007; Shibata et al., 2010). Overexpression of reticulons results in long

unbranched and mostly bundled ER tubules in both mammalian and yeast cells (Voeltz et al.,

2006). Deletion of all yeast reticulons disrupts peripheral tubular ER in stress conditions, but

otherwise leaves it largely intact, implying that they are not alone responsible for forming it

(Voeltz et al., 2006).

There are two Drosophila reticulons, reticulon-like 1 (Rtnl1) and reticulon-like 2 (Rtnl2).

Sequence analysis suggests that a single ancestral reticulon was duplicated independently in

fly and mammalian lineages, giving two paralogues in Drosophila and four in mammals.

Rtnl1 is widely expressed and is evolving relatively slowly, suggesting that it is the main

functional orthologue of all mammalian reticulons. Rtnl2 is evolving faster and so is less

functionally constrained, and its expression is restricted mainly to fat body and testis,

suggesting a more limited or specialized function (O’Sullivan et al., 2012). Rtnl1 deletion has

no overt effects on viability (Wakefield and Tear, 2006). However, Rtnl1 knockdown causes

expansion of ER sheets in epidermal cells and induces ER stress in epithelial and neuronal

cells. It also disrupts smooth ER and MTs in longer distal motor axons (O’Sullivan et al.,

2012). This finding is significant for HSP, providing a possible model for how ER-shaping

proteins may be essential for distal axon function – impaired ER organization might affect

physiological functions such as calcium signalling preferentially in these axonal regions.


REEP1 (SPG31)

REEP1 (receptor expression enhancing protein) mutations are the third most common

cause of autosomal dominant HSP, (Züchner et al., 2006). There are six REEP genes in

humans, REEPs 1-6, which all have two hairpin-loop domains in ER membrane, that enable

at least REEP1 to interact with atlastin-1 and spastin (Voeltz et al., 2006; Hu et al., 2008;

Park et al., 2010).

REEPs 1-4 have an MT-binding domain on their C-terminal cytoplasmic region, unlike

REEPs 5-6 (Park et al., 2010), and may therefore tether ER tubules with MTs (Park et al.,

2010). At least one HSP-causing mutation of REEP1 encodes a truncated protein that cannot

bind MTs and causes ER disruption (Park et al., 2010). REEP1 is predominantly localized to

tubular ER but it may also be mitochondrial (Saito et al., 2004; Behrens et al., 2006; Züchner

et al., 2006).

Drosophila has six REEP genes. Using the same criteria as with the reticulons, CG42678

(which we designate ReepA) appears to be the main functional orthologue of mammalian

REEPs 1-4, and CG8331 (which we designate ReepB) appears to be the main functional

orthologue of mammalian REEPs 5-6. Since Drosophila has two REEP proteins that

presumably do the job of six mammalian REEPs, it offers a system to analyse REEP function

with less redundancy than in mammalian models. However, there is no published information

on their roles in ER or axonal function in Drosophila.

Hairpin-Loop ER-Shaping Proteins: Conclusions

What is the relevance of ER hairpin protein function to axon degeneration? Axons have

extensive stretches of smooth ER tubules (Terasaki et al., 1991; Droz et al., 1975), and the

effects of Rtnl1 knockdown in Drosophila axons (O’Sullivan et al., 2012) hint at a role for

SPG proteins in its integrity. How could impairment of axonal ER lead to degeneration? The

continuous tubular organization of smooth ER suggests the model of a “neuron within a

neuron”, which can carry signals along axons and dendrites independently of action

potentials, and faster than MT-based transport (Berridge, 2002). Disruption of this network

could impair processes such as calcium or BMP signalling, that could lead to degeneration

preferentially in distal axons, which may be more dependent on continuity of this network.

Drosophila offers an exciting model to test some of these ideas. Spastin and atlastin

mutants clearly have synaptic defects, and loss of reticulon leads to ER defects in distal axons

that have not yet been ultrastructurally defined. Axonal ER is also a fascinating compartment

in its own right, and Drosophila offers many tools to investigate its role in neuronal function

and dysfunction.


HSP MUTATIONS AFFECT ADDITIONAL ER PROTEINS

Seipin (SPG17)

Mutations in BSCL2, which encodes seipin, cause an autosomal dominant complex HSP

that is known as Silver syndrome. Loss-of-function seipin mutations cause recessive

Berardinelli-Seip congenital lipodystrophy, characterized by loss or absence of body fat and

mental retardation, but without abnormalities in motor neurons (Agarwal and Garg, 2003,

2004; Fu et al., 2004).

Seipin is a homo-oligomer of about nine subunits located in ER membrane, mostly at

junctions with lipid droplets (Windpassinger et al., 2004; Szymanski et al., 2007; Fei et al.,

2008; Binns et al., 2010). It has cytosolic N- and C-termini, and two transmembrane domains

in the ER membrane (Lundin et al., 2006). Unlike the hairpin proteins above, seipin has a

glycosylated loop in the ER lumen. Dominant HSP results from mutations in the ER lumenal

domain, which cause an unfolded protein response (UPR) (Ito et al., 2008; Yagi et al., 2011).

Drosophila seipin loss-of-function mutants also show reduced lipid storage in the fat

body, and ectopic lipid droplets in the non-adipose salivary gland (Tian et al., 2011).

However, there is as yet no published work on HSP-related gain-of-function effects in

Drosophila.

Neuropathy Target Esterase (SPG39)

Mutations in neuropathy target esterase (NTE) cause autosomal recessive complex HSP

(Rainier et al. 2008; 2011). Modification of NTE by organophosphorus (OP) compounds also

causes neuropathy (Johnson and Glynn, 1995). NTE is a 150-kDa ER membrane protein, with

a short lumenal N-terminus, a transmembrane domain, and a large cytosolic C-terminus

(Akassoglou et al., 2004; Chang et al., 2010).

NTE has phospholipase and lysophospholipase activity in its cytosolic domain (Lush et

al., 1998; van Tienhoven et al., 2002; Quistad et al., 2003), and hence regulates membrane

phospholipid content (Akassoglou et al., 2004). It hydrolyses lysophosphatidylcholine (LPC),

protecting cell membranes from LPC accumulation (Zaccheo et al., 2004; Vose at al., 2008),

and degrades ER-associated phosphatidylcholine (Zaccheo et al., 2004). Deletion of NTE in

mouse brain causes neurodegeneration, with disrupted ER and vacuolation in nerve cell

bodies, and abnormal reticular aggregates (Akassoglou et al., 2004). While neuropathy or

neurodegeneration could be due to impaired phospholipid metabolism, the finding of

abnormal ER (albeit not axonal) could also be relevant to HSP.

The Drosophila NTE orthologue is swiss cheese, named after the extensive vacuolation

in mutant nervous systems, as both glia and neurons undergo cell death (Kretzschmar et al.,

1997). Swiss Cheese protein also localizes to ER, and mutants show elevated levels of

phosphatidylcholine (Mühlig-Versen et al., 2005). Whether axonal ER is affected is

unknown.


CYP7B1 (SPG5A)

Mutations in CYP7B1, which encodes a cytochrome P450, can cause either autosomal

recessive pure HSP with variable age of onset (Tsaousidou et al., 2008; Schule et al., 2009),

or liver failure (Stiles et al., 2009). CYP7B1 is a widely expressed 506-amino-acid enzyme,

thought to be localized in ER, that catalyzes 6- and 7-hydroxylation of some steroids and

oxysterols including cholesterol, and the variety of disease phenotypes may result from the

range of substrates that the enzyme can metabolize (reviewed by Stiles et al., 2009).

Gene duplication has given rise to many CYP7B1 orthologues in Drosophila

(www.flybase.org). This may be due to selection for the ability to metabolize numerous

substrates, perhaps ones encountered in the wild. However without better knowledge of which

substrates are most relevant to HSP, it is not clear which Drosophila CYP7B1 orthologues are

useful for modelling SPG5A.

FA2H (SPG35)

Mutations affecting fatty acid 2-hydroxylase (FA2H), cause autosomal recessive complex

HSP, with additional symptoms including leukodystrophy (myelin sheath defects), and

neurodegeneration with brain iron accumulation (Alderson et al., 2009; Dick et al., 2010).

FA2H is a membrane-bound ER enzyme (Eckhardt et al., 2005). There are four putative

transmembrane domains of human FA2H and the N- and C-terminals, including a cytochrome

b5 domain and an ER retention signal, are on the cytoplasmic side of the membrane (Haak et

al., 1997; Mitchell and Martin, 1997). FA2H catalyses 2-hydroxylation of sphingolipids and

straight chain fatty acids (Zöller et al., 2008; Maldonado et al., 2008). Sphingolipids with 2-

hydroxy fatty acids are abundant in myelin (Norton and Cammer, 1984), and FA2H is also

abundant in the oligodendrocytes that form the myelin sheath – so disease mechanisms may

involve myelin dysfunction rather than impaired neuronal ER function. Drosophila has a

single FA2H orthologue, which is a sphingolipid-specific fatty acid hydroxylase (Carvalho et

al., 2010), but there is little knowledge of its role in neuronal or glial function.

SLC33A1 (SPG42)

A missense mutation affecting the acetyl-CoA transporter SLC33A1 segregates with

autosomal dominant pure HSP in a large Chinese pedigree (Lin et al., 2008). SLC33A1 is

localized in ER membrane, where it is important for lumenal acetylation of ER proteins and

for cell viability (Jonas et al., 2010). SLC33A1 is induced by the ER unfolded protein

response. In its absence, failure to acetylate the lumenal domain of Atg9A leads to induction

of autophagy, and potentially to autophagy-mediated cell death (Pehar et al., 2012). Loss-of-

function mutations cause a human syndrome with cataracts, hearing loss and severe

developmental delay (Huppke et al., 2012), and SLC33A1 inhibition in zebrafish inhibited

axon outgrowth (Lin et al., 2008). The dominant HSP allele is predicted to disrupt the second

transmembrane domain, and potentially reverse the topology of all downstream domains (Lin

et al., 2008), and could therefore lead to either 50% loss-of-function, or to dominant gain-of-


function via a misfolded protein. SLC33A1 has a Drosophila homologue, CG9706. However,

no in-depth characterization of this gene has been published.

MICROTUBULE MOTOR PROTEINS

Two types of HSP are caused by mutations in different kinesin heavy chains: KIF5A

(SPG10), and KIF1A (SPG30). Kinesins are plus-end-directed MT motors that carry a variety

of cargoes such as synaptic vesicles and mitochondria in an anterograde direction in axons

(Hirokawa, 1998; Goldstein and Yang, 2000; Barkus et al., 2008). They are multi-subunit

complexes that include two identical heavy chains (kinesin heavy chains or KHCs) and two

identical light chains.

KIF5A (SPG10)

Mutations in KIF5A cause autosomal dominant complex HSP (Reid et al., 2002). This is

a component of kinesin-I. Mammals have three kinesin-I heavy chains, KIF5A, KIF5B and

KIF5C. KIF5A is expressed in all neurons and it is localized in the cytoplasm of cell body,

dendrites and axons (Kanai et al., 2000). Experiments with primary motor neuron cultures of

knockout mice suggest that KIF5A is required for neuronal survival and outgrowth, and

transport of mitochondria (Karle et al., 2012); other functions include anterograde

transportation of secretory vesicles to growth cones (Burgo et al., 2012).

KHCs contain three domains: a globular N-terminal motor domain, a -helical coiled-

coil stalk domain that mediates dimerization, and a C-terminal globular tail with roles in

light-chain and cargo binding (Wickstead and Gull, 2006; Lo Giudice et al., 2006). Mutations

in both the motor and stalk domains of KIF5A are found in HSP patients, and can cause

impaired axonal transport (Ebbing et al., 2008). Since the motor protein is made of two

chains, one non-functional chain can lead to non-functional kinesin.

Drosophila Khc (CG7765) is the single orthologue of human KIF5A, KIF5B and KIF5C.

Homozygous mutant larvae show impaired growth rate, and severe loss of muscle activity in

the posterior region, a striking similarity to human HSP. Adults also exhibit impaired sensory

and motor activity (Saxton et al., 1991) and defects in anterograde and retrograde axonal

transport (Hurd and Saxton, 1996). Drosophila has been useful in identifying additional

components of Khc-dependent axonal transport such as the mitochondrial adaptor Milton,

Unc-76 and Unc-51 kinase (Stowers et al., 2002; Gindhardt et al., 2003; Toda et al., 2008),

potential cargoes such as mitochondria and synaptic vesicles (Stowers et al., 2002; Hurd and

Saxton, 1996; Toda et al., 2008), and additional neuronal roles of Khc such as transport of

some cargoes to dendrites (Henthorn et al., 2011), that may also be relevant to the complex

disease phenotypes of SPG10.


KIF1A (SPG30)

Mutations in KIF1A, from the kinesin-3 subfamily, cause autosomal recessive complex

HSP (Erlich et al., 2011; Klebe et al., 2012). Its C-terminus has a lipid-binding Pleckstrin

homology (PH) domain, which is required for vesicle transport (Klopfenstein and Vale,

2004). KIF1A is the primary motor for fast axonal transport of neuropeptide-containing

dense-core vesicles, into axons and dendrites to pre- and postsynaptic sites (Scalettar, 2006;

Lo et al., 2011).

The Drosophila KIF1A orthologue is unc-104 (also known as imac). Like Khc it also has

a central role in axon transport, but appears to transport different cargoes. Whereas synaptic

vesicles accumulate in axons in khc mutants rather than the synapse (Hurd and Saxton, 1996;

Gindhardt et al., 2003), in unc-104 mutants, they fail to enter axons (Pack-Chung et al.,

2007), implying that these kinesins have complementary roles in synaptic vesicle transport. In

contrast to Khc, unc-104 has little role in axonal transport of mitochondria (Barkus et al.,

2008).

Therefore the KIF5/Khc and KIF1A/unc-104 kinesins transport different axonal cargoes

(impairment of which might lead to distal axon degeneration by different mechanisms), or

might play complementary roles in transport of the same cargoes that are essential to prevent

degeneration. Interestingly in Caenorhabditis elegans, unc-104 mutations redistribute the

smooth ER protein lunapark/Lnp-1 (Chen et al., 2012) from axons to cell bodies (Ghila et al.,

2008), hinting that SPG30 pathology might also involve axonal ER.

ENDOSOMAL SPG PROTEINS

Many SPG proteins are localized on components of the endosomal-lysosomal

endomembrane system. The significance of this for the disease is not clear, but may represent

roles for these proteins in intracellular signalling processes that are required for axonal

maintenance.

NIPA1 (SPG6)

Mutations in NIPA1 (non-imprinted in Prader-Willi/Angelman) cause autosomal

dominant HSP. It is a membrane protein with seven to nine transmembrane domains (Chai et

al., 2003; Lefevre, et al., 2004) and is predominantly localized in the early endosomal

pathway and peripheral surface (Goytain et al., 2007).

All known mutations are missense mutations at a limited number of amino acid residues

in diverse populations, which tentatively suggests a dominant gain-of-function mechanism.

These mutations could be affecting trafficking of NIPA1, and trapping of misfolded NIPA1 in

the ER could induce ER stress, giving the disease a gain-of-function feature (Goytain et al.,

2007; Beetz et al., 2008; Zhao et al., 2008). NIPA1 is also suggested to be a Mg2+

transporter

(Goytain et al., 2007). This role is potentially important since Mg2+

is the second most

abundant cation in the cell, but its relationship to HSP is unclear.


Spict (Spichthyin) is the Drosophila NIPA1 orthologue, and it is predominantly localized

on early endosomes. It is involved in the inhibition of BMP signalling, so it mediates the

growth of NMJ pre-synaptically. Spict can redistribute BMP receptors from the plasma

membrane to endosomes, and inhibit BMP signalling (Wang et al., 2007), properties shared

by NIPA1 in mammalian cells (Tsang et al., 2009). Loss of BMP signalling impairs axonal

transport (Aberle et al., 2002; Wang et al., 2007; Ellis et al., 2010); although the mechanisms

are not clear, this is a possible route by which SPG6 mutations could lead to spastic

paraplegia.

Three endosomal HSP proteins, spastin, spartin and NIPA1, and the ER protein atlastin-1,

are inhibitors of BMP signalling (Tsang et al., 2009; Fassier et al., 2010; Zhao and Hedera,

2012), although there is no indication of the mechanisms for spastin and spartin. However,

this shared involvement suggests abnormal BMP signalling as a possible mechanism for at

least some forms of HSP, a model that requires further investigation.

Strumpellin (SPG8)

Strumpellin mutations cause autosomal dominant pure HSP (Valdmanis et al., 2007).

Strumpellin is a subunit of the WASH complex, which regulates actin dynamics (Jia et al.,

2010). It interacts with the retromer complex, which traffics cargo from endosomes to Golgi,

and it promotes formation of endosomal tubules (Harbour et al., 2010). It is also localized to

ER (Clemen et al., 2010), and it is interesting to speculate whether it could promote extension

of ER tubules. Strumpellin has a Drosophila homologue (CG12272) but little is known about

its function.

Spartin (SPG20)

A frameshift mutation in spartin causes a syndromic autosomal recessive complex HSP

that is also known as Troyer Syndrome. Endogenous spartin is found in a cytoplasmic pool

that can be recruited to endosomes, lipid droplets, the cytokinesis midbody, and mitochondria

(Robay et al., 2006; Bakowska et al., 2007; Eastman et al., 2009; Edwards et al., 2009;

Renvoisé et al., 2010; Joshi and Bakowska, 2010), and it can regulate properties or trafficking

of some of these organelles.

Spartin has an N-terminal MIT domain that interacts with ESCRT-III (Ciccarelli et al.,

2003), and like spastin M87 this can explain its localization to endosomes and to the

midbody. At endosomes it functions in EGF receptor traffic and degradation (Bakowska et

al., 2007), and perhaps also in BMP receptor signalling (Tsang et al., 2009; Renvoisé et al.,

2012). It can also recruit ubiquitin ligases AIP4 and AIP5, and PKC- at lipid droplets

(Eastman et al., 2009; Edwards et al., 2009; Hooper et al., 2010; Urbanczyk and Enz, 2011),

and thus regulate properties including their subcellular distribution. Spartin is multiply

monoubiquitinated (Bakowska et al., 2007); although this appears to be constitutive, it could

allow spartin to act as an adaptor to recruit ubiquitin-binding proteins such as Eps15

(Bakowska et al., 2005). The multiple roles and localizations of spartin may contribute to the


syndromic phenotype of its loss, but their relationship to HSP is still unknown. Spartin has a

single Drosophila orthologue, CG12001, but little is known of its function.

Alsin (ALS2)

Mutations in alsin can cause complex infantile-onset ascending hereditary spastic

paraplegia (IAHSP), as well as early-onset forms of ALS or primary lateral sclerosis (PLS)

(Hadano et al., 2001; Yang et al., 2001; Eymard-Pierre et al., 2002; Panzeri et al., 2006).

Alsin has three predicted guanine exchange factor (GEF) domains: an N-terminal RCC

domain characteristic of Ran-GEFs, Dbl-homology and PH domains characteristic of Rho-

GEFs, and a C-terminal Vps9 domain characteristic of Rab5-GEFs. Rab5-GEF activity and

effects on endosome traffic have been demonstrated (e.g. Otomo et al., 2003; Topp et al.,

2004; Kunita et al., 2004; Devon et al., 2006; Deng et al., 2007; Kunita et al., 2007; Lai et al.,

2009), although a reported activity as a Rac1-GEF (Topp et al., 2004) might be accounted for

by being an effector of activated Rac1 (Kunita et al., 2007). The role of alsin in regulating

endosomal traffic is suggestive of a role in regulating one or more signalling pathways that

are relevant to axon degeneration (e.g. BMP?), but to date there is no information on this.

Drosophila has a single alsin orthologue, CG7158, but there is little information on its

function.

AUTOSOMAL RECESSIVE HSP WITH THIN CORPUS CALLOSUM, AND

THE AP5 ADAPTOR COMPLEX

Three autosomal recessive HSPs can be caused by mutations affecting proteins that are

members of the same multiprotein complex: SPG11 (spatacsin), SPG15 (spastizin) and

SPG48 (Stevanin et al., 2007; Hanein et al., 2008; Słabicki et al., 2010). At least SPG11 and

SPG15 have very similar complex phenotypes, HSP with thin corpus callosum (HSP-TCC;

the corpus callosum is the main tract linking the two brain hemispheres), and frequent

cognitive impairment. While this multiprotein complex was originally suggested to have

DNA repair function (Słabicki et al., 2010), it now appears to be a novel adaptor/coat protein

complex, AP-5 (Hirst et al., 2011).

Spatacsin (SPG11)

Spatacsin mutations are the most common cause of HSP-TCC (Stevanin et al., 2007), and

some spatacsin mutations are also causative for juvenile-onset ALS (Orlacchio et al., 2010)

or Parkinson’s Disease (Stevanin et al., 2008). Spatacsin is a large protein of some 2400

amino acid residues. Although it lacks obvious known domains, a sensitive homology search

suggests similarity to clathrin heavy chain (Hirst et al., 2011). It appears localized to vesicles,

ER, mitochondria and MTs (Murmu et al., 2011). It has a single Drosophila orthologue,

CG13531, about which little is known.


Spastizin (SPG15)

Mutations in spastizin cause a broadly similar HSP-TCC as spatacsin mutations.

Spastizin is also a large protein of some 2500 residues, and contains a C2H2 zinc finger, a

FYVE domain predicted to bind to endosomal membrane, and a possible leucine zipper

(Hanein et al., 2008). Spastizin partially co-localizes with spatacsin and both are found in the

same multi-protein complex (Hirst et al., 2011; Murmu et al., 2011). It has a single

Drosophila orthologue, CG5270, about which little is known.

SPG48

The multi-protein complex that contains spastizin and spatacsin was recently identified as

another adaptor complex, AP-5, for budding of a late endosomal compartment that is not yet

well defined (Hirst et al., 2011). Mutations in a large subunit of this adaptor, AP5-, are

found in a family with late-onset autosomal recessive SPG48 (Słabicki et al., 2010; Hirst et

al., 2011). Although this is a phylogenetically ancient gene, it appears to have been lost from

the protosomes, including Drosophila and most invertebrate phyla, together with other AP5

subunits (Hirst et al., 2011).

It is at first sight puzzling why AP5 should have been lost from the Drosophila lineage,

yet spatacsin and spastizin retained. Spatacsin and spastizin must therefore have roles in

addition to those in the AP-5 complex, that have been retained in Drosophila. If spatacsin is

indeed a distant homolog of clathrin, then it could function (aided somehow by spastizin) as a

coat protein for a number of adaptor complexes and not only AP-5, analogously to clathrin. If

HSP is caused by impaired AP-5 function, the roles of spatacsin and spastizin in Drosophila

may not be immediately relevant to HSP pathology, but may well be relevant to other disease

phenotypes of spatacsin and spastizin mutations, which appear to be more severe than those

of the single published AP5- mutation (Słabicki et al., 2010).

OTHER ENDOMEMBRANE SPG PROTEINS NOT FOUND

IN DROSOPHILA

In addition to SPG48, some other HSPs are caused by mutations affecting endomembrane

proteins that have no Drosophila orthologue.

Adaptor Complex AP-4 (SPG47)

Mutations affecting three components of the AP-4 adaptor complex give rise to a

complex condition that includes severe intellectual disability and spastic paraplegia (Abou

Jamra et al., 2010); one of these loci encoding the beta subunit, AP4B1, is designated SPG47.

AP-4 is a low-abundance adaptor associated with a subset of Golgi and other vesicles (Hirst

et al., 1999), which is also required for normal dendritic localization of AMPA receptor

complexes (Matsuda et al., 2008). AP-4 has a wide phylogenetic distribution that includes


vertebrates, sponges, and many plants, but appears to have been lost from protostomes

(including Drosophila).

Maspardin (SPG21)

SPG21 is an autosomal recessive complex HSP, caused by mutations in maspardin

(Simpson et al., 2003), a largely Golgi-associated protein that is a predicted hydrolase

(Zeitlmann et al., 2001). Maspardin is found in a wide range of animals including some

insects, but has been lost recently from the Drosophila lineage.

Erlin-2 (SPG18)

SPG18 is an autosomal recessive complex HSP, caused by mutations in erlin-2 (Alazami

et al., 2011), a putative hairpin-loop protein found in detergent-insoluble domains of the ER

(Browman et al., 2006). It has orthologues in a wide range of animals including some insects,

but has been lost from the Drosophila lineage.

MITOCHONDRIAL PROTEINS

Mitochondria are transported to distal axons via fast anterograde transport using kinesin

motors, principally the Khc1/KIF5 family (Hollenback, 1996; Tanaka et al., 1998; Kanai et

al., 2000). HSP could conceivably result from a limited energy supply in distal axons, and

indeed at least two HSPs are caused by mutations in mitochondrial proteins.

Paraplegin (SPG7)

Mutations in paraplegin cause an autosomal recessive complex HSP. Paraplegin is an

ATP-dependent m-AAA protease in the inner membrane of mitochondria (Hanson and

Whiteheart, 2005; Rugarli and Langer, 2006), involved in degradation of misfolded proteins,

cleavage of mitochondrial target sequences (Nolden et al., 2005), and maturation of

mitochondrial enzymes (Koppen et al., 2009). Drosophila CG2658 is the orthologue of

human paraplegin, but has not yet been studied in depth.

Heat Shock Protein 60 (SPG13)

Mutations in heat shock protein 60 (HSP60) cause autosomal dominant pure HSP

(Hansen et al., 2002) and autosomal recessive hypomyelinating leukodystrophy (Magen et al.,

2008). Hsp60 forms a large multisubunit complex with its co-chaperonin Hsp10 in the

mitochondrial matrix (Hansen et al., 2002), where it mediates protein folding, prevents

misfolding, and helps to clear misfolded and damaged non-functional proteins (Hansen et al.,


2008). Hsp60 has one Drosophila orthologue; although it is well studied (www.flybase.org),

little is known of its functions in motor neuron degeneration.

INTERACTIONS OF NEURONS WITH SUBSTRATES OR

NEIGHBORING CELLS

L1CAM (SPG1)

Loss-of-function mutations in L1CAM lead to X-linked hydrocephalus, MASA (Mental

retardation, Aphasia, Shuffling gait and Adducted thumbs) syndrome or complex recessive

HSP (Jouet et al., 1994). L1CAM is a single pass transmembrane protein with six Ig-like

domains and five fibronectin repeats extracellularly, and a short cytoplasmic tail (De Angelis

et al., 2002). Drosophila neuroglian is an orthologue of four human paralogues: neural cell

adhesion molecule, neurofascin, L1CAM, and neural cell adhesion molecule L1-like protein.

The structure of neuroglian is similar to L1CAM, but it also contains ankyrin and ezrin-

radixin-moesin (ERM) binding motifs in the cytoplasmic region (Davis and Bennnett, 1994;

Dickson et al., 2002; Cheng et al., 2005).

Drosophila neuroglian is involved in neurite growth, axon guidance, and sensory-neuron

migration (Bieber et al., 1989; Davis et al., 1994; Hall and Bieber, 1997; Dickson et al., 2002;

Islam et al., 2004; Cheng et al., 2005; Kristiansen et al., 2005; Godenschwege et al., 2006;

Chen and Hing, 2008). Its roles include axon fasciculation (Goossens et al., 2011), and

anchoring MTs in synaptic terminals during giant-synapse formation (Godenschwege et al.,

2006). However, given the complexity of these roles, and its orthology to four human genes,

it is not easy to see which neuroglian functions are most relevant for HSP.

PLP1 (SPG2)

Mutations in proteolipid protein 1 (PLP) cause Pelizaeus-Merzbacher disease (PMD) and

X-linked complicated spastic paraplegia (SPG2). SPG2 has milder symptoms than PMD,

involving demyelination rather than failure to form a myelin sheath (Gencic et al., 1989;

Woodward, 2008). PLP1 is an integral membrane protein and a major component of myelin.

M6 (CG7540) is the single Drosophila orthologue of PLP1 and of two other human

glycoproteins M6a and M6b. Depletion of M6 can lead to mild eye roughness and lowered

phototaxis (Zappia et al., 2012). However, Drosophila M6 may be of limited use to

understand the role of PLP1 in HSP, since Drosophila axons lack a myelin sheath.

CONCLUSION

Studying the function and mutant phenotypes of SPG products is a route to better

understanding both of HSP, as well as the mechanisms that maintain axonal function at

enormous distances from the cell body. Work in Drosophila has made major contributions to

understanding the roles of SPG proteins in processes including ER organization, axon


transport, and BMP signalling, and thus to the model that the wide spectrum of HSP genes

and phenotypes is due to a more limited number of common disease mechanisms.

Flies now offer great potential for the future. First, new SPGs continue to emerge, and

flies will continue to be an excellent model for understanding the basic function of these

genes. Furthermore, there is now a need to meet the next challenge, of understanding disease

mechanisms. Drosophila, as a simple genetic model system with well-developed tools to

study axonal transport, function and integrity, will increasingly provide insights into

pathomechanisms as well as into basic biological function of SPG proteins.

ACKNOWLEDGMENTS

We thank Niamh O'Sullivan for helpful discussions. B.Y. is supported by a Yousef

Jameel Scholarship.

ABOUT THE AUTHORS

Belgin Yalçın is a PhD student in the Department of Genetics, University of Cambridge.

Her first degree is from Istanbul Technical University, and her Ph.D. project aims to

understand the roles of Drosophila SPG proteins in organization of axonal ER.

Cahir J O'Kane is a Faculty member in the Department of Genetics, University of

Cambridge. He has helped to develop some of the tools used most widely in Drosophila

neurobiology, including enhancer trapping and targeted silencing of neurons by tetanus toxin.

He has a long-standing interest in neuronal membrane traffic, and his work on this aspect of

SPG function includes defining the role of the Drosophila NIPA1/SPG6 homolog in BMP

receptor traffic and signalling, and the role of Drosophila reticulon in organization of axonal

ER.

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Chapter 5

DROSOPHILA AS A MODEL FOR CMT PERIPHERAL

NEUROPATHY: MUTATIONS IN TRNA SYNTHETASES

AS AN EXAMPLE

Georg Steffes1 and Erik Storkebaum

1,*

1Molecular Neurogenetics Laboratory, Max Planck Institute

for Molecular Biomedicine, Muenster, Germany

ABSTRACT

Charcot-Marie-Tooth (CMT) disease is characterized by the degeneration of

peripheral motor and sensory neurons, leading to progressive muscle weakness and

wasting, and sensory loss. Electrophysiological and pathological criteria allow the

distinction between demyelinating, axonal and intermediate forms of CMT. The disease

is genetically heterogeneous, with currently more than 30 genes causally linked to CMT.

