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Research Collection
Doctoral Thesis
Function, regulation and involvement of Mtmr2 and Mtmr13/Sbf2in the hereditary human diseases CMT4B1 and CMT4B2
Author(s): Tersar, Kristian
Publication Date: 2008
Permanent Link: https://doi.org/10.3929/ethz-a-005767040
Rights / License: In Copyright - Non-Commercial Use Permitted
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https://doi.org/10.3929/ethz-a-005767040http://rightsstatements.org/page/InC-NC/1.0/https://www.research-collection.ethz.chhttps://www.research-collection.ethz.ch/terms-of-use
DISS. ETH.Nr. 17844
Function, Regulation and Involvement of Mtmr2 and MTMR13/Sbf2 in the Hereditary Human Diseases
CMT4B1 and CMT4B2
A dissertation submitted to
ETH ZURICH
for the degree of
Doctor of Science
presented by
Kristian Tersar
Dipl. Biol. Technische Universität Darmstadt, Germany
Born November 25, 1973 in Speyer, Germany
Citizen of Slovenia
accepted on the recommendation of
Prof. Dr. Ueli Suter, examiner
Prof. Dr. Sabine Werner, co-examiner
Dr. Philipp Berger, co-examiner
May 2008
1 THESIS OUTLINE 5
2 SUMMARY 6
3 ZUSAMMENFASSUNG 8
4 INTRODUCTION 10
4.1 The Nervous System 10
4.2 The peripheral Nervous System 11 4.2.1 Cells of the PNS 11 4.2.2 The Schwann Cell 11 4.2.3 Structure of the myelinated Axon 13 4.2.4 Myelin composition – Myelin Proteins 16
4.3 Charcot-Marie-Tooth diseases 17 4.3.1 CMT4B1 and CMT4B2 – clinical characteristics and pathology 19 4.3.2 Animal models of CMT4B1 19
4.4 The Myotubularin family 20 4.4.1 Myotubularin Related Protein 2 and Myotubularin Related Protein 13/SBF2 22 4.4.2 Myotubularin Related Protein 2 and intermediate filaments 25
4.5 Aim of the Study 27
5 MTMR13/SBF2-DEFICIENT MICE 28
5.1 Animal model for CMT4B2 28
5.2 Gene-trap disruption of Mtmr13/Sbf2 28
5.3 Expression analysis of Mtmr2, Mtmr13/Sbf2 and Dlg1/Sap97 in sciatic nerves of mutant animals 30
5.4 Behavioral analysis 33
5.5 Electrophysiology of peripheral nerves of Mtmr13/Sbf2-deficient mice 34
5.6 Progressive myelin abnormalities in peripheral nerves of Mtmr13/Sbf2-deficient mice 35
5.7 Motor and sensory nerves are affected in Mtmr13/Sbf2-deficient mice 39
5.8 Complex structures of misfolded myelin in Mtmr13/Sbf2-deficient mice 40
I
6 INTERACTION PARTNERS OF MTMR2 44
6.1 Principle of the Screening procedure and mass spectrometry-based identification of isolated proteins 44
6.2 Screening tool – Immunoprecipitation via monoclonal anti-Mtmr2 antibody 46
6.3 Identification of novel Mtmr2 interaction partners 47 6.3.1 In-gel digestion - Electrophoresis based screening 47 6.3.2 In-solution tryptic digestion – screening of total immunopreciptates 48
6.4 Expression of intermediate filament mRNA in cultured Schwann cells 51
6.5 Interaction of Mtmr2 and intermediate filaments 52 6.5.1 Immunoprecipitation with anti-intermediate filament antibodies 52 6.5.2 Coimmunoprecipitation with an anti-Mtmr2 antibody 54 6.5.3 Colocalisation of Mtmr2 and intermediate filaments 55
7 DISCUSSION 59
7.1 MTMR13/SBF2 deficient mice – an animal model for CMT4B2 59
7.2 Mtmr2 interacts with intermediate filaments 63
7.3 Conclusions and future directions 68
8 EXPERIMENTAL PROCEDURES 69
8.1 Generation of Mtmr13/Sbf2-/- mice 69
8.2 Genotyping PCR 69
8.3 Protein expression analysis on mutant and wt sciatic nerves 70
8.4 Rotarod test 70
8.5 Neurophysiology 70
8.6 Electron microscopy 71
8.7 Morphometric analysis and quantification of focally folded myelin 71
8.8 Cell Culture 72
8.9 Monoclonal anti-Mtmr2 Antibodies 72
II
8.10 Covalently coupling of antibodies to Protein A Sepharose 73
8.11 Immunoprecipitation for identification of new binding partners of MTMR2 74
8.12 Tryptic in-gel digestion of protein bands (IG) 75
8.13 Automated desalting of tryptic protein digests for MALDI measurements 75
8.14 Identification of of proteins from 1-D gels – MS/MS Acquisition and Mass spectrometry 76
8.15 Identification of of proteins in solution – Tryptic digest in solution (IS) 77
8.16 MS/MS Aquisition via LCQ Deca after tryptic in-gel digestion 77
8.17 Protein identification 78
8.18 Co-Immunoprecipitaion 78
8.19 RT PCR 79
8.20 Primary Rat Schwann Cell (RSC) Culture 82
8.21 Immunstaining of differentiated primary RSC 82
8.22 VSV-tagged intermediate filament Constructs 83
8.23 Specificity Test of mouse monoclonal anti-intermediate filament antibodies 85
9 LIST OF MATERIALS 88
9.1 General Solutions and Fixatives 88
9.2 Blocking solutions 88
9.3 Slides, Plastic ware, Mouting and Embedding Media 88
9.4 Semi Thin Staining solution 89
9.5 Antibody coupling solutions 89
9.6 Immunoprecipitation 89
9.7 Western Blotting 89
9.8 Tail lysis Solutions 90
9.9 Polymerase Chain Reaction 91
III
9.10 Agarose Gel Elecrtophoresis 91
9.11 Primary Antibodies 91
9.12 Secondary Antibodies 92
9.13 Mass spectroscopy 92
9.14 Instruments and Software 92
9.15 Mouse Lines 93
10 REFERENCES 94
11 LIST OF FIGURES 103
12 ACKNOWLEDGEMENTS 105
13 CURRICULUM VITAE FEHLER! TEXTMARKE NICHT DEFINIERT.
IV
Thesis outline
1 THESIS OUTLINE
This thesis is based on the following publications:
Tersar, K., Boentert, M., Berger, P., Bonneick, S., Wessig, C., Toyka, K.V., Young,
P., and Suter, U. (2007). Mtmr13/Sbf2-deficient mice: an animal model for CMT4B2.
Hum Mol Genet 16, 2991-3001.
Berger, P., Berger, I., Schaffitzel, C., Tersar, K., Volkmer, B., and Suter, U. (2006).
Multi-level regulation of myotubularin-related protein-2 phosphatase activity by
myotubularin-related protein-13/set-binding factor-2. Hum Mol Genet 15, 569-579.
All Experiments described in chapters 5 and 6 have been carried out by the author,
with the exception of results presented in chapter 5.5 (performed in collaboration with
Dr. Carsten Wessig and Prof. Dr. Klaus V. Toyka from the Department of Neurology,
University of Wurzburg, Wurzburg, Germany) and in chapters 5.6 – 5.8 (performed in
collaboration with Dr. Matthias Boentert and Dr. Peter Young from the Department of
Neurology and Interdisciplinary Center of Clinical Research, University of Munster,
Munster, Germany).
The thesis begins with a general introduction to the nervous system, Schwann cell
biology, the myotubularin family proteins MTMR2 and MTMR13/SBF2 and the related
CMT4B disease. The results of the first publication are described in chapter 5.
Chapter 6 describes the results of the screening for new Mtmr2 interaction partner
and contains an experiment from the second publication. Afterwards the results are
discussed in detail in chapter 7 and an outlook on future directions is provided.
Subsequent to the discussion the experimental procedures and materials used for
the experiments are described in chapters 8 and 9. Chapters 10 and 11 give a
detailed reference list and the list of figures.
5
Summary
2 SUMMARY
Charcot-Marie-Tooth (CMT) diseases comprise a large group of genetically
heterogeneous hereditary motor and sensory neuropathies (HMSN). With a
prevalence of 1:2500 they range among the most common inherited neurological
disorders affecting the peripheral nervous system (PNS). Mutations in the
Myotubularin-Related Protein-2 (MTMR2) or MTMR13/Set-Binding Factor-2 (SBF2)
genes are associated with the autosomal recessive disease subtypes CMT4B1 or
CMT4B2. Both CMT4B subtypes share similar pathological and clinical
characteristics, including a demyelinating neuropathy associated with reduced nerve
conduction velocity (NCV), focally folded myelin, and an onset of the disease in early
childhood. The proteins MTMR2 and MTMR13/SBF2 belong to the Myotubularin
protein family, which contains 8 active and 6 inactive members. MTM1, an active
member, is the founder of the Myotubularin family and responsible for the X-linked
Myotubular Myopathy. The active Myotubularin family members, including MTMR2,
are phosphoinositide D3-phosphatases. The inactive family members, including
MTMR13/SBF2, contain inactivating substitutions in their phosphatase domain. Two
animal models have been generated to demonstrate that CMT4B1 is caused by the
loss of MTMR2. To better understand the disease mechanism in CMT4B2 the
generation and analysis of a mouse model mimicking the human disease is of major
importance. For a better understanding of the cellular functions of MTMR2 it is
important to identify protein complexes this protein is involved in.
The first goal of of my thesis project was to generate and analyze a Mtmr13/Sbf2-
deficient mouse line. Therefore I used embryonic stem cell line XH212 from
BayGenomics carrying a gene trap plasmid between exon 14 and 15. Only mice
homozygous for the gene trapped Mtmr13/Sbf2 gene displayed a phenotype, as was
expected in comparison to the human disease and the disease models for CMT4B1.
These animals reproduced myelin outfoldings and infoldings in motor and sensory
peripheral nerves as the pathological hallmarks of CMT4B2, concomitant with
decreased motor performance. The number and complexity of myelin misfoldings
increased with age, and was associated with axonal degeneration and decreased
compound motor action potential amplitude. Prolonged F-wave latency indicated a
6
Summary
mild NCV impairment. Loss of Mtmr13/Sbf2 did not affect the levels of its binding
partner Mtmr2 or the Mtmr2-binding Dlg1/Sap97 in peripheral nerves. With the
Mtmr13/Sbf2-deficient mouse line I generated a suitable animal model for the human
disease CMT4B2, and provided further evidence that MTMR13/SBF2 is the disease-
causing gene in CMT4B2.
