Regulation of Schwann Cell Differentiation and Peripheral

Regulation of Schwann Cell

Differentiation and Peripheral Myelination

by Src-like Kinases, p38 MAPKs and Rho

GTPases

Shireen Hossain

Department of Pharmacology and Therapeutics

McGill University, Montreal, Canada

June, 2010

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

TABLE OF CONTENTS LIST OF FIGURES ...... 7 LIST OF TABLES ...... 9 ABSTRACT ...... 10 RÉSUMÉ ...... 12 PREFACE ...... 14 ORIGINAL CONTRIBUTION TO KNOWLEDGE ...... 17 ACKNOWLEDGEMENTS ...... 20 LIST OF ABBREVIATIONS ...... 22 CHAPTER 1: INTRODUCTION TO SCHWANN CELLS AND PERIPHERAL MYELINATION ... 29 1.1 HISTORICAL PERSPECTIVE ...... 30 1.2 TYPES OF SCs ...... 31 1.3 PERIPERAL MYELIN ...... 32 1.3.1 Dorsal root ganglion neurons ...... 33 1.3.2 Properties of myelin ...... 35 1.3.3 Function of myelin ...... 36 1.3.4 Architecture of a myelinated internode ...... 36 1.3.4.1 Node of Ranvier ...... 39 1.3.4.2 Paranode ...... 41 1.3.4.3 Juxtaparanode ...... 43 1.3.4.4 Internode ...... 44 1.3.5 Lipids of myelin sheath ...... 46 1.3.6 Proteins of Myelin Sheath ...... 47 1.3.6.1 Myelin associated glycoprotein (MAG) ...... 48 1.3.6.2 Myelin basic protein (MBP) ...... 49 1.3.6.3 Protein zero (P0) ...... 50 1.3.6.4 Periaxin ...... 52 1.3.6.5 Peripheral myelin protein 22 kDa (PMP22) ...... 53 1.3.6.6 Peripheral myelin protein‐2 (PMP‐2) ...... 55 1.3.6.7 Connexins ...... 55 1.3.6.8 Proteolipid protein (PLP)/DM20 ...... 57 1.3.6.9 2’, 3’‐cyclic nucleotide 3’phosphodiesterase (CNP) ...... 59

1.4 SC DEVELOPMENT AND DIFFERENTIATION ...... 59 1.4.1 Origin and development of SCs ...... 59 1.4.2 SC differentiation and myelination ...... 64 1.4.2.1 Cell cycle exit ...... 64 1.4.2.2 Cell adhesion molecules ...... 66 1.4.2.3 Transcription factors ...... 68 1.5 REGULATION OF SC MYELINATION: POSITIVE AND NEGATIVE REGULATORS ...... 77 1.5.1 Positive regulators of differentiation ...... 77 1.5.1.1 Axon‐Glia interactions: cell contact and secreted factors ...... 78 1.5.1.2. Autocrine Growth factors ...... 80 1.5.1.3 Basal lamina assembly ...... 80 1.5.2 Negative regulators of differentiation ...... 81 1.6 BASAL LAMINA: COLLAGEN AND LAMININ ...... 82 1.6.1 Collagen ...... 82 1.6.1.2 Receptors: ...... 84 1.6.2 Laminins ...... 85 1.6.2 Receptors ...... 87 1.7 INTEGRIN SIGNALING: Focal adhesion complex formation ...... 91 1.7.1 Protein Kinases ...... 92 1.7.1.1 Focal adhesion kinase (FAK) ...... 92 1.7.1.2 Src‐Like Kinases (SLK) ...... 93 1.7.2 Phosphatases ...... 95 1.7.3 Rho GTPases ...... 96 1.8 SIGNALING PATHWAYS INVOLVED IN PERIPHERAL MYELINATION ...... 100 1.8.1 Neuregulins/ErbB ...... 100 1.8.2 Phosphoinositide 3‐kinase/Akt ...... 103 1.8.3 Mitogen‐activated protein kinases ...... 106 1.8.3.1 Extracellular regulated kinase (ERK)‐1/2 ...... 107 1.8.3.2 Jun amino‐terminal kinases (JNK)...... 108 1.8.3.3 p38 MAPK ...... 109 1.8.3.4 Kinase targets of MAPKs: MAPK‐activated protein kinases (MK) ...... 111 1.9 DEMYELINATING DISEASES ...... 114

1.9.1 Charcot‐Marie‐Tooth (CMT) disease ...... 114 1.9.2 Guillain‐Barre‐Strohl (GBS) Syndrome ...... 116 1.9.3 Leprosy ...... 118 CHAPTER 2: RATIONALE AND OBJECTIVES ...... 120 2.1 RATIONALE ...... 121 2.2 HYPOTHESIS ...... 124 2.3 SPECIFIC OBJECTIVES ...... 125 2.3.1 Specific Objective 1: Characterize the role of the SLKs in peripheral myelination125 2.3.2 Specific Objective 2: Determine whether p38 MAPK regulates transcription factors expression in SCs to ultimately modulate expression of myelin genes associated with SC terminal differentiation and peripheral myelination ...... 125 2.3.3 Specific Objective 3: Characterize the role of Rho GTPases in peripheral myelination involving activation of p38 MAPK ...... 126 CHAPTER 3: MATERIALS AND METHODS ...... 127 3.1 MATERIALS ...... 128 3.1.1 Cell culture reagents...... 128 3.1.2 Inhibitors ...... 128 3.1.3 siRNA transfection reagents ...... 128 3.1.4 Adenoviruses ...... 129 3.1.5 Antibodies ...... 129 3.1.6 Other reagents ...... 130 3.2 METHODS ...... 130 3.2.1 Primary cell culture ...... 130 3.2.2 Secondary cell culture ...... 132 3.2.3 siRNA Transfection ...... 132 3.2.4 Western Blot Analysis ...... 133 3.2.5 Electron Microscopy ...... 133 3.2.6 Immunofluorescence ...... 134 3.2.7 RNA isolation, reverse transcription and Real‐time quantitative PCR ...... 134 3.2.8 Adenovirus construction and expansion ...... 135 3.2.8.1 Rho A construction ...... 135 3.2.8.2 Expansion of adenoviruses ...... 137

3.2.9 Drug Treatments ...... 137 3.2.10 Statistical Analysis ...... 137 CHAPTER 4: RESULTS ...... 138 4.1 SRC‐LIKE KINASES ARE INVOLVED IN PERIPHERAL MYELINATION ...... 139 4.1.1 Inhibition of SLKs decreases accumulation of several myelin proteins in SC‐ DRGN co‐cultures in a dose‐dependent manner ...... 139 4.1.2 Fewer and shorter myelinated internodes are formed in the presence of PP2145 4.1.3 Myelin formed in the presence of PP2 appears normal at the ultrastructural level with clustering of sodium channels and caspr in axonal domains ...... 151 4.1.4 Krox‐20 protein expression is decreased by PP2 treatment: Roles for Fyn and Lyn ...... 154 4.1.5 SLK activation lies upstream of Akt, ERK and p38 MAPK as PP2 reduces the activation of these kinases by extracellular matrix (ECM) ...... 159 4.2 p38 MODULATES TRANSCRIPTION FACTORS KROX‐20, SCIP, SOX10 AND CREB TO REGULATE SCHWANN CELL DIFFERENTIATION AND PERIPHERAL MYELINATION ...... 161 4.2.1 p38 alpha is the predominant isoform expressed by SCs ...... 161 4.2.2 PD169316‐treatment arrests SCs at a promyelinating stage ...... 161 4.2.3 p38 regulates Krox‐20 expression in myelinating SCs ...... 165 4.2.3 p38 MAPK regulates SCIP ...... 170 4.2.4 p38 MAPK regulates transcription of Sox10 during myelination ...... 173 4.2.5 p38 MAPK regulates p27kip1 expression ...... 175 4.2.6 p38 MAPK does not regulate Krox‐20‐independent periaxin gene expression178 4.2.7 p38 MAPK does not regulate Sox2 expression ...... 182 4.2.8 Krox‐20 overexpression reverses the effect of p38 inhibitor on MAG expression185 4.2.9 MAPK‐activated proten kiase‐2a (MK2) is a downstream effector of p38, regulating Krox‐20 expression and myelin protein accumulation ...... 188 4.2.10 CREB and p38 MAPK phosphorylation induced by ECM is blocked by p38 inhibitor and an MSK‐1 inhibitor ...... 191 4.3 RHO GTPASES REGULATE SC MORPHOLOGY IN PERIPHERAL MYELINATION ...... 196 4.3.1 Altering RhoA activity abrogates myelination of DRGNs by SCs ...... 196 4.3.2 RhoA is involved in early stages of myelination ...... 201 4.3.2 RhoA regulates length of SCs process and cell survival ...... 201 4.3.3 WT‐ RhoA overexpressing SCs retain the ability to myelinate ...... 201

4.3.4 Rho Kinase (ROCK) regulates myelin protein and Krox‐20 expression ...... 204 4.3.5 ROCK is not an activator of p38 MAPK or CREB ...... 207 4.3.5 Altering Rac1 and Cdc42 activity abrogates myelination in SC‐DRGN co‐cultures and alters SC morphology ...... 209 4.3.6 Rac1 is not an activator of p38 MAPK ...... 213 CHAPTER 5: DISCUSSION ...... 214 5.1 Discussion of Results ...... 215 5.2 Summary of Results and General Discussion ...... 234 Implications for Therapy ...... 237 5.3 Concluding Remarks ...... 238 REFERENCES ...... 240

LIST OF FIGURES

Figure 1.1 A schematic illustrating a myelinated axon ...... 33 Figure 1.2 Axo‐glial functional domains of a peripheral myelinated internode ...... 38 Figure 1.3 SC Lineage...... 61 Figure 1.4 Factors regulating SC development...... 63 Figure 1.5 Integrin activation and FAC formation...... 92 Figure 1.6 Model depicting Rho‐GTPases cycling between active “on” and inactive “off” states ...... 98 Figure 3. 1 Schema outlining the procedure to obtain in vitro cultures...... 131 Figure 4.1.1 Inhibitors of SLK cause a dose‐dependent decrease in MBP accumulation142 Figure 4.1.2 Recovery of myelin protein levels following removal of PP2 in SC‐DRGN co‐ cultures ...... 144 Figure 4.1.3 Chronic PP2‐treatment of SC‐DRGN co‐cultures reduces the number of myelinated internodes...... 147 Figure 4.1.4 Chronic exposure of SC‐DRGN cultures to PP2 reduces both the number and the length of myelinated internodes formed...... 149 Figure 4.1.5 Chronic SLK inhibition (PP2 treatment) does not affect clustering of either sodium channels or caspr and myelin is normally compacted...... 153 Figure 4.1.6 SLK‐regulated Krox‐20 protein levels were reduced by PP2 treatment. .... 156 Figure 4.1.7 Fyn and Lyn regulate Krox‐20 protein expression...... 158 Figure 4.1.8 PP2 blocks ECM‐induced phosphorylation of Akt, ERK and p38 MAPK. .... 160 Figure 4.2.1 p38 MAPK isoforms expressed by SCs...... 162 Figure 4.2.2 PD169316 arrests SCs at a pro‐myelinating stage...... 164 Figure 4.2.3 p38 regulates Krox‐20 expression...... 168 Figure 4.2.4 Inhibiting p38 MAPK reduces Krox‐20, SCIP and Sox10 transcription but does not alter Sox2...... 169 Figure 4.2.5 Inhibiting p38 decreases the number of SCIP+ SCs...... 172 Figure 4.2.6 Inhibiting p38 reduces Sox10 protein levels...... 174 Figure 4.2.7 PD169316 reduces VC and ECM‐induced p27kip1 expression...... 177 Figure 4.2.8 PD169316 reduces the number of periaxin+ SCs...... 181 Figure 4.2.9 p38 MAPK does not regulate Sox2 expression...... 184

Figure 4.2.10 Overexpression of Krox‐20 reverses the inhibitory effect of PD169316 on MAG expression...... 187 Figure 4.2.11 MK2 regulates Krox‐20 and myelin genes expression...... 190 Figure 4.2.12 p38 MAPK regulates CREB activation...... 194 Figure 4.3.1 Overexpressing dominant‐negative RhoA in SC‐DRGN co‐cultures abrogates myelination...... 198 Figure 4.3.2 Isolated SCs infected with WT‐ and DN‐RhoA adenovirus seeded onto DRGNs do not myelinate...... 200 Figure 4.3.3 RhoA is involved in early stages of myelination...... 202 Figure 4.3.4 SCs infected with WT‐RhoA adenovirus retain potential to myelinate...... 203 Figure 4.3.5 ROCK inhibitor Y27632 decreases Krox‐20 and myelin proteins expression...... 206 Figure 4.3.6 Y27632 does not alter p38 or CREB phosphorylation induced by extracts of ECM...... 208 Figure 4.3.7 SCs infected with DN‐ and CA‐Rac1 do not myelinate...... 211 Figure 4.3.8 SCs infected with DN‐ and CA‐Cdc42 do not myelinate...... 212 Figure 4.3.9 Rac1 inhibitor, NSC23766, does not alter p38 or CREB phosphorylation induced by ECM...... 213

LIST OF TABLES

Table 4.1.1: Shorter internodes form in the presence of PP2…….………………………………150

Table 4.2.1: MSK‐1 inhibitor H89 reduced % of p27kip1+, Krox‐20+ SCs………………………..195

ABSTRACT Myelin, a complex lipid-rich membranous structure, enables rapid conduction of nerve impulses. Schwann cells (SCs) myelinate axons of the peripheral nervous system through a stringently controlled process, requiring basal lamina assembly. Laminin, an essential constituent of this matrix, directs SC function, development and myelination by differentially activating cell surface receptors such as integrins. We previously demonstrated the phosphorylation of mitogen-activated protein kinase (MAPK) p38 following addition of exogenous laminin to SC-dorsal root ganglion neuron (DRGN) co-cultures. This activation is indispensible to the onset of myelination and is postulated to occur through integrin activation.

The objective of this thesis was to identify effectors of basal lamina- integrin signaling resulting in p38 MAPK activation. We thus investigated the involvement of two known participants of integrin signaling, src-like kinases (SLKs) and Rho GTPases. Contrary to conclusions drawn from SLK-null mice, we identify roles for SLKs in peripheral myelination using SC-DRGN co-cultures and SLK-specific inhibition. Using siRNA knockdown, we pinpoint Fyn and Lyn as regulators of Krox-20, a transcription factor key to PNS myelin formation and maintenance. Furthermore, using the SLK-specific pharmacological inhibitor, PP2, we demarcate SLKs as upstream activators of p38 MAPKs, Akt and ERKs, three protein kinases implicated in SC development, survival and proliferation, respectively.

Rho GTPases regulate actin remodeling, linking integrins to the cytoskeleton. Through ectopic overexpression, we show RhoA, Rac1 and Cdc42 are modulators of SC morphological homeostasis. Pharmacologically, we identify Rho kinase (ROCK), a downstream effector of RhoA, as a regulator of Krox-20 but not as an activator of p38 MAPK. Similarly, results obtained by inhibiting Rac1, indicate that it does not mediate p38 activation.

Finally, we report that p38 acts on multiple transcription factors and the cell cycle inhibitor, p27kip1, to direct SC transition from an immature to a myelinating state. It specifically regulates the expression of Krox-20, as well as

SCIP and Sox10, two regulators of Krox-20 expression. Moreover, we identify MAPK-activated protein kinase-2 and CREB as downstream effectors of p38.

Hence, we define a molecular mechanism of SC differentiation mediated by the basal lamina, whereby p38, partly activated by SLKs, regulates cell cycle exit and transcription factor expression to ultimately promote peripheral myelination.

RÉSUMÉ La myéline, une structure membraneuse complexe, et riche en lipides, permet la conduction rapide des impulsions nerveuses. Les cellules de Schwann (CS) myélinisent les axones du système nerveux périphérique (SNP) à travers un processus strictement contrôlé qui requière l’assemblage de la lame basale. La laminine, un constituent essentiel de cette matrice, dirige la fonction, le développement et la myélinisation des CS en activant différents récepteurs de surface tels que les intégrines. Nous avons précédemment démontré la phosphorylation de la mitogènes activée par des agents protéine kinase (MAPK) p38 après l’ajout de laminine exogène à des co-cultures CS-neurone ganglionnaire de la racine dorsale (NGRD). Cette activation est indispensable au commencement de la myélinisation et se produit présumément à travers l’activation des intégrines.

L’objectif de cette thèse a été d’identifier les effecteurs de la signalisation lame basale-intégrine causant l’activation de la MAPK p38. Nous avons donc regardé l’implication de deux participants connus à la signalisation des intégrines, src-like kinases (SLKs) et les Rho GTPases. Contrairement aux conclusions dérivées des souris SLK-nulle, nous avons identifié des rôles pour SLKs dans la myélinisation périphérique en utilisant des co-cultures CS-NRDG et une inhibition spécifique des SLKs. En utilisant un knockdown par RNAi, nous avons identifié Fyn et Lyn comme régulateurs de Krox-20, un facteur de transcription clé pour la formation et la maintenance de la myéline du SNP. De plus, en utilisant un inhibiteur pharmacologique spécifique pour SLK, PP2, nous avons demarqué les SLKs comme des marqueurs en amont des MAPKs p38, Akt et ERKs, trois protéines kinases impliquées respectivement dans le développement, la survie et la prolifération des CS.

Les Rho GTPases régulent le remodelage de l’actine en liant les intégrines au cytosquelette. En utilisant une approche génétique, nous avons demontré que RhoA, Rac1 et Cdc42 sont des modulateurs de l’homéostasie morphologique des CS. Pharmacologiquement, nous avons identifié Rho kinase (ROCK), un effecteur en aval de RhoA, comme régulateur de Krox-20, mais pas comme activateur de la MAPK p38. Similairement, nos résultats obtenus en inhibant Rac1 indiquent qu’il ne participe pas à l’activation de p38.

Finalement, nous avons trouvé que p38 agit sur de multiples facteurs de transcription et sur l’inhibiteur de cycle cellulaire p27kip1 pour diriger la transition des CS de cellules immatures à des cellules capables de myéliniser. La MAPK p38 régule spécifiquement l’expression de Krox-20 aussi bien que celles de SCIP et Sox10, deux régulateurs de l’expression de Krox-20. De plus, nous avons identifié MAPK-activée protéine kinase-2 et CREB comme effecteurs en aval de p38.

En conclusion, nous avons défini un méchanisme moléculaire de différenciation des CS dirigé par la lame basale, par lequel p38, partiellement activée par les SLKs, régule la sortie du cycle cellulaire et l’expression de facteurs de transcription pour ultimement promouvoir la myélinisation périphérique.

PREFACE This thesis is written in full format as permitted in the guidelines of the McGill Faculty of Graduate Studies.

The data appearing in Chapter 4: Section 4.1 has been accepted by the Journal of Experimental Neurology and appears as follows:

S Hossain, G Fragoso, W E Mushynski and G Almazan. 2010. Src-like kinases are involved in peripheral myelination. 2010. J. Experimental Neurology. In Press.

The data presented in Chapter 4: Section 4.2, is in preparation for submission to the Journal of Cell Biology shortly. S Hossain, M-A Morcillo-de-la- Cruz, D B Parkinson, W E Mushynski and G Almazan. Mitogen-Activated Protein Kinase p38 regulates Krox-20 and CREB activation to modulate Schwann cell differentiation.

The data in Chapter4: Section 4.3, is being written as a manuscript for submission. S Hossain, H-N Liu, J Bamburg, W E Mushynski and G Almazan. Molecular regulation of Schwann cell differentiation by Rho GTPases.

Contribution of Authors:

I performed the majority of the work presented in Chapter 4 and wrote the manuscripts under the supervision of Dr. Guillermina Almazan.

The following are the contributions of co-authors:

In Chapter 4, Section 4.1, Dr. Gabriela Fragoso performed some experiments regarding short-term stimulation of SC-DRGN co-cultures with ECM and inhibition using the SLK inhibitor, PP2, to assess the phosphorylation state of p38, and the results are presented in Figure 4.1.8.

In Chapter 4, Section 4.2, Dr. David Parkinson kindly provided us with the Krox-20 overexpressing adenovirus used in results presented in Figure 4.2.9,

14 while Miguel-Angel de la Cruz-Morcillo performed the qRT-PCR reactions presented in Figure 4.2.4.

In Chapter 4, Section 4.3, Dr. Hsieh-Ning Liu constructed the RhoA wild type, constitutive active, dominant negative adenovirus constructs, while Dr. James Bamburg kindly provided us with the constitutive active, dominant negative and wild-type constructs of the Rac1 and Cdc42 GTPases.

Lastly, Dr. Walter E. Mushynski extensively reviewed the manuscripts.

I dedicate this thesis to:

My parents, Dr. Afzal Hossain and Zakiya Akhter.

Words cannot express my gratitude for all the love and support they have provided and their many sacrifices to make this possible.

They truly are an inspiration to me in my every walk of life.

My brothers, Sheehab, Ahmed and Omar for being there for me through everything and for being so supportive.

Most importantly, to Allah for blessing me with this opportunity, providing me with the tools, the drive, the courage and the inspiration for making this possible.

ORIGINAL CONTRIBUTION TO KNOWLEDGE The following findings presented in this thesis represent original contributions to knowledge:

 Treatment of SC-DRGN with the Src-like kinase (SLK) inhibitors (PP2 and SU6656) caused a dose-dependent reduction in myelination as assessed by the accumulation of the myelin proteins MBP, MAG and P0, without altering integrins β1 and 4.  Treatment of SC-DRGN with the SLK inhibitor PP2 caused a time- dependently reduction in the myelin proteins MBP, MAG and P0.  The effect of short-term treatment with PP2 on myelin protein expression was not fully reversible in cultures allowed to recover for more than 1 week.  SLKs regulate myelin thickness, myelinated internode number and length, as evidenced by immunocytochemistry with MBP and electron microscopy of the SC-DRGN cultures treated with PP2.  Fyn and Lyn are the most predominantly expressed SLKs in SC-DRGNs and regulate protein levels of the critical transcription factor Krox-20, as demonstrated using siRNA targeted gene knockdown and immunofluorescence.  Extracts of ECM induced phosphorylation of Akt and ERK-1/2 in SC- DRGNs.  SLKs regulate phosphorylation of p38 MAPK, Akt and ERK1/2 induced by ECM extract.  Isoforms p38 α, γ and δ are expressed by SCs while DRGNs expressed all four.  p38 α is the most abundantly expressed isoform by both SCs and DRGNs, followed by the γ isoform.  PD169316 treatment arrested SCs in SC-DRGN co-cultures at a promyelinating state as observed ultrastructurally by electron microscopy.  p38 α/β selective inhibitor PD169316 blocked VC- and ECM-induced Krox-20 and SCIP expression.

 p38 α/β selective inhibitor PD169316 decreased Krox-20, Sox10 and SCIP expression at both the protein and mRNA levels.  Addition of VC to SC-DRGN cultures reduced Sox2 expression. While PD169316 did not reverse the effect of VC on Sox2.  PD169316 reduced p27kip1 nuclear protein expression at the onset of myelination.  Overexpressing of Krox-20 with an adenovirus vector can reverse the inhibitory effect of PD169316 on MAG expression by SCs in myelinating cultures  MAPKAPK-2a (MK2a) inhibitor (CMPD1) dose-dependently reduced accumulation of myelin proteins MBP, P0 and MAG in SC-DRGN cultures.  CMPD1 reduced VC-induced Krox-20 expression.  Addition of ECM extracts to SC-DRGN cultures induced phosphorylation of CREB.  The p38 inhibitor PD169316 blocked ECM-induced CREB- and p38- phosphorylation.  The MSK-1 inhibitors, H89 and Ro 31-8220, blocked CREB phosphorylation induced by ECM in SC-DRGNs.  The MSK-1 inhibitor H89 blocked ECM-induced p27kip1 and Krox-20 expression in 3-day myelinating co-cultures.  The Rho GTPase RhoA is involved in early stages of myelination as early infection of SC-DRGN with a dominant negative (DN)-RhoA form blocked myelination, and it also reduced MBP accumulation.  SCs expressing DN-RhoA have hyper-elongated cellular processes.  SCs expressing WT-RhoA that do not form myelin, still possess myelination potential as they express Krox-20 following addition of VC to co-cultures.  Rho kinase (ROCK) regulates Krox-20 and myelin proteins gene expression as determined by treatment of myelinating SC-DRGN cultures with the selective inhibitor Y27632.  ROCK is not downstream of p38 and does not regulate CREB phosphorylation induced by ECM.

 Rac1 and Cdc42 DN and CA isoforms produce aberrant SC morphologies, and their expression abrogates myelination of SC-DRGN cultures.  Rac1 is not an upstream regulator of p38 MAPK phosphorylation, induced by ECM, as assessed by treatment of SC-DRGN cultures with the selective pharmacological inhibitor NSC 23766.

ACKNOWLEDGEMENTS I would like to express my sincerest gratitude and appreciation to my supervisor and friend, Dr. Guillermina Almazan. She has been a role model as a scientist and as an individual. Even more inspiring is that she has remained true to her kind, gentle, caring nature in spite of the challenges faced as a woman in science. Her good nature, encouragement, patience, and guidance proved to be remarkable sources of assistance throughout the various stages of my PhD studies. Moreover, I cannot thank her enough for her kindness, support throughout all my personal challenges.

I would like to thank Dr. Walter E Mushynski for co-supervising me and providing me with advice regarding the SC-DRGN co-cultures, and suggestions for experiments but for extensively editing my manuscripts. His fantastic sense of humor and infectious enthusiasm made it easy to maintain interest.

I would also like to thank Dr. Melissa Vollrath of the Physiology department, for allowing me to use her culture facilities during the long period of renovations in our own.

I would like to acknowledge all my lab mates, past and present, Shirley Liu, Qiao-Ling Cui, Gabriela Fragoso, Sandy Hemdan, Olivia Bibollet-Bahenas, Eli Fogle, Manuelle Rongy, Jeffery Gross, Jeffrey Haines, Jun Fang, Benoit Gentil, and Derek Kastner. I would like to acknowledge Jun Fang for his help in the preparation of some of the SC-DRGN co-cultures and computer assistance. I particular, I would like to thank Olivia, Eli, Sandy and Manuelle for their friendship and continuous support.

I would like to thank Jacynthe Laliberte and Johanne Ouelette not only for their expert help and guidance in confocal and electron microscopy, but also for their friendship.

I would like to thank the Department of Pharmacology and Therapeutics, and the Chair, Dr. Hans Zingg, for making this such a pleasant memorable experience. I would like to thank the administrative staff past and present, for their help, encouragement and kindness. I am very thankful to my thesis advisory

20 committee members for their time, discussion of my project and valuable input. I would like to thank my official advisors, Drs. Brian Collier and Daniel Bernard, for their constant support, time, reassurance and interest throughout my PhD. I appreciate all the help and encouragement from all my professors and colleagues. In particular, I would also like extend a special thank you to two of my ‘unofficial’ advisors and mentors, Drs. Terry Hebert and Alfredo Ribeiro-da-Silva. I am very thankful to Dr. Terry Hebert for his constant support, time, and for our many coffee discussions about science and my PhD. Also I would like to give a special thank you to Dr. Alfredo Ribeiro-da-Silva, as I am indebted to him for going above and beyond the call of duty with his remarkable enthusiasm, motivational speeches, advice on my confocal and electron microscopy queries, and his unnecessary but most-welcomed assistance with all my computer problems. I greatly appreciate the financial support from the Multiple Sclerosis Society of Canada and Canadian Institute of Health Research.

I have utmost gratitude for my family in being my ultimate rock of support. I extend my deepest thanks to my parents who have sacrificed much to ensure me the opportunity of a good education and for their consistent encouragement in my academic progress. Their love and relentless confidence in my abilities have been fundamental to my accomplishments. I would like to thank my three brothers and musketeers, Sheehab/Shonal, Ahmed/Sabah and Omar for their constant encouragement and support. Their collective encouragement, confidence in my abilities and help in these final stages of this degree have helped me persevere.

I thank my friends within the department for making this experience so memorable and fun, while providing scientific ‘food for thought’. I commend my friends outside the department for their patience and support throughout. In particular, I would like to thank my life-long friend, Bonny Choy, and Sophie-Anne Lamour. I would like to thank Sophie and Olivia for translating my abstract.

Lastly, but most importantly, I would like to express my sincerest gratitude to Rudolf Virchow, Santiago Ramon y Cajal, Robert Remak and Theodore Schwann, for their pivotal discoveries and studies centuries ago pertaining to myelin and to the glial cell of the periphery, making this thesis work possible.

LIST OF ABBREVIATIONS

ADAM22 A disintegrin and metalloproteinase 22

AIDP Acute inflammatory demyelinating polyradiculoneuropathy

AMAN Acute motor axonal neuropathy

AMSAN Acute motor and sensory axonal neuropathy

AP-1 Activator Protein-1

ARE AU-rich element

AREBP AU-rich element binding protein

ATP Adenosine tri-phosphate

BDNF Brain-derived neurotrophic factor

BMP Bone morphogenic protein

BSA Bovine serum albumin

Brn1 Brain1

Brn2 Brain2

CA Constitutive Active

CAM Cell adhesion molecule

CaMK Calmodulin-dependent kinase

cAMP cyclic adenoside monophosphate

CDK Cyclin dependent kinase

CGT Ceramide galactosyltransferase

CHN Congenital hypomyelinating neuropathy

CMT Charcot-Marie-Tooth

CNP 2’-3’-Cyclic nucleotide-2’-phosphodiesterase

CNS Central nervous system

CPE Cytopathic effect

CRE cAMP response element

CREB cAMP response element binding protein

CST Cerebroside sulfotransferase

CTKD C-terminal kinase domain

Cx Connexin

CYP51 7-dehydrocholesterol reductase, and lanosterol 14-demethylase

DAPI 4’, 6-diamidino-2-phenylindole

DG Dystroglycan

DGC Dystrophin-glycoprotein complex

Dhh Desert hedgehog

DMEM Dulbecco’s modified enriched medium

DN Dominant negative

DRGN Dorsal root ganglion neuron

DRP Dystrophin-related protein

DSP Dual specificity phosphatase

DSS Dejerine-Sottas syndrome

EAN Experimental allergic neuritis

ECM Extracellular matrix

EGR Early growth response eIF-4E Eukaryotic initiation factor-4E

EM Electron microscope

ERK Extracellular-signal regulated kinase

ERM Ezrin radixin moeisin eSC Embryonic Schwann cell

FAC Focal adhesion complex

FAK Focal adhesion kinase

FAT Focal adhesion targeting

FERM Erythrocyte band four, one ezrin-radixin-moeisin

GalC Galactocerebroside

GAP GTPase activating protein

GBS Guillain-Barre-Strohl

GDAP Ganglioside-induced differentiation associated protein

GDI Guanidine dissociation inhibitor

GDNF Glial-cell derived neurotrophic factor

GDP Guanine di-phosphate

GEF GTPase exchange factor

GFP Green fluorescent protein

GGF Glial growth factor

GPI Glycosylphosphatidylinositol

GSK Glycogen synthase kinase

GTP Guanine tri-phosphate

HEK-293 Human embryonic kidney cell-293

HMG High mobility group

HMGCR 3-hydroxyl-3-methylglutaryl coenzyme A reductase

HNPP Hereditary neuropathy with liability to pressure palsy

hsp Heat shock protein

Ig Immunoglobulin

IGF-1 Insulin-like growth factor-1

ILK Integrin linked kinase

ISE Immature Schwann cell enhancer

JIP-1 JNK-interacting protein-1

JNK c-Jun N-terminal kinase

Kv Potassium voltage gated channel

LPA Lysophosphatidic acid

MAG Myelin associated glycoprotein

MAPK Mitogen-activated protein kinase

MBP Myelin basic protein

MDC1A Merosin-deficient congenital muscular dystrophy

MFN2 Mitofusin2

MK Mitogen activated protein kinase activated protein kinase

MK2 Mitogen activated protein kinase activated protein kinase-2

MK3 Mitogen activated protein kinase activated protein kinase-3

MK5 Mitogen activated protein kinase activated protein kinase-5

MKP Mitogen activated protein kinase phosphatase

MNK MAPK-interacting kinase

MPK MAPK phosphatases

MSC 80 Murine Schwann cell 80

MSE Myelin Schwann cell enhancer

MSK Mitogen- and stress-activated kinase

NAB NGFI-A/Egr-binding

NCC Neural crest cell

NCID Notch intracellular domain

NCV Nerve conduction velocity

Necl Nectin-like

NES Nuclear export signal

NF Neurofilament

NF-155 Neurofascin-155 kDa

NF-186 Neurofascin 186 kDa

NFAT Nuclear factor of activated T cells

NF-κB Nuclear factor-κ B

NGF Neuronal growth factor

NLS Nuclear localization signal

NRG-1 Neuregulin-1

NrCAM Neuronal cell adhesion molecule

NT Neurotrophin

NTD Noncollagenous N-terminal domain

NTKD N-terminal kinase domain

OLG Oligodendrocyte

P2 Protein 2

PAK p21-activated kinase

PBS Phosphate buffered saline

PDGF Platelet derived growth factor

PDK1 3’-phosphoinositide dependent kinase 1

PGL-1 Phenolic glycolipid-1

PKA Protein kinase A

PKC Protein kinase C

PI Phosphoinositol

PI3-K Phosphoinositide 3-kinase

PLP Proteolipid protein

PMD Pelizeaus-Merchbacher

PMP-2 Peripheral myelin protein-2

PMP22 Peripheral myelin protein-22 kDa

PNS Peripheral nervous system

P0 Peripheral myelin protein zero

Pro Proline

PTEN Phosphatase tensin homolog

PTP Protein tyrosine phosphatase

Rb Retinoblastoma

ROCK Rho kinase

RSK p90 ribosomal S6 kinases

RT-PCR Reverse transcription polymerase chain reaction

SC Schwann cell

SCD Stearoyl CoA desaturase

SCIP Suppressed cAMP inducible protein

SDS Sodium dodecyl sulphate

SG Sarcoglycan

SLK Src-like kinases

SLI Schmidt-Lantermann incisure

Ser Serine

SOX SRY box-containing

SRY Sex determining region on the Y chromosome

SW Shah-Waardenburg syndrome

TAB-1 Transforming growth factor β-activated protein kinase-1 binding protein

TAG-1 Transient axonal glycoprotein

TGF-β Transforming growth factor-β

Thr Threonine

Tr Trembler

Tyr Tyrosine

VC Vitamin C

VGSC Voltage-gated sodium channel

VZG-1 Ventricular zone gene-1

WT Wild-type

CHAPTER 1: INTRODUCTION TO SCHWANN CELLS AND PERIPHERAL MYELINATION

1.1 HISTORICAL PERSPECTIVE The nervous system has central and peripheral branches, and the main cell types are neurons and glia. In the central nervous system (CNS), the glial cells consist of three types: oligodendrocytes (OLGs), astrocytes and microglia. The peripheral nerves are made up of axons surrounded by the glial cells of the peripheral nervous system (PNS), Schwann cells (SC), and fibroblasts. Axons are differentially supported by SCs according to size. Large caliber axons of motor or sensory neurons are surrounded by a myelin sheath, synthesized by SCs. On the other hand, sensory fibers that are smaller in caliber, are unmyelinated but are surrounded by SCs, forming a Remak bundle. The structure of myelinated axons was well described by the 1906 Physiology and Medicine Nobel prize winner, Santiago Ramon y Cajal, in his three volumes entitled Textura del sisterna nervioso del hombre y de los vertebrados in 1897-1899 (French translation published in 1909 and 1911) 561. However, the existence of myelin as a structural entity has been known since the mid-19th century, when Rudolf Virchow reported the presence of sheaths surrounding nerve fibers. Myelin is synthesized by glial cells – OLGs and SCs produce myelin in the CNS and PNS, respectively. Glial cells as a whole were simply viewed as ‘support’ cells and the ‘glue’ between neurons, participating in a ‘monologue’. However, findings over the last few decades successfully dispelled that notion. Glial cells are now viewed as active participants in a rather interesting ‘dialogue’ with neurons, maintaining an inter-dependent ‘relationship’. In 1838, Robert Remak first described the ‘tubulus primitivus’ (now called myelinated fibers) and organic fibers or ‘nonmedullated fibers’ (now called Remak bundles) found in peripheral nerves. His findings were subsequently confirmed by Theodore Schwann in 1839. Despite Remak’s initial discovery, this structure was subsequently referred to as the sheath of Schwann, after the German physiologist. Once it was recognized that the sheath was produced by a cell and not secreted, the cell came to be named the Schwann cell (SC). The establishment of an in vitro model of peripheral myelination, the SC- Dorsal root ganglion (DRGN) organotypic co-cultures, by Richard and Mary Bunge 87 was fundamental to the field of peripheral myelination and SC biology. The system has greatly facilitated the study of SC biology, particularly in the pursuit of uncovering molecular pathways regulating cell growth, survival and

30 peripheral myelination. Moreover, advances in molecular biology and genetics have led to the discovery of gene mutations affecting SCs and neurons, which manifest as peripheral neuropathies of varying severity. Myelin is indeed an architecturally complex specialized membrane. With each new scientific discovery, it becomes more evident that myelin formation and its regulatory mechanisms are far more intricate than originally described. Thus, despite many important advancements, these regulatory pathways and molecular ‘dialogues’ which decide the fates of neurons and SCs, remain largely enigmatic.

1.2 TYPES OF SCs

The SC, originating from neural crest precursor cells in the developing embryo (for a detailed developmental description, please see section 1.4.1), form close associations with axons of motor, sensory and autonomic neurons. As such, SCs provide support to the axons. The SC can be classified according to morphology, biochemistry and the neuronal types and axonal site of association as myelinating, non-myelinating, perisynaptic (or terminal) SCs, or satellite cells of peripheral glia (reviewed in Corfas et al, 2004 140). They are briefly described below: A myelinating SC wraps around all large diameter axons, including sensory and motor neurons to form a myelin sheath. They express several proteins typically found in the myelin sheath, including protein zero (P0), myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and myelin and lymphocyte protein (MAL). They modulate the formation of the nodes of Ranvier, axonal structure, and axon caliber through neurofilament spacing and phosphorylation 302,303. This is perhaps the best characterized SC.

A non-myelinating or ensheathing SC typically surrounds several axons from small caliber c-fibers from sensory and post-ganglionic sympathetic neurons to form a Remak bundle. Individual axons are separated by thin extensions of the SC body. They express high levels of the protein glial-fibrillary acid protein (GFAP), the low affinity neurotrophin Trk receptor p75NTR and the cell adhesion molecule L1. These ensheathing SCs have been shown to play important roles in unmyelinated axons: maintenance, nociception, and electrical conductivity in the adult 119.

The perisynaptic SC is found in the neuromuscular junction (NMJ), covering the presynaptic terminals of motor axons. The perisynaptic SC together with the synapse and postsynaptic muscle membrane, form a ‘tripartite synapse’ 25. They actively participate in the synaptogenesis, synaptic transmission and maintenance of the neuromuscular junction 202. They are also involved in axonal regeneration following injury. Although specific biochemical markers for PSCs have not been identified, perisynaptic SCS can be readily distinguished using electron microscopy.

Satellite glial cells may originate from neural crest-derived boundary cap cells. These SCs associate with neuronal cell bodies in peripheral ganglia and are positioned at the spinal cord surface at nerve root entry and exit zones 423.

This literature review will focus on the development of the myelinating SC. The architecture and molecular constituents of peripheral myelin will first be described, followed by an overview of the essential factors regulating SC commitment to a myelinating phenotype, with particular emphasis on molecules of the extracellular matrix, and key transcription factors involved. The major signaling pathways regulating SC differentiation from an immature SC to a myelinating phenotype will also be discussed. Finally, the major hereditary and acquired peripheral demyelinating diseases will be described.

1.3 PERIPERAL MYELIN

Rapid communication between nerve cells can be enhanced by increasing the diameter or by insulating axons. Myelin is a specialized lipid-rich cytoplasmic structure formed by SCs in the PNS and OLGs in the CNS. It represents a unique and fundamental adaptation of vertebrates to permit rapid ‘node’ to ‘node’ jumping or saltatory conduction of nerve impulses (shown in a schematic in Figure 1.1). However, myelin-like wrappings do exist in the ventral nerve chords of certain species of two invertebrate phyla (Annelida oligochaetes 271,275,396, and some Crustacea decapods 276,286,439). Beginning with cartilaginous fish to present-day vertebrates, central and peripheral axons of all classes are myelinated, except the most ancestral surviving species of fish, agnathan cyclostomata as lamprey (Petromyzon marinus) and hagfish (Eptatretus stouti),

32 which possess unmyelinated nerves. Many of its axons are, however individually embedded within one or more glial cells 86. It is thus postulated that true myelin originated within vertebrates. This may have been an important driving force during early vertebrate evolution and natural selection.

Figure 1.1 A schematic illustrating a myelinated axon

Shown in a, is a myelinated axon emanating from a neuronal cell body, with the initial segment, nodes of Ranvier and four myelinated internodes. Shown in b, is a close up of a myelinated internode surrounded on either side by nodes of Raniver. Arrows indicate the influx of sodium channels, and subsequent membrane repolarization (+) involved in the conduction of action potentials, “jumping” from one node to the next, along an axon. Adapted by permission from Elsevier [Current Opinion in Neurobiology] 528, copyright (2000).

