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Review

Apoptosis in amyotrophic lateral sclerosis:a review of the evidence

S. Sathasivam*, P. G. Ince²

and P. J. Shaw*

Departments of *Neurology and ²Neuropathology, University of Shef®eld, Shef®eld, UK

S. Sathasivam, P. G. Ince and P. J. Shaw (2001) Neuropathology and Applied Neurobiology 27, 257±274

Apoptosis in amyotrophic lateral sclerosis: a review of the evidence

Amyotrophic lateral sclerosis (ALS) is a progressive

neurodegenerative disease primarily affecting the upper

and lower motor neurones of the central nervous

system. Recently, a lot of interest has been generated bythe possibility that a mechanism of programmed cell

death, termed apoptosis, is responsible for the motor

neurone degeneration in this condition. Apoptosis is

regulated through a variety of different pathways which

interact and eventually lead to controlled cell death.

Apart from genetic regulation, factors involved in the

control of apoptosis include death receptors, caspases,

Bcl-2 family of oncoproteins, inhibitor of apoptosis

proteins (IAPs), inhibitors of IAPs, the p53 tumour

suppressor protein and apoptosis-related molecules. The

®rst part of this article will give an overview of the

current knowledge of apoptosis. In the second part of

this review, we will examine in detail the evidence for

and against the contribution of apoptosis in motor

neurone cell death in ALS, looking at cellular-,

animal- and human post-mortem tissue-based models.

In a chronic neurodegenerative disease such as ALS,

conclusive evidence of apoptosis is likely to be dif®cult

to detect, given the rapidity of the apoptotic cell death

process in relation to the relatively slow time course of

the disease. Although a complete picture of motor

neurone death in ALS has not been fully elucidated,there is good and compelling evidence that a

programmed cell death pathway operates in this

disorder. The strongest body of evidence supporting

this comes from the ®ndings that, in ALS, changes in

the levels of members of the Bcl-2 family of

oncoproteins results in a predisposition towards apop-

tosis, there is increased expression or activation of

caspases-1 and -3, and the dying motor neurones in

human cases exhibit morphological features reminis-

cent of apoptosis. Further supporting evidence comes

from the detection of apoptosis-related molecules and

anti-Fas receptor antibodies in human cases of ALS.

However, the role of the p53 protein in cell death in

ALS is at present unclear. An understanding of the

mechanism of programmed cell death in ALS may

provide important clues for areas of potential ther-

apeutic intervention for neuroprotection in this

devastating condition.

Keywords: apoptosis, amyotrophic lateral sclerosis, apoptosis-related molecules, Bcl-2 family members, caspases,

death receptors, morphology of motor neurones, p53 pathway, TUNEL/ISEL staining

Introduction

Amyotrophic lateral sclerosis (ALS) is a rapidly progres-

sive neurodegenerative disease that primarily affects

the motor neurones of the cerebral cortex (upper motor

neurones), brain stem and spinal cord (lower motor

neurones) [112]. It is one of the most common neuro-

degenerative diseases of adult onset, with an incidence

of one to two per 100 000 of the population. Approxi-

mately 10% of cases are familial and one-®fth of these

are associated with dominantly inherited missense

Correspondence: Professor P. J. Shaw, Department of Neurology,

E Floor, Medical School, University of Shef®eld, Beech Hill Road,

Shef®eld S10 2RX, UK. E-mail: Pamela.Shaw@shef®eld.ac.uk

Neuropathology and Applied Neurobiology (2001), 27, 257±274

# 2001 Blackwell Science Ltd 257



mutations in the gene on chromosome 21 encoding the

free radical-scavenging enzyme copper/zinc superoxide

dismutase-1 (SOD1) [27,102]. Animal and cellular

models of ALS may provide useful information on the

molecular pathways leading to cell death of motor

neurones [38]. Transgenic mouse models of ALS have

been produced by overexpressing either glycine 37

to arginine (G37R), glycine 93 to alanine (G93A), or

glycine 85 to arginine (G85R) mutant SOD1 alleles

in a normal mouse genetic background [63]. These

transgenic models replicate many of the clinical and

pathological hallmarks of familial ALS (FALS) [38].

Features in the former category include normal motor

function at birth, the development of weakness typically

starting in the hind limbs and the progression to paralysis

and death over weeks to months. Neuropathological

changes in the transgenic mouse model, which are

also observed in human ALS cases, include the presenceof neuro®lament, ubiquitin and SOD1-positive inclusions

within motor neurones. Cellular models in the form

of motor neuronal cell lines, and cultures of primary

embryonic motor neurones and neonatal spinal cord

slices from rodents can also be used to investigate

cell death pathways which operate in motor neurone

degeneration. These in vitro models offer the advantage

of easy access to manipulation, for example by gene

transfections or therapeutic interventions. However, we

still need to exercise caution when extrapolating results

from experimental models to human disease, as the

accelerated time course of motor neurone degeneration

in these models compared to humans may critically affect

certain pathogenetic mechanisms of motor neurone

injury. Several recent studies have suggested that

the programmed cell death pathway of apoptosis may

be responsible for motor neurone degeneration in ALS.

In this article, we review the evidence currently available

supporting and refuting the contribution of apoptosis

in motor neurone cell death in ALS.

In 1972, Kerr et al. [66] proposed the term àpoptosis',

used in Greek to describe the `dropping off of petals from

¯owers or leaves from trees', to de®ne the regulatedremoval of cells. Although there are overlapping features,

apoptosis can be distinguished from necrosis on the basis

of different biochemical and morphological character-

istics (Table 1). It is important to note that apoptosis

represents an active process, requiring energy to pro-

ceed, unlike necrosis which does not need energy for

its occurrence. In apoptosis, scattered single cells are

affected. Morphologically, apoptosis is characterized by

plasma membrane blebbing with preservation of its

integrity, cytoplasmic condensation, compaction of cyto-

plasmic organelles, chromatin condensation and nuclear

fragmentation. There is absence of an acute in¯amma-

tory response in apoptosis. In contrast, clusters of con-

tiguous cells are affected in necrosis. In necrosis, there is

early plasma membrane disruption, swelling and destruc-

tion of cytoplasmic organelles and nuclear chromatin

lysis. Necrosis is accompanied by an in¯ammatory

response including chemotaxis of neutrophils.