An important issue in understanding the characteristics of CMT4B is to know the
protein interactors of MTMR2 in Schwann cells, as such interactors might integrate
and control the functions of MTMR2 and explain the involvement of MTMR2 in the
CMT4B disease. I used mass spectrometry (MS)-based proteomics to screen for
protein complexes MTMR2 is involved in. In an MS-based screen I could confirm the
myotubularin family members Mtmr5/Sbf1, Mtmr13/Sbf2, and Mtmr12/3-PAP as
binding partners of Mtmr2. Besides the already described binding partners of
MTMR2, I could confirm the interaction of MTMR2 with proteins of the intermediate
filament family. Using co-immunoprecipitation and western blot analysis I showed
that Vimentin, Desmin, Peripherin, Glial fibrillary acidic protein (GFAP) and the
already described binding partner Neurofilament light chain protein (NF-L) interact
with MTMR2 in lysates of mouse Schwann cell line MSC80 cells.
7
Zusammenfassung
3 ZUSAMMENFASSUNG
Charcot-Marie-Tooth (CMT) Krankheiten beschreiben eine grosse Gruppe
vererbbarer motorischer und sensorischer Neuropathien mit einem heterogenen
genetischen Hintergrund. Mit einer Prävalenz von 1:2500 gehören sie zu einer der
meist verbreiteten neurologischen Krankheiten die das periphere Nervensystem
(PNS) betreffen. Mutationen im Myotubularin Related Protein-2 (MTMR2) oder
MTMR13/Set-Binding Factor-2 (SBF2) Gen führen zu den autosomal rezessiven
Krankheitssubtypen CMT4B1 oder CMT4B2. Beide CMT4B Krankheitssubtypen
haben sehr ähnliche pathologische und klinische Ausprägungen mit einem
Krankheitsbeginn im Kindesalter, gekennzeichnet durch eine demyelinisierende
Neuropathie, welche mit einer verminderten Nervreizleitgeschwindigkeit (NCV) und
örtlich auftretenden Myelinfaltungen einher geht. Das MTMR2 Protein und das
MTMR13/SBF2 Protein gehören beide zur Myotubularin Familie, einer sogenannten
dual-spezifischen Phosphatase-Familie, die 14 aktive und inaktive Proteine
beinhaltet. MTM1, ein aktives Mitglied, ist das Gründerprotein der Myotubularin
Familie und verantwortlich für die sogenannte X-chromosomal vererbte myotubuläre
Myopathie. Die aktiven Mitglieder der Myotubularin Familie, zu denen auch MTMR2
gehört, sind Phosphoinositid-D3-Phosphatasen. Die inaktiven Familienmitglieder, zu
denen MTMR13/SBF2 gehört, haben eine inaktivierende Substitution in ihrer
Phosphatase Domäne. Es wurden zwei Tiermodelle hergestellt um zu zeigen, dass
die CMT4B1 Krankheit durch eine Null-Mutation im MTMR2 Protein hervorgerufen
wird. Um den Mechanismus der CMT4B2 Krankheit besser zu verstehen, ist es von
grösster Bedeutung, ein Maus Modell herzustellen, welche die menschliche
Krankheit imitiert. Um die zellulären Funktionen von MTMR2 besser zu verstehen, ist
es ebenso wichtig die Proteinkomplexe zu identifizieren, an welchen es beteiligt ist.
Der erste Teil meiner Doktorarbeit war es, eine Maus Linie mit einem fehlerhaften
Mtmr13/Sbf2 Gen herzustellen. Dafür habe ich die embryonale Stammzelllinie XH212
von Baygenomics, welche ein sogenanntes „Gen-Trap“ Plasmid zwischen den Exons
14 und 15 trägt, verwendet. Nur Mäuse welche homozygot für das „Gen-Trap
insertierte“ Mtmr13/Sbf2 Gene waren, zeigten eine phänotypische Ausprägung, wie
es auch von der menschlichen Krankheit und den beiden CMT4B1 Mausmodellen
8
Zusammenfassung
her bekannt ist. Diese Tiere bildeten Myelin Ein- und Ausfaltungen in den
motorischen und sensorischen Nerven aus, dem Kennzeichen der CMT4B2
Krankheit, begleitet durch einen Rückgang in der motorischen Leistungsfähigkeit. Die
Anzahl und Vielschichtigkeit der Myelin Fehlfaltungen stieg mit zunehmendem Alter
der Tiere, einhergehend mit axonaler Degeneration und einen verminderten
Amplitude des Muskel- und Nervensummenpotentials. Eine längere F-Wellen Latenz
weist auf eine leichte Beeinträchtigung der Nervreizleitgeschwindigkeit (NCV) hin.
Der Verlust des Mtmr13/Sbf2 Proteins beeinträchtigte jedoch nicht den Pegel des
Bindepartners Mtmr2, sowie den Pegel des an Mtmr2 bindenden Proteins
Dlg1/Sap97 in Nerven des peripheren Nervensystems. Mit der Mtmr13/Sbf2
defizienten Maus Linie habe ich ein entsprechendes Mausmodell für die menschliche
Krankheit CMT4B2 hergestellt und gleichzeitig bewiesen, dass das MTMR13/SBF2
Gen, das Gen ist, welches für die CMT4B2 Krankheit verantwortlich ist.
Um die Eigenheiten der CMT4B Krankheiten zu verstehen, ist es wichtig die
Proteinbindepartner von MTMR2 in der Schwann Zellen zu kennen. Dies ist wichtig,
da solche Bindepartnerproteine die Funktion von Mtmr2 integrieren und kontrollieren
und die Beteiligung von MTMR2 an der CMT4B Krankheit erklären können. Daher
habe ich eine massenspektrometrische Raster-Methode für Proteine verwendet, um
nach Proteinkomplexen zu suchen an denen MTMR2 beteiligt ist. Mit der
massenspektrometrische Raster-Methode konnte ich alle bekannten Bindepartner
von MTMR2 die zur Myotubularin Familie gehören, nachweisen. Neben den schon
beschrieben Bindepartnern von MTMR2, konnte ich die Bindung von MTMR2 an
Proteine der Intermediärfilament-Familie nachweisen. Durch Zuhilfenahme der Co-
Präzipitationsmethode und anschliessender Western Blot Analyse, zeigte ich, dass
die Proteine Vimentin, Desmin, Peripherin, Glial fibrillary acidic Protein (GFAP) und
der unlängst beschriebene Bindepartner Neurofilament light chain Protein (NF-L) in
Lysaten aus den Maus Schwann Zellen MSC80, mit MTMR2 interagieren.
9
http://dict.leo.org/ende?lp=ende&p=wlqAU.&search=Vielschichtigkeit
Introduction
4 INTRODUCTION
4.1 THE NERVOUS SYSTEM
The mammalian nervous system is divided into the central nervous system (CNS)
and the peripheral nervous system (PNS). The CNS consists of the brain and the
spinal cord, while the PNS is made up of nerves that connect theCNS with peripheral
structures. The nerves of the PNS innervate skeletal, cardiac, and smooth muscle, as
well as the glandular epithelium. Sensory fibers of the PNS connect the CNS with the
surrounding environment of the organism, and with its internal state. The nervous
system is composed of two major cell types, neurons and glial cells. As the basic
structural and functional units of the nervous system, neurons are specialized to
receive information, transmit electrical impulses, and influence other neurons and
effector tissues (Haines et al., 2002). Glial cells provide neurons with structural
support and maintain the appropriate environment that is essential for neuronal
function.
The fast transmission of electrical impulses in the mammalian nervous system is
achieved by saltatory conduction of action potentials along the axon towards their
target cell. Axons are enwrapped by glial cells, which built up a myelin segment
called an internode. The nodes of Ranvier are myelin-free segments located between
adjacent internodes. Thereby axons are discontinuously insulated. Only at the nodes
of Ranvier are the axons electrically charged relative to the fluids surrounding them.
Thus the electrical impulses (action potentials) jump from node to node, achieving a
conduction up to 100m/sec, which some 10 times faster than in an unmyelinated
axon (Brown, 2002).
10
Introduction
4.2 THE PERIPHERAL NERVOUS SYSTEM
4.2.1 CELLS OF THE PNS
The glial cell types in PNS are the Schwann cells and the satellite glia. These may be
compared with the major glial cell types of the CNS, the astrocytes and
oligodendrocytes. The astrocytes are responsible for nutrition of the neurons and are
involved in the control of the blood brain barrier (Kandell et al., 2000). The
oligodendrocytes enwrap with their numerous processes different axon and built up
the myelin sheath in the CNS.
The PNS contains myelinating as well as non-myelinating Schwann cells
(Kuhlenbaumer et al., 2005). The Schwann cells insulate axons (with a diameter
more than 1µm) by building up a myelin sheath. In contrast to the oligodendrocytes of
the CNS, Schwann cells do not myelinate multiple axons, but rather establish a one-
to-one relationship at the promyelinating state. Non-myelinating Schwann cells show
similarities to astrocytes and are likely to have metabolic and mechanical support
functions of small caliber axons (smaller than 1µm in diameter) (Jessen, 2004).
Besides Schwann cells and axons, fibroblasts are found, (Berger et al., 2002b) and
also macrophages (Maurer et al., 2002). Macrophages play a role as antigen-
presenting cells and as effector cells that phagocytose damaged myelin (Craggs et
al., 1984).
4.2.2 THE SCHWANN CELL
The Schwann cells of the peripheral nerves originate mainly from the neural crest
cells, except for a fraction derived from boundary cap cells at the margin of the neural
tube. Neural crest cells (NCC) emigrate after the closure of the neural tube from the
most dorsal part of the neural tube. These highly migratory and multipotent stem cells
migrate along two major streams, in a lateral stream and a ventral stream (Bhatheja
and Field, 2006; Jessen and Mirsky, 2002). The lateral stream gives rise to
melanocytes in skin and the ventral stream gives rise to sensory neurons in the
dorsal root ganglia, to autonomic neurons, and to glia cells. The NCC give also rise to
smooth muscle cells and fibroblasts (Jessen and Mirsky, 2005; Lobsiger et al., 2002).