1.3.1 Dorsal root ganglion neurons Spinal sensory dorsal root ganglia neurons (DRGNs) are a heterogeneous population of cells that convey sensory information from peripheral targets to the CNS 376. They originate from neural crest cell (NCC) progenitors, which migrate outwards from the neural crest to their final target sites, where, in the presence of environmental cues, they differentiate to form ganglia organized along the spinal cord. Sensory neurons extend two axonal

33 processes from the cell bodies in opposite directions, innervating peripheral and central regions of the organism. Placodes, specialized tissue or thickenings of the embryonic ectodermal epithelial layer, can also give rise to cranial sensory ganglia, forming large neurons. This is contrasted with the NCC-derived smaller neurons. Thus, mammalian primary sensory neurons can be divided into two major neuronal subtypes according to their size and origin – large ‘light’ ventrolateral and small diameter ‘dark’ dorsomedial neurons. The latter neuronal type contains relatively larger amounts of granular endoplasmic reticulum (ER) and ribosomes.

The axon cytoskeleton is made of three polymers: microtubules, microfilaments and neurofilaments (NFs). NFs, are intermediate filaments found in neurons. They are the most abundant structural component of large myelinated axons 291. NFs establish axon caliber and promote axon growth over long distances 290. NFs are obligate heteropolymers of three intermediate filament proteins, neurofilament light (NF-L) (61 kDa), medium (NF-M) (90 kDa) and heavy (NF-H) (110 kDa) subunits. NF-L forms heteromeric coiled-coiled units with NF-M, or NF-L, which then associate to form tetramers, and ultimately a polymeric structure. Synthesized in the neuronal soma and transported into the axon by slowed axonal transport, NFs are involved in establishing and maintaining the three-dimensional array of axoplasm. The NF-M and NF-L proteins contain multiple lysine-serine-proline (KSP) repeat motifs at the C-tail regions. These regions are phosphorylated after entering the axon 41,488. Phosphorylation regulates axon caliber by controlling transport, local accumulation and spacing of NFs. There are several proline-directed protein kinases that phosphorylate NF-M and NF-L, including cyclin-dependent kinase-5, extracellular regulated kinases (ERK)-1/2, glycogen synthase kinase-3 (GSK-3), and stress-activated protein kinase γ (SAPKγ) or c-Jun N-terminal Kinase-1 (JNK1) 337. NF-M tails were shown to primarily influence the dynamics of microtubules in the axon, which are essential for radial growth of both motor and sensory axons. NF-M has also been shown to affect NFs organization in the axon 562. However, a recent study using mutated Serine to Alanine replacement in the KSP consensus sequence of NF-M, suggests KSP phosphorylation is not required for radial axon growth 231.

An in vitro model of peripheral myelination was established where embryonic DRGNs are cultured on collagen-coated dishes. Both light and electron microscope in-depth comparative analyses of longterm (3 month) cultured rat DRGNs, by Bunge et al. affirmed the neurons closely resembled those in vivo in terms of content (nuclear and cytoplasmic) as well as in its organization 87. Nonetheless, neuron size, myelinated fiber diameter and internode length were smaller than values reported in vivo. Moreover, SCs originating from this dissociated DRGN culture, proliferate in vitro, and when co- cultured with DRGNs, can be induced to myelinate. Addition of ascorbate/Vitamin C (VC) to mature, confluent myelination-competent SC-DRGN co-cultures synchronizes many SCs to ensheathe or myelinate axons by promoting basal lamina assembly 184. SCs synthesize and secrete Type IV pro-collagen 102. In this instance, VC functions as a co-factor for the enzyme pro-collagen hydroxylase, which hydroxylates proline residues of the secreted pro-collagen. This leads to the assembly of triple-helical collagen fibrils, forming a mesh onto which molecules of the basal lamina, such as laminin, are entrapped. Close examination of these in vitro myelinating cultures affirmed that SC relationships with the myelinated or unmyelinated axons, and properties of myelin mimic that in vivo 87. In addition, axons of cultured DRGNs can be myelinated by OLGs in vitro 212,755.

Thus, co-culturing DRGNs with glial cells (SCs or OLGs) in vitro is an invaluable scientific tool in studying neuronal and glial cell development and peripheral and central myelination.

1.3.2 Properties of myelin Peripheral and central myelin is similar with respect to structural and molecular organization, despite originating from different cells. 72-78% of peripheral myelin consists of lipids, and only 20-30% of the myelin’s dry mass is protein. At least 60% of the proteins are glycoproteins and 20-30% are basic proteins (reviewed by Garbay et al. 228). The organization, length and thickness of the myelin sheath formed around axons are important determinants of electrical conductance along peripheral and central nerves 1,736. A measure used universally to assess the

35 myelination state of an axon (hypo- or hypermyelinated) is the g-ratio. It is defined as the ratio between the diameter (d) of the axon alone and the diameter of the myelinated axon.

g-ratio= daxon /daxon+myelin The closer the ratio is to one, the more hypomyelinated the axon is. The optimal ratio permitting maximal conductance, found in normal nerves is typically between 0.60-0.65 267,451,736.

1.3.3 Function of myelin The principal role of the myelin sheath is to allow the faster propagation of action potentials along the axons it surrounds. Interestingly, axonal insulation by myelin not only facilitates rapid nerve conduction but also regulates axonal transport and protects against axonal degeneration 179,774. Myelination has also been shown to modulate the caliber of axons 751 as a bi-directional signaling exists between the axon and the SC. They mutually promote survival and trophic support establishing a tight intertwined relationship.

1.3.4 Architecture of a myelinated internode A myelinated internode is comprised of a myelin sheath containing both compacted and non-compacted regions, that center around the node of Ranvier. Each internode is surrounded by a continuous basal lamina membrane that is deposited by the SC. The SC membrane facing the basal lamina is referred to as basal or abaxonal, while the membrane facing the axon is called referred to apical or adaxonal. A myelinated axon is organized into polarized axo-glial functional domains: the node of Ranvier, the paranode, the juxtaparanode and compacted myelin of internode (reviewed by Salzer, JL 600,601). Most of the myelin sheath is compacted myelin. Paranodes, Schmidt-Lantermann incisures (SLI) and Cajal bands are non-compacted regions of myelin. Each domain is distinct structurally and biochemically, as illustrated schematically in Figure 1.2. A unique complex of ion channels, cell adhesion molecules, adaptor proteins and cytoskeletal molecules are selectively localized to either the glial or neuronal membranes 27,528,608. The molecular composition and structure of each domain collectively enables rapid saltatory conduction of action potentials along the

36 axon. Complex interactions and bi-directional signaling between the myelinating glial cell and the axon directs the proper formation of these polarized structures. Mutations affecting the localization of any of the proteins can alter domain organization, yielding functional consequences. Described below are the structures and unique molecular composition of these domains:

Figure 1.2 Axoglial functional domains of a peripheral myelinated internode

a, the node (blue), paranode (green), juxtaparanode (purple) and internode (red) functional axo-glial regions are shown in this schema of the longitudinal-section of a myelinated internode from the PNS. In the internode from the PNS, a basal lamina membrane covers the entire axo-glial unit, while incisures channels spiral throughout the compacted myelin of the internode. Moreover, the outermost layer of the myelinating SC ends in cytoplasmic projections or microvilli in the nodal region, which contacts the axon. In the paranodal region, the compacted myelin opens up to form a series of cytoplasm-containing paranodal loops which invaginate and appose the axolemma through paranodal septate-like junctions, which is rich in NF-155. The unique set of molecules (cell adhesion molecules (CAMs), channels and cytoskeleton-associated proteins) localized to each region of the SC and axon are summarized in the table in b. Adapted by permission from Elsevier [Glia] 601, copyright (2008).

1.3.4.1 Node of Ranvier The node of Ranvier can be described as the gap between two internodes located beneath the SC basal lamina, where hundreds of microvilli extend from the outer collar of adjacent SC, interdigitate and make contact. The size of the node of Ranvier is typically 1-2 µm in length and the axonal caliber of this region is reduced since NFs are less heavily phosphorylated and are slowly transported 158. It is approximately one tenth the axon caliber found directly beneath the myelinated internode. This functional region is critical to generating action potentials and for its saltatory propagation. It is characterized by a high concentration of voltage-gated sodium channels (VGSCs) and is surrounded by ‘nodal gap substance’, a specialized extracellular basal lamina. Nodes are of particular interest due to their links to several diseases including neuropathies where nerve conductance is compromised, such as Charcot-Marie-Tooth (CMT) disease 46,663.

Axolemma: VGSCs mediate inward sodium currents that are required for nodal membrane depolarization involved in action potential generation and propagation. They are found at a higher density at nodes (1000-2000 channels/µm-2) than at the adjacent internodal axolemma (100-200 channels/µm- 2) 58,131,585,638. VGSCs are encoded by about a dozen genes. Each channel is composed of a pore-forming α subunit that is essential for conduction, and auxiliary β subunits 175. Each channel exhibits relatively unique physiological characteristics (for example, a low versus high-frequency firing rate). Moreover, VGSC gene expression is highly dynamic owing to its transcriptional and post- transcriptional regulation. In pathological conditions or following nerve injury, the 737 expression of these genes can be dysregulated . VGSC type Nav1.6 is primarily found in the axolemma of myelinated axons of peripheral nerves. Additionally, it is found along non-myelinated PNS axons such as DRG C-fibers, nerve endings of corneal C-fibers, where it participates in continuous conduction 58. The Na+/K+ ATPases and Na+/Ca2+ ion exchangers are also found in the nodal axolemma 249,738, and in conjunction with sodium channels, help produce and maintiain the ionic gradients necessary to generate action potentials.

In addition, the nodal axolemma contains several adhesion molecules, including the immunoglobulin cell adhesion molecules 186 kDa isoform of neurofascin (NF-186) and neural cell adhesion molecule (Nr-CAM), along with the scaffolding proteins ankyrinG 361 and βIV spectrin 358. Ankyrin-G interacts with

VGSCs, Kv channels, Nr-CAM, and neurofascin, tethering them to the actin cytoskeleton via βIIV spectrin. Thus, ankyrin-G links these proteins to the nodal axolemma. It has been reported that neurofascin and Nr-CAM appear temporally before ankyrin and VGSCs 371, and therefore may potentially mediate VSGC localization to the nodal region.

Glial membrane: Several lines of evidence support a model where node formation in the PNS requires glial contact with the axon 546,600. The SC microvilli are believed to play a critical role in organizing and stabilizing nodal domains during development. Microvili are rich in ezrin, radixin, moesin (ERM) proteins, ‘linkers’ between the plasma membrane and the actin cytoskeleton 611, and Ezrin binding phospho-protein-50 kDa (EBP50) 445. While devoid of myelin proteins 445, microvilli are also enriched in the GTPase RhoA, the proteoglycans syndecans (3 and -4) 256, extracellular matrix receptor dystroglycans and specific dystrophins 499,598. Similar to the perinodal astrocyte in the CNS 59, SCs participate in potassium ion buffering 450. Inwardly rectifying potassium channels (IRK1 and 3) are highly concentrated at microvilli and may allow the SC to actively engage in neuronal signaling. Sodium channels have been detected in SC cytoplasm and in the plasma membranes, including the perinodal processes surrounding the nodes of Ranvier 579. This data support the hypothesis that glial cells may act as an extraneuronal source of sodium channels. Gliomedin, a ligand for neuronal neurofascin (NF-186) and NrCAM, is an adhesion molecule expressed by SCs in both a membrane-bound form at the microvilli and as a soluble free form 188. The soluble form of gliomedin alone (in the absence of SCs) was able to induce node-like sodium channel clusters in the axolemma. However, the membrane-bound form of gliomedin works in conjunction with NrCAM to first cluster nodal sodium channels at heminodes during early node formation 188,198. Furthermore, gliomedin binds axonal NF-186 and leads to its accumulation at the distal ends of the myelinating SC. Hence, it

40 serves as a docking site for ankyrin-G to retain Nav channels, Kv channels and βIV spectrin to this region 198. Thus, clustering of sodium channels in peripheral nodes is contingent on direct SC contact with the nascent axolemma. This induces the enrichment of ankyrin-G 445. ERM protein activation and Rho signaling at tips of SC are also implicated in peripheral node formation by modulating actin cytoskeletal dynamics 234.

1.3.4.2 Paranode The compacted myelin sheath opens up to form a series of SC cytoplasmic (paranodal) loops. These extensions of the SC membrane spiral around in close apposition with the axolemma, separated only by 2.5-3 nm. This non-compacted region of myelin flanks the node of Ranvier in the central and peripheral nervous systems. The paranodal loops contain free ribosomes, tubules of agranular reticulum, and a few mitochondria; these loops are separated from the node of Ranvier by helically-wound septate-like tight junctions (paranodal junctions) that form with the axolemma. These are the largest vertebrate junction adhesion complexes 170. The paranodal junctions serve several functions: a) provide partial electrical insulation to the internodal region, b) act as physical barriers by preventing the lateral mobility of sodium channels (node) and potassium channels (juxtaparanode) in the axolemma, c) may contribute to axo-glial signaling d) play important roles in myelination 170 and e) regulate mitochondrial transport and function in the axoplasm of nodal/paranodal region of myelinated axons, and thus, potentially modulate the local regulation of energy metabolism in the nodal region 180. Upon paranodal disruption, sodium and potassium channels become mislocalized. These disruptions take place early in several neuropathies and dysmyelinating disorders 52,77,577,634. Moreover, aberrant swollen and large mitochondria accumulate in the nodal region of the peripheral nerves of mice deficient in the transmembrane protein, Caspr with disrupted paranodal junctions 180. Several adhesion molecules found on the axolemma and glial membrane contribute to the formation and the maintenance of the paranode.

Axolemma: The first paranode-specific protein discovered was Caspr (paranodin), localized to the paranodal axolemma 447,527. Caspr has an extensive extracellular domain, a single transmembrane segment and a short cytoplasmic C-terminal proline-rich sequence that binds SH3-domain bearing proteins 182,527. Another protein localized to the paranodal axolemma is Contactin (F3), a glycosylphosphatidylinositol protein (GPI)-anchored cell adhesion molecule of the Ig superfamily. Contactin binds Caspr through its Ig domain, and targets Caspr to the plasma membrane. Otherwise, Caspr remains associated with the ER 194. Caspr and Contactin are often found as a heterodimeric complex. This dimerization is essential to paranode formation. Finally, a novel axonal specialized cytoskeletal complex consisting of ankyrin B, αII spectrin, and βII spectrin was recently discovered in the paranode region 501. Organization is modulated through the interactions of axon-glial specific cell adhesion molecules. In the PNS, paranodal markers appear temporally later than nodal markers and sodium channel clustering 445. Other proteins of this region and other potential binding partners of Caspr remain elusive.

Glial membrane: The L1 subgroup of the IgG superfamily member, a 155kDa splice isoform of neurofascin (NF-155) was the first protein isolated from the glial membranes of paranodal loops. It is a binding partner involved in axo-glial contact. Several other proteins are enriched in the glial membrane, including the adhesion molecule, E-cadherin, the gap junction forming Connexin32 (CX32), and myelin associated glycoprotein (MAG). E-cadherin, MAG and L1 participate in SLI formation (discussed in section 1.6.2B) while Cx32 connects adjacent SC membranes in myelin. The axo-glial association of glial NF-155 with the axonal heterodimeric contactin-Caspr complex is critical for the formation and maintenance of the paranodal junction 546,669. Gangliosides, components of lipid rafts, are also important in the stabilization and integrity of paranodal junctions and ion channel clustering in myelinated axons 661.

1.3.4.3 Juxtaparanode This is the region (5-15 µm long) of the axon beneath compacted myelin that is adjacent to the paranode. The axolemma is characteristically enriched in voltage gated Shaker-type delayed rectifying potassium channels (Kv1 channels) and of several cell adhesion proteins (Caspr2) 449.

Axolemma:

Kv1 channels are multi-protein complexes, consisting of four membrane spanning and pore-forming α subunits and as many as four cytoplasmic β 563 subunits . In the periphery, α subunits Kv1.1 and Kv1.2 and cytoplasmic Kvβ2 449 subunits are expressed . Kv channels appear to help regulate axonal firing during early PNS development 714 and play a role during disease, injury, and remyelination 74,477,565,679,680. Caspr2, another member of the neurexin family of cell adhesion molecules, is expressed in this region 544. This 180-kDa cell adhesion protein has a short cytoplasmic tail that binds protein 4.1 family members, and a PDZ-binding domain in its C-terminal tail (not found in Caspr) 51,93,404,447. The GPI-anchored transient axonal glycoprotein (TAG-1)/Axonin- 1/Contactin-2 is also localized to the juxtaparanodal region of the axon 697. It can 698 form a complex with Caspr2 . TAG-1 is also postulated to associate with Kv1 channels through a PDZ-domain containing protein 547. Finally, a disintegrin and metalloproteinase 22 (ADAM22) was discovered to be a component of the

juxtaparanode. It colocalizes with Kv1.2, and membrane-associated guanylate kinases PSD-93 and PSD-95 in myelinated nerves 299,500. ADAM22 is a transmembrane protein with an extracellular disintegrin and catalytically inactive metalloproteinase domains believed to participate in cell-cell and cell-matrix interactions 746. Mutations of Lgi4, an ADAM22 binding partner, cause hypomyelination and axon sorting deficits 49. On the other hand, knockout of ADAM22 causes severe hypomyelination and death shortly after birth 594.

Glial membrane: The adhesion protein TAG-1 is also expressed by SCs. It is first expressed by ensheathing SCs early in development and then is maintained throughout adulthood 697. Moreover, a member of the connexin family, Connexin- 29 (Cx29), is found in the glial membrane of the juxtaparanodal region 17. A

43 function for Cx29 has yet to be elucidated. However, it has been proposed that Cx29 forms a hemichannel to aid in the removal of potassium ions following 17 release from Kv1 channels . Caspr additionally plays an essential part in the formation of the juxtaparanodal junction as Caspr2 localization is dependent on axon-glia interaction and junction formation 545. Subsequently, Caspr2 organizes the juxtaparanode structure. It binds to axonal TAG-1 and cytoskeletal adapter protein 4.1B through its cytoplasmic tail and forms a membrane scaffold 298,547. 698 The Caspr2-TAG-1 complex can then bind to glial TAG-1 . Thus, the Kv1 channels complexed with Caspr2, TAG-1, ADAM22, PSD-93, PSD-95, and protein 4.1 in the axolemma, are localized to the juxtaparanode as a macromolecular protein complex. This complex can bind TAG-1 found on the glial membrane.

1.3.4.4 Internode The axolemma of the node, paranode and juxtaparanode domains are defined by the enrichment of specific molecules. In contrast, the axolemma directly beneath the compacted region of the myelinated internode is not demarcated by any such molecular localization. This region of myelin is characterized by the multi-lamellae nature of compacted myelin with periodic interruptions by the Schmidt-Lantermann incisures (SLI), areas of non- compacted membrane specializations the. The outermost (abaxonal) SC cytoplasm outside myelin is polarized into two domains: the transverse domains (or appositions/patches) and the Cajal bands, trabeculae (Figure 1.3). ‘Patches’ are regions of the SC membrane in closest apposition to the myelin sheath (little cytoplasm). The Cajal bands are areas of abundant cytoplasm that form channels along the length of the internode. The ‘patches’ contain high levels of DRP2 and the myelin protein, L-periaxin 14,146,633. In contrast, the cytoplasmic Cajal bands contain large amounts of microtubules. These microtubules are postulated to be involved in the transport of nutrients and modulation of proper SC elongation that makes rapid impulse transmission possible 145,146. The formation of these compartments involves the interaction of the extracellular matrix molecule, laminin-2, with its receptor, dystroglycan (DG) 145.

SLIs are unique to the PNS. The membranes of the SC form a series of cytoplasmic channels continuous with the SC cytoplasm at the inner and outer areas of the myelin internode. These channels facilitate the rapid diffusion of nutrients and molecules radially between the adaxonal cytoplasm of myelin and the periaxonal region of the internode 37. This structure is rich in actin and several myelin proteins (CNP, DM-20, MAG) but not the structural proteins (PLP or P0), calcium channel protein S100, Cx32 and E-cadherin 20,47,197,230,263,435,551,608,614,699. Adjacent layers of the compacted myelin sheath are linked to one another through the adherens junctions, formed by E-cadherin, and the reflexive gap- junctions, formed by Cx32. An X-linked form of CMT disease is caused by mutations in the Cx32 gene 47, emphasizing the importance of incisures to nerve

conduction. Shaker-type Kv1 channels have also been localized to the incisures and inner adaxonal membrane 28. The number of SLIs is increased in MBP- and sulfatide-deficient mice 254,300 and are absent in P0 deficient mice 776. Moreover,

strands of Caspr and Kv1 channels form circumferential rings apposing the innermost aspect of the incisures 28 .

Figure 1.3 The complex architecture of a myelinated fiber.

In the immunohistochemical staining of a teased nerve fiber (a), laminin- α5 (green) is seen to localize to the nodal region, to constitute the nodal gap substance (arrowhead). The cytoplasm-rich and cytoplasm-poor regions that constitute the outermost layer of a myelinating SC are shown in b. Cytoplasm-rich cajal bands are identified by phalloidin (actin) (red) and tubulin (blue) staining, which identify the intricate cytoskeleton network of the SC. Moreover, the cytoplasm-poor regions of the SC where the membranes are in close apposition with one another are Drp2 positive ‘patches’ (green). These patches help demarcate and maintain the integrity of the Cajal bands. Distinct functional axo-glial regions (node of Ranvier (NR), compacted myelin (M), paranodal loops, Cajal Band (CB), trabeculae (T), apposition (APP)) of a myelinated fiber surrounded by the basal lamina (BL) are shown schematically in c. Also shown are the SC microvilli (Mv) extending into and contacting the NR region. Adapted by permission from Elsevier [Current Opinion in Neurobiology], 147, copyright (2006)

1.3.5 Lipids of myelin sheath The low capacitance, high resistance and insulating properties of myelin are largely due to the vast abundance of lipids and its unique lipid composition. Myelin is rich in cholesterol and glycosphingolipids. Cholesterol accounts for over one fourth of the total lipid content, compared to less than 20% in other plasma membranes 465. Expression of the rate-limiting enzyme in cholesterol synthesis, hydroxymethylglutaryl-coenzyme A reductase (HMGCR), developmentally increases during myelination closely resembling the expression pattern of myelin protein P0 222. In addition to its

46 structural and insulator function in myelin, a novel regulatory role for cholesterol in myelination was recently discovered. High levels of this sterol are specifically required for the ER exit of P0 and trafficking to the growing SC myelin membrane 596. Thus, cholesterol is the first membrane component shown to be central for SC myelination. In contrast to most plasma membranes, myelin contains a high proportion of glycosphingolipids, specifically, galactosylceramide (GalC) and its sulfate derivative, sulfatide. They constitute almost one-third of the total lipid mass of myelin. UDP-galactose, ceramide galactosyltransferase (CGT) is an enzyme required for the synthesis of both GalC and sulfatide, while cerebroside sulfotransferase (CST) synthesizes sulfatide. Paranodes are abnormal in CGT- and CST-null mice and they exhibit progressive dysmyelination 71,133,208,296. Sulfatide is important for normal axon-glial domain formation and maintenance. It binds to extracellular laminin in developing nerves, then polymerizes and anchors them to the SC surface. Thus, it enables basement membrane assembly 401. In CST-deficient mice, the peripheral myelin exhibit disordered microvilli alignment, abnormal paranodal junctions, mildly elongated nodes, and axonal protrusions at nodes containing mitochondria and abnormally large vesicles. Moreover, there are fewer clusters of paranode-localized Caspr and NF155, and increased numbers of incisures 296,300. In conclusion, GalC and sulfatide play important roles in the organization of peripheral myelin functional domains and in the maintenance of normal myelin. Additionally, cholesterol and glycosphingolipids form dynamic cellular membrane micro-domains called ‘lipid rafts’ together with specific membrane proteins. These specialized microenvironments serve as platforms for protein sorting and signal transduction 642. It has recently been suggested that myelin membranes form through the accumulation of myelin-specific rafts where cholesterol is closely associated with myelin membrane proteins. This was shown to take place in central myelination.

1.3.6 Proteins of Myelin Sheath Although proteins represent only a small fraction of the constituents of myelin, mutations in specific proteins results in demyelinating diseases such as

Charcot-Marie-Tooth (CMT) disease. Proteins of peripheral myelin include glycoproteins (MAG, P0, PMP22, periaxin), basic proteins (MBP, P2) and other proteins (PLP, CNP). The functional roles of myelin components in distinct steps of myelination have been illustrated using perturbing antibodies, specific antisense RNAs and mice carrying genetic mutations or deficient in particular proteins. For instance, specific proteins are involved in the formation of spiral-like extensions around the axon, myelin compaction, myelin thickness and the maintenance of compacted myelin.

1.3.6.1 Myelin associated glycoprotein (MAG)

MAG is a ~100 kDa transmembrane glycoprotein of the immunoglobulin (Ig) superfamily. It is expressed exclusively by myelinating SCs and OLGs (reviewed by Quarles 555). MAG constitutes less than 1% of the proteins found in peripheral myelin 554. It exists as two isoforms, a large (L) or a small (S) isoform, the result of alternative mRNA splicing from the Mag gene. MAG consists of five extracellular immunoglobulin-like domains, eight N-linked oligosaccharides, a single transmembrane domain and a cytoplasmic domain with two different C termini (forming L- or S-MAG). MAG can undergo glycosylation, sulfation of the oligosaccharide moeities, acylation of the transmembrane domain and phosphorylation of the cytoplasmic domain of L-MAG. Phosphorylation of the cytoplasmic domains enables MAG to participate in signal transduction 555. S-MAG is the predominant isoform expressed by SCs although a small amount of L-MAG is present. In contrast, OLGs express L-MAG early in myelinogenesis and S-MAG expression increases with maturity of myelin, such that both isoforms are equally expressed in the adult 223. In the periphery, MAG appears early during myelination in the growing tips of myelin sheath and in mature fibers where it is restricted to regions of non-compacted myelin (paranodal loops, periaxonal cytoplasm, nodes of Ranvier and SLIs). It is involved in the organization of the periaxonal space and the collar 397,398,702,703. However, MAG does not appear essential for the formation of compacted myelin in the periphery, since MAG-deficient SCs myelinate DRGN axons in culture 100. In addition, myelination occurs normally in the peripheral nerves of mice lacking the L-MAG 223. It is however, involved in the maintenance of peripheral myelin 99.

Furthermore, since MAG is localized to the periaxonal membranes of myelin, it participates in axo-glial interactions and bi-directional signaling. It provides trophic support to axons and influences axon caliber growth in the periphery. MAG-null mice exhibit both reduced Nf spacing and phosphorylation, paranodal myelin tomaculae and ultimately axonal degeneration in the periphery 775. Moreover, despite exhibiting normal gross motor coordination, fine motor coordination is affected in these mice 398. Lack of a hypomyelinating phenotype in the peripheral nerves is in direct contrast with the CNS, where MAG is essential for myelination 223. The phosphorylation site in L-MAG is targeted by the src-like kinase (SLK) Fyn, whose activity is required for CNS myelination. In fact, the absence of both MAG and Fyn resulted in a more severe CNS hypomyelination than mutants lacking each individual molecule 55,326.

1.3.6.2 Myelin basic protein (MBP) MBP is a member of the family of intrinsically unstructured proteins (reviewed by Boggs 65). It is encoded by the Golli (Genes of Oligodendrocyte Lineage) gene complex (11 exons in mice and 10 in humans) which gives rise to multiple golli products found in both the nervous (MBP) and hemopoietic system (Hemopoietic (H)-MBP) 250,342,432. MBP is found in both peripheral and central myelin, constituting 15-20% and 35% of the myelin proteins, respectively 2. Several isoforms of MBP are produced from differential splicing of a single mRNA transcript: 21.5, 20.2, 18.5, 17.24, 17.22 and 14 kDa (mouse); 21.5, 20.2, 18.5, 17.2 kDa (human) 39,65,160,279,344. In the rat PNS, 21.5, 18.5, 17 and 14 kDa isoforms are expressed at sufficiently high levels detectable by Western blotting 38,246,260,261,743. All isoforms contain exons I, III, IV, and VII. Exon II is found in 21.5, 20.5, and 17.22 kDa isoforms, while exon VI is found in all except 17.2 and 14 kDa isoforms. Several lines of evidence support the possibility that MBP gene expression in OLGs and SCs is primarily regulated at the level of initiation of transcription, by transcription factors and regulatory elements expressed exclusively by either glial cell. Indeed, distinct enhancers dictate glial cell-specific MBP expression in SCs and OLGs 257. In SCs, an enhancer (Mod4) was found to contain several binding sites for the two transcription factors, Sox and Krox-20. The Krox-20 binding is believed to amplify, rather than initiate MBP expression

169. There is stable accumulation of MBP mRNA in peripheral nerves throughout maturity 379,648,654. The 18.5 kDa MBP isoform is abundantly expressed in the CNS and exists as a series of charged isomers (C1-C8), with reduced positive charge. It accounts for 2% of the total myelin proteins in rat 259. Moreover, MBP is regulated post-transcriptionally and undergoes post-translational modifications including phosphorylation, deimidation, deimination/citrullination, acylation and methylation 2. These modifications are postulated to play a role in partitioning and relocalization of MBP to membrane microdomains 164. The high net charge and low hydrophobicity of MBP contributes to its role as a multi-functional protein. It is thus implicated in signaling, interactions with the cytoskeleton, and regulating functions of other myelin proteins (reviewed by 65,279). MBP interacts with lipid monolayers and bilayers through electrostatic and hydrophobic interactions 305,455,578. The distribution of basic residues over its entire length may enable it to form tight adhesion of the two lipid bilayers and act as a scaffolding protein. Hence, MBP is localized to the major dense line of both PNS and CNS myelin 503. Genetic deletion of 5 out of 7 MBP gene exons, yielded the naturally occurring autosomally recessive Shiverer (shi/shi) mutant. It lacks most CNS compact myelin 567 and has characteristic whole body tremors 125,581,770. This illustrates a critical role for MBP in myelin compaction of the CNS, with a lesser role in compaction of peripheral myelin due to the presence of other structural proteins including P0, PMP22 and P2 430,586,587. However, MBP does play an important role in PNS since morphological abnormalities are found in the PNS of Shiverer mice: reduced axon caliber and myelin sheath thickness, aberrant SC-axon contacts, and ~ double the number of SLIs in compacted myelin 254,534,587. Moreover, studies in Shiverer mice support a role for MBP in regulating gene expression in the PNS. MBP appears to exert an effect on both Cx32 and MAG at a post-transcriptional level 647.

1.3.6.3 Protein zero (P0) A third protein found in peripheral myelin is protein zero (P0), a single transmembrane protein belonging to the immunoglobulin gene superfamily of membrane proteins. It is encoded by the Mpz gene. P0 is a 28 kDa glycoprotein, consisting of a hydrophobic glycosylated extracellular domain, a transmembrane

50 and a basic intracellular domain which may contribute to the formation of the major dense line of myelin. It was initially the primary structural protein of both the CNS and PNS, appearing first ~440 million years ago in cartilaginous fish 352,593,725,780. However, with the appearance of reptiles and aves, the function of PLP became fully established and P0 was eradicated from CNS myelin 725,780. P0 now comprises approximately 70% of PNS myelin protein 391 and is highly conserved across species 2,780. It is the major structural protein of PNS myelin, expressed exclusively by myelinating SCs. However, there is basal expression during embryonic development by neural crest, SC precursors and embryonic SCs 54,385,792. Constitutive basal P0 expression during embryogenesis is attributable to Sox10 regulation. The profound upregulation of P0 during the course of myelination occurs via the cooperative binding of Krox20 and subsequent transactivation of the first intron of the Mpz gene in conjunction with Sox10 380. Furthermore, exit of P0 from the ER requires a high level of cholesterol 596, which adds another level of complexity. Moreover, P0 can be modified post-translationally through glycosylation, phosphorylation, sulfation and acylation 2. P0 is a pleiotropic protein. P0’s most important role is in adhesion, participating in myelin compaction through homophilic interactions 210. Both P0 molecules of the homophilic pair are glycosylated 209. This modification and pairing is required for myelin formation and maintenance. On the other hand, heterophilic interactions likely stabilize the axon-SC interface 621. In the absence of P0, uncompacted myelin and severe dysmyelinating peripheral neuropathy is exhibited in null mice 245. P0 may regulate expression of other myelin genes and modulate myelin morphogenesis. For example, in P0 null mice, PMP22 is downregulated, MAG and PLP are upregulated, and the quaking proteins, QKI-6 and -7 are absent. Moreover, there is accumulation of PLP/DM-20 and MAG, in the ER of SCs and the redundant loops of uncompacted myelin, respectively 763. SCs in the adult null mice MPZ mutations have been identified in a significant number of peripheral neuropathies 663. Moreover, severe and early onset demyelinating neuropathy (myelin decompaction, degeneration and axonopathic changes) occurs in P0-null mice, thus illustrating its pertinence to myelin compaction 245.

P0 is critical for SLI formation and correct localization of VGSCs, paranodal and juxtaparanodal molecular constituents. Yin et al (2008) demonstrated that SLIs are absent from peripheral myelin in P0-null mice. Substituting PLP with P0 in CNS myelin, induces SLI formation 776. Using homologous recombination to produce P0-null mice, a deficiency of P0 was demonstrated to lead to dysregulation of nodal VGSC isoforms and to alter localization of molecules of the paranode and juxtaparanode 709. Finally, normal PNS myelination requires strict dosage of P0. For instance, transgenic mice with additional copies of Mpz (30-80% overexpression) exhibit a dose-dependent dysmyelinating neuropathy 556,759. P0 accumulates inappropriately in domains of the plasma membrane, inhibiting spiral mesaxon growth and myelination 777.

1.3.6.4 Periaxin Periaxin is a PDZ-domain containing cytoskeleton-associated protein expressed by myelinating SCs. It represents 5% of total peripheral myelin protein 639. The PRX gene encodes two isoforms, L- (147 kDa) and S- (16 kDa) periaxin, generated by alternative splicing 178. L-periaxin is nuclear as of E14.5 in mice. With the onset of myelination, by E17.5 periaxin is redistributed and becomes primarily associated with the SC plasma membrane – the adaxonal membrane. It is subsequently upregulated as myelination progresses postnatally and becomes localized to the abaxonal membranes, SLIs and paranodal membranes 612,632. The pattern of expression for periaxin is similar to that of P0 during development. Interestingly, periaxin expression prior to myelination occurs in a Krox-20- independent manner, and during onset of myelination, it is robustly upregulated by Krox-20 514,515. Moreover, expression is regulated by axonal contact. Nerve transection or crush decreases periaxin expression 612. PRX mutation causes recessive DSS 64, Type 4F CMT 270,340 and degenerating peripheral neuropathies. Similarly, periaxin-null mice myelinate but suffer progressive demyelination in adulthood 248. Hence, periaxin is believed to stabilize the myelin sheath, likely by complexing with dystrophin-related protein DRP2 and β-dystroglycan, two receptors of extracellular matrix molecules in the SC membrane 633. Moreover, disruption of periaxin in null mice results in an absence of Cajal bands,

52 longitudinal and transverse bands of cytoplasm, thus impairing SC elongation during nerve growth 146.

1.3.6.5 Peripheral myelin protein 22 kDa (PMP22) PMP22 is a 22 kDa integral membrane glycoprotein belonging to a small family of highly related molecules including epithelial membrane proteins-1, 2 and 3 and the lens-specific membrane protein 20. With four membrane-spanning domains, PMP22 is also a member of a large superfamily of tetraspan proteins associated with myelin that includes PLP/DM20. Additionally, it has two extracellular domains and a carbohydrate chain. PMP22 gene expression is complex. It is regulated transcriptionally from two distinct promoters 664, yielding an SC-exclusive CD25 RNA transcript (exon 1A), and a more ubiquitous SR13/growth arrest-specific gene3 (gas3) transcript (exon 1B) 73. Additionally, PMP22 is regulated at several levels post-transcriptionally 72,719. PMP22 transcripts and protein have been detected in brain, intestine, lung, heart and motor neurons 518,522,683. This differential expression of the CD25 and SR13/gas3 transcripts may account for dual roles of PMP22 in myelination and in cell growth, respectively. PMP22 additionally regulates cell spreading, cell migration and apoptosis and is found early at intercellular junctions of the developing blood-nerve and blood-brain barriers 81,82,192,495,589,590,603.

In SCs, PMP22, found in compacted myelin, accounts for 2-5% of total myelin protein. CD25 transcript follows an expression pattern, similar to that of several other myelin genes in vivo during development and nerve regeneration, as well as in vitro 73,368. Duplications, deletions, and point mutations affecting PMP22 are responsible for several peripheral neuropathies (Charcot-Marie- Tooth-1A (CMT1A), hereditary neuropathies with liability to pressure palsy (HNPP), and a subtype of Dejerine-Sottas syndrome (DSS)). A gene dosage effect is implicated in the incidence of PMP22-linked disease. Genetic duplication of a 1.5 megabase-intrachromosomal on chromosome region 17p11.2 accounts for the most common form of CMT1A, affecting 70-80% of CMT patients 410,436,580. The deletion of this region is linked to mild HNPP 113. Thus, patients with a) one copy of the gene develop a mild phenotype with nerves liable to HNPP, b) two gene copies are normal, c) three copies develop CMT1A and d) four copies

53 present a severe phenotype of DSS (Hanemann and Muller, 1998). Furthermore, animal models under- or over-expressing PMP22 have helped pinpoint the roles of PMP22 in myelination. Onset of myelination is delayed in mice devoid of PMP22. They develop tomacula or focal hypermyelination at a young age, followed by severe demyelination, axonal loss and functional impairment later in life 8. These null mice exhibit walking difficulties at P14 due to progressive paralysis of hind limbs, with occasional mild tremors and stress-induced convulsions.

Missense mutations in any of the four hydrophobic transmembrane domains are associated with demyelinating neuropathies in humans and mice 486. The discovery of mice with naturally occurring mutations in the PMP22 gene has played a key role in elucidating functions of PMP22 in myelination. Three naturally occurring mice mutants exist: Trembler (Tr), Trembler-Jackson (Tr-J) and Tr-Ncnp. Mice carrying the autosomal dominant Tr missense mutation, display severe hypomyelination and continuous SC proliferation, forming tomaculae. In Tr-J mice, PMP22 gene mutations prevent the maintenance of normal myelin in the mature PNS 473. Furthermore, the intracellular trafficking of both Tr-J and Tr-mutant PMP22 is impaired, where the mutated protein is retained in the ER/Golgi complex 153,472,692. In sciatic nerves of PMP22-null mice, myelin protein is expressed but at lower levels 8.

PMP22 also forms homodimers. It associates with P0 in peripheral myelin 152, forming a functional complex that participates in the adhesion of adjacent SC membranes, in stabilizing compaction, and later in the maintenance of compacted myelin. Mutations in either glycoprotein could interfere with proper complex formation and lead to subsequent demyelination. This may represent the common pathological pathway in patients with CMT1A and 1B. Accordingly, PMP22 is involved in early stages of myelination, influencing myelin thickness, myelin maintenance and axonal integrity 473.

The transcript SR13, which is more ubiquitously expressed, is inversely correlated with proliferation. SR13 overexpression in SCs leads to the arrest of proliferation 797, and to cell death in fibroblasts 192,798. In neonates, CD25 levels

54 steadily increase from low levels to a maximum at postnatal day 14, while SR13 levels are elevated at birth but decline throughout adulthood 73.

1.3.6.6 Peripheral myelin protein2 (PMP2) PMP-2, formerly known as Protein P2, is an extrinsic membrane protein, which first appeared in tetrapods. This small ~15 kDa basic protein is found in the cytoplasmic spaces of peripheral myelin lamellae 700 and in the CNS myelin of some species 246,314,701. The concentration and distribution of PMP-2 in myelin is highly variable according to species and fiber tract (reviewed by Sedzik 626). This is distinct from the other major myelin proteins including MBP, PLP and P0. PMP-2 constitutes <1% of the total protein of mouse or rat sciatic nerve and 5- 15% of human, bovine and rabbit nerve root myelin proteins 2. In the mouse, the PMP-2 gene was sequenced and found to contain four exons, encoding separate peptide segments 476. PMP-2 is the least studied of all the myelin proteins. Accordingly, an exact role in myelination has not been fully deciphered. PMP-2 is a member of the family of fatty acid binding proteins, sharing basic secondary and tertiary structures with several homologous paralogues. It is postulated to participate in intracellular lipid solubilization and transportation of fatty acids or retinoids. Consequently, PMP-2 is thought to contribute to the assembly, remodeling and maintenance of myelin. Recently, PMP-2 with MBP was shown to synergistically cause the stacking of brain lipid bilayers ie. myelin sheath formation 660. PMP-2 is neuritogenic and is both chemically and immunologically distinct from MBP. It has been implicated in a number of neurological diseases including autoimmune diseases of both CNS and PNS. It is shown to induce experimental allergic neuritis (EAN), an autoimmune demyelinating disease of the PNS, following the injection of Lewis rats with Freund’s adjuvant 313. EAN is a proposed animal model for Guillain-Barre-Strohl (GBS) syndrome, an acute inflammatory neuropathy in humans.