The concept of an apoptosis±necrosis morphological

continuum has been proposed [96,97]. Excitotoxic

activation of N -methyl-D-aspartate (NMDA) and non-

NMDA glutamate receptors in the newborn rat brain

has been shown to cause neuronal cell death ranging

from apoptosis to necrosis [96]. The three main struc-

turally different forms of dying cells identi®ed in theimmature rat brain are a classically apoptotic form, a

classically necrotic form and a vacuolated form (the

latter is thought to be a precursor of the apoptosis

stage). In fact, neuronal death from excitotoxicity in the

newborn rat striatum is morphologically indistinguish-

able from that found in developmental apoptosis.

However, in the adult rat striatum, excitotoxic NMDA

receptor activation results in neurodegeneration that is

morphologically necrotic, whereas non-NMDA receptor-

mediated neuronal death is somewhere between classic

apoptosis and classic necrosis [97]. Non-NMDA receptor

excitotoxic neurodegeneration in the adult rat brain

results in a shrunken, dark perikaryon and discrete

round clumps of chromatin which are somewhat simi-

lar, but not identical, to apoptosis occurring naturally

in the developing rat brain. Therefore when evaluating

the mechanism of cell death in a system, it is crucial

to understand that the classi®cation into apoptosis or

necrosis is not a strict one, but rather a recognition of

features that point towards one end of the spectrum

or the other of the apoptosis-necrosis morphological

continuum.

Recently, an alternative non-apoptotic form of pro-grammed cell death, or paraptosis, has been described,

which does not conform morphologically to either apop-

tosis or necrosis [116]. The pathway is driven by

caspase-9 activity which is independent of apoptosis-

activating factor-1 (Apaf-1). Features of this form of

cell death include prominent cytoplasmic vacuolation

in the absence of nuclear fragmentation, chromatin

258 S. Sathasivam, P. G. Ince and P. J. Shaw

# 2001 Blackwell Science Ltd, Neuropathology and Applied Neurobiology, 27, 257±274



condensation and the formation of apoptotic bodies.

Certain of these morphological criteria have been

reported in neuronal development, such as the embryo-nic cell death in chick ciliary ganglia [95], and

neurodegeneration, such as the G93A transgenic

mouse model of ALS [23].

Molecular control of apoptosis

Genetic regulation

The recognition of the genetic regulation of apoptosis was

characterized through a series of experiments in the

nematode Caenorhabditis elegans [39]. These studies

revealed that, during the normal development of

C. elegans, two genes (ced-3 and ced-4) are necessary for

the initiation of apoptosis. A third gene (ced-9) inhibits

the action of the previous two genes [13,109,115,134].

Upon receiving a death commitment signal, the protein

product CED-4 activates CED-3 by binding to the

inactive CED-3. On the other hand, CED-9 prevents the

activation of CED-3 by binding to CED-4. The result is

that there are exactly 131 cell deaths from a total of

1090 cell births in the development of the adult

nematode. The signi®cance of these ®ndings was realizedwhen it was discovered that there were structural and

functional homologies between the genes ced-3 and

ced-9 and the mammalian genes interleukin converting

enzyme (ICE) [141] and bcl-2 [58], respectively. The ced-4

gene encodes a protein which is homologous to the

human protein Apaf-1 that participates in cytochrome

c-dependent activation of caspase-3 [143].

Caspases and death receptors

The C. elegans gene ced-3 encodes a cysteine proteasehomologous to the mammalian interleukin-1b

(IL-1b)-converting enzyme which cleaves and activates

the in¯ammatory cytokine IL-1b. Approximately 14

members of the CED-3/ICE family of proteases, termed

caspases, are now recognized and ®rmly established as

effectors of apoptosis [79]. The caspases are synthesized

as inactive precursors (zymogens) which need to be

proteolytically processed to generate active subunits. The

natural substrates of the caspases are essential cyto-

skeleton and regulatory proteins of cells. Processing of

the proenzymes and substrates usually occurs bycleavage at a speci®c aspartate residue in the P1 position.

Three main pathways triggering activation of caspases

(Figure 1) have been identi®ed. One pathway involves

the activation of cell death receptors of the tumour

necrosis factor (TNF) family, including the Fas and type I

TNF receptor (TNFR1) [131] by death receptor ligands.

Adaptor proteins, such as Fas-associated death domain

(FADD), form bridges between caspases and upstream

regulators of apoptosis [137,142]. The recruitment of

the adaptor protein FADD by the death receptor Fas via

interactions between death domains (DDs) has been

shown to promote apoptosis [5]. Subsequently, FADD

binds to caspase-8 via a homotypic interaction involving

death effector domains (DEDs). This leads to the acti-

vation of caspase-8 which, in turn, directly or indirectly

activates effector caspases such as caspase-3, -6 and -7

[90,117]. Although the activation of caspase-8 is not

fully understood, it is believed that the crowding of

Table 1. Differences between apoptosis and necrosis

Apoptosis Necrosis

Cells affected Isolated single cells Clusters of cells

Cell volume homeostasis Preserved in early stages Lost in early stages

Plasma membrane Preservation of integrity, blebbing Loss of integrity

of cell surface

Cytoplasmic organelles Compaction and contraction, Swelling of structures

formation of àpoptotic bodies'

Nuclear chromatin Condensation and fragmentation Karyolysis and pyknosis

DNA Internucleosomal fragmentation Random degradation (`smear'

(`ladder' pattern) pattern)

Cellular response No in¯ammation or neutrophilic Marked in¯ammatory response with

in®ltration leukocytic in®ltration

Rate of progression Depending on the insult, follows an Depends on the nature of the insult

àll-or-none' phenomenon in a `dose±response' principle

Apoptosis in amyotrophic lateral sclerosis 259




zymogens resulting from recruitment by the membrane-

bound complexes of death receptors and their ligands

in some way promotes the mutual cleavage and acti-

vation of the proenzyme molecules [106]. However, in

drug-induced apoptosis of B-lymphoid cells, it appears

that caspase-8 activation occurs independently of Fas

receptor±ligand interaction and occurs downstream of

caspase-3 [133]. Therefore it appears that caspase-8





may act as an executioner caspase in certain cases. In

the second pathway, cytochrome c, which is often

released from the mitochondria into the cytosol during

apoptosis [74], induces the activation of caspase-9 via

interaction with the adaptor protein Apaf-1 [72].