11
Introduction
Before a Schwann cell (either myelinating or non-myelinating) is formed two, other
cell types appear in this lineage, namely the Schwann cell precursor cells (SPCs) and
the immature Schwann cells (Fig. 4.1). Numerous molecules have been identified
regulating Schwann cell development. The Sox 10 protein is essential for the
generation of the peripheral glia from NCC, and is expressed by all neural crest cells
(Britsch et al., 2001). Neuregulin1 (NRG1) derived from axons is important for SPC
survival and promotes the SPC to Schwann cell transition (Garratt et al., 2000). To
maintain the survival of immature Schwann cells, NRG1, Laminin, and ETS
transcription factors have to be expressed (Fig. 4.1). Myelination is promoted by
NRG1, BRN2, KROX20 and OCT-6. The transition from SPCs to Schwann cells
come along with major changes in the cytoarchitecture of the peripheral nerves. The
immature Schwann cells exit the cell cycle and start to form myelinating and non-
myelinating Schwann cells (Fig. 4.1). The fate of the immature Schwann cells
(myelinating or non-myelinating) is determined by the diameter of the axon which
they contact.
Figure 4. 1 The mouse Schwann cell lineage. The developmental transitions of the main cell types in Schwann cell development is illustrated from embryonic day 9 (E9) to postnatal day 15 (P15). The dashed lines indicate reversible (postnatal) transitions. The cell fates are illustrated from the neural crest cell (NCC) to the myelinating and non-myelinating Schwann cell. Bold letters indicate the proteins that are important for the transition steps or maintain a cell fate. The proteins expressed by myelin forming and non-myelinforming Schwann cells are depicted in bold/italic letters (adapted and modified from Jessen and Mirsky, 2005).
12
Introduction
If a Schwann cell contacts a large caliber axon (with a diameter more than 1µm) then
myelination occurs. When a Schwann cell ensheaths small diameter axons (with a
diameter less than 1µm), it become a non-myelinating cell (Jessen and Mirsky,
2005). Myelination involves a combination of down-regulation and up-regulation of
proteins. Marker proteins expressed in myelinating Schwann cells are the myelin
proteins P0, MAG, MBP or PMP22. Non-myelinating Schwann cells show expression
of p75, L1, O4 and GFAP (Fig. 4.1, bold italic) (Jessen and Mirsky, 2002).
4.2.3 STRUCTURE OF THE MYELINATED AXON
The precise arrangement of Schwann cells along axons is an important event before
myelination starts. Initially Schwann cells select individual axons from a nerve bundle
and establish a 1:1 relationship through a process termed "radial sorting" (Simons
and Trotter, 2007). Basal lamina is produced by the Schwann cells before they start
to wrap around an axon. Adhesion molecules (Necl1 and 4) establish axon-glia
contact, and the intracellular asymmetrically distributed Par-3 protein together with
the basal lamina establish a radial axis (Fig. 4.2. A)
Figure 4. 2 Glia-axon recognition in the PNS (A) Necl4 and Necl1 establish glial-axon contact. The asymmetric distribution of the intracellular protein Par-3 and the basal lamina on the outer side establish radial axis. (B) MAG and Necl4 establish the longitudinal polarity and form the internode. Gliomedin, NF-155 and TAG-1 accumulate to establish the presumptive node of Ranvier. (adapted and modified from Simons and Trotter, 2007)
13
Introduction
The longitudinal axis is defined by accumulation of MAG and Necl4 proteins at the
axon-glial junction, which establishes the region forming the future internode.
Accumulation of the proteins Gliomedin, NF-155 and TAG-1 occurs at or around the
future node of Ranvier (Fig. 4.2 B) This compartmentation of the myelin membrane
reaches its maximum extent in the fully myelinated axon (Simons and Trotter, 2007).
The myelin sheath is formed by extension of the Schwann cell plasma membrane in
a spiral growing around the axon. Thereby the Schwann cell nucleus is located
ouside the myelin sheath. Only a small amount of cytoplasma persists at the outer
myelin compartment, which is called the abaxonal domain. The adaxonal domain is
the innermost wrap of the Schwann cell containing cytoplasm adjacent to the axon.
Adaxonal and abaxonal domains are linked via cytoplasmic channels in the Schmidt-
Lanterman incisures (Fig. 4.2, A). They enable transport of small molecular weight
substances between the inner and the outer cytoplasmic domains of the Schwann
cell. The adaxonal domain mediates the contact to the axon. The abaxonal domain
expresses extracellular matrix receptors (Bhat, 2003; Previtali et al., 2001).
Figure 4. 3 Schematic presentation of myelin sheath compartments. (A) Schematic depiction of the longitudinal organization of the PNS myelin. Node, Paranode, Juxtaparanode and Internode are depicted. The coloured axon represents the compartmentation borders and shows important proteins expressed by the axon. Neurofascin 155 indicates the paranodal region. Microvilli, the basal lamina and the incisures are depicted. (B) The myelinating Schwann cell in an “unrolled” presentation. The compartments of the Schwann cell are depicted. Also the regions of compact and non-compact myelin. (adapted and modified from Scherer and Arroyo, 2002)
14
Introduction
The compartment of the myelin sheath can be divided into compact and non-compact
myelin. The internodal compartment consists mostly of compact myelin. The length of
an intermodal compartment is (depending on the axon diameter) about 0.5mm, with
a diameter of 2.5 – 2.8 µm (Salzer, 2003). The compact myelin sheath is formed by
fusion of adjacent Schwann cell membranes. Thereby the major dense line is formed
by cytoplasmic membrane leaflets, while extracellular leaflets form the interperiod
lines (Scherer and Arroyo, 2002). The compact myelin inhibits ion exchange during
nerve conduction by having low capacitance and high resistance. The Schmidt-
Lanterman incisures (Fig. 4.2, A and B) radially traverse the compact myelin. A
cytoplasmic channel extends as well over the entire length of the internode,
containing the outer mesaxon (comprising the membranes that connect the outer,
abaxonal Schwann cell membrane and compact myelin). A comparable channel is
present at the inner surface containing the inner mesaxon. The major dense line
opens towards the node and the paranodal loops contact the axon and form the non-
compact myelin (Fig. 4.3 and 4.4.). The axo-glial junctions are mediated in the
paranodal region via the proteins Caspr and Contactin on the axonal side, and by
neurofascin (NF) 155 on the Schwann cell side. In the juxtaparanodal region
(adjacent to the paranodal region) a protein called Caspr2 is enriched, as are the
potassium channels Kv1.1 and Kv1.2 (Rasband et al., 1998; Schafer and Rasband,
2006) (Fig. 4.4). The node of Ranvier is located between two paranodal junctions and
its length depends on the axon diameter (1-5µm). Voltage gated Na+ and K+
channels are accumulated at the node of Ranvier and mediate the transmembrane
currents to enable rapid saltatory conduction. The axonal side at the node of Ranvier
contains the voltage gated channels Nav1.6 (main representative) as well as Nav1.2,
Nav1.8, Nav 1.9, and the cytoskeletal and scaffolding proteins AnkyrinG βIV and
Spectrin that cluster the sodium channels. Other important proteins are the CAMs
neurofascin-186 and neuri-glia related NrCAM (Susuki and Rasband, 2008).
Schwann cell microvilli contact the node via gliomedin, which binds to axonal NF-186
(Schafer and Rasband, 2006).
15
Introduction
Figure 4. 4 Node, Paranode and Juxtaparanod. Composition and structure of the nodal region and the adjacent paranodal and the juxtaparanodal regions. The regions can be defined morphologically and via their protein compositions (adapted and modified from Scherer and Arroyo, 2002).
4.2.4 MYELIN COMPOSITION – MYELIN PROTEINS
The dry mass of of the PNS myelin is characterized by high lipid (70 – 85%) and low
protein (15 – 30%). Though there are no “myelin-specific” lipids, myelin is enriched
for cerebrosides and cholesterol (Garbay et al., 2000). Lipids play a crucial role in
assisting nerve conduction and provide an inert insulation (Menon et al., 2003). The
PNS myelin contains a high portion of glycoproteins (~60% of total protein) and is
enriched with basic proteins (~20% of total protein). Glycoproteins are Protein zero
(P0) and Peripheral myelin protein 22 (PMP22). The major basic protein is the
Myelin-basic protein (MBP) (Berger et al., 2002b; Suter and Scherer, 2003) (Fig. 4.5).
16
Introduction
Figure 4. 5 Proteins of the compact and non-compact myelin Compact myelin contains P0, MBP and PMP22. Non-compact myelin contains MAG, DM20, E-cadherin Cx32, and claudin (of unknown subtype) (adapted and modified from Scherer and Arroyo, 2002).
P0 is the major protein in the PNS myelin, constituting about 50 to 60% of the total
myelin protein, and interacts directly with PMP22. PMP22 is also part of the compact
myelin. PMP22 has major impact on myelination and maintenance of the myelin
sheath. The function of PMP22 is highly dosage dependent (Suter and Scherer,
2003). MBP is a minor compact myelin component. The loss of MBP is a reliable
marker for demyelinaton (Martini and Schachner, 1997). Myelin-associated
Glycoprotein (MAG) is a protein of the non-compact myelin. MAG is also located in
the paranodal loops and is important for axonal growth and regeneration (Hu and
Strittmatter, 2004). Connexin 32 (Cx32), also a protein of the non-compact myelin, is
located in the gap junctions of Schwann cells. These gap junctions mediate radial
diffusion across incisures (Meier et al., 2004). E-cadherin is localized in the
paranodes, the Schmidt Lanterman incisures, and the outer mesaxon. There it forms
adherens junctions with α- and β-catenin (Young et al., 2002).
4.3 CHARCOT-MARIE-TOOTH DISEASES
J.M. Charcot and P. Marie and, independently, H.H Tooth described in the late 19th
century a hereditary peripheral neuropathy. What is now called Charcot-Marie-Tooth
(CMT) disease or Hereditary Motor and Sensory Neuropathies (HMSN), comprise a
genetically heterogeneous group of inherited disorders affecting myelinated axons in
the peripheral nervous system (Berger et al., 2006b; Dyck et al., 1993; Niemann et
17
Introduction
al., 2006) with a prevalence of approximately 1:2500 (Skre, 1974). The disease is
characterized by progressive distally accentuated muscle weakness and atrophy.
Based on clinical, electrophysiological and histological data, CMT has been
subdivided into demyelinating (CMT1 CMT3 and CMT4) and axonal (CMT2) forms
(Berger et al., 2002b). The demyelinating subtypes CMT4 belong to the autosomal
recessive forms of CMT. CMT4 forms account for about 5% of CMT neuropathies in
western countries and often apperar as isolated cases because of small numbers of
siblings (Niemann et al., 2006; Suter and Scherer, 2003). Demyelinating
neuropathies are diagnosed by reduced nerve conduction velocity (NCV). Axonal
loss and muscle atrophy are also observed, most likely as secondary effects due to
the tight interaction and communication between myelinating Schwann cells, axons,
and muscle cells (Suter and Scherer, 2003). Axonal forms of CMT are characterized
by a reduction of the compound muscle action potential (CMAP) amplitude due to a
loss of myelinated axons (Zuchner and Vance, 2006). Dissection of the cellular
functions of the gene products altered in CMT (Fig. 4.6) as well as the generation of
detailed pathophysiological models are of crucial importance to understand the
underlying common as well as distinct disease mechanisms which may affect
Schwann cells, axons, or both.