The molecular underpinnings of the peripheral motor and sensory neuropathy are poorly

understood, and there is no effective drug treatment available. In this chapter, we discuss

the use of Drosophila melanogaster as a genetic model organism for CMT. Major

advantages include the possibility to study the effect of CMT-associated mutant proteins

on motor and sensory neurons in their physiological context, and its suitability to perform

genetic screens. To illustrate the usefulness of Drosophila as a model for CMT, we

highlight forms of CMT that are associated with mutations in tRNA synthetases. These

enzymes ligate amino acids to their cognate tRNA, and therefore catalyze an essential

step in protein synthesis. Mutations in the genes encoding tyrosyl-tRNA synthetase

(YARS), glycyl-tRNA synthetase (GARS), alanyl-tRNA synthetase (AARS), and possibly

lysyl-tRNA synthetase (KARS) and histidyl-tRNA synthetase (HARS) give rise to axonal

and intermediate forms of CMT. Loss of aminoacylation activity per se is not the cause

of the disease, although the possibility that altered subcellular localization of

aminoacylation-active mutants could lead to defects in local protein synthesis cannot be

excluded at the present moment. Current evidence suggests that the disease may be

caused by a gain-of-toxic function mechanism, the molecular nature of which remains

elusive. The future use of Drosophila CMT models in genetic screens for disease-

modifying genes may be of great value to unravel the molecular mechanisms of disease,

and to identify possible therapeutic targets.

Georg Steffes and Erik Storkebaum 122

Keywords: Charcot-Marie-Tooth disease, peripheral motor and sensory neuropathy, axonal

degeneration, tRNA synthetase, aminoacylation activity, genetic screen

INTRODUCTION

Charcot Marie Tooth disease (CMT) is the most common inherited neuromuscular

disorder and has an estimated prevalence of 1 in 2500 individuals (Martyn and Hughes,

1997). CMT is characterized by distal muscle weakness and atrophy, sensory loss, decreased

reflexes, and foot deformities. These classical symptoms are due to degeneration of peripheral

motor and sensory nerves, whereby the longest nerves are preferentially affected. Hence,

CMT is also referred to as hereditary motor and sensory neuropathy. The disease usually

occurs in the first two decades of life and is subsequently slowly progressive over decades

(Dyck, 1993).

CMT is both clinically and genetically heterogeneous. Traditionally, two main forms are

distinguished, namely demyelinating and axonal forms of CMT. Clinically, the distinction is

based on electrophysiological criteria: nerve conduction velocities (NCVs) lower than 38m/s

are classified as demyelinating, whereas NCVs higher than 38m/s are considered axonal

forms (Reilly et al., 2011). Demyelinating forms of CMT are pathologically characterized by

segmental demyelination and remyelination with so called onion bulb formations - concentric

arrangements of supernumerary Schwann cells around an incompletely remyelinated axon.

Demyelinating forms are classified as CMT1 if the inheritance pattern is autosomal dominant,

and CMT4 in case of autosomal recessive inheritance (Patzko and Shy, 2011). Many CMT1-

and CMT4-associated genes are expressed in myelinating Schwann cells but not in neurons.

However, the primary demyelination in these forms of CMT ultimately leads to (secondary)

axonal degeneration, and the classical CMT symptoms can be mainly attributed to this axonal

degeneration, rather than the demyelination itself (Pareyson and Marchesi, 2009). Whereas

demyelinating CMT accounts for the majority of CMT cases, axonal forms (CMT2) give rise

to about 20% of CMT cases (Ajroud-Driss et al., 2011). Electrophysiologically, axonal CMT

is characterized by normal or mildly slowed NCVs, but with reduced compound action

potential amplitudes. Pathologically, evidence of chronic axonal degeneration and

regeneration is found. The majority of CMT2 is autosomal dominant, but autosomal recessive

forms have been described (Pareyson and Marchesi, 2009). More recently, it has become

evident that a clear distinction between demyelinating and axonal forms of CMT is not

always possible, and intermediate forms between CMT1 and CMT2 are recognized. These

forms of intermediate CMT are characterized by intermediate NCVs (25-45 m/s) and

pathological features of both demyelination and axonal degeneration (Nicholson and Myers,

2006). Apart from autosomal dominant and autosomal recessive forms of intermediate CMT,

X-linked CMT often shows intermediate NCVs and pathological evidence of axonal loss and

some demyelination, with few onion bulbs (Kleopa and Scherer, 2006).

Further subdivision of CMT types is based on causative genes or assigned loci. To date,

more than 30 genes have been associated with CMT (Table 1). These genes encode proteins

with often very different molecular functions suggesting that derailment of multiple

Drosophila as a Model for CMT Peripheral Neuropathy 123

molecular pathways can give rise to peripheral motor and sensory neuropathy. Molecular and

cellular pathways that may be involved in CMT molecular pathogenesis include myelination

and myelin maintenance, axonal transport, mitochondrial dynamics, endosomal trafficking,

axon-Schwann cell interaction, transcriptional regulation and protein chaperone activity

(Ajroud-Driss et al., 2011; Patzko and Shy, 2011). To increase complexity even further,

CMT-associated proteins have very different expression patterns and subcellular localizations

(Table 1). Indeed, some CMT gene products are selectively expressed in Schwann cells or

neurons, whereas others are ubiquitously expressed. This raises the question why mutations in

such ubiquitously expressed genes give rise to the specific peripheral neuropathy phenotype.

Moreover, CMT proteins can be localized to the cytoplasm, the nucleus, mitochondria, ER,

endosomes, the plasma membrane, myelin or neuronal cytoskeleton (Pareyson and Marchesi,

2009).

Thus, the exact molecular mechanisms by which CMT-associated genes lead to

peripheral neuropathy are still enigmatic, and there is no effective drug treatment available for

CMT. Clearly, there is an urgent need for better insights into the molecular pathogenesis of

CMT, to identify therapeutic targets, and to test potential therapeutic agents. Animal models

for CMT, including Drosophila models, can be instrumental to achieve these goals.

CELLULAR AND ANIMAL MODELS FOR CMT

Studies on CMT patients and patient-derived samples allow genetic analysis to identify

causative genes, analysis of genotype/phenotype correlation, clinical trials to test the

efficiency of candidate (drug) treatments and neuropathological analysis of post-mortem

tissue samples. However, despite the relevance of directly conducting studies on CMT

patients, such studies have several limitations. These include (i) the fact that invasive studies

are not possible, so that e.g. neuropathology in early disease stages cannot be assessed, (ii) the

often very limited number of patients with certain genetic mutations, and (iii) the fact that

clinical trials are expensive and need to be based on good preclinical evidence in cellular or

animal models. For these reasons, cellular and animal models are of key importance to study

the molecular pathogenesis of CMT.

Cellular Models for CMT

Advantages of cellular models for CMT are the relative ease of studying cellular

processes (e.g. axonal transport), and their suitability for genetic (e.g. using small interfering

RNAs) or pharmacological manipulation and screening. An obvious drawback is that the

physiological, in vivo context is missing when culturing cells in a dish. The use of co-

cultures, e.g. of neurons and Schwann cells, addresses, but does not solve this problem.

Cellular CMT models can be divided into patient-derived and non-human, typically mouse

(model) derived cells.


The use of patient-derived cells is obviously very relevant, because one works in the

disease genetic background. Traditionally, blood sample-derived lymphocyte cultures or

fibroblasts obtained from skin biopsy are used. Limitations are that these cells can only be

used if the CMT-associated gene is expressed in lymphocytes or fibroblasts and obviously

these cells are not the affected cell types in CMT. It recently became possible to overcome

these limitations by the use of induced pluripotent stem cells (iPSCs). These cells can be

derived from fibroblasts, allow unlimited proliferation, and can be differentiated into all cell

types of the body, including motor and sensory neurons and Schwann cells (Dolmetsch and

Geschwind, 2011; Marchetto et al., 2011). Although this technology holds great promise for

studying molecular mechanisms of disease and identifying possible therapeutic compounds,

the procedure to generate iPSCs and subsequent differentiation is elaborate, time-consuming,

and technically challenging. Furthermore, at the present moment it is not yet possible to

obtain "pure" cultures of the CMT-relevant cell types, and the obtained cells often correspond

to "embryonic" stages, which is a disadvantage when studying adolescent or adult-onset

diseases (Dolmetsch and Geschwind, 2011; Marchetto et al., 2011). In contrast to patient-

derived cells, derivation of cells from animal models allows the generation of primary

neuronal or Schwann cell cultures (or co-cultures), as well as the derivation of these cell types

from embryonic stem cells (ESCs) or iPS cells. CMT rodent models are typical sources for

these cells.

Animal Models for CMT

CMT animal models have the major advantage that the disease-relevant cell types can be

studied in their physiological context, and that the effect of disease-associated mutations on

animal behaviour and physiology can be evaluated. However, the price to pay for this are the

anatomical and genetic differences between animal models and humans, which become more

prominent with increasing evolutionary distance. Indeed, when constructing an animal model

for CMT, one hopes not only to recapitulate the hallmark disease phenotypes, but also the

cellular and molecular mechanisms that lead to disease in patients. If this is not the case,

studying the animal model in question will never allow to decipher the molecular

pathogenesis underlying the human disease. Therefore, we think it is important that the

animal model has an orthologue of the studied CMT-associated gene, in order to maximize

the chances that the molecular pathways that lead to CMT in humans are conserved in the

animal model. Obviously, even if this is the case, one should keep in mind that the CMT-

associated gene may have acquired additional functions during evolution. If alteration of this

additional function would be the molecular cause of the disease, the animal model will not be

suitable for cracking the "CMT code".

The most popular animal models for neurodegenerative diseases are - with increasing

evolutionary distance - rodent models (mouse and rat), zebrafish, Drosophila melanogaster

and C. elegans. When modelling CMT in such animal models, the genetic approach will be

determined by the mode of inheritance of the CMT form studied.

Table 1. Different forms of CMT and associated genes

Type Subtype OMIM Gene Inheritance Location Molecular function Atypical clinical phenotypes

CMT1

CMT1A 118220 PMP22 autosomal dominant compact myelin myelination, cell growth, differentiation

CMT1B 118200 MPZ autosomal dominant compact myelin cell adhesion

CMT1C 601098 LITAF/SIMPLE autosomal dominant Schwann cells transcription factor

CMT1D 607678 EGR2 autosomal dominant Schwann cells transcription factor

CMT1E 118300 PMP22 autosomal dominant compact myelin myelination, cell growth, differentiation hearing loss

CMT1F 607734 NEFL autosomal dominant neuronal cytoskeleton regulation of axonal diameter, axonal

transport

CMT2

CMT2A1 118210 KIF1B autosomal dominant ubiquitous axonal transport

CMT2A2 609260 MFN2 autosomal

dominant/recessive

mitochondrial membrane

and ER

fusion of mitochondria; mitochondria-ER

interactions

CMT2B 600882 RAB7 autosomal dominant late endosomes regulates vesicular transport

CMT2B1 605588 LMNA autosomal recessive nuclear lamina nuclear stability, chromatin structure and

gene expression

CMT2B2 605589 MED25 autosomal recessive nucleoplasm transcriptional regulation

CMT2C 606071 TRPV4 autosomal dominant cell membrane ion channel, mediates calcium influx diaphragmatic and vocal cord paresis

CMT2D 601472 GARS autosomal dominant cytoplasm, mitochondrial

matrix

protein translation

CMT2E 607684 NEFL autosomal dominant neuronal cytoskeleton regulation of axonal diameter, axonal

transport

CMT2F 606595 HSPB1 autosomal dominant cytoplasm stress resistance, actin and intermediate

filament organization, chaperone activity,

anti-apoptotic activity, proteasome activation

CMT2G 608591 unknown autosomal dominant

CMT2H 607731 GDAP1 autosomal recessive outer mitochondrial

membrane

regulation of mitochondrial dynamics pyramidal features

CMT2J 607736 MPZ autosomal dominant compact myelin cell adhesion hearing loss and pupillary

abnormalities

CMT2K 607831 GDAP1 autosomal dominant/

recessive

outer mitochondrial

membrane

regulation of mitochondrial dynamics

CMT2L 608673 HSPB8 autosomal dominant cytoplasm chaperone activity



CMT2M 606482 DNM2 autosomal dominant cytoplasm endocytosis

CMT2N 613287 AARS autosomal dominant cytoplasm protein translation

CMT2O 614228 DYNC1H1 autosomal dominant cytoplasm axonal transport

CMT2P 614436 LRSAM1 autosomal

dominant/recessive

cytoplasm E3 ubiquitin-protein ligase

CMT3 CMT3 145900 MPZ, EGR2,

PMP22,PRX

autosomal

dominant/recessive

Schwann cells multiple Dejerine-Sottas neuropathy

CMT4

CMT4A 214400 GDAP1 autosomal recessive outer mitochondrial

membrane


CMT4B1 601382 MTMR2 autosomal recessive cytoplasm phosphatase activity, dephosphorylates

PI3P and PI3,5P2

CMT4B2 604563 SBF2/MTMR13 autosomal recessive cytoplasm pseudophosphatase; dimerizes with

MTMR2, thereby increasing its

enzymatic activity

early-onset glaucoma can occur

CMT4C 601596 SH3TC2 autosomal recessive Schwann cell plasma

membrane and perinuclear

endocytic recycling

compartment

endocytic recycling pathway

CMT4D 601455 NDRG1 autosomal recessive cytoplasm growth arrest and cell differentiation hearing loss

CMT4E 605253 EGR2 autosomal recessive Schwann cells transcription factor congenital hypomyelinating neuropathy

CMT4F 145900 PRX autosomal recessive Schwann cells myelin maintenance

CMT4G 605285 unknown autosomal recessive

CMT4H 609311 FGD4 autosomal recessive cytoplasm guanine nucleotide exchange factor for

the Rho GTPase CDC42; F-actin

binding and crosslinking activity

CMT4J 611228 FIG4 autosomal recessive endosome membrane PI3,5P2 phosphatase

CMT5 CMT5 600361 multiple autosomal dominant pyramidal features

CMT6 CMT6 601152 MFN2 autosomal dominant mitochondrial membrane and

ER

fusion of mitochondria; mitochondria-

ER interactions

optic atrophy

DI-CMT DI-CMTA 606483 unknown autosomal dominant

DI-CMTB 606482 DNM2 autosomal dominant cytoplasm endocytosis



DI-CMTC 608323 YARS autosomal dominant cytoplasm protein translation

DI-CMTD 607791 MPZ autosomal dominant compact myelin cell adhesion

DI-CMTE 614455 INF2 autosomal dominant cytoplasm actin dynamics focal segmental glomerulonephritis

RI-CMT

RI-CMTA 608340 GDAP1 autosomal recessive outer mitochondrial

membrane


RI-CMTB 613641 KARS autosomal recessive cytoplasm, mitochondria protein translation developmental delay, self-abusive

behaviour, dysmorphic features and

vestibular Schwannoma

CMTX

CMTX1 302800 GJB1 (Cx32) X-linked dominant Schwann cells and

oligodendrocytes

gap junction protein

CMTX2 302801 unknown X-linked recessive

CMTX3 302802 unknown X-linked recessive

CMTX4 310490 unknown X-linked recessive deafness and mental retardation

CMTX5 311070 PRPS1 X-linked recessive purine and pyrimidine biosynthesis optic atrophy, deafness, polyneuropathy

CMT types and subtypes, inheritance pattern, associated genes with their subcellular localization pattern and molecular function, and atypical clinical

phenotypes are listed.


In case of a recessive inheritance pattern, the disease is most likely caused by partial or

full loss of gene function. Therefore, inducing loss-of-function mutations in the orthologous

gene may provide an animal model for CMT in these cases. In case of dominant inheritance,

overexpression of the mutant human gene, or of the orthologous gene that carries the

corresponding disease-causing mutation, may result in a suitable CMT animal model. For

both modes of inheritance, "knock-in" models will best recapitulate the genetic situation in

human patients. However, one caveat is that knock-in models carry the inherent risk that the

introduced (often subtle) mutations may not result in CMT-associated phenotypes during the

relatively short life span of the animal model.

All CMT-associated genes known to date have orthologues in mice, and mouse models

are now available for 17 genetic forms of CMT (Table 2). These mouse models often

recapitulate several disease phenotypes, and are invaluable to study cellular and molecular

mechanisms of disease and to evaluate potential pharmacological treatments (Fledrich et al.,

2012). However, because of the high cage costs, space requirements and life cycle duration,

genome-wide unbiased genetic screens to identify disease-modifying genes seem practically

impossible. Given the fact that the molecular pathogenesis of CMT is poorly understood, and

that the disease may be caused by toxic gain-of-function mechanisms, in particular for

dominantly inherited forms of CMT, such genetic screens may be necessary to elucidate the

molecular mechanisms of disease. Indeed, if CMT-associated mutations cause the protein to

interfere with molecular pathways that are distinct from the normal functions of the protein

(e.g. when the mutant protein acquires novel protein-protein interactions), it is very unlikely

that hypothesis-driven research will be able to uncover the disease-causing molecular

mechanisms. Therefore, a key advantage of small non-vertebrate model organisms such as

Drosophila melanogaster and C. elegans is that they allow conducting genetic screens for

disease-modifying genes. Identification of these genes may not only provide insights into the

molecular pathogenesis of CMT, but may also identify putative therapeutic targets.

DROSOPHILA AS A MODEL FOR CMT

Apart from its suitability for genetic screens, experimental advantages of working with

Drosophila are its short life cycle (10 days at 25°C from fertilized egg to reproducing adult),

the ease and cheapness of its culture, the large number of offspring, the ease of genetic

manipulation and the multitude of genetic tools available (Venken and Bellen, 2005).

Furthermore, the organisational principles of the nervous system are remarkably conserved

between flies and vertebrates (including humans).

The Drosophila central nervous system (CNS) consists of brain and ventral nerve cord

(VNC), the latter being the homologous structure to the spinal cord in mammals (Figure 1).

Lower motor neurons in Drosophila have their cell bodies in the VNC, and project axons to

peripheral muscles, where they form neuromuscular junctions (NMJs). Sensory neurons have

their cell bodies in the periphery, and project into the central nervous system. Basic

neurophysiological principles (e.g. conduction of action potentials, transmission of signals by

release of neurotransmitters packaged in synaptic vesicles, the synaptic vesicle cycle, etc.) are

conserved.


Table 2. Mouse models for CMT

Human Gene

Symbol Subtype Human Gene

Mouse

Gene

Mouse model

for CMT

available

"Kind" of model

PMP22

CMT1A,

CMT1E,

CMT3

peripheral myelin protein 22 Pmp22 YES

PMP22 transgenic

mice, spontaneous

Pmp22 point

mutations

MPZ

CMT1B,

CMT2J,

CMT3, DI-

CMTD

myelin protein zero Mpz YES Null and knock-in

LITAF

(SIMPLE) CMT1C

lipopolysaccharide-induced

tumour necrosis factor Litaf NO

Litaf null mice do

not display

peripheral

neuropathy

EGR2

CMT1D,

CMT3,

CMT4E

early growth response 2 Egr2 YES

knock-out and

conditional

knock-out

NEFL CMT1F,

CMT2E

neurofilament, light

polypeptide Nefl YES

NEFL(P22S)

transgenic mice

KIF1B CMT2A1 kinesin family member 1B Kif1b YES

Kif1b

heterozygous

mice

MFN2 CMT2A2,

CMT6 mitofusin 2 Mfn2 YES

Mfn2 T105M

transgenic mice

RAB7 CMT2B RAB7, member RAS

oncogene family Rab7 NO

LMNA CMT2B1 lamin A/C Lmna YES Null

MED25 CMT2B2 mediator complex subunit 25 Med25 NO

TRPV4 CMT2C

transient receptor potential

cation channel, subfamily V,

member 4

Trpv4 NO

GARS CMT2D glycyl-tRNA synthetase Gars YES

ENU-induced

Gars point

mutations

HSPB1 CMT2F heat shock 27kDa protein 1 Hspb1 YES mutant HSPB1

transgenic mice

GDAP1

CMT2H,

CMT2K,

CMT4A,

RI-CMTA

ganglioside-induced

differentiation-associated-

protein 1

Gdap1 NO

HSPB8 CMT2L heat shock 22kDa protein 8 Hspb8 NO

DNM2 CMT2M,

DI-CMTB dynamin 2 Dnm2 NO

knock-in model

for centronuclear

myopathy

available, but no

peripheral

neuropathy model



Human Gene

Symbol Subtype Human Gene

Mouse

Gene

Mouse

model for

CMT

available

"Kind" of

model

AARS CMT2N alanyl-tRNA synthetase Aars NO

Sticky mouse

displays

cerebellar ataxia

but no

peripheral

neuropathy

DYNC1H1 CMT2O dynein cytoplasmic 1 heavy

chain 1 Dync1h1 YES

missense

mutations in

Loa and Cra1

mice, 9

nucleotide

deletion in Swl

mice

LRSAM1 CMT2P leucine rich repeat and sterile

alpha motif containing 1 Lrsam1 NO

PRX CMT3,

CMT4F periaxin Prx YES Null

MTMR2 CMT4B1 myotubularin related protein 2 Mtmr2 YES Null and E276X

knock-in

SBF2

(MTMR13) CMT4B2 SET binding factor 2 Sbf2 YES Null

SH3TC2 CMT4C SH3 domain and

tetratricopeptide repeats 2 Sh3tc2 YES Null

NDRG1 CMT4D N-myc downstream regulated

gene 1 Ndrg1 YES Null

FGD4 CMT4H FYVE, RhoGEF and PH

domain containing 4 Fgd4 NO

FIG4 CMT4J FIG4 homologue, SAC1 lipid

phosphatase domain containing Fig4 YES

'pale tremor'

mice contain a

homozygous

transposon

insertion in

intron 18 of the

Fig4 gene

YARS DI-CMTC tyrosyl-tRNA synthetase Yars NO

INF2 DI-CMTE inverted formin, FH2 and WH2

domain containing Inf2 NO

KARS RI-CMTB lysyl-tRNA synthetase Kars NO

GJB1 CMTX 1 gap junction protein, beta 1,

connexin32 Gjb1 YES

Null and Cx32

R142W

transgenic mice

PRPS1 CMTX5 phosphoribosyl pyrophosphate

synthetase 1 Prps1 NO

Mouse homologs of CMT-associated genes and available mouse models for the different CMT

subtypes are listed.

Because of its relevance for modelling CMT, the development and anatomy of the

Drosophila neuromuscular system is described in more detail in the next paragraphs.


Figure 1. A, B, The neuromuscular system in Drosophila larvae. (A) Confocal image of an abdominal

segment of the larval body wall showing the innervation pattern of the muscle fibers by motor neurons.

Motor neurons are labelled in magenta and muscle fibers are labelled in green. (B) Schematic diagram

of the innervation pattern of the intersegmental nerve (ISN) and the segmental nerve (SN) pathways;

the transverse nerve (TN) is not displayed. C, D, The adult neuromuscular system. (C) Schematic

representation of the adult nervous system with the brain (B), ventral nerve cord (VNC) and peripheral

nerves. (D) Schematic representation of the adult muscle system. In the head, the rostral retractor is

labelled in orange and the pharyngeal dilators are labelled in purple. In the thorax, the dorsal

longitudinal muscles (DLMs) are labelled in blue, the dorsoventral muscles are labelled in yellow, and

the tergotrochanteral muscle (TTM) is labelled in green. E, Schematic drawing of a cross-section

through a peripheral nerve (Rodrigues et al., 2011).


The Drosophila Neuromuscular System

During Drosophila development, two distinct phases of neurogenesis occur: the first

during embryonic development, and the second during larval life and early metamorphosis

(Lin and Lee, 2012). Embryonic neurogenesis begins at stage 9 of 17 embryonic stages, with

the delamination of neuronal stem cells, called neuroblasts, from the ectodermal germ layer.

Neuroblasts subsequently produce a series of ganglion mother cells. At stage 13, the progeny

of ganglion mother cells begins to differentiate into neurons and glial cells (Lin and Lee,

2012). The Drosophila embryo can be subdivided into 3 thoracic (T1-T3) and 8 abdominal

(A1-A8) segments. Each abdominal hemisegment from A1-A7 contains 30 muscle fibers,

which can be subdivided into ventral and lateral musculature (Fernandes and Keshishian,

1999). During embryonic development, the axons of 36 motor neurons per hemineuromer

project through 3 principal nerves into the muscle field: two main nerves, the intersegmental

nerve (ISN) and segmental nerve (SN), and a minor one, the transverse nerve (TN), which

contains two motor axons and projects along the segment border (Landgraf and Thor, 2006).

Embryonic motor neurons mediate embryonic contractions and larval hatching.

In the larval stage, neuromuscular junctions progressively enlarge as the animal grows, so

that by the end of the third instar the muscles are 10 times their embryonic lengths, and motor

neurons show a significant expansion in synaptic branching and bouton number. The larval

neuromuscular system consists of a segmentally repeated array of dorsal, ventral and lateral

muscle fibers, with variations in specific thoracic and abdominal segments (Figure 1A and B).

The motor neurons are segmentally repeated, and mediate larval behaviours such as crawling,

feeding and moulting. Drosophila motor neurons are glutamatergic, whereas the presynaptic

interneurons are predominantly cholinergic (Fernandes and Keshishian, 1999).

During metamorphosis, almost all of the larval musculature is histolyzed and replaced by

proliferating muscle progenitor cells that were set aside by the end of embryogenesis. In

contrast, the vast majority of adult motor neurons derive from functional larval motor

neurons, which are structurally re-specified to innervate adult muscle targets (Fernandes and

Keshishian, 1999). In contrast to motor neurons, almost all larval sensory neurons degenerate

during metamorphosis and are replaced by adult neurons, which develop from imaginal discs.

Likewise, most adult interneurons are formed during metamorphosis, although other

interneurons are remodelled larval interneurons, which may carry out new tasks during

adulthood (Tissot and Stocker, 2000).

In the adult, there are distinct sets of muscles in the head, thorax, and abdomen that

mediate adult-specific motor functions such as flight, walking, feeding and copulation (Figure

1). The largest muscles are located in the thorax, and are involved in walking and flight. Each

hemithorax has about 80 muscle fibers that are divided in dorsal and ventral sets. Much of the

dorsal thorax is occupied by the indirect flight muscles (IFMs), consisting of 13 muscle fibers

per hemithorax. Six of these comprise the dorsal longitudinal muscles (DLMs), which are

innervated by five identified mesothoracic motor neurons. The remaining seven fibers make

up the dorsoventral muscles (DVMs) (Figure 1), which are mononeuronally innervated by

seven motor neurons. DLMs are the wing depressors, whereas DVMs are the wing elevators.

The alternative contraction and relaxation of these muscles generates the wing beat during

flight. The other group of thoracic muscles are the direct flight muscles (DFMs), 17 in

number. DFMs are smaller than IFMs, and are located at the base of the wing. They are

responsible for steering functions of the wing during flight. The largest tubular muscle in the


thorax is the jump muscle, also known as tergotrochanteral muscle (TTM). The TTM is a

ventral mesothoracic muscle, which executes the jump motion and is innervated by the

tergotrochanteral motor neuron (TTMn) (Figure 1). Among the prominent muscles in the

head are the pharyngeal dilators involved in feeding, the rostral retractors, which control

movements of the proboscis, and the ptilinum retractors, which are used during adult

emergence (Figure 1). In contrast to thoracic and head musculature, adult abdominal muscles

have a simple organization and bear a closer similarity to larval musculature (Figure 1). There

are sets of dorsal, ventral, and lateral muscle fibers in segments A1-A6. A few segment-

specific specializations of muscle fibers are also evident in the adult abdomen, including the

male-specific muscle (MSM) in the fifth abdominal segment. The terminal abdominal

segments also show sex-specific muscle fiber patterning variations, and there are specific

muscles associated with the ovary and the testes.

Which Types of CMT Can Be Modeled in Drosophila?

Particularly relevant for CMT is the anatomy of Drosophila peripheral nerves. As can be

seen in Figure 1E, axons are ensheathed by wrapping glia, considered to be analogous to

vertebrate Schwann cells. Peripheral nerve bundles are surrounded by subperineurial and

perineurial glia, which form the blood-nerve barrier (Stork et al., 2008). However, despite the

similarities between Drosophila and vertebrate peripheral nerves, Drosophila peripheral

nerves lack myelin, and do not possess saltatory nerve conduction. In accordance with that,

Drosophila lacks close homologs of all CMT1-associated genes (PMP22, MPZ, LITAF,

ERG2 and NEFL). For these reasons, we think that Drosophila is not a suitable model to

study the demyelinating forms of CMT.

However, we believe that Drosophila is well suited to model axonal and intermediate

forms of CMT, as axonal morphology and function is conserved, and the vast majority of

axonal and intermediate CMT-associated genes have close homologs in Drosophila (Table 3).

Mutants of the Drosophila homologs of CMT-associated genes can provide insight into the

endogenous function of these genes, and - in case of (occasional) recessive inheritance - may

provide a Drosophila CMT model. Remarkably, most of the Drosophila homologs of CMT-

associated genes have not or only to a very limited extent been studied. Only for Drosophila

homologs of LMNA, DNM2 and DYNC1H1 more than 10 original research papers are

available (Table 3).

In case of a dominant inheritance pattern, the disease may be caused by haplo-

insufficiency, a dominant negative mechanism, gain-of-wild-type-function or gain-of-toxic-

function. Expression of a human CMT-mutant protein in Drosophila, or overexpression of the

homologous Drosophila protein with the CMT-associated mutations introduced in the

homologous amino acid residues, may provide a Drosophila CMT model, except in case of a

haplo-insufficient mechanism. Indeed, in the latter case, 50% reduction of gene dosage

(inactivation of one allele by the CMT mutation) is sufficient to cause the disease.

Overexpression of such a protein with a loss-of-function mutation in an otherwise wild-type

Drosophila background (the Drosophila homolog of the disease gene is intact) may not

induce phenotypes.


Table 3. Drosophila homologues of CMT-associated genes

Human Gene

Symbol Subtype Human gene name

Drosophila

homologue Gene name CG number

Papers on

gene function

in Drosophila

PMP22

CMT1A,

CMT1E,

CMT3

peripheral myelin

protein 22 NO n.a. n.a. n.a.

MPZ

CMT1B,

CMT2J,

CMT3,DI-

CMTD

myelin protein

zero NO n.a. n.a. n.a.

LITAF

(SIMPLE) CMT1C

lipopolysaccharide

-induced tumour

necrosis factor

NO n.a. n.a. n.a.

EGR2

CMT1D,

CMT3,

CMT4E

early growth

response 2 NO n.a. n.a. n.a.

NEFL CMT1F,

CMT2E

neurofilament,

light polypeptide NO n.a. n.a. n.a.