Activated caspase-9 eventually leads to the activation

of the downstream caspases [143]. Finally, it has recently

been shown that stress to the endoplasmic reticulum (ER)

leads to the activation of caspase-12 and this plays a role

in amyloid-b neurotoxicity in primary cortical neurone

cultures [91]. Thereafter, other caspases are probably

activated downstream from caspase-12 to complete the

apoptotic cascade.

Bcl-2 family of oncoproteins

The bcl-2 family of protooncogenes is a group of apoptosis

regulatory genes. In this group the antiapoptotic genes

are bcl-2, bcl-xL, bcl-w, a1, mcl-1 and boo, whereas the

proapoptotic genes are bax, bcl-xS , bad , bak, bid , bik, hrk,

bim, blk and mtd [81,121]. Membership of the family of

Bcl-2-related proteins is de®ned by distinct domains of

homology (BH1-BH4). During apoptosis, cytochrome c is

released from the mitochondria into the cytosol, possibly

through membrane channels comprising Bax [3]. Bcl-2

and Bcl-xL block cytochrome c release from the mito-

chondria [67,136], possibly by interfering with mem-

brane insertion and pore formation by the Bax protein [3]

or by disrupting the membrane potential and volumehomeostasis of mitochondria [127]. Further evidence of

mitochondrial involvement in apoptosis comes from

recent ®ndings by Shimizu et al. [114] that the

proapoptotic proteins Bak and Bax promote the opening

of the mitochondrial voltage-dependent anion channel

(VDAC) and the release of cytochrome c. On the other

hand, the antiapoptotic protein Bcl-xL closes VDAC by

directly binding to it, and inhibits cytochrome c release.

Therefore the binding of the Bcl-2 family of proteins to

VDAC appears to play a role in regulating mitochondrial

membrane potential and the release of cytochrome c,

both keys steps in the apoptotic cell death pathway. In

addition, it has been shown that Bcl-xL associates with

both caspase-9 and Apaf-1, resulting in the inhibition of

Apaf-1-dependent caspase-9 activation [62]. The homo-

logous domains of the Bcl-2 family members are involved

in the regulation of interactions between its members.

For example, the formation of Bax homodimers promotes

apoptosis by binding to and disrupting the permeability

transition pore complex, leading to mitochondrial

dysfunction which includes both the reduction of the

mitochondrial membrane potential and production of

reactive oxygen species [52,64,82,134]. On the other

hand, when Bax forms heterodimers with Bcl-2, theantiapoptotic effect of Bcl-2 is blocked [78].

Inhibitor of apoptosis proteins

Another group of antiapoptotic proteins is the inhibitor of

apoptosis proteins (IAPs). IAPs were originally identi®ed

in baculoviruses [17,18], where they have been found to

inhibit caspase activation [65,108]. The IAP family of

proteins are characterized by a domain of y70 amino

acids, the baculoviral IAP repeat (BIR). Human IAP

homologues that have been described include theneuronal apoptosis inhibitory protein (NAIP), c-IAP1,

c-IAP2, X-linked IAP (XIAP), survivin and BIR repeat

containing ubiquitin-conjugating enzyme (BRUCE)

[2,35,54,55,73,103,105,126]. Genetic inactivation of

the NAIP gene, initially identi®ed because its deletion

is associated with spinal muscular atrophy [105], is

Figure 1. Several pathways are involved in apoptotic cell death. Activation of a death receptor (e.g. Fas) results in recruitment of a speci®c

adaptor protein (e.g. FADD) via death domains. The adaptors, directly or indirectly through other adaptors, recruit initiator caspases

(e.g. caspase-8). Active initiator caspases directly activate the effector caspases (e.g. caspase-3, -6 and -7) or act on the mitochondria

and Bcl-2 homologues to induce caspase-9 activation, both pathways leading to apoptosis. Under certain conditions, caspase-8 may act as

a downstream caspase. Bcl-2 homologues regulate the release of cytochrome c from the mitochondria by altering the permeability transition

pore complex. Stress on the ER leads to activation of the initiator caspase-12, which probably results in activation of other downstream

caspases, causing apoptosis. IAPs interfere with apoptosis by inhibiting cytochrome c-induced activation of caspase-9 and directly

inhibiting active caspase-3. Inhibitors of IAPs (e.g. Diablo/Smac) block the action of IAPs, promoting cytochrome c/Apaf-1/caspase-9

pathway activation and increasing the activity of effector caspases. The p53 protein, when transactivated, increases expression of bax and

downregulates bcl-2, promoting apoptosis via the mitochondrial pathway. Par-4 interacts with PKCf which may suppress the activation

of NF-kB. NF-kB is thought to mediate its antiapoptotic actions through IAPs, MnSOD and calbindin. Apaf-1=apoptosis-activating factor-1,

ER=endoplasmic reticulum, FADD=Fas-associated death domain, IAP=inhibitor of apoptosis protein, MnSOD=manganese superoxide

dismutase NF-kB=nuclear factor kappaB, Par-4=prostate apoptosis response-4, PKCf=protein kinase Cf.





associated with neuronal cell death [73]. XIAP, c-IAP1

and c-IAP2 have been shown to inhibit cytochrome

c-induced activation of caspase-9, thereby preventing

the activation of caspases-3, -6 and -7 [29,104].

In addition, these IAPs arrest the proteolytic cascade

initiated by caspase-8 by directly inhibiting active

caspase-3, blocking the downstream execution of the

apoptotic cascade (Figure 1). Survivin, which is pro-

minently expressed in a variety of tumour and embryonic

cells [2], binds speci®cally to and directly inhibits the

terminal effector cell death proteases, caspase-3 and -7

[122]. It is not yet known if the BRUCE protein is

involved in preventing apoptosis. However, since there

is a functionally intact ubiquitin-conjugating domain in

BRUCE [54], it is possible that there may be a relationship

between apoptosis proteins and protein degradation

involving the ubiquitin/proteasome proteolytic pathway

[28,54].