Figure 4. 6 Schematic overview highlighting proteins that are mutated in CMT. The figure depicts the locations of the wild-type proteins encoded by the genes that are mutated in CMT. Proteins have been assigned to Schwann cells and/or neurons, respectively, when expression and the observed form of CMT overlap (adapted and modified from Niemann et al., 2006)
18
Introduction
4.3.1 CMT4B1 AND CMT4B2 – CLINICAL CHARACTERISTICS AND
PATHOLOGY
The gene responsible for the severe autosomal recessive CMT type 4B1 has been
identified as the Myotubularin Related Protein-2 gene (MTMR2; (Bolino et al., 2000)).
The disease onset is in early infancy, and the symptoms are those typical for a
demyelinating neuropathy. Patients exhibit reduced nerve conduction velocity, and
the histological analysis revealed typical focally folded myelin sheaths and
demyelination (Previtali et al., 2007). Muscle atrophy and weakness proceed towards
the proximal muscles and a wheelchair is needed from late childhood on. Intelectual
functions are not affected. The nerve conduction velocities are markedly reduced and
range from 9-20m/sec. The distal latency is prolonged and the amplitude is markedly
reduced (0.7 – 1mV) (Houlden et al., 2001). The autosomal recessive form CMT type
4B2 also shows focally folded myelin sheaths, which is due to mutations in the
MTMR13/Set-binding-Factor-2 (SBF2) gene. The phenotype in human CMT4B2
patients is usually less severe than in CMT4B1. The disease onset is around the age
of 8, involving motor and sensory defects. The neurophysiology is similar to the 4B1
subtype, although a NCV of 22m/sec suggests a milder phenotype. In some families
the neuropathy segregates with early onset glaucoma (Azzedine et al., 2003; Hirano
et al., 2004; Senderek et al., 2003).
The histological hallmark of both diseases is focally folded myelin from the outer
myelin sheaths (myelin outfoldings). The protrusions contain axon as well as
Schwann cell cytoplasm (Quattrone et al., 1996). Inward protrusions are present
extending towards the axon and are called “myelin infoldings”. Demyelination and
remyelination events can be inferred from numerous Schwann cell processes or
basal lamina structures encircling some fibers ("onion bulbs") (Quattrone et al.,
1996).
4.3.2 ANIMAL MODELS OF CMT4B1
Two MTMR2 "knockouts" have been generated as animal models of CMTB41 (Bolino
et al., 2004; Bonneick et al., 2005). In addition one conditional Mtmr2-null mouse
model was generated using the Cre/loxP system (Bolis et al., 2005). The conditional
19
Introduction
null mice were viable and showed no significant functional impairment. Behavioral
and electrophysiological tests suggest a neuromuscular defect. Motor and sensory
nerves are affected, showing typically myelin outfoldings, mainly at the paranodal
loops, starting at 3-4 weeks after birth. 12 month-old Mtmr2-null animals show myelin
outfoldings also at Schmidt-Lanterman incisures. These mice also show a
spermatogenesis defect, consistent with one CMT4B1 family (Bolino et al., 2004).
The mouse model produced by Bonneick and colleagues (Bonneick et al., 2005) also
mimics a mutation found in one familial CMT4B1 case. The animal model has been
produced by inserting an E276X mutation in exon 9. Mice homozygous for this
mutation show myelin outfoldings similar to those observed in the Mtmr2-null mouse
model produced by Bolino and colleagues. No electrophysiological and behavioral
alterations were observed and the testis appeared normal. Axonal loss was observed
in later stages (15 months) in distal nerves (Bonneick et al., 2005). The mouse model
based on the Cre/loxP system was used to generate two conditional Mtmr2-null lines
with specific ablation in Schwann cells or motor neurons. Only the Schwann-cell
specific ablation displayed the phenotype also achieved with the Mtmr2-null mouse
model (Bolis et al., 2005). It is possible to conclude that Mtmr2 in Schwann cells is
sufficient and necessary to provoke a condition resembling CMT4B1 with myelin
outfoldings, although Mtmr2 still might have a function in motor neurons (Previtali et
al., 2007).
4.4 THE MYOTUBULARIN FAMILY
MTMR2 and MTMR13/SBF2 both belong to the myotubularin family, which in turn
belongs to the tyrosine/dual-specific phosphatase superfamily (PTP/DSP). The
myotubularin family consists of 14 members in humans (Fig. 4.7, A). The
myotubularin family comprises both catalytically active and inactive phosphatases.
Inactive myotubularin phosphatases have divergent residues in the catalytically
active Cys-X5-Arg motif in the catalytic pocket (containing substitutions in the
Cysteine and Arginine residues) (Laporte et al., 2003). Proteins of the MTM family
share the same protein domain core. The structural hallmarks of myotubularins are a
PH-GRAM (pleckstrin homology glucosyltransferases, Rab-like GTPase activators
and myotubularins) domain, a large Phosphatase domain (PTP/DSP) and a coiled
coil domain. The Phosphatase domain is a large structural unit. It contains N-
20
Introduction
terminally a RID (Rac-induced recruitment domain) and C-terminally a SID (SET-
interacting domain) motif (Fig. 4.7, B) (Begley et al., 2003; Robinson and Dixon,
2006).
Figure 4. 7 The human Myotubularin family Panel (A) shows the phylogenetic tree of the MTM protein family. (B) Domains within the family members are indicated from N- to C-terminus. DENN (purple), PH-G (red), Phosphatase (dark blue) or inactive Phosphatase (blue), coiled-coil (green), FYVE (grey), PH (orange in hMTMR13 and hMTMR5), and PDZ binding motif (yellow in hMTMR1 and hMTMR2). MTMs with active phosphatase domains are shown in bold (adapted and modified from Clague and Lorenzo, 2005).
The phosphatase domain of MTM1 was found to catalyze the removal of 3-phoshate
from PtdIns3P at the D3 position of the inositol ring (Taylor et al., 2000).
Subsequently it was shown that also the MTMR proteins 1, 2, 3, 4, 6, and 7 hydrolyze
the 3-phoshate from PtdIns3P (Berger et al., 2002; Kim et al., 2002; Laporte et al.,
2002; Schaletzky et al., 2003). MTM1, MTMR1, MTMR2 and MTMR6 have been
reported to hydrolyze the 3-phosphate from PtdIns(3,5)P2 (Begley et al., 2003;
Berger et al., 2002; Schaletzky et al., 2003). Three members of the MTM family are
known to be involved in human diseases. Myotubularin, the founding member of the
family, was originally identified as the disease-causing gene in X-linked myotubular
myopathy (Laporte et al., 2003). Several heteromeric interactions of MTM family
members have been described. It seems to be common that an active Myotubularin
family member interacts with an inactive family member. It has been shown that
MTM1 and MTMR2 interact with MTMR12/3-PAP (Nandurkar et al., 2003), MTMR6
and MTMR7 with MTMR9 (Mochizuki and Majerus, 2003). MTMR2 also interacts with
MTMR5/Sbf1 and MTMR13/Sbf2 (Berger et al., 2006; Kim et al., 2003; Robinson and
Dixon, 2005). MTMR2 has also been shown to dimerize through its coiled-coil
domain (Berger et al., 2003).
21
Introduction
4.4.1 MYOTUBULARIN RELATED PROTEIN 2 AND MYOTUBULARIN
RELATED PROTEIN 13/SBF2
Mutations in the MTMR2 gene and MTMR13/SBF2 gene lead to CMT4B1 and
CMT4B2 respectively. For a detailed list of mutations in MTMR2 and MTMR13/SBF2
gene see: http://www.molgen.ua.ac.be/CMTMutations/Home/Default.cfm.
These two CMT forms are clinically indistinguishable, suggesting that these two
proteins have related functions. MTMR2 and MTMR13/SBF2 proteins have been
shown to exist as a complex (Berger et al., 2006; Robinson and Dixon, 2005). The
domain structure of both proteins is shown in Figure 4.8.
Figure 4. 8 Schematic presentation of the MTMR2 and MTMR13/SBF2 domain structure. DENN (white, in MTMR13/SBF2), PH-G (yellow), Phosphatase (green) or inactive Phosphatase (green – crossed through), coiled-coil (orange), RID (grey, in MTMR2), SID (blue) PH (off-white, in MTMR13/SBF2) and PDZ (red, in MTMR2). The indicated RID and SID sub-domains belong structurally to the Phosphatase binding motif. Some selected mutations are depicted that lead to CMT4B1 and CMT4B1.
The pleckstrin homology-GRAM (PH-G) domain, present in MTMR2 and
MTMR13/SBF2, mediates the membrane attachment by binding to
phosphoinositides. The PH-G domain of MTMR2 binds to PI(4)P, PI(5)P, PI(3,5)P2,
and PI(3,4,5)P3. The PH-G domain of MTMR13/SBF2 can bind to the same
phosphoinositides as MTMR2 (Berger et al., 2006; Berger et al., 2003).
MTMR13/SBF2 contains a C-terminal PH domain. A consensus motif of this PH
domain is required for binding to PI(3,4,5)P3 (Robinson and Dixon, 2005). The
function of the DENN domain of MTMR13/SBF2 is not understood. Several DENN
domain-containing proteins have been shown to regulate or associate with Rab
family GTPases, suggesting involvement in membrane trafficking. The coiled-coil
dimerization module is involved in membrane association of MTMR2 (Berger et al.,
2003). The coiled-coil domains do not directly mediate the interaction of MTMR2 and
22
Introduction
MTMR13/SBF2, and therefore further domains must be involved (Berger et al.,
2006). Also present in both proteins is a SET-interacting domain (SID) that was
initially described as mediating the interaction of MTMR5 with the SET-domain of
ALL1, human orthologue of trithorax. This suggests a possible nuclear localization of
MTMR2 and MTMR13/SBF2, which has already been reported in some studies (Cui
et al., 1998). MTMR2 contains an additional PDZ (PSD-95/DLG1/ZO-1) binding site
at the C-terminus (Robinson and Dixon, 2005). The RID sub-domain is the putative
binding site for the class IV intermediate filament neurofilament light chain (NF-L)
protein. The interaction of MTMR2 and NF-L was shown in Schwann cells as well as
neurons (Previtali et al., 2003a). The phosphatase activity of MTMR2 shows a
substrate specificity towards PI3P and PI(3,5)P2, and dephosphorylates the inositol
ring at the D3 position (Berger et al., 2002). Berger et al., also demonstrated with an
elegant expression system that MTMR2 and the MTMR2//MTMR13/SBF2 complex
use PI3P and PI(3,5)P2 as specific substrates (Fig. 4.9). The activity towards other
phosphoinositides was at background level. The activity of the
MTMR2//MTMR13/SBF2 complex is more than 25 fold increased towards PI(3,5)P2
compared to the activity of MTMR2 alone. The activity of the
MTMR2//MTMR13/SBF2 complex towards PI3P increases 10 fold when compared to
the activity of MTMR2 alone.