KIF1B CMT2A1 kinesin family

member 1B YES unc-104 CG8566 4

MFN2 CMT2A2,

CMT6 mitofusin 2 YES

Marf CG3869 5

fzo CG4568 2

RAB7 CMT2B

RAB7, member

RAS oncogene

family

YES Rab7 CG5915 6

LMNA CMT2B1 lamin A/C YES Lam CG6944 41

LamC CG10119 13

MED25 CMT2B2 mediator complex

subunit 25 YES MED25 CG12254 0

TRPV4 CMT2C

transient receptor

potential cation

channel, subfamily

V, member 4

YES

nan CG5842 7

iav CG4536 12

GARS CMT2D glycyl-tRNA

synthetase YES Aats-gly CG6778 1

HSPB1 CMT2F heat shock 27kDa

protein 1 multiple multiple multiple n.a.

GDAP1

CMT2H,

CMT2K,

CMT4A,

RI-CMTA

ganglioside-

induced

differentiation-

associated-protein

1

YES CG4623 CG4623 0

HSPB8 CMT2L heat shock 22kDa

protein 8 multiple multiple multiple n.a.

DNM2 CMT2M,

DI-CMTB dynamin 2 YES shi CG18102 44

AARS CMT2N alanyl-tRNA

synthetase YES Aats-ala CG13391 1

DYNC1H1 CMT2O

dynein

cytoplasmic 1

heavy chain 1

YES Dhc64c CG7507 35


Human Gene

Symbol Subtype Human gene name

Drosophila

homologue Gene name CG number

Papers on

gene function

in Drosophila

LRSAM1 CMT2P

leucine rich repeat

and sterile alpha

motif containing 1

NO n.a. n.a. n.a.

PRX CMT3,

CMT4F periaxin NO n.a. n.a. n.a.

MTMR2 CMT4B1 myotubularin

related protein 2 YES mtm CG9115 1

SBF2

(MTMR13) CMT4B2

SET binding factor

2 YES Sbf CG6939 2

SH3TC2 CMT4C

SH3 domain and

tetratricopeptide

repeats 2

NO n.a. n.a. n.a.

NDRG1 CMT4D

N-myc

downstream

regulated gene 1

YES MESK2 CG15669 0

FGD4 CMT4H

FYVE, RhoGEF

and PH domain

containing 4

YES RhoGEF4 CG8606 2

FIG4 CMT4J

FIG4 homolog,

SAC1 lipid

phosphatase

domain containing

YES CG17840 CG17840 0

YARS DI-CMTC tyrosyl-tRNA

synthetase YES Aats-tyr CG4561 0

INF2 DI-CMTE

inverted formin,

FH2 and WH2

domain containing

YES form3 CG33556 1

KARS RI-CMTB lysyl-tRNA

synthetase YES aats-lys CG12141 0

GJB1 CMTX 1

gap junction

protein, beta 1,

connexin32

NO n.a. n.a. n.a.

PRPS1 CMTX5

phosphoribosyl

pyrophosphate

synthetase 1

YES CG6767 CG6767 0

The number of papers on the function of the Drosophila gene listed in FlyBase are indicated. This

number gives an indication on how well the respective Drosophila genes have been studied.

As already indicated for mouse models, introducing the CMT-associated mutations in the

endogenous locus of the Drosophila orthologue by homologous recombination (knock-in

approach) is an alternative strategy to generate models for both dominantly and recessively

inherited mutations.

Apart from tyrosyl-tRNA synthetase (discussed in the next paragraph), only one other

CMT-associated protein has been expressed in Drosophila, namely mitofusin 2 (Mfn2).

Eschenbacher et al. studied the functional consequences of rare non-synonymous sequence

variants within the heptad repeat 1 (HR1) domain of Mfn2, two of which are predicted to be

potentially damaging based on bioinformatical analysis (M393I and R400Q) (Eschenbacher et

al., 2012). Expression of these mutant Mfn2 proteins in the Drosophila eye results in reduced

eye area, whereas expression of wild-type Mfn2 does not. Furthermore, RNAi knock-down of

the Drosophila Mfn2 homolog Marf also results in a reduced eye area, which could be

rescued by co-expression of wild-type Mfn2, but not by the mutant Mfn2 proteins. Although


these findings are interesting, the studied Mfn2 mutations have not been found in CMT

patients, and most of the 31 Mfn2 mutations that were previously linked to CMT are located

in the GTPase domain. It would therefore be interesting to evaluate the effect of CMT-

associated Mfn2 mutations, and not only on eye morphology but also on motor behaviour,

axonal morphology, and electrophysiology.

A DROSOPHILA MODEL FOR CMT ASSOCIATED WITH MUTATIONS IN

TYROSYL-TRNA SYNTHETASE

The only published bona fide Drosophila CMT model to date models dominant

intermediate CMT type C (DI-CMTC), which is caused by mutations in the YARS gene,

encoding tyrosyl-tRNA synthetase (TyrRS or YARS) (Storkebaum et al., 2009). This enzyme

aminoacylates tyrosyl-tRNA (tRNATyr

) with tyrosine in a two-step reaction (Figure 2A). Like

all tRNA synthetases, YARS is expressed ubiquitously and every cell in the body depends on

its aminoacylation activity for protein translation. YARS forms homodimers, and only the

dimeric form is enzymatically active. Interestingly, apart from YARS, dominant mutations in

GARS, AARS and possibly HARS, and recessive mutations in KARS also result in axonal and

recessive intermediate forms of CMT (Abe and Hayasaka, 2009; Antonellis et al., 2003; Del

Bo et al., 2006; Dubourg et al., 2006; James et al., 2006; Latour et al., 2010; Lin et al., 2011;

McLaughlin et al., 2012; McLaughlin et al., 2010; Rohkamm et al., 2007; Vester et al., 2012)

(Table 1). These 5 tRNA synthetases all contain an aminoacylation domain and an anticodon

recognition domain, which are essential for their canonical aminoacylation function (Figure

2B). Apart from these common domains, many tRNA synthetases have acquired additional

functional domains during evolution, conferring non-canonical functions to these proteins

(Brown et al., 2010; Guo et al., 2010). These non-canonical functions are, however, different

for distinct tRNA synthetases. From a genetic point of view, the fact that 5 of the 20

cytoplasmic tRNA synthetase genes are associated with CMT suggests that alteration of a

common function of these enzymes - probably tRNA aminoacylation - may be the cause of

the disease.

Expression of Mutant Tyrosyl-tRNA Synthetase Recapitulates Features of

Human CMT in Drosophila

To generate a model for DI-CMTC, the UAS/GAL4 system was used to express human

YARS (hYARS) in Drosophila. This binary expression system relies on UAS transgenic

lines, which, when crossed to a specific GAL4 transgenic line, will result in transgene

expression in the cell population in which the yeast transcription factor GAL4 is expressed

(Brand and Perrimon, 1993). As a large number of GAL4 lines are available, this expression

system allows transgene expression in virtually any cell type or tissue of interest. Transgenic

lines were generated that allow expression of wild-type or three CMT-associated hYARS

mutants: two missense mutations (hYARS_G41R and hYARS_E196K) and one in frame

deletion that results in the deletion of 4 amino acids in the YARS protein (hYARS_153-

156delVKQV) (Jordanova et al., 2006). As random transgene insertion was used to generate


the transgenic lines, transgene expression levels of several wild-type and mutant hYARS

expressing lines were determined, in order to select lines with similar expression levels for

further studies (Storkebaum et al., 2009). More recently, it became possible to use site-

specific transgenesis, whereby attachment sites (attP/attB) are used to target UAS-transgenes

to specific landing sites in the genome (Fish et al., 2007).

Figure 2. A, Two-step aminoacylation reaction catalysed by tyrosyl-tRNA synthetase. In the first step,

tyrosine is activated by ATP to form a TyrRS (Tyr-AMP) intermediate with simultaneous release of

pyrophosphate PPi. In the second step, the activated tyrosyl moiety is transferred from AMP to tRNATyr

to give rise to Tyr-tRNATyr

and AMP. B, Schematic representation of the 5 tRNA synthetases that have

been implicated in CMT: tyrosyl-tRNA synthetase (YARS), glycyl-tRNA synthetase (GARS), alanyl-

tRNA synthetase (AARS), lysyl-tRNA synthetase (KARS) and histidyl-tRNA synthetase (HARS).

CMT-associated mutations and their positions relative to the protein functional domains are shown.

Mutations represented in green segregate with disease in a pedigree, mutations in black were found in a

single patient, mutations in orange give rise to dominant peripheral neuropathy phenotypes in mice, and

mutations in blue were detected in compound heterozygous state in a single patient with intermediate

CMT. *The AARS E778A mutation was found in a family with rippling muscles and cramps, and also

in one patient with axonal CMT (McLaughlin et al., 2012).**The AARS D893N mutation was found in

a family with dominant distal hereditary motor neuropathy (dHMN) (Zhao et al., 2012).


This strategy eliminates confounding influences of the surrounding genomic DNA

environment on transgene expression levels and patterns, making it ideally suited to evaluate

the phenotypic effect of subtle mutations in the transgene (structure/function analysis).

Strong ubiquitous expression of mutant - but not wild-type - hYARS resulted in

developmental lethality, with no or reduced numbers of adult flies eclosing from their pupal

cases. This mutant-selective toxicity was transgene dosage-dependent, so that flies with lower

expression levels could be tested for motor performance. Mutant hYARS expressing flies

displayed motor deficits in a negative geotaxis climbing assay, as well as in a jump and flight

assay. These motor performance deficits were progressive over time. Also neuron-selective

expression of mutant - but not wild-type - hYARS induced motor performance defects,

showing that mutant hYARS is intrinsically toxic to neurons (Storkebaum et al., 2009). As

DI-CMTC is characterized by both demyelination and axonal degeneration (Jordanova et al.,

2003), this finding indicates that the axonal degeneration is not just secondary to

demyelination, as is the case in many demyelinating forms of CMT.

Figure 3. Schematic representation of the Drosophila giant fiber system. For reasons of simplicity, the

representation is unilateral. The giant fiber neuron (GF) has its cell body in the brain, and projects

through the cervical connective to the VNC, where it synapses with the tergotrochanteral motor neuron

(TTMn) and the peripherally synapsing interneuron (PSI). The TTMn innervates the tergotrochanteral

jump muscle (TTM), whereas the PSI synapses in the periphery with the dorsal longitudinal motor

neurons (DLMns), which innervate the dorsal longitudinal muscles (DLMs). The position of

stimulation and recording electrodes for electrophysiological evaluations are indicated. Brain

stimulation activates the GF, which then activates motor neurons. Thoracic stimulation excites the

TTMn and DLMn directly (Godenschwege et al., 2002).


Finally, expression of hYARS transgenes in the giant fiber (GF) system was used to

assess the effect of hYARS expression on axonal morphology and to evaluate the occurrence

of electrophysiological defects. The GF system mediates an escape response consisting of a

jump and subsequent flight (Allen et al., 2006). It consists of the GF neurons, which are 2

symmetrical neurons in the fly CNS that have their cell bodies in the brain and project their

giant axon to the VNC (Figure 3). In the VNC, the GF axons synapse with the peripherally

synapsing interneuron (PSI) and the tergotrochanteral motor neuron (TTMn). The PSI in turn

synapses with the dorsal longitudinal motor neurons that innervate the dorsal longitudinal

flight muscles. The TTMn synapses with the tergotrochanteral "jump" muscle in the legs of

the fly. This organisation of the network ensures that, when a shadow (e.g. of an approaching

predator) falls over the eye of the fly, the visual input will activate the GF neurons, what will

result in a jump followed by flight.

The GF system is ideally suited to study the effects of CMT-associated (mutant) proteins,

as the giant fiber neurons have particularly long axons, the system is well characterized, and

GAL4 driver lines are available that allow expression in all GF system neurons (A307-

GAL4), as well as selective expression in the GF neurons (C17-GAL4) or the TTMn (ShakB-

GAL4). Furthermore, it allows evaluation of GF morphology, either by expressing marker

transgenes, or - preferentially - by dye filling approaches. Finally, it also allows

electrophysiological approaches that evaluate the response latencies between activation of the

GF neurons (by brain stimulation) and the tergotrochanteral or dorsal longitudinal muscles. If

the synapse between the GF neurons and the TTMn or PSI are dysfunctional, the response

latencies will be increased. Similarly, the ability to follow high-frequency (100 Hz) stimuli

one-to-one can be evaluated (Allen et al., 2006). Expression of mutant hYARS proteins in the

GF system was found to induce both terminal axonal degeneration and electrophysiological

defects (Storkebaum et al., 2009). As selective expression in the GF neurons was sufficient to

induce these phenotypes, one can conclude that mutant hYARS has cell-autonomous toxic

effects in neurons. In conclusion, expression of mutant - but not wild-type - hYARS in

Drosophila resulted in progressive motor performance defects, terminal axonal degeneration

and electrophysiological defects. Thus, several hallmarks of human CMT are recapitulated in

the fly model.

The Drosophila genome contains a single gene encoding cytoplasmic tyrosyl-tRNA

synthetase (Aats-tyr, further referred to as dYARS). The dYARS protein is 68% identical and

80% similar at the amino acid level to human YARS (hYARS), and all amino acid residues

that are mutated in DI-CMTC are identical between dYARS and hYARS. Importantly,

overexpression of dYARS transgenes containing the DI-CMTC mutations induced similar

phenotypes as the hYARS transgenes, indicating that the hYARS phenotypes are not simply

the consequence of expressing a human protein in Drosophila (Storkebaum et al., 2009).

Interestingly, the Drosophila DI-CMTC model has been shown to be useful to predict the

pathogenicity of newly identified mutations in the YARS gene. This was illustrated by a novel

K265N substitution in the YARS anti-codon recognition site, which was identified in one

CMT patient and one control individual. Ubiquitous or pan-neuronal expression of a dYARS

transgene carrying the corresponding amino acid change (UAS-dYARS_K264N) did not

induce motor performance defects or developmental lethality, showing that this substitution is

a benign polymorphism (Leitao-Goncalves et al., 2012).


Peripheral Neuropathy Phenotypes are Independent of Aminoacylation

Activity of Mutant YARS Proteins

The Drosophila DI-CMTC model was further used to assess the effect of the CMT-

associated mutations on aminoacylation activity. A genetic complementation experiment was

performed, whereby an RNAi transgene targeting dYARS was expressed in sensory organ

precursor (SOP) cells (scabrous-GAL4). These cells give rise to the sensory organs, including

bristles, which are sensory hairs that cover the body of the fly and detect the direction of the

airflow during flight. Expression of dYARS-RNAi in SOPs induced shortening or loss of the

four scutellar bristles on the posterior part of the thorax, which are always long in control

flies. To assess the effect of CMT mutations on aminoacylation activity, hYARS transgenes

were expressed in the dYARS-RNAi background. Expression of wild-type hYARS fully

rescued the bristle phenotype, indicating that dYARS and hYARS are functional homologs.

This again underscores the relevance of expressing hYARS in Drosophila, and is conform

with the observation that dGARS and hGARS are functional homologs (Chihara et al., 2007).

Expression of hYARS_E196K in the Sca-GAL4>dYARS-RNAi background also fully rescued

the bristle phenotype, suggesting that this mutant has retained aminoacylation activity. In

contrast, expression of hYARS_G41R or hYARS_153-156delVKQV did not rescue the

bristle phenotype, which is suggestive for loss of aminoacylation activity. These findings

were confirmed by an in vitro aminoacylation assay and by genetic complementation in S.

cerevisiae (Storkebaum et al., 2009), as well as by a biochemical study (Froelich and First,

2011). From this it was concluded that hYARS_E196K retains aminoacylation activity,

hYARS_153-156delVKQV has severely reduced activity, and hYARS_G41R displays loss of

enzymatic activity. These findings indicate that loss of aminoacylation activity is not

necessary to cause peripheral motor and sensory neuropathy. 50% loss of aminoacylation

activity is also not sufficient to induce peripheral neuropathy phenotypes, as dYARS

hemizygous flies did not develop motor performance defects (Storkebaum et al., 2009).

These findings were rather unexpected, as all identified disease-causing YARS mutations

are located in the aminoacylation domain of the protein, raising the possibility that the disease

could be due to partial loss of aminoacylation activity (Figure 2). Furthermore, the fact that

mutations in 5 different tRNA synthetase genes all give rise to CMT suggests that (partial)

loss of a common function (probably the canonical aminoacylation function) could be the

cause of the disease. However, the findings are consistent with the fact that for both GARS

and AARS, some CMT-associated mutations result in loss of aminoacylation activity,

whereas others do not alter enzymatic activity (Achilli et al., 2009; Antonellis et al., 2006;

Cader et al., 2007; McLaughlin et al., 2012; Nangle et al., 2007; Seburn et al., 2006; Xie et

al., 2007). However, the fact that loss of aminoacylation activity per se is not necessary to

cause the disease does not exclude the possibility that aminoacylation-active mutants may be

mislocalised in peripheral motor and sensory neurons, hence resulting in defects in local

protein translation and terminal axonal degeneration.

Indeed, mislocalisation of mutant YARS and GARS proteins has been reported in mouse

neuroblastoma (N2A), human neuroblastoma (SH-SY5Y) and mouse motor neuron (MN-1)

cell lines (Antonellis et al., 2006; Jordanova et al., 2006; Nangle et al., 2007). However,

another study reports no alterations in subcellular localization of mutant GARS proteins in

ESC-derived motor neurons and spinal cord sections of GarsP234KY/+

mice, as well as in teased

fiber preparations of sciatic nerves of GarsC201R/+

mice (Stum et al., 2011). Also,


AARS_E778A displays a similar subcellular localization as wild-type AARS in MN-1 cells

(McLaughlin et al., 2012). Finally, although HARS_R137Q - but not wild-type HARS -

induces axonal morphology defects and locomotor defects when expressed in C. elegans

motor neurons, HARS_R137Q displays a similar subcellular localization as wild-type HARS.

Thus, further studies are needed to clarify the potential role of subcellular mislocalisation of

mutant tRNA synthetases in CMT pathogenesis. Ultimately, direct assessment of neuronal

protein translation rates in an animal model would be the best way to evaluate the effect of

tRNA synthetase mutations on (local) protein translation in vivo.

Another caveat when interpreting the available data on aminoacylation activity of mutant

tRNA synthetases is that these studies have been performed either in vitro, using purified

proteins, or in Drosophila or S. cerevisiae in vivo, where the endogenous orthologous tRNA

synthetase is either missing or its levels are strongly reduced. As a consequence, these studies

have evaluated the aminoacylation activity of homodimers of mutant tRNA synthetases.

However, since for YARS, GARS and AARS the disease is dominantly inherited,

heterodimers between WT and mutant tRNA synthetase subunits can be formed (Jordanova et

al., 2006). At this moment, no data on aminoacylation activity of such heterodimers has been

reported. A possible involvement of (hetero-)dimerization is also suggested by the fact that all

pathogenic GARS mutations reported so far all localize near the dimer interface (He et al.,

2011; Nangle et al., 2007). The effect of the mutations on dimer formation has been

investigated, and although some mutations do not alter dimer formation, others either

strengthen or weaken dimer formation (He et al., 2011; Marchetto et al.; Nangle et al., 2007).

Overall, the potential role of heterodimers in disease pathogenesis is currently not clear and

deserves further investigation.

Finally, both Drosophila and mouse models for CMT associated with mutations in tRNA

synthetases can be used to study the genetic mechanisms of disease. The fact that dYARS

hemizygous flies or GARS heterozygous mice do not develop peripheral neuropathy

phenotypes argues against haplo-insufficiency as the disease mechanism (Seburn et al., 2006;

Storkebaum et al., 2009). Furthermore, mice heterozygous for ENU-induced P234KY and

C201R mutations in the murine GARS gene display a peripheral neuropathy phenotype,

which is not modified by overexpression of wild-type GARS (Motley et al., 2011). This

argues against both dominant negative and gain-of-wild-type-function mechanisms.

Furthermore, wild-type GARS transgenic mice that are homozygous or transheterozygous for

the GARS mutant alleles have a more severe phenotype than heterozygous mice, indicating

that the phenotypic strength depends on mutant allele dosage (Motley et al., 2011).

Overall, these genetic findings suggest a gain-of-toxic function as the basis of the disease.

The molecular mechanism of such a gain-of-toxic function is currently unknown, but novel,

mutation-induced protein-protein interactions may well be involved. In this respect, the

finding that each of five spatially dispersed GARS mutations induce the same conformational

opening of a consensus area that is mostly buried in the wild-type protein may provide a

structural basis for newly acquired protein-protein interactions (He et al., 2011). Along with

other approaches, genetic screens in Drosophila models for mutant YARS- and GARS-

associated CMT would be ideally suited to unravel the molecular nature of the gain-of-toxic

function mechanism.


CONCLUSION

Drosophila may be of great value to model forms of CMT that involve axonal

degeneration. Arguments in favor of Drosophila are that (i) the basic organizational

principles and functional properties of the neuromuscular system are conserved, (ii)

Drosophila homologs for most forms of axonal and intermediate CMT exist, and (iii) the

effect of CMT mutant proteins on neurons can be studied in their physiological context,

whereby behavioral phenotypes, neuronal morphology and electrophysiology can be

evaluated. Finally, the fact that Drosophila CMT models can be used in genetic screens for

disease modifying genes raises the hope that these models may be instrumental in deciphering

the molecular pathogenesis of this incurable disease, and in the identification of possible

therapeutic targets.

As a Drosophila model for CMT associated with mutations in tyrosyl-tRNA synthetase is

the only published Drosophila CMT model to date, there is a window of opportunity to

generate models for many other forms of CMT and to study the function of the Drosophila

homologs of CMT-associated genes in more detail. Thus, the use of Drosophila as a model

for CMT is only in its initial stages, and future expansion and broadening of this field is

expected.

ACKNOWLEDGMENTS

We would like to thank Brigitte Sass for help with the figures, Daniel Banovic and

Hermann Aberle for providing the figure panels illustrating the larval neuromuscular system,

and Tanja Godenschwege for providing the figure illustrating the giant fiber system. We

thank Arzu Celik and Christian Klämbt for critical reading of the manuscript. E.S. is funded

by the state North Rhine Westphalia and supported by a grant from the Frick Foundation for

ALS Research, SFB629 programm project grant from the German Research Council (DFG),

and a Minna-James-Heineman-Stiftung Minerva research grant.

ABOUT THE AUTHORS

Georg Steffes graduated in Biology at the University of Muenster, Germany, and

performed his doctorate research at the same university in the Institute of Neurobiology and

Behavioural Biology. He did postdoctoral research at Sanger Institute in Cambridge, UK, and

at the Max Planck Institute for Molecular Biomedicine, Muenster, Germany. His research

interests are the genetics of sensory systems and neurodegenerative disease modelling.

Erik Storkebaum graduated in Pharmaceutical Sciences at the University of Leuven,

Belgium, and obtained his Ph.D. at the Vesalius Research Center, VIB, Leuven, Belgium. He

has been working as a postdoctoral researcher in the Laboratory for Developmental Genetics,

University of Leuven, Belgium. Since 2010 he is an independent research group leader at the

Max Planck Institute for Molecular Biomedicine, Muenster, Germany. His research aims at

deciphering the molecular pathogenesis of the motor neurodegenerative disorder amyotrophic

lateral sclerosis (ALS) and CMT peripheral neuropathy.


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Chapter 6

LESSONS FROM DROSOPHILA IN

NEURODEGENERATION: MECHANISMS OF

TOXICITY AND THERAPEUTIC TARGETS IN SPINAL

AND BULBAR MUSCULAR ATROPHY

Adrienne M. Wang University of Washington, Department of Pathology, Seattle, WA, US

ABSTRACT

Spinal and bulbar muscular atrophy (SBMA) is one of nine polyglutamine (polyQ)

repeat diseases caused by an abnormal expansion of a glutamine tract in the affected

gene. In each of the nine diseases, the expansion occurs in unrelated genes, and affects

distinct neuronal populations. Despite these differences, polyglutamine diseases are all

characterized by the build-up of misfolded proteins into nuclear aggregates, and selective

neuronal loss. Drosophila models of polyglutamine diseases have yielded many insights

into disease pathology. In SBMA, the polyQ expansion occurs in the first exon of the

androgen receptor (AR), leading to a partial loss of endogenous function as well as a

toxic gain-of-function. Studies using fly models of SBMA helped to establish both the

glutamine length-dependent and ligand-dependent toxicity that are characteristic of the

disease. Additionally, flies have given us numerous insights into molecular mechanisms

of toxicity, revealing a myriad of cellular processes that are altered in disease

pathogenesis, such as transcription, RNA processing, axonal trafficking and

mitochondrial function. With such a wide array of cellular mechanisms affected by the

presence of the mutant AR, treating dysfunction in individual pathways is likely to be

ineffective, and a promising therapeutic target is the modulation of the cell’s innate

protein quality control pathways to clear mutant protein upstream of its toxic effects.

While polyglutamine diseases currently lack efficacious treatment, Drosophila is a

promising model organism suited for straightforward genetic and pharmacologic

manipulation in the hunt for therapeutic targets that prevent or halt neurodegeneration.

Keywords: Polyglutamine, androgen receptor, neurodegenerative disease, protein quality

control

Adrienne M. Wang 148

INTRODUCTION

A diverse group of neurodegenerative diseases affecting the aging population are

characterized by accumulations of abnormally processed or mutant proteins that misfold and

aggregate. Among these are nine genetic disorders caused by expansions of a trinucleotide

CAG repeat within the coding regions of disease-causing genes (Table 1). Since CAG codes

for glutamine, this group of diseases is referred to as polyglutamine repeat diseases (polyQ

diseases).

For each of the nine disorders, the polyglutamine expansion occurs within unrelated

genes and selectively affects different neuronal subtypes, yet these polyglutamine diseases

share several clinical and pathological features (Zoghbi and Orr, 2000). As a group,

polyglutamine diseases preferentially affect the basal ganglia, brainstem nuclei, cerebellum,

and spinal motor nuclei, often leading to impaired motor function. Disease onset occurs most

often in middle age despite lifelong expression of mutant protein, and exhibits an inverse

correlation with CAG repeat length. The longer the glutamine expansion, the earlier the

patient becomes symptomatic, and the more acute the disease progression. Unfortunately,

these expanded polyglutamine repeats are also highly unstable, making them prone to genetic

anticipation, in which instability leads to an increase in length with each successive

generation. The expanded repeat is inherited in an autosomal dominant manner in all

polyglutamine diseases except for spinal bulbar muscular atrophy (), which is X-linked,

suggesting that pathology is due in part to a toxic gain-of-function (Zoghbi and Orr, 2000)

The unstable polyglutamine expansion can adopt an abnormal -sheet conformation

leading to the formation of insoluble aggregates, and these polyglutamine diseases are

characterized by the accumulation of nuclear and/or cytoplasmic protein inclusions. These

inclusions are typically composed of the mutant protein, heat shock proteins (HSPs) and

ubiquitin (Li et al., 1998). There is considerable debate in the field as to whether the presence

of aggregates are toxic or protective, or whether they simply reflect the end stage of

accumulation, with the toxic species being the precursors to the aggregates including

oligomers or proteolysed monomers (Adachi et al., 2005; Arrasate et al., 2004; Saudou et al.,

1998). Regardless, the presence of aggregates is associated with the late stages of disease

pathogenesis, implying that the accumulation of misfolded toxic proteins may be a key step in

degeneration.

Drosophila have proven to be an invaluable tool in the study of polyglutamine diseases,

with their single gene mutations lending themselves to direct genetic modelling. Although

outwardly very different from humans, the architecture and function of the fly body and

organs mirrors our own, and numerous cellular processes are conserved between flies and

mammals. Genetically, 50% of fly genes exhibit homology with human genes, and 75% of

human disease-causing genes have a homologue in Drosophila (Bier, 2005). Flies also exhibit

complex behaviour that is reflective of their highly organized neuronal network, allowing

them to be used as a model for cellular and higher-order behavioural studies, both aspects

affected by neurodegeneration. With such genetic similarity in the package of such a

malleable model system, flies offer an attractive system in which to manipulate endogenous

and exogenous gene expression.

Lessons from Drosophila in Neurodegeneration 149

Table 1. Polyglutamine Diseases

Disease Protein

Normal

repeat

length

Pathogenic

repeat

length

Brain region/neurons

affected

SBMA Spinal Bulbar

Muscular

Atrophy

androgen

receptor

6-36 38-62 Lower motor neurons and

dorsal root ganglion

HD Huntington’s

disease

huntingtin 11-34 40-121 Striatum and cortex

DRPLA Dentatorubral-

pallidoluysian

atrophy

atrophin-1 7-34 49-88 Globus pallidus, dentate

and subthalamic nucleus

SCA1 Spinocerebellar

ataxia type 1

ataxin-1 6-39 40-82 Purkinje cells, dentate

nucleus and brainstem


ataxia type 2

ataxin-2 15-24 32-200 Cerebellum, pontine nuclei,

substantia nigra

SCA3

(MJD)

Spinocerebellar

ataxia type

3/Machado-

Joseph disease

ataxin-3

13-36 61-84 Substantia nigra, globus

pallidus, pontine nucleus,

cerebellar cortex


ataxia type 6

P/Q-type

calcium channel

subunit 1A

4-20 20-29 Cerebellum, brainstem


ataxia type 7

ataxin-7 4-35 37-306 Photoreceptor and bipolar

cells, cerebellum, brainstem


ataxia type 17

TATA-box-

binding protein

25-42 47-63 Gliosis and Purkinje cell

loss

The UAS/Gal4 system developed by Brand and Perrimon (Brand and Perrimon, 1993) has

proven to be an indispensible tool for fly geneticists. It allows for expression of genes in a

tissue and time-specific manner, where the gene of interest is placed just down-stream of an

up-stream activating sequence (UAS), and binding of GAL4 to the UAS is required for gene

transcription. By driving expression of GAL4 in a tissue or developmental time-specific

manner, researchers can spatially or temporally control expression of the UAS-linked gene of

interest. In particular, the glass-multimer reporter (GMR-Gal4) driver line has been used

extensively in studies of neurodegeneration. This driver is expressed in the fly compound eye,

allowing for expression of UAS linked proteins in photoreceptor neurons and accessory

pigment cells of the developing eye discs. This is an attractive target for expression of toxic

proteins as the eye is non-essential in the fly, and allows researchers to circumvent

developmental or pathological effects of their target protein.