Inhibitors of IAPs

In the fruit¯y Drosophila melanogaster , IAPs are inhibited

by interactions with signalling proteins called Reaper,

Hid and Grim, allowing the caspase cascade to be

activated [120,132]. Now, a mammalian mitochondrial

protein known as Diablo [128] or Smac [33], has been

shown to promote the cytochrome c/Apaf-1/caspase-9

pathway activation and increase the activity of the

effector caspase-3 and -7 by binding to multiple human

IAPs, including c-IAP1, c-IAP2, XIAP and survivin

[11,119]. In addition, Diablo/Smac appears to increase

the sensitivity of certain types of cells to TRAIL death

receptor-induced apoptosis by neutralizing IAP inhibition

of effector caspases (Figure 1) [119]. Although Diablo/

Smac appears to be a functional analogue of Reaper, Hid

and Grim, there seems to be no structural similarity to the

Drosophila proteins, raising the possibility of convergent

evolution [26].

Tumour suppressor protein p53

The tumour suppressor gene p53 encodes a protein that

is involved in various pathways, including those

regulating cell proliferation and apoptosis. Wild-type

p53 has been shown to induce apoptosis [111,142] or

increase susceptibility to apoptosis [10,43,76,77] in

tumour cells or in cells exposed to radiation. p53

upregulation is mostly post-transcriptional, leading to

an increase in translation and half-life [32]. Phosphory-

lation of p53 is crucial for its activation [98,113], while

nuclear translocation of the protein is associated with

its activation [46,110]. Other factors involved in p53

regulation include the balance between protein synthesis

and degradation since the level of p53 in¯uences the

effect it causes and diverse protein±protein interactions,

for example the binding of p53 to mdm2 promotes

p53 destruction via ubiquitin-mediated proteolysis [98].

A further clue on the importance of p53 in cell death

comes from the fact that p53-related apoptosis has been

shown to be associated with increased expression of the

bax gene with concomitant downregulation of bcl-2 gene

expression (Figure 1) [86±88].

Apoptosis-related molecules

LeY

antigen Apoptosis has been associated with cellsurface glycosylation changes [59]. Hiraishi et al.

demonstrated that the expression of the LeY carbohy-

drate antigen, which results from the cooperative

action of a1(r)2 and a1(r)3 fucosyltransferases, corre-

lated well with the presence of apoptosis in both

normal and tumour tissue. However, it must be pointed

out that not all LeY-positive cells showed signs of apop-

tosis. In contrast, necrosis was not associated with LeY

antigen expression. Therefore it can be concluded that

LeY expression is a useful phenotypic marker predictive

of apoptosis.

Prostate apoptosis response-4 protein The prostate apop-

tosis response-4 (Par-4) protein is another apoptosis-

related molecule containing a leucine zipper domain

within a death domain that has been shown to be

upregulated in prostate tumour cells [107], and in

both PC12 cells and primary hippocampal neurones

when these two cell groups are induced to undergo

apoptosis by trophic factor withdrawal and exposure to

amyloid-b peptide [53]. The exact mechanism by

which Par-4 causes neuronal death is uncertain. It has

been shown in tumour cells that Par-4 interacts withatypical isoforms of protein kinase C (PKCf), inhibiting

the kinase activity [31]. The interaction of Par-4 with

PKCf may suppress the activation of the antiapoptotic

transcription factor nuclear factor kappaB (NF-kB) [30].

NF-kB is thought to mediate its antiapoptotic actions

through IAPs, the enzyme manganese-SOD (MnSOD) and

the calcium-binding protein calbindin (Figure 1) [83].





Detection of apoptosis

The detection of internucleosomal DNA cleavage, result-

ing in the appearance of a ladder formation of DNA on

agarose gel electrophoresis, is widely considered as a

molecular indicator of apoptosis [21]. The DNA cleavage

is attributed to the actions of Ca2+/Mg2+-dependent

endonucleases [70], deoxyribonuclease I [4,94] or

deoxyribonuclease II [7]. However, internucleosomal

DNA fragmentation may not always be seen in conjunc-

tion with the morphological characteristics of apoptosis

[19,41]. To further complicate matters, internucleosomal

DNA fragmentation may also occur in some cells

undergoing necrosis [45,97]. Therefore detection of

internucleosomal DNA fragmentation needs to be sup-

ported by a morphological study to con®rm the presence

of apoptosis occurring in a particular model system of cell

death [20]. With in situ end-labelling (ISEL) techniques,detection of DNA fragmentation to assess neuronal via-

bility can be done by end-labelling DNA strand breaks

using the terminal transferase-mediated dUTP nick

end-labelling (TUNEL) method [6,40,61] or the DNA

polymerase 1-mediated biotin-dATP nick translation

(PANT) method [14]. The former method is probably

the most widely used method to detect apoptosis. How-

ever, it is important to note that these methods cannot

speci®cally distinguish between apoptosis (double-strand

DNA breaks) and necrosis (single-strand DNA breaks)

[12,48,51]. Both apoptosis and necrosis can give a

positive reaction with TUNEL staining which, although

different, may be very similar. Therefore, only careful

study of the pattern of TUNEL staining of affected cells in

conjunction with an ultrastructural study can reliably

differentiate apoptosis from necrosis.

Apoptosis and motor neurone cell death

There is a body of robust evidence that apoptosis does

occur in developmentally regulated motor neurone death

and in the death of motor neurones following axotomy.

For example, during normal vertebrate development,20±80% of neurones regularly die [16,50]. The amount

of nerve growth factor present is the limiting factor in

determining the survival of these fetal neurones. On the

other hand, overexpression of Bcl-2 protects against

motor neurone death from nerve transection in new-

born mice, lending support for the contribution of apop-

tosis in axotomy-induced motor neurone degeneration

[9,34,42]. Raoul et al. have recently provided evidence

that programmed cell death of motor neurones during

development may be triggered through the Fas cell

surface death receptor with caspase-8 activation [101].

Indices of apoptosis in amyotrophic lateralsclerosis

In a chronic neurodegenerative disease like ALS,

conclusive evidence of apoptosis is likely to be dif®cult

to detect given the rapidity of the apoptotic cell death

process in relation to the relatively slow time course of

the disease. In in vivo models of apoptotic neuronal death

in the brain induced by excitotoxicity [96], target

deprivation/axotomy [1] or deafferentation [57], the

visible signs of an apoptotic cell might be expected to last

from a few to a maximum of 24 h. This means that in

a disease like ALS, only one in several thousand motor

neurones might be expected to manifest signs of ongoing

apoptosis at any one time in a temporally static post-

mortem specimen. Thus, many sections must be exam-

ined in the search for apoptotic indices in post-mortem

tissue from ALS cases.