Figure 4. 9 Phosphatase activity of MTMR2 and MTMR2//MTMR13/SBF2 complex. In this experiment only PI3P and PI(3,5)P2 were dephosphorylated by the MTMR2 and the MTMR2//MTMR13/SBF2 complex. The complex formation of MTMR13/SBF2 and MTMR2 strongly increases the phospahtase activity towards PI3P and PI(3,5)P2 (adapted and modified from Berger et al., 2006).
23
Introduction
The MTMR2//MTMR13/SBF2 complex has an approximately two times higher activity
PI(3,5)P2 than towards PI3P (Berger et al., 2006). The MTMR2 substrate PI3P is
highly enriched on early endosomes in mammalian cells. If PI3P is depleted the
trafficking of a number of proteins through the early endosomes is delayed. There
PI3P plays an important role in endosome function and recruits effector proteins to
the endosomal membranes (Gruenberg and Stenmark, 2004). The enzyme
producing the other MTMR2 substrate PI(3,5)P2, the PIKfyve kinase, is located on
the late endosomes (Sbrissa et al., 1999). Changes in the level of PI(3,5)P2,
provoked through over-expression of the kinase or dominant-negative mutants of the
kinase, produce in yeast and in mammalian cells swollen endosomes and
vacuolization. This implicates a function of PI(3,5)P2 in membrane homeostasis
(Robinson and Dixon, 2006). When MTMR2 and MTMR13/SBF2 were expressed in
COS-7 cells a broad but incomplete overlap of MTMR2 and MTMR13/SBF2 was
observed in the cytoplasm. PI(3,5)P2 levels increase in COS-7 cells during hypo-
osmotic stress. When hypo-osmotic conditions were applied to COS-7 cells
expressing MTMR2 and MTMR13/SBF2, MTMR13/SBF2 was bound to the
membranes of the vesicles (formed by the hypo-osmotic conditions) and MTMR2
remained in the cytoplasm (Berger et al., 2006). This experiment shows the nature of
intracellular vesicle compartments to which MTMR2 and MTMR13/SBF2 bind.
Changes in the phosphoinositide levels might subsequently de-localize MTMR2 and
MTMR13/SBF2 also in Schwann cells. The events that might lead to these changes
need to be assessed. The interaction of MTMR2 and MTMR13/SBF2 is most likely
the molecular basis for the identical phenotypes when the gene encoding either
protein is mutated, showing that these proteins act together in an important pathway.
Loss of MTMR2 and MTMR13/SBF2 function lead to a disruption of the
MTMR2//MTMR13/SBF2 complex, leads to a lack of phosphatase activity, and might
be responsible for the human diseases CMT4B1 and CMT4B2. Thereby
MTMR13/SBF2 functions as a regulator fo the phosphatase activity of Mtmr2 (Berger
et al., 2006).
24
Introduction
4.4.2 MYOTUBULARIN RELATED PROTEIN 2 AND INTERMEDIATE
FILAMENTS
Mutations in neurofilament light chain gene NF-L are associated with dominantly
inherited axonal CMT type 2E and dominant demylinating CMT type 1F forms. The
identification of Neurofilament light chain protein (NF-L) interacting with MTMR2
draws additional attention to the intermediate filament family (Perez-Olle et al., 2005;
Previtali et al., 2003a). The mutations in NF-L appear as an axonal or an intermediate
form showing features of axonopahty and demyelination, with rare excessive myelin
which resemble myelin outfoldings (Zhu et al., 1997). NF-L is mainly expressed in
neurons NF-L mRNA is also upregulated after injury in Schwann cells and is present
during development. Whether the MTMR2/NF-L interaction also contributes to the
CMT4B1 disease isnot yet clear (Previtali et al., 2007). IFs are important cytoskeletal
polymers and the proteins are encoded by a large family of differentially expressed
genes. They are important for intracellular organization, provide structural support in
the cytoplasm and nucleus, and account for a large number of genetic human
diseases. In this summary I will focus on the IFs of class III and class IV type (Kim
and Coulombe, 2007) (Table 3.1).
IF name type Size
(kDa)
Main tissue distribution
Vimentin III 55 Mensencymal, Fibroblasts, endothelium,
Schwann cells
Desmin III 53 Muscle
GFAP III 52 Asctrocytes, Schwann cells
Peripherin III 54 Peripheral neurons
NF-L IV 68 Neurons, Schwann cells
Lamin A/C V 62-68 Ubiquitous expression in differentiated cells Table 4. 1. Class III and Class IV IF proteins Subset of intermediate filamentsimplicated in CMT diseases and Schwann cell biology (adapted and modified from Kim and Coulombe, 2007).
General features of intermediate filament proteins are the presence a central rod
domain. The rod domain of the individual molecules in subdivided into the coil
segments 1A, 1B, 2A, and 2B. L1, L12 and L2 are linker segments between the coil
25
Introduction
domains (Fig. 4.10). These domains are flanked by a N-terminal head domain and a
C-terminal tail domain.
Figure 4. 10 Domain signature of cytoplasmic class III and calss IV IFs. Head and Tail domain in green. The α-helical rod domain is the major determinant for self assembly. The heptate repeat-containing segments within the rod domain are indicated with 1A, 1B, 2A, and 2B, the flexible linker regions with L1, L12 and L2. Highly conserved rod domain boundaries are indicated in orange.
The rod domain boundaries consist of highly conserved amino acid regions (15-20
amino acides). These amino acids are frequently mutated in human disease and are
important for the polymerization of the IFs. The linker regions provide flexibility to the
stiff coiled-coil structure (Herrmann et al., 2007; Kim and Coulombe, 2007). IFs have
important roles in tissue integrity and cell-shape determination in mammalian cells.
IFs also coordinate mechanical forces and have diverse functions in embryonic
development, growth and maturation of specific tissues. GFAP and Vimentin are both
upregulated in the Schwann cell during nerve regeneration. They interact physically
in two signaling pathways involved in proliferation and regeneration. GFAP regulates
mitotic signals after nerve damage via αVβ8 integrin. Vimentin binds to α5β1 and
regulates thereby proliferation and differentiation later in regeneration (Triolo et al.,
2006). Recent publication provides a new link between disease-linked MTMR2 and
MTM1 mutations and NF-L assembly. The co-expressed disease mutant proteins of
MTMR2 and MTM1 produce missassembly of NF-L and aggregation of NF-L occurs.
The over expression of the wild type proteins have no effect on the assembly of NF-L
(Goryunov et al., 2008). Individual mutations can affect the biophysical properties of
the desmin filaments and afterwards interfere with cellular functions. Cellular
response is changed upon physiological alterations in the affected cell-type. These
cellular events lead to tissue-wide pathogenic changes like skeletal muscle atrophy
(Myopathy) or heart failure (Herrmann et al., 2007). An interesting link is also the
involvement of intermediate filament associated proteins (IFAPs) in the formation of
26
Introduction
axonal membrane domains at nodes and paranodes, as Vimentin is a known binding
partner of Ankyrin and Spectrin (Green et al., 2005). Ankyrin B and Spectrin (αII and
βII) mediate the correct positioning of the paranodal loops. Ankyrin G and the βV-
Spectrins are involved in positioning of the sodium channels at the node of Ranvier
(Schafer and Rasband, 2006).
4.5 AIM OF THE STUDY
The major aim of the first part of the study was to prove that MTMR13/SBF2 is the
disease-causing gene in CMT4B2 and provide a suitable animal model using gene-
trap disruption of MTMR13/SBF2. The mutant mice were used to analyze the protein
expression in sciatic nerves. The new mouse model for CMT4B2 also helped to
analyze in detail the behavioral, electrophysiological and histological changes that
occur upon a mutation in MTMR13/SBF2. The MTMR13/SBF2 deficient animals will
help to unravel the disease mechanism of CMT4B and to elucidate the critical
functions of protein complexes that are involved in phosphoinositide-controlled
processes in the peripheral nerves. In combination with the MTMR2 mutant animals
the MTMR13/SBF2 mutant mice will give rise to new insights into the mechanism of
hereditary neuropathies.
In the second part of the project, a mass-spectrometry based screen was performed
to identify additional binding partners of MTMR2 using co-immunoprecipitation from
the mouse Schwann cell line MSC80.
Specific mouse monoclonal antibodies were used for the precipitation of
endogenously expressed MTMR2. New identified interaction partners will be tested
for coexpression and colocalization to elucidate their influence on the complex and a
possible role in disease mechanisms of CMT4B.
27
Mtmr13/Sbf2-deficient mice
5 MTMR13/SBF2-DEFICIENT MICE
5.1 ANIMAL MODEL FOR CMT4B2
Mutations in the Myotubularin-Related Protein-2 (MTMR2) or MTMR13/Set-Binding
Factor-2 (SBF2) genes are associated with the autosomal recessive disease
subtypes CMT4B1 or CMT4B2 (Azzedine et al., 2003; Senderek et al., 2003). Both
forms of CMT share similar features including a demyelinating neuropathy associated
with reduced nerve conduction velocity (NCV) and focally folded myelin. To unravel
the disease mechanism in CMT4B the known Mtmr2-deficient animals were of major
value (Bolino et al., 2004; Bonneick et al., 2005). To prove that MTMR13/SBF2 is the
disease causing gene in CMT4B2 and to provide a suitable animal model, we have
generated Mtmt13/Sbf2 deficient mice. The CMT4B2 animal model should help to
elucidate the critical functions of protein complexes that are involved in
phosphoinositide –controlled processes in peripheral nerves. The possibility to
generate also Mtmt2//Mtmt13/Sbf2 double deficient mice will give us the possibility to
dissect the role of this pair of myotubularins in health and disease.