1.3.6.7 Connexins The connexins (Cx) are a multigene family of proteins that form intercellular channels clustered at gap junctions, dynamic plasma membrane

55 structures, which form aqueous channels connecting the cytoplasms of adjacent cells. They permit the rapid transfer of ions, small water-soluble molecules (<1,000 Daltons) and small molecular weight nutrients. Each channel is an oligomeric structure, comprised of two opposing hemichannels or connexons, each anchored in its respective plasma membrane, and each hemichannel is composed of a homomeric or heteromeric combination of six subunits of connexins 4. Furthermore, a functional channel can be formed by the homotypic or heterotypic pairing of connexons consisting of one type of connexin 4. Each connexin has four alpha helical transmembrane domains (TM1 to TM4), intracellular N- and C-terminals, a cytoplasmic loop connecting TM2 and TM3 and extracellular loops connecting TM1 to TM2, TM2 to TM3. In the peripheral nerves of rodents, Cx26, -32, -37, -43 and -46 have been detected 114,421,782,794. Although Cx26 and Cx43 are reportedly expressed by myelinating SCs 782,794, they are barely detected. Cx32 and 29 appear to be the best-characterized Cxs. Cx32, was the first connexin to be cloned through screening of a liver cDNA library, and hence can be considered a founding member of the connexin family. This 32 kDa gap junction channel protein possesses several domains: 1) four hydrophobic membrane spanning, 2) two extracellular (involved in docking) and 3) three cytoplasmic (N-terminal region, loop between transmembrane domains 2 and 3, C-terminal tail) 372. Cx32 is expressed in a number of tissues, including the CNS and PNS by both SCs and OLGs. mRNA and protein expression profiles of Cx32 in myelinating glia parallels that of myelin genes. Although, in peripheral nerves, during development and regeneration, Cx32 expression appears to lag behind MAG and its expression requires maintained axonal interaction and can be induced by cAMP analogs 609. In the CNS, it is confined to OLG bodies and processes but not compact myelin 609. On the other hand, in the PNS, it is confined to non-compacted myelin, forming reflexive gap junctions between paranodal loops and SLI 365,609. These reflexive channels located between multiple layers of the myelin sheath formed by the same SC, permits the bidirectional radial diffusion between the adaxonal membrane and the perinuclear cytoplasm 4,37,47,85,484,502,523,572. This diffusion pathway is at least 300 times shorter than the circumferential pathway through the myelin sheath 4. Transmission of essential axo-glial signals during the initiation of myelination or in the maintenance of myelin is greatly facilitated. Typically, patients carrying

56 mutations in the Cx32 gene, associated with the X-linked form of CMT (CMTX), predominantly present a peripheral phenotype 47,70, although clinical CNS deficits were also detected in a subset of Cx32 mutations 470. To date, a number of Cx32 mutations (missense, frameshift, deletion, non-sense) affecting nearly every portion of the protein has been detected in humans. These mutations affect Cx32 trafficking within SC, channel formation, disruption of electrical conductance and disruption of channel opening and permeability 4. Interestingly, Cx32 does not appear to be involved in initiation of peripheral myelin formation, but it is critical for its maintenance. Cx32-null mice, using a Mpz-driven promoter, initially form normal myelin but eventually develop a late-onset demyelinating peripheral neuropathy characterized by thinly myelinated axons, onion bulb formation, abnormal organization of non-compacted myelin and abnormally thick periaxonal collars, or ‘blown-up’ non-compacted myelin regions, and profiles indicative of axonal degeneration and regeneration 22,426. In fact, SCs in transgenic Cx32-null mice expressing human Cx32 using a rat Mpz-driven promoter, rescued the phenotype of Cx32-null mice 613. Demyelination of motor axons followed by remyelination along their length was mostly prevented or delayed by expressing normal human Cx32. Cx29, a novel connexin, is expressed by both OLGs and SCs 17. Cx29 has been identified as a marker for SC lineage commitment during development, as it is expressed when NCCs form precursor SCs 399. Cx29 is highly expressed in SCs of the cochlea and is essential for normal function and mutation is linked with nonsyndromic hearing loss 295,675. Moreover, in peripheral myelin, it is found enriched in regions adjacent to the inner mesaxon, in the inner aspects of the paranodes and juxtaparanodes, and is more uniformly distributed to incisures 17. This is juxtaposed with the enrichment of Cx32 in the outer aspects of paranodes and incisures.

1.3.6.8 Proteolipid protein (PLP)/DM20 Proteolipid protein (PLP), a hydrophobic tetraspan protein, is a family member of proteolipids, which despite being found in plants and animal membranes, is most abundant in myelin. The PLP gene gives rise to two proteins by alternative splicing: a longer PLP and a shorter DM20. PLP has four

57 transmembrane domains, three small hydrophilic loops, a third extracellular loop structured by two disulfide bridges. DM20 lacks a 35 amino acid sequence encoding a portion of the second intracellular loop, referred to as the ‘PLP- specific region’ 228. PLP is the major structural protein in the CNS, accounting for ~40% of proteins in the CNS 454 and DM20, expressed at high levels during embryogenesis, is only a minor constituent of mature myelin 617,690. PLP expression was previously believed to be exclusive to the CNS, however, it was later identified in peripheral myelin 13,230, comprising <1% of the total myelin protein. PLP/DM20 is found in the peripheral compact myelin of teleosts and amphibians 780. Juxtaposed with the PLP/DM20 expression ratio in central myelin, DM20 isoform appears to be the predominant PLP isoform in peripheral myelin 535. Moreover, studies using direct subfractionation of the peripheral nerve indicate both PLP/DM20 are found in compacted myelin 13, while studies using immunohistochemistry, indicate differential subcellular localization where PLP is additionally found in the perinuclear cytoplasm and DM20 is localized only to areas of uncompacted myelin 264,551. PLP expression profile follows that of the other myelin-specific proteins of the PNS and expression requires continuous SC-axon contact 331. On the other hand, SCs express DM20 independent of the presence of axons 331. In the CNS, like PMP22 in the PNS, gene dosage of PLP is essential for normal myelination, as mutations in this gene and gene duplication cause a spectrum of diseases including Pelizeaus-Merzbacher disease (PMD), a primarily CNS-targeted disease 288. PMD patients with missense mutations outside the PLP-specific region and gene duplication affecting PLP did not manifest abnormal peripheral nerve function and structure, leading to the belief that PLP did not to play an important role in PNS myelination 289. Notably, patients carrying null PLP mutations or mutations within PLP-specific domain, leading to PLP truncation without affecting its alternatively spliced variant, DM20, exhibit a mild peripheral neuropathy syndrome 229,640. These results indicate PLP is required for proper function of myelinated peripheral nerves and not DM20. This hydrophobic transmembrane protein is found in both the major dense line and the intraperiod line of compacted myelin, where it is believed to play a structural role by maintaining the integrity of the intraperiod line of myelin 310. Furthermore, several mutations causing misfolding and inhibiting the trafficking of both PLP and DM20

58 did not affect peripheral nerve function. This implies that PLP/DM20 can either function within the ER of SCs, where it accumulates or sufficient quantities of mutant protein is expressed at the cell surface, allowing normal function 640. Thus, the molecular mechanisms describing the exact role of PLP/DM20 in peripheral myelination remain undefined but are an avid area of investigation.

1.3.6.9 2’, 3’cyclic nucleotide 3’phosphodiesterase (CNP) This enzyme hydrolyzes 2’, 3’-cyclic nucleotides to produce exclusively 2’ nucleotides. CNP is localized to the outer perimeter of the peripheral myelin sheath, non-compacted myelin (SLI and periaxonal region), outer mesaxon, SC surface membrane and cytoplasm 783,784. CNP accounts for less than 0.5% of PNS myelin proteins 712. CNP is expressed as two isoforms – CNP1 (46 kDa) and CNP2 (48 kDa), which are derived from a single gene from two separate promoters 173,174. The function of CNP has yet to be elucidated, although it has been associated with maintaining axo-glial interactions at nodes of Ranvier in the CNS 564.

In summary, the numerous proteins constituting peripheral myelin, each plays a specific role either in the creation of this architecturally complex multilamellar sheath or in its maintenance thereafter.

1.4 SC DEVELOPMENT AND DIFFERENTIATION

1.4.1 Origin and development of SCs

Neural crest cells (NCCs) are transient migratory multipotent stem cells. During embryogenesis, they migrate out from the dorsal region of the neural plate as it curves and closes to form the neural tube. They travel along distinct pathways while proliferating, and arrive at their final destinations. In the presence of extrinsic environmental cues, post-migratory NCCs progress through step-wise developmental stages and commit to particular lineages. They give rise to a plethora of cell and tissue types, including glial cells of the PNS. Although the majority of SCs arise from the NCC, some may originate from the placodes and

59 ventral neural tube, as well as neural crest boundary cap cells 24,423. In addition, NCCs are a source for melanocytes, connective tissues of the head, and the neuronal (dorsal root sensory and autonomic) and several non-SC glial cell types of the PNS (teloglia of the somatic motor terminals, satellite glial cells associated with neuronal cell bodies in sensory and sympathetic/parasympathetic ganglia and enteric glia in ganglia of the gut) 235,237,377,510. Thus, given the pluripotent nature of the NCC, the signals regulating initial lineage decisions are of utmost interest and are only now beginning to be elucidated. Three signaling systems implicated in specification of the SC lineage (SC precursors) from NCCs are Delta/Notch, Neuregulin-1 (NRG1) and the transcription factor Sox10. As migratory NCCs travel outwards from the dorsal aspect of the neural tube, they come into contact with nascent embryonic peripheral axons emerging from the ventrolateral aspect of the neural tube and differentiate to form SC precursors (embryonic day 12 (E12) in mouse, E14/15 in rat). Whether NCCs are committed to the SC-fate (SC precursors) prior to or become committed upon encountering embryonic axons, remains obscure. Soluble factors, such as neurotrophins and Notch, influence this differentiation (summarized in Figure 1.3). SC precursors express basal levels of P0, Desert hedgehog (Dhh), Bone morphogenic proteins-2 and 4 (BMP-2/4) and Cx29 57,384,399,519. Moreover, they continue proliferating and migrating along the developing axons when they first loosely ensheath developing axons. They send out cytoplasmic projections to progressively package axons into bundles. Meanwhile, SC precursors differentiate into immature SCs (E17/18 in rat; E15-17 in mouse).

Figure 1.3 SC Lineage.

Schematic illustrating the development of a SC. SCs originate from neural crest cells. Myelination initiates around birth and non-myelinating SCs appear approximately 2 weeks after birth. The red arrow indicates the de-differentiation of SCs from a myelinating phenotype to an immature SC. The green arrow indicates the reverse differentiation and terminal differentiation of an immature SC, which has made a 1:1 contact with an axon greater than 1 µm in diameter. Adapted by permission from Macmillan Publishers Ltd: [Nature Reviews Neuroscience] 329, copyright (2005).

Immature SCs continue proliferating while they begin synthesizing a range of growth factors and a basal lamina. Their survival at this stage is primarily mediated by autocrine factors, and contact with both basal lamina constituents and axons. The numbers of SCs is negatively regulated through activation of the p75NTR neurotrophin receptor and transforming growth factor β (TGF-β). Moreover, during late embryogenesis and neonatal stages, immature SCs continue their rapid proliferation, migration, and begin radial axonal sorting. Immature SCs exit the cell cycle to adopt two phenotypes according to the size of the axon they associate with. If an SC ensheathes a single large caliber axon (greater than 1 µm), it will become a myelinating SC. On the other hand,

61 immature SCs that adopt a non-myelinating (ensheathing) phenotype, invest multiple small caliber axons, separating each unmyelinated axon by a cytoplasmic process, to form a Remak bundle.

An immature SC associated with a large caliber axon, passes through a transient pro-myelinating stage prior to elaborating a myelin sheath. A basal lamina is assembled. Expression of multiple myelin transcription factors, myelin- specific proteins and lipids are induced, with concomitant downregulation of immature SC/non-myelinating SC-associated markers including NCAM, L1 and Sox2. Extensive time-lapse imaging by Bunge and collaborators revealed that the SC extends the inner adaxonal cytoplasmic tip as it begins wrapping around the axon to ultimately form a multi-layered membrane structure 91. As the SC continues spiraling around the axon, the cytoplasm from the outer layers of myelin is extruded, and the myelin sheath extends longitudinally along the axon. Ultimately mature compacted myelin is formed. Thus, the development and differentiation of SCs may be traced according to the upregulated or downregulated expression of cell surface proteins and transcription factors, as well as the SC’s characteristic responses to soluble survival factors. This is summarized in Figure 1.4.

Several extracellular cues can stimulate or inhibit myelination. Thus, immature SCs can be influenced by positive regulators to differentiate to mature myelinating SCs, or fully differentiated, mature SCs can dedifferentiate to an immature precursor state in the presence of negative regulators, as occurs in injury (reviewed by Jessen and Mirsky, 2008 330). Moreover, de-differentiated SCs retain the ability to re-differentiate and myelinate. It is indeed this unique plasticity of the SC that lends itself to the current efforts to exploit this in developing therapies aimed at stimulating central myelination in such instances as spinal cord injury and the CNS demyelinating disease, multiple sclerosis involving SC transplantation 60.

Figure 1.4 Factors regulating SC development.

In the cartoon depicting the developmental lineage of myelinating SC from neural crest cell, numerous positive regulators (green arrows) of SC development from the neural crest cell are indicated above the green arrows. Moreover, several negative regulators, promoting SC dedifferentiation are noted and denoted by the red arrows. Adapted by permission from Macmillan Publishers Ltd: [Nature Reviews Neuroscience] 329, copyright (2005).

1.4.2 SC differentiation and myelination

1.4.2.1 Cell cycle exit In most cells, differentiation requires both the establishment of G1 arrest and the induction of the maturation program. Immature SCs are rapidly proliferating during early pre-natal development in the presence of embryonic developing axons and soluble factors. SC mitotic activity in the mouse peaks during late embryogenesis and declines thereafter. Accordingly, very little cell division is detected in major peripheral nerves during the second postnatal week, coinciding with cell cycle exit of immature SCs as they adopt the divergent myelinating and non-myelinating phenotypes 602,656. Therefore, initiating terminal differentiation of SCs and onset of myelination is preceded by cell cycle exit. Briefly, the four stages of cell cycle followed by proliferating eukaryotic somatic cells are gap phase1 (G1), synthesis (S), gap phase 2 (G2) and mitosis (M). Non-proliferating, terminally differentiated cells exit the cell cycle to reside in the G0 phase. Cell cycle progression is tightly controlled by several regulators and repressors which act at either the G1 to S or G2 to M cell-cycle checkpoints. Cyclin-dependent kinases (cdk) positively regulate cell cycle progression while cdk inhibitors negatively regulate it. Moreover, cdk kinase activity can be repressed or activated through phosphorylation. Cdks form a complex with cyclin proteins604. Cyclins are the regulatory unit of the cyclin/CDK complex and are transiently expressed during different stages of the cell cycle. They are classified in three groups according to the cell cycle phase at which they act: G1 cyclins (C, D, E), S phase cyclins (A) and G2/M phase cyclins (B1, B2, A). cdks partner with only specific cyclins: cdk2/cyclinE, cdk2/cyclinA, cdk4/cyclinD, cdk5/cyclinD, cdk6/cyclinD. Hence, cyclin/cdk complexes integrate extracellular and intracellular cues to determine cell cycle progression. They bind retinoblastoma (Rb), a protein that regulates E2F factors to control transcription in S phase synthesis (reviewed by Santamaria and Ortega 604). It is postulated that prenatal and postnatal SC proliferation is differentially regulated. SCs express cdk2 and 4 during development and in adult nerves 31. Using cdk-deficient mice, it was shown that cdk4 is essential for post-natal

64 proliferation, while cdk2 and cdk6 are not 31. Moreover, D-type cyclins regulate the transition through G1 in SCs in response to growth factors. They assemble with cdk2 and cdk4 to form a holoenzyme, which binds the Rb kinase to facilitate G1 cell cycle exit. There are three cyclin D types (D1, D2 and D3) that share substantial sequence homology. There may be functional redundancy in immature SCs during development as cyclin D1-knockouts do not display SC abnormalities. However, in postnatal nerves, proliferation of healthy adult mature SCs is strictly cyclin D1-dependent 348. Moreover, subtype-specific cyclin D knockout mice illustrate that cyclin D1 is required for SC growth following nerve injury 348. Two classes of cdk inhibitors modulate cdk-cyclin complex activity: inhibitors of cdk4 (INK4) family and the cip/kip inhibitors. The first group of cdk inhibitors, the INK4 family, specifically regulates G1 progression. The 4 members, p15INK4b, p16INK4a, p18INK4c and p19INK4d, bind only cdk4 and cdk6, thus, preferentially targeting cyclinD/cdk4 and cyclinD/cdk6 complexes 506,635. Accordingly in SCs, p16INK4a is required for proper cell cycle withdrawal postnatally at early and late stages to maintain quiescence. In fact, subcellular compartmentalization of this inhibitor appears to play a role in modulating its function. Soon after birth, p16INK4a is cytoplasmic (postnatal day 7) but at later stages of development (postnatal day 21), it becomes localized to the nucleus 32. The Cip/Kip family of cdk inhibitors is more broad-acting. They interact with complexes containing cyclins -D, -E, -A, and cdk2,-4, -6 to prevent cell cycle progression. Three family members, p27kip1, p21cip1, p57kip2, are implicated in SC cell cycle progression 505. p21cip1 inhibits cyclinE/cdk2 activity, is essential for proper withdrawal from cell cycle, and its subcellular relocalization is associated with maintaining quiescence of SCs at late postnatal stages 32. p27kip1 expression is associated with cell cycle arrest at the G1-S checkpoint (early G1 and late G1) in response to anti-mitotic signals, at G0 and the G2-M checkpoint 139,630. p27kip1 is highly regulated transcriptionally and post-translationally, which may dictate its role by altering its protein-protein interactions, subcellular localization and protein stability 139,630. It is expressed at high levels in the nuclei of cells in the G0 stage, including myelinating SCs 514,515. However, p27kip1 functions extend beyond simple cell cycle regulation as overexpression in OLGs enhanced MBP promoter activity 457. It has also been shown to regulate the cytoskeleton. In SCs, p27kip1

65 expression is linked with periaxin gene expression 515. In contrast to p21cip1 and p27kip1, p57kip2 is a key negative regulator of SC differentiation and hence, of myelination 283,284,364. In summary, cdk4, Cyclins (D), the INK4 family and the cip/kip family of cdk inhibitors have thus far been implicated in coupling SC cell cycle arrest and differentiation.

1.4.2.2 Cell adhesion molecules Myelination is achieved by a sequence of events that involve surface contact of the axon and SC with its environment and with one another. Cell adhesion molecules found on both axon and SC play key roles in proper cell-cell interactions involved in SC-axon recognition, adhesion, ensheathment, and formation of functional axo-glial myelin domains. Moreover, many of these cell adhesion molecules are spatio-temporally expressed during development. SCs express a number of cell adhesion molecules including glial TAG-1, gliomedin, neurofascin-155 (NF-155), Ig superfamily members (glial L1, N-CAM, MAG, P0), cadherin (E- and N-) and Nectin-like (Necl) proteins. These are described briefly below. As described earlier, NF-155, TAG-1, gliomedin, expressed by myelinating SCs, mediate their interaction with axons (section 1.5.3). They are critical for the formation of the node of Ranvier and polarization of the axolemma surrounding it. The neural cell adhesion molecules L1, neural cell adhesion molecule (NrCAM), and MAG, members of the immunoglobulin superfamily, are involved in early stages of myelination. L1 is an integral membrane adhesion molecule expressed in both the central and peripheral nervous systems. It has binding affinity for several extracellular ligands. L1 and NCAM are expressed by both SCs and neurons. They share a L2/HNK-1 carbohydrate epitope and are engaged in contact and/or recognition between axons and SCs at an early stage of myelination 670. Both are expressed by premyelinating SCs, but L1 appears earlier than NCAM. Axonal-L1 binds matrix receptors β1 integrins on SCs, ensuring proper axon ensheathment and myelination 320. It is involved in the extension of SC processes along axons, ensheathment and radial sorting of

66 axons, and the induction of myelin-specific components within the SC 756. SCs downregulate NCAM and L1 expression to undetectable levels after forming 1 ½ layers of compact myelin around the axon, and concomitantly upregulate MAG expression 428,429,670. Nonetheless, NCAM and L1 continue to be weakly expressed by non-myelinating SCs in the adult 193. Heterophilic L1 adhesion of axon to a molecule on the SC surface is essential for normal SC-axon interaction and for myelination. Axon-specific L1 knockout mice exhibit abnormalities; non- myelinating SCs fail to correctly ensheath small caliber axons 154,277. Thus, although L1 and NCAM are both involved in early stages of myelination, L1 is indispensable 756. A second family of cell adhesion molecules is the cadherins. These transmembrane glycoproteins have five cadherin domains. They participate in calcium-dependent homophilic (cadherin-cadherin) interactions. Binding of calcium to the interface of adjacent cadherin domains stabilizes their conformation, permitting homophilic adhesion. Two members of the classical cadherin family expressed by SCs are E- and N-cadherins, and participate in heterophilic binding with integrins. Intracellularly, cadherins interact with a number of proteins, such as α and β catenins, which are the intermediates between cadherins and the actin cytoskeleton. SC precursors express high levels of N-cadherin 733 during embryonic development and facilitate axonal growth 353. However, as they differentiate to form immature SCs, N-cadherin is downregulated while E-cadherin increases. Most axons at this stage have made contact with their targets. E-cadherin forms the core of the adherens-junctions between adjacent paranodal loops at the edge of myelin sheaths in SCs 197. Adherens junctions are abundant in non-compacted myelin, such as paranodes, Cajal bands and SLIs. E-cadherin is required for the maintenance of SLIs. β- and γ-catenins bind to the cytoplasmic domains of E-cadherins, linking it to the actin cytoskeleton by recruiting α-catenin 773. Also, P120 catenin binds to E-cadherin and modulates its adhesion. During myelination, P120 catenin is essential for the formation of mature adherens junctions and a normal myelin sheath 532. In fully formed myelin, P120 catenin is required to maintain SLI integrity 704. Lastly, SCs and axons express Necl (SynCAM), a protein member of a small group of Ig CAM superfamily. They mediate axo-glial interactions along the internode and also are integral to PNS myelination 437. Neurons express Necl-1

67 and -2, on the axolemma beneath the internode, while SCs express Necl-4 and lower amounts of Necl-2. Moreover, Necl-1, -2, and -4 are enriched at the SLIs. Axonal Necl-1 binds to SC-Necl-4 437,653. Both ablating Necl-4 expression and disrupting Necl-1-Necl-4 interaction reduces myelination 437,653. In conclusion, cell adhesion molecules regulate many aspects of SC- axon, SC-environmental interactions to regulate peripheral myelination.

1.4.2.3 Transcription factors Axon-SC and SC-basal lamina interactions activate multiple signaling pathways that converge onto the nucleus and regulate transcriptional programs to determine the phenotype of a SC (myelinating vs non-myelinating). As such, the distinct SC morphological changes that take place during peripheral myelination require coordinated gene expression. To date, several transcription factors critical to SC development are expressed at discrete stages and collectively form a hierarchical network through which SC development is modulated. The most important genetic regulators of peripheral myelination are described below (reviewed by Svaren and Meijer (2008) 666).

1.4.2.3.1 POU family of transcription factors The family of POU transcription factors, a subgroup of homeo-domain proteins, is characterized by the POU domain. The homeo-domain is connected by a short linker region to an N-terminally located POU-specific domain (reviewed by Ryan et al, 1993 592). This family consists of several members, of which Suppressed cAMP Inducible Protein (SCIP)/Oct-6/Pou3f1/Tst-1 and Brain2 (Brn2) play important roles in SC development and myelination. Brn5, on the other hand, has been found to be expressed by myelinating SCs and induced by neuregulin (NRG) 760. However, its relevance to myelination remains to be elucidated. SCIP, a 58 kDa protein, is expressed by embryonic stem cells and in developing brain and adult testis 665. Agents that elevate intracellular cAMP, such as the adenylate cyclase activator forskolin, strongly upregulate SCIP expression in cultured SCs 463,464. This inducible transcription factor is transiently expressed

68 by immature SCs 61,799 upon axonal contact 610. SCIP is detected as early as embryonic day 15.5 61,323, it reaches maximal expression in promyelinating SCs at birth, and then gradually declines 464. SCIP induces Krox-20 expression 239. Peak expression coincides with the induction of Krox-20, while its gradual downregulation is inversely matched with the maximal upregulation of Krox-20 48,281. In other words, the progression from early promyelinating to late promyelinating to myelinating SC development follows the phenotypic expression of SCIP+Krox-20- to SCIP+Krox20+ to SCIP-Krox20+ 61,799. Deletion of SCIP in mice causes SCs to transiently stall at the promyelinating stage 48,323; however, albeit temporally delayed, SCs still proceed to myelinate 323. These results suggest possible compensatory mechanisms at play. Indeed, recent studies have shown that Brn2 and Brn5 share important functions with SCIP. Thus, SCIP is an important regulator of the timing and rate of transition from promyelinating to myelinating SCs. Similarly, SCIP appears to be an important early regulator of oligodendrocyte differentiation, as it is highly expressed in promyelinating OLGs but is rapidly attenuated following initiation of myelination 136. Brn2 is a 50 kDa transcription factor expressed by SCs. It exhibits a developmental profile similar to SCIP, but their expression is independent322. Notably, Brn2 exhibits only a partial functional redundancy with SCIP since Brn2- deficient mice do not exhibit a SC phenotype 322. Moreover, Brn1 is a POU transcription factor expressed by promyelinating OLGs and absent altogether from SCs 136,622. Genetic studies conducted by ‘knocking-in’ Brn1 into the SCIP locus, demonstrate that replacement of SCIP allows full myelin formation by SCs in a timely manner, like wild type mice, without aberrations 220. Brn5 was found to be expressed in a subset of cortical neurons 19 and in the RT-4 neuronal cell line 171. Using differential display reverse transcription polymerase chain reaction (RT-PCR), Brn5 was found follow a pattern of expression inverse to SCIP; it is absent in promyelinating SCs but is stably and abundantly present in myelinating SCs 760. Moreover, neuregulin Glial Growth Factor-2 (GGF2) induced expression of Brn-5 in a dose- and time-dependent manner in culture. Following sciatic nerve injury, Brn5 was downregulated. Interestingly, Brn-5 was not detected in the recovery phase following peripheral nerve injury and Wallerian degeneration, suggesting differential roles of Brn-5

69 during injury and peripheral myelination. Nevertheless, genes regulated by Brn5 remain undefined. POU proteins function in conjunction with Sox proteins to induce gene transcription. Although predominantly nuclear, SCIP possesses an additional leucine-rich nuclear export signal (NES) within its homeodomain, which enables it to shuttle frequently between the nucleus and cytoplasm. This nucleocytoplasmic shuttling protein has been shown to cooperatively act with Sox10 to induce gene expression 83,524. Moreover, SCIP, Brn2 and Sox10 were discovered to bind to the myelin Schwann cell enhancer (MSE) element, found in the regulatory segment of the essential transcription factor, Krox-20. They collectively induce Krox-20 expression 238. Thus, Brn2 and SCIP in concert regulate SC transition from the promyelinating to myelinating stage. Furthermore, Brn5 may potentially participate in modulating the expression of myelin genes cooperatively with Sox proteins.

1.4.2.3.2 Early growth response (Egr) family This family of immediate early genes, zinc finger transcription factors, consists of four closely related genes: Kruppel box-24 (Krox-24) (Egr1/NGFI- A/Zif268), Krox-20 (Egr2), Pilot (Egr3) and NGFI-C (Egr4). They exhibit differential tissue distribution and function . Krox-24 and Krox-20 are strongly implicated in SC development, while roles for Egr3 and 4 in myelination have not yet been elucidated. Krox-20 was originally described as a serum response gene in NIH 3T3 cells 115. Null mice revealed an important developmental role for Krox-20 in hindbrain segmentation post-natally 620, endochondreal bone formation 394,395, and, more importantly, as a master regulator of SC differentiation and peripheral myelination 695. EGR2 mutations have been found in patients diagnosed for inherited peripheral neuropathies including Charcot-Marie-Tooth disease type I (CMT1) and Dejerine Sottas Syndrome (DSS) 43,62,471,512,689,734,781. Targeted Krox- 20 gene disruption in mice blocks peripheral myelination by arresting SCs at a promyelinating stage, where they only make one and a half turns around the axon 695. Furthermore, SCs maintain SCIP expression and remain in the cell cycle 799 although the early myelin marker MAG is expressed 695. Moreover,

70 ectopic expression of Krox-20 in SC cultures induces endogenous Mpz and Mag genes in the absence of axonal contact 474,515. Microarray expression profiling of these SCs ectopically expressing Krox-20, pinpointed Krox-20 as key coordinator for expression of multiple genes, including myelin proteins (Mag, Mpz, Mbp, Pmp22, Cx32, Prxn) and enzymes required for synthesis of critical myelin lipids (3-hydroxyl-3-methylglutaryl coenzyme A reductase (HMGCR), Stearoyl CoA desaturase (SCD), 7-dehydrocholesterol reductase, and lanosterol 14- demethylase (CYP51)) 381,474,515. Furthermore, it binds to and directly activates Dhh, Mag, Mbp 325 and Mpz 380 expression. Moreover, Krox-20 regulates expression of multiple genes in conjunction with other transcription factors or indirectly. For instance, Krox-20 cooperatively activates Mpz and Mag gene expression with Sox10. Adjacent binding sites have been identified for these two transcription factors in the promoter regions for these myelin genes. Neuropathy-associated dominant negative Krox-20 mutants were found to antagonize Sox10 binding at specific sites, thus disrupting the genetic control of myelination 382. Additionally, Krox-20 forms a complex with the NGFI-A/Egr-binding (NAB) co-repressors (Krox-20/NAB complex), which directly represses expression of genes, including Id2, an inhibitor of DNA binding/differentiation 415. Moreover, Krox-20 regulates SC proliferation and cell death by upregulating expression of the scaffolding protein, JNK-interacting protein-1 (JIP-1). JIP-1 is an inhibitor of JNK activity. Thus, Krox-20 modulates JIP-1 levels to ultimately suppress JNK-c-Jun activity, which is involved in both proliferation and cell death 514. Furthermore, Krox-20 controls SCIP expression, cell cycle exit and the susceptibility of developing myelinating SCs to apoptosis 799. It was also recently shown to suppress Notch signaling 757 Recently, using conditional Krox-20 knockouts, a critical role in the maintenance of peripheral myelin was unveiled 166. Specific inactivation of Krox- 20 in adult mice results in severe demyelination, involving rapid SC dedifferentiation and increased proliferation. Subsequently, SCs attempt to remyelinate but are arrested at the promyelinating stage. Collectively, these studies clearly illustrate the importance of Krox-20 at all stages of PNS myelination - at the onset of myelination and during the maintenance of myelin integrityKrox-20 is expressed by SCs in two waves: firstly, in boundary cap cells at E10.5, and secondly, along the peripheral nerve at E15.5, where expression is

71 maximal 695 and is subsequently maintained throughout life. Krox-20 expression is dependent on constant axonal contact 469,694,799. Two distinct regulatory sequences, immature SC element (ISE) and MSE, control Krox-20 expression in an axon-dependent manner at different phases during SC development. ISE is active in SC precursors, while MSE is active in promyelinating SCs. There are several binding sites for SCIP in the MSE 239. Using transgenic mice and a cell culture system, SCIP and Brn2 were shown to directly bind this enhancer and activate Krox-20 transcription. On the other hand, Sox10 synergizes on the MSE to drive expression of SC myelination and demyelination programs 238. Thus, regulation of Krox-20 gene expression is complex.

Krox-24 was first identified by differential hybridization in NGF-treated rat PC12 cells 453. It is involved in the development and physiology of the anterior pituitary 389,694. Krox-24 is the best characterized member of this family for its role in neural plasticity and memory formation 157,356,497. It is specifically required for the consolidation of long term memory using mutant mice 78-80,335. In contrast to Krox-20, expression of Krox-24 is restricted to non-myelinating and proliferating SCs in the adult 389,694. Krox-24 is induced after nerve injury and is integral for SC expression of the low-affinity p75 nerve growth factor receptor for SCs subsequent to nerve injury 487. Further studies into regulation of Krox-24 gene expression and its role in SC development are required.

1.4.2.3.3 SRY boxcontaining (SOX) family transcription factors (Sox2, Sox10) Transcription factors of the SRY (Sex determining region on the Y chromosome) box-containing (SOX) family not only play essential roles during embryogenesis, sex determination and development, but also control homeostasis in adults. They possess a DNA binding domain similar to the high- mobility group (HMG) domain of SRY 244,266. To date, over twenty Sox proteins have been identified in mammals and are classified into six groups (A-G) according to sequence homology; Group B is further subdivided into B1 (activators) and B2 (repressors) 607. They possess three functional domains: a) the HMG domain which binds to the minor groove of the DNA helix to induce a large bend, characteristic of Sox proteins 607,740, b) an activation or repression

72 domain, near the C terminus, and c) a region that includes part of the HMG, which interacts with partner transcription factors 343. Essentially, Sox proteins, which are widely distributed and have overlapping patterns of expression, partner off with specific transcription factors by interacting with their DNA-binding domains. As such, they synergistically activate gene expression. Sox proteins promiscuously cooperate with several transcription factors, including POU domain and Egr family members 334,359. This contributes to their pleiotropic functions. These partner proteins tend to demonstrate more tissue-specific expression. Of the Sox proteins, Sox10 and Sox2 are implicated in SC development and PNS myelination. Sox10 is expressed by NCCs during early stages of development. This Class E member is involved in the pluripotency and survival of NCCs 349. As development progresses, Sox10 becomes restricted to NCC derivatives (sensory and enteric ganglia, melanocytes and glia of the central and peripheral nervous systems (OLGs and SCs)) 366,458, where it regulates cell specification and differentiation 83. In humans, SOX10 mutations are associated with Shah- Waardenburg syndrome (SW4) 539, a disease where two neural crest-derived cell lineages, melanocytes and intestinal ganglia cells are affected. It is also linked with Hirschsprung disease 318. These two diseases have been linked to neurocristopathies, syndromes arising from the maldevelopment of NCCs (deficiencies in the migration, proliferation, and differentiation) 68. Additionally, patients with heterozygous Sox10 mutations or SW4, presented with central and peripheral myelin defects 319,541,696. Sox10 mutant mice exhibit the early loss of SCs 83, associated with demyelinating peripheral neuropathies 46. Sox10 controls the expression of the neuregulin receptor ErbB3 in NCCs 83, activation of which promotes the differentiation of NCCs into glia 631. Sox10 can bind DNA as monomer or a dimer 525 and is an active nucleocytoplasmic shuttle protein 569. It binds several sites in the Mpz promoter and is required for embryonic expression in developing SCs 524. Additionally, binding sites were identified in the promoter regions of Cx32 69,524,525, and the MSE 238,239. Furthermore, Sox10 cooperates with a variety of DNA-binding proteins, including Pax3, SP1, and POU domain proteins to regulate gene expression 185,214,323,366,367,446,695. Partnered with Krox- 20, as described earlier, Sox10 regulates the expression of Mag, Mpz, Cx32 myelin genes 69,382. On the other hand, while pairing with SCIP 366 or NFAT4c 346,

Sox10 synergistically induces Krox-20 expression in SCs 570,571. As such, Sox10 not only functions in the specification of myelin genes expression by embryonic SCs, but in the maintenance of myelin gene levels in myelinating SCs.

Sox2, a member of Group B1, participates in maintaining the self-renewal and pluripotent state of cells such as embryonic stem (ES)/EC cells, differentiation of neural tissues and lens cells. Like Sox10, Sox2 can complex with Oct-3/4 to cooperatively regulate gene expression 343. In ES cells, Sox2 is required to maintain Oct3/4 levels and hence stabilizes them in a pluripotent state 434. Moreover, Sox2-null mice are lethal at peri-implantation 34. Sox2 is expressed by undifferentiated precursors in the peripheral region of developing DRG, the glial sub-lineages of NCCs (SCs and satellite glia of the PNS, and the prospective oligodendrocyte precursors in the CNS of avian embryo) 727. Sox2 plays multiple roles in avian embryo. It is downregulated in the neural plate when the neural crest segregates from dorsal neural tube. Therefore, levels are low during NCC migration, but are subsequently upregulated and restricted to some glia. Sox2 is important for the neural plate development 354, by regulating proliferation and differentiation of migratory/post-migratory neural crest-derived cells 726. Mice homozygous for the hypomorphic Egr2 allele (Egr2lo), where a PGK-Neo cassette is inserted into the Egr2 locus to reduce Egr2 expression, have a longer lifespan than Egr2-null mice 695. This is a model of congenital hypomyelinating neuropathy (CHN). Mice display severe tremors and impaired coordination while sciatic nerves at P14 are thin and translucent. Microarray gene expression profiling on P14 sciatic nerves and RT-PCR demonstrates that Sox2 is expressed by immature SCs, and is downregulated in myelinating cells 378. Enforced Sox2 expression in forskolin treated SCs, suppresses the expression of several myelin-related genes including Egr2, Mpz, Prx and Ndrg1 but does not alter expression of nonmyelinating markers, such as L1 and NCAM. It also enhances the proliferative response of SCs to β-neuregulin stimulation. Additionally, SCs continuously expressing Sox2 are maintained in an undifferentiated state and do not myelinate DRG axons 378. It is upregulated following nerve injury and co-expressed with c-Jun 378,513. In summary, Sox2 is essential to maintain SCs in an immature state during development and following nerve injury.

1.4.2.3.4 Pax3 Pax (paired-box)-3 is one of nine members of the highly conserved family of helix-turn-helix class transcription factors that mediate embryogenesis and development. Pax-3 is expressed exclusively during embryogenesis, in the dorsal region of the neuroepithelium, in neural crest derivatives and somatic mesoderm critical for the closing of the neural tube and morphogenesis of the neural crest and myoblast lineages 255. Pax3 mutants have significant neural crest and neural tube/CNS defects 748. Hypomorphic Pax3 null mice die in utero, with defects of the neural tube closure, myogenesis, melanocytes, SC and a subpopulation of mesenchymal cells in outflow tract. Pax3 expression by embryonic SCs (eSCs) may depend on SC-axon interaction 350; it is implicated in the transition from embryonic SC to non-myelinating SC or myelinating SC. In adult sciatic nerve, Pax3 expression is present only in non-myelinating SCs. SCs ectopically expressing Pax3 in microinjected cells, downregulate MBP expression, while upregulating NGFR, GFAP, L1 and N-CAM, markers characteristic of nonmyelinating SCs 350.

1.4.2.3.5 Nuclear factor kappalightchain enhancer of activated B cells (NFkB) The immediate-early gene Nuclear Factor κB (NFκB) is a family of structurally related proteins consisting of p65/RelA, c-Rel, RelB, p50 and p52, which form homo- and heterodimers. These dimers are reversibly repressed in the cytoplasm by its endogenous inhibitor, IκBα. The phosphorylation of IκBα leads to its ubiquitination and subsequent degradation, thus, permitting the nuclear translocation of the DNA binding subunits of NFκB 240. NFκB is activated by a variety of extracellular signals, including cytokines, growth factors and extracellular matrix proteins 509 including neurotrophins 112, NRG-1 403 and collagen matrix of basal lamina 762. Activated p65 was detected in sciatic nerves of neonatal rats at high levels at P0-8 and declines after P12. SN50, a competitive inhibitor of NFκB nuclear translocation, arrested SCs at a premyelinating stage. Moreover, myelination in SC-DRGN co-cultures established from p65-null mice was abrogated and NFκB was found to be

75 required for SCIP expression 485. Post-translational modification of p65 subunit (phosphorylation, acetylation and ubiquitination) can modulate NFκB-dependent transcription 95,96. Protein kinase A (PKA) activation in SCs, likely through NRG-1 403, led to p65 phosphorylation at Ser276, which subsequently enhanced transcriptional activity. Mutation of this serine residue attenuated both phosphorylation and SC differentiation and myelination 779.

1.4.2.3.6 Nuclear factor of activated T cells (NFAT) The transcription factor NFAT participates in critical biological processes including cell growth, differentiation and development 258. It is highly phosphorylated and resides cytoplasmically in resting cells 148,292. Dephosphorylation of NFAT, mediated by calcineurin phosphatase, permits its nuclear localization. Within the nucleus, it interacts with other NFAT partners, to effectively initiate transcription. To terminate NFAT-mediated gene transcription, it requires rephosphorylation to facilitate cytoplasmic retention, or nuclear export. NFAT was recently shown to play a role in the differentiation of NCCs to SCs and in PNS myelination 346. NRG1 increases cytoplasmic calcium levels, leading to calcineurin activation, and ultimately, NFAT signaling. In fact, NFAT in conjunction with Sox10, activates Krox-20 expression leading to SC precursor differentiation to a myelinating phenotype.

1.4.2.3.7 cJun Jun is an inducible transcription factor that was first described as the oncogene of the avian sarcoma virus 17 110, followed by the identification of cellular Jun (c-Jun) from human cells 67. c-Jun is part of a basic leucine zipper transcription factor family, along with JunB and JunD. The immediate-early gene c-Jun is the major component of the Activator Protein-1 (AP-1) complex that is comprised of homo or heterodimers of Jun and Fos transcription factor families. The AP-1 complex is implicated in proliferation, survival, growth, differentiation, apoptosis, cell migration and cell transformation 720. Jun contains a docking site for Jun N-terminal kinases (JNKs). Phosphorylation of cJun on Ser63 and -73 positions by JNK enhances both its transcriptional potential and stability 646. Thus, c-Jun forms the terminal component of the Jun-N-terminal kinase (JNK)

76 pathway 66. c-Jun is a positive regulator of the cell cycle and is a strong activator of cyclin D1441. c-Jun and phosphorylated-c-Jun is found in proliferating SCs at E17 and neonatal nerves prior to myelination 514, but is absent in adult nerves 35,159,641,655. In SCs, c-Jun functions as a negative regulator of SC differentiation, acting primarily at the transition between immature and terminally differentiated stages 513. Continuous ectopic Krox-20 expression in SCs, blocks TGF-β and NRG-1- induced c-Jun phosphorylation and reduces c-Jun protein levels 514. In contrast, c-Jun inhibits Krox-20 expression and the associated induction of myelin genes expression 513.