Structural morphology of motor neurones

Martin [80] elegantly describes the morphology of motor

neurone degeneration in the lumbar cord of human ALS

cases as structurally resembling apoptosis when exam-

ined by light microscopy (Figure 2). The three major

consecutive morphological stages of degeneration identi-

®ed are chromatolysis, somatodendritic attrition and

apoptosis. The initial stage of chromatolysis is character-

ized by dispersion of the Nissl substance without nuclear

condensation. In the attritional stage, where affected

motor neurones seem to spend the longest time, the

cytoplasm becomes homogenous and the nucleus con-

denses, even though the nucleolus remains apparent.

In the terminal apoptotic stage, when the affected

motor neurone is approximately a ®fth of its normaldiameter, only an extremely condensed nucleus and

contracted cytoplasm remain. In addition, Troost et al.

detected the presence of apoptotic bodies usually within

macrophages in the motor cortex, brain stem and spinal

cord of ALS cases [123,124]. Apoptotic bodies are con-

tracted vesicular units of cytoplasmic organelles, such

as lysosomes and mitochondria.





TUNEL/ISEL staining

Several studies have used nick-end labelling to detectDNA fragmentation, indicative of apoptosis, in ALS.

In one study on post-mortem human tissue, nick-end

labelling was detected in motor neurones in the high

cervical region in ALS cases but not at all in control cases

which consisted of progressive supranuclear palsy, lacu-

nar stroke, polyarteritis nodosa or non-neurological con-

ditions [140]. In two other studies, DNA fragmentation

was detected by positive TUNEL staining in the motor

cortex [37], brain stem [37], cervical cord [37], thoracic

cord [37,85] and lumbar cord [37] of cases with ALS. Inthese two latter studies, TUNEL staining was also detected

in control cases (all of whom died from non-neurological

related causes except for one individual who had a

diagnosis of polyneuropathy and diabetes mellitus), albeit

to a lesser degree than that in ALS cases. These ®ndings

differ from the absence of apoptosis in all control speci-

mens reported by Yoshiyama et al. [140]. Using TUNEL

a

d

b

g

e

h

c

f

i

Figure 2. Martin [80] .has proposed that the morphological features present can identify the different stages of motor neurone death in ALS.Using H&E-stained paraf®n-embedded sections of the lumbar cord, the likely progression of motor neurone degeneration is shown. For

comparison, a normal motor neurone (a) is shown alongside motor neurones from ALS cases (b±i). The ®rst stage of motor neurone

degeneration is chromatolysis (b,c) which is characterized by swelling of the cell body, dispersion of the Nissl substance and an

eccentrically placed nucleus. Prominent cytoplasmic hyaline body inclusions are seen in some neurones in the chromatolytic stage (c). The

occurrence of cytoplasmic and nuclear basophilia marks the change from the chromatolytic to the attritional stages. Features of the second

stage of motor neurone degeneration, the attritional stage (d±f ), are a progressively small cell, and homogenously dark and shrunken

cytoplasm and nucleus. In the ®nal apoptotic stage of degeneration, the motor neurones become very condensed and shrunken, assuming

a fusiform or round shape (g±i). Scale bar in a±i=11 mm (Reproduced with the permission of Martin).





staining, Martin [80] detected internucleosomal DNA

fragmentation in subsets of pyramidal neurones in the

motor cortex, and cervical and lumbosacral spinal cord of

ALS cases. Positive TUNEL staining was not detected in

the somatosensory cortex. In the affected motor neu-

rones, DNA fragmentation was only detected in the

somatodendritic attrition and apoptotic stages of neuro-

nal death, but not in the chromatolytic stage (Figure 3).

Although TUNEL-positive non-neuronal cells were

found in both cases of ALS and controls (all in the

latter group did not have neurological disease), TUNEL-

positive motor neurones were speci®c for ALS cases.

In addition, internucleosomal DNA fragmentation was

detected by gel electrophoresis in DNA samples obtained

from the anterior horn grey matter of the spinal cord and

motor cortex of ALS cases, but not in the somatosensory

cortex of ALS cases or in the anterior horn grey matter

of the spinal cord and motor cortex of control cases.However, other groups have failed to provide evidence

of internucleosomal cleavage of DNA in post-mortem

tissue from human ALS cases or from animal models of

the disease. Migheli et al. [85] failed to demonstrate any

evidence of apoptosis in sections from the motor cortex

and spinal cord (level not given) of human ALS cases

using in situ nick translation (involving the use of DNA

polymerase I) and TUNEL staining techniques. Similarly,

no DNA fragmentation was detectable by TUNEL stain-

ing, or TUNEL and ubiquitin double staining, in diseased

motor neurones of lumbar cord segments of human ALS

cases [56]. Immunoreactivity to the protein ubiquitin is

a sensitive marker of an affected motor neurone in ALS

[49,75]. Another study, again using the ISEL technique,

of cervical and lumbar cord sections from a G93A SOD1

transgenic mouse model of FALS did not detect evidence

of apoptosis, even though widespread motor neurone

degeneration was revealed by antibodies to ubiquitin

[84]. Here, it is useful to note that only some of these

studies in ALS employing TUNEL/ISEL techniques to

detect apoptosis used morphological criteria to ensure

proper discrimination between apoptotic and necrotic

cells [56,80,84,85,124].

Expression of apoptosis-related molecules

Another body of evidence supporting the contention that

motor neurones die via an apoptotic pathway comes from

the ®nding of increased expression of certain apoptosis-

related molecules in ALS. Expression of the carbohydrate

a

e

b

c d

Figure 3. Martin.[80] has shown that TUNEL staining of motor

neurones in ALS reveals the presence of DNA fragmentation during

the attritional and apoptotic stages of cell death, but not in the

chromatolytic stage. TUNEL allows in situ detection of DNA

fragmentation (brown staining within the nucleus). The cresyl

violet counterstain is used to exhibit Nissl substance. During

chromatolysis (a), no DNA fragmentation is detected. During the

somatodendritic attrition stage (b), prominent TUNEL-positive

staining is seen in the nucleolus. In the ®nal apoptotic stageof motor neurone degeneration (c,d), the condensed nucleus

is strongly TUNEL-positive. An overall view (e) shows that DNA

fragmentation occurs in some (neurones shown in brackets),

but not all motor neurones (arrowheads). Scale bar in a±d=5 mm.