5.2 GENE-TRAP DISRUPTION OF MTMR13/SBF2
We have used mouse embryonic stem cells carrying a gene trap insertion in the
Mtmr13/Sbf2 locus (XH212; Baygenomics Gene Trap Resource) for the generation of
an Mtmr13/Sbf2-deficient mutant mouse line using established procedures (Bonneick
et al., 2005). The insertion site of the gene trap cassette was mapped 1267 bp
downstream of exon 14 of Mtmr13/Sbf2 (Fig. 5.1 A). Based on this information,
primers I, II and III were designed to discriminate between different alleles and for
genotyping (Fig. 5.1 B). Western blot analysis of sciatic nerve lysates of
Mtmr13/Sbf2-deficient and wt littermates revealed that the Mtmr13/Sbf2 protein was
absent (Fig. 5.1 C). Mice with a disrupted Mtmr13/Sbf2 allele are viable and were
born according to Mendelian expectations. No obvious signs of impaired
spermatogenesis were observed, in contrast to some Mtmr2 mutants (Bolino et al.,
2004). Having this Mtmr13/Sbf2 allele at hand, we also generated
28
Mtmr13/Sbf2-deficient mice
Mtmr2//Mtmr13/Sbf2-double deficient mice by appropriate cross-breeding with Mtmr2-
deficient mutant animals (Bonneick et al., 2005).
Figure 5. 1 Gene trap disruption of Mtmr13/Sbf2. A, Ideogram of the Mtmr13/Sbf2 protein structure (first row) and the Mtmr13/Sbf2 gene (second row). The gene trap vector and the locus of the gene-trap integration into intron 14 of Mtmr13/Sbf2 is schematically depicted in rows three and four, respectively. Arrows marked with I and II represent the forward primers for the wt and trapped Mtmr13/Sbf2 alleles, respectively, and III the reverse primer for the genotyping PCR (SA, splice acceptor; beta-geo, beta-galactosidase and neomycin-resistance fusion gene; pA, polyadenylation site). B, Genotyping PCR for homozygous (-/-), or heterozygous (+/-) Mtmr13/Sbf2 mutant mice, or wt (+/+). C, Western blot analysis of sciatic nerve lysates of Mtmr13/Sbf2-deficient (-/-) and wt (+/+) control mice. A rabbit polyclonal antibody was used to detect the 210 kDa Mtmr13/Sbf2 protein. Purified CBP-tagged Mtmr13/Sbf2 protein from a baculovirus expression system served as positive control (Berger et al., 2006). Bands below 210 kDa represent degradation products of Mtmr13/Sbf2.
These Mtmr2//Mtmr13/Sbf2-double deficient mice were also viable and born
according to Mendelian ratios. Upon visual inspection, the behavioral phenotype of
both Mtmr13/Sbf2-deficient and Mtmr2//Mtmr13/Sbf2-double deficient mice appeared
normal compared to control littermates. Starting at the age of two months, however,
both mutant lines showed an unusual but very mild hind limb clamping upon tail
suspension (data not shown). Double-heterozygous Mtmr2//Mtmr13/Sbf2 mutant
29
Mtmr13/Sbf2-deficient mice
animals appeared indistinguishable from their wt littermates up to fifteen months of
age (latest time point examined).
5.3 EXPRESSION ANALYSIS OF MTMR2, MTMR13/SBF2 AND DLG1/SAP97
IN SCIATIC NERVES OF MUTANT ANIMALS
In a first step, we analyzed whether alterations in Mtmr2 or Mtmr13/Sbf2 expression
alter the protein levels of their respective binding partners in the sciatic nerve of
mutant animals. Western blot analysis of sciatic nerve lysates from twelve month-old
animals revealed that Mtmr2 levels were unchanged in Mtmr13/Sbf2-deficient mice
(Fig. 5.2 A). Similarly, Mtmr13/Sbf2 levels remained unaltered in Mtmr2-deficient
mice (Fig. 5.2 B). Bolino et al. (Bolino et al., 2004) and Bolis et al. (Bolis et al., 2005)
have reported an interaction of Mtmr2 with Sap97. They also detected reduced
expression of Sap97 in the sciatic nerves of their strain of Mtmr2-deficient mice.
Here, we confirmed these findings in our strain of Mtmr2-mutant mice (Bonneick et
al., 2005). We continued to test whether loss of Mtmr13/Sbf2 would also reduce the
levels of Sap97 by reasoning that loss of the Mtmr2 interaction partner Mtmr13/Sbf2
might affect indirectly the interaction between Mtmr2 and Sap97 within a putative
larger complex. However, the levels of Sap97 were not significantly different
compared to wt in Mtmr13/Sbf2-deficient mice (Fig. 5.2 C). Consistent with these
findings, we found a comparable reduction of Sap97 in Mtmr2//Mtmr13/Sbf2-double
deficient mice as in Mtmr2-single mutants (Fig. 5.2 D). We conclude that the
interaction of Mtmr13/Sbf2 with Mtmr2 and the interaction between Mtmr2 and Sap97
are unlikely to be intimately connected.
30
Mtmr13/Sbf2-deficient mice
Figure 5. 2 Western blot analysis of the relative Mtmr13/Sbf2, Mtmr2 and Dlg1/Sap97 levels in sciatic nerve lysates from twelve month-old wt, Mtmr2-deficient (Mtmr2-/-), Mtmr13/Sbf2-deficient (Sbf2-/-), and Mtmr2//Mtmr13/Sbf2-double deficient (MTMR2-/- Sbf2-/-) mice. Each pool contains the sciatic nerves from two or three animals. Protein levels of Mtmr13/Sbf2, Mtmr2 and Sap97 were quantified by normalizing the relative protein levels to beta-actin, illustrated in a bar chart. A, The relative protein level of Mtmr2 does not differ significantly between wt and Mtmr13/Sbf2-deficient (Sbf2-/-) sciatic nerves. B, The Mtmr13/Sbf2 expression level shows no difference in Mtmr2-deficient (Mtmr2-/-) compared to wt control lysates of sciatic nerves. The Sap97 protein levels in Mtmr2-deficient (Mtmr2-/-) lysates are significantly reduced (p
Mtmr13/Sbf2-deficient mice
This conclusion was also supported by co-immunoprecipitation experiments revealing
no apparent differences in the interaction of Mtmr2 with Sap97 between wt and
Mtmr13/Sbf2-deficient sciatic nerves (Fig. 5.3).
Figure 5. 3 Co-Immunoprecipitation of Sap97 with mouse monoclonal anti-Mtmr2 antibody Each lysate pool contains sciatic nerves from three animals. The immunoprecipitation was performed with mouse monoclonal anti-Mtmr2 antibody covalently coupled to Protein A sepahrose. Western blot was performed with mouse monoclonal anti-Sap97 antibody and polyclonal rabbit anti-Mtmr2 antibody. Mtmr2 and Sap97 were present in the IPs from wild-type and Mtmr13/Sbf2 deficient sciatic nerves. Mtmr2 was not precipitated from Mtmr2 deficient sciatic nerves and Sap97 was not co-precipitated.
32
Mtmr13/Sbf2-deficient mice
5.4 BEHAVIORAL ANALYSIS
Visual examination of both Mtmr13/Sbf2-deficient and Mtmr2//Mtmr13/Sbf2-double
deficient mice revealed no obvious signs of tremor or major functional disability,
similar to what we had observed in the Mtmr2-deficient model of CMT4B1 (Bonneick
et al., 2005). Therefore, we performed a Rotarod test to assess whether a behavioral
difference related to motor function was detectable using this assay.
Figure 5. 4 Rotarod analysis of wt, Mtmr13/Sbf2-deficient (Sbf2-/-) and Mtmr2//Mtmr13/Sbf2-double deficient (Mtmr2-/- Sbf2-/-) mice.
33
Mtmr13/Sbf2-deficient mice
Mice were tested four times per day on four consecutive days, and the time spent on the rotating rod was plotted versus the trial number. For statistical analysis Students t-test was used. Error bars show the Standard Error of the Mean (S.E.M.). A, Analysis of four month-old Mtmr13/Sbf2-deficient (Sbf2-/-) and wt control mice (n=6). No significant difference between the two groups was detected. B, Analysis of five twelve month-old Mtmr13/Sbf2-deficient (Sbf2-/-) mice and six wt control animals. Trials 2, 5, 6, 7, and 9-15 were significantly different for p
Mtmr13/Sbf2-deficient mice
Figure 5. 5 Motor nerve conduction studies of sciatic nerves of four months-old (A-C) and twelve months-old animals (D-H). Error bars indicate the Standard Error of the Mean (S.E.M.). A, At four-months of age, Mtmr13/Sbf2-deficient (Sbf2-/-) mice showed mild but not significant NCV slowing compared to wt mice (n=6). B, In comparison to wt animals, Mtmr13/Sbf2-deficient (Sbf2-/-) mice showed no significant difference in CMAP amplitudes at this age. C, F-wave latency was slightly but significantly increased in Mtmr13/Sbf2-deficient (Sbf2-/-) mice compared to wt (*p
Mtmr13/Sbf2-deficient mice
Figure 5. 6 Histological analysis of cross sections of sciatic nerves at four months of age. Wt (A, C) and Mtmr13/Sbf2-deficient (Sbf2-/-) (B, D) mice were compared. Mtmr13/Sbf2-deficient (Sbf2-/-) mice show numerous nerve fibres with redundant myelin loops scattered across the nerve section. Affected nerve fibres exhibit different morphologies including myelin sheath outfoldings and infoldings (white arrows in D), and likely degradation of the axon-Schwann cell unit (white arrowhead in D). Scale bars for A,B: 100 µm; for C,D: 25 µm.
Abnormalities included both infoldings and outfoldings of the entire myelin sheath,
which particularly affected large caliber fibres but also smaller, thinly myelinated
fibres. Non-myelinated fibres appeared normal. At higher magnification, sciatic nerve
cross sections of mutant mice were littered with various degrees of dysmyelination
ranging from focal budding of the myelin sheath to multiple or combined infoldings
and outfoldings. Abnormal myelin structures were first but rather sporadically
observed in the sciatic nerves of Mtmr13/Sbf2-deficient mice already at the age of
three weeks (Fig. 5.7 A, B).