The fly eye is a highly ordered organ, with each eye containing around 800 units called

ommatidia. These ommatidia are organized in a crystalline pattern with interommatidial

bristles studding each junction at a discrete angle (Figure 1, EtOH). Each ommatidium

contains eight photoreceptor neurons, organized in an invariant fashion. Changes or

degeneration of this highly organized structure are readily seen under the light microscope

and are often referred to as a ‘rough-eye phenotype’ due to the roughness introduced when

the lattice of the eye is disrupted, making it particularly useful for genetic screens (Figure 1,

DHT). In fact, numerous genetic screens for modifiers of the polyglutamine induced rough-


eye phenotype have informed our current hypotheses about mechanisms of toxicity in

polyglutamine diseases, including transcriptional dysregulation, altered RNA processing,

mitochondrial dysfunction, and defects in axonal transport (Bilen and Bonini, 2007;

Fernandez-Funez et al., 2000; Kazemi-Esfarjani and Benzer, 2000; Nedelsky et al., 2010).

Flies expressing expanded polyglutamine repeats were the first neurodegenerative models

successfully created in Drosophila using human transgenes (Jackson et al., 1998; Warrick et

al., 1998). Dr. Nancy Bonini’s group at the University of Pennsylvania and Dr. Larry

Zipursky’s group at the University of California, Los Angeles both expressed truncated

fragments of human disease causing genes in the fly, publishing their models in the same

year. Directed expression of truncated fragments of expanded polyQ human ataxin-3 and

huntingtin, the disease causing proteins in spinocerebellar ataxia type 3 (SCA3) and

Huntington’s disease (HD) respectively, recapitulate several aspects of human polyglutamine

disease. These flies show glutamine length-dependent nuclear inclusions and neuronal

degeneration, and were used to demonstrate the glutamine length-dependence of the disease

pathology. These groups used the rough-eye phenotype to monitor degeneration and showed

that targeting expression of the toxic polyglutamine protein to the fly eye leads to

degeneration of the eye as monitored by age-related changes in eye morphology such as loss

of pigment, disruption of the gross ommatidial array and photoreceptor degeneration. Such

early studies helped to establish Drosophila as a highly tractable model of neurodegenerative

disease in vivo.

Figure 1. DHT-induced rough eye phenotype in a fly model of SBMA. Scanning electron micrographs

of eyes from flies expressing the expanded human AR52Q under control of the GMR-Gal4 promoter

fed control food (EtOH, upper panels) or food supplemented with dihydrotestosterone (DHT, lower

panels). High magnification images are shown in panels on the right. DHT induces hormone and

glutamine length dependent degeneration in the fly eye causing a rough-eye phenotype that is

characterized by ommatidial disarray and fusion, in addition to abnormal and extra interommatidial

bristles.


Figure 2. Androgen receptor domain structure. The AR protein consists of 3 functional domains: the N-

terminal domain containing the activation function-1 (AF-1) surface and the glutamine tract (PolyQ),

the DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD) containing the

activation function-2 (AF-2) region. The nuclear localization signal (NLS) is found within the hinge

region that links the DBD and the LBD.

In SBMA, fly models have been indispensible to our understanding of disease pathogenesis,

answering many fundamental questions about requirements for disease initiation and

progression, the extent to which normal androgen receptor (AR) function is involved in

toxicity, and genetic modifiers of toxicity, paving the way for valuable insights into

polyglutamine pathogenesis and therapeutics.

SBMA PATHOLOGY

SBMA is a progressive neuromuscular disorder that is caused by a toxic expansion of the

glutamine tract in the AR (La Spada et al., 1991). This polyglutamine disease affects only

men, and is characterized by degeneration of proximal limb, mouth, and throat muscles in

patients (Kennedy et al., 1968). Symptom onset is typically between 30-60 years of age, but

muscle weakness is often preceded by muscle cramping and tremor. The clinical features of

SBMA correlate with loss of lower motor neurons in the brainstem and spinal cord, with

marked myopathic and neurogenic changes in skeletal muscle (Sobue et al., 1989). Patients

may also exhibit partial androgen insensitivity such as enlarged breasts, testicular atrophy and

decreased fertility (Dejager et al., 2002). Disease progression is slow, but many patients

eventually require assistance to walk, and risk for aspiration pneumonia increases as bulbar

paralysis develops. Currently, very few treatment options exist for SBMA patients, the most

promising of which are testosterone blockade therapies (Banno et al., 2009; Fernandez-

Rhodes et al., 2011; Katsuno et al., 2006b).

The endogenous function of the AR has been well characterized, making it an ideal

context in which to study the effects of a toxic polyQ tract. In SBMA, the toxic glutamine

tract expansion occurs in the first exon of the AR. The repeat length found in the normal

population is 9-36 glutamines, with an expansion to a length between 38 and 62 glutamines in

SBMA patients. In addition to the glutamine tract in the N-terminal transactivation domain,

the AR contains a nuclear localisation sequence (NLS) and a central DNA binding domain

(DBD) linked to the C-terminal ligand-binding domain (LBD) by a hinge region (Figure 2).

Two interaction surfaces, activation function-1 (AF-1) in the N-terminal domain, and

activation function-2 (AF-2) in the LBD, recruit nuclear co-regulators to modulate

transcription. Without ligand, the nuclear localization signal is masked, and the AR is

localized to the cytoplasm where it is held in a high-affinity ligand binding state by molecular


chaperones. Binding of testosterone to the ligand-binding domain exposes a nuclear

localization signal within the hinge region of the AR, allowing for the trafficking and import

of the AR into the nucleus (Cutress et al., 2008). Once in the nucleus, ligand-bound AR

dimerizes, interacts with co-regulators via exposed AF-1 and AF-2 domains, and binds to

androgen responsive elements (AREs), triggering transcription or repression of androgen-

dependent genes (He et al., 2002; Wong et al., 1993) (Figure 3).

In the presence of an abnormally expanded glutamine tract, the AR suffers from both a

partial loss of endogenous function as well as a toxic gain-of-function. Expansion of the

polyglutamine tract mildly suppresses transcriptional activities of the AR (Chamberlain et al.,

1994; Kazemi-Esfarjani et al., 1995; Lieberman et al., 2002), likely contributing to the partial

testosterone insensitivity seen in SBMA patients. However, patients with other loss-of-

function mutations in the AR gene, as well as mouse models with similar loss-of-function

mutations in the AR, show androgen insensitivity and testicular feminisation without the

motor impairment observed in SBMA (Sato et al., 2003). In addition, evidence for a gain-of-

function by the pathogenic AR is robust.

Figure 3. Mechanisms of toxicity in SBMA. PolyQ AR affects many cellular processes, both nuclear

and cytoplasmic. In the absence of ligand, the nuclear localization sequence (NLS) is masked, and the

AR is retained in the cytoplasm. Heat shock protein-90 (HSP90) stabilizes the receptor, priming it for

ligand binding. Upon binding of ligand, the NLS is exposed, allowing trafficking of the AR into the

nucleus where it leads to toxicity by altering numerous cellular processes. Pathogenic AR with

expanded polyQ tract is prone to misfolding, interaction with heat shock protein-70 (HSP70), and

subsequent degradation by the proteasome. Proteasomal degradation is shown occurring in the

cytoplasm, but also occurs within the nucleus. Abbrevations: DBD: DNA binding domain, LBD: ligand

binding domain, Ub: ubiquitin, ARE: androgen responsive elements, PGC1: PPAR coactivator-1,

DCTN1: dynactin 1, ROS: reactive oxygen species.


Several SBMA mouse models have been created in which transgenic expression of the

aberrant AR leads to muscular atrophy and motor dysfunction in the continued presence of

the wild-type protein (Abel et al., 2001; Katsuno et al., 2002; McManamny et al., 2002;

Sopher et al., 2004). Similarly, exogenous expression of the mutant AR in transformed cell

lines and primary neurons leads to altered cellular processes and cell death (Merry et al.,

1998; Mhatre et al., 1993). Data from fly models of polyglutamine disease are consistent with

a toxic gain-of-function, as expression of expanded polyglutamine tracts in the absence of

protein context and ectopic expression of the human expanded AR lead to toxicity (Kazemi-

Esfarjani and Benzer, 2000; Kazemi-Esfarjani and Benzer, 2002; Marsh et al., 2000;

Takeyama et al., 2002). Taken together, these data indicate that the polyglutamine tract

expansion in the AR leads to both a toxic gain-of-function and partial loss of normal function,

and suggest that toxic effects of the mutant protein are the main drivers of disease

pathogenesis.

DROSOPHILA MODELS OF SBMA

Although flies do not have a direct AR orthologue, the nuclear hormone receptor system

is well conserved between humans and flies, with fly hormone receptors containing AF-1 and

AF-2 (two activating domains in the human androgen receptor) domains and interacting

proteins. The first Drosophila model of SBMA was created by Dr. Ken-Ichi Takeyama and

colleagues at the University of Tokyo, by expressing a UAS-responsive full-length human

AR with either a normal (AR12Q) or expanded (AR52Q) polyQ tract under control of the

GMR promoter (Takeyama et al., 2002). The authors show that in this system, the human AR

translocates to the nucleus, and can activate GFP when driven by an androgen responsive

element (ARE). Importantly, this is done in a ligand-dependent manner, requiring dietary

ingestion of dihydroxytestosterone (DHT). A later paper from the same group further verified

that modulation of Drosophila homologues of mammalian AR co-regulators alters human AR

transactivation in flies, establishing this model as a functionally relevant and tractable means

to probe normal and abnormal AR function (Takeyama et al., 2004).

When flies ectopically express the human AR in the eye with either a non-toxic

glutamine tract or with an expanded glutamine tract in the absence of DHT, no abnormalities

or degeneration can be seen. This is in contrast with other polyQ disease models where the

expansion occurs in non-hormone responsive genes, exhibiting toxicity in a glutamine length-

dependent manner only (Kazemi-Esfarjani and Benzer, 2000; Tsai et al., 2004; Warrick et al.,

2005), and indicating a hormone requirement for disease progression in SBMA, an idea that

was debated at the time. Two possible mechanisms had been proposed, the first being that

females were protected from disease pathogenesis by random X-inactivation in which one of

the two copies of the X chromosome in females is inactivated, theoretically reducing motor

neuron toxicity by 50%. The second hypothesis posited that the toxicity was ligand-

dependent, and that higher levels of androgens in males increased their susceptibility to

disease pathogenesis (Lloyd and Taylor, 2010). This debate was resolved with help from the

humble fruit fly.

Dr. Takeyama’s model helped to establish the ligand dependence of SBMA. Expression

of non-toxic glutamine repeat length (AR12Q) in the fly eye leads to DHT-dependent nuclear


translocation and expression of an ARE responsive GFP, without abnormalities or

degeneration. Flies expressing a toxically expanded glutamine tract (AR52Q) in the eye show

the same DHT dependent transactivation, but also exhibit ommatidial disarray, pitting, and

fusion, in addition to abnormal and supernumerary interommatidial bristles when reared on

DHT containing food (Figure 1). The authors establish that these abnormalities are due to

degeneration rather than developmental effects by showing that the rough eye phenotype can

be delayed and induced in naïve adult flies exposed to DHT. These data suggest that

testosterone-dependent activation of the AR is a critical step in disease pathogenesis.

This model is further supported by the fact that deletion of the LBD and constitutive

transactivation of a truncated AR causes degeneration in the absence of DHT (Takeyama et

al., 2002). Consistent with this, retaining the mutant AR in the cytosol by deletion of the NLS

and/or insertion of a nuclear export sequence (NES) abolishes toxicity (Takeyama et al.,

2002). Ligand dependent degeneration was confirmed by a mouse model of SBMA published

simultaneously, as well as in independently-generated fly models (Chevalier-Larsen et al.,

2004; Pandey et al., 2007). In this way, Drosophila served to definitively establish glutamine

length-dependent and hormone-dependent degeneration in SBMA, today considered two

fundamental features of the disease.

In addition to exhibiting the characteristic glutamine length and hormone dependence of

SBMA when mutant AR is expressed in the fly eye, these aspects of the disease are

recapitulated when the mutant polyQ AR is expressed in a variety of tissues. Larvae

expressing the expanded polyglutamine AR in the developing salivary gland exhibit

significant decrease in size in response to DHT, while larvae expressing the expanded

polyglutamine AR in motor neurons show hormone-dependent motor defects as assessed by

larval crawling (Nedelsky et al., 2010). Further, adult flies expressing the expanded polyQ

AR in motor neurons exhibit an eclosion defect when reared on DHT-supplemented food that

is consistent with motor neuron impairment (Wang et al., 2012).

MECHANISMS OF TOXICITY

Although the exact mechanism by which the mutant androgen receptor exerts a toxic

gain-of-function is unclear, the ligand dependence of SBMA highlights the AR’s nuclear

functions in effecting disease pathogenesis. Ligand induced transactivation of the AR is

further implicated in disease pathogenesis in the SBMA fly, with both nuclear import and

DNA binding being an upstream requirement in disease progression (Nedelsky et al., 2010).

This section will discuss several mechanisms of toxicity implicated in SBMA and other

neurodegenerative diseases summarized in Figure 3.

Transcriptional Dysregulation

Several polyglutamine diseases require nuclear localization of the mutant protein and

exhibit altered transcription prior to phenotypic onset (Lin et al., 2000; Wyttenbach et al.,

2001). A spinocerebellar ataxia 1 (SCA1) mouse model in which the polyQ Ataxin-1 is

cytoplasmically retained shows neither the ataxia nor Purkinje cell loss characteristic of the


disease. Alternatively, allowing nuclear transport in the absence of aggregation leads to

pathogenesis, establishing nuclear events as a requirement for polyglutamine toxicity

(Klement et al., 1998). Similarly, cytoplasmic retention of the AR rescues the disease in

SBMA fly and mouse models, while nuclear localization of an unliganded AR is insufficient

to cause the toxicity seen in the presence of ligand (Montie et al., 2009; Nedelsky et al., 2010;

Takeyama et al., 2002).

The endogenous function of the AR as a nuclear transcription factor lends added weight

to the idea that transcriptional dysregulation underlies disease pathogenesis in SBMA. The

mutant AR abnormally interacts with transcriptional co-regulators, and has been shown to

alter transcription in both fly and cellular models (Irvine et al., 2000; Kazemi-Esfarjani et al.,

1995; Lieberman et al., 2002; Mhatre et al., 1993; Nedelsky et al., 2010). Several of these co-

regulators are transcription factors that modify the acetylation state of the histones that

package and compact nuclear DNA, thus modifying the accessibility of DNA to the

transcriptional machinery. Furthermore, the acetylation state of the AR itself has been

suggested to regulate the subcellular localization and misfolding of the receptor (Thomas et

al., 2004), as well as interaction with co-regulators (Fu et al., 2002).

One of these co-regulators, CREB-binding protein (CBP), is a histone acetyltransferase

and an important co-activator that is functionally sequestered in the nuclear inclusions seen in

cell culture, animal models, and tissue derived from SBMA patients. Trapping CBP in

nuclear inclusions is thought to result in down-regulation of CBP mediated transcription

(McCampbell et al., 2000). In support of this, overexpression of CBP rescues polyQ mediated

toxicity in cell and Drosophila models of polyglutamine disease, restoring normal

transcription and histone acetylation levels (McCampbell et al., 2000; Taylor et al., 2003).

Genetically or pharmacologically inhibiting histone deacetylases rescues lethality and

ommatidial degeneration in a Drosophila model of HD (Steffan et al., 2001), and has also

been shown to decrease the toxicity of a truncated fragment of the expanded AR

(McCampbell et al., 2001), further implicating transcriptional dysregulation as one

pathogenic mechanism in SBMA.

Post-transcriptional Dysregulation

Post-transcriptional changes in gene expression also affect protein expression and can

produce dysfunction in much the same way as altered transcription. Toxic forms of RNA

have been implicated in other repeat expansion diseases, such as in select spinocerebellar

ataxias and in myotonic dystrophy, where trinucleotide expansions in noncoding mRNA

confer neurotoxicity. The expanded repeats confer toxicity and alter RNA splicing by

affecting expression of splicing factors and/or by sequestering RNA binding proteins (Ranum

and Day, 2004). Aberrant RNA processing can have widespread effects on many transcripts,

and can amplify existing transcriptional dysregulation. RNA missplicing has also been shown

to contribute to SBMA pathogenesis in a knock-in mouse, where changes in RNA binding

protein expression and mRNA splicing have been observed (Yu et al., 2009). Expressing

expanded, non-coding CAG repeat RNA in the fly eye leads to disorganized ommatidia and

interommatidial bristles, a rough-eye phenotype which can be modulated by expression of

RNA binding proteins (Mutsuddi et al., 2004). In fact, numerous RNA binding proteins have

been implicated as modifiers of polyglutamine toxicity and have been identified through


screens using Drosophila (Fernandez-Funez et al., 2000; Kazemi-Esfarjani and Benzer, 2000;

Li et al., 2008). More recently, expanded CAG RNAs have been shown to alter ribosomal

RNA (rRNA) transcription, resulting in apoptosis (Tsoi et al., 2012).

Mitochondrial Dysfunction

Mitochondrial dysfunction has also been implicated in polyglutamine disease

pathogenesis. Glial expression of the mitochondrial anion transporter uncoupling protein 5

(UCP5) increases metabolism and rescues lifespan and motor deficits in a Drosophila model

of HD without altering neuronal loss (Besson et al., 2010). Perturbations in RNA

transcription discussed above result in cell death due to mitochondrial localization of p53, and

subsequent release of cytochrome C and caspases. In vitro and in vivo models of SBMA show

altered expression of peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-

1), a transcription factor that governs mitochondrial biogenesis and function. Mitochondria in

cells expressing pathogenic polyQ AR are lower in number and exhibit decreased

mitochondrial membrane potential, occurring in conjunction with higher levels of reactive

oxygen species (ROS) (Ranganathan et al., 2009). Mitochondrial abnormalities are also

detectable in the leukocytes of SBMA patient samples, highlighting the role of mitochondrial

bioenergetics in polyglutamine disease (Su et al., 2010).

Axonal Trafficking

Neurons, and especially motor neurons, with their long axonal projections, are highly

reliant upon fast axonal transport to shuttle nutrients and molecular signals between the

nucleus and the cell periphery, and defects in axonal transport have been reported in several

neurodegenerative and polyglutamine diseases (Gunawardena et al., 2003; Piccioni et al.,

2002; Szebenyi et al., 2003). Transport deficiencies can be caused by both physical blockage

of the narrow axons and by altered protein interactions leading to sequestration of necessary

equipment away from normal function or altered expression. Specific disruption of this

pathway by dynactin mutations causes an inherited form of motor neuron disease (Puls et al.,

2003), and flies expressing polyglutamine tracts in neurons show glutamine length-dependent

organelle blockages (Gunawardena et al., 2003; Lee et al., 2004).

Controversy exists as to the extent of axonal trafficking defects in SBMA models. Axonal

trafficking defects caused by the polyQ AR occur in isolated squid axons, where impaired fast

transport occurs secondary to activation of JNK signalling (Morfini et al., 2009; Szebenyi et

al., 2003). Defects have also been documented in several, but not all, SBMA mouse models

that have been studied. Dynactin 1, a regulator of retrograde axonal transport, is expressed at

lower levels in SBMA transgenic mice that show perturbed axonal trafficking (Katsuno et al.,

2006a). Early deficits in retrograde labelling also occur in both SBMA knock-in mice and a

myogenic mouse model that overexpresses wild-type AR exclusively in muscle (Kemp et al.,

2011). In contrast, human polyQ-expanded AR yeast artificial chromosome (YAC) transgenic

SBMA mice show no change in retrograde transport or dynactin levels (Malik et al., 2011),

suggesting that these defects may not be necessary for the occurrence of a disease phenotype.


PROTEIN DEGRADATION AS A THERAPEUTIC TARGET IN SBMA

With such a diverse array of cellular processes affected by the presence of the mutant

AR, therapeutic treatments targeting individual pathways are likely to be unsuccessful (Figure

3). The common upstream factor in all the toxic mechanisms is the activation of the abnormal

AR. Therefore, diminishing levels of the misfolded protein could abrogate multiple

downstream effects. Perhaps the most promising route to achieving this is to enhance the

cell’s natural protein quality control pathways. This machinery contains several distinct and

interacting components, including molecular chaperones, the ubiquitin-proteasome system

and autophagy.

The two main protein degradation pathways in eukaryotic cells are the ubiquitin-

proteasome system (UPS) and autophagy. Both of these degradation pathways serve to

modulate protein homeostasis and have been identified as potential therapeutic targets in

neurodegenerative disease. The UPS degrades damaged or misfolded proteins in both the

cytoplasm and nucleus, while autophagy is responsible for bulk degradation of long-lived

proteins and organelles in the cytosol. In SBMA, it is unclear whether the toxic form of the

AR is a monomeric receptor that would be degraded by the UPS, or an oligomeric or

aggregated form which would require clearance through autophagy. However, the nuclear

localization of the testosterone-bound AR in SBMA keeps the toxic receptor sequestered from

the cytosolically located autophagic machinery, suggesting that modulating UPS activity may

provide a promising therapeutic target.

The Ubiquitin Proteasome System

The UPS is a selective and tightly regulated process of degrading soluble cytosolic and

nuclear proteins into short peptide chains for recycling. By selectively degrading short-lived

and misfolded or damaged proteins, the UPS is able to govern localized protein

concentrations, allowing for regulation of cell cycle and growth regulators, signal

transduction, metabolic enzymes and general housekeeping functions (Hershko and

Ciechanover, 1998). Degradation of a protein through the UPS requires 2 steps: the covalent

attachment of multiple ubiquitin molecules and the degradation of the ubiquitinated protein

by the proteasome.

Ubiquitination of the target protein is achieved in an ATP-dependent stepwise process.

Through a series of transient associations of the ubiquitin molecule with the ubiquitin E1-

activating, E2-conjugating, and E3-ligating enzymes, the ubiquitin molecule is conjugated to

a lysine on the targeted substrate (Hershko et al., 1983). This process repeats itself, allowing

for sequential addition of ubiquitins. Once a protein incurs a chain longer than four ubiquitins,

it is targeted to the 26S proteasome for degradation (Chau et al., 1989).

The 26S proteasome is a multi-subunit, multi-catalytic protease found in both the nucleus

and cytoplasm. It is comprised of a 20S core catalytic complex flanked by two 19S regulatory

components forming a barrel-shaped structure (20S) with a lid (19S). In a highly energy

dependent manner, the 19S subunit functions to identify and bind polyubiquitinated proteins,

feeding unfolded substrates through the 20S proteasome for degradation into oligopeptides

ranging from 4-25 amino acids which can then be reused by the cell.


Components of the UPS, as well as the molecular chaperones HSP70 and HSP40, are

found in the nuclear aggregates of SBMA models and patients, indicating cellular recognition

of the misfolded protein and cellular attempts to degrade the aggregate-prone protein prior to

inclusion formation (Abel et al., 2001; Adachi et al., 2001; Li et al., 1998). This accumulation

of UPS components in aggregates has also been implicated in proteasome inhibition, where

sequestration of the components in inclusions would lead to downregulation of proteasomal

degradation (Bence et al., 2001). However, the activity of the UPS in an SBMA mouse model

is preserved and even up-regulated in late stages of disease (Tokui et al., 2008).

The importance of the proteasome as the primary degradation pathway of the AR is

shown in a Drosophila model of SBMA, where proteasome inhibition further increases the

toxicity of a truncated fragment of the expanded polyQ AR in the fly eye. This suggests that

steroid hormone receptor degradation occurs through the proteasome (Chan et al., 2002), and

implies that methods to stimulate this innate degradation pathway could be a promising

therapeutic strategy for the clearance of toxic polyQ AR. As discussed below, manipulating

the expression or function of certain molecular chaperones and chaperone-dependent

ubiquitin ligases ameliorates the disease phenotype in SBMA mice and flies, consistent with

the notion that clearance of the polyQ AR through the UPS is critically important to the

disease.

Autophagy

The cell’s alternative degradation pathway allows for bulk cytoplasmic degradation of

larger organelles and long-lived proteins. Autophagy, or “self eating”, is a process in which a

cell is able to engulf contents of cytoplasm and deliver them to the lysosome for degradation

and recycling (Klionsky and Emr, 2000). Activation of autophagy involves tightly regulated

stepwise induction of a double membraneous autophagic vesicle that grows to engulf

cytoplasmic components that the proteasome cannot degrade, such as mitochondria and

protein aggregates, as well as long-lived proteins. This mechanism consists of several discrete

steps executed by autophagy related (Atg) proteins that are analogous to the cascade required

to attach ubiquitin to target proteins. Once the autophagosome is completed, it fuses with

lysosomes where the contents of the autophagosome are enzymatically broken down for reuse

by the cell (Ravikumar et al., 2010).

Upstream of the formation of the autophagic vesicle, autophagy is regulated by two main

proteins, the mammalian target of rapamycin (mTOR), which negatively regulates autophagy

(Ravikumar et al., 2004), and Beclin-1, which is required for both autophagosome formation

and autophagic flux (Liang et al., 2006). mTOR is responsive to cellular signals conveying

the energy and nutrient status of the cell to cellular processes. In times of excess, mTOR

inhibits autophagy; inhibition of mTOR through nutrient deprivation or rapamycin treatment

leads to a release of this inhibition, allowing for autophagy induction and an increase in

nutrients via this recycling pathway.

Due to its ability to degrade organelles and cytosolic aggregates, the role of autophagy in

protein aggregation diseases offers a potentially promising therapeutic target. In fly and

mouse models of HD, mTOR inhibition has been shown to be protective against

neurodegeneration through increased autophagy and decreased protein translation (Ravikumar

et al., 2004). A fly model of another polyglutamine disease, SCA3, displays increased


autophagy and degeneration that is exacerbated when autophagy is inhibited (Bilen and

Bonini, 2007) and inhibiting autophagy in an SBMA fly model increased the severity of the

rough eye phenotype (Pandey et al., 2007). Paradoxically, inhibiting autophagy in a mouse

model of SBMA increases lifespan and decreases muscle wasting (Yu et al., 2011). This

discrepancy may be due to context and model specific differences between ectopic expression

of the mutant AR in the invertebrate eye and endogenous expression of the mutant AR in

mammalian neurons and muscle cells, or due to variations in the extent to which autophagy is

disrupted.

Crosstalk between protein quality control pathways has been suggested, and several lines

of evidence imply cross-regulation and compensation when some aspect of protein

homeostasis is perturbed. Temperature-sensitive dominant-negative proteasome mutant flies

exhibit increased autophagy, a compensatory response that requires the microtubule

associated deacetylase, HDAC-6 (Pandey et al., 2007). Compensatory regulation between

autophagy and another protein quality control pathway, the unfolded protein response (UPR),

has also been implicated. The UPR is an integrated signal transduction pathway that transmits

information about protein folding within the endoplasmic reticulum (ER) lumen to the

nucleus and cytosol to regulate protein synthesis and folding, influencing cell survival (Ron,

2002; Ron and Walter, 2007). The UPR is activated in skeletal muscle in SBMA patients and

mice expressing AR113Q, and deficiencies in this signalling pathway lead to increased

autophagy (Yu et al., 2011), although this link has yet to be shown in Drosophila models.

Molecular Chaperones

In the absence of ligand, the AR resides in the cytoplasm where it forms a multi-protein

heterocomplex, bound to heat shock proteins, co-chaperones, and tetratricopeptide repeat

(TPR)-containing proteins (Pratt and Toft, 1997). The HSPs are molecular chaperones that

bind to the receptor, and either stabilize the AR in a high-affinity state primed for ligand

binding (Fang et al., 1996), or target the AR for degradation by the proteasome. The

upregulation of specific proteins in response to heat shock was first described in Drosophila

salivary cells (Tissieres et al., 1974), and soon after was recognized as a response across

species from bacteria to human (Schlesinger, 1990).

HSP90

HSP90 is an abundant molecular chaperone that controls the maturation, function, and

turnover of its client proteins. The regulation of AR proteostasis by HSP90 makes it an

HSP90 “client” protein, and puts the AR in a class with many other steroid hormone receptors

and signalling molecules. Binding of HSP90 to its client protein is preceded by the binding of

HSP70 to hydrophobic residues found on partially unfolded proteins. Once HSP70 has

primed the steroid binding cleft, HSP90 binds and stabilizes the binding cleft in an open

conformation with high affinity for ligand (Pratt and Toft, 2003). Both HSP70 and HSP90

have intrinsic ATPase activity that is required in the stepwise assembly of the chaperone

machinery. The binding affinity of these molecular chaperones to their protein substrates is

mediated by their nucleotide binding state, with the ATP-bound form being the low-affinity

state and the ADP-bound form being the high-affinity state (Brehmer et al., 2001). Once the

ligand binds to the accessible ligand-binding cleft of the primed AR, interaction with HSP90


is required to help traffic the client protein to the site of its action, the nucleus (Pratt et al.,

2004; Thomas et al., 2004; Thomas et al., 2006).

HSP90 inhibitors, which target the ATPase activity of HSP90, have been shown to have

beneficial effects in several models of SBMA, but are yet to be tested in Drosophila. 17-AAG

and geldanamycin, both HSP90 inhibitors, ameliorate disease pathology in cell culture and

mouse models of SBMA (Tokui et al., 2008; Waza et al., 2005). These drugs promote

degradation of the mutant AR protein through the ubiquitin-proteasome system by inhibiting

HSP90’s ATP-dependent progression towards the stabilized heterocomplex. They decrease

accumulation of aggregated mutant receptor by enhancing its degradation, and ameliorate the

motor phenotype of SBMA mice (Tokui et al., 2008; Waza et al., 2006). These drugs also

serve to induce a stress response, which upregulates the levels of several heat shock proteins,

including the inducible form of HSP70 (Sittler et al., 2001). However, the beneficial effects of

HSP90 inhibition is independent of the heat shock response, as mouse embryonic fibroblasts

(MEFs) deficient in HSF-1 (the HSP90-regulated transcription factor required to induce a

heat shock response) still clear AR113Q after treatment with HSP90 inhibitors, even in the

absence of a stress response (Thomas et al., 2006).

HSP70

While HSP90 binds to and stabilizes client proteins in their native conformation, HSP70

binds to hydrophobic residues exposed by partially unfolded or misfolded proteins in non-

native conformations, targeting them for degradation by the ubiquitin proteasome system. In

fact, HSP70 is required for both ubiquitination and the subsequent degradation of

ubiquitinated proteins (Bercovich et al., 1997). The best-studied chaperone-dependent

ubiquitin E3 ligase is the CHIP (carboxy terminus of HSP70-interacting protein). CHIP

interacts with HSP70 through its amino-terminal TPR domain and with E2 ubiquitin

conjugating enzymes through a carboxy-terminal U-box domain (Ballinger et al., 1999). In

this manner, it is thought that CHIP is able to initiate the ubiquitination and promote the

degradation of proteins that have been identified as unfolded or misfolded by HSP70. CHIP is

also associated with nuclear inclusions characteristic of polyglutamine disease, including

SBMA (Morishima et al., 2008), and overexpression of CHIP has been shown to rescue both

a Drosophila model of SCA1 (Al-Ramahi et al., 2006) and a mouse model of SBMA (Adachi

et al., 2007). Notably, although CHIP plays an important role in client protein degradation,

other chaperone-dependent ligases, such as Parkin, can function redundantly with CHIP to

promote client protein degradation (Morishima et al., 2008).