Scale bar in e=57 mm (Reproduced with the permission of Martin).





antigen LeY in apoptotic cells of both normal and tumour

tissues suggests that changes of cellular glycosylation

pattern is closely correlated with the process of apoptosis

[59]. Positive expression of the LeY antigen has been

demonstrated in motor neurones in high cervical sections

of ALS cases but not those of control cases [140].

A study by Pedersen et al. examined the levels of the

apoptosis-related Par-4 protein in the lumbar spinal cords

of patients with ALS and controls with no neurological

disease [93]. Although Par-4 protein was detectable in

all control and ALS cases, it was signi®cantly increased

in the ALS group. In the same study, Par-4 levels were

increased in the lumbar cords of G93A SOD1 transgenic

mice when compared to wild-type mice. In addition, the

exposure of primary mouse spinal cord motor neurones

or NSC-19 motor neurone cells to oxidative insults, for

example ferrous sulphate (FeSO4) or 4-hydroxynonenal

(HNE), caused large increases in Par-4 levels precedingapoptosis. More supportive evidence suggesting a role

for Par-4 in the pathogenesis of ALS came from the fact

that a Par-4 antisense oligodeoxynucleotide blocked the

increase in Par-4 levels caused by the exposure of spinal

cord motor neurone cultures to FeSO4 or HNE, with

inhibition of apoptosis. In NSC-19 cells undergoing

oxidative stress when exposed to staurosporine, FeSO4

or HNE, pretreatment with the Par-4 antisense DNA

reversed the mitochondrial dysfunction and prevented

apoptosis. Therefore Par-4 appears to signi®cantly con-

tribute towards apoptotic motor neurone degeneration

in MND.

Alteration in the balance of

Bcl-2 family members

Cellular models

The deleterious effect of the mutant SOD1 enzyme

underlying one form of FALS is now considered to be

due to a toxic gain of function. The four major hypotheses

for this toxic gain of function are: (1) the formation of

hydroxyl radicals; (2) the nitration of protein tyrosineresidues by peroxynitrite derivatives; (3) copper and zinc

toxicity; and (4) protein aggregation [22]. More than

one of these mechanisms may operate simultaneously.

It has been demonstrated that mutant SOD1 expression

in neural cells stimulates apoptosis by converting the

enzyme from an antiapoptotic protein to a proapoptotic

one under conditions of oxidative stress induced by

serum or growth factor withdrawal [100], or possibly by

the formation of intracellular aggregates of mutant SOD1

[36]. On the other hand, wild-type SOD1 expression has

been shown to inhibit nitric oxide-mediated apoptosis in

a neuronal cell culture, possibly due to higher and more

stable Bcl-2 expression and decreased intracellular

release of reactive oxygen species [15]. It has been

shown in several types of differentiated neural cells

expressing mutant SODs that apoptotic cell death can be

signi®cantly reduced by overexpression of Bcl-2 [47].

SOD1 transgenic mice

In a G93A SOD1 transgenic mouse model of FALS cross-

bred with transgenic bcl-2 mice, overexpression of the

Bcl-2 protein has been shown to delay disease onset,

prolong survival and reduce spinal cord motor neurone

degeneration. However, the disease duration itself did notalter [68]. In another study using the same transgenic

mouse model of FALS, the expression of the antiapoptotic

proteins Bcl-2 and Bcl-xL were reduced, while the

expression of proapoptotic proteins Bad and Bax were

increased in symptomatic mice [129]. In asymptomatic

transgenic mutant SOD1 mice, expression of Bcl-2, Bcl-

xL, Bad and Bax did not differ from those in non-

transgenic mice. Although overexpression of Bcl-2 did

not prevent upregulation of Bax in transgenic mice, it

increased the formation of Bax±Bcl-2 heterodimers which

lack proapoptotic properties. Vukosavic et al. has also

demonstrated that overexpression of the antiapoptotic

Bcl-2 protein delayed the activation of caspases-1 and -3

as well as prolonging the survival time of transgenic

SOD1 mice by 20% [130]. Using a G86R SOD1

transgenic mouse model of ALS, de Aguilar et al.

showed decreased antiapoptotic Bcl-xL expression in

conjunction with increased proapoptotic Bcl-xS and

Bax immunoreactivity in the lumbar cords of the G86R

mice compared to those of the wild-type controls [24].

Human central nervous system tissue

Troost et al. reported that although the motor cortex and

spinal cord of human ALS and control cases expressed

the oncoprotein Bcl-2 to the same degree, the expression

of Bcl-2 was increased in the postcentral gyrus bordering

the affected motor cortex of ALS cases compared to the

corresponding anatomical area of controls [123,124].

No inverse relationship between apoptosis and Bcl-2





expression was demonstrated. Mu et al. showed that

expressed bcl-2 mRNA was signi®cantly decreased and

bax mRNA was signi®cantly increased in ALS lumbar

cord motor neurones compared to controls that consisted

of cases with non-neurological diseases, Alzheimer's

disease and Parkinson's disease [89]. No changes of bcl-2

and bax mRNA levels were seen in neurones of the

sensory nucleus of the dorsal horn. These ®ndings sug-

gest that alteration in the balance of proapoptotic and

antiapoptotic proteins contributes to spinal motor neur-

one degeneration in ALS. However, one study of thoracic

cord motor neurones in human ALS and control cases

only demonstrated an upregulation of the proapoptotic

protein Bax, without any alteration in the expression

of the antiapoptotic protein Bcl-2 [37].

More recently, results of experiments by Martin have

further suggested that altered compartmental expression

of Bcl-2 family members plays a role in the cell death of motor neurones in ALS [80]. In this study, the expression

of Bcl-2, Bcl-xL, Bax and Bak was examined in the

cytosolic- and mitochondrial-enriched membrane com-

partments. In the motor cortex and spinal cord anterior

horn, the levels of the proapoptotic proteins Bax and Bak

were increased in the mitochondrial-enriched membrane

compartment, but reduced or unchanged in the cytosol.