36
Mtmr13/Sbf2-deficient mice
Figure 5. 7 Time course of morphologic changes indicates a progressive neuropathy in Mtmr13/Sbf2-deficient (Sbf2-/-) mice. A qualitative and quantitative analysis of sciatic nerve cross section was performed at the age of three weeks, four months, and fifteen months. Three animals were analyzed per age and genotype, and representative images are shown. At 3 weeks (A, B), mutant animals already show focally folded myelin (white arrows in B), ranging from mere “budding” (oblique white arrow in B) to a major outfolding of the myelin sheath (horizontal white arrow in B). At four months (C, D) and fifteen months (E, F), mutant mice exhibit numerous nerve fibers with redundant myelin loops (oblique white arrows in D and F). Progression of the neuropathy is reflected by an increasing number of affected fibers, a higher morphological complexity of myelin abnormalities, and signs of additional axonal degradation. Focal folding of the myelin sheath may be also observed, albeit very rarely, in wt littermates, possibly reflecting age-dependent dysmyelination. Scale bars: 25 µm.
Irregular myelin folds were easily detectable although of low complexity. Quantitative
analysis of these pathological structures revealed a significant increase in numbers
compared to wt animals (Fig. 5.8 A). Next, since CMT is usually associated with a
37
Mtmr13/Sbf2-deficient mice
clinically progressive time course, we followed the qualitative and quantitative
progression of the pathology over time. Thus, we examined sciatic nerves at the age
of four months and fifteen months (Fig. 5.7 C-F; Fig. 5.8 A).
Figure 5. 8 Quantification of myelin misfoldings, whole fiber morphometry and relation between axon diameter and myelin sheath thickness. A, Quantification of myelinated fibers at three weeks, four months-, and fifteenth months of age. For each stage, the sciatic nerves were isolated from three mice per genotype. Two semithin sections per nerve, taken at an interval of 1.5 mm, were used for quantification. After the total number of fibers had been determined, the proportion of myelinated fibers exhibiting misfolded myelin was calculated. B, Relation between axon diameter and myelin sheath thickness of three week- and four month-old mice. For each age, the sciatic nerves of three animals per genotype were analyzed. Myelin thickness was determined and plotted as a function of the corresponding axon diameter. Each line represents the trend line for one mouse. Slope and intercept did not differ significantly between wt and Mtmr13/Sbf2-deficient (Sbf2-/-) mice at either age. For statistical analysis a Student`s t-test was used (*, p
Mtmr13/Sbf2-deficient mice
signs of axonal damage were recognized although we did not observe an obvious
major loss of myelinated axons. Next we reasoned, considering the suggested
molecular function of myotubularins, that the misfoldings of myelin observed in
Mtmr13/Sbf2-deficient mice might be due to altered vesicular trafficking and myelin-
membrane overgrowth. This could potentially lead to generally altered myelin sheath
thickness as we have observed in myelin mutants with multi-folded myelin extensions
in the central nervous system (Thurnherr et al., 2006). However, using computer-
aided morphometry, we did not detect significant alterations in myelin thickness and
axon diameter in Mtmr13/Sbf2-deficient sciatic nerves (Fig. 5.8 B,C), consistent with
our identical previous findings in Mtrm2-deficient mice (Bonneick et al., 2005). In
agreement with these data, we did not observe Schwann cell onion bulb formation as
the classical indicator of demyelination and remyelination.
5.7 MOTOR AND SENSORY NERVES ARE AFFECTED IN MTMR13/SBF2-
DEFICIENT MICE
CMT4B2 is classified as a motor and sensory neuropathy. Thus, we analyzed
whether these mixed symptoms were reflected in pathological aberrations in both
motor and sensory nerves. We chose to examine the ventral roots containing
exclusively axons derived from motor neurons and dorsal roots for sensory axons. In
both locations, the pathological hallmarks of myelin misfoldings were barely
detectable at the age of four months (Fig. 5.9 A, B), in contrast to the more distally
(with respect to the neuronal cell bodies) located sciatic nerve (Fig. 5.9 D) which
contains both motor and sensory axons. These findings indicate that the pathology is
more severe in distal compared to proximal parts of PNS nerves. At the age of fifteen
months, myelin misfoldings were prominently visible in ventral and dorsal roots
suggesting that both motor and sensory nerves become affected in a progressive
manner over time (Fig. 5.9 C, D).
39
Mtmr13/Sbf2-deficient mice
Figure 5. 9 Involvement of proximal motor and sensory nerves in Mtmr13/Sbf2-deficient mice. Histological analysis of ventral and dorsal roots from mutant mice at four and fifteen months of age. On cross sections from four months-old mutants (A, B), focally folded myelin is only scarcely present. At fifteen months, both infoldings and outfoldings of the myelin sheath are present in dorsal and ventral roots (arrows in C, D), reflecting a progressive involvement of proximal nerves in the neuropathy. Scale bar for (A–D): 25 µm
5.8 COMPLEX STRUCTURES OF MISFOLDED MYELIN IN MTMR13/SBF2-
DEFICIENT MICE
In order to gain more detailed insights into the fine structure of aberrant Schwann
cell-axon units in our mutant mice, we performed ultrastructural analysis using
electron microscopy. Figure 5.10 shows a collection of pictures to provide a sampling
of the different aberrant structures that we have observed. The myelin misfoldings
invariably originated from compacted myelin and showed an identical number of
myelin lamellae in both myelin misfoldings and the myelin sheath they originated from
(Fig. 5.10 D, quantitative data not shown). Within myelin misfoldings, we observed
normal compaction and periodicity of the myelin sheath. In general, the impression of
pathological alterations was dominated by myelin outfoldings with strongly variable
complexity (Fig. 5.10 A-D).
40
Mtmr13/Sbf2-deficient mice
Figure 5. 10 Electron microscopic analysis of focally folded myelin and axonal degeneration in sciatic nerve of Mtmr13/Sbf2-deficient mice. The figure shows a collection of different morphologies at age P21 (A-D), four months (E-F, M, O ,P), and 15 months (G-L, N). Outfolding of the myelin sheath is the most frequent type of dysmyelination (A-C). Single or multiple redundant myelin loops are visible adjacent to the original myelinated fibre. The Schwann cell membrane surrounds both the outfoldings and the Schwann cell-axon unit they arise from. Simple outfoldings and the original myelin sheath share the same periodicity and number of lamellae (D). Infoldings of the myelin sheath may severely affect the axonal shape by leading to constriction of the axonal cytoplasm (arrow in E) or by forming extensions giving the cross-sectioned fibre a target-like appearance (F,G). The double circles in F and G likely reflect the retrograde inversion of infolded myelin loops. Note that the interspaces between the inner and outer infolding and the original myelin sheath show the structure of axonal cytoplasm (insert in F). Some fibres exhibit
41
Mtmr13/Sbf2-deficient mice
both infoldings and outfoldings (G). Apart from abnormalities of the myelin sheath, some fibres show disintegration or even degradation of the entire Schwann cell-axon unit. We observed widening of the periaxonal space (white arrow in H), vacuolar disruption of the inner myelin layers (I), compression and lateralization of the axon by massive infoldings (J), and various stages of axonal degeneration (K, L). The white arrow in L points to residual myelin. Longitudinal sections (M-P) revealed preferential location of myelin abnormalities in the nodal and paranodal segments (white arrow in M). Note that the aberrant myelin loops ensheath axonal processes and lead to massive disruption of the normal architecture of the node, which here is forced off the cutting plane (white arrow in N). Infoldings and outfoldings also occur in the internodal segment of the myelinated fibre (white arrowhead in O) or near the Schmidt-Lantermann incisures (arrows and white arrowhead in P). Scale bars for D: 1 µm, L-P: 5 µm.
The most common formation consisted of one or multiple outfoldings of different size
adjacent to a myelinated large caliber axon, and multiple outfoldings showed a
tendency to form groups (Fig. 5.10 C). Aberrant myelin loops were always
ensheathed by the plasma membrane of the related Schwann cell (Fig. 5.10 A).
Myelin infoldings were also prominent. They usually presented as finger-like
inversions of the myelin sheath (Fig. 5.10 E) or circular inclusions within the
myelinated nerve fiber (Fig. 5.10 F). Entraining Schwann cell cytoplasm on their outer
surface, they protrude far into the axon and displace the axoplasm (Fig. 5.10 E). The
formation of double circles (Fig. 5.10 F,G) is most likely due to the retrograde
inversion of a single infolding since, at higher magnifications, we observed axonal
material in the gap between the inner and outer infolding. Alternatively, the nesting of
two distinct infoldings may have led to the double-circle appearance.
On electrophysiological examination, older Mtmr13/Sbf2-deficient mice showed a
reduction of the CMAP indicating axonal loss or damage. Thus, we also carefully
looked for axonal pathology. Degeneration of whole Schwann cell-axon units (Fig.
5.10 K,L) was not observed in young mutants, but was sporadically present at four
months and rather frequent at fifteenth months. Lateral dislocation of the axon by
myelin infoldings and vacuolar alteration of the axoplasm (Fig. 5.10 I,J) was often
observed. We occasionally found also myelinated nerve fibers not affected by myelin
misfoldings but with a conspicuous widening of the periaxonal space (Fig. 5.10 H).
The amazing complexity of myelin misfoldings and the consequences for the affected
Schwann cell-axon units, however, can be best appreciated on longitudinal sections
(Fig. 5.10 M-P). Complex myelin formations are preferentially, although not
exclusively, located in the nodal and paranodal regions. At the same time misfoldings
contain axoplasm-like structures suggesting the entrainment of axonal parts during
their formation (Fig. 5.10 M, P). Occasionally, misfoldings of the myelin sheath had a
larger diameter than the original nerve fibre, or lead to spatial disarrangement of the
42
Mtmr13/Sbf2-deficient mice
normal fibre anatomy like affecting the node of Ranvier (Fig. 5.10 N). Relating these
structural changes back to the molecular and cellular functions (and misfunctions in
disease) of MTMR13/SBF2 will be a major challenge for the future. On a pure
morphological level, the pathological observations in Mtmr13/Sbf2-deficient mice are
very reminiscent of our observations in Mtmr2-deficient animals consistent with the
biochemical finding suggesting a crucial role for a high-molecular complex containing
both MTMR13/SBF2 and MTMR2 in the biology of myelinated peripheral nerves,
possibly in the regulation of membrane trafficking.
43
Interaction partners of Mtmr2
6 INTERACTION PARTNERS OF MTMR2
6.1 PRINCIPLE OF THE SCREENING PROCEDURE AND MASS
SPECTROMETRY-BASED IDENTIFICATION OF ISOLATED PROTEINS
To date several interactions between Mtmr2 and other myotubularin family members
have been characterized (Berger et al., 2006; Lorenzo et al., 2006). Furthermore
Mtmr2 has been shown to interact with NF-L (Previtali et al., 2003a) and Dlg1/Sap97
(Bolino et al., 2004). Proteins usually do not act alone, but fulfill their cellular roles in
complex interactions with other proteins (Pandey and Mann, 2000). In recognition of
this we wanted to identify additional binding partners to better understand the role of
MTMR2 in the pathogenesis of the peripheral neuropathy CMT4B. Mass-
spectrometry based proteomics is becoming an indispensable tool for molecular and
cellular biology.