Like HSP90 inhibition, HSP70 over-expression has been shown to have beneficial effects

in a variety of neurodegenerative diseases, including the polyglutamine diseases. HSP70, in

conjunction with its co-chaperone HSP40, inhibits aggregation of the truncated huntingtin

protein in both in vitro and cellular and yeast models of polyglutamine aggregation

(Muchowski et al., 2000). HSP70 alters solubility of the abnormal protein, and suppresses

polyglutamine-induced neurodegeneration when overexpressed in SCA3 and SBMA flies

(Chan et al., 2002; Chan et al., 2000; Warrick et al., 1999). Further, when HSP70 is

overexpressed in a mouse model of SBMA, there is decreased aggregated and soluble AR,

indicating that increased levels of HSP70 promote degradation of the AR. Importantly, these

mice also showed improved survival and motor phenotype when compared to SBMA mice

that were not overexpressing HSP70 (Adachi et al., 2003).


HSP70 interacts with several co-factors or co-chaperones in the course of recognizing,

binding and targeting misfolded proteins to the proteasome. These co-factors serve to

modulate HSP70 activity and substrate binding affinity. In particular, HSP40, a well-

characterized family of HSP70 co-chaperones, affect substrate binding by enhancing HSP70’s

ATPase activity (Freeman et al., 1995). In the opposite direction, Hip (Hsc70-interacting

protein) is a co-chaperone that interacts with the ATPase domain of HSP70/Hsc70 and

stabilizes it in its ADP bound state (Hohfeld et al., 1995), and thus increasing the affinity of

the chaperone for client proteins. Experimental modulation of HSP70 activity has proven

difficult, and small molecules specifically targeting HSP70 have been identified only recently

(Jinwal et al., 2009; Leu et al., 2009; Wang et al., 2010). Due to this, much of the evidence

probing HSP70 as a modulator of neurodegeneration has come from overexpression studies.

More recently, modulation of HSP70 has been shown to be a promising therapeutic target,

where inhibiting HSP70’s ATPase activity genetically, through overexpression of Hip, or

pharmacologically, through small molecule inhibition, enhances client protein ubiquitination,

stimulates clearance of the polyQ AR, and rescues toxicity in a Drosophila model of SBMA


CONCLUSION

Drosophila as a model organism has been invaluable in increasing our understanding of

polyglutamine disease pathogenesis and especially SBMA. These small organisms have given

us a huge insight into the molecular mechanisms of disease pathogenesis and modifiers of

these mechanisms. Their simplified genetic similarity, ease of screening, and powerful

numbers have allowed a much greater understanding of disease mechanisms than would be

possible or ethically viable in mammalian models or humans. However, despite huge

advances in our knowledge of disease aetiology, efficacious therapies remain elusive.

Evidence from numerous models indicates that harnessing cells' protein quality control

pathways to degrade toxic proteins is an attractive therapeutic target. Disease progression

affects numerous cellular pathways, and increasing degradation of the disease-causing protein

may serve to halt disease progression upstream of several pathological features of protein

aggregation diseases. Fly models have proven to be useful tools to probe the effects of

potentially therapeutic compounds, and are ideal for larger scale screens of compounds, easily

providing information about toxicity and beneficial effects. Their usefulness in identifying

potentially therapeutic compounds is illustrated by work done looking at HDAC inhibitors

and small molecule modulators of HSP70 (Steffan et al., 2001; Wang et al., 2012). In both of

these papers, authors were able to identify therapeutic targets using Drosophila models to

verify these targets both genetically and pharmacologically. The potential of the fly to inform

new therapies in neurodegeneration has not been fully explored, but I am convinced they will

prove even more fruitful in the future.


ACKNOWLEDGMENTS

This chapter has benefited from the comments and edits from several people, including

Dr. Catherine Collins, Dr. Susan Klinedinst and especially Ian Whiteford who gave assistance

in both editing and figure design. I am grateful for your time and help. I am also very grateful

to Joshua Tuininga for his excellent design skills, and to Alissa Tuininga for facilitating this

work. Finally, I would like to thank Dr. Andrew Lieberman for his years of mentorship and

for allowing my investigation to lead me to fly models.

ABOUT THE AUTHOR

Adrienne M. Wang is currently a postdoctoral fellow in Dr. Matt Kaeberlein’s lab at the

University of Washington in Seattle. She received her PhD in Neuroscience from the

University of Michigan, where she worked with Dr. Andrew Lieberman studying protein

quality control in cellular, mouse, and fly models of SBMA. Her current research utilizes

Drosophila to understand the role of mitochondrial quality control in aging and age-related

diseases.

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Neurosci. 23:217-247.



Chapter 7

SPINAL MUSCULAR ATROPHY:

INSIGHTS FROM THE FRUIT FLY

Stuart J. Grice,1 Kavita Praveen,

2,3

A. Gregory Matera2,3,4

and Ji-Long Liu1,

1MRC Functional Genomics Unit, Department of Physiology,

Anatomy and Genetics, University of Oxford, Oxford, UK 2Program in Molecular Biology and Biotechnology,

University of North Carolina, Chapel Hill, NC, US 3Department of Biology, University of North Carolina,

Chapel Hill, NC, US 4Department of Genetics, University of North

Carolina, Chapel Hill, NC, US

ABSTRACT

Spinal muscular atrophy (SMA) is the most common genetic cause of childhood

mortality. SMA is caused by deletion or mutations in the survival of motor neuron 1

(SMN1) gene, resulting in inadequate levels of the SMN protein. Conserved from yeast to

human, the SMN protein is best known for its critical role in small nuclear

ribonucleoprotein biogenesis and RNA splicing. However, one of the puzzles in the SMA

field is how the reduction of SMN, a housekeeping gene, causes SMA, a motor neuron

specific disease. The fruit fly Drosophila melanogaster has proven to be a powerful

model for human biology and disease. Here we discuss recent progress in SMA disease

modelling in the fruit fly, which has provided unprecedented insights into the

pathological mechanism of SMA.

Keywords: Drosophila; Survival motor neuron (SMN); Uridine-rich small nuclear

ribonucleoprotein (U-snRNP)

Corresponding authors: A. Gregory Matera (E-mail: [email protected]. (AGM)) and Ji-Long Liu (E-mail: jilong.liu

@dpag.ox.ac.uk. (JLL)).

Stuart J. Grice, Kavita Praveen, A. Gregory Matera et al. 172

INTRODUCTION

Spinal muscular atrophy (SMA) is the most prevalent genetic cause of infant mortality.

SMA is characterized by the degeneration of motor neurons in the anterior horn of the lower

spinal cord, leading to symmetrical paralysis, atrophy of the proximal muscles, and loss of

motor function. In most cases, SMA is caused by an insufficient amount of the survival of

motor neuron (SMN), a protein best known for its function in the assembly of Uridine-rich

small ribonucleoproteins (U-snRNPs).

SMA AND SMN IN HUMANS

With a carrier frequency of 1 in 50 and an incidence rate of 1 in 6000-10,000, SMA is the

most common autosomal recessive disorder in the population after Cystic Fibrosis (Ogino and

Wilson, 2002). SMA can be classified into three types based on the severity of the phenotype

(Ogino and Wilson, 2002). Type I SMA, also known as Werdnig-Hoffman disease (Nicole et

al., 2002; Ogino S, 2004), is the most common (60%) and the most severe form where

symptoms can be apparent as early as in utero. Affected infants experience progressive

muscle weakness and hypotonia (reduced muscle tone), and die from complications such as

respiratory failure by 2 years of age. Type II SMA patients become symptomatic between 6

and 18 months of age, show developmental motor delays, and although able to sit

unsupported, they are unable to stand or walk. Their life expectancy is between 2-30 years.

Type III SMA is the mildest form with an age of onset after 2 years. Most type III patients are

able to stand and walk, but can become wheelchair bound in adulthood, and have a normal

life expectancy. In 1995, Lefebvre et al. identified a novel gene on the long arm of

chromosome 5, survival of motor neuron 1 (SMN1), as the causative gene in SMA (Bussaglia

et al., 1995; Rodrigues et al., 1995; van der Steege et al., 1995; Chang et al., 1997).

Over 95% of SMA patients have homozygous deletions involving exon 7 of SMN1

(Lefebvre et al., 1995; Campbell et al., 1997). Further characterization of the 5q region

revealed the existence of at least two copies of the SMN gene in most people. The telomere-

proximal copy (SMN1) and the centromere-proximal copy (SMN2) arose as a result of a 500-

kb inverted duplication.

SMN1 generates fully functional full-length SMN protein, whereas SMN2 predominantly

produces a truncated and unstable protein. This is due to a translationally silent C-to-T

transition, which causes regular skipping of exon 7. Mutations in, or loss of, SMN1 lead to

SMA, while loss of SMN2 alone has no adverse effect. SMN2 resides in a labile region of the

chromosome and is often found in duplicate. Since SMN2 produces low levels of SMN,

SMN2 copy number and disease severity are inversely correlated. SMA is a disease caused by

low SMN levels and not loss, which leads to embryonic lethality.

THE DROSOPHILA SMN COMPLEX AND RNP BIOGENESIS

Studies over the last decade have shown that the majority of SMN in mammalian cells

exists as part of a large multimeric complex consisting of eight additional proteins: Gemins 2-

Spinal Muscular Atrophy: Insights from the Fruit Fly 173

8 and Unrip (a cytoplasm-specific complex member) (Baccon et al., 2002; Carissimi et al.,

2006; Charroux et al., 1999; Charroux et al., 2000; Grimmler et al., 2005; Gubitz et al., 2002;

Pellizzoni et al., 2002a). Orthologues for Gemin2 and Gemin3 have been identified in the

Drosophila genome, and shown to function in U-snRNP biogenesis, similar to their

mammalian counterparts (Cauchi, 2011; Cauchi, 2012; Cauchi et al., 2008; Cauchi et al.,

2010; Cauchi et al., 2011; Kroiss et al., 2008; Shpargel et al., 2009).

Although a putative Drosophila orthologue of Gemin5, Rigor Mortis, can be identified

bioinformatically and loss-of-function mutants phenocopy Gemin3 mutations (Gates et al.,

2004; Shpargel et al., 2009), a physical interaction between Gemin5 and SMN has not been

detected in flies (Kroiss et al., 2008).

The best-characterized function of the SMN complex is in the biogenesis of the core

components of the spliceosome, the Uridine-rich small nuclear ribonucleoproteins (U-

snRNPs). The life cycle of the Sm-class U-snRNAs, U1, U2, U4, U11, U12 and U5, involves

both nuclear and cytoplasmic maturation steps. Sm-class U-snRNAs are transcribed by RNA

polymerase II and acquire a 7-methylguanosine (m7G) cap (Cougot et al., 2004).

Following transcription and 3' end processing, the pre-snRNA is bound by the cap-

binding complex (CBC) (Izaurralde et al., 1995). The pre-snRNA then transits through

nuclear structures called Cajal Bodies (CBs), where it is recognized by the phosphorylated

adaptor for RNA export (PHAX) (Frey et al., 1999; Frey and Matera, 1995; Frey and Matera,

2001; Suzuki et al., 2010). PHAX recruits CRM1/RanGTP to the pre-snRNA, and this

complex is exported to the cytoplasm. The pre-snRNA is released upon phosphorylation of

PHAX in the cytoplasm (Ohno et al., 2000).

The SMN complex then facilitates the binding of seven proteins, SmB/B’, SmD1, SmD2,

SmD3, SmE, SmF and SmG (the Sm proteins), onto a conserved motif called the ‘Sm site’ on

the pre-snRNA (Golembe et al., 2005; Meister et al., 2001; Yong et al., 2004; Yong et al.,

2002) in an adenosine triphosphate (ATP) dependent reaction. The SMN complex provides

specificity and speed to this reaction, which can also occur spontaneously and non-

specifically in vitro (Pellizzoni et al., 2002b).

After assembly with Sm proteins, the pre-snRNA is trimmed at the 3’ end (Kleinschmidt

and Pederson, 1987; Seipelt et al., 1999; Will and Luhrmann, 2001) and the m7G cap is

hypermethylated to a trimethylguanosine (m3G) cap by the enzyme TGS1 (Huang and

Pederson, 1999; Mattaj, 1986; Mouaikel et al., 2002). The import adaptor, Snurportin (SPN),

and Importin (Imp) take the partially assembled pre-snRNA, and the SMN complex, into

the nucleus (Huber et al., 1998; Narayanan et al., 2004; Palacios et al., 1997).

Interaction between SMN and Imp, and observations that U-snRNP import is defective

in the presence of some SMN mutations, indicate that SMN may also function in facilitating

snRNA import (Narayanan et al., 2004; Narayanan et al., 2002).

In the nucleus, the snRNA localizes to CBs, where it is released from the SMN complex,

modified, and bound by other U-snRNP-specific proteins. The mature U-snRNP then

localizes to either regions of active transcription and splicing (perichromatin fibrils) (Fakan,

1994) or to nuclear domains called speckles, where U-snRNPs are thought to be ‘stored’

while not participating in splicing (Sleeman and Lamond, 1999).


DROSOPHILA SMN MUTANTS AS A MODEL FOR SMA

Integral aspects of cell and developmental biology in humans have been conserved in

Drosophila. Approximately 75% of disease-causing loci in humans have homologues in the

fly (Reiter et al., 2001). This conservation allows us to model and study human disorders in

an organism that is likely to respond with similar pathology to disease-causing mutations.

Furthermore, the availability of a sequenced genome, a multitude of genetic tools and a short

generation time make the fruit fly a particularly attractive model organism. Neuromuscular

development in adult flies resembles that of vertebrates (Fernandes and Keshishian, 1999),

thus making Drosophila suited for study of disorders such as SMA.

The most well-conserved regions between human and fly SMN are the Gemin2 binding

site near the N-terminus, the Tudor domain and the YG box (Miguel-Aliaga et al., 2000). The

Drosophila SMN complex participates in the assembly of Sm proteins onto snRNAs,

indicating that the function of human SMN in U-snRNP biogenesis is conserved in the fly

(Rajendra et al., 2007).

In 2003, Chan et al. identified two point mutations in the YG (self-oligomerization) box

of Drosophila SMN (dSMN) through a small-scale ethyl methane sulphonate (EMS)

mutagenesis screen (SmnA and Smn

B). Smn

A animals survived only until the late larval stages

and showed increasing loss of mobility and coordination. Using genetic mosaic techniques to

create homozygous SMN mutants specifically in the germ line of female flies, the authors

showed that survival of zygotic SMN mutants beyond embryogenesis was due to a large

maternal contribution of dSMN (Chan et al., 2003). The SmnA and Smn

B point mutations are

thought to destabilize the protein as flies carrying these mutations have very low levels of

dSMN (Shpargel et al., 2009).

Several additional Smn mutants are available that have been generated via transposon

mediated mutagenesis (Rajendra et al., 2007; Shpargel et al., 2009). These vary in the severity

of their phenotype based on the location of the insertion (Table 1). Specifically, the SmnD and

SmnC mutations are null mutations, as defined by the inability to detect any zygotic dSMN

protein in these animals (Shpargel et al., 2009). Chang et al. (2008) created a new Smn null

mutation by generating a microdeletion derived from a parental line carrying a transposon

insertion upstream of the Smn gene, SmnE. The Smn

X7 deletion removes the promoter, open

reading frame and part of the 3` UTR of Smn (Chang et al., 2008) and homozygous mutants

are lethal at larval stages. A detailed characterization of SmnX7

/SmnD trans-heterozygous

larvae shows that the lethal phase is broad, with majority of larvae dying between 4-5 days

post egg laying. A small fraction of the remaining larvae are able to survive for 2-3 weeks

without undergoing metamorphosis (Praveen et al., 2012). Smn null larvae also display

locomotion defects that have been characterized using a number of different assays (Chan et

al., 2003; Shpargel et al., 2009; Praveen et al., 2012; Imlach et al., 2012).

An SMN hypomorphic mutant was isolated by the imprecise excision of the SmnE

insertion in a screen for adult flies with neuromuscular phenotypes (Rajendra et al., 2007).

These mutants, referred to as SmnE33

(excision 33), are viable and fertile, but unable to fly or

jump. Severe atrophy of the flight muscles was observed in these animals, indicated by a

complete disorganization of the flight muscles.

Table 1. Drosophila SMN mutants


This was accompanied by routing and branching defects in the dorsal longitudinal motor

neurons of the flight muscles (Rajendra et al., 2007). Interestingly, SmnE33

flies show a

decrease in the levels of dSMN only in thorax, and can be considered a regio-specific

hypomorph.

Several RNAi fly strains targeting Smn have also been developed. Chang et al. (2008),

describe three transgenic lines carrying UAS-based RNAi constructs targeting the N-terminus

(SmnN4

), the C-terminus, (SmnC24

), and the full-length (SmnFL26B

) dSMN. The most severe of

these, SmnN4

, is lethal at early pupal stages when expressed using the actinGAL4 driver

(Chang et al., 2008). The SmnC24

and SmnFL26B

transgenic flies have milder phenotypes with a

proportion of SmnFL26B

animals reaching adulthood. Two additional RNAi fly lines,

SmnGL00581

and SmnJF02057

, targeting Smn have been generated by the Transgenic RNAi

Project (TRiP). The TRiP lines express a short hairpin (SmnGL00581

) and long hairpin

(SmnJF02057

) RNAi constructs targeting the 3' UTR and open reading frame of Smn,

respectively. Preliminary characterization shows that flies expressing SmnJF02057

construct

using a ubiquitous driver die as pupae (our unpublished observations).

Praveen et al. (2012), recently described a new Drosophila model of SMA that mimics a

point mutation identified in SMA patients. The authors created the equivalent of the human

SMA mutation, T274I, in the fly Smn gene (SmnT205I

), and expressed this transgene from its

native Smn promoter in an Smn null background. The majority of SmnT205I

animals die as

pupae, which is consistent with this mutation being associated with milder forms of SMA in

humans. The variety of Drosophila SMA models that exist and that can be made with relative

ease make this model organism very valuable to the study of SMA at an organismal level.

SMA in humans is the result of a decrease in SMN levels below a tolerable threshold.

However, all SMA patients have enough SMN protein produced from the SMN2 locus to

rescue embryonic lethality. Similarly, the considerable amount of maternally contributed

dSMN in flies rescues the embryonic lethality, extending their lives to larval stages and

allowing us to investigate phenotypes relevant to human SMA. In addition, with the

versatility of Drosophila genetic tools it is possible to transgenically express a low level of

dSMN in SMN mutant animals to mimic the human SMA situation.

USING DROSOPHILA TO UNDERSTAND

THE ROLE OF SMN IN DEVELOPMENT

A number of developmental defects have been described in patients with severe SMA

including congenital heart defects, multiple contractures, bone fractures, respiratory

insufficiency and sensory neuropathy (Garcia-Cabezas et al., 2004; Kelly et al., 1999; Menke

et al., 2008; Rudnik-Schoneborn et al., 2008; Vaidla et al., 2007).

As one of the most genetically tractable organisms, Drosophila has contributed a great

deal to the molecular and cellular understanding of development. Drosophila undergoes four

distinct developmental stages over the course of two weeks. The first, embryogenesis,

involves the organogenesis and neurogenesis required for larval life. SMN is maternally-

contributed and is highly expressed in the early embryo (Miguel-Aliaga et al., 2000). Female

fruit flies have a pair of ovaries. In Drosophila melanogaster, each ovary comprises 16-20

finger-like ovarioles, which are strings of oval-shaped egg chambers.


There are three major cell types in each egg chamber: 15 nurse cells, 1 oocyte and about

1000 follicle cells. The maternal SMN contribution is supplied by nurse cells in the egg

chamber, which generate large amounts of proteins and RNAs. SMN can be found in CBs in

the nucleus of nurse cells and oocytes (Liu et al., 2006a; Liu et al., 2006b; Liu et al., 2009).

However, SMN predominantly localizes to the cytoplasm and is highly enriched in the U-

snRNP body (U-body) along with other members of the SMN complex (Liu and Gall, 2007;

Cauchi et al., 2010). U-bodies are subcellular structures that contain components of the

spliceosome such as Uridine-rich snRNAs and associated proteins. U bodies invariably

contact cytoplasmic processing bodies (P-bodies), which contain proteins involved in mRNA

processing, RNA silencing and transport (Liu and Gall, 2007).

Knocking out SMN specifically in the germline alters the organization of the P-body,

while, reciprocally, the mutation of P-body components changes the aggregation of SMN in

U-bodies (Buckingham and Liu, 2011; Lee et al., 2009). Furthermore, the nurse cell

chromosomes fail to disperse in Smn and gemin3 mutants – a phenotype also observed in the

mutants of P-body components (Buckingham and Liu, 2011; Cauchi, 2012; Lee et al., 2009).

Chromosome dispersal occurs prior to the generation of maternal proteins and RNAs that will

be loaded into the oocyte ready for embryogenesis (St Johnston et al., 1991). Embryos

derived from egg chambers with SMN selectively removed die very early (Lee et al., 2009),

showing that, as seen with other model organisms, SMN protein is essential at the very early

stages of development (Monani et al., 2000).

Wild-type embryos hatch into larvae, which go through three progressive moults.

Drosophila larvae feed continuously, driving the growth of imaginal tissues as well as a

second wave of neurogenesis required for adult life. In holometabolous insects, imaginal discs

are present in the larvae and are the tissue precursors that form the majority of the adult body

structures. There are 10 sets of imaginal discs, each set producing a body structure (e.g. eyes,

legs and wings). The voracious feeding behaviour of larvae coincides with, and is supported

by, a 10-fold increase of the larval neuromuscular system. The majority of Smn mutants are

larval lethal, display poor growth and have reduced motor function. Morphological defects at

the larval neuromuscular junctions (NMJs) have been reported (Chan et al., 2003; Chang et

al., 2008), however, this phenotype is controversial. Chan et al. (2003) observed enlarged

boutons (the synapse between neuron and muscle) with no change in bouton number in Smn

mutants, while Chang et al. (2008) reported a decrease in the number of boutons with no

change in size. Most recently, Imlach et al. (2012) reported that they did not find any

morphological defects in the NMJs of Smn mutant larvae. They did show an increase in the

amplitude of evoked excitatory postsynaptic potential (eEPSP) at the Smn mutant NMJs

compared to wild-type NMJs. However, the relevance of this phenotype to motor function or

viability of these mutants is unclear.

Although ubiquitously expressed in the larval central nervous system (CNS), SMN

protein is enriched in the postembryonic neuroblasts (pNbs). pNbs generate the neurons and

glia required for the adult during 2nd

and 3rd

larval stages as well as early pupation. With stem

cells having the highest levels of SMN, the protein then forms a concentration gradient in the

CNS that is inversely proportional to the state of differentiation. In Smn mutants, the CNS

displays growth and maturation defects (Grice and Liu, 2011).

SMN deficiency reduces stem cell division and can lead to stem cell loss (Grice and Liu,

2011). The daughter cells of these dividing stem cells have reduced levels of the U-snRNPs

U2 and U5 that are required for splicing.


In addition, the tight localization of Miranda protein during metaphase at the basal

membrane of the neuroblast is lost (Grice and Liu, 2011). Miranda forms complexes with

RNA-binding proteins, including Staufen, which binds to mRNAs that will become

partitioned into the daughter cells (Broadus et al., 1998; Li et al., 1997). The Miranda-Staufen

complex has been implicated in facilitating the transport of RNP complexes in the neuroblast,

axon and germline (Roegiers and Jan, 2000).

In addition to the nervous system, defects in germline stem cell development have been

observed in Smn mutants. Like in the CNS, germline stem cells are enriched with SMN, and

these levels again form a differentiation/concentration gradient. SMN loss leads to the

retardation of germline stem cell division and promotes premature stem cell loss.

Overexpression of SMN alters the timing of germ cell development in the male germline

(Grice and Liu, 2011).

Furthermore, ectopic expression of SMN in the testis, in the region across the wild-type

SMN gradient, changes the timing of cell differentiation and leads to a build-up of immature

cyst cells (Grice and Liu, 2011). Together, these results suggest that SMN levels need to be

fine-tuned during development, and that alterations in SMN levels can lead to abnormal

development and modify the capability of stem cells to generate new cells during

development and throughout adult life.

Once a critical size is reached, ecdysone expression stimulates pupa formation and the

start of metamorphosis. During pupariation, the adult body plan is generated and the mature,

adult nervous system is formed from immature neurons generated during the larval wave of

neurogenesis.

Both SMN reduction and overexpression have an effect on pupation and metamorphosis.

Ectopic overexpression of human SMN, which is dominant negative in Drosophila, leads to

pupal lethality with malformation of the head, legs and wings (Miguel-Aliaga et al., 2000).

Overexpression of Drosophila wild-type SMN shortens the time to pupation, but at 25oC,

does not have an overall negative effect on survival.

Further growth and pupation defects have been observed in the less severe Smn mutants

(including SmnB), which have an extended larval period but fail to reach the wild-type size,

and form pseudopupae (Shpargel et al., 2009).

THE QUESTION OF CELL-AUTONOMY IN SMA

Whether SMA results from disruption of a cell-autonomous or non-autonomous function

of SMN remains a highly contested area of research.

To this end, researchers have attempted to identify tissues most important in the

pathology of SMA. The approach adopted is to analyse the level of rescue of SMA-like

phenotypes in mice and Drosophila by selective restoration of SMN levels in specific tissues

or cell types.

Studies in the mouse have used the Cre-LoxP system to selectively ablate or restore SMN

through the use of muscle and neuron specific promoters driving Cre recombinase expression

(Cifuentes-Diaz et al., 2001; Vitte et al., 2004; Gavrilina et al., 2008; Park et al., 2010;

Martinez et al., 2012).


Figure 1. Overview of Drosophila research into SMN function and SMA.

Initial reports of neuronal (vs. muscular) restoration of SMN having greater effect in

rescuing SMA-like phenotypes in mice are difficult to interpret due to the leaky expression of

Cre in muscle cells (Gavrilina et al., 2008).

In Drosophila, tissue-specific expression of SMN was achieved using the Gal4-UAS

system, where expression of Gal4 in neuronal or muscular tissues can restrict expression of

SMN to those tissues. There are hundreds of Drosophila Gal4 driver strains available making

it easier to find those that express in a highly tissue-specific manner. Using this system, two

groups showed that complete rescue of the lethality resulting from loss of SMN, required

expression of SMN in both neurons and the mesoderm. In addition, expression in the

mesoderm alone resulted in a higher level of rescue than neurons alone (Chan et al., 2003;

Chang et al., 2008). The question of a cell-autonomous role for SMN has recently been

addressed at greater resolution using the Drosophila system. Taking advantage of the large

number of well-characterized Gal4 drivers available, Imlach et al. (2012) restored SMN

expression in Smn null flies in various tissues, including several different neuronal subtypes.

They analysed the impact of SMN restoration on electrophysiological phenotypes at the

neuromuscular junction as well as on muscle size and larval locomotion.


Surprisingly, they found that restoration of SMN in motor neurons alone did not rescue

any of these phenotypes. However, expressing SMN in cholinergic neurons (including

sensory and interneurons, but not motor neurons) did rescue larval phenotypes. This finding

suggests that SMN can function in a non-autonomous manner to influence other cell types. It

is important to note that rescue in neuronal or muscles cells alone did not rescue the viability

of the null mutants, consistent with previous data from studies in Drosophila. In contrast to

mice, Drosophila have proved especially useful for such analyses due to their relatively short

generation time and affordability, allowing an extensive analysis of several different patterns

of SMN expression.

CONCLUSION AND FUTURE

Drosophila models offer many advantages for understanding the biology of SMA. Many

parallels between the Drosophila SMA models and vertebrate systems have been identified

including defects in neurotransmission and synaptic architecture, as well as the conservation

of SMN binding partners and basic function.

In addition, Drosophila has provided novel insights into SMA including SMN’s function

in muscle structure, stem cell biology and neuronal circuit formation. The Drosophila genetic

tool kit is becoming ever more advanced, giving scope for the continued analysis of the

processes that drive neuronal developmental and homeostasis. In turn, this versatility allows

for an increasingly refined interrogation of the fundamental cellular defects that cause motor

neuron dysfunction, and can help elucidate the many functions of SMN in the cell.

ABOUT THE AUTHORS

Stuart J. Grice is a postdoctoral researcher at the MRC Functional Genomics Unit,

University of Oxford. He works with Dr. Ji-Long Liu on Drosophila models of SMA,

Charcot-Marie-Tooth-neuropathy and autism. He is particularly interested in how defects in

neurogenesis can lead to neurodevelopmental disease.

Kavita Praveen is a postdoctoral fellow in the Program for Molecular Biology and

Biotechnology at the University of North Carolina, Chapel Hill, US. She works with Dr.

Gregory Matera on developing and characterizing novel models of SMA in Drosophila. Her

particular interest is in understanding the aetiology of SMA at the molecular level.

Gregory Matera is a Professor in the departments of Biology and Genetics at the

University of North Carolina, Chapel Hill, US. He is interested in studying the biogenesis and

functions of non-coding RNAs, including small nuclear RNAs, using the Drosophila

melanogaster model system.

Ji-Long Liu is a programme leader at the MRC Functional Genomics Unit, University of

Oxford. He is interested in intracellular compartmentation in Drosophila. Currently his group

studies three aspects related to RNA: cytoophidia, long noncoding RNAs, and SMA

modelling.

These authors contributed equally to this work: Stuart J. Grice, Kavita Praveen.


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Chapter 8

GENETIC SCREENS IN DROSOPHILA AND THEIR

APPLICATION IN MOTOR NEURON DISEASE MODELS

Liya E. Jose,1,2

Patrik Verstreken1,*2

and Sven Vilain1,2,†

1VIB Center for the Biology of Disease, Leuven, Belgium 2KU Leuven, Center for Human Genetics and Leuven Research Institute for

Neurodegenerative Diseases (LIND), Leuven, Belgium

ABSTRACT

The dearth of powerful therapeutic treatments for patients diagnosed with motor

neuron diseases (MNDs) emphasizes the significance of research that focuses on

understanding the causes of these diseases. Identification of the genes responsible for

these diseases enables the study of their function in model organisms. Drosophila

melanogaster is renowned for its powerful genetic tools for studying neurological

diseases.