In contrast, the levels of the antiapoptotic protein Bcl-2

was decreased in the mitochondrial-enriched membrane

compartment in ALS motor cortex and spinal cord

anterior horn, but increased in the cytosol. On the other

hand, Bcl-xL protein levels did not differ signi®cantly in

the mitochondrial-enriched membrane fractions or

cytosolic fractions of ALS motor cortex or spinal cord.

Immunoblotting for the expression of Bax, Bak, Bcl-2

and Bcl-xL was only carried out in the membrane frac-

tions of the somatosensory cortex where no signi®cant

changes were observed between ALS and control cases.

Co-immunoprecipitation studies revealed greater Bax±

Bax interactions in the mitochondrial-enriched mem-

brane compartment of ALS motor cortex than in controls,

in contrast to lower Bax±Bcl-2 interactions in the

membrane compartment of ALS motor cortex than incontrols.

Relevance of the p53 pathway

There are con¯icting reports on the relevance of the

p53 protein in promoting apoptosis in ALS. Increased

immunoreactivity of p53 was reported by de la Monte

et al. in the motor cortex and anterior horn of the spinal

cord (lumbosacral segments more so than cervical

segments) in human post-mortem ALS tissue compared

to controls [25]. In another study using the G86R SOD1

transgenic mouse model of ALS, there was increased

p53 immunoreactivity noted in the nuclei of lumbar cord

neurones in the G86R mice compared to predominant

immunoreactivity in the cytoplasm of the wild-type

mice [24]. In the same study, PC12 cultured cells over-

expressing G86R mutant SOD1 showed both increased

expression and phosphorylation of p53, compared to cells

overexpressing wild-type SOD1. The selective subcellular

localization of p53 in the nucleus indicating its activa-

tion, enhanced expression and phosphorylation of

p53, and alteration of the Bcl-x : Bax ratio (as explained

in the previous section) in the presence of the G86R

mutated form of the Cu/Zn SOD1 gene support the

concept of p53-associated apoptosis in ALS. However, ina cross-breeding experiment between G93A SOD1 trans-

genic mice and mice lacking both p53 alleles, no sig-

ni®cant differences were observed in the time of onset

of motor dysfunction, disease progression, mortality or

lumbar anterior horn motor neurone cell counts [69].

This study suggested that activation of the p53 pathway

is not essential for motor neuronal cell death occurring in

the context of a SOD1 mutation. Similarly, in another

cross-breeding experiment between G93A SOD1 trans-

genic mice and p53-knockout mice, it was demonstrated

that the absence of p53 did not offer protection from ALS

[99]. Moreover, histological study of presymptomatic

G93A mice showed ALS-associated vacuolation within

the dendrites of motor neurones which was independent

of the p53 status.

Role of antibodies to the death receptors

Yi et al. have recently demonstrated that sera from more

than a quarter of patients with sporadic ALS induced in

vitro apoptosis of a human neuroblastoma cell line and

most contained anti-Fas antibodies [138]. In contrast,

sera from patients with Alzheimer's disease rarelyinduced apoptosis and did not contain detectable levels

of anti-Fas antibodies. In addition, ALS sera were shown

to induce apoptosis in motor neurones in mixed cultures

of rat embryonic and spinal cord cells. The affected

neurones were identi®ed as motor neurones based on

their positive staining with the monoclonal antibody SMI

32. Furthermore, the expression of Fas was only found in





the SMI 32-positive neurones. The authors of the study

suggest that autoimmunity related to anti-Fas antibodies

may contribute to neuronal cell death in a proportion

of patients with ALS.

Caspase activation/activity

Cellular models

In vitro studies using the rat phaeochromocytoma PC12

cell line have shown that SOD1 downregulation results

in an increase in IL-1b (also known as caspase-1) pro-

duction [125]. There was also increased IL-1b release

after exposure of the PC12 cells to nerve growth factor

(NGF), although NGF is known to prevent rather than

cause cell death. Therefore it was concluded that apop-

tosis brought upon by SOD1 downregulation is not dueto increase IL-1b alone but due to increased susceptibility

to the effects of this molecule. PC12 cells, in the presence

of downregulated SOD1, could be rescued from apoptosis

by blocking antibodies against both IL-1b or the IL-1

receptor antagonist (IL-1Ra), suggesting that downregu-

lation of SOD1 expression induces caspase-dependent

neuronal apoptosis. In the aforementioned study and

in another study by Ghadge et al. [47] using PC12 cells

transfected with two FALS-related mutant SODs (A4V

and V148G), two irreversible caspase inhibitors, benzyl-

oxycarbonil-Val-Ala-Asp(O-methyl)-¯uoromethylketone

(ZVAD-FMK) and acetyl-Tyr-Val-Ala-Asp-chloromethyl-

ketone (Ac-YVAD-CMK), were shown to exert a pro-

tective effect against cell death.

Pasinelli et al. demonstrated increased caspase-1

activity in the presence of oxidative stress generated

by xanthine/xanthine-oxidase (X/XO) and cleavage of

caspase-1 associated with apoptotic morphology in

mouse neuroblastoma N2a cell lines transfected with

mutant (G37R, G41D and G85R) SOD1 cDNAs [92].

The mutant SOD1 by itself was insuf®cient to fully activ-

ate the death process. Oxidative stress was necessary for

fully inducing caspase-1 cleavage and activity, resultingin apoptosis. In addition, this study showed that it is likely

that there are other caspases involved in the death

process, as inhibitors not selective for ICE, for example

ZVAD-FMK, completely blocked apoptosis, whereas ICE-

speci®c inhibitors, for example acetyl-Tyr-Val-Ala-Asp-

aldeide (Ac-YVAD-CHO), prevented only approximately

50% of the induced cell death.

SOD1 transgenic mice

Pasinelli et al. also reported cleavage and activation of

caspase-1 in two transgenic mouse models of FALS

(G37R and G85R), but not in the wild-type SOD1 mice

[92]. In support of the role of caspase-1 in SOD1-

mediated motor neurone death, cross-breeding experi-

ments have shown that the expression of a dominant

negative inhibitor of caspase-1 in a G93R SOD1

transgenic mouse model of FALS slows the progression

of motor weakness and delays mortality, but has no effect

on the timing of disease onset, in the affected mice [44].