1.5 REGULATION OF SC MYELINATION: POSITIVE AND NEGATIVE REGULATORS

SC development is directed by the integration of intrinsic timing mechanisms and extrinsic factors (e.g. growth factors, neuronal signals, extracellular matrix) that culminate in promoting SC differentiation (positive regulation) or SC de-differentiation (negative regulation). In this section, we will review some positive and negative regulators of SC differentiation. Furthermore, several key signal transduction pathways modulating SC differentiation/dedifferentiation that have come to light in the last few years, will be discussed in Section 1.8.

1.5.1 Positive regulators of differentiation

Immature SCs require positive cues to exit the cell cycle and to initiate myelination. In order for myelination to proceed, two important conditions must be met; 1) SCs must make and maintain contact with a large caliber axon, and 2) extracellular matrix (ECM) or basal lamina must be assembled. In addition a number of autocrine or paracrine factors are also important. These are described below:

1.5.1.1 AxonGlia interactions: cell contact and secreted factors Axons and SCs form a mutually beneficial relationship to support their survival and differentiation during development. Signals provided by axons promote SC proliferation and survival so that their numbers match the axonal requirement. Likewise, SCs provide trophic support to axons and potentiate axonal targeting. Moreover, this bi-directional communication is partly mediated through the release of soluble neurotrophic, mitogenic and survival factors by axons and SCs, and the activation of cell-surface receptors through direct contact.

Soluble factors: SCs secrete numerous soluble neurotrophic factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4, glial-cell line-derived neutrophic factor (GDNF) and Insulin growth factor (IGF) 89,90,219,225,262,285,328. Meanwhile, in response to several SC-derived neurotrophic factors, sensory and motor axons secrete the growth factor neuregulin-1 (NRG-1) isoforms type I (glial growth factor2 (GGF2) and type II in a dose-dependent manner 189. NRG-1 Type I has been implicated in migration of developing SCs along axons as they begin to bundle axons 418.

The neurotrophin family of growth factors (NGF, BDNF, NT-3, NT-4) primarily act through a small family of receptors, the Trk tyrosine protein kinases and the p75 NT receptor (p75NTR). p75NTR is a common receptor that can bind all neurotrophins with similar affinity. NGF binds most selectively to trkA, BDNF and NT-4 to trkB, and NT-3 to trkC 165. Studies on differentiation of SCs have reported that neurotrophins influence myelination in the periphery. SCs express p75 and trkC, but only low levels of fulllength trkB 143. Activation of both receptors in SCs has opposing roles in myelination; p75NTR promotes myelination while TrkC receptor activation inhibits it 143. BDNF binds p75NTR while NT-3 binds TrkC to elicit its promyelinating and inhibitory pro-migratory effects in immature/premyelinating SCs, respectively. Moreover, neurotrophin secretion from developing SCs leads to the activation of Protein kinase C δ (PKCδ) in neurons through TrkC, to induce the cleavage and release of NRG-1 189,190. NGF

78 and GDNF are the most potent in inducing NRG-1 release from sensory DRGNs, while GDNF and BDNF maximally induce release from motor neurons. Moreover, injection of high doses of GDNF into sciatic nerves induces robust proliferation of non-myelinating SCs, radial sorting of many unmyelinated axons, formation of 1:1 SC:axon association, with subsequent myelination of these small-diameter axons 293. As such, neurons and SCs mutually support their survival and differentiation during development.

Contact: Axonal contact also directs SC behavior by inducing its proliferation and survival, primarily through the NRG-1 type III protein expressed on its membrane (reviewed in section 1.7.1). Interaction of neuronal NRG-1 with SC surface receptors, ErbB, induces rapid SC proliferation partly by elevating cAMP levels 393 and cJun expression 641. It is well established that continuous cAMP levels in cultured SCs results in the upregulation of myelin gene expression 392,466. Thus, forskolin, an activator of adenylate cyclase 625, and cAMP analogues mimic some of the effects of axonal contact on SCs and induce SC differentiation towards a myelinating phenotype. Myelin proteins P0, MAG, and MBP are upregulated, with concomitant downregulation of non-myelinating markers such as GFAP and N- CAM 461,558. In zebrafish, a G-protein coupled receptor Gpr126, may additionally modulate elevations in cAMP levels 462, independent of the NRG-1-ErbB pathway. Moreover, NRG-1 type III neuroaxonal expression is also an essential signal for ensheathment and axonal myelination 681. Also, SCs synthesize progesterone in response to axon contact 582,623. Progesterone can increase myelin genes expression 269,416,623. Similarly, SC contact with the axon can help direct the architecture of functional axo-glial domains of myelinated internodes. For instance, axonal contact with SC microvilli helps reorganize the axonal cytoskeleton, leading to a redistribution of several molecules, including sodium and potassium channels in the axolemma, formation of distinct functional domains (described in section 1.3.4.1). Additionally, the close SC-neuron interactions influence axonal caliber, electrical properties and neuronal morphologies by modulating a kinase- phosphatase system which acts on Nfs phosphorylation and possibly a number of other substrates. For instance, the axonal diameter of regions covered by the

79 myelinated sheath is larger than regions in the narrow unmyelinated nodes of Ranvier since the C-terminal tails of Nfs are phosphorylated 303,336. This is more evident in the case of demyelinated axons, where axonal transport is slowed; there is reduced Nf phosphorylation, axonal caliber and electrical conduction 158.

1.5.1.2. Autocrine Growth factors Intriguingly, post-natal immature SCs possess an alternative axon- independent autocrine survival loop, which is absent in SC precursors whose survival is contingent upon axonal-derived signaling. This characteristic of developed SCs is an essential intrinsic property to promote axonal regeneration in injured nerves. Immature SCs around E17-18 in rat begin to synthesize various growth factors, including insulin-like growth-factor (IGFs), platelet derived growth factor-BB (PDGF-BB) and NT-3124,442. SCs express their respective receptors (TrkC, PDGF-β, IGF-R). These factors primarily act through the mitogen-activated kinase (MAPK) and Akt pathways to synergistically block SC death 442,517. Moreover, the bioactive phospholipid, lysophosphatidic acid (LPA) has been shown to promote SC survival in vitro, paralleling the effect of NRG, activating PI-3K-Akt pathway, downstream of the G protein-coupled receptor ventricular zone gene-1 (VZG-1) 742

1.5.1.3 Basal lamina assembly During embryonic development, a basal lamina assembles, surrounding each axon-SC unit. This is embedded within a matrix of collagen fibrils (endoneurial collagen), which, together form the endoneurium or the extracellular matrix in the peripheral nerve 102. Numerous studies have shown that ECM formation (extracellular fibril and SC basal lamina assembly) and myelination are tightly linked 183. Assembly of basal lamina and SC adhesion to this matrix is a pre-requisite for myelin-specific gene expression and PNS myelination 183,184. Interactions with ECM molecules have a direct influence on important cellular events including morphological changes, migration, proliferation and differentiation of SCs. Moreover, it is essential for the early events of SC ensheathment and progression of myelination 92,204.

The ECM of peripheral nerve contains a diverse set of macromolecules, including collagens (types IV and V), and proteoglycans (heparin sulfate proteoglycans) and non-collagenous glycoproteins (entactin/nidogen, fibronectins and laminins) 542. However, focus will be drawn to the two major players in peripheral nerve myelination: collagens and laminins in section 1.6.

1.5.2 Negative regulators of differentiation In addition to the positive, forward-drivers of myelination program, active negative regulators also instruct quiescent, fully differentiated, non-myelinating or myelinating SCs to de-differentiate and re-enter the cell cycle, thus, reverting to an earlier immature state. In SCs, several known negative regulators of myelination include the transcription factors, c-Jun, Pax3 and Sox2, as described in section 1.4.2.3. Notch is a novel negative regulator of SC differentiation, recently identified, while Id2, Krox-24 and Egr3 are potential negative regulators of myelination, but roles for each have not yet been deciphered.

Notch is a family of heterodimeric transmembrane proteins, composed of four cell surface receptors (Notch 1-4), which regulate the differentiation, maturation and function of a number of cell types during embryogenesis through paracrine signaling 330,339. The extracellular region of Notch consists of epidermal growth factor (EGF)-like repeats and L1N12/Notch repeats (LNR), which bind Notch ligands and prevents ligand-independent activation, respectively. The intracellular portion contains the RAM domain, ankyrin repeats, nuclear localization sequences and a transactivation domain (TAD) with phosphorylation sites, and a C-terminal PEST sequence. RAM domain and ankryin repeats interact with transcription factors. Moreover, TADs are potential sites of modulation by other intracellular pathways. Jagged1/2, Delta1/3/4, contactin/F3 are Notch ligands 29,304. The binding of ligand located on an adjacent cell to Notch, triggers receptor signaling. This involves cleavage of Notch by an ADAM metalloprotease to release the Notch intracellular domain (NICD) and by a γ- secretase complex to induce NICD nuclear translocation. NCID is the active intracellular region of Notch, which modulates biological effects. Extracellular, cytoplasmic and nuclear proteins, such as Fringe, Numb, Deltex, Sel-10, can

81 additionally modulate Notch signaling positively or negatively. During development, Notch positively regulates SC development from NCCs to immature SCs, by upregulating ErbB2 receptor expression to maximize sensitivity to NRG-1, and increase proliferation 757. However, in myelinating SCs, Notch expression is downregulated by Krox-20. Moreover, expression of Notch is believed to signal the initiation of myelination. NCID enforced expression in SCs inhibited myelination. Infection of myelinated SC-DRGN co-cultures and intact healthy myelinated nerves in vivo induced demyelination.

Hence, this idea of negative regulation of myelin differentiation is emerging as a major aspect of SC biology, with particular relevance to injury and regeneration, demyelinating pathologies, and SC development.

1.6 BASAL LAMINA: COLLAGEN AND LAMININ

1.6.1 Collagen Collagen is the major structural protein of both the basal lamina and the endoneurium, the connective tissue surrounding the nerve fibers within a bundle 360. These extracellular proteins are characterized by their triple helical structure. There are 29 different collagen types (I-XXIX), each formed by three α chains (homo- or heterotrimers of two or three different α chains). Each defined collagen type can have various isoforms with distinct attributes and functions. Collagen trimers can assemble to form suprastructures to be further classified into: fibril-, basement membrane-, network-, beaded filament-, and anchoring fibril-forming as well as transmembrane proteins. Moreover, specific proteins possess collagen domains, including gliomedin 309. In addition to its architectural role during development, where collagens primarily direct the formation of fibrillar and microfibrillar networks of ECM and basal membranes, they are also determinants of cellular phenotype during development, including SC terminal differentiation. Accordingly, inhibiting collagen synthesis prevents ECM assembly and myelination 183. Two families of collagens are expressed by SCs: fibril-forming (types-I, III and V) and basement membrane (type-IV).

When SCs make contact with and ensheath axons 88,132, they begin synthesis of and secrete pro-collagen Type IV 102. Six genes encode Type IV collagen, exhibiting tissue specific expression and patterns of heterotrimer 456 assembly. In the periphery, the main subunits are α1(IV)2, α2(IV)1 . As described earlier, in vitro, VC is required by collagen hydroxylase to post- translationally hydroxylate pro-collagen fibrils at proline residues, cross-linking the subunits into trimers which then self-assemble to form a matrix 184. Other molecules of the ECM are then deposited onto this matrix or basement membrane, ultimately forming the basal lamina that surrounds SC-axon units. Inhibition of collagen synthesis, prevents the assembly of the ECM and hence, myelination. In the in vitro system of myelination, collagen IV synthesis and assembly by SCs requires vitamin C (VC) 184. Collagen type IV trimer formation is indispensible to basal lamina formation despite the secretion of other ECM proteins by SCs 129,468. SCs synthesize the fibril-forming Type I/III collagens. These are localized to the outer aspect of SC basal lamina 507 and the endoneurial space, possibly providing strength and flexibility to the matrix. Additionally, SCs constitutively secrete collagen type V 127,588. In culture, collagen type V is secreted as a stable trimer in the presence of VC 128. During development, it first appears between embryonic day 15 and 18 and then in adult nerve following nerve injury 128. Unlike collagen types I and III, collagen type V retains its noncollagenous N-terminal domain (NTD), which is normally cleaved. It is the NTD of the α3(V) chain which determines collagen V adhesion or receptor binding 186. Interestingly, both the rat gene product, called α4(V), and a 200 kDa protein, called p200, secreted by SCs 128, were discovered to be the α3(V) chain following further studies by Imamura et al. 317. α3(V) was found on the basal lamina of SCs, co-localizing with collagen types I and III 126, and in the nodes of Ranvier 443. Thus, collagen type V is found in the basal lamina of SC-myelinating units and in the ECM 126. Binding to α3(V) collagen, modulates SC spreading, actin cytoskeleton assembly, tyrosine phosphorylation, ERK protein kinases activation, and adhesion 128,186. Assembly of collagen V is important for SC myelination as knockdown of expression or abrogation of assembly, greatly perturbs SC myelination 126,588.

1.6.1.2 Receptors: SCs express two main heparin sulfate-proteoglycans that act as receptors for collagen type-V (NTD-α3 (V) collagen chain), syndecan-3 and glypican-1, these are described below.

1.6.1.2.1 Syndecans Syndecan-3 (also known as N-syndecan) belongs to a family of transmembrane proteoglycan receptors, which consists of a protein core, covalently linked to heparan sulfate chains. Typically, the heparan sulfate chains bind to growth factors and molecules of the ECM, while the protein core and cytoplasmic C-terminal tail can initiate signaling by interacting with integrins or tyrosine kinase receptors. Originally cloned from neonatal rat SCs 103, syndecan- 3 is the principal member of the syndecan family expressed post-natally in the PNS 101,103. SCs also express syndecans-1 and -4 106,256. However, thus far, collagen type V has been shown to bind to the heparan sulfate proteoglycan syndecan-3 with high affinity 186. Syndecan-3, along with syndecan-4, is localized to the SC microvilli or perinodal processes at the node of Ranvier 256. Syndecan- 3 is detected with sodium channel aggregates very early on in development, and subsequently becomes heavily concentrated at nodes. Similarly, collagen V is concentrated at both the microvilli and the nodes. Moreover, nodes form normally in syndecan-3 knockout mice 443. Thus, although syndecan-3 and collagen V may be involved in the early axo-glial interactions during nodal development, syndecan-3 is not essential for myelination.

1.6.2.1.2 Glypican1 Collagen V (α3(V)) also binds to the GPI-anchored cell surface heparan proteoglycan sulfate glypican-1 588 through the α3(V)-NTD high affinity heparan- binding site 186. It is anchored to the cell surface through GPI 104,105, and lacks a cytoplasmic domain, allowing it to directly participate in intracellular signaling by binding to structural or signaling proteins. It is found on the surface of SCs on small cylindrical membrane extensions resembling filopodia and regions contacting basement membranes 105. The significance of glypican-1 to peripheral myelination is underscored by the reduction of myelination (number of fibers and steady state MBP levels) in myelinating SC-DRGN co-cultures following siRNA- mediated knockdown of either glypican-1 or collagen V 126. SC binding to,

84 extension along or radial sorting of axons are not affected. Moreover, suppressing glypican-1 expression inhibits collagen type V binding to SC surface and incorporation into the SC basal lamina. This provides yet another signal transduction pathway for ECM-dependent signaling. Furthermore, glypican-1 may be the major receptor mediating α3(V) collagen binding in SCs as inhibiting syndecan-3 expression neither alters myelination nor SC adhesion to α3(V) 126. Glypican-1 also regulates actin cytoskeleton assembly and SC spreading 186. This may be carried out through its association with lipid rafts 130. Finally, glypican-1 also binds laminin in vitro 104, leading to the postulate that laminin binding may be modulated by interacting with integrins. However, glypican-1 knockdown in SCs does not alter cell surface laminin binding or clustering, and assembly of basal lamina containing laminin 126. Thus far, a significant role for glypican-1 in peripheral myelination has been demonstrated, however the precise role in myelin assembly remains to be elucidated. Future studies aimed at elucidating and discerning the signaling pathways initiated through binding of collagen to these receptors are required.

1.6.2 Laminins Laminin is a second important constituent of the basal lamina. It is a family of large (400-600 kDa) heterotrimeric glycoproteins composed of an α, β and γ chain which polymerize to form a cross-like structure. Five α, three β and three γ isoforms exist in mammals and are distributed in a tissue-specific manner. Each chain has a large globular G domain at one end and three short arm-like domains at the other. Moreover, each isoform also exists in spliced- forms; the arm-like domains can undergo differential proteolytic cleavage as part of post-translational post-secretion modifications that can be species-dependent. Laminins trimers can either self-assemble or combine with other laminins to form a large polymeric structure. Moreover, laminins can selectively interact with non- laminin ECM molecules including perlecan, collagens, nidogen, to ultimately form a polymeric matrix essential for basement membrane assembly. In the periphery, laminins form an essential constituent of the basal lamina surrounding SC-axon units, operating not only as structural scaffolding proteins but interchangeably as a modulator of cellular properties. For instance, upon binding to receptors, laminin can redistribute within the plasma membrane,

85 participate in redistribution of intracellular signaling proteins, cytoskeletal rearrangement, receptor and matrix rearrangement or distribution as well. Laminins directly affect SC function, development and myelination. Addition of exogenous laminin to culture enhances SC attachment, cell growth and induces SC elongation 440. Moreover, adding exogenous laminin alone to SC-DRGN co- cultures in the absence of VC, induces normal basal lamina assembly 183 and stimulates myelination 183,213. SCs express laminins-2 (α2β1γ1), -8 (α4β1γ1), -10 (α5β1γ1) as part of the basal lamina, and are secreted by SCs upon ensheathment of axons in vivo 88,92,141, and in vitro following VC addition to SC-DRGN co-cultures 440. SCs bind exogenous laminin on their cell surface and form laminin clusters 705. In fact, the membrane-bound glycolipid sulfatide binds the LG domain of laminins-1 and 2 and anchors them to the SC surface, helping in the assembly of the basal lamina 401. Moreover, the basal lamina is polarized; laminin-10 (α5β1γ1) is exclusively in the matrix surrounding the node of Ranvier (Figure 1.5), and together with laminin-2, constitute the ‘nodal gap substance’. In contrast, laminins-9 (α4β2γ1) and -11 (α5β2γ2) are in the peripheral nerve endoneurium. Spontaneous mutations and targeted genetic manipulations (disruption or overexpression) have provided insight into the specific roles of these laminins in SC development and myelination. The importance of laminin to myelin formation was first underscored by myelin aberrations detected in dystrophic mice (dy/dy) and humans with laminin α 2 (LAMA-2)-associated congenital muscular dystrophy. Laminin-2, formerly known as merosin, is required throughout development, obviated by the phenotypes of dystrophic mice (merosin-deficient congenital muscular dystrophy (MDC1A) 550,771. Studies using SC-DRGN cultures from dy/dy mice, demonstrated that laminin-2 is essential to establish and maintain SC-axon interactions in normal and regenerating nerves 713. Mice devoid of laminin-2 (α2-null or dyJ mice) are hypomyelinated 771. Moreover, Yang et al used mice devoid of laminin-2 or -8 alone, or a double laminin-2 and - 8 null, to demonstrate unique roles for each laminin during peripheral myelination. While both are involved in radial sorting of axons, laminin-2 appears to be more important in sorting in the spinal roots than laminin-8, while sorting in distal nerve is dependent on both. Thus, laminins-2 and -8 act in concert to complete radial sorting in distal nerves. Moreover, laminins-2 and -8 appear to be

86 essential for SC proliferation associated with radial sorting perinatally. The subunit γ1 is part of all laminins expressed by SCs; accordingly, γ1-specific ablation in SCs using conditional knockout mice depletes all expressed laminins. The hypomyelinated peripheral nerves of these conditional null mice further underscore the importance of laminins in radial sorting, myelination, survival and proliferation. SCs in these mice do not extend processes to initiate sorting 122 and there is increased apoptosis coupled with decreased proliferation 786. In addition to its role in the development of myelinating SCs, laminins are critical for the development and the differentiation of ensheathing SCs and to maintain normal nociceptive function. γ1-null SCs in conditional knockout mice are arrested at the immature stage of development 787. No Remak bundles are formed, axons are not sorted by SCs, and fewer C-fibers are present in sensory neurons. Thus, laminins are critical to the development and maturation of myelinating and non-myelinating SCs alike.

1.6.2 Receptors The multifaceted roles of laminins are attained through the activation of specific receptors. Dystroglycans and Integrins are the primary families of laminin receptors expressed by SCs.

1.6.2.1 Dystroglycans The receptor Dystroglycan (DG), originally isolated from skeletal muscle as an integral membrane component of the dystrophin-glycoprotein complex (DGC), is highly expressed in peripheral nerve and at low levels in several tissues such as kidney and brain 135. DG-knockout is embryonic lethal 750. DG is composed of an α and a β subunit 187, which are formed from post-translational cleavage of the DAG1 gene product 315. α-DG is a highly glycosylated extracellular peripheral membrane protein that binds molecules of the ECM. It is anchored to the plasma membrane by the transmembrane subunit β-DG. DG binds laminin-2 and agrin in SCs 764, and perlecan, biglycan and neurexin in other cell types 76,658,671. Immature SCs express DG perinatally, just prior to onset of myelination. It localizes to the abaxonal membrane of the SC 549 and the nodal SC microvilli 433,597,765. It plays important roles in node formation, organization of myelin and at later stages in myelin maintenance.

DG is essential for myelin maintenance. The cytoplasmic domain of β-DG interacts with dystrophin, which directly binds the actin cytoskeleton 598. L- periaxin, found on the SC surface, homodimerizes and forms a transmembrane dystrophin-glycoprotein complex (DGC) in the plasma membrane of internodal region with dystrophin-related protein-2 (DRP2), another member of the dystrophin family, and DG 338. Two other members of the dystrophin family, utrophin (DRP-1) and DP116, are expressed by SCs, and form part of this L- periaxin-DG-DRP2 DGC complex. Utrophin binds actin through its actin-binding site. As such, this L-periaxin-DG-DRP2 complex is thought to participate in myelin maintenance. Periaxin-deficient mice display late onset demyelinating neuropathy and loss of DRP2 clustering 633. Accordingly, DG or DRP2-SC- specific ablation also exhibit delayed hypomyelination 248,633. Moreover, they lack compartments, smaller internodal lengths and hence, reduced conduction velocity 598. The compartments of the abaxonal SC - patches and Cajal bands differ in DGC complex constituents 146. The actin cytoskeleton of the cytoplasm is first segregated into distinct domains, creating actin-rich and actin-free regions (pre-patterning), and then later during development the actin-free regions become enriched in DRP2 (patches). These regions are subsequently maintained by actin and tubulin associates through linkage with the DGC. Patches, containing little cytoplasm, are highly enriched in DRP2 and periaxin but are devoid of microtubules, while Cajal bands are rich in actin and microtubules (Figure 1.5). Thus, integrity of Cajal bands and trabeculae requires actin and microtubule-networks, respectively, which maintain DRP2-localization to patches in check. Disrupting either cytoskeletal network leads to DRP2 diffusion into the surrounding cytoplasm, with consequent shrinkage and disappearance of either the Cajal bands or the trabeculate. Furthermore, deleting utrophin, an actin- binding dystrophin localized to Cajal bands, reduces DRP2 patches, abrogates myelin compartmentalization and reduces internode length. Both presence of laminin-2 and DG, and their interaction are critical for normal DRP2 patch formation; smaller patches form otherwise. Therefore, the laminin-2-DG-utrophin- actin axis is essential for polarization of the actin cytoskeleton into cytoplasmic domains, proper compartmentalization and elongation of myelin sheaths 145. As described earlier (section 1.3.4.1), direct physical interaction of SC microvilli with nodal axolemma coordinates sodium channel clustering to nodes

88 of Ranvier. Laminin and DG participate in sodium channel localization 145. Also, as mentioned, laminin-10 is uniquely localized to the nodal area with laminin-2. This may specifically interact with DG found on the SC microvilli to direct clustering of channels on the nodal axolemma 499. Accordingly, ablating DG in SCs alone impairs sodium channel clustering and disorganizes microvilli (disrupted interaction between adjacent microvilli and with nodal axolemma), significantly reducing nerve conduction velocity, which is similarly observed in laminin-2-deficient SCs 499. Moreover, in SCs, DG complexes with DP116, and utrophin in the nodal region, in contrast to the internodal region, where L-periaxin and DRP2 are additionally present in the complex 598. Also, DP116 is exclusively localized to the microvilli. Collectively, this is suggestive of a specialized DG- dystrophin association exclusive to this region. Thus, laminin-DG- DRP2/DP116/utrophin axis likely modulates sodium channel clustering and nodal structure formation. Moreover, DG is co-localized with moesin, one of the ERM proteins found in SC microvilli 499. Since ERM proteins interact with the actin cytoskeleton, this may be another means by which SCs use laminin-DG to coordinate sodium channel clustering. Also, the marked morphological abnormalities in myelin sheath are potentially due to the disrupted linkage between the ECM and SC cytoskeleton. DG-null SCs do not display plaque-like laminin clusters at the cell surface 445,611. DG also forms a macromolecular DGC complex with sarcoglycan (SG), involved in stabilizing the myelin sheath 598. SGs are a family of transmembrane proteins consisting of α, β, δ, γ, ε and ζ. In sciatic nerve homogenates, β-, δ-, ζ- and ε-SG are detected, along with DG, L-periaxin, DRP2, DP116 and utrophin 94,316. DG forms a DGC with β-, δ-, ε- and ζ-SGs and DP116 at the abaxonal SC membrane 94,598. Although SGs are expressed prior to myelination, induced by neurons, they continue to be expressed thereafter 94,316. They help stabilize DP116 and DG localization to the SC abaxonal membrane. Knocking out SGs destabilizes DP116 and αDG; mice display altered myelin sheaths and disorganized SLIs. Thus, interaction of DG with SG plays an important unforeseen role in maintenance of peripheral myelin integrity. In summary, laminin-induced DG activation is critical for initial sodium channel clustering and for myelin maintenance.

1.6.2.2 Integrins The family of cell surface heterodimeric (αβ) integrins is the second class of laminin receptors expressed by SCs. These are transmembrane glycoproteins that are involved in cell-cell and cell-ECM interactions, modulating outside-in and inside-out signaling. They have been shown to mediate cell migration, proliferation and differentiation in many cell types. Each subunit is a transmembrane glycoprotein with an extracellular ligand-binding domain that interacts either with molecules of the extracellular matrix or counter receptors on adjacent cells, and an intracellular domain which is the interface between the external and internal cellular environments. Integrin activation and affinity are modulated both by ligand binding and receptor aggregation 94 as well as lipid rafts, where integrins can be found 241,242,624. Six α and eight βintegrin transmembrane subunit isoforms exist; the subunits associate through non- covalent bonds to form at least 22 heterodimers, described thus far. Moreover, receptor composition confers specificity of ligand interaction. α3/6/7 integrins pair with β1 subunit, or α6 pairs with β1 or β4 to generate laminin-specific integrins. SCs express 11, 61, 64 and 71 in a developmentally regulated manner 549,550. SCs express 61 before birth, 64 perinatally just prior to onset of myelination, and α7β1 postnatally 548. Specific roles for each integrin subunit in SC development and myelination were studied primarily using transgenic mice and in vitro SC cultures. β1 integrins are randomly distributed on undifferentiated SCs, as shown using immunogold labeling of SC-DRGN co-cultures. At the onset of myelinating, these integrins become localized to the SC surface contacting the basal lamina 548. Furthermore, several lines of evidence illustrate that β1 integrin subunit is the most critical integrin in myelination. Dominant-negative β1 integrin expressing mice have region-specific myelination defects in the CNS 206, while SCs deficient in 1 integrin fail to myelinate, resulting in severe neuropathy with impaired radial sorting of axons 383. Moreover, application of a function-blocking β1 integrin antibody to SC-DRGN co-cultures prevents basal lamina adhesion to the SC surface and subsequent myelination 199. Recently, β1 integrin has been shown to modulate SC migration; astrocyte-produced aggrecan disrupts β1 integrin-laminin interaction to abrogate the migration of transplanted SCs in spinal cords 206,542.

In contrast, ablating β4 integrin in null 10 and conditional-null 215 mice in SCs does not hinder myelination, thereby reinforcing a central role for 1 integrin in the development of peripheral myelin. However, α6β4 integrin in conjunction with DG, cooperatively stabilizes myelin sheaths in the periphery 200. This is in keeping with the upregulation of β4 integrin postnatally. Integrin β4 appears perinatally and is polarized only subsequent to myelination. Thus, over the course of myelination,1 is downregulated while 4 is upregulated. Integrin β4, PMP22, and laminin form a complex involved in mediating the interaction of SCs with the extracellular environment during myelination 492. Moreover, β4 integrin levels are decreased in PMP22 knockout mice. α7 subunit appears postnatally and α7β1 forms the last laminin receptor expressed by differentiating SCs 18. This receptor was found to be dispensable for peripheral nerve development and myelination, however, it may be involved in regeneration.

1.7 INTEGRIN SIGNALING: Focal adhesion complex formation Integrins, unique in their lack of intrinsic protein kinase activity, participate in inside-out and outside-in signaling through the formation of a focal adhesion complex (FAC) (schematically represented in Figure 1.6) at its intracellular C- terminal domains or by regulating lipid-rafts microdomains 548. They serve as molecular linkages between the extracellular milieu and the cell, transducing signals modulating cell attachment, migration, proliferation, differentiation, and gene expression. Over 50 focal adhesion proteins have been identified, including structural, enzymatic and adaptor proteins that interact with the actin cytoskeleton. This complex contains protein kinases, phosphatases and their recruited substrates 167. The family of src-like kinases (SLKs) and focal adhesion kinase (FAK) are two of the many families of tyrosine protein kinases recruited to these sites of adhesion. Phosphorylation of these proteins creates docking sites for SH2-containing adapter and signaling molecules. Moreover, these kinases can phosphorylate other proteins to recruit and to activate the family of Rho GTPases to FAC to modulate cytoskeletal changes and produce the desired biological effect. Several of these components are described below.

Figure 1.5 Integrin activation and FAC formation.

Activation of integrins (αβ), a, leads to receptor heterodimerization, b, in conjunction with recruitment of kinases (FAK, SLKs) and other substrates to form a focal adhesion complex at the cytosolic tail of the β integrin subunit. Collectively these factors modulate actin polymerization.

1.7.1 Protein Kinases

1.7.1.1 Focal adhesion kinase (FAK) Focal adhesion kinase (FAK), a 125 kDa cytoplasmic non-receptor tyrosine kinase, with PYK2, comprises the FAK family of kinases. FAK is expressed by most tissue and cell types and is rapidly autophosphorylated in response to integrin signaling. Moreover, FAK plays important roles during development in many cell systems and in the formation and turnover of FACs, especially during migration 758. Also, it can regulate Rho GTPases activation 739. FAK is composed of a centrally-located catalytic kinase domain, an N- terminal erythrocyte band four, one ezrin-radixin-moesin (FERM) homology domain and a C-terminal region rich in protein-protein interaction sites that includes a focal adhesion targeting (FAT) sequence. FAT allows its recruitment to newly or existing FACs, and acts as a binding site for paxillin, an actin adapter protein. FAK is regulated by tyrosine phosphorylation. In fact, phosphorylation on Tyr397 is a binding site for the SH2 binding site of SLKs 693, PI3-K, PLC, and Grb7. Two other FAK phosphorylation sites following integrin clustering and activation yields phospho-tyrosine docking sites for several classes of signaling

92 molecules. Phosphorylation of this tyrosine residue has been shown to take place following integrin activation FAK binds numerous signaling molecules including Shc, Grb2 and SLKs 606,761. Four Serine phosphorylation sites in the C- terminus (Ser722, Ser843, Ser846 and Ser910) may represent another mechanism of FAK regulation 520,521. Furthermore, FAK also contains a FAK-related-non-kinase domain (FRNK), which may serve as a site for negative regulation in certain cells. In myelinating SCs, FAK localizes to the abaxonal SC membrane, upon ECM deposition, and is part of a FAC containing β1 integrin, Fyn and paxillin 411. Since FAK knockout is embryonic lethal, a conditional mutant was used to study the role of FAK in SC-mediated myelination 117,207. FAK deficient SCs display an inability to sort axons and hence, FAK conditional mice have many areas of amyelination 268, similar to the phenotype of conditional β1 integrin-deficient mice 268. Recently, a role for FAK was also elucidated in OLG-mediated myelination; it is postulated to be involved in the timing of myelination and efficiency as fewer fibers are myelinated in transgenic mice 199.

1.7.1.2 SrcLike Kinases (SLK) A second family of kinases recruited to FAC is the Src-like kinases (SLK). This is the largest family of non-receptor tyrosine kinases, composed of nine family members including Fyn, Lyn, Src, Blk, Lck, Yes, Hck, and Fgr that share similar homology and structure. They range between 52-62 kDa in size and have four conserved domains: 1) Src homology (SH)-1 domain, 2) SH2- binding site, 3) SH3-binding site, and 4) SH4 domain. SLKs bind to other proteins through their SH2 or SH3 domain. SH1 domain, located towards the carboxy (C)- terminal tail, contains both the catalytic domain (ATP binding site and substrate- binding site) and a negative regulatory tail. The Tyr416 site of the catalytic domain must be auto-phosphorylated for maximal kinase activity. The negative regulatory tail is about 20 amino acids long and contains a conserved tyrosine residue, Tyr508, that when phosphorylated is folded back onto the SH2 domain to produce an inactive conformation. Conversely, dephosphorylation of this residue promotes enzyme autophosphorylation and therefore, increased kinase activity. Negative regulation is carried out by the Csk-related enzymes. SLKs also contain

93 a ‘unique’ region, located immediately after the N-terminal. It contains a Ser/Thr site that is specific to each member 211. Binding through SH2 domain allows SLK localization to FACs by binding proteins containing phospho-Tyrosine motifs. On the other hand, SH3 domain is critical for inter- and intramolecular interactions modulating kinase catalytic activity, and binds substrates with proline-rich motifs. The SH4 domain at the N-terminus, contains consensus sequences for lipid modifications (myristoylation and palmitoylation), enabling membrane translocation of the SLKs. Thus, SLKs can localize to FACs following either SH4 domain myristoylation or interaction of its SH2 domain with tyrosine phosphorylated proteins, which localize to FAC, such as FAK. As mentioned earlier, following integrin signaling, SLKs can positively regulate FAK activity by phosphorylating and potentiating FAK Tyr397 autophosphorylation 687. While some family members are ubiquitously expressed, such as Fyn, Src and Yes, others exhibit tissue-specific distribution such as Lck that is found in lymphocytes. Moreover, unique-combinations of SLK-integrin association by cells may be used to modulate specific cell functions 108. SCs express Fyn, Lyn, Src and Yes 26,138. This family of tyrosine kinases has been implicated in many cell processes including cell proliferation, survival, migration, and myelination of the CNS. Fyn is critical for OLGs development and central myelination. Kinase- deficient mice are hypomyelinated 651. In OLGs, Fyn is involved in the transcription 651,711 and post-transcriptional regulation 710,711 of MBP, as well as in maturation 407, differentiation 407,651,652, process extension 508 and compaction of myelin sheaths 355. More recently, integrins were shown to direct SLKs (Fyn and Lyn) in regulating CNS myelination 627. The brains and spinal cords of Fyn-null mice are deficient in myelin by ~40-50% 138. Also, Fyn is reported to phosphorylate the C-terminal tail of MAG in OLGs 407,711. Peripheral myelin formation, however, is neither delayed nor aberrant in Fyn-null mice, highlighting a dichotomy in the relevant signaling molecules required for peripheral and central myelinogenesis. β1 integrin, FAK, paxillin, and Fyn kinase form an actin- associated complex in SCs adhering to basal lamina in the presence of axons, which is postulated to be critical in initiating SC differentiation to a myelinating cell 55,223,326. Fyn also modulates phosphorylation of potassium Kv1.5 and 2.1 channels, whereby the channel α subunits are constitutively phosphorylated and are associated with this SLK in cultured SCs and in sciatic nerve 117. Moreover,

94 this link between Fyn and the delayed rectifier channels may be involved in SC proliferation and the onset of myelination. Lyn, which is typically involved in the immune system 649, was recently discovered to play a role in CNS myelination during integrin-directed OLG development 138. Fyn associated with α6β1 integrin enhances growth factor survival signaling, triggers the differentiation program and myelin membrane formation; it is activated through lowered Csk phosphorylation of its negative Tyr. On the other hand, Lyn associates with αVβ3 integrin for progenitor proliferation. It is activated through increased autophosphorylation of its tyrosine residue. Knockouts of Lyn exhibit immune system or hematopoietic cells deficiencies but no peripheral myelin defects 138. Several lines of evidence support a role for Src in modulating myelination by SCs, although kinase-null mice display no peripheral myelin aberrations 651. For instance, Src activity is increased in SCs following peripheral nerve injury 651. Finally, very little research has been conducted in elucidating a role for Yes in peripheral myelination, especially the kinase-null mice had no peripheral defects, similar to the results reported for Fyn-, Lyn- and Src-null mice by Sperber et al 794 .

1.7.2 Phosphatases Equally important as tyrosine kinases to FAC signaling and in maintaining steady-state levels of tyrosine phosphorylation, are phosphatases. This superfamily of protein tyrosine phosphatases (PTP), both receptor-like and cytoplasmic forms, regulates the activity of many kinases by reversing the phosphorylation of tyrosine residues. Since tyrosine phosphorylation is involved in signaling pathways linked to many processes including cell growth and differentiation, PTPs are essential modulators of their kinase counterparts, and represent potential therapeutic drug targets. They are positive or negative regulators of numerous cellular processes including cell cycle control, signal transduction and development. Focal adhesion disassembly has been linked to increased PTPase activity 651. In peripheral myelination, several PTPs have been implicated: receptor-like PTPs (-α and -ε) and the non-receptor cytoplasmic PTP Shp2.

PTP-α activates SLKs by dephosphorylating the C-terminal regulatory Tyr527 residue 573. PTP-α is involved downstream of integrins activation, leading to both FAK and SLK activation 168,796. In the CNS, PTP-α modulates Fyn activity in OLGs, promoting differentiation and myelination 118,280,591,790. Moreover, it is involved in regulating Kv channel activity together with PTP-ε 730. PTP-ε knockout mice exhibit severe hypomyelination 691. PTP-ε is similar to PTP–α, where it regulates Kv channel activity and its subunit phosphorylation in SCs, as studied in vivo using knockout mice 531. In contrast, PTP-β-null mice display no obvious aberrations 691. Another tyrosine phosphatase implicated in peripheral myelination, is Shp2. It is critical for force-dependent strengthening of initial integrin- cytoskeleton linkages (FAC) where dynamics of α-actinin-assembly are downregulated 282. It is also involved in NRG-1 signaling mediating SC differentiation 723. It can regulate the phosphorylation of regulators of Csk, to modulate Csk activity, and ultimately, SLK activity 267. Thus, PTPs, another constituent of the FAC, participate in signaling downstream of integrins.

1.7.3 Rho GTPases As described, integrins are a critical link between the ECM and the actin cytoskeleton. Coordinated changes in the actin cytoskeleton are essential for the numerous biological processes in which integrins are implicated, including myelination. This is often accomplished through the family of Rho GTPases. Rho GTPases belong to the Ras superfamily of small GTPases. They are comprised of 22 members in mammals and range in size between 20-30 kDa. These ubiquitously-expressed proteins have been shown to participate in transcriptional activation, membrane trafficking, signaling pathways (c-jun N- teminal kinases and p38 mitogen activated protein kinases), G1 cell-cycle progression and assembly of cadherin cell-cell contacts 793. However, their highly acclaimed role is directing actin and microtubule cytoskeletal dynamics in response to a wide range of extracellular signals. Rho GTPases have been shown to be coupled to integrins and tyrosine kinase receptors activation 324,351. Thus, numerous actin-mediated morphological cell processes in eukaryotic cells

96 include cell migration, cell-cell and cell-extracellular matrix adhesion, polarity, neuronal development, and myelination are indeed modulated through Rho GTPases 616,693. As molecular switches, Rho GTPases cycle between an active/‘on’ and inactive/’off’ states when GTP is hydrolyzed to GDP. Binding of a Rho GTPase to a GTP, alters the conformation, turning it ‘on’, such that the active GTP-bound GTPase can interact with downstream effector proteins to initiate signaling and modulate cellular processes. Each Rho GTPase possesses a slow intrinsic GTP hydrolytic activity, where GTP hydrolysis to GDP turns ‘off’ the GTPase, restoring it to an inactive conformation and consequently terminating signal transduction. Additionally, Rho GTPase activity is influenced by three types of regulatory proteins: 1) GTP exchange factors (GEFs) promote the exchange of bound GDP with GTP to activate the GTPase; 2) GTPase activating proteins (GAP) stimulate the intrinsic kinase activity of the GTPase, promoting GTP hydrolysis and hence, turning ‘off’ the GTPase downstream signaling cascade; and 3) Guanine dissociation inhibitors (GDIs) inhibit binding of Rho GTPases to the plasma membrane, thus, preventing their activation. C-terminal post-translational modifications (prenylation or palmitoylation) can further modulate Rho GTPase activity by enabling membrane localization to alter its subcellular distribution, phosphorylation, and ubiquitination. Strict regulation of Rho GTPase activity is essential. Mutations in GEFs, GDIs or GAPs may lead to Rho GTPase disregulation and even disease. For instance, patients with a mutant Rho GEF10 exhibits slowed conduction and thin myelination of peripheral nerves 324,363,409.