Most human disease genes have at least one functional orthologue in flies and

transgenic expression of human disease genes can recapitulate features of the disease.

The power of Drosophila for unbiased genetic screens to identify novel genes that cause

neurodegeneration or, to identify modifiers of disease genes, holds promise to uncover

the molecular mechanisms and the (parallel) pathways involved in disease progression

and neurodegeneration.

Throughout Drosophila’s rich genetic screen history, different screen approaches

have been used and different phenotypes have been screened for. The aim of this chapter

is to introduce the various screening approaches and phenotypes that have been used in

the past for high-throughput screening of neuronal defects in MND and we will present

additional available genetic tools that have the potential for uncovering further molecular

mechanisms of MNDs.

Keywords: Enhancer, suppressor, RNAi, mutagenesis, phenotypes

* Patrik Verstreken: [email protected], Herestraat 49 - bus 602, 3000 Leuven, Belgium, Tel:

+32 (0) 16 330018, Fax: +32 (0) 16 330589. † Sven Vilain: [email protected], Herestraat 49 - bus 602, 3000 Leuven, Belgium, Tel: +32 (0) 16

330018, Fax: +32 (0) 16 330589.



Liya E. Jose, Patrik Verstreken and Sven Vilain 186

INTRODUCTION

Drosophila is recognized as a powerful model for analysing the function of human

disease genes (Bonini, 2001; Fernandez-Funez et al., 2000; Ghosh and Feany, 2004; Kazemi-

Esfarjani and Benzer, 2000; Marsh and Thompson, 2004; Outeiro et al., 2007). About 75% of

all known human disease genes have a very well conserved fly orthologue (Bier, 2005; Reiter

et al., 2001).

Furthermore, even when the human gene orthologues are not present in the fly genome,

expression of the human gene or its mutant form can recapitulate characteristics of human

disease, suggesting that the underlying pathways are conserved (Feany and Bender, 2000).

These observations emphasize the ability to understand diverse and complex biological

problems associated with neurological disorders through the use of fruit flies including

pathways involved in motor neuron disease (MND) (Auluck et al., 2002; Canizares et al.,

2000; Chang et al., 2008; Chen et al., 2011; Chihara et al., 2007; Djagaeva et al., 2012; Elia et

al., 1999; Estes et al., 2011; Feany and Bender, 2000; Furutani et al., 2005; Hanson et al.,

2010; Joyce et al., 2011; Leitao-Goncalves et al., 2012; Li et al., 2011; Mockett et al., 2003;

Nedelsky et al., 2010; Praveen et al., 2012; Puccio, 2009; Rajendra et al., 2007; Rubin et al.,

2000; Sang and Jackson, 2005; Sasayama et al., 2012; Storkebaum et al., 2009; Trotta et al.,

2004; Watson et al., 2008).

MNDs are neurological disorders characterized by progressive loss of motor neurons that

result in extensive disability and early death (Lambrechts et al., 2007). Despite numerous

pathological studies in humans, mechanistic insights into the pathology and molecular

mechanisms associated with disease onset are still largely lacking (Carrasquillo et al., 2009;

Lesage and Brice, 2009; Mougeot et al., 2009). Loss-of-function studies in combination with

expression of (human) disease proteins in Drosophila and well-designed genetic screens, can

yield further insight into MND at unprecedented resolution (Chang et al., 2008; Kazemi-

Esfarjani and Benzer, 2000; Suzuki et al., 2009). Fruit flies are thus routinely used to screen

for genes that modify MND-associated phenotypes, helping to understand the mechanisms

underlying these diseases.

Genetic screening holds promise for unravelling the molecular mechanisms of disease

pathology, and for identifying novel therapeutic targets. The short generation time (<2 weeks)

and the ability to maintain large collections of mutant Drosophila in the lab has enabled

researchers to screen for mutants that affect development, neuronal function and behaviour

(Benzer, 1967; Pak, 1995; St Johnston, 2002) and more recently also for modification of

phenotypes induced by disease related genes, including MND. Drosophila can be used to find

enhancers and suppressors of Drosophila models of human MND and to uncover the

pathways and molecular mechanisms involved in the pathology of these disease models

(Chang et al., 2008; Sen et al., 2011; Suzuki et al., 2009). An increasing number of

sophisticated genetic screening strategies are being developed that allow to conduct more

focused genetic screens, including analyses in mosaic animals (Xu and Rubin, 1993), or in

animals that use temporal and spatially controlled gene expression (Brand and Perrimon,

1993). Hence, both classical screen approaches but also more advanced methodologies can be

employed to conduct nonbiased genetic screens to identify in vivo modifiers of MND

pathological mechanisms.

Genetic Screens in Drosophila and Their Application … 187

In this chapter, we give an overview of the genetic tools that have been created to

facilitate genetic screens in Drosophila and discuss some of the phenotypes that are amenable

to such screens. Indeed, the choice of ‘screen-phenotype’ is of paramount importance for two

reasons.

First, the phenotype that is screened for defines the pathway(s) in which mutations will

be isolated; choosing a focused (more ‘molecular’) phenotype therefore usually results in the

isolation of genes in a limited number of processes compared to more broad phenotypes (e.g.

behaviour).

Second, if individual flies are being tested in a screen, the phenotype assessed should be

highly penetrant; this is less of an issue when a population of flies that carries a particular

mutation can be assessed. Taking these ‘limitations’ into account, numerous MND-related

phenotypes that have been described are well-suited for screening such as defects in

locomotion, problems with neurotransmission and neurodegeneration, akin to defects seen in

humans (Chang et al., 2008; Li et al., 2010; Watson et al., 2008). In fact, several of these

phenotypes have been used successfully in the past to screen for genetic modifiers (Benzer,

1967; Harris and Stark, 1977; Pak, 1995).

In the next sections we will describe different screening strategies. With respect to MND,

such genetic screens can be used either to isolate mutations with phenotypes similar to the

MND models that exist, or they can be used to isolate modifiers of the phenotypes exhibited

by MND fly models. In the latter case, the screening approach is performed in the background

of the MND model, screening for enhancement or – ideally – suppression of the MND-

model-induced phenotype.

CLASSIC GENETIC SCREENS

Thanks to the discovery of potent mutagens, high-throughput screens in Drosophila

became feasible. Ethyl Methane Sulphonate or EMS1 is a popular mutagen used in

Drosophila (but also other species) due to its effectiveness and relatively low toxicity to flies

(Ashburner, 2005).

However, the compound is relatively non-specific and can result in the disruption of

almost all genes in the genome. EMS creates point mutations or small deletions in the

genome and mapping the molecular lesions created by EMS can be labour intensive.

Nonetheless, the advent of novel mapping technology (Chen et al., 2008; Parks et al., 2004;

Ryder et al., 2007; Ryder et al., 2004; Thibault et al., 2004; Zhai et al., 2003) in combination

with next generation whole genome sequencing (Wang et al., 2010) greatly facilitate mapping

efforts and bring new life to EMS-based mutagenesis screens. Furthermore, it is possible to

use EMS (or other mutagens) to generate and maintain a large collection of flies carrying

mutations as they can be ‘stably maintained’ with balancer chromosomes without the need to

genotype individual animals every generation. Hence, balancer chromosomes facilitate the

upkeep of vast amounts of mutant flies as ‘lab-stocks’.

1 EMS is an alkylating agent and it affects only one strand of the double helix. Since only one DNA strand is

mutated, the mutated DNA strand might segregate during the first zygotic division from the wild-type strand

before mismatch repair changes the wild-type strand to a mutant strand. Therefore the F1 progeny can be

mosaic for the mutation. This only matters in F1 screens where the mutation could be present in the somatic

tissues but absent in the germline, hence, not passed on to the next generation.


Another strategy used to disrupt genes involves transposable elements (TEs), which are

DNA sequences that are mobile within the genome of single cells, including germ cells, thus

allowing transmission of novel TE insertions through the germline. While disrupting genes

with TEs is not very efficient in comparison to EMS, the insertion of a TE in a gene provides

a molecular tag such that the disrupted gene can be readily identified.

The most commonly used TEs are P-elements. P-elements insert preferentially at specific

loci, called hot spots, while they fail to insert in some other loci (Bellen et al., 2004;

Spradling et al., 1995). Nonetheless, numerous genes can be disrupted by P-element

mutagenesis and in an attempt to target a larger number of genes, other types of TEs have

been used in fruit flies including piggyBacs (Hacker et al., 2003; Handler and Harrell, 1999;

Horn et al., 2003; Thibault et al., 2004) and Minos elements (Bellen et al., 2011; Metaxakis et

al., 2005; Venken et al., 2011). Given that creating novel TE insertions is relatively

inefficient, it is more straightforward to screen existing collections of TE-bearing flies

generated for example by the Berkeley Drosophila Genome Project (Bellen et al., 2004). It

should be noted that TE insertions are often weak alleles that result in reduced protein

expression while phenotype-causing EMS-induced mutations usually cause more severe gene

disruption.

Nonetheless, the collection of fruit flies that harbour a TE with molecular information of

the TE insertion site is rapidly expanding and can be used to screen for mutants affecting the

biological process of interest without much need to map lesions (however, see ‘second site

hits’ below).

To isolate recessive mutations, the most straightforward, yet also most labour intensive

screening methodology is a classical “F3 screen”. This strategy allows one to generate

multiple (several thousand) populations of flies where all flies within one population carry the

same mutant chromosome. Intercrossing animals in the F2 generation within a population

results in the creation of homozygous animals that can be screened in the F3 generation

(Figure 1.a). This strategy holds the advantage that homozygous lethal or sterile animals can

be screened in the F3 generation while maintaining the mutations heterozygous over a

balancer as a stock.

If a phenotype is observed, the stock is maintained. This crossing strategy has been

extensively and successfully used by numerous fly researchers around the globe (Nusslein-

Volhard and Wieschaus, 1980; Salzberg et al., 1994) and is well-suited for unbiased screening

approaches.

One disadvantage is that only early roles of a given gene can usually be assessed and

potential later functions are not easily revealed, for example because of lethality or severe

developmental defects induced by the mutation. These drawbacks can be circumvented by

using a clonal screen approach where only part of the fly is rendered homozygous mutant

while the rest of the tissues remain heterozygous, thus precluding lethality induced by the

mutation (see below).

While classical F3 screens are labour intensive, the isolation of viable and fertile

recessive mutations on the X chromosome is quite straightforward. Here, virgin female flies

that carry a pair of attached X chromosomes and a Y chromosome (Benzer, 1967) is crossed

to mutagenized males (that carry an X and Y chromosome). Given that the Y chromosome in

the female fly and the X chromosome in the mutagenized male fly segregate independently,

the male offspring will be hemizygous for the mutagenized X chromosome and carry the Y

chromosome they received from their mother (Figure 1.b).


Figure 1. Examples of crossing schemes used in EMS mutagenesis screens. a) Typical F3 crossing

scheme: Males are mutagenized by EMS and then crossed to female virgin flies carrying a balancer

chromosome. Single balanced flies putatively carrying a mutation (red asterisk) are crossed back to the

balanced flies to create a mutant stock. In the next generation, mutant males and females can be crossed

and homozygous flies can be retrieved in the F3 generation. b) F1 screen for viable hemizygous male

mutant flies: Males are mutagenized and crossed with female flies that carry a compound X

chromosome and that carry a Y chromosome. Since XXX and YY flies are lethal, in the next generation

all male flies will have the mutant X chromosome (red asterisk) from the father and the Y chromosome

from the mother. c) EMS screen for dominant modifiers: males carrying a mutation that yields a

phenotype that can be easily screened for (black asterisk) is mutated and crossed back to females

carrying the same mutation. In the next generation dominant modifiers of the phenotype can be

investigated.

Hence, these males can be screened, mutants isolated and crossed. This strategy allows

the identification of viable and fertile mutants on the X chromosome in a single generation

(Figure 1.b).

CLONAL GENETIC SCREENS

F3 screening strategies are labour intensive and the early roles of some genes precludes

screening for a potential later function. These issues can be circumvented by performing

clonal screens (Figure 2.a-e). Here, homozygous tissue is generated in an otherwise

heterozygous animal. The homozygous mutant ‘patch’ does not interfere with viability or

fertility, but allows to assess the phenotype of a given mutation, and depending on when in

the lifetime of the organism the tissue is rendered homozygous, ‘later’ functions of a given

gene can also be assessed.

To generate such animals, mitotic recombination between homologous chromosomes is

induced using the Flp/FRT system (Xu and Rubin, 1993). Consequently, at least one cell

division is needed to generate homozygous mutant cells using this methodology, precluding

the generation of homozygous mutant tissue in the post-mitotic phase of a cell. Both

chromosomes, the mutagenized chromosome from the father fly and a specially ‘engineered’

chromosome from the mother fly, harbour FRT sites (Flp Recognition Target) (Duffy et al.,

1998; Golic and Lindquist, 1989).


Figure 2. Clonal screens in Drosophila melanogaster using Flp. When the two FRT sites are on the

same identical chromosomal position, close to the centromere, inducing expression of Flp will cause

efficient recombination between the FRT sites and exchange of the distal part of the chromosome arms.

a) Mutagenized male flies that carry chromosomes with FRT sites are crossed to female flies that

express Flp and also harbour an FRT site with a dominant marker and a recessive cell lethal mutation.

b) When Flp is not present, mitotic recombination does not occur. c) When the Flp is present, mitotic

recombination between the FRT sites results in cells homozygous for the mutant chromosome, as well

as ‘twin spot’ cells for the recessive cell lethal mutation. The latter cells die, thus resulting in large

patches of homozygous mutant tissue. d) When eyFlp is used, almost the entire eye is rendered mutant

using this system (Newsome et al., 2000; Stowers and Schwarz, 1999). e) Similar experiments can be

done in the thorax of the flies, where yellow mutant clones can be generated (removing the bristles in

this drawing).

During normal mitosis one chromatid from each homologous chromosome would

segregate into each daughter cell upon cell division (Figure 2.b); however, the tissue specific

expression of Flipase (Flp) following DNA replication induces recombination between the

FRT sites such that now, identical chromatids segregate into one daughter cell (Figure 2.c).

The ‘erroneous’ segregation of chromosomes results in cells homozygous for the

mutagenized chromosome or cells homozygous for the ‘engineered’ chromosome. During

subsequent cell divisions, a homozygous patch of cells is created. The latter can carry a

visible marker (e.g. GFP or w+) and a recessive cell lethal mutation such that homozygous

cells die. The patch of remaining cells that carry the homozygous mutant chromosome can be


screened for phenotypes. While more complicated in setup, this screening strategy holds the

advantage that homozygous mutant tissue can be screened in the F1 generation (see previous

footnote on EMS, specifically the possibility that EMS creates mosaic animals) and recessive

homozygous lethal as well as viable mutants can be isolated. Furthermore, numerous

transgenic Flp expressing flies have been generated including an eye specific Flp (ey3.5-Flp)

(Iris Salecker and colleagues), an eye/optic lobe expressing Flp (ey-Flp) (Newsome et al.,

2000), a thorax Flp (Ubx-Flp) (Jafar-Nejad et al., 2005), and heat-shock inducible Flp (hs-

FLP) (Duffy et al., 1998), allowing for tissue specific screens (Figure 2.d and 2.e).

The Flp/FRT system has been used very extensively in the Drosophila eye using ey-Flp

(Newsome et al., 2000), where the Flp cDNA was placed under transcriptional control of an

eye-specific promoter from the eyeless (ey) gene. Despite the fact that the ey promoter

fragment used is predominantly expressed in the developing eye, expression was also

detected in the optic lobes (Newsome et al., 2000). If photoreceptor (PR)-specific expression

is desired, the more restricted ey3.5-Flp may be a better choice (Hiesinger et al., 2005).

Screening using the ey-Flp system has allowed to isolate novel genes involved in axon

guidance (Newsome et al., 2000), neurotransmitter release (Babcock et al., 2003; Stowers and

Schwarz, 1999; Verstreken et al., 2003), mitochondrial function (Guo et al., 2005; Stowers et

al., 2002; Verstreken et al., 2005) and neurodegeneration (Bayat et al., 2012; Simpson et al.,

2009; Zhai et al., 2006). While such clonal screens have extensively been performed to isolate

novel players in the aforementioned processes, limited work has been done to exploit the

methodology to isolate novel players in relation to MNDs, yet the technology holds potential

to identify novel key players in MND-relevant pathways. Unfortunately, it may be difficult to

generate homozygous mutant motor neuron clones in fruit flies; however, given the

conservation of many pathways across neuronal subtypes, it may be possible to screen other

types of neurons (e.g. PRs) to discover novel concepts and pathways governing specific

aspects of MND in fruit flies.

DOMINANT GENETIC SCREENS

The methodologies described so far aim at generating homozygous mutants or tissue that

can be used to screen for modification of MND-relevant phenotypes. However, a very

popular and successful strategy involves screens for dominant modification of a phenotype

(Raftery et al., 1995; Simon et al., 1991) including MND-relevant phenotypes. Provided the

heterozygous mutants alone do not modulate the phenotype screened, the isolated mutants are

dominant enhancers or suppressors of the process under investigation (Figure 1.c) (reviewed

in St Johnston, 2002). Enhancers of lethal mutants can also be found through screening for

synthetic lethality. Synthetic lethality is a phenomenon by which hetero-allelic combinations

of two interacting genes leads to organismal lethality while each heterozygous mutant is

viable. Since only one generation is needed to screen for synthetic lethality it is a quick way

to screen for interactors of lethal mutations and has been used in the past to find modifiers in

an MND model (Chang et al., 2008). Enhancer/suppressor screens have proven their merit in

the past in the isolation of pathway members involved in developmental signalling and they

are now also making their way into neurodegenerative disease research (Chang et al., 2008;

Rimkus et al., 2008; Sen et al., 2011; Vos et al., 2012). In this context, dominant suppressors


are particularly interesting as drug targets: only one mutant allele is sufficient for suppression

(or enhancement).

While dominant screens may be performed with collections of TEs or EMS-induced

mutations (Chang et al., 2008; Rimkus et al., 2008; Sen et al., 2011; Vos et al., 2012), it is

also possible to use a collection of overlapping deficiencies for dominant suppression or

enhancement of a phenotype (Weiss et al., 2012). This methodology holds two advantages:

First, one deficiency uncovers tens to hundreds of genes at the same time and in a single

cross, the involvement of all genes is tested at once. Second, modifiers are usually contained

within the interacting deficiency thus saving time on mapping compared to EMS-induced

mutations. To identify the causative modifying gene within the deficiency, the individual

genes within the deficiency can then be knocked-down or mutants in these genes can be

tested for phenotype modification. Despite this and several other successful deficiency

screens, a number of important drawbacks need to be taken into account as well: first, the

genes contained within a deficiency sometimes interact, confounding the observed effect;

and, second, similar to EMS and TE bearing chromosomes, the chromosomes that harbour the

deficiency may also contain additional ‘second site’ mutations, and these may also modulate

the phenotype under investigation, thus complicating the identification of the causative gene.

This particular issue may be circumvented by performing linkage analysis to test if the

modifier (suppression or enhancement of the phenotype) is genetically linked to the

deficiency. Hence, while deficiency screening for dominant modifiers allows for the rapid

identification of modifying loci, pinpointing the individual gene that modifies the phenotype

is not always trivial and it is essential to complement the isolation of modifying genes with

rescue experiments where a wild-type copy of the identified gene, ideally expressed under

endogenous promoter control, is placed back into the mutant background.

UAS/GAL4-BASED SCREENS

It is often desirable to generate mutant tissue in an otherwise wild-type background. As

described above, mitotic recombination is a powerful tool, but tissue specific Flp expression

is needed and at least one (and ideally more than one) cell division is required. Hence the

observed phenotype could be the sum of early phenotypes, occurring early after cell division

and later independent phenotypes occurring at a later stage; earlier phenotypes can even

preclude the detection of later phenotypes. Expression of transgenes via the UAS/Gal4 system

alleviates many of these drawbacks. Gal4, a yeast transcription factor that does not have a

deleterious effect in Drosophila (Fischer et al., 1988), binds UAS (Upstream Activator

Sequence) and activates transcription of downstream responder constructs (genes, RNAi)

(Brand and Perrimon, 1993). When flies bearing a transgene that allows for tissue specific or

temporally controlled Gal4 expression are crossed to flies bearing a transgene with a UAS

responder construct, this construct will be transcribed in a tissue- and time-controlled manner.

The UAS/Gal4 system allows control of expression levels, since the system is inherently

temperature-sensitive with minimal activity at low temperature (16°C) and maximum activity

at high temperature (29°C) (Duffy, 2002). However, it is important to note that function and

morphology of the fly NMJ are temperature dependent and carefully designed control

experiments are needed. Further, numerous collections of Gal4 and UAS-responder lines exist


and countless variations to control the expression of Gal4 have been developed (reviewed in

Duffy, 2002). Hence, the UAS/Gal4 system enables researchers to manipulate gene expression

(overexpression or RNAi-mediated knockdown; see below) in a spatially- and temporally-

controlled manner.

Different libraries of transgenic flies have been generated for overexpression of a large

collection of genes in the genome. Several TEs that have been used in gene disruption

projects harbour UAS sites. If such a TE inserts in the vicinity of a gene, the insertion itself

may disrupt gene expression, but adding Gal4 may induce (overactivate) gene expression

(Figure 3.a). Relevant TE collections are the EP P-elements, the “Exelixis collection” (XP

and WH lines) and the “GS” collection (available at the Bloomington Drosophila Stock

Centre [BDSC] and Harvard Medical School) (Beinert et al., 2004; Bellen et al., 2004; Rorth,

1996; Staudt et al., 2005; Thibault et al., 2004; Toba et al., 1999). Using these TE collections

it becomes possible to perform MND-modifier screens based on overexpression. Such a

screen was recently successfully used to screen for modification of a hypomorphic smn allele.

smn is the Drosophila orthologue of Survival Motor Neuron1 (SMN1), mutations in which

cause spinal muscular atrophy (SMA). Screening the “Exelixis collection” identified several

modifying loci, including members of the BMP and FGF signalling pathways, and further

studies indeed linked defective BMP and FGF signalling to the defects observed in smn

mutant NMJs (Chang et al., 2008; Sen et al., 2011). Several other overexpression screens

have also been performed in relation to MND (Guo et al., 2011; Li et al., 2010) and similarly,

over- or mis-expression screens for genes that modulate axon guidance and synaptogenesis at

the larval neuromusculature have been performed (Kraut et al., 2001). While conceptually

straightforward, it is important to take into account that gene expression may be forced in

ectopic tissues and mis-expression artifacts should be controlled for. Furthermore, TEs that

bear UAS sites are often located in between genes, and it is not always clear which gene or

group of genes are being overexpressed. Nonetheless, mis- or over-expression screens are

fairly simple to set up as well as execute and numerous tools are already available.

The advent of genome-wide RNAi collections to target almost all genes in the

Drosophila genome not only addresses the limitations of limited mutability of particular

genomic regions that are refractory to EMS or TE insertion, but also allows researchers to

perform temporally and spatially controlled knockdown of gene expression (Fortier and

Belote, 2000; Kennerdell and Carthew, 1998; Kennerdell and Carthew, 2000; Lam and

Thummel, 2000; Martinek and Young, 2000; Misquitta and Paterson, 1999). Hairpin RNAi

constructs against different regions of cDNAs in the Drosophila genome exists and are

expressed under control of the UAS/Gal4 system.

Different collections, optimized for RNAi targeting efficiency, have been constructed and

are available from various stock centres (http://www.shigen.nig.ac.jp/fly/nigfly/;

http://stockcenter.vdrc.at/control/main; http://www.flyrnai.org/TRiP-HOME.html) (Dietzl et

al., 2007; Ni et al., 2009). These RNAi constructs can be used to knockdown gene expression

in the entire animal, but they can also be expressed in specific neuronal networks or neuronal

subtypes using specific Gal4 driver lines (Figure 3.a and 3.b, b). Furthermore, RNAi

expression can be employed to knock-down gene expression in multinuclear cells, including

muscles, relevant to MND. While conceptually simple there are important drawbacks to be

considered. First, numerous RNAi constructs are known to harbour off-target effects that are

not straightforward to assess in vivo, thus complicating interpretation of the results (Dietzl et

al., 2007; Ni et al., 2009; Ni et al., 2008; Perrimon et al., 2010).


Figure 3. Using the UAS/Gal4 system to perform tissue specific overexpression experiments or target

the gene function with RNAi. a) Crossing the UAS-RNAi collection or a UAS-GeneX collection with

flies that express Gal4 in a tissue specific manner (green in the drawings) allows one to generate flies

that harbour tissue specific knock-down or overexpression of a gene in a tissue specific manner.

b) Neuronal specific Gal4 lines allow for gain- or loss-of-function experiments in subsets of neurons.

One way to control for off-target effects is to conduct rescue experiments using strategies

that allow for the translation of normal wild-type protein from mRNA that is not recognized

by the RNAi machinery (Kondo et al., 2009; Langer et al., 2010; Schulz et al., 2009). Second,

in most cases, RNAi expression causes only partial gene knockdown that may be insufficient

to produce or affect the phenotype.

One solution can be to co-express the RNAi with Dicer2, improving the effectiveness of

some RNAi lines, but Dicer2 expression also increases the incidence of off-target effects

(Dietzl et al., 2007). Although genome-wide RNAi based screens are labour intensive, the

methodology allows to screen sub-collections, for example RNAi lines targeting kinases,

phosphatases etc. Given the ease to apply RNAi in Drosophila, it has in recent years become

a popular tool that can be additionally used to conduct screens.

SCREENABLE PHENOTYPES

When performing large-scale screens it is important to be able to rely on phenotypes that

are specific enough for the process under investigation, yet robust and sensitive such that they

can be easily and quickly quantified, thus limiting the number of individual animals that need

to be analysed.


A very commonly used neuronal system to conduct genetic interaction screens is the

Drosophila compound eye since it is not essential for viability or fertility. The fly eye is

composed of an array of 800 ommatidia, each containing eight PR neurons seven of which are

visible in tangential sections (Katz and Minke, 2009; Montell, 2012).

Many genes involved in cell-fate determination and differentiation will yield a rough-eye

phenotype when overexpressed or mutated in the fly eye (Brumby et al., 2002; Chanut et al.,

2000; Firth et al., 2000; Mao and Freeman, 2009; Thao et al., 2012). Similarly, many disease

genes when mis-expressed in the eye induce a rough-eye phenotype. However, it is not

always clear how this phenotype arises, whether the defects are caused by abnormal mis-

expression and what the relationship of the eye phenotype to the disease gene is. Nonetheless,

this phenotype has often been used in modifier screens, also in relation to MND-relevant

genes (Suzuki et al., 2009).

Spinobulbar muscular atrophy (SBMA) is a toxic gain-of-function polyglutamine disease

caused by expanded trinucleotide repeats in the androgen receptor (AR) gene. Expression of

full-length AR with expanded poly-glutamine repeats in the Drosophila eye causes a rough-

eye phenotype that is dependent on the presence of the ligand and worsens when an AR

protein with longer repeats is expressed (Chan et al., 2002; Pandey et al., 2007; Takeyama et

al., 2002).

The toxicity of the mutant protein seen in SBMA patients can thus be recapitulated by

eye-specific expression in flies and this tool makes for a good model to screen for genes that

are involved in mutant AR-induced toxicity (Chan et al., 2002). Screening such expanded AR

expressing flies using a set of 2000 genes that were co-overexpressed in the eye (GS strains)

revealed that the Drosophila homolog of the Retinoblastoma protein, Rbf, is a modifier of

AR-dependent SBMA (Suzuki et al., 2009). Thus, by screening 2000 flies for modulation of

AR-induced neurodegeneration in flies, a link between polyQ-AR toxicity and

Retinoblastoma protein function was proposed.

Rough-eye phenotypes can also be the result of degeneration of cells other than PRs,

including the cornea and pigment cells. PR-specific degeneration can be easily assessed using

a rapid optical neutralization technique called the deep pseudo-pupil method that does not

require dissection, antibodies or dyes (Kirschfeld and Franceschini, 1968) and was introduced

in studies of neurodegenerative disease in Drosophila (Jackson et al., 1998). PRs elaborate a

membranous organelle, the rhabdomere which is a photosensitive structure and within one

ommatidium, the rhabdomeres are organized in a very stereotypic manner. Degeneration of

the PRs can thus be assessed by analysing the rhabdomeric organization and structure. In the

deep pseudo-pupil assay, the virtual images of a number of rhabdomeres are superimposed

and the properly organized rabdomeric structure is seen as a magnification of a single

ommatidium (Figure 4.e). Mis-organization or degeneration of the ordered array of

rhabdomeres results in disruption of the pseudo-pupil image, signifying morphological

(degenerative) defects.

However, subtle defects in one or a few ommatidia may not be obvious to detect using

this methodology. Nonetheless, this assay enables fast screening of the gross morphology of

ommatidia, signifying retinal degeneration in a somewhat more subtle manner than screening

for external rough-eye phenotypes.

A commonly used readout to assess the functionality of the PRs is the electroretinogram

(ERG) (Figure 4.f) (Heisenberg, 1971; Pak, 1995), which allows for very fast and efficient

measurement of the electrical response of the fly eye to light stimulus. ERGs measure


differences in extracellular potential between PRs and the fly body during a short light

stimulus (e.g. 1s).

In controls, ERG recordings show a de- and re-polarization of the PRs, reflecting an

intact phototransduction mechanism of the PRs, and ‘on’ and ‘off’ transients at the onset and

conclusion of the light stimulus. The presence of the ‘on’ and ‘off’ transients indicate that the

PRs can activate postsynaptic neurons while the depolarization of the PRs signifies the

presence of healthy PR cells that efficiently depolarize in response to light. Recording ERGs

is relatively fast and this assay has been used to screen vast amounts of flies to isolate mutants

involved in neuronal communication (‘on’ and ‘off’ defects) (Hiesinger et al., 2005; Zhai et

al., 2003) and in neurodegeneration (depolarization defects) (Bayat et al., 2012). In relation to

MND, screens based on ERG recordings led to the identification of the fly orthologue of elp3,

a gene whose expression levels are associated with ALS in humans (Simpson et al., 2009).

Hence, ERG recordings are a valuable high-throughput tool in the search for genes that affect

neuronal communication and survival.

Figure 4. Drosophila melanogaster behaviour, retinal morphology and neuronal function can be used to

study mutations affecting it, in a relative fast and easy way. a) During Drosophila melanogaster

development various behavioural traits can be studied. b) During the larval stage, crawling of the larvae

can be assessed whereas c) during the adult state, climbing or flight can be investigated. d) Schematic

of larval fillet preparation with larval motor neurons in grey reaching the muscles and neuromuscular

junction (inset), which allows for functional studies of various synaptic processes. e) Schematic of a

Drosophila melanogaster compound eye with the typical trapezoid shape of the rhabdomeres as can be

assayed with the pseudo pupil method. f) Schematic of the setup used to record electroretinograms

(ERGs) of the fly eye and example of a wild-type ERG.