Li et al. studied the functional role of caspase-1 and -3 in

a G93A SOD1 transgenic mouse model of FALS [71].

They detected activated caspase-1 and -3 in neurones

within the anterior horn of the spinal cord in sympto-

matic, but not in presymptomatic, mice. The broad-

spectrum caspase inhibitor ZVAD-FMK non-signi®cantlyprolonged survival of the mutant SOD1 transgenic mice.

The cerebral intraventricular administration of this agent

resulted in signi®cantly greater numbers of surviving

motor neurones in the cervical, but not the lumbar,

region of the spinal cord compared to vehicle-treated

mice. Li et al. also suggested that caspase-1 seemed to

be involved early in the disease process of ALS, while

caspase-3 probably mediated the terminal stages of

apoptosis as caspase-1 mRNA was upregulated before

that of caspase-3. Further evidence of sequential activa-

tion of caspase-1 followed by caspase-3 in the spinal cord

of SOD1 transgenic mice has been demonstrated with

immunoblotting by Vukosavic et al. [130]. The same

group has also shown by immunohistochemistry that

cleaved caspase-3 is localized within the motor neurones

of the anterior horn of these transgenic mice. Spooren

et al. using two different lines of transgenic mice with the

mutated G93A SOD1 gene (low and high copy numbers),

reported a statistically signi®cant increase in caspase-

3-like activity in both the upper and lower half of the

spinal cord in animals in advanced stages of the disease

[117]. No difference in caspase-3-like activity was

observed between lines with low and high copies of themutated G93A SOD1 gene.

Human central nervous system tissue

Further evidence implicating caspases in the pathoge-

nesis of ALS comes from two other studies in human

post-mortem tissue. Martin reported that there was





signi®cantly increased caspase-3 activity in human ALS

spinal cord anterior horn and motor cortex, but not in the

somatosensory cortex, compared to controls [80]. Li et al.

described a more than 80% increase in caspase-1 activity

in the spinal cord of ALS cases when compared to

controls [71].

Conclusion

There is good evidence (Table 2) that, in ALS, motor

neurones die by a programmed cell death or apoptotic

pathway. The morphological studies on human ALS

post-mortem tissue provide strong evidence of apoptosis,

such as nuclear and cytoplasmic condensation and the

formation of apoptotic bodies. Although not all the

studies reported have detected DNA fragmentation by

TUNEL/ISEL staining, this may be due to the fact that

since several thousand motor neurones need to be

examined to detect one motor neurone undergoing

apoptosis at a particular snapshot on a tissue section,

it is not inconceivable that apoptosis may be missed.

The increased expression of the apoptosis-related mole-

cules LeY and Par-4 in the spinal cord of ALS compared

to control cases further strengthens the fact that apop-

tosis occurs in this disease. The role of the p53 protein

in apoptosis in ALS is less clearcut as the evidence

uncovered so far has been contradictory. Arguably the

most compelling evidence that apoptosis is indeed

the mechanism involved in the pathogenesis of motor

neurone degeneration in ALS comes from the ®ndings

of alteration of the levels of the members of the Bcl-2

family of oncoproteins and the increased expression or

activation of caspases in the cellular- and tissue-based

models of ALS studied. The changes in expression of

the Bcl-2 family members generally result in a predis-

position towards apoptosis within the motor neurones

of ALS cases compared to controls. On the other hand,

caspases-1 and -3 have consistently been shown to play

an important role in the death of motor neurones in

ALS. The role of the death receptors in ALS is still sparse

at present. Although the picture is far from complete,

the weight of currently available evidence does indicate

that apoptotic motor neurone death occurs in ALS.

Therefore, to increase our understanding of the cell death

process in ALS and to develop potential therapeutic

interventions for the disease, it is imperative that further

Table 2. Evidence for apoptosis in amyotrophic lateral sclerosis

Human tissue Transgenic mice

Structural morphology Troost et al. 1995 [124]* Migheli et al. 1994 [85]

Martin 1999 [80]* Migheli et al. 1999 [84]He and Strong 2000 [56]

TUNEL/ISEL Yoshiyama et al. 1994 [140]* Migheli et al. 1994 [85]

Troost et al. 1995 [124]* Migheli et al. 1999 [84]

Ekegren et al. 1999 [37]*

Martin 1999 [80]*

He and Strong 2000 [56]

Apoptosis-related molecules Yoshiyama et al. 1994 [140]* Pedersen et al. 2000 [93]*

Pedersen et al. 2000 [93]*

Bcl-2 family of oncoproteins Troost et al. 1995 [123]* Kostic et al. 1997 [68]*

Troost et al. 1995 [123]* Vukosavic et al. 1999 [129]*

Mu et al. 1996 [89]* de Aguilar et al. 2000 [24]*

Ekegren et al. 1999 [37]* Vukosavic et al. 2000 [130]*

Martin 1999 [80]*

p53 expression de la Monte et al. 1998 [25]* de Aguilar et al. 2000 [24]*

Kuntz et al. 2000 [69]Prudlo et al. 2000 [99]

Death receptors Yi et al. 2000 [138]* Yi et al. 2000 [138]*

Caspase activation/activity Martin 1999 [80]* Friedlander et al. 1997 [44]*

Li et al. 2000 [71]* Pasinelli et al. 1998 [92]*

Li et al. 2000 [71]*

Spooren et al. 2000 [117]*

Vukosavic et al. 2000 [130]*

*a positive study.





investigation continues to unravel the complicated

programmed cell death pathways involving cytosolic,

organelle, cell surface and nuclear compartment mole-

cules [60]. For further study, as highlighted by Beckman

et al., there is a `¼ need for a combined approach

including human tissues, transgenic animals, neuronal

culture models, and in vitro biochemistry' [8].

Acknowledgements

Professor Pamela J. Shaw is supported by the Wellcome

Trust and the Motor Neurone Disease Association.

Professor Paul G Ince is supported by the Medical

Research Council, UK. Dr S Sathasivam is supported by

the University of Shef®eld Moody Endowment Fund.

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Received 19 October 2000

Accepted after revision 2 May 2001



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