Figure 1.6 Model depicting RhoGTPases cycling between active “on” and inactive “off” states

Guanine dissociation inhibitors (GDIs) sequester inactive GDP-bound Rho-GTPases (Rho) in the cytoplasm. Upon release from Rho-GDIs, Rho-GTPases are targeted to the plasma membrane, where guanine exchange factors (GEFs) modulate their activation, promoting GDP-GTP exchange, and GTPase activation protein (GAPs) promote GTP hydrolysis to GDP and consequent inactivation.

Of the 22 mammalian Rho GTPases, three key family members have been extensively studied: Ras homologous member A (Rho A), Ras-related C3 botulinum toxin substrate-1 (Rac1) and cell division cycle 42 (Cdc42). Activation of each yields distinct cytoskeletal structures: stress fibers and focal adhesions, lamellipodia and filopodial microspikes, respectively, as initially elucidated by overexpression studies carried out in fibroblasts 717. Although this family of Rho GTPases is activated by numerous receptors including integrins and receptor tyrosine kinases such as the NRG receptor ErbB2 274,489,490 to carry out specific but varied biological functions. Specificity of how these interactions take place remain elusive, although it is partially dictated through targeting of specific GEFs following receptor activation, and effector molecules by each active GTPase and cell type. In general, an individual Rho GTPase can activate multiple effectors, but different GTPases may activate the same effectors, while some effectors are activated exclusively by specific GTPases. For instance, Rho kinase (ROCK) and Dia are effectors exclusively activated by RhoA, while the p21-activated kinase (PAK) family of Ser/Thr kinases can be activated by both Rac1 and Cdc42. This

98 divergence in effectors activated by the three Rho GTPases is reflected in the pathways activated and functions. Rac1 and Cdc42 often have overlapping functions, distinct from Rho. Cdc42 is often associated with cell proliferation and directionality, while Rac1 is involved in migration 616. In peripheral myelination, Rac1 and Cdc42 are both critical for radial sorting as displayed by conditional knockouts 324. As described earlier, interaction of laminin and β1 integrins are required by SCs to radially sort axon bundles, as shown to be mediated through SC-specific conditional ablation of β1- integrin and Rac1 45,493. Thus, Rac1 is activated downstream of 1 integrin, and is essential for SCs that have migrated to the axons and that have elongated, to extend and to generate radial lamellae during axonal sorting 45,199. With time, however, nerves of Rac1-deficient mice undergo delayed myelination, as reflected in the levels of delayed induction of SCIP and Krox-20 expression. In contrast, Cdc42-deficient sciatic nerves are and remain severely hypomyelinated, with no recovery of SCIP or Krox-20 expression with time 493. Cdc42 is activated by NRG-1, and is required for normal SC proliferation during radial axon sorting as fewer SCs were present. These findings using tissue-specific conditional gene ablation are drastically different from those in OLGs 45, where proliferation, migration and differentiation are unaffected. Rac1 and Cdc42 synergistically regulate central myelination, as gene-specific ablation results in aberrations of the inner tongue. Additionally, Rac1 is implicated in regulating SC migration and motility in response to neurotrophin-3 688. SCs express Rho A, B 766-769, however roles for each isoform have not been extensively studied. RhoA is likely the most abundant isoform diffusely distributed throughout the cytoplasm, while B appears to be primarily localized to the perinuclear region, with a potential role in modulating gene transcription. It is postulated that Rho acts downstream of β1 integrin, however a β1 integrin complex isolated from myelinating SCs did not contain Rho 682. Rho coordinates SC myelination through its effector, ROCK 682. Prior to onset of myelination, Rho is diffusely distributed throughout the cytoplasm, and subsequently becomes localized to sites of non-compacted myelin - the nodal microvilli and SLIs in adult sciatic nerve 444. Using pharmacological inhibitors it has been shown that ROCK regulates the initial SC membrane wrapping around the axon, axo-domain formation (nodes, paranodes), internode length, but not SC proliferation or

99 differentiation. Early events of wrapping involve ROCK activation and phosphorylation of myosin light chain. In ROCK-inhibited SC-DRGN co-cultures, SCs produce multiple short myelin segments with small filopodial-like protrusions emanating from the outer surface, some segments have multiple nodes and paranodes. Recently, using SC-specific ablation, integrin-linked kinase (ILK), was shown to negatively regulate Rho/ROCK to promote SC process extension during radial sorting 444. ILK is a 52 kDa Ser/Thr kinase found in FACs at cytoplasmic tails of activated β1 or β3 integrins, linking the receptor to the actin cytoskeleton 530. In vivo studies of Rho akin to tissue-specific gene ablation of Rac1 and Cdc42 have not been carried out thus far. Thus, we have in some detail, described the main receptors bound by the two key molecules of the SC basal lamina, collagen and laminin, and the signal transduction pathways delineated thus far. Indeed, this is very complex process and only some light has been shed on the molecular mechanisms triggered by molecules of the basal lamina, which direct SC maturation, differentiation and myelination. For instance, in other cell systems, integrins have been shown to couple with tyrosine receptor kinases to carry out cellular processes in response to extracellular signals 390. This remains a distinct and unexplored possibility in SCs.

1.8 SIGNALING PATHWAYS INVOLVED IN PERIPHERAL MYELINATION A major expansion in understanding SC biology has seen a thrust in pinpointing the molecular mechanisms and pathways that control SC development, myelination and myelin maintenance, and in defining the molecules mediating interactions between SCs and their environment (axons and the ECM). Here we review the major signaling pathways involved in the differentiation of immature SC to myelinating SCs and the process of myelination activated by neuregulins (NRGs)/ErbB, PI3-K/Akt, ERK, JNK/cJun and p38 MAPK.

1.8.1 Neuregulins/ErbB NRGs are a large family of diverse but structurally related glycoproteins, encoded by Nrg1, Nrg2, Nrg3 and Nrg4 650 of which NRG-1 is the best

100 characterized. It is a pleiotropic factor, known to be a critical player in neural, mammary and cardiac development 107,196,252,278,294,422,526,744,791. Over 15 secreted and membrane-bound isoforms of NRG-1 are produced from the Neuregulin-1 gene from different promoters and alternative splicing 478,745. They share a common EGF-like domain that binds to its receptors to elicit activation 478. This EGF domain is alternatively spliced to produce two forms: α and β, which differ in potency as mitogenic and survival factors 745. β NRG are primarily responsible for SC ErbB activation 172. The three well-characterized β NRG isoforms are Types I- III. Type I (also known as Neu differentiation factor, heregulin, Acetylcholine receptor inducing activity) and II (also known as glial growth factor2 (GGF2)) are produced as single transmembrane proteins, while Type III (also known as Sensory and motor-neuron derived factor, cysteine-rich domain neuregulin-1) has two transmembrane domains. All three isoforms undergo proteolytic metalloproteinase cleavage, in response to receptor binding, to produce the mature forms. Types I and II are secreted by neurons while Type III is membrane-bound at the neuronal surface 448, such as that of DRGNs. NRG-1 types I and III are the most abundant. These key axon-derived factors are SC mitogens and differentiation factors, directing all stages of SC development including the specification of SCs from NCCs in culture 195, proliferation and terminal differentiation of immature SCs, and peripheral myelinogenesis. Also, Nrg1 conditional gene ablation in sensory neurons (small-diameter DRGN, with small unmyelinated axons, a population of large-diameter DRGNs with thinly myelinated axons), demonstrates NRG-1 is essential for normal sensory function in adults 631. It modulates signaling between axons and myelinating and non- myelinating SCs. Ablation also results in aberrant Remak bundle formation, myelination of large diameter axons, slowed conduction and reduced sensitivity to noxious mechanical stimuli. NRG-1 binds to tyrosine kinase ErbB receptors to elicit its action. There are four ErbB receptors: ErbB2, ErbB3 and ErbB4, members of the EGF receptor superfamily. SCs express ErbB2 and ErbB3 218. ErbB2 does not bind NRG efficiently while ErbB3 lacks kinase activity but strongly binds NRG-1 716. Thus, ErbB2 and ErbB3 form functional heterodimeric receptors, where NRG-1 binding induces ErbB3 and ErbB2 receptor dimerization and initiates signaling by receptor cross-phosphorylation, recruitment of SH3-containing adaptor molecules

101 and downstream signaling to induce myelination 645. Moreover, erbin, a protein that contains leucine rich repeats and a PDZ-domain, modulates ErbB2 stability by binding to ErbB2 through its PDZ domain in SCs and thus, plays a pivotal role in NRG-1 signaling and peripheral myelination 7,232,645. Although ErbB2 signaling is not critical to maintain myelin, it is essential for immature SCs differentiation to myelinating/non-myelinating phenotypes 676. SC-specific ablation of ErbB2 in adult mice does not detectably alter myelinated peripheral nerves.

NRG-1 Type III/ErbB ensheathment NRG-1 Type III directs myelination in the PNS as expression dictates neuronal ensheathment, and level of expression by the axon decides subsequent myelination 33. Moreover, it is a critical dictator of myelin sheath thickness in vivo, as neurons overexpressing NRG-1 Type III are hypermyelinated. This effect is graded according to the amount of NRG-1 expressed 681. Also hypermyelination is more pronounced in smaller axons in mice overexpressing NRG-1 Type III. Furthermore, this effect of NRG-1 may be influenced by integrins 451,681. Additionally, P0 expression 137 and glial cholesterol synthesis appear to be under axonal control as NRG-1/ErbB signaling activates the HMG-CoA reductase gene in SCs 120. Thus, axonal NRG-1 induces cholesterol synthesis in SC, such that when cholesterol levels surpass a specific threshold, myelin gene expression is stimulated, permitting P0 export into the growing myelin sheath and enabling myelin compaction 533. Disrupting NRG-1 Type III-ErbB signaling using CNP-DN- ErbB4 mice does not appear to affect the maturation of non-myelinating SCs or Remak bundle formation 596. These effects on immature SCs are in direct opposition to the effect on SC precursors, where proliferation and survival are promoted 120 and in adult SCs where proliferation is inhibited while promoting survival and stability 172,232,478,667.

NRG-1 Type III/ErbB/cAMP/PKA/NFkB axis Binding of SCs to membrane bound NRG-1 type III through ErbB2-ErbB3 receptors and activation of Protein kinase A (PKA), induces Ser phosphorylation of the p65 subunit of the transcription factor, NFkB at Ser276, enhancing its transcriptional activity 119,478. This phosphorylation by PKA is required for myelin

102 formation. SCs expressing a mutant form of p65 blocking the phosphorylation, reduces the number of myelinated fibers 403,610,779.

NRG-1/ErbB/PKA/PI-3K/Akt axis: NRG-1 (soluble and membrane-bound) stimulates various cascades including PI-3K and Akt that are involved in SC survival and proliferation, downstream of ErbB2/3 receptors 779. ErbB signaling was shown to regulate internode length as well 438. NRG-1 participates in SC proliferation 120 during radial sorting through Cdc42 233. The NRG-1 isoform type II, GGF2, upregulates Cx-32 expression in SCs 45. It also robustly induces expression of Brn5, a transcription factor expressed exclusively by myelinating SCs, in a time and dose-dependent manner 216. Addition of exogenous GGF to SC-DRGN co-cultures inhibits myelination by preventing axonal sorting and ensheathment, without affecting basal lamina formation 760. NRG-1 signaling involves PKA/ cAMP-mediated transduction pathways, among other signaling mechanisms. Also, NRG-1 induces prolonged CREB Ser133 phosphorylation in cultured SCs, likely mediated by MAPKs ERK1/2 and not CaMK 788

1.8.2 Phosphoinositide 3kinase/Akt In addition to NRG-1, several other growth factors, such as IGF-1 and PDGF, have been shown to activate this phosphoinositide 3-kinases (PI3-K)/Akt pathway to promote SC survival and proliferation 668. Additionally, PI3-K/Akt regulates several other fundamental cell functions including growth, transcription, translation, cell cycle and apoptosis 500, as shown in other systems.

Addition of NRG-1 Type II (GGF2) and neurite membranes (likely containing NRG-1 Type III) to SC cultures induces Akt phosphorylation and SC proliferation, which is reversibly blocked by an inhibitor of PI3-K (LY294002) 97,715. Similarly, addition of NRG-1 type II to cultures rescues SCs from serum- deprived cell death, which is reversed by a PI3-K pharmacological inhibitor. Neurites also promotes SC survival in a contact-dependent manner, through PI3- K, where NRG-1 Type III is likely the active constituent 438. Hence, NRG-1 Type II and III mediate SC proliferation and survival through PI3-K/Akt. Inhibiting PI3-K

103 activity at the onset of myelination in SC-DRGN co-cultures grown in serum- containing media, blocked SC elongation and myelination, but not when added at a later time (day 4) 438. Laminin deposition is unaltered following PI3-K inhibition 438. Moreover, selectively activating PI3K in SC-DRGN co-cultures increased myelination 500. Also, inactivating the downstream target of Akt, glycogen synthase kinase-3β (GSK-3β), using lithium chloride enhances SC differentiation and myelination in SC-DRGN co-cultures 500. Therefore, the signal transduction pathway by NRG-1/PI3-K/Akt/GSK-3β is critical for the proliferation, differentiation and onset of myelination by SCs. Moreover, ErbB signaling can activate MEK-dependent MAPK. Axonal NRG-1 type III binding and activation of ErbB2/3 receptors, induces PKA, which enhances levels of cAMP 500. cAMP potentiates the NRG-1 effect on SC proliferation by prolonging the duration of MEK-ERK activation and enhancing activation of Akt signaling pathway, to accelerate G1-S cell cycle progression 460. Thus, cAMP and NRG-1 synergistically regulate SC proliferation. PI3-Ks metabolize inositol lipids by phosphorylating the 3’OH position of the inositol ring of the inositol phospholipids. Three classes of PI3-Ks exist in mammalian cells (Classes I-III), of which Class I PI3-K is typically coupled with receptors regulating signal transduction associated with extracellular stimuli. Class II PI3-K are larger proteins and Class III PI3-Ks phosphorylate PI alone to produce PI(3)P. It likely modulates vesicular transport within cells. Class I alone possesses a regulatory adaptor subunit (p85) in addition to its p110 catalytic subunit.

The Class I PI3-Ks are coupled to tyrosine receptor kinases. There are four isoforms of the p110 catalytic subunit (α, β, δ, γ) and three isoforms of the regulatory adapter p85 subunits (α, β, γ). Upon receptor activation, the PI3-K is recruited to the receptor through the p85 regulatory subunit. p85 regulatory binds tyrosine phosphorylated sites on the receptor through its SH2 domain. Thus, targeting of the p110 catalytic subunit to the plasma membrane, allows

phosphorylation of phosphatidylinositol (PI)-3,4 biphosphate (PI(4,5)P2), and subsequent production of phosphatyidylinositol (3,4,5) tri-phosphate PI(3,4,5)P3 production. PI (3,4,5)P3 acts as a second messenger to modulate downstream signaling by binding to pleckstrin homology (PH) domain-containing proteins,

104 including Akt and 3’-phosphoinositide dependent kinase 1 (PDK1). This PI3-K pathway is negatively regulated by lipid phosphatase tensin homolog (PTEN), which dephosphorylates 3’-phosphorylated phosphoinositides (PI(3,4,5)P3 to PIP2) 461.

Akt (protein kinase B (PKB)), the cellular homologue of the viral oncogene, v-Akt, is a 57 kDa Ser/Thr kinase. The three isoforms of Akt exist - Akt1, Akt2, Akt3 (PKBα, β and γ, respectively) and, display broad tissue distribution. Akt consists of an N-terminal PH domain, a central kinase domain (activation loop) and C-terminal regulatory tail. For full activation, both Thr308 residue in the activation loop and Ser473 residue in the regulatory tail need to be phosphorylated. This can be carried out by PDK1, ILK, and Akt itself 731. Akt 155 preferentially binds PI (3,4)P2 and PI(3,4,5)P3 through the PH domain . It is primarily cytosolic, but upon phospholipid binding, it translocates to the plasma membrane to be activated. It then activates a large number of protein substrates via phosphorylation and effectively modulates their different biological functions. Akt substrates have the consensus sequence RXRXXpS/T, where X is any amino acid 98,155,715. Moreover, Akt maintains cell survival by inhibiting apoptosis. For instance, Akt can phosphorylate the pro-apoptotic factor BAD to inhibit its interaction with the anti-apoptotic factor, Bcl-XL. It thereby prevents mitochondrial cytochrome c release, an event intimately linked with irreversible apoptosis 715. Akt can also regulate cell cycle progression by phosphorylating cyclins, CDK inhibitors such as p27kip1, p21cip1 and regulating cyclin D stability 155. Activated Akt can also directly phosphorylate transcription factors to modulate gene expression 414.

GSK-3β is a well-characterized substrate of Akt. It regulates cell fates in development by transmitting signals from various growth factors such as insulin and IGF-1. Kinase activity is in part, negatively regulated by phosphorylation at its N-terminal Ser9. Thus, when phosphorylated by Akt, it is inactivated while its effector molecules become activated 715. It regulates transcriptional activities of CREB and activator protein-1 or the stability of cyclin D1 and c-myc 134.

In summary, PI3-K/Akt is the main signaling pathway activated by various growth factors to regulate survival and proliferation of SCs.

105

1.8.3 Mitogenactivated protein kinases The family of MAPKs are intracellular Ser/Thr protein kinases that are activated in response to a wide range of stimuli, including growth factors, inflammatory cytokines or physical stress. Activation is achieved through a kinase cascade of three evolutionarily conserved Ser/Thr kinases – MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK). Phosphoprotein phosphatases also modulate the MAPK cascade positively or negatively by dephosphorylating the Tyr or Ser/Thr sites in the activation loops of MAPK, MAPKK, MAPKKKs, or both sites in MAPKs (dual-specificity phosphatase (DSP). Phospatases regulating MAPKs include protein phosphatase 2A and B (PP2A, B), while MAPK phosphatases (MKPs), VH5 are DSPs 134,265. Activation of MAPKs through phosphorylation of their activation loop, typically leads to its translocation within the cell (often from the cytoplasm to the nucleus). Activated MAPK subsequently recognizes and binds to Ser/Thr-Pro phospho-acceptor sites in the downstream targets. MAPKs phosphorylate a number of substrates to elicit its function, including transcription factors, MAPK- activated protein kinases (MKs) 66,504,559. Transcription factors are key targets of MAPK signaling used to effectively control gene expression 66,504,559,591. Transcription factor activity can be directly altered by phosphorylation, impacting its intracellular localization, protein levels (expression or stability), binding to DNA and interactions with regulatory proteins. Moreover, MAPKs can indirectly influence transcription factor activity by targeting coregulatory factors.

Another group of substrates of MAPK signaling protein kinases, which are activated by phosphorylation is MAPK-activated protein kinases (MKs) 772. This family is comprised of p90 ribosomal S6 kinase (RSKs), mitogen and stress activated kinases (MSKs), MAPK interacting kinase (MNKs), MKs (described in section 1.8.3.4). Collectively, MAPKs regulate gene expression, mitosis, metabolism, motility, survival, apoptosis and differentiation. There are five major families of MAPKs: the extracellular signal-regulated kinases (ERKs), c Jun N-terminal- linked kinases (JNKs), ERK5 (or Big MAP kinase-1 (BMK1)) and p38. ERK, JNK

106 and p38 are, however, the most extensively studied. In fact, they are implicated in SC development and in peripheral myelination, and they are described below.

1.8.3.1 Extracellular regulated kinase (ERK)1/2 ERK1 and ERK2 (or p42 and p44) is the classic MAPK but is often still referred to as MAPKs. Mapk3 and Mapk1 encode ERK 1 and 2, respectively, and there are two splice variants (ERK1b, ERK1c). ERK1/2 share 83% amino acid identity and are expressed ubiquitously, distributed throughout the cell 591. In an unstimulated state, many constituents of the cascade are found in membrane compartments (lipid rafts, caveolae), while ERK1/2 is likely bound to cytoplasmic microtubules, associated with membrane receptors and transporters, until nuclear translocation of ERK is induced by activation 121. The kinase cascade to activate ERK consists of A-Raf, B-Raf, and Raf-1 as MAPKKKs, MEK1 and MEK2 as the dual-specificity MAPKKs. Double phosphorylation of ERK1/2 on Thr183 and Tyr185, on its highly conserved motif Tyr-Glutamine (Glu)-Tyr in the activation loop renders it active. Also, the ERK/MAPK cascade is positively regulated by PP2A, and negatively by the DSP MKP 559. Once activated, ERK1/2 phosphorylates a number of substrates including membrane proteins (such as calnexin), nuclear substrates (such as Pax6, NFAT, STAT3, c-Myc), cytoskeletal proteins (Nfs and paxillin) and the MKs RSKs, MSKs, MNKs 559. Growth factors, serum, phorbol esters are potent activators of ERK1/2, while cytokines, osmotic stress, microtubule disorganization are modest activators. Studies with knockout mice implicate ERK1 in immune system development, learning and adipocyte differentiation, and ERK2 in earlier stages of development (placental development, trophoblast formation and mesodermal differentiation) 591. There appears to be a threshold of kinase activity that determines the outcome - transient versus sustained activation of ERK signaling pathway. In many cell systems, transient activation is associated with proliferation, while sustained activation is associated with cell cycle exit and differentiation 23. In SCs, ERK1/2 is activated following NRG1 stimulation 424. Sustained Ras/Raf/ERK signaling in fact, drives the dedifferentiation of SCs 438. Interestingly, Mycobacterium leprae exploits this pathway during early stages of pathogenesis (described in section 1.9) 281. Also, instead of an all-or-none phenomenon, it has been proposed that the balance between Akt and ERK

107 activation in response to specific stimuli determines if an SC will myelinate or dedifferentiate 677,678.

1.8.3.2 Jun aminoterminal kinases (JNK) JNKs were originally named stress-activated protein kinases (SAPKs). Mapk8, Mapk9, Mapk10 encode the three isoforms of JNK, JNK1, JNK2, JNK3, respectively 500. These proteins range between 46 and 55 kDa in size, and exist as 10 or more splice variants which are over 85% identical. JNKs 1 and 2 are ubiquitously expressed, while JNK3 is primarily expressed in brain and testes. JNKs are activated by dual phosphorylation of the conserved Thr-Pro-Tyr motif in their activation loop. Several MAPKKKs (MKK1-4, mixed lineage kinase 2/3 (MLK2/3), TPL-2, TAO1/2, TAK1, ASK1/2) can activate the MAPKKs MKK4/7. Also, MKPs 1-7, DSP2 and VH5 are the DSPs reported to modulate JNK cascade activity, and the phosphor-Ser/Thr protein phosphatase (PP5) 559. Moreover, ubiquitination is another means of regulating JNK activity 559. MEKK1 activation leads to its ubiquitination, in addition to the phosphorylation of downstream MEK1 and MEK4. Ubiquitination prevents the MEKK1-mediated phosphorylation and activation of MEK1/4, thus downregulating JNK pathway activation 408. Similar to ERK, activated JNKs can phosphorylate substrates in the cytoplasm, membrane, nucleus and on the cytoskeleton. It targets a number of transcription factors including Jun family, ATF, JDP2, Elk-1, c-Myc, p53, NFAT, STAT and Pax 408. A major target of JNK signaling is the activation of the transcription factor, activator protein-1 (AP-1). It is homo- or hetero-dimers of jun- jun, jun-fos, jun-atf family members, which have been implicated in regulation of proliferation, survival, differentiation, growth, apoptosis, cell migration and transformation. Activation is mediated in part through phosphorylation of c-Jun to enhance transcriptional activity 66. JNKs are potently induced in response to cytokines, UV irradiation, growth factor deprivation, DNA-damaging agents, and some GPCRs, serum, and growth factors. Thus, the JNK family of MAPKs is involved in cytokine production, immune system stress-induced and developmentally programmed apoptosis, actin reorganization, cell transformation and differentiation 552,646. In SCs, JNKs

108 have been implicated in modulating cell survival, proliferation and motility. For instance, JNK activation is implicated in serum-withdrawal SC apoptosis, as IGF- 1 rescue represses this pathway 121. Moreover, in immature SCs, NRG-1 and Transforming growth factor β (TGF-β), a death signal in immature SCs 123, activate JNK/c-Jun pathway to promote cell proliferation and death during development, respectively 516. Krox-20 suppresses this pathway by acting on the JNK interacting protein-1 (JIP-1), a scaffolding protein which can inactivate JNK. Krox-20 elevates JIP-1 levels to selectively inactivate JNK induction by NRG-1 and TGF-β, to enhance survival, cell cycle arrest and turn on myelin genes expression in immature SCs 514. Additionally, NT-3 activation of TrkC induces JNK signaling pathway to modulate SC migration 514. TrkC directly phosphorylates the exchange factor Dbs to enchance SC myelination 767. Thus, in SCs, JNK activates c-Jun to control SC proliferation, dedifferentiation and cell apoptosis.

1.8.3.3 p38 MAPK The family of p38 MAPKs are proline (Pro)-directed Ser/Thr kinases. There are four isoforms of p38, α, β, γ and δ, encoded by the MAPK14, MAPK11, MAPK13 and MAPK12, respectively 766. The Thr-Gly-Tyr dual phosphorylation motif is common to all isoforms. However, differences between family members in terms of homology, distribution and function exist and are currently being investigated. p38α and β share 74 % sequence homology, while p38γ and δ are only 63% homologous 559. p38α isoform appears to be ubiquitously expressed, while the others are distributed in a tissue-specific manner. Furthermore, there are two splice variants of p38α that have unique properties 236. The MAPK p38 activation module consists of several MAPKKKs (MEKK1- 4, MLK2 and -3, DLK, ALK1, Tpl2 and Tak1) and MAPKKs (MEK3/MKK3, and MEK6/MKK6). MKK3/6 can also exhibit isoform-selectivity: MKK6 activates all isoforms, while MKK3 can preferentially activate p38α and p38β. Moreover, p38α/β can also be activated in an MKK-independent manner. Following the interaction with the adaptor or scaffolding protein, transforming growth factor- beta-activated protein kinase 1-binding protein 1 (TAB-1), p38α can auto-activate 559. Also, both p38α/β can auto-activate proceeding the phosphorylation of a non- canonical Tyr323 site 345. Furthermore, the intracellular distribution of p38 appears

109 to be controversial. In quiescent cells, p38 is localized both to the cytoplasm and the nucleus but upon activation, it can translocate to the nucleus. Activated p38 MAPKs Ser/Thr can phosphorylate a vast number of cellular substrates including molecules regulating cytoskeleton reorganization, transcription factors (ATF1/2, MEF2A Sap1, Elk-1, NF-kB, Ets-1, p53), and several MKs (MSK1/2, MNK1/2, MK2/3) (reviewed in Turjanski et al, 2007 538), to either enhance or inhibit its activity. Thus, p38 MAPKs can regulate gene expression at the level of transcription by modulating transcription factor activity, chromatin remodeling, or at a post-transcriptional level by regulating mRNA stability and mRNA translation. Moreover, it is involved in chromatin remodeling and protein synthesis p38 MAPK regulates gene expression transcriptionally or post- transcriptionally, as p38 can modulate transcription factor activity and mRNA transcript stability 706. The stability of mRNAs is important in the control of gene expression, as shown with cytokine genes in inflammation 163,504,591. AU-rich elements (AREs), located in the 3’ untranslated region of transcripts, are well known to promote rapid mRNA decay 217. ARE-binding proteins (AREBPs) bind these regions and either confer stability or promote transcript degradation. p38 MAPK stabilizes mRNA by inhibiting destabilizing AREBPs or by enhancing the function of stabilizing AREBPs at the AREs 116. For instance, Sox9 mRNA in human articular chondrocytes is regulated by p38 MAPK activation and mRNA stabilization 163.

Genetic inactivation of p38α is embryonic lethal 686. Isoform-specific functions in some cell types are being elucidated using conditional tissue-and isoform-specific ablation 5,672. The use of pyridinyl-based p38 α/β isoform – selective inhibitors (SB203580 and PD169316) has accelerated the process of elucidating roles for these two isoforms in many different biological processes. Moreover, isoforms exhibit substrate-specificities, for instance p38α/β can phosphorylate MK2, while p38 δ/γ cannot 529. MAPK p38 are regulators of several cellular functions including proliferation and apoptosis, which appears to be a cell type-dependent phenomenon. For instance, p38 MAPK has been shown to positively influence cell cycle progression in Swiss 3T3 cells induced by FGF-2, but inhibits cyclin D1 expression to attenuate CCL39 and NIH 3T3 fibroblast proliferation 227,591.

110

Similarly, p38 positively and negatively promotes survival, according to the cell type, and these effects may also be p38 isoform-dependent. For instance, p38α activation appears to induce apoptosis in cardiomyocytes, Jurkat cells and HeLa cell, while survival is promoted by p38β 375,419,459. Nevertheless, its emerging role as a central modulator in development and differentiation appears to be of greatest interest. Indeed, p38 signaling is implicated in adipogenesis, neurogenesis, chondrogenesis, erythroid differentiation, myogenesis and myelination 413,481,732. In SCs, the laboratory of Dr. Almazan previously demonstrated that addition of exogenous laminin to SC-DRGN co-cultures led to a time-dependent phosphorylation of p38 MAPK. Inhibiting p38 MAPK blocked changes associated with early stages of peripheral myelination, including morphological changes and induction of myelin genes 273,347,480,789.

1.8.3.4 Kinase targets of MAPKs: MAPKactivated protein kinases (MK) There are 11 members of this family of MAPK activated kinases (MKs), which are activated by ERK1/2, JNK and p38 signaling. They can be classified into five sub-groups according to sequence homology: RSKs, MSKs, MNKs, MK2/MK3, MK5, and are briefly described below.

The p90 ribosomal S6 kinases (RSKs) family, the first MK identified, belongs to the calcium-, and calmodulin-dependent kinase (CaMK) group of kinases. It is cited as exclusively activated by ERK1/2 213. In humans, there are four isoforms (RSK1-4). They contain two distinct functional kinase domains – the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD) joined by a 100 amino acid linker region. In RSK1 and 2, the NTKD is involved in substrate phosphorylation and both NTKD and CTKD are important for RSK auto-activation 21. RSKs interact with ERK through a C-terminally located docking site. ERK1/2 binding can enhance RSK activation by inducing plasma membrane translocation and activation by membrane-associated kinases 591. RSKs are additionally activated by PDK1 at the NTKD 574, and repressed by 14-3-3β proteins 327,575. RSKs 1-3 are usually expressed in the cytoplasm of quiescent cells, but upon stimulation, a significant portion translocate to the nucleus. RSK regulates gene expression at a transcriptional level by phosphorylating multiple

111 transcription factors such as CREB, and is involved in inducing immediate-early genes and immediate early gene products (c-Fos, c-Jun) to enhance stability 111.

Moreover, it can regulate cell cycle progression at G1- and G2-phases. For instance, RSK1/2 has been shown to phosphorylate p27kip1 to inhibit its activity, and hence, promote cell proliferation. RSKs can also modulate survival 591.

A second family of MKs targeted by MAPKs is Mitogen- and Stress- activated kinase (MSK), which is comprised of MSK1 and MSK2. MSKs are activated downstream of ERKs and p38 MAPKs only 591. They are nuclear Ser/Thr protein kinases highly homologous to RSKs (40% identity) 161,537. MSKs possess an NTKD and a CTKD joined by a linker region. CTKD mediates ERK and p38 MAPK docking and activation. Moreover, activation of CTKD mediates autophosphorylation and the activation of the NTKD, which carries out substrate phosphorylation and the further autophosphorylation 591. Single and double knockout mice of MSK1/2 are viable and fertile, presenting no apparent abnormalities 591,718. MSKs are implicated in immunity and neuronal function. There are a number of substrates, including the transcription factors CREB, ATF1, NFκB, STAT3, factors associated with chromatin remodeling and gene activation HMGN1 and histone H3 109,747. Moreover, MSKs may be involved in regulating apoptotic proteins (Bad) and initiators of translation (4E-BP1) 30.

MAPK-interacting kinases (MNK1/2) are activated by both ERK and p38 MAPK 718. The mnk1 gene encodes one isoform and mnk2 gene encodes two spliced isoforms (MNK2A, MNK2B). MNK1 contains the full sequence used by ERK and p38 to dock, while the docking domain of MNK2A differs slightly but only permits interaction with ERK1/2. Moreover, MNK2B lacks a region in C- terminus containing the docking domain used by both ERK and p38 MAPKs 735. Similarly, the three isoforms exhibit differential intracellular localization in quiescent cells. MNK1 and MNK2A are found primarily in the cytoplasm while MNK2B is enriched in the nucleus. This can be attributed to the absence of a nuclear export signal. While all three MNKs possess an N-terminal nuclear localization signal, and only MNK1 and MNK2A have a nuclear export signal. MNKs contain only one catalytic kinase domain that belongs to the CaMK family of kinases. MNK1/2 modulate general regulation of protein translation by targeting the eukaryotic translation initiation factor (eIF)4E 591. Although MNK2B

112 has been shown to bind estrogen receptor β, it has not been shown to modulate its function 417,553.

The family of MAPK activated protein kinases (MK) signal downstream of p38α/β MAPKs. It is composed of three structurally related proteins, MK2 (MAPKAPK2), MK3 (3pK) and MK5 (p38-regulated and –activated kinase (PRAK)).

MK2: The mk2 gene encodes two alternatively spliced MK2 isoforms a shorter MK2A isoform lacking a C-terminally located nuclear localization signal (NLS) and a longer MK2B isoform. This NLS domain is also a docking D domain for p38α/β. Moreover, MK2B has an NES that modulates nuclear export to the cytoplasm following activation, a regulatory phosphorylation site in the hinge region near the kinase domain a C-terminal. Also, MK2 possesses a proline-rich stretch containing two SH3-binding domains 644. Thus, in quiescent cells, MK2B is primarily nuclear, while MK2A is cytoplasmic. MK2 forms a stable complex with p38α in the nucleus, and upon p38 activation, MK2 is phosphorylated on its activation loop, unmasking the NES binding, and inducing cytoplasmic targeting 227,591. MK2 phosphorylates many proteins involved in mRNA stability, transcription factors (CREB, serum response factor), the scaffolding protein 14-3- 3ζ, and modulators of actin cytoskeleton remodeling (p16Arc, heat shock protein- 27 (HSP27) and vimentin). Akt, glycogen synthase and tyrosine hydroxylase are also targeted by MK2. Moreover, MK2 is involved in post-translational regulation, and cell cycle regulation 227,251.

MK3 is encoded by mk3. It has only one functional kinase domain. Like MK2B, MK3 possesses a C-terminal NLS and NES, a p38α/β docking site, and an SH3-binding domain 227. As such, MK3 is predominantly nuclear in unstimulated cells, but rapidly translocates to the cytoplasm following activation. MK2 and MK3 share several targets, such as the eEF2 factor involved in mRNA translation, and the basic helix-loop-helix transcription factor E47 227.

MK5 is the least characterized MK. The mk5 gene encodes two alternatively spliced isoforms (MK5A, MK5B). Both isoforms possess a functional NLS, NES and a docking D domain that is preferentially bound by p38α/β 227,591. Moreover, MK5 may have a lower affinity for p38 than MK2/3 227,482,591. However,

113 a docking site for the ERK3 and ERK4 was recently shown in MK5. ERK3/4 use this site to bind, translocate and activate MK5 637. MK5 is localized to the nucleus in quiescent cells, but upon activation, translocates to the cytoplasm. Substrates of MK5 are common to MK2, including HSP27 and glycogen synthase 3.

Thus, MKs collectively regulate gene expression at a transcriptional and post-transcriptional level, cell cycle arrest, cytoskeletal arrangement. They are very diverse in terms of their functions and regulatory means.

1.9 DEMYELINATING DISEASES As described thus far, SCs and neurons are in a highly regulated interdependent relationship, whereby SCs and the myelin sheaths are essential to axonal integrity of axons, conduction and trophic support, while neurons regulate the survival, proliferation and differentiation of SCs. The significance of one to another and to the organism is underscored by the clinical symptoms of pathological conditions resulting from SC dysfunction and the progressive demyelination of peripheral nerves. An array of inherited and acquired (toxic, infectious, metabolic or traumatic-induced) neuropathies affecting motor and/or sensory neurons exists. The primary hereditary demyelinating and axonal peripheral neuropathies include a wide spectrum of neuropathic phenotypes, including Charcot-Marie-Tooth (CMT) disease, Hereditary neuropathy with liability to pressure palsy (HNPP), Dejerine-Sottas syndrome (DSS), congenital hypomyelinating neuropathy (CHN) and giant axonal neuropathy 227,591. HNPP is a milder form of demyelinating peripheral neuropathy than CMT, while DSS and congenital hypomyelinating neuropathy are clinically more severe. Guillain-Barre- Strohl syndrome (GBS) and leprosy are acquired demyelinating neuropathies. The most common form of inherited neuropathy, CMT, and the acquired neuropathies are discussed below.

1.9.1 CharcotMarieTooth (CMT) disease CMT disease, or hereditary motor and sensory neuropathies (HMSN) encompasses a genetically heterogenous group of disorders, originally described by Charcot, Marie, Tooth and Herringham in the 1880’s. It is the most common

114 inherited disorder of the PNS, affecting approximately 1 in 2,500 individuals 63. These disorders can be classified into CMT type 1 (CMT1) or type 2 (CMT2) according to primary or secondary axonal impairment. Mutations for both types can be autosomal-dominant, autosomal-recessive or X-linked. CMT1 is more prevalent and typically has an earlier age of onset, in the first or second decade of life. This is a primary demyelinating or dysmyelinating disease whereby myelinating SCs are afflicted, with secondary axonal degeneration (reduced axon caliber, axonal transport, neurofilament phosphorylation and density, axolemmal ion channel distribution 643). Cells of the immune system are also implicated in disease pathogenesis 425,528. Clinically, significant reduction in nerve conduction velocity (NCV) (forearm NCV less than 38 m/s) is indicative of dysmyelination and/or demyelination 46,431,663. Normal conduction velocity, attributable to the presence of myelin, is 40-50 m/s 46,663. Moreover, nerve biopsies of CMT1 patients reveal segmental demyelination and remyelination, onion bulbs formation and axonal loss 800. In contrast, CMT2 usually displays later onset and is associated with loss of myelinated axons. Accordingly, axonal neuropathies are characterized by normal or near normal NCV (forearm motor NCV greater than 38 m/s) 663 but decreased conduction amplitudes due to the decreased number of total functional axons 663. Thus far, mutations of several genetic loci in over a dozen genes have been found to cause different forms of CMT with varied degrees of severity 800. The mutations in specific genes are not necessarily CMT1- or -2-exclusive, except genes associated with axonal transport that are CMT2. Thus, there is overlap between the types of neuropathies according to the location and type of mutation. Myelin structural proteins (P0, PMP22, Cx32 and periaxin) constitute one group of genes affected in those with compromised myelin stability. This is illustrated by gene dosage effect of two structural proteins P0 and PMP22 in CMT1 and CMT2 patients. Mutations of the gene regulatory transcription factors (Krox-20, Sox10) produce differing phenotypes of CMT1 depending on the genetic location. In addition, genes related to the intracellular vesicular transport, synthesis and degradation of myelin proteins (myotubularin-related lipid phosphatase- myotubularin protein 2 (MTMR2) and dynamin-2) were mutated in patients with CMT1 and CMT2, emphasizing the significance of strict control of myelin proteins levels in myelination. Mutations in mitochondrial morphology-

115 related genes (mitochondrial fission factor, mitofusin2 (MFN2), and ganglioside- induced differentiation associated protein 1 (GDAP1)) were found in patients of both types of neuropathies. Identification of these mutated genes highlights the pertinence of the mitochondria to PNS myelination. However, mechanisms surrounding disease pathogenesis and the primary cellular target (SC or neuron) remain obscure. Mutations of the mitochondrial chaperone proteins heat-shock proteins-22 and -27 (Hsp-22, -27) genes are also found in CMT2 patients. Furthermore, in CMT2, mutations of genes encoding proteins of the axon cytoskeleton (neurofilament light chain and kinesin-1B) and axonal transport result in axonal degeneration. As such, in patients with these mutations, the distal ends of axons are maximally afflicted. Finally, there are several genes of unknown function mutated in CMT patients, such as N-myc downstream- regulated gene 1 product and protein KIAA1985. Therefore, mechanisms of disease pathogenesis require further study.

1.9.2 GuillainBarreStrohl (GBS) Syndrome GBS refers to a group of acute inflammatory peripheral neuropathies causing reversible myelin damage, that manifest clinically as acute flaccid paralysis 46,301,486,663,800. Symptoms of GBS include generalized muscle weakness, ascending from lower to upper limbs, while facial and respiratory muscle weakness may also occur. More often than not, it is a single phase illness, but some patients may relapse. GBS may occur several weeks following infection (Epstein-Barr virus, cytomegalovirus, Mycoplasma pneumonia, Campylobacter jejuni), vaccination, surgery and head trauma 724. There are several subtypes, exhibiting distinct geographical distribution: acute inflammatory demyelinating polyradiculoneuropathy (AIDP), acute motor axonal neuropathy (AMAN), and acute motor and sensory axonal neuropathy (AMSAN). AIDP is more prevalent in Western countries, while the axon-directed AMAN and AMSAN diseases are more common in Asia, South and Central America 724.