Other assays that are often used to describe neurodegenerative phenotypes in flies are

lifespan assays (Van Voorhies et al., 2004) and assays to study sensitivity to oxidative stress

(Shukla et al., 2011) that is often associated with neuronal degeneration. Sensitivity to

oxidative stress can be assayed by exposing the flies to rotenone or paraquat, both of which

are inhibitors of mitochondrial complex I (Li et al., 2003; Sriram et al., 1997); hydrogen

peroxide, which is a more general oxidative stress inducer (Nickla et al., 1983; Wang et al.,

2008); or menadione sodium bisulfite, a mild oxidative stress inducer that persist longer when

mixed in the food and is effective at inducing chronic oxidative stress (Jordan et al., 2012).

Assaying survival in the presence or absence of these compounds is relatively easy, but time-

consuming. Furthermore, the genetic interactors identified will likely improve organismal

health but may yield quite broadly defined and potentially less specific components of the

pathways under study. With respect to MND, secondary screens are thus likely needed to

further group interactors into relevant and defined pathways.

Assays that are more directly linked to the motor system encompass locomotion and

activity-related tests as well as morphological and functional analyses of neuromuscular

endplates. Several external stimuli can be used to trigger locomotion related responses in

adult fruit flies or in larvae, including light (Borst, 2009), gravity (Kamikouchi et al., 2009),

temperature (Sayeed and Benzer, 1996), humidity (Liu et al., 2007), odours and taste

(Vosshall and Stocker, 2007) as well as sound (Kamikouchi et al., 2009) (Figure 4.a-c). More

directly assessing motor behaviour, screens based on flight (Benzer, 1973; Drummond et al.,

1991; Pesah et al., 2004; Vos et al., 2012), locomotion geotaxis, phototaxis (Armstrong et al.,

2006; Desroches et al., 2010; Gargano et al., 2005; Hirsch, 1959; Inagaki et al., 2010;

Kamikouchi et al., 2009; Le Bourg and Buecher, 2002; Pak et al., 1969; Seugnet et al., 2009;

Strauss and Heisenberg, 1993; Toma et al., 2002; Vang et al., 2012), as well as activity

monitoring over a prolonged period using an activity monitoring system (TriKinetics)

(Pfeiffenberger et al., 2010) have been successfully used and several of these assays are well-

described in video-based publications (Ali et al., 2011; Chiu et al., 2010; Nichols et al.,

2012).

The third instar larval NMJ has emerged as a model for neuronal cell biology

(Featherstone and Broadie, 2000), and also in the context of MND, this synapse has yielded

invaluable insight into the mechanisms of several diseases (Chang et al., 2008; Pennetta et al.,

2002; Ratnaparkhi et al., 2008; Sen et al., 2011; Sherwood et al., 2004; Xia et al., 2012).

Several features of this synapse are very stereotyped and could be used in a screen setting as

well (Collins and DiAntonio, 2007). Indeed, several models of MND show defects at this

synapse, for example mutations in fly VAP33, the fly orthologue of ALS8 (Nishimura et al.,

2004) show morphological and functional changes at the NMJ (Pennetta et al., 2002) and the

protein has been linked to Ephrin signalling (Tsuda et al., 2008) that is implicated in ALS

(Van Hoecke et al., 2012). While in most cases, larvae need to be dissected to gain access to

the neuromusculature, it is also possible to visualize NMJs through the cuticle by virtue of

transgenic expression of GFP fusion proteins (Rasse et al., 2005; Zito et al., 1999), thus

making screening of vast numbers of mutants possible using this system. However, given

practice, assays based on dissected preparations in combination with live imaging to monitor

synaptic function (Verstreken et al., 2008) or morphological assays based on expression of

GFP fusion proteins (Pilling et al., 2006) or immunohistochemistry are feasible as well in

medium-throughput screens. Such assays have indeed been used in the past to isolate mutants

that affect the NMJ morphology (Aberle et al., 2002) as well as NMJ synaptic function


(Featherstone et al., 2000; Rohrbough et al., 2004) and are attainable as well to use in the

context of MND (Figure 4.d).

CONCLUSION

Several phenotypes have been observed in models of MND in fruit flies that are

particularly amenable to genetic screening. Phenotypes range from larval lethal, pupal lethal,

retinal degeneration to defects in locomotion behaviour and specific defects at the NMJ.

Given the considerations discussed above, different types of screens either focused on NMJ

dysfunction, or using alternative tissues such as the PRs are feasible and may yield invaluable

insights into ’the pathways that lead to MND.

ACKNOWLEDGMENTS

We apologize for the omission of any relevant publication due to the specific focus of

this chapter. We are grateful to Tillman Achsel for critical reading of the manuscript and

Helena Renders for help. Work in the Verstreken lab is supported by an ERC starting grant,

FWO grants, IWT, FCT, the research fund KU Leuven, the Hercules foundation and VIB. SV

is an FWO fellow and LJ is supported by VIB.

ABOUT THE AUTHORS

Liya Elsa Jose obtained her masters degree in 2006 from Amrita University in India. In

2006 she joined a Biotech firm in India and worked as Junior Research Fellow since 2009. In

2009 she got an International VIB PhD Scholarship and joined the lab of Prof. Patrik

Verstreken (Laboratory of Neuronal Communication) to pursue her PhD. After joining the lab

she studied the role of elp3 in the synapse and she is presently focusing on the role of elp3 in

motor neuron disease.

Patrik Verstreken performed a post-doc at Howard Hughes Medical Institute, BCM,

Houston, USA. He performed several Ethyl Methane Sulphonate (EMS)-based screens to

identify novel genes involved in neuronal communication. He is a group leader at VIB since

2007 and his lab focuses on the study of several genes that affects synaptic communication in

health and disease. Researchers in his lab use novel EMS screens to isolate novel components

of this process. Interestingly, several genes that his group has identified have been linked to

neurological disease; including Parkinson’s disease and amyotrophic lateral sclerosis (ALS),

further underscoring the central involvement of neuronal communication in neuronal disease.

Sven Vilain obtained his Ph.D. in 2009 at the Katholieke Universiteit Leuven, Leuven,

Belgium. At the Center for Human Genetics, in the laboratory of neurogenetics led by

Bassem Hassan, he studied the function of the gene atonal by means of a genetic screen and

he participated in the creation of a novel toolkit for the study of atonal. After joining the

Laboratory of Neuronal Communication led by Patrik Verstreken, he studied the role of

mitochondrial Complex I in the Parkinson’s disease related gene pink1 and he participated in


a genetic screen for dominant modifiers of pink1 mutants. Currently he is focusing on the

synaptic function of the Parkinson’s disease related gene LRRK.