Figure 6. 1 Screening scheme for the detection of new binding partners of Mtmr2 Mouse monoclonal anti-Mtmr2 antibody was covalently coupled to Protein A Sepharose. This antibody matrix was used to isolate protein complexes from cell or tissue extracts. Afterwards detection of precipitated proteins was performed by Western blotting, MALDI TOF/TOF MS or LC-MS analysis.
44
Interaction partners of Mtmr2
Therefore we used a screening scheme (Figure. 6.1) based on immunoprecipitation
followed by MALDI TOF/TOF mass spectrometry (MS) (Gstaiger et al., 2003; Yart et
al., 2005) or liquid-chromatography integrated ESI (Electrospray ionization)-MS
analysis (Aebersold and Mann, 2003).
The MS-based screening procedure consists of five stages as described elsewhere
(Aebersold and Mann, 2003). In the first stage protein complexes were isolated from
lysate of a mouse Schwann cell line (MSC80) by affinity selection using a specific
mouse monoclonal anti-Mtmr2 antibody covalently coupled to Protein A Sepharose
beads (see Experimental Procedures 8.11) to isolate the Mtmr2 interaction partners
(Figure 6.1). Afterwards the isolated protein complex was either separated on a one-
dimensional gel by electrophoresis and proteins were excised prior to digestion, or
the isolated protein complex was used for digestion in solution (IS). In the first
method the excised proteins are enzymatically digested by trypsin (in-gel digestion,
IG). For the second method the proteins were enzymatically digested by trypsin in
solution. The difference between these two procedures is that separating the isolated
protein complex on a one-dimensional gel by electrophoresis defines a sub
proteome, e.g. on a 10% SDS-PAGE only proteins between 250 kDA and 37 kDA
can be separated. The digestion of the isolated protein complex in solution has no
cut-off of proteins. In the third step the peptides obtained by one-dimensional gel
electrophoresis and in-gel digestion were processed and spotted on a MALDI plate
(see Experimental Procedures 8.14) prior to MALDI TOF/TOF MS (Granvogl et al.,
2007). The peptide mixture obtained from the isolated protein complex by in-solution
digestion was separated by high-preasure liquid chromatography (HPLC) in fine
capillaries and eluted into an ion source where they were nebulized in small, highly
charged droplets (Aebersold and Mann, 2003). In the fourth stage the mass spectra
of the peptides are recorded. In the fifth stage a computer prioritized list of these
peptides is generated and a series of tandem mass spectra (MS/MS) is produced. A
given peptide ion is thereby isolated and fragmented by energetic collision with gas
and an MS/MS spectrum is recorded. The last step to identify the proteins from the
in-gel (IG) or from the in-solution (IS) digestion experiment was performing a
database search with GPS (Global Proteome Server) Explorer Software version 3.5
(Applied Biosystems) and Mascot version 2. 1. 0 (Matrix Science, London, UK) being
utilized as the search engine.
45
Interaction partners of Mtmr2
6.2 SCREENING TOOL – IMMUNOPRECIPITATION VIA MONOCLONAL ANTI-
MTMR2 ANTIBODY
The isolation of interaction partners of Mtmr2 was done using immunoprecipitation. A
highly specific affinity selection was obtained by using mouse monoclonal anti-Mtmr2
antibodies (Berger et al., 2006) covalently coupled to Protein A Sepharose with
dimethylpimelimidate (Yart et al., 2005).
Figure 6. 2 Immunoprecipitation with a monoclonal anti-Mtmr2 antibody (Berger et al., 2006) Immunoprecipitation was performed from mouse sciatic nerve and various Schwann cell lines, followed by Western blotting. In the upper panel polyclonal rabbit anti-Mtmr2 antibody was used to detect Mtmr2. In the lower panel polyclonal rabbit anti-Sbf2 was used to detect co-precipitated Sbf2. As controls purified Mtmr2 and Sbf2 were used. Sciatic nerve extracts show besides the full length Mtmr2 also major degradation products.
In the experiment shown in the upper panel of Figure 6.2 endogenously expressed
Mtmr2 was precipitated from lysates from Schwann cell lines and sciatic nerve using
the mouse monoclonal anti-Mtmr2 antibody 4H10 (see Experimental Procedures
8.10). Endogenously expressed Mtmr13/Sbf2 was co-precipitated from sciatic nerve
and Schwann cell lysates (Fig. 6.2 lower panel). In this experiment we show for the
first time the endogenous interaction of Mtmr2 and Mtmr13/Sbf2 and the suitability of
the mouse monoclonal anti-Mtmr2 antibody (Berger et al., 2006). MSC80 cells
express antigens of myelin-forming Schwann cells such as S-100 and Laminin
(Boutry et al., 1992). On the basis of this experiment MSC80 cells were used to
screen for new binding partners of Mtmr2.
46
Interaction partners of Mtmr2
6.3 IDENTIFICATION OF NOVEL MTMR2 INTERACTION PARTNERS
6.3.1 IN-GEL DIGESTION - ELECTROPHORESIS BASED SCREENING
To find new protein complexes that contain endogenous Mtmr2, a one-step
immunoprecipitation with mouse monoclonal anti-Mtmr2 antibody covalently coupled
to Protein A sepharose was carried out. After extensive washings the bound proteins
were eluted from the beads by boiling the samples with 1x SDS-sample buffer,
electrophoresed on a 10% SDS-PAGE and the separated proteins were stained with
colloidal blue (Fig. 6.3).
In this ideal situation the endogenous protein Mtmr2 serves as the bait for the affinity
purification with the monoclonal mouse anti-Mtmr2 antibody. Thereby a number of
proteins should be enriched in the immunoprecipitation with anti-Mtmr2 antibody. In
parallel a control immunoprecipitation with monoclonal mouse anti-Myc antibody was
performed. The anti-Myc antibody belongs to the same IgG subclass as the anti-
Mtmr2 antibody, namely the IgG1 subclass. In the control immunoprecipitation the
enriched proteins, especially from ca. 120 to 150 kDa, appeared as background with
a very low Protein Score. The relevant bands were excised from the gel (Fig. 6.3, IP:
mouse anti-Mtmr2) and processed for tryptic digestion within the gel material. The
peptides obtained by tryptic digestion were eluted and subjected to MALDI TOF/TOF
mass spectrometry analysis. This analysis provided several proteins that precipitated
together with the bait Mtmr2. Among them were known interaction partners of Mtmr2
belonging to the myotubularin family, Mtmr5/Sbf1 (Kim et al., 2003) and Mtmr13/Sbf2
(Berger et al., 2006; Robinson and Dixon, 2005). Mtmr12/3-PAP, also a member of
the myotubularin family, was to date not shown to interact with Mtmr2. All proteins
belonging to the Myotubularin family appeared on the SDS-PAGE according to their
molecular mass described in the Swiss-Prot database, http://expasy.org/. Another set
of proteins that was of particular interest due to their Protein Score were Plectin,
Spectrin, F-Actin cross linking protein, Peripherin, Vimentin Desmin and beta-Actin.
The Protein Score specifies a confidence interval for those proteins that are
considered to be significant. The more MS/MS spectra are recorded from one protein
on the basis of the MS spectrum, the higher the Protein Score and significance
becomes for identified proteins. Besides their high Protein Score these proteins also
appeared on the SDS-PAGE according to their molecular mass described in the
Swiss-Prot database, http://expasy.org/.
47
http://expasy.org/
Interaction partners of Mtmr2
Figure 6. 3 Separation of immunoprecipitated proteins by 1D SDS-PAGE and identification by MALDI TOF/TOF MS. The proteins were precipitated from MSC80 lysate with monoclonal mouse anti-Mtmr2 antibody and as negative control with monoclonal mouse anti-Myc antibody covalently coupled to Protein A Sepharose (both antibodies belong to the IgG1 subclass). The immunoprecipitates were separated on a 10% SDS-PAGE, stained with GelCodeBlue (Pierce) and the protein bands were excised with a 1D gel spotting pencil. After tryptic in-gel digestion of the proteins each band was analyzed with a MALDI TOF/TOF MS system. The collected MS and MS/MS data were used to identify the proteins that correspond to the excised bands. The control immunoprecipitation with a mouse monoclonal anti-Myc antibody revealed none of the proteins that were precipitated with the anti-Mtmr2 antibody. Polypeptides that yielded unambiguous mass spectrometry spectra are indicated. The major protein bands in both IP lanes at about 55 kDa represent IgG heavy chains of anti-Mtmr2 and Anti-Myc antibody. In the control IP lane with anti-Myc also the light chain of the antibody appears at about 38 kDa.
6.3.2 IN-SOLUTION TRYPTIC DIGESTION – SCREENING OF TOTAL
IMMUNOPRECIPTATES
In comparison to the electrophoresis based screening procedure (see Results 6.3.1)
two things are changed in the screening procedure of the total immunoprecipitates.
48
Interaction partners of Mtmr2
First, the proteins that were affinity purified with mouse monoclonal anti-Mtmr2
antibody covalently coupled to Protein A Sepharose and co-precipitated with Mtmr2
were eluted with Glycine pH 2.5, immediately neutralized and prepared for tryptic in-
solution digestion (see Experimental procedures 8.16). Second, prior to mass
spectrometry the peptides were separated with an integrated HPLC-System. This
procedure allows a more rapid and generic analysis of the precipitated proteins
(Gingras et al., 2007), since the whole immunoprecipitates are analyzed and no cut-
off occurs due to SDS-PAGE limited size exclusion. Also the integrated liquid-
chromatography ESI-MS systems are preferred to analyze complex samples
because all components are analyzed in one experiment. A limiting factor is that the
false-positive error rates can be generally large, but can be reliably estimated at the
level of the whole data set (Rinner et al., 2007) and are also backed up by the
electrophoresis-based screening method (see Results 6.3.1). In the screening of the
total immunoprecipitates all proteins were found that were also identified in the
electrophoresis based screening (Fig. 6.3). In addition five new proteins were
identified, namely Ankyrin G, Lamin A and Voltage-gated potassium channel subunit
beta-1 (see Table 5.1).
Of particular interest were the proteins Vimentin, Desmin and Peripherin, because
they appeared in both screening experiments, feature a significant Protein Score,
and display high protein-sequence coverage by their identified peptides. These three
proteins belong to the type III intermediate filament family; they provide cel