AIDP is characterized by demyelination of peripheral axons and axonal degeneration in severe cases. Mononuclear infiltrates are commonly found throughout the PNS, and myelin sheaths are damaged by invading macrophages. Serum antibodies to P0, PMP22, P2 and Cx32 are present in

116 many AIDP patients, suggestive of development of autoimmunity to peripheral myelin proteins 311. Moreover, patients with GBS also display α6β4 integrin immunoreactivity. Thus, circulating fragments of myelin-associated α6β4 integrin can be used as a novel biomarker for extensive peripheral myelin damage, to evaluate disease course and activity 15,226,370.

AMAN is a purely motor neuron disorder, characterized by extensive axonal degeneration with little demyelination. Pathological studies suggest the motor axon is primarily targeted by autoimmune system, where activated macrophages are found to invade the space between SCs and axon, beneath the myelin sheath 629. By contrast, both motor and sensory neurons are afflicted in AMSAN. In the early stages of disease, muscle and sensory action potentials are reduced. However, it is often difficult to discern between AMSAN and severe cases of AIDP involving axon degeneration 312 using electrophysiological approaches. As such, conducting serological tests would help accurate diagnosis.

As conduction deficit is the hallmark of GBS, investigations into altered sodium channel clustering as disease mechanisms were conducted. Studies using two models for AIDP and AMAN, experimental allergic neuritis against peripheral myelin (EAN-PM) and the neuritogenic P2 peptide (EAN-P2) point to disrupted nodal sodium channel clustering 312. Firstly, gliomedin and NF-186, two molecules involved in sodium channel clustering expressed by SCs and axons at the node of Ranvier, respectively, appear to be selectively targeted by auto- antibodies prior to onset of disease in AIDP 405,496,662. Complement is deposited at nodes of Ranvier and on the external surface of SCs, while high titers for anti- ganglioside (GM-1) antibodies are found in the cerebrospinal fluid. Anti-GM1 antibodies effect complement-mediated disruption of SC-axonal interaction at nodes of Ranvier, thereby inhibiting the voltage gated channels clustering modulated by gangliosides 405. GM-1 auto-antibodies are additionally isolated from AMSAN patients 662. Since several other CNS disorders mimic signs often used for GBS diagnosis, nerve conduction studies and cerebrospinal fluid analysis should be performed to confirm GBS diagnosis.

117

1.9.3 Leprosy Leprosy or Hansen’s disease is a prime example of an infectious neurodegenerative disease of the PNS. Prevalence of this chronic disease has declined in the last 50 years primarily in developed countries, but remains largely rampant in developing countries such as Africa, Asia and Latin America. The cornerstone of this disease, peripheral neuropathy, is caused by the bacteria Mycobacterium leprae (M.leprae) 312 and affects sensory, motor and autonomic fibers. The acid-fast pathogen is an obligate intracellular bacillus that targets SCs. Its mode of transmission remains partly obscure. Humans are the principal reservoirs of M. leprae. Nasal droplet infection appears to be the primary route of transmission. Contact with infected soil, direct implantation (tattooing) are other means of infection. Skin to skin transfer of M.leprae is believed to be highly unlikely.

M. leprae induces early demyelination by binding and activating the ErbB2 tyrosine receptor on SCs to trigger de-differentiation. It bypasses the obligate heterodimerization with ErbB3 to activate the Ras-Raf-MEK-ERK pathway via son of sevenless (SOS) 12. Upon SC dedifferentiation, M. leprae invades these immature SCs by binding to the SC α-DG laminin receptor. Phenolic glycolipid-1 (PGL-1), found in the cell wall of M.leprae, is unique to this pathogen. PGL-1 binds the G domain of α2 chain of laminin-2, found in the basal lamina surrounding the SC-axon units 677. This PGL-1-α2-laminin-2 complex then binds to α-DG receptor on SCs to induce internalization 483,636. Once inside, M. leprae subsequently drives SCs to proliferate by inducing the protein kinase C-ε - Lck pathway to activate ERK once again. M. leprae can then replicate within these host SCs 483,560. Non-myelinating SCs are also infected by this pathogen to further expand its reservoirs for infection. Using a similar mechanism, M.leprae invades macrophages and monocytes, two cells of the immune system. It first forms a complex with complement C3, to bind cell-surface complement receptors on macrophages and monocytes to modulate its internalization 678.

M.leprae multiplies very slowly and requires a temperature of 27-30oC for growth. Hence, superficial and cooler areas including skin, nerves, testes and upper respiratory tracts are primarily afflicted. The incubation period is approximately 5 years. Symptoms may only manifest 20 years post-infection.

118

Biopsies of nerve lesions indicate a chronic or subchronic inflammatory infiltrates in the endoneurium, perineurium and epineurieum, which progressively damage myelinated and non-myelinated fibers. Fibrotic tissue eventually replaces these nerve regions once filled by fibers, and complete destruction of nerves due to the immune response may occur if untreated. Clinically, M. leprae-infected patients present a diverse set of symptoms, which are classified according to the number of skin lesions and the number of bacilli found on slit-smear examinations. Skin manifestation, together with neurological involvement, is indicative of the stage of disease progression 483,618. Moreover, host cell-mediated immunity (genetic predisposition) contributes to the susceptibility to infection and disease manifestation. As leprosy is a curable disease, early detection is vital for treatment. In this regard, the order of afflicted types of axon fiber is a hindrance. Unmyelinated sensory c-fibers (detect pain, temperature) are significantly affected prior to the larger myelinated (detect vibration, position, motor) sensory fibers.

In summary, attaining a better understanding of SC biology, development and mechanisms regulating peripheral myelination will help gain insight into combating and preventing of some of these peripheral neuropathies. For instance, in leprosy, understanding the biology of SC myelination/dedifferentiation, allowed the insight into molecular mechanisms of disease pathogenesis. As a result, developing drugs aimed at blocking SC dedifferentiation through ErbB2 receptor are being investigated 12.

119

CHAPTER 2: RATIONALE AND OBJECTIVES

120

2.1 RATIONALE Myelin, synthesized by SCs in the PNS, is formed through a complex process. Several hereditary demyelinating or axonal neuropathies are caused by faulty expression of several myelin proteins (PMP-22 and P0), transcription factors (EGR2), proteins of the axon cytoskeleton (NFL), proteins related to intracellular vesicular transport and myelin protein degradation (MTMR2), mitochondrial chaperone proteins (HSP-22) and mitochondrion morphology- related genes (MFN2) 494 . Thus, although many regulatory factors directing SCs and myelin formation have been unveiled, how myelination takes place and how it is so precisely coordinated remains largely enigmatic. As described, three factors are of utmost importance for an SC to myelinate: continuous contact with the axon, the presence of specific secreted soluble factors and the assembly of a basal lamina surrounding each axo-glial unit.

Immature SCs integrate signals originating from the basal lamina and axons to initiate differentiation and formation of myelin around large caliber axons of the PNS 46,301,486,663,800. Adhesion to basal lamina is a pre-requisite for myelin- specific gene expression in SCs 92,204, while interactions with ECM molecules influence morphological changes, migration and differentiation. Laminin, an essential constituent of the basal lamina, is required for myelination in vivo 183,184 and in vitro 771. Previous studies in Dr. Almazan’s laboratory showed that addition of laminin or exogenous ECM to SC-DRGN cultures caused a time-dependent phosphorylation of p38 MAPK, and HSP27, a downstream effector of p38 involved in actin cytoskeleton remodeling. In contrast, p38-selective inhibitors blocked HSP27 phosphorylation and reduced laminin accumulation as well as ECM-induced changes in SC shape and axonal alignment. Furthermore, inhibition of p38 reduced mRNA steady state levels for the myelin proteins MAG, MBP, and Po and blocked myelination of DRGN by SCs 183,213. Collectively, these results suggested that p38 is an important molecule for PNS myelination. However, it is unclear as to how the signals from laminin are transduced intracellularly to activate p38 and myelination.

Laminin signals through heterodimeric (αβ) integrin receptors, of which SCs express α1β1, α6β1, α6β4 and α7β1 in a developmentally regulated manner 213. SCs deficient in β1 integrin fail to myelinate, resulting in severe neuropathy

121 with impaired radial sorting of axons 549,550. Moreover, application of a function- blocking β1 integrin antibody to SC-DRGN cultures prevents basal lamina from attaching to the SC surface and subsequently blocks myelination 199. In contrast, conditional ablation of β4 integrin in SCs did not hinder myelination 206,542 , thus reinforcing a central role for β1 integrin in peripheral myelination. α6β4 integrin in conjunction with dystroglycan, another laminin receptor expressed by SCs, cooperatively stabilize myelin sheaths 200.

Integrins lack intrinsic protein kinase activity and participate in inside-out and outside-in signaling by forming focal adhesion complexes containing protein kinases, including FAK and phosphatases, such as Shp2, recruited to intracellular C-terminal domains. FAK and Shp2 are involved in SC development and myelination 492. The family of SLKs is among the protein kinases recruited to these sites. In OLGs, Fyn is involved in transcriptional 267,268 and post- transcriptional regulation 710,711 of MBP, maturation 407, differentiation 407,651,652 and myelination 355,508. Moreover, Fyn is involved in signaling downstream of L-MAG, which is essential for myelin maintenance in OLGs 138,407,627,711. In contrast, a precise role for SLKs in peripheral myelin formation remains elusive as single kinase knockouts present no overt peripheral defects and further studies to identify potential roles for SLKs have not been conducted. Although this might highlight yet another difference between central and peripheral myelin, compensatory mechanisms could also occur in vivo since SCs express Src, Fyn, Lyn and Yes 55. In SCs undergoing basal lamina-stimulated differentiation, several cytoskeletal and signaling proteins were shown to complex with β1 integrin and Fyn 651. Furthermore, following peripheral nerve injury, Src induction was coupled with remyelination 117,498 pointing to a potential role for SLKs in this biological process. Another family of molecules known to form part of FACs at the cytoplasmic domains of integrins and to propagate this integrins-mediated intracellular signaling in other systems is the Rho GTPase. The Rho GTPases Rac1 and Cdc42 were previously shown by other labs to participate in radial axonal sorting by SCs 794. Recent work conducted in vivo, examining the roles of Cdc42 and Rac1 in both central and peripheral myelination suggest unique roles for each of these GTPases. Conditionally ablating either Cdc42 or Rac1 in the

122

CNS, revealed that both GTPases synergistically regulate and are required for modulating proper myelin sheath formation by OLGs 45,493. Tissue-specific conditional ablation of Cdc42 or Rac1 in vivo indicated that Cdc42 is required for normal SC proliferation, while Rac1 regulates SC process extension and stabilization, for efficient radial sorting of axon bundles 688. Rac1 activation occurred in a β1-integrin dependent manner. In Drosophila melanogaster, using gain-of-function and loss-of-function mutants, important roles for RhoA and Rac1 were identified in peripheral glial cell migration and nerve ensheathment 45,493. Furthermore, ROCK was shown to regulate correct axo-glial functional domain formation, as well as the number and length of myelinated internodes 628. No effect of the ROCK inhibitor, Y27632, was reported on the accumulation of myelin proteins or on Krox-20 expression. Also, MAPKs are among the downstream targets of Rho GTPases, including p38 444,722. Our laboratory previously demonstrated that p38 MAPK is required for early morphological changes in SCs associated with myelination, including axonal alignment, and HSP27 phosphorylation following addition of laminin to SC-DRGN cultures 684,685.

Finally, we previously demonstrated the significance of p38 MAPK activation to myelin protein gene expression (MBP, P0 and MAG) using inhibitors of p38 MAPK activity 213. However, the molecular mechanisms by which myelin genes are regulated remain obscure. p38 MAPK has been shown to regulate both the expression and the activity of several transcription factors. Control of gene expression can occur at the transcriptional and post-transcriptional levels. Transcripts containing AU-rich elements (ARE) are more labile, and, p38 MAPKs act on ARE-BPs to modulate the stability of transcripts. In addition, p38 can phosphorylate transcription factors to modulate transactivation properties. In SCs, the coordinated expression of transcription factors regulate morphological changes associated with the transition from the immature to the myelinating state, including Krox-20/EGR2, SCIP, NF-κB, Sox10 and NFAT (reviewed in Jessen and Mirsky 213; Svaren and Meijer 329). Krox-20, a zinc-finger transcription factor, is critical for SCs to myelinate axons and for the maintenance of mature compacted myelin 666. The SCs from EGR2 knockout mice, which lack peripheral myelin, are arrested at a pro-myelinating state but continue to proliferate 166,695. Krox-20 either directly regulates expression of several myelin proteins 695, or cooperates with Sox10, an

123

HMG transcription factor to do so 380,475. Expression of Krox-20 is upregulated following increases in cAMP and axonal contact 334,382. Moreover, members of the POU domain transcription factor family are integral in regulating Krox-20 expression and myelination. Expression of SCIP, induced in response to cAMP 615,799, can regulate Krox-20 expression together with Brn2 and Sox10 464. Moreover, Krox-20 was shown to share cross-antagonistic roles with the basic leucine zipper protein, c-Jun, and Sox2. The JNK-c-Jun pathway is involved in both proliferation and cell death of immature SCs 238,322,323. c-Jun blocks Krox-20- mediated gene expression, pushing SCs towards an undifferentiated state 469,514, while Krox-20 suppresses this pathway to promote cell survival and differentiation 513. Sox2, another HMG transcription factor, expressed by immature SCs, negatively regulates differentiation and myelination. It promotes expression of genes of the immature and pro-myelinating SCs (cyclin D3, cdk4) while repressing pro-myelination genes (Mpz, Prx, Cx32) 514. Moreover, it is postulated that some of the effects of c-Jun may in part be carried out through Sox2 378.

Hence, we hoped to shed light on the mechanisms by which p38 MAPK regulates the expression of myelin genes at the onset of myelination and to study the roles of SLKs and Rho GTPases, two families known to form part of the FACs at the cytoplasmic domains of integrins to propagate intracellular signaling, in myelination involving p38 MAPK activation.

2.2 HYPOTHESIS The working hypothesis in this thesis is that laminin, acts through β1 subunit-containing integrins to initiate and to coordinate myelination of peripheral axons by SCs, involving activation of SLKs and Rho GTPases, which activate p38 MAPKs, leading to the expression or activation of key transcription factors.

124

2.3 SPECIFIC OBJECTIVES

2.3.1 Specific Objective 1: Characterize the role of the SLKs in peripheral myelination

Specific Aims:

a) Determine the effect of specific pharmacological inhibitors of SLKs on myelin protein accumulation (SC differentiation), cell death, proliferation b) Evaluate the effect of SLK-specific pharmacological inhibitors on DRGN axonal structure, organization and neurofilament expression c) Assess the reversibility of the effect of SLK inhibition on myelin protein levels d) Characterize the morphology of myelinated fibers formed in the presence of the SLK inhibitor, by assessing length and number of internodes, the g- ratio that relates the thickness of the axon to that of myelin lamella, and the formation of functional axo-glial domains (nodes and paranodes) e) Assess the role of SLKs on expression of the essential transcription factor, Krox-20 f) Determine whether SLKs regulate phosphorylation of p38, Akt, ERK-1/2, as induced by constituents of the basal lamina or ECM

2.3.2 Specific Objective 2: Determine whether p38 MAPK regulates transcription factors expression in SCs to ultimately modulate expression of myelin genes associated with SC terminal differentiation and peripheral myelination

Specific Aims:

a) Characterize and compare p38 MAPK isoforms expressed by SCs and DRGNs b) Characterize the ultrastructure of SC-DRGNs treated with the p38 inhibitor PD169316

125

c) Characterize the effect of p38 MAPK inhibition on mRNA and protein levels of several key transcription factors (Krox-20, SCIP, Sox2 and Sox10) in SC differentiation and peripheral myelination using SC-DRGN co-cultures d) Determine the isoform-specific p38 function in SC differentiation e) Assess the effect of p38 inhibition on expression of the cell cycle inhibitor, p27kip1 f) Assess the role of MK2 in expression of peripheral myelin proteins and transcription factor Krox-20 g) Determine whether CREB is phosphorylated following addition of extracts of ECM h) Identify possible upstream activators of CREB such as p38 MAPK, MK2 or MSK-1

2.3.3 Specific Objective 3: Characterize the role of Rho GTPases in peripheral myelination involving activation of p38 MAPK

Specific Aims:

a) Characterize the role of RhoA in SC morphology and myelination following genetic overexpression of constitutive active, dominant negative, or wild type RhoA constructs in SC-DRGN co-cultures, and in pure SCs seeded onto mature dissociated DRGNs b) Assess the effect of expressing RhoA constructs of on Krox-20 expression by SCs c) Determine the role of the RhoA downstream effector, ROCK, on expression of myelin proteins and the transcription factor Krox-20 d) Assess the effect of ROCK inhibition on phosphorylation of p38 MAPK and CREB induced by extracts of ECM e) Characterize the roles of Rac1 and Cdc42 in SC morphology and myelination following genetic overexpression of constitutively active, dominant negative, and wild type constructs in SC-DRGN co-cultures f) Assess the involvement of Rac1 in phosphorylation of p38 MAPK and CREB induced by extracts of ECM

126

CHAPTER 3: MATERIALS AND METHODS

127

3.1 MATERIALS

3.1.1 Cell culture reagents Dulbecco's modified Eagle medium/Ham’s F12 medium (DMEM/F12), Hanks Balanced Salt Solution (HBSS), Leibovitz’ medium (L-15), phosphate buffered saline (PBS), 7.5% bovine serum albumin fraction V, soybean trypsin inhibitor, trypsin (0.025%) and penicillin/streptomycin were from Invitrogen (Burlington, ON, Canada). Bovine transferrin, bovine insulin, putrescine, laminin, L-ascorbate, and extracellular matrix were from Sigma-Aldrich (Oakville, ON, Canada). Nerve growth factor (NGF 2.5S) was purchased from Alomone Labs (Jerusalem, Israel).

3.1.2 Inhibitors The inactive analogue of PP2, PP3 (4-amino-7-phenylpyrazolo [3, 4-d] pyramidine), the SLK inhibitors PP2 (4-amino-5-4-chloropheynyl)-7-(t –butyl) pyrazolo (3, 4-d) pyrimidine) and SU6656 (2,3-Dihydro-N,N-dimethyl-2-oxo-3- [(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide), the p38 α/β MAPK inhibitor PD169316 (4-(4-Fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)- 1H-imidazole), the MK2a inhibitor CMPD1 (4-(2′-Fluorobiphenyl-4-yl)-N-(4- hydroxyphenyl)-butyramide), the protein kinase A/MSK-1 specific inhibitor H-89 (N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, 2HCl), the MSK-1 inhibitor Ro 31-8220 the Rac1 inhibitor NSC23966, and the Rho Kinase inhibitor Y27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)- cyclohexanecarboxamide, 2HCl) were all from EMD Chemicals (San Diego, CA, USA).

3.1.3 siRNA transfection reagents The siMPORTER siRNA transfection kit was purchased from Upstate Biotechnology Inc. (Millipore, Billerica, MA, USA). . Two Alexa Fluor 488-labeled siRNAs directed against Fyn (fyn-1 and fyn-2) and Alexa Fluor 488-labeled non- specific negative controls were designed and produced by Qiagen (Mississauga, ON, Canada), as previously published 513. The siRNA sequences for fyn-1 and

128 fyn-2 covered nucleotides 482–502 and 1307–1327, respectively (NCBI accession number U35365), and for the non-specific negative control was AAT TCT CCG AAC GTG TCA CGT. Predesigned anti-lyn (M-093726-00, and anti- Mapk14 (p38α) (M-080059-00) SMARTpool siRNAs and the siGLO-Green (FAM)/Red (DY-547) transfection indicators were purchased from Dharmacon Biotechnology (Lafayette, CO, USA).

3.1.4 Adenoviruses David Parkinson kindly provided us with control GFP (GFP Ad) and GFP- tagged Krox-20 adenovirus (Krox-20 Ad). Also, James R. Bamburg (Fort Collins, CO, USA) provided us with adenoviruses expressing GFP-tagged constitutive active (CA, V12 mutation), dominant-negative (DN, N17 mutation) and wild-type (WT) constructs of Rac1 and Cdc42. The RhoA cDNA allelic pack containing wild type, activated (Q63L mutation; V14) and dominant negative (T19N substitution; N19) was ordered from Upstate The AdEasy-1 kit and the adenovirus packaging Human Embryonic Kidney (HEK) 293 cell line were purchased from Stratagene (La Jolla, CA, USA).

3.1.5 Antibodies Primary antibodies were obtained from the following suppliers: rabbit polyclonal anti-pan-sodium channel, -laminin from Sigma-Aldrich (Oakville, ON, Canada); rabbit polyclonal anti-phospho-Akt (Ser473), -phospho-ERK (Thr202/Tyr204), -phospho-p38 (Thr180/Tyr182), -phospho-CREB (Ser133), -p38α, – p38δ, -p38γ, -Akt from New England Biolabs (Mississauga, ON, Canada); - mouse p38β from Zymed Laboratories (San Francisco, CA, USA); -mouse monoclonal MBP (SMI99), -neurofilament (n52), -CNP (SMI91) from Chemicon (Temecula, CA, USA); rabbit polyclonal anti-Krox-20 from Cedarlane (Toronto, ON, Canada); rabbit polyclonal anti–Fyn (FYN3) and -Lyn, goat anti--actin, -β1 and -β4 integrin, -SCIP/Oct-6, –Sox10 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); mouse monoclonal v-Src (327) from Calbiochem/EMD Biosciences (LaJolla, CA, USA); rabbit polyclonal anti-Sox2, mouse anti-p27kip1 from BD Pharmingen (Mississauga, ON); chicken anti-P0 and –GFP were from Novus Biologicals (Littleton, CO, USA). Anti-caspr and -P0 were generous gifts

129 from Dr. David Colman, anti-MAG (Myelin associated glycoprotein) was from Dr. Peter Braun, and David Parkinson kindly provided us with rabbit anti-periaxin. Horseradish peroxidase (HRP)-, fluorescein isothiocyanate (FITC)- or Texas Red-conjugated secondary antibodies were from Cedarlane or BIO-RAD Canada (Mississauga, ON, Canada), Jackson Immunoresearch Laboratories (Mississauga, ON, Canada), Southern Biotechnology (Hornby, ON, Canada) or Aves Labs (Tigard, OR, USA). Anti-chicken, -rabbit, and -goat Alexafluor-488, - 563, -647 were from Invitrogen (Burlington, ON, Canada).

3.1.6 Other reagents Protein assay reagents were obtained from BIO-RAD. The bicinchoninic acid protein assay kit, Triton X-100 and 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium bromide (MTT) were from Sigma-Aldrich (Oakville, ON, Canada). X-OMAT Kodak x-ray film was purchased from Mandel Scientific (Guelph, ON, Canada). Sodium cacodylate was from EMD Chemicals (San Diego, CA, USA). SYBR1 Green PCRMaster Mix was from Roche (Missisauga, ON, Canada). RNA isolation kit and DNase were from Qiagen. Superscript II first strand synthesis system was from Invitrogen. ECL Western blotting detection reagents were from Perkin-Elmer (Boston, MA, USA). All other reagents were obtained from VWR (Mont-Royal, QC, Canada) or Fisher Scientific (Whitby, ON, Canada).

3.2 METHODS

3.2.1 Primary cell culture Primary Schwann cell-Dorsal root ganglion neuron (SC-DRGN) co- cultures were derived from the DRGs of embryonic day 15-16 Sprague-Dawley rats, as described (Figure 2.1b) 151. Briefly, under sterile conditions, in L-15 media, embryos were removed from their sacs and their spinal cords isolated. DRGs associated with the spinal cord were then individually removed, transferred to a 15 mL tube and centrifuged. Subsequently, DRGNs were thoroughly dissociated by trituration following 15 min trypsinization (0.025%) at 370C and treatment with a soybean trypsin inhibitor (5mg/mL in HBSS). The

130 dissociated DRGNs were suspended in N1 serum-free medium containing NGF and plated on rat tail collagen-coated dishes. Approximately 40-50 DRGs were obtained from each embryo and each DRG contains about 5000 sensory neurons.

SCs (Figure 2.1a) and DRGNs (Figure 2.1c) were purified from mixed SC-DRGN co-cultures. SCs were enriched using a modified protocol of the cold jet method 213,243, whereby dissociated DRGNs were plated on un-coated petri dishes and grown for two weeks in N1 medium containing NGF. Upon reaching full confluency, SC-DRGNs were trypsinized, resuspended in 1-containing media, filtered to remove DRGNs and plated onto poly-L-lysine-coated dishes.In contrast, mixed SC-DRGN co-cultures were treated 48 h and 7 d after plating with an anti-mitotic for 2-3 days to yield pure DRGN cultures.Cultures were characterized immunocytochemically using cell type-specific antibodies.

Figure 3. 1 Schema outlining the procedure to obtain in vitro cultures. a) pure SC, b) mixed SC‐DRGN and c) pure DRGNs in vitro cultures from rats. The procedure is described in the text above in greater detail.

131

All experimental protocols for the preparation of primary cell cultures were approved by the McGill Faculty of Medicine Animal Care Committee in accordance with Canadian Council on Animal Care guidelines.

3.2.2 Secondary cell culture Two cell lines were used: the Murine Schwann cell 80 (MSC 80) and Human Embryonic Kidney cells-293 (HEK-293). The MSC 80 cell line was established by Boutry et al 332 and expresses markers of myelinating SCs. MSC 80 cells were grown in DMEM media containing 10% fetal bovine serum (FBS) containing Hepes, while HEK 293 cells were grown in 10% FBS alone. Cells were passaged every two to three days according to confluency. Cells were washed once with warmed PBS, trypsinized for 5 minutes at 370C until they detached from the dishes. The trypsin was subsequently inactivated using serum-containing media. Cells were counted using a hemocytometer and plated to acquire the required density.

3.2.3 siRNA Transfection SC-DRGN co-cultures and MSC 80 cells were transfected with siRNA using the siMPORTER transfection kit to study the effect of gene knockdown on expression levels of proteins using immunocytochemistry and Western blotting, respectively.

SC-DRGNs were transfected with 100 nM control, anti-Fyn (fyn1, -fyn2), lyn, or -p38α (Mapk14) siRNAs. Transfection medium was removed 18 h or 24 h later and myelination was stimulated with the addition of vitamin C (VC). 48 h after initiation of myelination, cells were fixed, immunolabeled for Krox-20 expression and the nuclei stained with 4’, 6’-diamidino-2-phenylindole (DAPI). Three independent experiments were performed in duplicate and at least 30 fields from each independent experiment were imaged using confocal microscopy. The total number of siRNA-transfected SCs expressing Krox-20 was quantified and expressed as a percentage of total SCs transfected for each condition. Fluorescently tagged control siGLO-Green or -Red was used as a marker for transfection. SC-DRGNs transfected with anti-Fyn or –Lyn siRNA

132 were also harvested for Western blotting 48 h after transfection. Samples were blotted for total Fyn, Lyn and actin.

MSC 80 cells were also transfected with 100 nM control or anti-Mapk14 (p38 α) siRNA in suspension in DMEM containing 3% FBS and plated onto 6-well dishes. Cells were allowed to attach; the medium was changed 18 h later. Cells were re-transfected with 100 nM siRNA 6 h later, and subsequently harvested for Western blotting 48 h after the initial transfection.

3.2.4 Western Blot Analysis Cells were harvested in either Tris-HCl (62.5 mM, pH 6.8 and 2% SDS) or in RIPA (150 mM sodium chloride, 50 mM Tris (pH 8.0), 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) lysis buffer. The protein concentration in samples was determined using the bicinchoninic acid protein assay kit or Bio-Rad protein assay, respectively. Protein extracts were resolved using SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by standard protocols. Blots were blocked with 5 % dry milk in Tris- buffered saline containing 0.1% Tween-20 and then incubated with primary antibodies. The membranes were incubated with the appropriate HRP- conjugated secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence and quantified by densitometry using AlphaEaseFC software

3.2.5 Electron Microscopy Samples were fixed in 2.5% glutaraldehyde/2.5% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2, post-fixed in 4% osmium-tetroxide, stained with 1% uranyl acetate, dehydrated in graded ethanol series and embedded in epon. Semi-thin sections were cut and stained with methylene blue. Selected regions were used for ultrathin sections, which were subsequently stained with uranyl acetate and lead citrate and examined using a JEOL 2000FX electron microscope. The g-ratio was calculated as the ratio between the axonal diameter and the myelinated fiber (axon plus myelin) diameter.

133

3.2.6 Immunofluorescence Cells grown on collagen-coated glass coverslips, were fixed in 4% paraformaldehyde in PBS, pH 7.4, followed by further fixation in ice-cold methanol, blocked with 10% goat serum in TBS-Tween and immunostained with a primary antibody followed by incubation with a fluorescent-conjugated secondary antibody. All coverslips were stained with DAPI to visualize nuclei and subsequently mounted using ThermoMount mounting medium. Slides were imaged at room temperature using Zeiss LSM 510 confocal microscopes (McGill Life Sciences Imaging Facility and Department of Pharmacology (two-photon) with PlanFluor 40x 0.75 water objectives and Zeiss LSM acquisition software (Zeiss). DAPI staining was taken with either the 405 line or the two photon laser. Exposure time, gain, laser intensity were maintained for each experiment, per condition and per antibody staining. All images were acquired as z-stacks and slices compiled to form composite images. These composite images were subsequently processed using LSM 5 Image Browser software and Adobe Photoshop (version 7.0). The quantitative analysis was then performed using ImageJ analysis software (National Institutes of Health).

3.2.7 RNA isolation, reverse transcription and Realtime quantitative PCR

Total RNA was obtained from SC-DRGN co-cultures treated for 48 h using the Qiagen RNA isolation kit, following the manufacturer protocol.

The isolated RNA was subsequently treated with DNase to remove any contaminating genomic DNA. Reverse transcription was performed using 1µg of RNA in 20 µl of reaction volume of Superscript II first strand synthesis system. Samples were stored at 20 ºC until their utilization. cDNA (5 µl of diluted reverse transcription product) was amplified using SYBR1 Green PCRMaster Mix in the presence of primer oligonucleotides specific for each gene.

Changes in the mRNA expression of different genes were examined by real-time quantitative PCR using a Rotor gene 6000 Sequence Detection System (Corbett life science). The PCR conditions were as follows: 95ºC for 10 min,

134 followed by 40 cycles consisting of 95 ºC for 20 s, 59 ºC for 20 s, and 72ºC for 20 s. The quantification was performed by the comparative Ct (cycle threshold) method, using the GADPH RNA expression level as an internal control. Primers for all target sequences were designed using the Primer 3 input (Version 0.4.0). In all cases, only one amplification product was obtained. Chosen PCR primers were as follows:

Transcription factors: Krox-20 sense 5’-CTACCCGGTGGAAGACCTC-3’ Antisense 5’-AATGTTGATCATGCCATCTCC-3’ SCIP sense 5’-GTTCTCGCAGACCACCATCT-3’ antisense 5’-CTTTGACACCCACCTCAAT-3’ Sox2 sense 5’-CACAACTCGGAGATCAGCAA-3’ antisense 5’-CTCCGGGAAGCGTGTACTTA-3’ Sox10 sense 5’-ATGTCAGATGGGAACCCAGA-3’ Antisense 5’-GTCTTTGGGGTGGTTGGAG-3’

Myelin genes Connexin32 sense 5’-CCCTGCAACTCATCTTGGTT-3’ antisense 5’-CGGAACACCACACTGATGAC-3’ MAG sense 5’-TCGCCTCACTGTACTTCACG-3’ antisense 5’-CTGAGTTGGGAATGTCTCCTG-3’ MPZ sense 5’-CTGGTCCAGTGAATGGGTCT-3’ antisense 5’-CATGTGAAAGTGCCGTTGTC-3’ Periaxin sense 5’-GAGCCTCAGTTTGCAGGAAG-3’ antisense 5’-GCCCTTCATCTCGTATCCAG-3’; GAPDH sense 5’-TCGTGGAAGGACTCATGACCA-3’ antisense 5’-CAGTCTTCTGGGTGGCAGTGA-3’.

3.2.8 Adenovirus construction and expansion

3.2.8.1 Rho A construction A myc tag was added to the 5’ ends of RhoA WT, CA, DN cDNAs (allelic pack from Upstate) using polymerase chain reaction (PCR). The inserts and the

135 pVAX vector were extracted by phenol/chloroform, digested, and subsequently purified using Qiaquick gel extraction column. The inserts were cloned into the pVAX vector, transformed into competent E.coli XL-1 blue cells, and plated. Colonies were screened for insert sequence and orientation. Colonies correctly expressing the insert were selected, amplified and purified. Next, Rho-pVAX vectors containing the insert were cloned into the Track vector containing GFP. Adenovirus expressing RhoA WT, CA and DN were constructed using the Ad-Easy1 system from Stratagene. RhoA WT, CA, DN-Track plasmids were linearized with PmeI, then electroporated into BJ5183-AD-1 bacteria containing the adenoviral backbone vector Ad-Easy1 plasmid in 2.0 mm cuvettes using Bio- Rad Gene pulser electroporator at 2500V, 200 Ohms, 25 µFD. Bacteria were suspended immediately in sterile TB broth, transferred to sterile 1.5 mL eppendorf tubes and placed on shaker for 1 h at 370C. The suspension was then spread on LB agar plates containing kanamycin and incubated overnight at 37oC. Colonies were screened for inserts by first performing mini-preps, isolating the plasmid DNA using DNA extraction kit, digesting with enzyme and then running it on agarose gel. Clones exhibiting a similar pattern as Ad-Easy-1 with a slighty shifted band were selected, screened and sequenced. DNA from the top ten potential recombinants was linearized, purified and transfected into the packaging cell line, HEK 293, in 24 well plates to yield GFP- expressing adenovirus particles. Transfection and viral particle production were monitored using GFP expression. Cells producing a large amount of viral particles, show signs of cytopathic effect (CPE), where they have rounded up and detached from the plate. A single CPE was selected and used to infect cells plated in a 24 well plate. Cells were then harvested as crude viral stocks upon exhibiting 100% GFP expression and 50% CPE. These stocks were then plaque purified using SeaPlaque agarose. Cells plated in 6 well plates were infected with a dilution of each stock, the next day, overlaid with 5% SeaPlaque agarose mixed with DMEM and covered with media. Single isolated plaques exhibiting strongest GFP expression and 50% CPE were selected and used to again infect cells in a 24 well plate. Clones with the strongest GFP and 50% CPE were harvested for Western blotting. Samples were probed for myc expression; the adenovirus clone expressing the strongest myc was chosen for amplification.

136

3.2.8.2 Expansion of adenoviruses Adenovirus was expanded in HEK 293 cells. Briefly, HEK 293 cells grown in a 10 cm petri plate to 40% confluency were infected with 5-10 µL of adenovirus stock. When cells began rounding up, they were gently scraped off using a rubber policeman and transferred to a sterile conical tube. The tubes were then centrifuged to pellet the cells, and frozen at -800C. In order to lyse the cells and to release the viral particles, frozen cells were subjected to freeze/thaw cycle three times by first thawing at 370C, freezing in ice-cold bath consisting of methanol mixed with dry ice, then thawing again. Cell membranes were subsequently pelleted by centrifugation. The multiplicity of infection (MOI) was determined, through titration. HEK 293 cells plated in a 24 well plate were infected with several serial dilutions of the virus (1x10-9 to 1x10-2). The MOI is defined as the lowest concentration at which one cell is infected. This was determined by visualizing the dish using an inverted fluorescent microscope.

3.2.9 Drug Treatments Twenty-one day old SC-DRGN co-cultures were induced to myelinate by the addition of ECM (2-4 µL of stock (8-12 mg/mL according to manufacturer’s approximation)) or VC (50 µg/mL). SC-DRGN co-cultures were pretreated with PP2, SU6656, PD169316, CMPD1, H89, Ro 31-8220, NSC23966 or Y-27632 for 30 min then stimulated with either ECM or VC. For longterm experiments, fresh drug and medium were replaced every two days in the cultures.

3.2.10 Statistical Analysis Results are expressed as means  SEM (Standard Error of mean). Statistical comparisons were made by either unpaired Student’s t-test followed by Bonferroni correction, ANOVA Tukey-Kramer or Dunnett’s post hoc test to identify differences between treatments. Chi-squared test was used to determine differences in frequency. P values < 0.05 were considered significant.

137

CHAPTER 4: RESULTS

138

4.1 SRCLIKE KINASES ARE INVOLVED IN PERIPHERAL MYELINATION

4.1.1 Inhibition of SLKs decreases accumulation of several myelin proteins in SCDRGN cocultures in a dosedependent manner To determine whether SLKs play a role in peripheral myelination, 21-d myelination-competent SC-DRGN co-cultures were treated with aninhibitor selective for members of the SLK family, PP2 (0.1-7.5 µM), for 10 days in the presence of vitamin C (VC, (50 µg/mL). VC is required for collagen synthesis and formation of basal lamina to initiate myelination in the cultures. The level of myelin basic protein (MBP) was assayed as a measure of myelin formation. PP2 decreased the accumulation of all four MBP isoforms in a dose-dependent manner (Figure 4.1.1a), without exhibiting any apparent isoform-selectivity. Significant reductions in MBP were observed with concentrations of PP2 greater than 1 µM. In contrast, continuous treatment of cultures with the inactive analog of PP2, PP3 (5 µM), for 10 days did not alter MBP levels (Figure 4.1.1b). These findings were confirmed using SU6656, another selective inhibitor of SLKs (Figure 4.1.1c), which also decreased MBP levels in 10-day myelinating co- cultures. The effect of SU6656 on MBP expression was dose-dependent starting at a 1 µM concentration and with a total inhibition at 240 µM. The effects of PP2 treatment on levels of laminin, the β1 and β4 integrins, two extracellular matrix receptors found on the SC surface that play important roles during SC myelination 75, and the neurofilament medium subunit (NFM) were also examined. The relative levels of these proteins were not consistently altered (Figure 4.1.1a, 4.1.2b). Moreover, laminin deposition assessed by immunofluorescence (data not shown) and Western blotting (Figure 4.1.1a), was not affected in PP2-treated co-cultures. Furthermore, to assess whether chronic SLK inhibition (2.5 µM PP2) altered cell survival, SC nuclei in 10-d myelinating co-cultures were quantified using DAPI. There was neither a significant reduction in the total number of SC nuclei per field (VC = 1499.8 ± 46.8; PP2+VC = 1416.4 ± 70.8 (n=60, two-tailed t test, ns)) nor a significant increase in the number of fragmented nuclei (VC= 2.64 ± 0.73 %; PP2+VC= 3.6 ± 0.57 % (n= 60, two-tailed

139 t test p=0.57, ns)). Thus, the 2.5 µM concentration of PP2 was used for the majority of the experiments.

140

141

Figure 4.1.1 Inhibitors of SLK cause a dose-dependent decrease in MBP accumulation

SC-DRGN co-cultures were exposed to PP2 (0.1-7.5 µM) (a), PP3 (5 µM) (b) or SU6656 (1-1000 nM) (c) for 10 d in the presence of vitamin C (VC). In b, CO represents a control in the absence of VC. Medium and drug were changed every two days. Levels of laminin, NFM, integrins (β1 and β4) and MBP were examined by Western blotting. Actin or ERK served as controls of equal protein loading. Shown are representative autoradiographs of single or duplicate samples. No differences in levels of laminin, NFM or integrins (a) were detected. A dose-dependent decrease in MBP accumulation was clearly seen (a, c) with PP2 and SU6656, while chronic treatment with PP3 (5 µM), an inactive analog of PP2, did not reduce MBP levels.

142

Next, to assess whether the effect of PP2 was reversible, myelinating SC- DRGN co-cultures were treated with PP2 (2.5 M) for several time periods, starting on day 0, corresponding to initiation of myelination by addition of VC), followed by drug removal and recovery for 1-9 days. All groups were harvested on day 10 to determine MAG, MBP and protein zero (P0) levels by Western blotting. Continuous treatment with PP2 for 10 d maximally decreased MBP level (>90%, p0.001) (Figure 4.1.2), while MAG and P0 decreased by ~65% (p0.001 and p0.01, respectively). Interestingly, full recovery of MBP expression was not observed even in cultures treated for only 1 day at the beginning of myelination (30% lower than in VC control, p0.05). When drug treatment extended beyond 3-5 days, the level of expression of all three myelin proteins did not fully recover by day 10. These results suggest that PP2 most significantly affects the synthesis of MBP as compared to P0 or MAG, or perhaps that MBP synthesis is delayed, requiring a longer time to recover.

143

Figure 4.1.2 Recovery of myelin protein levels following removal of PP2 in SC-DRGN co-cultures

Cells were treated with VC, to induce myelination, in the presence of 2.5 µM PP2 on day 0. PP2 was removed on days 1 to 9, and all experimental groups were harvested on day 10. Cell lysates were immunoblotted for the myelin proteins P0, MAG and MBP. Actin served as a control of equal protein loading per well. Top: representative Western blots from one experiment are shown while the graph below indicates the mean ± SEM of protein accumulation, expressed as a percent of control (VC) from three independent experiments performed in duplicate. a, p<0.05, b, p < 0.01 and c, p < 0.001, Dunnett’s post-hoc ANOVA.