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INDEX

A

AAA, 26, 51, 87, 89, 91, 94, 105, 112, 113, 115, 116

access, xi, 197

accessibility, 155

accounting, 4

acetylation, 71, 99, 155, 164, 168

acid, 10, 12, 14, 99, 108, 110, 139

acidic, 39

actin dynamics, 102, 127

action potential, x, 97, 122, 128

activity level, 205

adaptation, 43

adenosine triphosphate, 173

adhesion(s), 9, 12, 16, 44, 90, 93, 106, 108, 109, 111,

125, 127, 204

adipose, 98

ADP, 159, 161

adulthood, 132, 172, 176

adults, 2

aetiology, 80, 161, 180

age, ix, 14, 18, 25, 57, 60, 99, 148, 150, 151, 162,

172, 201

age-related diseases, 162

aggregation, 12, 16, 41, 59, 60, 61, 75, 76, 77, 81,

82, 83, 109, 155, 158, 160, 161, 163, 165, 166,

168, 169, 177, 202, 203, 210

aging population, 148

agonist, 42

akinesia, 3

Aldrich syndrome, 112

allele, 23, 69, 99, 133, 141, 192, 193, 201

ALS2, 5, 7, 17, 25, 88, 92, 103, 113, 115

alsin, 5, 7, 25, 33, 55, 58, 88, 92, 103, 109, 110, 115

alters, 31, 74, 153, 160, 177, 178

amino acid(s), x, 6, 8, 16, 28, 38, 53, 66, 94, 95, 101,

103, 121, 133, 136, 139, 157

amplitude, 54, 177

amyotrophic lateral sclerosis (ALS), v, viii, ix, xii, 1,

2, 3, 4, 5, 6, 7, 8, 9, 13, 19, 20, 21, 22, 23, 24, 25,

26, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,

54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,

81, 82, 103, 11, 142, 196, 197, 198, 201, 202,

203, 204, 206, 209, 210

amyotrophy, 2, 13, 16, 27, 86

anatomy, x, 130, 133

anchoring, 51, 106

androgen, xi, 18, 19, 147, 149, 151, 152, 153, 154,

162, 163, 164, 165, 166, 167, 168, 169, 195, 201,

204, 208

angioplasty, 48

anterograde transportation, 100

anticodon, 136, 144

antigen, viii, 36, 42

AP-5 complex, 104

apoptosis, ix, 45, 58, 61, 70, 72, 82, 109, 156, 164,

168, 203, 210

Arabidopsis thaliana, 114

arrest, 168

aspiration, 151

aspiration pneumonia, 151

assessment, 141, 146

assets, 70

ataxia, 8, 11, 28, 70, 81, 117, 130, 149, 150, 154,

167, 199, 200, 205

atlastin, 12, 87, 92, 94, 95, 96, 97, 102, 110, 115,

116, 119

ATP, 70, 105, 137, 157, 159, 160, 173, 183

atrophy, vii, xi, 1, 2, 3, 4, 17, 18, 20, 22, 25, 26, 27,

28, 30, 37, 52, 122, 126, 127, 143, 147, 148, 149,

151, 153, 162, 163, 164, 165, 166, 167, 168, 171,

172, 174, 181, 182, 183, 184, 193, 195, 200, 204,

205, 207

attachment, 137, 157

attractant, 44

autism, 180

Index 212

autopsy, 58

autosomal dominant, 2, 6, 10, 11, 13, 15, 17, 26, 28,

34, 46, 94, 95, 96, 97, 98, 99, 100, 101, 102, 105,

110, 111, 112, 119, 122, 125, 126, 127, 148

autosomal recessive, xi, 2, 6, 10, 11, 13, 17, 18, 29,

98, 99, 101, 102, 103, 104, 105, 113, 114, 122,

125, 126, 127, 172

avian, 17

axon, viii, ix, x, 12, 14, 15, 16, 29, 31, 32, 44, 48, 51,

54, 75, 85, 86, 96, 97, 99, 101, 103, 106, 110,

111, 113, 114, 119, 122, 123, 139, 178, 191, 193,

203, 204

axon regeneration, 96, 111

axonal Charcot-Marie-Tooth disease, vii, 1, 24, 27,

144

axonal degeneration, xi, 7, 12, 14, 18, 25, 86, 122,

138, 139, 140, 142, 146

axonal microtubule-based transport, x, 85

axonal pathology, 15

axons, ix, x, 1, 3, 4, 11, 14, 15, 16, 20, 28, 30, 67, 75,

85, 86, 95, 96, 97, 100, 101, 105, 106, 112, 115,

128, 132, 133, 139, 143, 156, 205

B

Baars, 113

bacteria, 114, 159

basal ganglia, 148

base, 36, 132

Belgium, xii, 142, 185, 198

beneficial effect, 160, 161

benign, 139

Berardinelli-Seip congenital lipodystrophy, 98, 111

bias, 65

biochemistry, 47, 50, 53

bioinformatics, 67

biopsy, 124

biosynthesis, 127

biosynthetic pathways, 107

biotechnology, 203

blood, 18, 41, 124, 133, 146

blood-brain barrier, 18, 146

BMA, 17

body fat, 74, 98

bone, x, 11, 70, 85, 176, 182, 184

bone morphogenetic protein (BMP), x, 85

boutons, 40, 68, 69, 95, 96, 177

brain, 8, 14, 36, 52, 62, 78, 98, 99, 103, 113, 114,

116, 128, 131, 138, 139, 199, 201, 207, 208

brainstem, 3, 148, 149, 151

branching, 11, 31, 78, 109, 132, 176

breakdown, 164

budding, 104

by-products, 58

C

Ca2+, 42, 48, 51, 70

calcium, 11, 15, 39, 49, 54, 96, 97, 125, 149

CAM, 109, 110, 111

cancer, 70, 76, 77

candidates, 47

carboxyl, 39, 113, 208

cargoes, x, 7, 51, 85, 100, 101

cascades, 9, 15, 42, 43

caspases, 156

catabolism, 8, 14

catalysis, 6

catalytic activity, 109

cation, 13, 101, 129, 134

CBP, 155

cDNA, 191

cell biology, 53, 180, 181, 182, 184, 197, 205, 207

cell body, ix, 4, 85, 86, 100, 106, 138

cell culture, 124, 155, 160, 168

cell cycle, 88, 108, 157, 206

cell death, 18, 21, 31, 45, 81, 98, 99, 153, 156, 162,

170

cell differentiation, 126, 145, 178

cell division, 94, 177, 178, 182, 189, 190, 192

cell fate, 74, 143, 163, 181, 200

cell line(s), 61, 77, 140, 153

cell membranes, 98

cell signaling, 143

cell size, 205

cell surface, 12, 15, 43

central nervous system,

central nervous system (CNS), 14, 27, 28, 44, 51, 77,

90, 95, 128, 139, 145, 177, 178

centromere, 172, 190

centrosome, 94

ceramide, 39, 41, 53

cerebellum, 15, 148, 149

cerebral palsy, 29

cerebrospinal fluid, 77

certification, 47

challenges, vii, ix, 36, 85, 86

chaperones, 11, 16, 45, 76, 152, 157, 158, 159, 161,

167

cheese, 98, 108, 113, 115

childhood, 144, 171

children, 2

cholesterol, 14, 99, 118

chromatid, 190

chromosome, 7, 49, 50, 53, 75, 78, 80, 113, 144,

156, 169, 172, 188, 189, 190, 199, 202

Index 213

circadian rhythms, 200

classes, 4

classification, 3, 117

clathrin, 15, 31, 103, 104

cleavage, 61, 66, 83, 105

clinical presentation, 58

clinical symptoms, 2

clinical trials, ix, 123

clustering, 40, 42, 118

coding, 8, 65, 94, 148, 155, 180

codon, 94, 139

cognitive function, vii

cognitive impairment, 103

collaboration, 59

communication, 40, 86, 196, 198

compaction, 16

compensation, 14, 159

competition, 42

complement, 192

complexity, vii, 44, 59, 106, 123

complications, 172

composition, 14, 32, 76

compound eye, 149, 195, 196

compounds, 71, 72, 98, 124, 161, 197

conduction, x, xii, 14, 122, 128, 133

conjugation, 163, 200

consensus, 141

conservation, 65, 71, 95, 110, 174, 180, 191

contracture, viii, 1, 2, 3, 4, 17

controversial, 177

controversies, 74

COOH, 164

coordination, 174

copper, 6, 17, 18, 79

copulation, 132

cornea, 195

corpus callosum, 32, 103, 117

correlation, 27, 123, 148

correlations, 52

cortex, 3, 149

cortical neurons, 11, 31

courtship, 79

cross-sectional study, 79

crystal structure, 47

crystalline, 149

CST, 2, 3, 11, 12

cues, viii, 35

culture, 128

cure, 58

cuticle, 197

CYP7B1, 9, 14, 32, 89, 93, 99, 117, 118

cyst, 178

cystic fibrosis, xi

cytochrome, 99, 114, 117, 156

cytochrome P450, 99, 117

cytokinesis, 31, 102, 109, 116

cytoplasm, 8, 19, 62, 63, 67, 69, 70, 100, 123, 125,

126, 127, 151, 152, 157, 158, 159, 173, 177

cytoplasmic phase, xi

cytoplasmic tail, 106

cytoskeleton, 2, 9, 10, 12, 16, 17, 42, 44, 47, 71, 95,

123, 125

D

database, 52

defects, viii, xi, 8, 11, 12, 15, 20, 25, 33, 35, 37, 40,

43, 44, 45, 46, 58, 66, 68, 69, 72, 79, 94, 97, 99,

100, 115, 116, 119, 121, 138, 139, 140, 141, 150,

154, 156, 168, 174, 176, 177, 178, 180, 183, 185,

187, 188, 193, 195, 196, 197, 198, 205

deficiency(ies), 14, 16, 18, 19, 29, 30, 33, 107, 156,

159, 177, 192, 206, 209

degenerate, 132

degradation, ix, 5, 6, 9, 12, 13, 16, 17, 20, 37, 61, 67,

70, 72, 90, 102, 105, 113, 152, 157, 158, 159,

160, 161, 163, 165, 167, 169

dementia, 2, 6, 7, 11, 24, 26, 31, 70, 78, 79, 86, 117

demyelination, 14, 29, 106, 122, 138

dendrites, 97, 100, 101, 112

dendritic spines, 62, 65, 76

dephosphorylation, 47

depolarization, 196

deprivation, 158

depth, 100, 105

deregulation, 9

detectable, 156

detection, 192, 202, 207, 208

detoxification, 204

developmental process, 43

dimerization, 100, 141, 169

disability, 1, 2, 29, 104, 186

discs, 132, 149, 177

disease gene, vii, ix, 13, 46, 65, 74, 124, 133, 185,

186, 195, 200

disease model, 79, 142, 153, 171, 186, 210

disease progression, xi, 148, 153, 154, 161, 185

diseases, vii, ix, x, xi, 4, 36, 66, 77, 85, 109, 124,

147, 148, 150, 154, 155, 156, 158, 160, 161, 169,

185, 186, 197

disorder, xi, 18, 29, 36, 37, 46, 57, 58, 111, 114, 122,

142, 144, 151, 169, 172

distal hereditary motor neuropathy (dHMN), viii, 4,

20, 137

distress, 19, 25

distribution, 16, 26, 43, 102, 104, 115, 144, 145, 167

Index 214

diversity, 86, 145

DNA, viii, 4, 5, 8, 19, 23, 31, 37, 55, 57, 58, 61, 65,

78, 79, 103, 117, 138, 151, 152, 154, 155, 168,

169, 182, 187, 188, 190, 207

DNA damage, 65, 168, 207

DNA repair, 103, 117

domain structure, 70, 151, 182

dopaminergic, 209

dosage, 53, 133, 138, 141, 205

double helix, 187

down-regulation, 155

drawing, 131, 190

drug discovery, 66, 79

drug targets, 36, 192

drug treatment, x, 121, 123

drugs, 160

D-serine, 8

dyes, 195

dynamin, 12, 15, 34, 87, 95, 112, 115, 129, 134

E

egg, 128, 174, 176, 177, 184

electrodes, 138

electron, 15, 43, 45, 150, 209

elongation, 44

embryogenesis, 40, 132, 174, 176, 177

embryonic stem cells (ESCs), 124

EMG, 54

EMS, xii, 174, 187, 188, 189, 191, 192, 193, 198

encoding, x, 7, 8, 12, 14, 18, 19, 23, 25, 32, 33, 55,

58, 76, 104, 111, 112, 115, 117, 119, 121, 136,

139

endocrine, 118, 164

endoplasmic reticulum (ER), viii, 35, 41, 85, 86, 159

endosomal traffic, x, 5, 7, 11, 13, 85, 86, 88, 89, 92,

93, 103, 123

endosomes, 7, 11, 15, 88, 89, 94, 102, 110, 123, 125

energy, 43, 105, 157, 158, 163

energy supply, 105

engineering, 209

England, 48, 49, 50, 54

environment, 73, 138

environmental conditions, 67

environmental factors, 57, 58, 59, 70

enzymatic activity, 126, 140

enzyme(s), x, 6, 14, 16, 17, 18, 19, 58, 99, 105, 121,

136, 143, 157, 160, 173

epigenetics, 59

epilepsy, 50, 86

equipment, 156

ER stress, viii, 12, 35, 37, 41, 45, 46, 47, 52, 53, 81,

96, 101

ERLIN2, 10, 21, 90, 93, 108

ESCRT-III, 11, 90, 94, 102, 116, 119

eukaryotic, 48, 157, 164

eukaryotic cell, 157

evidence, viii, xi, 1, 4, 11, 12, 14, 16, 20, 24, 42, 44,

45, 46, 60, 65, 68, 70, 71, 77, 121, 122, 123, 145,

152, 159, 161, 209

evolution, 124, 136

excision, 174

excitotoxicity, 8, 12, 37, 42

exclusion, 8

exocytosis, 7, 202, 206

F

families, vii, 12, 108, 116, 119

family members, 39, 49, 116

fasciculation, 4, 106

fat, 62, 74, 96, 98

fat body, 96, 98

fatty acid 2-hydroxylase (FA2H), 99

fatty acids, 99, 114

fertility, 151, 189, 195

fertilization, 40, 55

fiber(s), 55, 131, 132, 133, 138, 139, 140, 142

fibrinolysis, 48

fibroblasts, 124, 160

filament, 16, 24, 125

financial, 72

financial support, 72

fish, 59

fission, 15, 41, 43

flight, xii, 132, 138, 139, 140, 174, 176, 196, 197

fluorescence, 50

follicle, 177

food, 150, 154, 197

formation, 6, 7, 8, 9, 12, 14, 16, 24, 25, 49, 51, 53,

61, 65, 76, 94, 96, 102, 106, 109, 110, 111, 114,

115, 118, 141, 144, 148, 158, 163, 168, 178, 180,

181, 182, 202, 204, 205, 206, 208

fractures, 176, 182, 184

Fragile X mental retardation protein (FMRP), 61

fragments, 41, 62, 66, 150

frameshift mutation, 102

functional analysis, 44

functional changes, 197

funding, 20, 72

fused in sarcoma (FUS), viii, 8, 37, 80, 207

fusion, 7, 15, 43, 44, 52, 88, 96, 114, 115, 125, 126,

150, 154, 197

Index 215

G

gait, 11, 12, 106

ganglion, 132, 149

GEF, 88, 91, 103

gene expression, xi, xii, 8, 19, 54, 61, 67, 74, 77, 80,

125, 143, 148, 155, 163, 169, 186, 193, 200

gene silencing, 201, 203

gene targeting, 199

gene therapy, 47

genes, vii, viii, x, xii, 1, 5, 6, 7, 8, 9, 11, 14, 15, 16,

17, 18, 19, 20, 37, 38, 39, 45, 55, 58, 62, 65, 71,

81, 85, 86, 87, 92, 97, 106, 107, 109, 121, 122,

123, 125, 128, 130, 133, 134, 136, 140, 142, 147,

148, 149, 150, 152, 153, 164, 166, 181, 182, 185,

186, 187, 188, 189, 191, 192, 193, 195, 196, 198,

199, 201, 203, 208, 209

genetic background, 68, 76

genetic disease, 117

genetic disorders, 86, 148

genetic diversity, 20

genetic factors, 36, 58

genetic mutations, 123

genetic screening, 186, 198

genetics, 14, 21, 48, 49, 52, 55, 74, 75, 76, 77, 78,

79, 81, 82, 112, 142, 143, 144, 163, 182, 183,

184, 200, 201, 202, 203, 204, 205, 207, 208

genome, 44, 62, 66, 70, 77, 81, 117, 128, 137, 139,

173, 174, 186, 187, 188, 193, 194, 201, 202, 203,

204, 205, 207

genomic regions, 193, 201

genomics, 202, 206

genotype, 52, 123, 187, 208

geotaxis, 138, 197, 201, 202, 208

germ cells, 188

germ layer, 132

germ line, 174

Germany, x, 39, 121, 142

gerontology, 201

gestation, 18

glaucoma, 126

glia, ix, 45, 61, 71, 98, 133, 163, 177

glial cells, 1, 21, 60, 61, 62, 63, 109, 132

globus, 149

glomerulonephritis, 127

glucose, 39

glutamate, 12, 37, 40, 41, 42, 60, 77

glutamic acid, 201

glutamine, xi, 147, 148, 150, 151, 152, 153, 154,

156, 167, 168, 169, 195

glycine, 8, 61, 62, 64, 80

glycoproteins, 106

glycosylation, 12

Golgi, 5, 7, 11, 14, 39, 53, 95, 102, 104, 105, 115,

116

grants, xiii, 198

granules, 61, 63, 65, 67, 73

gravity, 197, 202, 203

growth, viii, xii, 11, 12, 35, 40, 44, 49, 68, 69, 76,

77, 82, 88, 100, 102, 106, 107, 110, 113, 125,

126, 129, 134, 157, 177, 178, 199, 210

growth arrest, 126

growth cones, 100

growth factor, 77, 82

growth rate, 100

GTPase, 12, 15, 34, 87, 95, 96, 111, 115, 119, 126,

136, 202

GTPases, 12, 91, 112, 116

guanine, 7, 25, 33, 54, 55, 91, 103, 113, 115, 118,

126, 207

guanine exchange factor (GEF), 91, 103

guidance, viii, 35, 44, 51, 54, 106, 113, 191, 193,

203, 204

gynecomastia, 3, 18

H

hairless, 200

hairpins, 96

HDAC, 159, 161

health, 197, 198

hearing loss, 99, 125, 126

heart disease, 184

heat shock protein, 11, 16, 20, 27, 105, 148, 152,

159, 160, 163, 165, 167

heat shock protein 60 (HSP60), 11, 105

hereditary spastic paraplegia (HSP), vii, 4

heterogeneity, x, 30, 85, 86, 145

histone, 71, 155, 168

histone deacetylase, 71, 155

histones, 155

history, vii, 145, 185

HIV-1, 61

homeostasis, 9, 19, 39, 41, 49, 90, 115, 118, 157,

159, 180

homologous chromosomes, 189

hormone, 150, 153, 154, 158, 159, 164, 169

host, 58

hot spots, 188

hotspots, 108, 109

HSP60, 11, 89, 93, 105

hub, 112

human, vii, ix, x, xi, 21, 27, 33, 35, 39, 40, 41, 45,

47, 48, 52, 53, 54, 57, 59, 62, 65, 66, 67, 68, 69,

70, 71, 72, 73, 74, 76, 79, 80, 82, 83, 85, 86, 95,

99, 100, 105, 106, 110, 111, 112, 114, 117, 123,

Index 216

124, 128, 133, 136, 139, 140, 143, 144, 146, 148,

150, 153, 156, 159, 162, 163, 164, 165, 168, 171,

174, 176, 178, 182, 183, 184, 185, 186, 200, 201,

202, 204, 206, 208, 209

human brain, 143

human genome, vii

human immunodeficiency virus, 79

humidity, 197

hydrocephalus, 26, 106, 113

hydrogen, 58, 197

hydrogen peroxide, 58, 197

hydrolysis, 118

hydrops, 3

hyporeflexia, 14

hypothesis, 37, 44, 57, 59, 60, 61, 70, 71, 128, 153

I

ideal, 151, 161

identification, ix, 9, 16, 37, 59, 61, 62, 65, 109, 142,

183, 189, 192, 196, 208, 209

identity, 66, 92, 93

image, 131, 195

images, 150, 195

immune system, 21

immunoglobulin, 25, 40, 108

immunoglobulin superfamily, 108

immunohistochemistry, 197

immunophilins, 167

immunoreactivity, 81

impairments, 86

in utero, 16, 172

in vitro, 41, 58, 60, 96, 140, 141, 160, 163, 164, 173,

183, 207

in vivo, vii, 33, 41, 58, 61, 66, 70, 78, 81, 96, 123,

141, 146, 150, 156, 164, 186, 193, 206, 207

incidence, 58, 172, 194

India, 198

individuals, 37, 57, 77, 122

inducer, 81, 197

induction, 47, 54, 76, 99, 158, 169, 201

infant mortality, 18, 172

infantile-onset ascending hereditary spastic

paraplegia (IAHSP), 103

infants, 2, 172

ingestion, 153

inheritance, 6, 11, 18, 122, 124, 127, 128, 133

inherited disorder, 9, 18

inhibition, 4, 43, 61, 99, 102, 158, 160, 161, 169

inhibitor, 47, 70, 96, 111, 165, 169, 203

initiation, 151

injury, viii, ix, 20, 36, 43

inositol, 11, 19, 39, 70, 77

insect(s), 105, 146, 177, 202

insertion, 130, 136, 154, 174, 188, 193, 209

integration, 75, 168

integrity, 32, 39, 86, 97, 107, 210

Intellectual Disability, 21

intellectual impairment, 86

interface, 115, 141, 145

interference, 203, 204

interneuron, 138, 139

interneurons, 3, 132, 180

interphase, 182

intervention, 46

intron, 130

introns, 62, 65

inventions, 144

ions, 6

iron, 99

iron accumulation, 99

Islam, 106, 111, 112

isolation, 187, 188, 191, 192

issues, 189

Italy, 78

J

Japan, 47

Jordan, 165, 197, 202

K

KIF1A, 88, 92, 100, 101, 110, 113, 114

KIF5A, 10, 12, 15, 30, 33, 88, 92, 100, 110, 114, 116

KIF5B, 100

KIF5C, 100, 113

kinase activity, 209

kinesin, 10, 13, 30, 112, 116, 201, 205

kinesin-I, 100

kinetics, 210

L

L1CAM, 9, 12, 90, 93, 106, 111

labeling, 209

laminar, 3

larvae, xii, 95, 100, 131, 154, 174, 177, 196, 197

larval development, 182

larval stages, 174, 176, 177

lateral sclerosis, vii, 1, 2, 3, 4, 17, 24, 36, 48, 50, 57,

58, 73, 74, 76, 77, 78, 103

LDL, 49

Index 217

lead, viii, ix, xi, 9, 19, 37, 40, 46, 58, 61, 62, 85, 86,

97, 99, 100, 101, 102, 106, 121, 123, 124, 153,

158, 159, 162, 172, 177, 178, 180, 198

learning, 47, 199, 203

legs, 16, 139, 177, 178

lending, 148

lesions, 54, 187, 188

leucine, 104, 130, 135

leukemia, 165

leukocytes, 156

leukodystrophy, 99, 105, 114

life cycle, 128, 173

life expectancy, 57, 172

lifetime, 189

ligand, viii, xi, 35, 42, 43, 44, 54, 109, 119, 147, 151,

152, 153, 154, 155, 159, 195, 201, 209

light, viii, xii, 13, 16, 100, 129, 134, 149, 195, 196,

197

limb weakness, 3

lipid metabolism, viii, 2, 9, 10, 11, 17, 20, 46

lipids, 14, 39, 86, 90, 118

lipodystrophy, 98, 107, 111, 117

liposomes, 96

liver, 14, 21, 99

liver failure, 99

localization, xi, 40, 51, 62, 63, 67, 69, 76, 77, 102,

104, 109, 115, 116, 119, 121, 127, 140, 151, 152,

154, 155, 156, 157, 165, 178, 181, 182

loci, x, 6, 11, 57, 58, 59, 85, 104, 122, 174, 188, 192,

193, 201

locomotor, 11, 12, 15, 17, 59, 66, 68, 69, 71, 82, 141,

199, 200, 201, 208

locus, 21, 31, 48, 58, 118, 135, 176, 200

longevity, 66, 209

Lou Gehrig's disease, 4

lumen, 98, 159

lunapark/Lnp-1, 101

lymphocytes, 124

lysine, 157, 163

lysosome, 7, 21, 158

M

machinery, ix, 15, 62, 63, 75, 85, 86, 96, 143, 155,

157, 159, 167, 194

majority, vii, 8, 11, 61, 62, 64, 66, 122, 132, 133,

172, 174, 176, 177

mammalian cells, 50, 66, 102, 172

mammals, 42, 95, 96, 128, 148

man, 24, 114

management, 145

manipulation, xi, 45, 123, 128, 146, 147, 165

mapping, 23, 187, 192, 199, 200

MASA (Mental retardation, Aphasia, Shuffling gait

and Adducted thumbs) syndrome, 106

maspardin, 11, 89, 93, 105

mass, 41, 205

mass spectrometry, 41

matrix, 11, 105, 112, 125

measurement, 195

median, 14

medical, 52, 182, 183, 184

medicine, vii, 36, 48, 50, 54, 201, 209

membranes, 7, 96, 115, 118

memory, 79, 199

menadione, 197

mental retardation, 11, 61, 65, 77, 82, 95, 98, 127

mentorship, 162

mesoderm, 179

messenger RNA, 74

metabolism, ix, 6, 8, 10, 14, 39, 41, 50, 53, 57, 58,

59, 62, 74, 89, 90, 93, 98, 156, 163

metabolites, 43

metamorphosis, 132, 174, 178

metaphase, 178

meter, 86

methodology, 188, 189, 191, 192, 194, 195

methylene blue, 169

Mg2+, 101, 111

mice, 2, 6, 7, 11, 12, 14, 15, 16, 19, 21, 23, 24, 25,

27, 31, 33, 37, 42, 45, 47, 53, 54, 59, 62, 75, 81,

82, 86, 100, 109, 116, 128, 129, 130, 137, 140,

141, 156, 158, 159, 160, 162, 165, 168, 170, 178,

179, 180, 183, 207

microcephaly, 29

microinjection, 40

microRNA, 8, 77

microscope, 149

microtubule (MT), 86

migration, 12, 42, 106

miniature, 40

mitochondria, viii, 11, 15, 24, 35, 41, 43, 44, 47, 52,

55, 58, 87, 100, 101, 102, 103, 105, 113, 115,

116, 118, 123, 125, 126, 127, 156, 158, 202, 205,

208, 209

mitochondrial DNA, 43

mitosis, 116, 190

mixing, 43

model system, x, 2, 57, 58, 59, 65, 107, 148, 180

modelling, vii, 20, 65, 71, 72, 99, 124, 130, 148, 180

models, vii, viii, ix, xi, xii, 20, 27, 32, 43, 45, 54, 59,

60, 65, 67, 68, 70, 71, 72, 76, 77, 79, 80, 81, 97,

119, 121, 123, 124, 128, 129, 135, 136, 141, 142,

144, 147, 150, 151, 152, 153, 154, 155, 156, 158,

159, 160, 161, 162, 165, 166, 167, 176, 180, 186,

187, 197, 198, 199, 201, 203, 205, 207, 209

Index 218

molecular biology, 20, 47, 55, 202

molecular medicine, 81, 204

molecular oxygen, 58

molecular weight, 8

molecules, 65, 74, 109, 157, 159, 161

monomers, 148

morphogenesis, 12, 15, 52, 78, 115, 116

morphology, xii, 11, 12, 15, 16, 37, 43, 44, 62, 68,

69, 76, 94, 95, 118, 133, 136, 139, 141, 142, 144,

150, 192, 195, 196, 197

mortality, 59, 82, 171

mosaic, 174, 186, 187, 191

mother cell, 132

motif, 8, 13, 27, 39, 50, 51, 62, 64, 91, 130, 135,

164, 173

motor activity, 100

motor behavior, 24, 109

motor control, 4

motor neuron degeneration, vii, viii, ix, xii, 7, 14, 15,

19, 20, 21, 25, 30, 43, 48, 59, 68, 75, 106, 165,

168, 169, 207

motor neuron disease, 4, 6, 27, 30, 33, 48, 50, 55, 57,

58, 59, 61, 62, 71, 72, 79, 116, 156, 167, 185,

186, 198

motor neurons, vii, viii, ix, xi, 1, 2, 4, 6, 12, 14, 19,

20, 22, 35, 36, 42, 43, 44, 45, 49, 53, 58, 60, 61,

66, 67, 68, 69, 72, 81, 86, 98, 113, 128, 131, 132,

138, 139, 140, 149, 151, 154, 156, 172, 176, 180,

183, 186, 196

motor system, xii, 85, 197

mRNA, ix, 8, 19, 29, 30, 46, 61, 62, 63, 65, 71, 73,

76, 77, 80, 144, 155, 177, 181, 184, 194

mRNAs, 19, 61, 65, 67, 78, 178

mtDNA, 51

muscle atrophy, xii, 3, 18, 36, 58, 59

muscles, 14, 18, 43, 49, 96, 128, 131, 132, 137, 138,

139, 151, 172, 174, 176, 180, 193, 196

muscular dystrophy, 32

muscular tissue, 179

mutagen, 187

mutagenesis, xii, 174, 185, 187, 188, 189, 202

mutant, viii, ix, x, xi, 2, 6, 12, 15, 16, 18, 19, 22, 23,

25, 26, 27, 32, 33, 35, 37, 40, 41, 43, 44, 45, 46,

48, 51, 52, 54, 59, 63, 65, 66, 67, 68, 69, 75, 76,

77, 78, 81, 82, 85, 86, 95, 98, 100, 106, 113, 114,

121, 128, 129, 133, 135, 137, 138, 139, 140, 141,

142, 143, 144, 145, 147, 148, 153, 154, 155, 157,

159, 160, 162, 163, 174, 176, 177, 182, 186, 187,

188, 189, 190, 191, 192, 193, 195, 201, 202, 204,

209

mutant proteins, x, 37, 121, 142, 148

mutation, viii, x, 7, 19, 21, 23, 28, 31, 32, 35, 37, 39,

40, 41, 43, 45, 48, 50, 51, 52, 53, 55, 59, 61, 64,

73, 76, 81, 97, 99, 104, 110, 111, 112, 114, 115,

116, 117, 128, 133, 137, 141, 143, 145, 146, 174,

176, 177, 187, 188, 189, 190, 200, 202, 204, 206

myelin, x, 14, 15, 16, 23, 27, 28, 86, 90, 99, 106,

110, 111, 114, 115, 119, 123, 125, 126, 127, 129,

133, 134

myelin metabolism, 111

myelin sheath, x, 86, 99, 106, 119

myelination, viii, x, 2, 14, 15, 28, 123

myocardial infarction, 48

myopathy, 70, 129

myosin, 40, 52

N

NAD, 210

National Academy of Sciences, 53, 54, 73, 74, 76,

77, 79, 118, 182, 183, 199, 202, 203, 204, 206,

207, 208, 210

necrosis, 129, 134

negative effects, 95

nematode, 39, 47, 55

nerve, 4, 14, 98, 122, 128, 131, 132, 133, 183

nervous system, viii, x, 23, 30, 43, 44, 71, 98, 109,

117, 128, 131, 177, 178

networking, 52

neural development, 119

neurobiology, 47, 75, 107, 200

neuroblastoma, 140

neuroblasts, 132, 177

neurodegeneration, xi, 12, 15, 19, 20, 47, 50, 59, 70,

73, 74, 75, 78, 81, 82, 98, 99, 108, 114, 147, 148,

149, 158, 160, 161, 163, 164, 166, 167, 168, 169,

170, 185, 187, 191, 195, 196, 199, 200, 201, 205,

206, 208, 209, 210

neurodegenerative diseases, 7, 46, 47, 60, 124, 148,

154, 160, 201, 204

neurodegenerative disorders, vii, xi, 60, 70, 71, 169

neurofilaments, 16

neurogenesis, 132, 176, 177, 178, 180

Neuroglian, 109, 111

neurological disease, 1, 2, 14, 36, 145, 185, 198

neurologist, viii, 36

neuromuscular function, vii, 116

neuromuscular junction (NMJ), 95

neuronal cells, 96

neuronal circuits, xii, 50

neuronal stem cells, 132

neurons, vii, ix, x, xii, 1, 3, 4, 6, 11, 12, 15, 16, 19,

36, 42, 44, 48, 59, 60, 61, 62, 63, 65, 66, 67, 68,

69, 71, 82, 85, 86, 98, 100, 107, 111, 114, 116,

118, 121, 122, 123, 124, 128, 131, 132, 138, 139,

140, 141, 142, 143, 145, 146, 149, 153, 154, 156,

Index 219

159,165, 177, 178, 179, 180, 184, 191, 194, 195,

196, 202, 209

neuropathy, viii, x, xi, 1, 2, 3, 4, 14, 17, 18, 20, 23,

24, 26, 27, 28, 31, 33, 34, 98, 108, 115, 116, 118,

119, 121, 122, 123, 126, 137, 140, 141, 143, 144,

145, 146, 176, 180, 208

neuropathy target esterase (NTE), 98

neuropeptides, 108

neurophysiology, 54

neuroscience, xii, 36, 46, 48, 52, 54, 73, 78, 79, 80,

81, 82, 200, 203, 206, 208, 209

neurosecretory, 117

neurotoxicity, 70, 75, 76, 77, 78, 155, 163, 169, 201,

202, 208

neurotransmission, 180, 187

neurotransmitter, 191

neurotransmitters, x, 128

neurotrophic factors, 8

next generation, 187, 189

NH2, 164, 169

NIPA1, 9, 11, 30, 88, 92, 94, 101, 102, 107, 108,

111, 118

NMDA receptors, 8, 48, 49

NTE, 90, 93, 98, 113

nuclear membrane, 48

nuclear stability, 125

nuclei, 3, 148, 149

nucleocytoplasmic shuttling, ix, 63, 70

nucleolus, 183

nucleoplasm, 125

nucleoprotein, 8

nucleus, xi, 8, 19, 61, 63, 67, 70, 109, 123, 149, 152,

153, 156, 157, 159, 160, 168, 173, 177

null, 7, 43, 66, 69, 95, 129, 174, 176, 179, 180

nutrient, 158, 181

nutrients, 156, 158

O

obesity, 74, 168

oligodendrocyte, 14

oligodendrocytes, 14, 99, 127

oligomerization, 113, 119, 174

oligomers, 96, 117, 148

omission, 198

ommatidium, 149, 195

oocyte, 40, 42, 48, 49, 52, 55, 177

oogenesis, 119, 181

organ, 109, 140, 149, 202

organelle(s), 6, 11, 15, 20, 43, 86, 102, 108, 156,

157, 158, 195

organism, vii, x, 15, 46, 66, 69, 121, 147, 161, 174,

176, 189

organs, 140, 148

orthologue, viii, ix, 66, 95, 96, 97, 98, 99, 100, 101,

102, 103, 104, 105, 106, 115, 124, 135, 153, 173,

185, 186, 193, 196, 197, 204

ovaries, 176

overlap, 15, 16, 18, 42, 58

ovulation, 52

ox, 171

oxidative damage, 6, 60

oxidative stress, 1, 7, 20, 58, 75, 197, 202, 207

oxygen, 204, 205

P

p53, 156

Pak3, 95

palliative, 58

paralysis, vii, 3, 4, 12, 16, 58, 110, 151, 172

paraplegin, 11, 24, 89, 93, 105

paresis, 6, 125

parkinsonism, 7

pathogenesis, 11, 17, 20, 21, 27, 35, 36, 39, 42, 43,

44, 45, 46, 55, 74, 80, 123, 124, 128, 141, 142,

147, 148, 151, 153, 154, 155, 156, 161, 167, 204

pathology, viii, ix, xi, 4, 6, 7, 8, 15, 22, 35, 36, 37,

40, 41, 43, 45, 46, 54, 57, 58, 59, 60, 67, 68, 69,

71, 72, 94, 95, 101, 104, 144, 147, 148, 150, 160,

163, 170, 174, 178, 186

pathophysiological, 66

pathophysiology, viii, ix, xii, 67, 68, 72, 167

pathways, vii, viii, ix, x, xi, 1, 3, 6, 11, 12, 20, 22,

35, 36, 37, 39, 40, 41, 42, 44, 46, 59, 65, 70, 71,

72, 85, 103, 111, 123, 124, 128, 131, 147, 157,

159, 161, 163, 185, 186, 191, 193, 197, 198, 200,

201, 208

Pelizaeus-Merzbacher disease (PMD), 106

peptide chain, 157

peptides, 166

peripheral nervous system, 111, 114

peripheral neuropathy, 14, 16, 21, 123, 129, 130,

137, 140, 141, 142, 143, 145

permeability, 49

pharmacological treatment, 128

pharmacology, 201

PHB, 91

phenocopy, 173

phenotype(s), ix, xii, 1, 11. 14. 8, 21, 23, 29, 30, 36,

40, 42, 43, 44, 51, 52, 65, 66, 67, 68, 69, 70, 71,

74, 86, 95, 96, 99, 100, 103, 104, 106, 111, 113,

115, 123, 124, 125, 126, 127, 128, 133, 137, 139,

140, 141, 142, 143, 149, 150, 154, 155, 156, 158,

159, 160, 162, 163, 170, 172, 174, 176, 177, 178,

Index 220

179, 180, 185, 186, 187, 188, 191, 192, 194, 195,

197, 198, 199, 200, 201, 206

phosphate, 17, 39

phosphatidylcholine, 98, 115, 119

phospholipids, 14

phosphorylation, 48, 58, 61, 82, 173, 183

phototaxis, 106, 197

physical interaction, 44, 173

physiological psychology, 202

physiology, vii, viii, xi, xii, 124, 167, 202

placebo, 164

plasma membrane, 12, 15, 50, 91, 102, 123, 126

plasmid, 144

plasticity, 201

platform, 144, 203

playing, 9

PLP, 91, 106

PLP1, 9, 14, 90, 93, 106

PLS, vii, 2, 3, 4, 6, 17, 103

point mutation, 35, 94, 129, 174, 176, 187

polarization, 196

polymerase, 74, 173

polymorphism(s), 44, 76, 139

polypeptide, 9, 129, 134

population, xi, 51, 78, 94, 136, 151, 172, 187, 188

pregnancy, 18

premature death, 2, 14, 18

preparation, 196

priming, 152

principles, vii, x, 128, 142

private sector, 47

probe, 153, 161

progenitor cells, 132

progressive bulbar palsy (PBP), vii, 4

progressive muscular atrophy (PMA), vii, 4

progressive neurodegenerative disorder, 35

project, 3, 107, 128, 132, 139, 142, 199, 207

proliferation, 8, 43, 124, 182

proline, viii, 37, 41

promoter, 21, 65, 114, 150, 153, 162, 174, 176, 191,

192

propagation, 20

proteasome, ix, 5, 6, 9, 61, 70, 72, 125, 152, 157,

158, 159, 160, 161, 169

protective role, 60

protein chaperone activity, x, 20, 123

protein folding, ix, 70, 72, 89, 105, 159

protein kinases, 91

protein misfolding, 16, 60

protein synthesis, x, 62, 121, 159

protein-protein interactions, 9, 128, 141

proteolipid protein, 9, 14, 27, 31, 91, 106, 111

proteolipid protein 1 (PLP), 106

proteolysis, 89

proteome, 75

proteostasis, viii, 2, 9, 16, 20, 60, 159

protrudin, 51, 89, 93

pruning, 30

pupa, 178

purification, 62, 63, 164

pyramidal cells, vii, 3

pyrimidine, 127

pyrophosphate, 130, 135, 137

Q

quality control, xi, 11, 147, 157, 159, 161, 162

R

Rab, 15

radicals, 58

reactions, 89

reactive oxygen, 6, 152, 156, 203

reactivity, 33

reading, 142, 174, 176, 198

receptors, viii, 11, 28, 35, 37, 40, 41, 42, 43, 44, 46,

49, 51, 54, 102, 114, 116, 153, 159, 169, 209

recognition, 8, 27, 61, 64, 136, 139, 158

recombination, 135, 189, 190, 192, 202

recommendations, iv

recruiting, 112

recycling, 126, 157, 158

redistribution, 70

redundancy, 97, 167

REEP1, 10, 12, 17, 18, 22, 34, 94, 97, 115, 120

reflexes, 4, 36, 122

regeneration, 96, 111, 122

relaxation, 132

relevance, 45, 97, 123, 130, 140, 165, 177

remodelling, 16, 44

remyelination, 122

repair, 8, 187

repellent, 44

replication, 190

repression, 61, 65, 152, 164

repressor, 61, 78

requirements, 128, 151

researchers, xii, 149, 178, 186, 188, 193

residues, 39, 94, 101, 103, 104, 133, 139, 159, 160

resistance, 125

resolution, 47, 50, 179, 186, 200, 205

respiration, 43

respiratory failure, 172

Index 221

response, 19, 44, 45, 47, 53, 54, 61, 66, 67, 98, 99,

129, 134, 139, 154, 159, 160, 168, 195, 196

restoration, 178, 179, 180

retardation, 106, 178

reticulon-2, 94

reticulum, viii, x, 33, 35, 36, 41, 47, 48, 49, 52, 53,

54, 55, 85, 86, 108, 114, 115, 117, 118, 159, 167,

168

retina, 70

ribosomal RNA, 156

ribosome, 115

risk, 24, 57, 60, 70, 75, 128, 151, 163, 183

risk assessment, 183

RNA, viii, ix, 2, 5, 6, 8, 17, 18, 19, 20, 23, 27, 30,

32, 37, 57, 58, 59, 60, 61, 62, 63, 64, 66, 67, 70,

71, 73, 74, 75, 76, 78, 80, 81, 122, 147, 150, 155,

156, 165, 167, 170, 171, 173, 177, 178, 180, 181,

182, 203, 204

RNA processing, viii, ix, 2, 5, 6, 8, 17, 18, 63, 64,

81, 147, 150, 155

RNA splicing, 8, 61, 75, 155, 170, 171

RNAi, xii, 31, 62, 65, 68, 69, 77, 81, 95, 117, 135,

140, 176, 185, 192, 193, 194, 201, 203, 204, 205,

207

RNAs, 62, 65, 123, 156, 177, 180, 184

rodents, 57, 59

root, 149

roughness, 106, 149

routes, 201

S

safety, 164

salivary gland, 98, 154, 169

salivary glands, 169

science, 47, 51, 205

secretion, viii, 20, 41, 45, 46, 109

secretory vesicles, 100

segregation, 182, 190

seipin, 12, 33, 89, 93, 98, 110, 112, 114, 117, 118,

119

selectivity, 163

self-assembly, 167

sensing, 52, 202, 203

sensitivity, 197, 202, 205

sensory systems, 142

sequencing, 50, 61, 62, 71, 76, 110, 187, 209

serine, viii, 8, 37, 41

serum, 41

services, 64

sex, 133, 165

shape, 94, 120, 196

shock, 10, 13, 17, 24, 26, 129, 134, 152, 159, 160,

162, 168, 191

short-term memory, 207

showing, 46, 95, 131, 138, 139, 154, 177

siblings, 183

signal transduction, 157, 159

signaling pathway, 48, 205

signalling, viii, x, 7, 9, 11, 15, 18, 19, 36, 40, 41, 42,

43, 44, 45, 47, 52, 65, 85, 86, 88, 94, 96, 97, 101,

102, 103, 107, 118, 156, 159, 167, 191, 193, 197,

204

signals, 44, 52, 64, 97, 111, 128, 156, 158

signs, 4

skeletal muscle, 3, 4, 11, 43, 49, 51, 55, 151, 159,

170

skin, 124

SLC33A1, 10, 28, 89, 93, 99, 112, 114, 116

SNP, 200

sodium, 197

solubility, 160, 163

solution, 194

spartin, 11, 31, 90, 93, 94, 102, 108, 109, 110, 116,

118

spastic, vii, x, 1, 2, 3, 4, 6, 9, 10, 11, 12, 21, 22, 23,

24, 26, 29, 30, 31, 32, 34, 85, 86, 102, 103, 104,

106, 107, 108, 109, 110, 111, 112, 113, 114, 115,

116, 117, 118, 119, 209

spastic paraplegia gene (SPG), 85

spasticity, x, 3, 4, 12, 86

spastin, 12, 32, 87, 89, 92, 94, 95, 96, 97, 102, 108,

109, 110, 114, 115, 116, 117, 118, 119, 207, 209

spastizin, 11, 89, 92, 103, 104, 115

spatacsin, 7, 11, 32, 88, 92, 103, 104, 115, 117

species, viii, 6, 20, 38, 39, 148, 152, 156, 159, 187,

203

spectroscopy, 50

sperm, viii, 35, 36, 39, 40, 42, 43, 47, 50, 52, 55

SPG1, 9, 26, 90, 93, 106, 113

SPG10, 10, 30, 88, 92, 100, 116

SPG11, 5, 7, 10, 32, 88, 92, 103, 117

SPG12, 10, 87, 92, 96

SPG13, 10, 25, 89, 93, 105, 112

SPG15, 10, 25, 89, 92, 103, 104, 111

SPG17, 10, 89, 93, 98

SPG18, 10, 21, 90, 93, 105

SPG2, 9, 90, 93, 106

SPG20, 10, 30, 90, 93, 102, 108, 110, 113, 116

SPG21, 10, 31, 89, 93, 105

SPG30, 88, 92, 100, 101, 113

SPG31, 10, 87, 92, 97

SPG33, 89, 93

SPG35, 10, 24, 90, 93, 99, 110

SPG39, 10, 90, 93, 98

Index 222

SPG3A, 9, 87, 92, 95, 115, 119

SPG4, 9, 87, 92, 94, 95, 108, 111, 114, 117

SPG42, 10, 28, 89, 93, 99, 114

SPG47, 10, 22, 104

SPG48, 10, 31, 103, 104, 117

SPG5A, 9, 89, 93, 99

SPG6, 9, 30, 88, 92, 101, 102, 107, 111

SPG7, 10, 89, 93, 105

SPG8, 10, 32, 88, 92, 102, 118

sphingolipids, 99, 119

spinal cord, vii, 3, 4, 6, 9, 12, 19, 36, 45, 47, 53, 55,

62, 94, 128, 140, 151, 172

spinal muscular atrophy (SMA), vii, xi, 4, 20, 183,

193

spindle, 16, 110

spine, 62, 76

spinobulbar muscular atrophy (SBMA), vii

spliceosomal Uridine-rich small nuclear

ribonucleoprotein biogenesis, xi

stability, 24, 61, 71, 113, 118, 165, 209

stabilization, 63

state, 137, 142, 151, 155, 159, 161, 177, 196

stem cells, 124, 145, 177, 178

sterile, 13, 130, 135, 188

steroids, 89, 99

sterols, 14

stimulation, xi, 138, 139

stimulus, 195, 196

stock, 188, 193

stoichiometry, 19

storage, 61, 98

strategy use, 188

stress, viii, ix, 5, 8, 12, 16, 33, 35, 37, 41, 45, 46, 47,

49, 52, 53, 60, 61, 63, 65, 67, 74, 75, 76, 78, 79,

80, 81, 96, 101, 125, 160, 167, 169, 181, 197, 205

stress granules, 8, 61, 63, 65, 67, 74, 75, 76, 78, 80

stress response, ix, 37, 45, 160, 167

structural protein, 14

structure, vii, 16, 39, 44, 47, 51, 69, 106, 115, 118,

125, 128, 138, 143, 149, 157, 177, 180, 183, 184,

195, 209

strumpellin, 11, 88, 92

substitution, 139

substitutions, 61, 95

substrate(s), 70, 99, 157, 159, 161, 163, 164

suppression, xii, 163, 165, 187, 192, 199, 201, 203,

207

surface area, 94

surveillance, 184

survival, xi, 19, 20, 22, 27, 43, 47, 50, 60, 66, 68, 69,

72, 75, 81, 82, 100, 159, 160, 171, 172, 174, 178,

181, 183, 196, 197, 205, 209

susceptibility, 1, 20, 42, 59, 153, 200, 203

swiss cheese, 98, 108, 113

symptoms, 4, 11, 85, 86, 99, 106, 122, 172

synapse, 3, 45, 49, 62, 68, 69, 70, 78, 86, 101, 106,

111, 113, 115, 139, 144, 177, 197, 198, 201, 206,

210

synaptic plasticity, 61, 79

synaptic strength, 95

synaptic transmission, 39, 111, 199

synaptic vesicles, 100, 101, 128

synaptogenesis, 193, 203

syndrome, viii, 1, 2, 3, 4, 9, 17, 26, 29, 30, 31, 33,

36, 65, 74, 79, 98, 99, 106, 108, 111, 113, 117,

119, 145

synthesis, xi, 39, 114, 121, 169, 210

T

Taiwan, 145

target, 4, 10, 14, 19, 65, 71, 78, 98, 105, 108, 113,

114, 115, 116, 118, 119, 137, 147, 149, 157, 158,

159, 160, 161, 188, 193, 194, 201

tau, 5, 7, 26, 165

TCC, 103, 104

techniques, 36, 174

technologies, 146

technology, 124, 187, 191

telangiectasia, 206

telomere, 172

temperature, 192, 197

temporal lobe, 50

temporal lobe epilepsy, 50

terminals, 16, 19, 40, 68, 71, 99, 106

testing, 2, 72, 183

testis, 73, 78, 96, 178

testosterone, 19, 151, 152, 154, 157

tetanus, 107

therapeutic agents, 123

therapeutic approaches, viii, xi

therapeutic interventions, 2

therapeutic targets, xi, 20, 95, 121, 123, 128, 142,

147, 157, 161, 186

therapeutics, xii, 32, 59, 151, 165, 201

therapy, ix, 26, 36, 47, 75, 169

thorax, 131, 132, 140, 176, 190, 191

thyroid, 169

time frame, 81

tissue, xii, 6, 33, 65, 71, 78, 123, 136, 149, 155, 177,

179, 189, 190, 191, 192, 194, 206, 207, 208

topology, 99, 114

toxic effect, xi, 139, 147, 153

toxicity, xi, 6, 8, 22, 32, 37, 48, 54, 60, 66, 67, 68,

69, 70, 76, 77, 79, 81, 83, 94, 138, 147, 150, 151,

Index 223

152, 153, 154, 155, 158, 161, 163, 165, 166, 167,

187, 195, 199, 200, 201, 202, 203, 205

toxin, 107

trafficking, viii, ix, x, xi, 2, 5, 6, 7, 9, 10, 11, 13, 15,

16, 17, 18, 24, 25, 39, 40, 49, 52, 85, 86, 87, 88,

89, 92, 93, 101, 102, 108, 109, 111, 113, 115,

119, 123, 147, 152, 156, 167, 168, 169, 204, 206

traits, 196

trajectory, 44

transcription, 8, 18, 48, 62, 63, 65, 125, 126, 136,

147, 149, 151, 154, 155, 156, 160, 168, 169, 173,

192, 201

transcription factors, 155

transcriptional regulation, ix, x, 8, 65, 88, 123, 125,

166

transcripts, 63, 67, 71, 155, 181

transduction, 204

transformation, 202

transgene, 54, 136, 138, 139, 140, 144, 176, 192

translation, ix, 15, 16, 18, 19, 45, 61, 62, 63, 65, 75,

78, 109, 125, 126, 127, 136, 140, 141, 143, 158,

182, 194, 200

translocation, 154

transmission, x, 39, 128, 188

transport, viii, ix, x, 2, 6, 7, 10, 11, 12, 13, 15, 16,

17, 18, 19, 20, 24, 25, 32, 33, 39, 43, 50, 51, 52,

53, 58, 60, 62, 63, 65, 71, 76, 85, 86, 88, 89, 90,

92, 94, 97, 100, 101, 102, 105, 107, 108, 110,

111, 112, 113, 114, 115, 117, 118, 123, 125, 126,

150, 155, 156, 164, 165, 166, 168, 177, 178, 184,

202, 205, 208

transport processes, 71

transportation, 100

treatment, 47, 58, 115, 147, 151, 158, 160

tremor, 130, 151

trial, 119, 163, 164

triggers, 7, 14, 51

Troyer Syndrome, 102

turnover, 110, 159

tyrosine, xi, 42, 48, 49, 51, 54, 136, 137, 201, 207

U

ubiquitin, 9, 10, 16, 19, 37, 60, 102, 110, 112, 126,

148, 152, 157, 158, 160, 163, 164, 169, 208

ubiquitin-proteasome system, 10, 37, 157, 160, 163

underlying mechanisms, 71

unfolded protein response (UPR), 98, 159

United Kingdom (UK), x, xi, 85, 142, 171

United States, viii, xi, 21, 53, 54, 73, 74, 76, 77, 78,

79, 82, 83, 109, 116, 118, 165, 166, 167, 169,

182, 183, 198, 199, 202, 203, 204, 206, 207, 208,

210

University of Cambridge, x, 85, 107

V

vacuolation, 98

validation, 72

variations, 132, 133, 159, 193, 202

vascular endothelial growth factor (VEGF), 8

vector, 202

VEGF expression, 8

versatility, 176, 180

vertebrates, 43, 105, 128, 145, 174

vesicle, x, 7, 11, 15, 16, 20, 51, 52, 53, 54, 101, 108,

111, 112, 113, 115, 128, 158, 183, 202, 204, 206,

209

vision, 200, 205

vulnerability, xii, 30, 80

W

walking, 50, 69, 132

water, 204

weakness, vii, x, 3, 4, 6, 9, 11, 14, 16, 18, 19, 36, 86,

121, 122, 151, 172

wealth, 20, 46

worms, 38, 41, 42, 57, 59

X

X chromosome, 153, 188, 189, 199

X-inactivation, 153

X-linked hydrocephalus, 106, 113

Y

Y chromosome, 188, 189

YAC, 156, 168

yeast, 38, 39, 44, 50, 59, 72, 74, 79, 96, 110, 119,

136, 156, 160, 171, 183, 192, 202

yield, 186, 195, 197, 198

Z

ZFYVE27, 89, 93

zinc, 6, 8, 62, 79, 91, 104

Documents

Drosophila melanogaster Models of Motor Neuron Disease 2013 (edited by Ruben Cauchi)