144

4.1.2 Fewer and shorter myelinated internodes are formed in the presence of PP2 In order to determine if the number of myelinated internodes was altered by persistent SLK inhibition, we performed immunocytochemical staining of 10 d myelinating cultures in the presence and absence of PP2 (2.5 M). Indeed, the numbers of MBP-, P0- and MAG-positive myelinated internodes were reduced by PP2, however, the internodes formed in the presence of the SLKs inhibitor co- expressed all three myelin proteins (Figure 4.1.3a and b). We next quantified the number of internodes per field in the presence and absence of PP2 and found ~65% fewer internodes (p0.001) present in SLK-inhibited myelinating cultures (Figure 4.1.4c). Moreover, myelinated internodes formed in the presence of PP2 appeared to be shorter than in the control (VC) (Figure 4.1.4). Thus, to determine whether inhibiting SLKs significantly altered the lengths of myelinated segments, we measured internode lengths of MBP-positive fibers in myelinating cultures in the absence or presence of PP2 and quantified their frequency. As shown in figure 4.1.4d, the frequency of myelinated internode lengths formed in myelinating VC controls was normally distributed with a mode of 36 µm - 42 µm and a range up to 73 µm in length, values in keeping with those reported by other groups 228,549,550. PP2 treatment appeared to shift the distribution to the left (Figure 4.1.4d) where twice as many short (< 30 µm), ~20% less medium (30-60 µm) and 5 times fewer large (>60 µm) myelin segments formed (Table 4.1.1). Moreover, abnormally short (< 10 µm) internodes were observed.

145

146

Figure 4.1.3 Chronic PP2-treatment of SC-DRGN co- cultures reduces the number of myelinated internodes.

SC-DRGN co-cultures, treated for 10 days with PP2 (2.5 µM) in the presence and absence of VC, were immunostained for MAG (red) (a), MBP (green in a and red in b), P0 (green) (b) and NF (purple) in b. Also in a, the merged images are the overlays of MAG and MBP. Shown are representative confocal images, scale bar = 50 µm. Myelinated internodes are indicated by white arrowheads. Nuclei are visualized with DAPI staining in blue.

147

148

Figure 4.1.4 Chronic exposure of SC-DRGN cultures to PP2 reduces both the number and the length of myelinated internodes formed.

Myelinating co-cultures untreated and treated with 2.5 µM PP2 for 10 d were immunostained for MBP (red, left panels of a, b), and SC nuclei were stained with DAPI (blue, right panels of a, b). Shown in a are representative confocal images, scale bar = 50 µm. The myelinated internodes were counted in 125 fields and results are illustrated in c. (VC (73.02 ± 2.89) vs. VC + PP2 (26.93 ± 1.45 internodes/field), a, p<0.001, Student’s t-test). The lengths of myelinated internodes were measured as shown by x in b. The distribution frequency of the internode lengths in both VC control (dotted line) and PP2-treated (solid line) is illustrated in d. The lengths of 369 and 598 myelinated internodes were quantified in VC control and PP2-treated cultures, respectivey.

149

Table 4.1.1: Shorter internodes form in the presence of PP2

NUMBER OF INTERNODES

(% of Total)

CATEGORY LENGTH (µm) (X) CONTROL +PP2

Small X ≤ 30 20.06 46.32

Medium 30 < X ≤ 60 73.98 52.67

Large 60 < X 5.96 1

The lengths of 369 myelinated internodes were quantified in 10-day control (VC) and 598 internodes from PP2-treated cultures. A Chi-squared test for frequency was performed to determine if short-, medium- or large-sized internodes formed with significantly more frequency than another, p<0.001.

150

4.1.3 Myelin formed in the presence of PP2 appears normal at the ultrastructural level with clustering of sodium channels and caspr in axonal domains Evidence from the CNS 444 suggest that SLKs may potentially be involved in compaction and/or maintenance of myelin in the PNS. Myelin compaction was assessed using electron microscopy in 10-d myelinating co-cultures maintained in the presence and absence of PP2 (2.5 µM). Figure 4.1.5 depicts the compacted multilamellar structure of myelin in control (panel a) and PP2-treated cultures (panel b). At higher magnification (panels to the right), the periodicity of compact myelin in control and PP2-treated cultures was comparable. However, the PP2-treated myelinated fibers appear to be hypomyelinated (g-ratio: VC control: 0.680 ± 0.019 (22 fibers) vs. PP2-treated: 0.782 ± 0.026 (25 fibers), p = 0.0034, Student’s t-test).

The unique architecture of myelinated axons is comprised of several functional domains: the node of Ranvier, the paranode, the juxtaparanode and the internode 627. The selective concentration of specific proteins in each region 27,528 facilitates both the conduction of action potentials along the myelinated axon and the maintenance of myelin. Thus, to assess whether these myelinated fiber domains formed in the presence of PP2 are normal, we used an indirect measure by examining the enrichment of nodal and paranodal proteins to these regions using anti-pan sodium channel and -caspr antibodies, respectively. Both sodium channel and caspr proteins appear to form clusters in 10-d PP2-treated cultures (Figure 4.1.5h, j), as compared with VC controls (Figure 4.1.5g, i), suggesting normal formation of nodes and paranodes. However, subtle aberrations cannot be detected using this approach.

151

152

Figure 4.1.5 Chronic SLK inhibition (PP2 treatment) does not affect clustering of either sodium channels or caspr and myelin is normally compacted.

SC-DRGN co-cultures untreated or treated with 2.5 µM PP2 in the presence of VC were harvested for electron microscopy (a, b) or immunostaining (c-n) at 10 d. Representative electron micrographs of myelinated axons in untreated (a) and drug-treated (b) cultures at low magnification are shown in the left panels of a (scale bar = 500 nm) and b (scale bar = 2 µm), and the multilamellar compacted nature of the formed myelin can be seen at higher magnification (scale bars = 200 nm), on the right. Myelin, axon and Schwann cell are denoted by M, A and SC, respectively. c, myelinated fibers were stained for MBP (red, c-f and k - n) and sodium channel or caspr clustering were visualized using anti- pan-sodium channel (g, h, k, l) or anti-caspr (i, j, m, n) antibodies (green), respectively. Scale bars = 5 µm.

153

4.1.4 Krox20 protein expression is decreased by PP2 treatment: Roles for Fyn and Lyn Expression of the transcription factor Krox-20/Egr-2 (Early grown response-2) is critical for the initiation and maintenance of myelination by SCs, as illustrated by hypomyelinating phenotypes of knockout mice 528. We therefore examined whether SLKs modulate Krox-20 protein levels, using an immunocytochemical approach, as Krox-20 is expressed selectively by SCs and not by DRGNs 166,469,695. Western blotting was not used since we found it masked any changes in Krox-20 upregulation due to background expression. As expected, under myelinating conditions VC increased the number of Krox-20- expressing SCs by 10-fold (as compared to non-myelinating controls, p0.001) at 3 days (Figure 4.1.6). Continuous treatment with PP2 reduced the number of Krox-20-positive SCs by half, providing evidence for a role for SLKs in the early stages of myelination.

We next examined by Western blotting the endogenous levels of three different SLKs, Src, Fyn and Lyn, which are reportedly expressed in SCs. Although all three SLKs were detected in cellular extracts from 10 d myelinating SC-DRGN co-cultures, Lyn and Fyn were more abundant (Figure 4.1.7a). To determine whether a particular SLK member was important in the modulation of Krox-20 expression, Alexafluor-tagged siRNAs selective for Fyn (fyn-1 and -2) and Lyn were used to knockdown protein levels (Figure 4.1.7b-f). In our hands only SC and not DRGNs are transfected with siRNA. Furthermore, the transfection efficiency in this very dense co-culture system is very difficult to estimate accurately, which is reflected in the small reduction in total Fyn and Lyn protein levels estimated by Western blotting (Figures 4.1.7b and 4.1.7c, respectively). Therefore, it was necessary to quantify the number of Krox-20- positive SCs as a percentage of transfected SCs by immunocytochemistry (Figures 4.1.7d). Both anti-Fyn (p0.001) and anti-Lyn (p0.01) siRNAs significantly reduced the number of SCs expressing Krox-20 as compared to the negative control siRNA. A greater reduction in Krox-20-positive SCs was observed with anti-Fyn siRNA (~two-thirds) than anti-Lyn siRNA (~one-half) (Figure 4.1.7e and f).

154

155

Figure 4.1.6 SLK-regulated Krox-20 protein levels were reduced by PP2 treatment.

SC-DRGNs were left untreated (CO) or were treated with VC alone (VC) or with VC and 2.5 µM PP2 (+PP2). After 3 days, cultures were immunolabeled with anti-Krox-20 antibody (red, left panels) and DAPI nuclear staining (right panels). Scale bar = 50 µm. Krox-20-positive nuclei in 30-60 fields were counted and expressed as a percentage of total SCs as shown in the graph at the bottom of the figure. a, p<0.001, Tukey-Kramer post-hoc ANOVA.

156

157

Figure 4.1.7 Fyn and Lyn regulate Krox-20 protein expression.

Fyn, Lyn and Src are expressed in SC-DRGN co-cultures. a, Relative levels of Fyn, Lyn and Src proteins were assessed in equal amounts of lysate (25 µg) from 10-d myelinating SC-DRGN co-cultures. b-f, SC-DRGNs were transfected with either control siGLO, anti-Fyn (alexa-488-tagged-fyn1 or -fyn2) or anti-lyn siRNAs for 18h. Co-cultures were harvested 48h after transfection and lysates immunoblotted for Fyn (b), Lyn (c) and actin. Total Fyn protein levels were decreased using both anti-fyn1 and -fyn2 siRNAs (b), while a SMARTPool of siRNAs reduced Lyn protein levels (c). d-f, 24 h after transfection, co-cultures were stimulated to myelinate with VC for 48 h and subsequently immunostained for Krox-20. Krox-20-positive Fyn- or Lyn siRNA-transfected SCs were counted in 30 fields and expressed as a percentage of transfected SCs in e and f, respectively. Scale bars = 50 µm (image) or 10 µm (inset) a, p<0.01 and b, p<0.001, Tukey- Kramer post-hoc ANOVA.

158

4.1.5 SLK activation lies upstream of Akt, ERK and p38 MAPK as PP2 reduces the activation of these kinases by extracellular matrix (ECM) Previous studies in our laboratory provided evidence that MAPK p38 plays a critical role in mediating the morphological changes in SCs corresponding to early stages of myelinogenesis (axonal alignment and basal lamina deposition) induced by both VC and the extracellular matrix component, laminin 695. Addition of exogenous laminin to SC-DRGN co-cultures resulted in a time-dependent phosphorylation of p38 MAPK 213 . For our experiments, cultures were stimulated with Matrigel Matrix (ECM), which is a solubilized basement membrane preparation extracted from EHS mouse sarcoma, a tumor rich in ECM proteins. Its major component is laminin, followed by collagen IV, heparan sulfate proteoglycans, and entactin. As expected, stimulation of SC-DRGN cultures with the ECM extract resulted in robust p38 MAPK phosphorylation (Figure 4.1.8). Interestingly, pretreatment of SC-DRGN cultures for 30 min with 10 µM PP2 blocked this phosphorylation. Furthermore, Akt and ERK are phosphorylated in SCs during differentiation and proliferation, respectively 213. As such, using phospho-specific antibodies, we also assessed whether molecules of the basal lamina stimulate ERK and Akt phosphorylation, and whether SLKs are upstream activators of these two kinases. Indeed, both ERK and Akt kinases were phosphorylated following ECM addition, and phosphorylation was blocked by PP2. Therefore, these results provide further evidence for a function(s) of SLKs in early stages of peripheral myelination, upstream of Akt, ERK and p38 kinases.

159

Figure 4.1.8 PP2 blocks ECM-induced phosphorylation of Akt, ERK and p38 MAPK.

SC-DRGN co-cultures were pretreated with 10 µM PP2 (30 min) prior to stimulation with ECM (30 min). Phosphorylation states of Akt, ERK 1/2 and p38 MAPK were assessed by Western blot analysis. Actin was used as a loading control. Shown are representative blots from one of three independent experiments performed in duplicate.

160

4.2 p38 MODULATES TRANSCRIPTION FACTORS KROX20, SCIP, SOX10 AND CREB TO REGULATE SCHWANN CELL

DIFFERENTIATION AND PERIPHERAL MYELINATION

4.2.1 p38 alpha is the predominant isoform expressed by SCs The family of p38 MAPK consists of four different isoforms: α, β, δ and γ. Therefore, we determined their relative levels of expression in enriched SC or purified DRGN cultures by Western blotting. The results show that mature (4 week old) DRGNs express all four isoforms while SCs from confluent cultures express ,  and  but not the p38 β proteins (Figure 4.2.1a). Moreover, the α isoform was most abundantly expressed by both SCs and DRGNs. The expression of p38α was confirmed by immunocytochemical staining performed on 21-day confluent myelination-competent SC-DRGN co-cultures, which revealed a predominant nuclear localization in SCs (Figure 4.2.1b).

4.2.2 PD169316treatment arrests SCs at a promyelinating stage As previously reported, myelination in myelin-competent SC-DRGN co- cultures was abrogated with PD169316 treatment, a selective inhibitor of p38 α and β isoforms 438,500. We used electron microscopy to ultrastructurally assess the stage of development at which SCs are arrested by inhibiting p38 in 10 d myelinating co-cultures (treated with 2.5 µM PD169316 from day 0). A normal myelinated axon is shown in cross-section in Figure 4.2.2a, and the multilamellar nature of myelin is shown at higher magnification in the inset. Continuous PD169316 treatment of cultures under myelinating conditions (presence of VC (50 µg/mL)) appeared to arrest SCs at the pro-myelinating stage of development. Thus, SCs formed the required 1:1 association with the axon, characteristic of the stage following radial sorting of axons, but they did not proceed further in the axonal wrapping (Figure 4.2.2b).

161

Figure 4.2.1 p38 MAPK isoforms expressed by SCs.

Protein extracts from pure SCs and 4 week DRGNs were immunoblotted using anti-p38α, β, δ and γ-selective antibodies, a. Total Akt was used as a loading control. b, 21 day SC-DRGN co- cultures were immunostained using an anti-p38α MAPK antibody (red), while SC nuclei were visualized using DAPI (blue). Scale bar = 50 µm.

162

163

Figure 4.2.2 PD169316 arrests SCs at a pro-myelinating stage.

Electron micrographs of 10 d myelinating SC-DRGN co-cultures treated with PD169316 (5 µM PD169316). a, Control and b, PD169316-treated myelinating coculture. Shown in a is the mutli- lamellar structure of myelin at higher magnification Scale bars for low magnification in a = 500 nm, and high magnification in the inset of a = 200nm. As seen in b, the cytoplasmic projection of the SC unable to complete its wrap (black arrow). Scale bar = 500nm. Myelin, axon, and Schwann cell are denoted by M, A and SC, respectively.

164

4.2.3 p38 regulates Krox20 expression in myelinating SCs In our previous study, treatment of SC-DRGN co-cultures with the p38 α/β inhibitors PD169316 and SB203580 reduced MBP, P0 and MAG mRNA expression, associated with the onset of myelination 213. Since expression of these myelin genes are primarily controlled by Krox-20 213, we examined whether p38 MAPK regulates expression of this master transcription factor. The number of Krox-20 positive SCs was determined by immunofluorescence in 3-d myelinating co-cultures grown in the absence or presence of VC (50 µg/mL) and p38 inhibitor. DAPI nuclear staining was used to estimate the total numbers of SCs in the cultures. Addition of VC to DRGN-SC co-cultures competent to myelinate increased the total number of Krox-20-positive SCs nuclei 10 fold from non-myelinating controls (10% of total SCs) (Figure 4.2.3f), while PD169316 (5 µM) co-treatment prevented this increase (p0.001). In addition, treatment of 2-d myelinating cultures with the same concentration of PD169316 reduced Krox-20 mRNA levels by half (as compared to VC-myelinating cultures) (p0.05; Figure 4.2.4a), as determined by qRT-PCR. Furthermore, the p38 inhibitor also decreased the transcription of several myelin proteins, including periaxin (80%, p0.001), MAG (70%, p0.001), CX32 (50%, p0.01) and MPZ (30%, p0.05) (Figure 4.2.4b), in keeping with our previous findings 475.

Furthermore, as the α/β-selective p38 MAPK inhibitor, PD169316, significantly reduced both Krox-20 mRNA and protein expression in myelinating cultures, and since α was found to be the most predominant isoform expressed by SCs, we assessed whether this isoform indeed regulates Krox-20 expression. A small interfering RNA was used to knock-down p38α (sip38α) levels. Cultures were transfected with 80 nM siRNA and analyzed 72 h later for the expression of p38α and Krox-20. Western blotting showed that p38α protein levels were reduced by 70% in sip38α-transfected cultures (Figure 4.2.3h). Three-day myelinating SC-DRGN co-cultures transfected with anti-p38α siRNA were immunolabeled for Krox-20 and nuclei visualized with DAPI staining. The number of transfected SCs expressing Krox-20 were counted. The total number of control siRNA (siCO)-transfected SCs expressing Krox-20 was ~5.5% (calculated as a percentage of the total number of SCs transfected per field) (Figure 4.2.3d, e, g). p38α knockdown reduced the number of Krox-20 positive transfected-SCs by half

165

(~3% of the total number of transfected SCs express Krox-20+), as compared to siCO-transfected SCs (p0.05).

166

167

Figure 4.2.3 p38 regulates Krox-20 expression.

(a-c) 21 day SC-DRGN co-cultures treated with PD169316 (5 µM) and induced to myelinate with VC, were stained for Krox-20 (red) and DAPI after 3 days. Representative images are shown from a, control (no VC), b, VC and c, PD169316 in the presence of VC. Scale bar = 50 µm. Krox-20 positive SCs per field were counted and the mean ± SEM as a percentage of total cells per field is shown in f. 30-60 fields per condition were quantified. c, p < 0.001, Tukey-Kramer post-hoc ANOVA. (d, e) SC-DRGN co-cultures were transfected with alexa-563nm-tagged control siRNA alone (siCO) (d), or siCO plus p38α (sip38α) (e) for 18 h. 24 h later, cells were induced to myelinate by adding VC for 48 h, then immunostained for both Krox-20 (green) and DAPI. Shown are representative confocal images, scale bars = 10 µm (inset) and 50 µm. Quantifications of Krox-20-positive transfected SCs are illustrated in g. h, Western blotting was carried out on lysates from MSC-80 cells transfected with anti-p38α siRNA for 48 h. Shown are representative immunoblots for p38 α and total Akt from an experiment performed in duplicate. Akt was used as a loading control. a, p<0.05, Student t-test in g and h.

168

Figure 4.2.4 Inhibiting p38 MAPK reduces Krox-20, SCIP and Sox10 transcription but does not alter Sox2.

qRT-PCR was used to assess the mRNA expression levels of transcription factors (a) Krox-20, SCIP, Sox2 and Sox10, and the myelin proteins (b) MAG, Cx32, periaxin and Mpz, in 21 d SC- DRGN co-cultures treated for 48 h with VC and VC+PD169316. Data presented are mean ± SEM of three independent experiments. a, p<0.05, b, p<0.01, and c, p<0.001, one way t-test.

169

4.2.3 p38 MAPK regulates SCIP Next, we evaluated the effect of p38 inhibition on SCIP levels in myelinating SC-DRGN co-cultures. Expression of this POU transcription factor is linked to promyelinating SCs, and has been shown to regulate both Krox-20 induction and peripheral myelination 213. Using immunofluorescence, we found that ~40% of total SCs in control 21-d myelination-competent cultures expressed SCIP protein (Figure 4.2.5a). Myelination induced by three-day treatment with VC increased the number of SCs expressing SCIP to 60% of total, while PD169316 (5 µM) treatment attenuated expression to 50% of total SCs (as compared with VC, p0.05) (Figure 4.2.5d). Interestingly, a more potent effect of p38 inhibition on SCIP was detected at the mRNA level, where PD169316 (5 µM) treatment reduced transcription by ~80% (p0.001) in 2-day myelinating co-cultures (Figure 4.2.4a). Therefore, it is possible that our methodological approach to estimate the expression levels of SCIP by counting the number of SCIP+ nuclei under experimental conditions does not fully reflect total protein. Future experiments will be designed to calculate both the number and intensity of immunofluorescently labeled nuclei.

170

171

Figure 4.2.5 Inhibiting p38 decreases the number of SCIP+ SCs.

SCIP expression by SCs was determined using immunocytochemistry on 21 day SC-DRGN co-cultures treated with PD169316 (5 µM) and induced to myelinate with VC for 3 d. Shown are representative images from a, control (no VC), b, VC and c, PD169316 in the presence of VC. Scale bar = 50 µm. The number of SCs, brightly positive for SCIP, per field was counted and mean ± SEM is shown in the graph, d, expressed as a percentage of total cells per field. 30-60 fields per condition were quantified. a, p<0.05, Tukey-Kramer post-hoc ANOVA.

172

4.2.4 p38 MAPK regulates transcription of Sox10 during myelination

Sox10, a member of the HMG transcription factor family, cooperatively interacts with Krox-20 to regulate myelin protein gene expression in SCs, such as Mpz 799. Moreover, Sox10 can partner with two other transcription factors, Brn2 and SCIP, to modulate Krox-20 expression 382. Since Sox10 is expressed by cells of the neural crest lineage, such as DRGNs and SCs, we examined the effect of p38 inhibition on Sox10 protein expression using immunofluorescence. 10 d myelinating co-cultures grown in the presence and absence of PD169316 (5 µM) were co-immunolabelled for Sox10 and Krox-20. PD169316 (Figure 4.2.6c) decreased Krox-20 levels, relative to myelinating (VC) cultures (Figure 4.2.6b). In terms of Sox10, the intensity of Sox10 immunofluorescence in non-myelinating control (no VC) and myelinating (VC) co-cultures were intense and similar. However, the intensity of Sox10 staining in cultures grown in the presence of PD169316 (5 µM) (Figure 4.2.6c) appears half as intense as either non-VC control or VC cultures. These results indicate p38 MAPK regulates the protein levels of Sox10. Moreover, we evaluated the effect of p38 inhibition on Sox10 mRNA levels. As such, qRT-PCR revealed a 40% reduction in Sox10 mRNA following PD169316-treatment of two-day myelinating cultures (p0.05, Figure 4.2.4a). Collectively, these results indicate Sox10 gene expression is regulated by p38 MAPK during myelination.

173

Figure 4.2.6 Inhibiting p38 reduces Sox10 protein levels.

Sox10 expression by SCs was determined using immunocytochemistry on 21 day SC-DRGN co-cultures treated with PD169316 (5 µM) and induced to myelinate with VC for 10 d. Cultures were immunostained with Sox10 (red) and Krox-20 (green). Nuclei were visualized using DAPI (blue). The merged images represent the co-expression of Sox10 and Krox-20. Inhibition of p38 MAPK appears to reduce the intensity of Sox10 (red) staining and the number of Krox-20 (green)-expressing SCs. Shown are representative images from a, control (no VC), b, VC and c, PD169316 in the presence of VC. Scale bar = 50 µm.

174

4.2.5 p38 MAPK regulates p27kip1 expression Terminal differentiation of an immature SC to a myelinating phenotype, involves the exit from the cell cycle, coupled with the expression of cell cycle inhibitors, including members of the cip/kip family such as p27kip1 238. Thus, we examined the effect of p38 inhibition on the expression of p27kip1 in VC-induced 3-day myelinating co-cultures using immunofluorescence (Figure 4.2.7). We concurrently assessed the co-expression of Krox 20 in these cultures, using it as a marker for myelinating SCs. As such, we determined the total number of SCs expressing p27kip1 or Krox-20 alone, and the number of SCs co-expressing p27kip1 and Krox-20. In myelinating (VC) controls, the total number of SCs expressing p27kip1 and Krox-20 were found to be 9 and 4% of total SCs per field, respectively. In PD169316–treated myelinating cultures, the total number of SCs expressing p27kip1 and Krox-20 were half as many as in myelinating (VC) cultures (total number of p27kip1+ and Krox-20+ SCs in PD169316+VC (% of total): 5 and 1%, respectively) (p<0.05) (Figure 4.2.7d-e, f).

Furthermore, the addition of extracellular matrix extracts (ECM) to SC- DRGN co-cultures potently induces myelination, as shown by Fragoso et al, 2003 514. Accordingly, relative to control, ECM addition increased the total number of p27kip1+ and Krox-20+-SCs by 10 and 5 fold, respectively (p<0.001). This increase was completely prevented by PD169316 treatment (Figure 4.2.7g) (p<0.001). Notably, in both VC- and ECM-induced myelinating co-cultures, we found only 50% of the p27kip1+ SCs co-expressing Krox-20, suggesting a temporal delay in the acquisition of the myelinating phenotype (Krox-20+) and p27kip1 is an earlier marker of myelinating SCs.

175

176

Figure 4.2.7 PD169316 reduces VC and ECM-induced p27kip1 expression.

21 d SC-DRGN co-cultures were induced to myelinate using ECM (b, c) or VC (d and e) for 3 d in the presence and absence of PD169316 (5µM), and double immunolabeled for p27kip1 (green in a-e) and Krox-20 (red in a-e). SC nuclei were visualized with DAPI (blue). Representative confocal images are shown. Scale bars = 50 µm. The number of SCs expressing p27kip1, Krox-20 and co- expressing p27kip1 and Krox-20 were determined and expressed as a percent of total SCs per field in the graphs in f and g. Data are from one to four independent experiments performed in triplicate. At least 30 images (taken using a 40 x objective) per treatment were quantified per experiment. Statistical significance was determined using a t-test with Bonferonni correction (ECM stimulated group was compared to control (unstimulated), PD169316-treated groups were compared to VC- or ECM stimulated groups): a, p<0.05, c, p<0.001.

177

4.2.6 p38 MAPK does not regulate Krox20independent periaxin gene expression Periaxin is a cytoskeleton-associated protein expressed by myelinating SCs, which has been shown to stabilize myelin sheaths 213. Over the course of development, periaxin becomes redistributed from the nucleus to the adaxonal membranes, and finally, with progression of myelination, it becomes localized to the abaxonal membrane and non-compacted regions of myelin postnatally 247,248. A dual mechanism of periaxin gene induction has been proposed: whereby an earlier, Krox-20-independent activation step takes place, where periaxin+ SCs co- express p27kip1, followed by a later, Krox-20-dependent step, which amplifies periaxin expression, such that SCs are periaxin/p27kip1/Krox-20+ at this stage 612,632. As we have thus far established the critical involvement of p38 MAPK in Krox-20-dependent myelin gene regulation, we wanted to explore its possible involvement in Krox-20-independent mechanisms, using periaxin gene expression as a marker. Thus, we evaluated the effect of p38 inhibition on Krox- 20-independent and Krox-20-dependent gene regulation by triple labeling of 4-d myelinating SC-DRGN co-cultures grown in the presence and absence of the PD169316 (5 µM) p38 inhibitor, with periaxin, p27kip1 and P0. Since periaxin+ myelinating SCs co-express the cell cycle inhibitor p27kip1 from early on, we evaluated the effect of PD169316 on the total number of SCs co-expressing both periaxin and p27kip1. This is what we have denoted as ‘Krox-20-independent’ gene expression. In VC-cells (control), ~85% of p27kip1+ SCs co-expressed periaxin (Figure 4.2.8a, c). PD169316 treatment did not significantly reduce this population of cells (Figure 4.2.8b, c), suggesting p38 does not modulate ‘Krox- 20-independent’ mechanisms of periaxin gene expression.

We next evaluated the effect of PD169316 on ‘Krox-20-dependent’ gene regulation. P0 was selected as an indicator of Krox-20 activity, since it is both directly regulated by Krox-20 and it appears prior to other myelin proteins regulated by Krox-20. Using these established criteria, we first characterized the number of p27kip1+ SCs co-expressing P0. In VC control cultures, ~40% of total p27kip1+ SCs co-expressed P0 (Figure 4.2.8a, c). This value is in keeping with our results presented in Figure 4.2.7, where ~50% of p27kip1+ SCs co-expressed Krox-20. As expected, PD169316-treatment reduced the number of p27kip1/P0+

178

SCs (Figure 4.2.8b, c) (p<0.05). Finally, we evaluated the number of SCs co- expressing all three proteins (p27kip1/periaxin/P0). In VC myelinating control, the total number of p27kip1+ SCs expressing periaxin and P0 was ~30%. The number of periaxin/p27kip1+ co-expressing P0 (p27kip1/periaxin/P0+ SCs) was also significantly reduced by p38 inhibition (~25% of p27kip1+ SCs)(p0.05), reiterating the role of p38 MAPK in Krox-20 gene regulation.

We also assessed the effect of PD169316 treatment on periaxin mRNA expression in 2-day myelinating co-cultures. qRT-PCR results demonstrated a 75% reduction in periaxin gene expression in PD169316 treated cultures (p0.001, Figure 4.2.4b). This likely corresponds to Krox-20 dependent amplification of periaxin gene expression.

Collectively, these results indicate a lesser role for p38 MAPK in modulating Krox-20-independent mechanisms, as evaluated by periaxin gene expression.

179

180

Figure 4.2.8 PD169316 reduces the number of periaxin+ SCs.

4-d myelinating SC-DRGN co-cultures in the presence and absence of PD169316 were triple-immunostained for periaxin (purple), P0 (green) and p27kip1 (red) in a and b. Shown are representative confocal images. Scale bar = 50 µm. 29-33 images taken with a 40 x objective. The number of p27kip1 positive SCs co- expressing P0, periaxin and both P0/periaxin were counted and expressed as a percentage of total p27kip1-positive SCs in c. Statistical significance between VC and VC+PD169316 was determined using Student t-test: a, p<0.05 .

181

4.2.7 p38 MAPK does not regulate Sox2 expression In the presence of negative regulators of myelination, fully differentiated myelinating SCs can dedifferentiate to an immature proliferating stage (reviewed by Jessen et al 515). As we have demonstrated thus far, PD169316 treatment arrested SC differentiation (Krox-20, p27kip1 expression). So we next examined whether these arrested SCs were consequently reverted to an immature state. The HMG transcription factor Sox2, a negative regulator of PNS myelination, is expressed by undifferentiated and immature SCs; it induces the expression of genes associated with proliferation, including Cyclin D 330. Moreover, Krox-20 has been shown to suppress Sox2 expression while promoting SC differentiation and myelination 378. Thus, we evaluated the expression of Sox2 by SCs in ECM- induced myelinating SC-DRGN co-cultures in the presence and absence of PD169316 using immunocytochemistry, while concurrently staining for p27kip1 as a marker for terminally differentiated myelinating SCs. As observed in Figure 4.2.8, the intensity of Sox2 immunofluorescence is higher in control p27kip1- negative non-myelinating cultures (a) than in p27kip1-positive ECM-induced 3-d myelinating cultures (b). Interestingly, PD169316 treatment did not enhance Sox2 expression while abrogating p27kip1 expression (c), as compared to myelinating cultures in b. These results were substantiated by Western blotting. In 10-d VC-induced myelinating SC-DRGN co-cultures, VC decreased Sox2 protein levels by 50% (of control) while increasing the accumulation of the myelin protein, MBP, 600% (of control) (Figure 4.2.8d and e). As expected, PD169316 (5 µM)-treatment completely blocked VC-induced MBP protein accumulation, but consequently did not increase Sox2 protein levels (50% of control) to those found in non-myelinating controls (Figure 4.2.8e). Furthermore, Sox2 mRNA expression in myelinating co-cultures treated for 48 h was unaltered by PD169316 (Figure 4.2.4a). Collectively, these results indicate that p38 MAPK does not directly promote SC differentiation and myelination by modulating expression of the negative regulator, Sox2 expression.

182

183

Figure 4.2.9 p38 MAPK does not regulate Sox2 expression.

Levels of Sox2, MBP and p27kip1 were assessed in myelinating SC-DRGN co-cultures in the presence and absence of PD169316 (5 µM) using immunocytochemistry (a-c) and immunoblotting (d). In a-c, co-cultures were stimulated to myelinate with ECM for 3 days in the absence (b) or presence of PD169316 (5 µM) (c), and subsequently immunolabeled for Sox2 (red) and p27kip1 (green). In d, VC-stimulated co-cultures treated with PD169316 (5 µM), were harvested after 10 days and samples immunoblotted. Representative autoradiographs are shown in d, while the means ± SEMs for each protein, as determined using densitometry, are illustrated in the graphs in (e). Results shown are from an experiment performed in duplicate.

184

4.2.8 Krox20 overexpression reverses the effect of p38 inhibitor on MAG expression We have thus far demonstrated that Krox-20 is a primary target of p38 during peripheral myelination. Moreover, previous studies have shown that overexpression of Krox-20 is sufficient to turn on myelin gene expression in a heterologous system 513. As such, we attempted to determine whether the inhibitory effects of PD169316 on myelin gene expression could be reversed by overexpressing wild type Krox-20 using a GFP-tagged adenovirus (Krox-20Ad) in SC-DRGN co-cultures. Cultures were infected with the virus 12 h prior to PD169316 treatment and subsequent addition of VC to initiate myelination. We assessed myelination 3 d later by examining the expression of an early marker, MAG, using immunofluorescence. Control uninfected myelinating cultures expressed Krox-20 and MAG (MAG+ SCs: ~3% of total SCs) (Figure 4.2.10a). Five µM PD169316 blocked Krox-20 expression (Figure 4.2.10b, middle column) and MAG (~0.3% of total SCs, Figure 4.2.9b, f), as expected. In infected cultures, we assessed the total number of infected GFP+SCs. We then characterized the number of infected SCs (GFP+) expressing MAG, as myelinating SCs. As such, Krox-20 overexpression induced ~93% of infected SCs to express MAG (Figure 4.2.10c, g). We also maintained these K20Ad infected SCs under myelinating conditions (presence of VC) and assessed whether it would potentiate the effect of K20Ad on the number of MAG+/GFP+ SCs. VC did not significantly increase the number of MAG+ cells beyond the K20Ad alone (Figure 4.2.10d, g) However, infection of PD169316 (5 µM)-treated SC-DRGN co-cultures with K20Ad, induced ~66% of SCs to myelinate (Figure 4.2.10e, g). Hence, these results suggest that overexpressing Krox-20 can reverse the effects of p38 MAPK inhibition on myelin proteins gene expression but not totally.

185

186

Figure 4.2.10 Overexpression of Krox-20 reverses the inhibitory effect of PD169316 on MAG expression.

Co-cultures infected with Krox-20 adenovirus (K20Ad) (c-e) for 12 h were then treated with 5 µM PD169316 (b, e). Myelination was induced by addition of VC (a, b, d, e). (c) K20 Ad alone, (d) K20 Ad + VC, (e) K20 Ad + PD169316 + VC. Shown are representative images for co-cultures immunostained for MAG (red), Krox-20 (a, b in green) and GFP (c-e in green). Nuclei were visualized using DAPI (blue). Scale bars = 50 µm. The number of MAG positive SCs per field were counted and expressed as a percentage of total cells (a, b) or as a percentage of GFP positive infected SCs (c-e) in f and g, respectively. Shown is data from one or two independent experiments performed in duplicate.

187

4.2.9 MAPKactivated proten kiase2a (MK2) is a downstream effector of p38, regulating Krox20 expression and myelin protein accumulation p38 MAPK signals through a number of downstream kinases including MK2, the MSKs, MK3, MNKs and RSKs. MK2, the best characterized substrate, is implicated in cytokine production, endocytosis, cell migration, transcriptional regulation, cell cycle control, differentiation including OLGs chromatin and cytoskeletal remodeling 514,515. More recently, MK2 has been implicated in cellular differentiation 584. p38 MAPK activates MK2 via multiple proline-directed phosphorylation on multiple amino acid residues (Ben-Levy et al. 1995). In turn, MK2 phosphorylates a number of substrates, including the heat shock protein-27 (hsp-27) 272. We previously demonstrated, using SC-DRGN co-cultures, a time- dependent phosphorylation of hsp-27 following the addition of exogenous laminin 149,657. HSP-27 is a downstream substrate of MK2. Therefore, in this study we wanted to assess whether MK2 is an essential effector of p38 in regulating peripheral myelination. We used an MK2-specific inhibitor (CMPD1), which binds the substrate-binding pocket of p38α, physically hindering the phosphorylation of MK2 213. CMPD1 dose-dependently decreased MBP and MAG protein levels in 10-d myelinating cultures (Figure 4.2.11a), without altering neurofilament levels (NFM). Moreover, 2.5 µM CMPD1 reduced the number of SCs expressing Krox- 20 in 3-d myelinating co-cultures (Figure 4.2.11b). Together, these data demonstrate that MK2 is an important effector of p38, regulating Krox-20 expression and consequently peripheral myelination.

188

189

Figure 4.2.11 MK2 regulates Krox-20 and myelin genes expression.

VC-induced myelinating SC-DRGN co-cultures were treated with an specific inhibitor of MK2, CMPD1 (0.5 – 2.5 µM), for 10 days then harvested for Western blotting, a. The levels of myelin proteins MBP and MAG were quantified and are depicted in the graph as a percentage of control. 3 day VC-induced myelinating SC-DRGN co-cultures in the presence (c) and absence (b) of CMPD1 (2.5 µM) were immunostained for Krox20 (green) and nuclei stained using DAPI. Images were taken using a 40 x objective. Representative images are shown. Scale bars = 50 µm.

190

4.2.10 CREB and p38 MAPK phosphorylation induced by ECM is blocked by p38 inhibitor and an MSK1 inhibitor Another potential effector of p38 is CREB, Cre-Responsive Element Binding Protein, a constitutively expressed transcription factor, whose activity is regulated through phosphoryation at Ser133 156. CREB can be phosphorylated by another group of p38 substrates, the MSKs, and the RSKs 333. In OLGs, CREB phosphorylation was linked to differentiation 591, and CREB form an immunoprecipitable complex with p38 and MSK1 53. To assess whether CREB activation is regulated by p38 at the onset of myelination, SC-DRGN co-cultures were stimulated with ECM for 30 min and immunoblotted for phosphorylated CREB. Indeed, ECM robustly induced CREB phosphorylation (~200% of control) (Figure 4.2.12a). This was completely blocked by 10 µM PD169316 (Figure 4.2.11a). Thus, CREB is a downstream effector of p38 MAPK. Interestingly, ECM also induced phosphorylation of p38 MAPK (400% of control), which was also reduced by PD169316 (300% of control) (Figure 4.2.11a). These results could be explained by the recent observation that p38 α and β homologues can undergo non-canonical autophosphorylation at Tyr323, leading to auto-activation 272. Their activation is regulated by phosphorylation of Thr180 and Tyr182 by the upstream kinases MKK3 and MKK6. Thus, PD169316 is potentially inhibiting p38 autophosphorylation, and may represent another level of regulation in SCs and peripheral myelination.

Next, to determine if p38 MAPK modulates CREB activity through MK2, we assessed the phosphorylation of CREB in CMPD1-treated cultures. Pretreatment of SC-DRGNs with CMPD1 (5 µM) did not reduce phosphorylated CREB levels as induced by ECM, as (Figure 4.2.11b). In contrast, H89, a general PKA/MSK-1 inhibitor, decreased ECM-induced CREB phosphorylation at 5 (p0.01) and 10 µM (p0.01). We also used an MSK-1-selective inhibitor, R0 31- 8220, and found a dose-dependent reduction in ECM-induced CREB phosphorylation (Figure 4.2.12d). This substantiated MSK-1 as a downstream effector of p38 MAPK activation. Additionally, we evaluated the effect of MSK-1 inhibition on p27kip1 and Krox-20 expression, as markers for differentiation and myelination. 5 µM H89 completely blocked ECM-induced p27kip1 and Krox-20 expression in 3-day myelinating co-cultures, as assessed using

191 immunofluorescence (Table 4.2.1) (p<0.001). These results indicate CREB is a downstream target of p38/MSK-1, and not p38/MK2, and it may regulate expression of cAMP-dependent genes critical for myelination including SCIP.

192

193

Figure 4.2.12 p38 MAPK regulates CREB activation.

SC-DRGN co-cultures were pretreated with PD169316 (10 µM) (a), CMPD1 (5 µM) (b), H89 (5 and 10 µM) (c), or Ro 31-8220 (0.1 and 5 µM) 1 h prior to stimulation with ECM for 30 min. Cells were then harvested and subjected to Western blot analysis using anti- phosphorylated-p38 (p-p38) and -phosphorylated-CREB (p- CREB). Total ERK (ERK) or actin were used to correct for equal loading. The levels of p-CREB and p-p38 were quantified and are depicted in the graphs as a percentage of control. Data shown are from one of one or three experiments performed in duplicate. Statistical significance was determined using one-way ANOVA, followed by Tukey-Kramer post-hoc test, a, p<0.05, b, p<0.01, c, p<0.001.

194

TABLE 4.2.1: MSK-1 inhibitor H89 reduced % of p27kip1+, Krox-20+ SCs

SCs expressing

(% of Total)

Treatment p27kip1+ Krox-20+ p

Value

Control (ECM) 4.46 ± 0.33 1.83 ± 0.19 p<0.001

+5 µM H89 0.47 ± 0.24 0.09 ± 0.09 p<0.001

The number of p27kip1 and Krox-20 positive SCs were quantified in at least 30 fields from 3-day control (ECM) and H89-treated SC-DRGN co-cultures. The numbers are expressed as a percentage of total SCs per field. A Student t-test was performed to determine if significantly fewer SCs were p27kip1 or Krox-20 positive in the presence of H89.

195

4.3 RHO GTPASES REGULATE SC MORPHOLOGY IN PERIPHERAL MYELINATION

4.3.1 Altering RhoA activity abrogates myelination of DRGNs by SCs RhoA is the most abundantly expressed isoform in SCs 538. While the roles of downstream effectors of RhoA 682 in peripheral myelination and SC differentiation have been examined, the precise role of RhoA in myelination itself has not been assessed. Hence, we sought to study its involvement in peripheral myelination and to determine whether it regulates the early stages of myelin formation. To this end, we used a genetic approach using GFP-tagged adenoviruses to ectopically express wild type (WT), dominant negative (DN) and constitutively active (CA)-isoforms of RhoA. DN- and CA-RhoA constructs contain a mutation in the GTP exchange (activation) site, preventing GDP/GTP exchange. We first assessed the effect of DN-RhoA. SC-DRGN co-cultures were first infected with control (GFP Ad) and DN-RhoA, following 24 h later by addition of VC to induce myelination. Cultures were maintained for 10 d under these conditions and myelination was assessed by immunostaining for MBP. In Figure 4.3.1a, it is shown that a control GFP-Ad-infected SC is myelinating. Expression of DN-RhoA substantially blocked the formation of MBP+ fibers by SCs, as no MBP+ fibers were GFP+ (Figure 4.3.1a).

We also found that neurons are easily infected by adenovirus. Given RhoA’s involvement in neuronal development 444, to eliminate a potential confounding factor, purified SC cultures first infected with either the DN-RhoA or WT-RhoA adenoviruses, were seeded onto 4-week old mature purified DRGNs 24 h later. Co-cultures were maintained for 10-d under myelinating conditions (presence of VC) then immunostained for MBP or neurofilament (NF) to visualize myelinated fibers and DRGN axons, respectively. Control uninfected, purified SCs myelinated (MBP+) the axons of mature DRGNs (green) (Figure 4.3.2a). In contrast, SCs infected with either DN - or WT-RhoA constructs did not myelinate axons (few GFP/MBP+ fibers in merge column in Figure 4.3.2c and e, respectively). We assessed myelination of these cultures in parallel, using

196

Western blotting. MBP accumulation in DN- and WT-RhoA infected SCs was significantly lower than that observed with uninfected control SCs (Figure 4.3.2f). Interestingly, the amount of laminin synthesized by infected SCs was also significantly reduced, suggesting abrogation of early steps of myelination involving basal lamina deposition. Notably, infected SCs did not appear to align or ensheath axons. Thus, these results confirm the findings obtained with infections of SC-DRGN co-cultures.

197

Figure 4.3.1 Overexpressing dominant-negative RhoA in SC-DRGN co-cultures abrogates myelination.

a, b, 21 d SC-DRGN co-cultures were infected with control GFP- tagged (GFP Ad) or GFP- and Myc-tagged dominant negative RhoA (DN-RhoA)-expressing adenovirus, media was changed 24 h later and myelination was induced by addition of VC. Cultures were maintained for 10 d and subsequently immunostained for MBP (red). A myelinated internode in a and b is indicated by the white arrowhead. Scale bar= 50 µm.

198

199

Figure 4.3.2 Isolated SCs infected with WT- and DN-RhoA adenovirus seeded onto DRGNs do not myelinate.

a, SCs were seeded onto 4 week old purified DRGNs and subsequently induced to myelinate (1 h later) with VC. In b-e, enriched SC cultures were infected with GFP-tagged DN-RhoA or WT-RhoA-expressing adenovirus, and 24 h later, seeded onto pure DRGNs. 1 h after seeding, SCs were induced to myelinate by adding VC. All SC-DRGN co-cultures were maintained for 10 d under non-myelinating (b, d) myelinating (a, c, e) conditions, followed by immunostaining for MBP (red, in a, c, e) and neurofilament (NF) (green in a, red in b and d). Infected SCs were identified according to GFP expression in the representative confocal images depicted in b-e. SC nuclei were visualized with DAPI. Scale bars = 50 µm. Shown in f, are representative immunoblots of uninfected (CO) or DN-RhoA/WT-RhoA-infected (DN or WT) SCs seeded onto DRGNs (N) and cultured for 10 d under myelinating conditions. Samples were immunoblotted for MBP, laminin, NFM and actin. Shown are representative blots of one of two experiments performed in duplicate.

200

4.3.2 RhoA is involved in early stages of myelination To determine whether RhoA is involved in early stages of myelination, we assessed the effect of inhibiting RhoA activity on the accumulation of myelin proteins such as MBP and laminin. As such, SC-DRGN co-cultures were induced to myelinate using VC on day 0, and cultures were infected on day 0, 1, 4 and 7 with DN-RhoA after VC addition (d 0). All cultures were maintained until day 10 under myelinating conditions, and harvested for Western blotting (Figure 4.3.3). As the WT- and DN-RhoA adenoviruses have myc tags, the level of infection was assessed by immunoblotting for myc. MBP accumulation and laminin deposition in cultures infected on d 0 or 1 were substantially lower than uninfected control myelinating cultures or cultures infected 4, 5, 7 d after initiation of myelination. These results indicate a role for RhoA during early (first four days) stages of myelination.

4.3.2 RhoA regulates length of SCs process and cell survival Interestingly, we noted that simple expression of each construct WT- or DN-RhoA yielded a unique and altered cellular morphology. SCs expressing DN- RhoA were bipolar, exhibiting hyper-elongated processes (extending 3-10 times the cell body length) (Figure 4.3.1b, 4.3.2b, c). SCs expressing WT-RhoA have medium to normal process length (Figures 4.3.2d and e, and 4a and b). These results suggest a homeostatic role for RhoA in the SC. In contrast, few SCs expressing CA- RhoA were viable, suggesting a role for RhoA in cell survival.

4.3.3 WT RhoA overexpressing SCs retain the ability to myelinate Since modifying RhoA activity alters SC morphology, we assessed whether SCs in the presence of VC still maintain the potential to myelinate. Thus, we immunostained SCs infected with WT- or DN-RhoA cultured under myelinating conditions (10 d) for Krox-20. No SCs infected with DN-RhoA acquired Krox-20 nuclear staining. In contrast, some WT-expressing RhoA SCs become Krox-20-positive, thus retaining the potential to myelinate (Fig 4.3.4b).

201

Figure 4.3.3 RhoA is involved in early stages of myelination.

21 d SC-DRGN myelination-competent co-cultures were induced to myelinate on day 0. Cultures were infected for 24h with DN- RhoA adenovirus on different days (0-7 d) after the initiation of myelination. Infected cultures were changed 24 h after infection. Moreover, all cultures were maintained until day 10 under myelinating conditions. Cultures were then harvested for Western blotting with antibodies to laminin, NFM, Myc, MBP and actin to control for equal loading. Representative blots of one out of three experiments performed in duplicate are shown.

202

Figure 4.3.4 SCs infected with WT-RhoA adenovirus retain potential to myelinate.

21 d SC-DRGN co-cultures infected with either GFP-tagged adenovirus expressing wild-type (WT)- (a, b) or DN-(c, d) RhoA, were cultured for 10 d in the absence (a, c) and presence (b, d) of VC. Cultures were immunostained for Krox-20 (red) (a-d). Shown are representative confocal images. Scale bars= 50 µm. In b, a Krox-20-expressing infected SC is indicated by a white box.

203

4.3.4 Rho Kinase (ROCK) regulates myelin protein and Krox 20 expression ROCK, a principal downstream effector of RhoA, modulates the actin cytoskeleton remodelling through myosin light chain phosphatase 357. A previous study has shown that ROCK regulates internode length, internode number and paranode formation without altering MBP or Krox-20 expression 576. Since we observed a decrease in MBP accumulation when expressing DN-RhoA adenovirus in myelinating SC-DRGN co-cultures, we reassessed the role of the ROCK in peripheral myelination. We examined the effect of the ROCK inhibitor (Y27632) on myelination and Krox-20 expression. Treatment with Y27632 dose- dependently reduced MBP accumulation, without any effect on CNP (Figure 4.3.5a). This may suggest there is differential regulation of myelin proteins by ROCK. It also decreased laminin deposition. We next assessed the effect of Y27632 on Krox-20 expression. Immunostaining 3-day myelinating SC-DRGNs cultured in the presence and absence of Y27632 revealed that ROCK inhibition decreased the number of SCs expressing Krox-20 by half, as compared to myelinating (VC) control (p0.001, SC Krox-20+ (% of total): control (no VC): 0.96 ± 0.199; VC: ~10.44 ± 0.63; Y27632+VC: 5.04 ± 0.43, Figure 4.3.5c vs d, e ). Thus, these results identify ROCK as a regulator of myelin gene transcription.

204

205

Figure 4.3.5 ROCK inhibitor Y27632 decreases Krox-20 and myelin proteins expression.

a, 21 day myelin-competent SC-DRGN co-cultures were induced to myelinate with VC in the presence and absence of several concentrations of the ROCK inhibitor, Y27632 (0.1-30 µM) for 10d. Cells were then harvested for immunoblotting for MBP, CNP and laminin. Actin was used for equal loading. In b-d, 3 day myelinating co-cultures in the presence and absence of 30 µM Y27632 were immunostained for Krox-20 (red) and nuclei were visualized with DAPI (blue). Shown are representative confocal images from b, control (no VC), c, VC and d, Y27632 in the presence of VC. Scale bar = 50 µm. The numbers of Krox-20 positive SCs per field were counted, and the mean ± SEM expressed as a percentage of total cells per field is shown in e. 30 fields per condition were quantified. c, p < 0.001. Tukey-Kramer post-hoc ANOVA.

206

4.3.5 ROCK is not an activator of p38 MAPK or CREB To further demarcate the p38 pathway involved in myelination, we sought to determine whether the ROCK, and consequently RhoA, is an upstream effector of ECM-induced activation of p38 MAPK. Indeed, p38 MAPK is among the downstream targets of Rho GTPases 444. Thus, 21 day myelination- competent SC-DRGN co-cultures treated for 1 h with the pharmacological inhibitor of ROCK (Y27632, 30 µM) and subsequently stimulated with ECM for 30 min were harvested for Western blotting (Figure 4.3.7). In accordance with our results described in Section 4.2.12, ECM robustly stimulated the phosphorylation of p38 and its downstream target, CREB. However, Y27632 did not alter the activation of either the kinase p38 or CREB. As such, these results do not indicate that ROCK is an upstream activator of p38 MAPK.

207

Figure 4.3.6 Y27632 does not alter p38 or CREB phosphorylation induced by extracts of ECM.

SC-DRGN co-cultures were pretreated for 1 h with 30 µM Y27632, a ROCK inhibitor, stimulated for 30 min with an extracellular matrix extract and subsequently harvested for immunoblotting. Samples were probed for phosphorylated CREB, p38 and actin as a marker for equal loading. Representative immunoblots are shown from one of three experiments performed in duplicate.

208

4.3.5 Altering Rac1 and Cdc42 activity abrogates myelination in SCDRGN cocultures and alters SC morphology Recent work conducted in vivo, examining the roles of Cdc42 and Rac1 in both central and peripheral myelination suggest unique roles for both of these GTPases. To determine the effect of expressing DN- or CA-constructs of Cdc42 and Rac1 on myelination, we infected SC-DRGN co-cultures with GFP-tagged adenovirus and maintained under myelinating and non-myelinating conditions for 10 d before immunostaining for MBP or NF. Expressing DN or CA constructs of Rac1 and Cdc42, abrogated myelination in SC-DRGN cultures (Figures 4.3.7 and 4.3.8, respectively).

Tissue-specific conditional ablation of Cdc42 or Rac1 in vivo indicated that Cdc42 is required for normal SC proliferation, while Rac1 regulates SC process extension and stabilization, for efficient radial sorting of axon bundles 684,685. We attempted to identify and attribute a specific phenotype to each construct (WT, DN or CA) of Rac1 or Cdc42. SC-DRGN co-cultures were infected for 24 h, and subsequently maintained in myelinating conditions. Cultures were harvested after 10 d and immunostained for MBP and NF. In contrast to the phenotype exhibited by DN-RhoA-expressing SCs where all infected SCs possess hyper-elongated processes (Figure 4.3.1), we found that the population of adenovirus-expressing SCs exhibited a mixture of morphologies. This suggests a slightly higher threshold of activation or inactivation of either GTPase, which may need to be surpassed to yield a distinct phenotype. This may be due to an overlap of function and signaling pathways between the two GTPases or compensatory mechanisms 45,493. Many SCs expressing DN-Rac1 are bipolar, although each main (primary) process can possess at least one secondary bifurcation (arrows in Figure 4.3.7a). CA-Rac1- expressing SCs tend to have longer processes, as compared to WT (data not shown), but the primary morphological dysfunction appears to be an agglomeration of cytoplasm at the process tip, giving it a ‘foot-like’ or pseudopodia-like appearance (double arrowheads in Fig 4.3.7c). In contrast, some SCs expressing DN-Cdc42 appear to extend multiple spike-like projections (arrow in Figure 4.3.8b), suggesting a loss of cell polarity, a function ascribed to Cdc42 in other cell systems 144. There are degrees of morphological

209 abnormalities associated with Rac1- and Cdc42- infected SCs, thus further studies quantifying the number of secondary processes and lengths are required.

210

Figure 4.3.7 SCs infected with DN- and CA-Rac1 do not myelinate.

21 d myelin-competent SC-DRGN co-cultures infected with either GFP-tagged adenovirus expressing DN-(a, b) or CA-(c, d) constructs of Rac1, were cultured for 10 d under myelinating conditions, before being immunostained for MBP (red) (a, c) or NFM (red) (b, d). Shown are representative confocal images. Secondary bifurcations and the accumulation of cytoplasm are indicated by an arrow and a double arrowhead in a and c, respectively. Scale bars= 50 µm.

211

Figure 4.3.8 SCs infected with DN- and CA-Cdc42 do not myelinate.

21 d myelin-competent SC-DRGN co-cultures infected with GFP- tagged adenovirus expressing DN-(a, b) or CA-(c, d) constructs of Cdc42, were cultured for 10 d under myelinating conditions, before being immunostained for MBP (red) (a, c) or NFM (red) (b, d). Shown are representative confocal images. Arrow in b indicates a SC with multiple cytoplasmic projections. Scale bars= 50 µm.

212

4.3.6 Rac1 is not an activator of p38 MAPK To determine whether the Rac1 is an activator of p38 MAPK during early stages of peripheral myelination induced by the basal lamina, SC-DRGN co- cultures were pre-treated for 1 h with the Rac1-specific pharmacological inhibitor, NSC 23766 (30 µM) (Figure 4.3.8) and subsequently stimulated with ECM for 30 min. Cell lysates were immunoblotted for phosphorylated p38 and CREB. NSC 23766 altered neither p38 MAPK nor CREB phosphorylation. These results suggest Rac1 is not a modulator of p38 activation following ECM stimulation.

Figure 4.3.9 Rac1 inhibitor, NSC23766, does not alter p38 or CREB phosphorylation induced by ECM.

SC-DRGN co-cultures were pretreated for 1 h with the Rac1 inhibitor, NSC23766 (30 µM), stimulated for 30 min with an extract of the extracellular matrix and subsequently immunoblotted for phosphorylated CREB and p38. ERK was used as a loading control. Representative immunoblots of one of three experiments performed in duplicate are shown.

213

CHAPTER 5: DISCUSSION

214

5.1 Discussion of Results

Peripheral nerve development is a complex process requiring the constant interaction and signaling between SCs and axons, as well as with components of the extracellular milieu. Secreted trophic and survival factors, and molecules of the basal lamina greatly impact and direct this process. The phenotypes of laminin-2 (merosin)-deficient mice and genetic mutations in patients with peripheral neuropathies provide testimony to the central role of the basal lamina constituent, laminin, to SC myelination 16,491. The studies presented in this thesis stem from previous work in Dr. Almazan’s lab where addition of laminin to SC-DRGN cultures induced p38 MAPK phosphorylation and myelination. Moreover, pharmacological inhibitors of p38 MAPK prevented myelination. Postnatally, prior to the onset of myelination, SCs express the dual collagen/laminin-specific α1β1 integrin, and α6β1 integrin, a laminin-exclusive receptor linked to F-actin. Consequently, myelinating SCs downregulate α6β1 integrin to express the laminin-exclusive α6β4 integrin, associated with intermediate filaments 201. As such, the working hypothesis is that laminin signals through β1-subunit containing integrins on the SC surface to yield morphological and gene expression changes in SCs associated with the initiation of myelination and activation of p38 MAPK. Furthermore, integrins rely on the formation of focal adhesion complexes at its cytosolic tail to propagate signals intracellularly (“outside-in” signaling) since they lack intrinsic catalytic properties 181,200,206,550. We have evaluated in this thesis the involvement of two families of molecules known to comprise focal adhesion complexes and to participate in integrin- mediated signaling, SLKs and Rho GTPases, in relation to p38 MAPK activation by the basal lamina during myelination. Secondly, we focused our attention towards unveiling the molecular mechanisms by which p38 MAPK regulates myelin gene expression during initiation of myelination. In particular, we examined the expression of several key transcription factors involved in SC development and myelination. The results presented in Sections 4.1-4.3 are summarized in Figure 5.1 and are discussed below, in light of prospective studies and followed by their potential implications for disease and therapeutics.

215

SLKs regulate peripheral myelination

In contrast to the CNS, where roles for SLKs (Fyn) are well documented, a paucity of findings to date exclude the involvement of SLKs in peripheral myelination. Hence, we sought to evaluate their putative role. In section 4.1, we demonstrate for the first time, a role for SLKs, specifically Fyn and Lyn, in the early stages of peripheral myelination. A short treatment of SC-DRGN cultures with the SLK inhibitor PP2 significantly decreased the levels of the myelin proteins, MBP, P0 and MAG. We postulated that these reductions were mediated by changes in Krox-20 expression since both inhibition by PP2 and kinase- specific knockdown with siRNAs significantly reduced the number of SCs expressing this critical transcription factor. Moreover, we showed that phosphorylation of p38 MAPK, ERK and Akt induced by basal lamina extracts are mediated by SLKs, highlighting their role during early myelination events (SC differentiation, proliferation and survival, respectively) (summarized in Figure 5.1). The shorter myelinated internodes formed in the presence of PP2 suggest that SLKs may also be involved in modulating cytoskeletal organization.

SCs reportedly express Yes, Lyn, Fyn and Src 241,242 and we found that Lyn and Fyn were abundantly expressed in SC-DRGN co-cultures. Using differentiating SC-DRGN co-cultures, we demonstrate that chronic treatment with selective SLK inhibitors (PP2 and SU6656) decreased accumulation of the myelin proteins, MBP, P0 and MAG. In this in vitro model of PNS myelination, addition of VC leads to the organization of collagen fibers and basal lamina assembly, thereby largely synchronizing SC myelination 40,651. This process can be approximated using a developmental time-line for these cultures 183,184, where events can be divided into an early (initiation) or a late (compaction, maintenance) phase. The effect on MBP accumulation was more pronounced with longer exposure to PP2. This may be because Fyn can regulate MBP both transcriptionally 583 and post-transcriptionally 710, as elucidated in OLGs. While the master transcription factor Krox-20 partially regulates Mbp expression, it significantly regulates Mpz and Mag expression 407.

In Krox-20 null mice, SCs are permanently arrested at a pro-myelinating stage 334,380, while in humans, several independent Krox-20 mutations are

216 associated with Charcot-Marie Tooth disease and severe peripheral neuropathies 695. Thus, we examined the effect of PP2 treatment and anti-Fyn and -Lyn siRNAs on the expression of this essential transcription factor in SC- DRGN cultures. Both approaches significantly downregulated Krox-20, in accordance with the observed reductions in MBP, MAG and P0 following PP2 treatment. These findings, together with the observation that the effect of 1-3 d PP2 treatment on MBP levels was not fully reversed when the cultures were allowed to grow for 7 more days in the absence of the inhibitor, suggest a role for SLKs during the early stages of myelination.

Another molecule essential for early stages of myelination is laminin, which is a component of the basal lamina deposited by SCs 43,452,689,734. Laminin- and VC-induced myelination requires p38 MAPK activation in SC-DRGN cultures 754,785. Both SB203580 and PD169316, p38 MAPK inhibitors, prevented laminin- and VC-induced SC elongation and alignment along axonal tracts, myelin gene expression and consequent axonal myelination. Here we found that PP2 treatment blocked the phosphorylation of p38 MAPK induced by ECM, indicating that SLK activation occurs upstream of p38 MAPK and further emphasizing the importance of SLK activation during early stages of myelination.

In our experiments, myelinated internodes formed in the presence of PP2 were compacted normally, without obvious aberrations. A role for Fyn in myelin compaction in the CNS 213 has been shown using Fyn knockout mice, while myelin in the PNS appeared normal. MBP is the major structural protein in CNS myelin, heavily involved in both compaction and maintenance and its expression is regulated by Fyn kinase activity. In contrast, P0 is the major structural protein in PNS myelin (reviewed by Garbay 627) and is involved in myelin sheath compaction. This may explain why a severe effect was not observed in peripheral nerves of Fyn knockout mice, although MBP is thought to participate in the maintenance of the major dense line and myelin sheath compaction 228. Thus, transgenic mice with an inactivated gene for each individual SLK (Fyn, Lyn, Src or Yes) did not exhibit an apparent phenotype in the periphery 427,503. Furthermore, Biffiger et al, reported that initiation of myelination and sheath maintenance were not impaired even in 9 month old fyn-null mice 55,651. Based on these results, it was concluded that SLKs were not involved in peripheral

217 myelination. However, it appears very likely that compensatory mechanisms or functional overlap between the different SLKs exists, as suggested by our results with anti-fyn and -lyn siRNAs, masking phenotypic perturbations in each kinase null mouse. In a model of regeneration, where mature SCs undergo processes of dedifferentiation and proliferation following crush or transection lesion of the sciatic nerve, expression and activation of Src were selectively increased, while c-Fyn was not altered 55. Recently, Src was implicated as a target of Shp2 and NRG-1-mediated SC proliferation 267. Collectively, these observations suggest differential roles for SLK family members in SCs where Src is involved in SC proliferation while Fyn and Lyn are involved in differentiation and myelination.

Moreover, in the presence of PP2, axons were found to be significantly hypomyelinated since the g ratio, which relates the thickness of the axon to myelin was found to be significantly increased. As demonstrated by Taveggia et al, and Michailov et al, axonal NRG-1 dictates not only the ensheathment and myelination, but also myelin thickness 267. Thus, NRG-1 secretion or signaling may be affected by SLK inhibition, contributing to the hypomyelinating phenotype observed in our drug-treated cultures. It would be interesting to measure NRG-1 levels secreted in culture in the presence and absence of PP2.

Additionally, we indirectly assessed the formation of functional axo-glial domains (node of Ranvier and paranode) through clustering of sodium channels and the axonal adhesion molecule caspr. Although obvious aberrations in terms of clustering (domain formation) were not detected through this method, ultrastructural studies would further substantiate our claims regarding normal myelin formation in the presence of PP2. Interestingly, we did observe a high frequency of heminodes in PP2-treated cultures and PP2-treated myelinated fibers appeared to have smaller nodes. Further examination of these parameters would be interesting, more so because of the implication of SLKs in modulating phosphorylation of Kv1.5 and Kv2.1 potassium channels 451,681, and hence, onset of myelination.

The maintenance of peripheral myelin is a dynamic process, requiring the constant expression of Krox-20 649. Adult mice in which Krox-20 was selectively inactivated in SCs using a conditional Cre-based strategy, suffered severe

218 demyelination of the sciatic nerve, accompanied by rapid SC dedifferentiation and increased proliferation. SCs subsequently attempting to remyelinate were arrested at the promyelinating stage, consistent with results from Krox-20 null mice 166. Evidently, constant expression of Krox-20 is required to maintain SCs in a myelinating state 378,695. Since the effect of PP2 on Krox-20 at later stages of myelination was not investigated in our studies, the potential involvement of SLKs in myelin maintenance cannot be excluded.

Apart from the significantly fewer myelinated internodes, inhibition of SLKs with PP2 also caused a significant reduction in their length. Another group of investigators 166 previously showed that an inhibitor of PI-3K affected early events in SC myelination, including axon segregation and internodal length, and Akt was involved as well. In our study, PP2 treatment clearly blocked ECM- induced Akt phosphorylation, indicating that in response to basal lamina signaling, SLKs are activated upstream of Akt. In addition to phosphorylation of Thr308 and Ser473 residues, tyrosine phosphorylation of Akt may be essential for its biological function 438. Moreover, activation of SLKs in many cellular systems including OLGs is critical for proliferation and survival 151,749. ERK activation typically prevents SC differentiation while promoting proliferation 150,151. A recent study by Grossmann et al identified SLKs as targets of the nonreceptor tyrosine phosphatase Shp2 in NRG-1/ErbB-mediated SC proliferation and signaling 500. In pure SC cultures established from E13.5 mice, PP2 blocked NRG-1-evoked ERK activation and proliferation, without altering Akt/PI3K phosphorylation. Similarly, in SC-DRGN co-cultures, we found that PP2 blocked ERK phosphorylation induced by ECM. Thus, although SLKs may be involved in proliferation induced by the basal lamina, we did not study this in depth. Moreover, in our hands, chronic treatment of mature 3-week cultures with lower concentrations of PP2 neither reduced SC number nor increased the incidence of fragmented nuclei. Collectively, these results have led us to postulate that the primary role of Fyn and Lyn, SLK family members, in SCs myelination is related to differentiation, while a possible role in proliferation and even perhaps migration remains to be explored further.

Our findings in this study also suggest that elongation of the developing myelin sheath along axons was affected by PP2 treatment. The outgrowth of

219

OLG processes is promoted by the interaction of Fyn with the cytoskeletal protein tau 267 and morphological differentiation of OLGs is postulated to involve signaling from Fyn to Rho 355. p190 RhoGAP was shown to be phosphorylated by Fyn and tightly linked with both Fyn activity and OLG process extension and differentiation. Phosphorylation increases RhoGAP activity with increases in Rho activity to regulate morphological changes that accompany OLG differentiation 402. In SCs, remodeling of actin is essential for SC shape changes 753 and involves Rho GTPase activity 204. Hence, the process of SC myelin sheath extension along axons may indeed be an actin-mediated event, modulated by SLKs.

Moreover, while we demonstrate a role for SLKs in early stages of myelination using short-term (3d) siRNA studies, it would be interesting to analyze the effects of gene knockdown during myelin formation and at later stages, such as compaction and maintenance. Unfortunately, levels of genes targeted by siRNA are only transiently reduced for a maximum of 72 h. Given that key myelin proteins only accumulate to detectable levels 3 to 4 days after initiation, and compaction takes place thereafter, alternative avenues to assess SLK roles at these later time points will have to be explored. Moreover, as we have only attained approximately 30% transfection efficiency with siRNAs in SC- DRGN co-cultures, a means to achieve a higher level of knockdown is required to facilitate future studies. To this end, the construction of conditionally ablated Lyn, Fyn and Src SC-specific mice represents the ideal means to tease out roles for these SLKs in peripheral myelination. However, the use of Lyn- or Fyn- genetically deficient SCs from null mice may suffice as an interesting possibility. For instance, Lyn- or Fyn-deficient SCs could be seeded onto pure wild-type DRGNs, and attachment, adhesion and myelin formation subsequently assessed. Alternatively, chronic abrogation of SLK activity would be a more feasible approach. In fact, attempts to construct adenoviruses expressing kinase dead and kinase active constructs of Fyn were made, but we were unsuccessful. In addition, as we have found experimental artifacts and toxicity associated with expression of genes using adenoviral vectors, a lentiviral approach seems more amenable to these studies.

220

Finally, myelin formation is a process requiring axon-glial contact and bidirectional signaling (reviewed in Nave and Trapp, 2008 493). In neurons, SLKs are activated during normal development 479. Fyn overexpression causes abnormal development of primary sensory neurons in Xenopus laevis embryos by modifying signals regulating axonal guidance cues and fasciculation 341, while Src is involved in proliferation and differentiation of central neurons 599. Although treatment with PP2 could potentially affect the function of DRGNs or the interaction between SC and neurons, with a consequent effect on myelination, a number of observations appear to suggest that the main effect of PP2 must be on SCs. These include: 1) DRGNs have matured at the time of VC/PP2 treatment (21 d cultures), 2) NF levels are unaffected, 3) ultrastructural studies demonstrated normal axon caliber and morphology, 4) there appeared to be normal clustering of sodium channels and caspr to nodes and paranodes, respectively, and 5) DRGNs were not transfected with fyn and lyn siRNAs.

In conclusion, our work illustrates that SLKs, specifically Fyn and Lyn, play important roles in the early events of peripheral myelination and SC differentiation, chiefly through their regulation of Krox-20 expression and p38 MAPK activation. Collectively, this work essentially represents the first concerted effort aimed at identifying a role for each distinct SLK family member during SC myelination.

MAPK p38 regulates transcription factor expression and activation to coordinate SC differentiation and peripheral myelination

A network of extra- and intracellular signaling pathways regulate the transcriptional programs of SC differentiation and the progression of cellular changes along the lineage. We previously demonstrated the critical nature of p38 MAPK activation to SC differentiation associated with basal lamina-dependent myelin formation 213. Early events associated with SC differentiation and myelination, in particular, the induction of myelin genes (MBP, P0 and MAG) are abrogated in SC-DRGN co-cultures treated with either SB203580 or PD169316, two p38 MAPK inhibitors. In section 4.2, we attempted to dissect the molecular

221 means by which p38 MAPK regulates gene expression in differentiating SCs. Accordingly, we evaluated its possible involvement in modulating the expression or activation of critical transcription factors associated with SC terminal differentiation and consequent myelination. Indeed, we uncovered a central role for p38α MAPK as the regulator of Krox-20, Sox10 and SCIP gene expression, three transcription factors critically involved in SC differentiation. Moreover, we identify CREB as a target of p38, consequently implicating it in SC differentiation, PNS myelination. Furthermore, we found MK2 to be a downstream effector of p38 in peripheral myelination, and as a regulator of Krox-20 and myelin gene expression. However, it does not appear to modulate CREB phosphorylation. On the other hand, MSK-1 is targeted by p38 to effect CREB activation and Krox-20 expression (summarized in Figure 5.1).

Myelination of axons by SCs does not occur in the absence of Krox-20 213. In humans, EGR2 mutations have been linked with several diseases including CMT (1D and 4E) and DSS 695. Expression of this zinc-finger transcription factor is induced shortly following the first appearance of the POU domain transcription factor SCIP. Krox-20 subsequently becomes restricted to the myelinating lineage and its expression is maintained into adulthood 43,452,689,734. During the pro-myelinating to myelinating transition in SCs, Krox-20 expression is directly triggered by SCIP and Brn2 acting on the transcriptional enhancer, MSE 694,799. SCs from Krox-20 knockout mice are permanently arrested at a pro-myelinating stage, while SCIP-deficient SCs are only transiently arrested at a pro-myelinating stage 238. As such, using PD169316 and anti-p38α siRNA, we have demonstrated that p38α regulates Krox-20. Moreover, results obtained from our ultrastructural studies using electron microscopy in 10-d myelinating cultures showed that PD169316 arrests SCs at a pro-myelinating stage, where SCs formed a one to one association with axons. Notably, this morphology presented by PD169316-arrested SCs is reminiscent of that reported in Krox-20-null mice 61,695, reinforcing the significance of p38 MAPK to Krox-20 gene regulation. Moreover, we demonstrated a regulatory role of p38 MAPK on SCIP at the RNA level. This may occur transcriptionally or post-transcriptionally, in view of the fact that p38 can also regulate mRNA transcript stability. The stability of mRNAs is important in the control of gene expression, as shown with

222 cytokine genes in inflammation 695. AREs, located in the 3’ untranslated region of transcripts, are renowned in promoting rapid mRNA decay 217. AREBPs bind to these regions and either confer stability or promote transcript degradation. As such, p38 MAPK has been shown to stabilize mRNA by inhibiting destabilizing AREBPs or by enhancing the function of stabilizing AREBPs at the AREs 116. Indeed, the Krox-20 mRNA transcript contains an ARE and this may be a potential site of p38 MAPK regulation 163 in SC differentiation.

Additionally, p38 MAPK may regulate expression of Krox-20 and Krox-20- dependent myelin proteins by yet another indirect means. Sox10 typically transactivates gene expression synergistically with other transcription factors, including SCIP 217. Sox10 specifically cooperates with SCIP and Brn2 in MSE- dependent enhancer activity 366,367, and synergizes with NFATc4 to activate Krox- 20 expression in SCs 238. In fact, mutations in Sox10 have been identified in patients with myelin deficiencies 346. Moreover, expression of P0, Cx-32 and PLP are under direct transcriptional control of Sox10 319,367,540, while synergy between Sox10 with Krox-20 is required for P0 expression 69,524. Interestingly, in OLGs, Sox10 enhances MBP gene promoter activity by interacting with Sp1 and p27kip1 334,382. We found PD169316 treatment decreased Sox10 mRNA by 10 %, and appears to regulate Sox10 protein levels. Due to the abundance of Sox10 protein in SC-DRGN co-cultures, it was difficult to quantitatively assess any changes in Sox10 protein using Western blotting as cells of neural crest lineage express Sox10 (reviewed in Svaren 741). However, immunocytochemical staining of cultures indicated significant differences in Sox10 protein accumulation with PD169316 treatment as we observed a reduction in fluorescence intensity. Furthermore, p38 MAPK may also regulate association of Sox10 with its multiple binding partners to, in turn, ultimately control myelin gene expression. Moreover, two other members of the Egr family, Krox-24 (Egr1) and Pilot (Egr3) may also be regulated by p38 MAPKs. These transcription factors are heavily implicated in neural plasticity and memory formation 666, but very little known regarding their function in relation to SC differentiation and myelination, We recently performed an Affymetrix microarray analysis of 48 h myelinating SC-DRGN co-cultures grown in the presence and absence of PD169316 (5 µM). Data obtained indicate a 30% reduction in levels of Egr1 (data not shown) and we are currently in the

223 process of validating these targets with qRT-PCR. Consequently, Krox-24 and perhaps Pilot represent attractive candidates as regulators of myelin gene expression in SCs.

Thus far, we have demonstrated the regulation of Krox-20 and Krox-20- regulated genes (MAG and P0) by p38 MAPK. However, a Krox-20-independent mechanism of periaxin gene expression has also been reported by Parkinson et al. 400,543. Periaxin is a PDZ-domain cytoskeleton-associated protein expressed by myelinating SCs. In concert with DG, the other laminin receptor expressed by SCs, and DRP2, periaxin stabilizes mature compacted myelin sheaths 515. Low levels of periaxin were found to be expressed prior to and independent of Krox- 20, but upon expression of Krox-20, higher levels of periaxin are detected, thus indicating that Krox-20 acts to amplify periaxin expression in SCs during myelination 247,248,543,632,633. We thus explored the possible involvement of p38 MAPK in regulating Krox-20-independent mechanisms of gene expression. We evaluated the effect of PD169316 on periaxin and p27kip1 expression by SCs under myelinating conditions, in comparison to Krox-20-dependent expression of P0. We did not observe a large reduction in p27kip1 and periaxin proteins by PD169316, as compared to the effect on other known Krox-20-regulated myelin proteins (P0, MAG (this study), MBP 515). Curiously, a greater decrease in periaxin mRNA with PD169316 was detected, resembling that observed for Cx32, MAG, Krox-20 mRNAs. Moreover, this likely coincides with the appearance of Krox-20 during myelination, inferring a block in Krox-20- dependent amplification of periaxin transcription by PD169316 rather than an effect on Krox-20-independent induction of periaxin expression per se. It is possible that periaxin was expressed by SCs at the time of our study (21 d co- cultures), when myelination was initiated by adding VC. Hence, although our results appear to preclude involvement of p38 MAPK in Krox-20-independent mechanisms of peripheral myelin gene regulation, further experimentation will allow us to endorse or disregard a role for p38 MAPK in this alternative mechanism of periaxin gene regulation.

CREB is constitutively expressed and bound to the CRE promoter element, but it is only upon phosphorylation of Ser133 that its full transcriptional activity is imparted, with the recruitment of CBP and p300 cofactors. CBP and

224 p300 possess acetylation activity, leading to histone acetylation, chromatin relaxation and ease of transcription factor binding (reviewed in Johannessen et al 213). In SCs, sustained CREB phosphorylation has been linked to axon-mediated cAMP increases 333. Furthermore, βNRG-1 induced CREB phosphorylation in cultured SCs which involved MAPK but not CaMK activity 386,387. Here, using PD169316, we demonstrated the activation of CREB (Ser133 phosphorylation) by ECM was effected by p38 MAPK. However, further experimentation involving luciferase reporter gene transcriptional assays would demonstrate the induction of CREB transcriptional activity by ECM, its consequent attenuation with p38 MAPK inhibition, and substantiate its role in early stages of peripheral myelination/SC differentiation. Moreover, it may be worthwhile to directly assess whether CREB regulates Krox-20 expression and accordingly, myelin genes expression.

p38 MAPK phosphorylates downstream MKs to ultimately regulate cellular biological processes. The Ser/Thr kinase MK2 is amongst several possible downstream MAPK-activated kinases targeted by p38 MAPK. Phosphorylated-MK2 complexes with p38 MAPK and permits its nuclear export 668. Here, we demonstrated a role for MK2 in peripheral myelination using the inhibitor CMPD1, as Krox-20 expression was decreased. We attempted to corroborate this finding with MK2-directed siRNA knockdown and subsequent assessment of Krox-20 expression, however currently available reagents for detecting total MK2 protein levels (Western blotting) were incompatible with our cell-culture system, although moderate success has been achieved in other cell types 44. As MK2 regulates RNA stability by phosphorylating ARE-BPs, as shown in other systems 272, it may also regulate Krox-20 at a post-transcriptional level. Notably, large effects on Krox-20 and myelin gene expression, comparable to that attained with PD169316, were not achieved using CMPD1. Moreover, although MK2 has been shown to phosphorylate CREB 287,362,420,752, in our hands, CMPD1 did not significantly reduce phosphorylation of CREB induced by ECM. Taken together, these results imply p38 MAPK, is likely modulates CREB activation and peripheral myelination by targeting MKs other than MK2. One potential target is RSK-1, also known to regulate CREB 673. The family of MSKs (- 1 and -2) is another effector of p38 MAPK, which regulates gene transcription at

225 multiple levels. MSKs, activated by both ERKs and p38 21, can activate CREB and direct gene expression 718. In OLGs, CREB can form an immunoprecipitable complex with p38 and MSK1 161. We discovered that treatment of SC-DRGN cultures with the general MSK-1/PKA inhibitor, H89, and the more selective MSK-1 inhibitor, Ro 31-8220, caused a dose-dependent decrease in ECM- induced CREB phosphorylation. H89 also significantly reduced the number of Krox-20 positive SCs in myelinating cultures. Thus, we report a role for MSK-1 in SC differentiation. Moreover, MNK is yet another direct substrate of p38 272. As such, we are currently exploring the roles of MNKs as possible as downstream effectors of p38 MAPK in the modulation of myelin gene activation.

As we have demonstrated thus far, p38 appears to primarily target Krox- 20 to induce SC differentiation and myelination. PD169316 treatment blocked the expression of Krox-20, abrogating PNS myelination. Thus, we attempted to promote myelination in drug-treated SC-DRGN co-cultures by overexpressing Krox-20. Ectopic expression of Krox-20 has been shown to induce expression of myelin genes such as P0, Cx32, MBP 224,735. In our hands, adenoviral mediated Krox-20 expression was in fact, able to induce MAG expression in p38-inhibited cultures, indicating a reversal of PD169316 effects. However, we did not observe formation of MBP-positive myelinated internodes in these infected cultures. This may be due to an artifact of the adenoviral vector. However, these results may also implicate the inhibition of other non-Krox-20-related processes regulated by p38 MAPK which are inhibited by PD169316, and are equally important for myelination to take place. For instance, p38 MAPK has been shown to regulate actin cytoskeleton remodeling in other cell systems 475,515. Hsp-27 is phosphorylated in a time-dependent manner following laminin stimulation 373. PD169316 treatment of SC-DRGN co-cultures at an early time, impeded laminin- induced SC elongation and alignment with axons 213, which are actin-dependent processes 213. Moreover, recent results from microarray analysis of myelinating SC-DRGN cultures grown in the presence and absence of PD169316 indicate p38 MAPK regulates expression of proteins associated with the actin cytoskeletal rearrangement, although these results have yet to be validated. For instance, Rho GTPases and their activators or substrates (RhoB, Rac GTPase1, Rho GEF, Rhotekin) and cytoskeletal associated protein4 (ckap4) were found to be

226 downregulated (data not shown). These results collectively suggest that although Krox-20 may be present, and myelin genes are expressed, SCs are unable to physically synthesize myelin when p38 activity is inhibited.

NFkB is yet another important transcription factor associated with SC differentiation and peripheral myelination 205. Moreover, p38 MAPK has been shown to be an upstream regulatory kinase of NFκB in other systems. In primary human astrocytes, p38 regulated the transcriptional activity of NF-κB p65 via acetylation by regulating the acetyltransferase activity of coactivator p300 485,779. Therefore, NFκB may potentially be targeted p38 MAPKs while promoting myelination.

Using PD169316, we effectively blocked the differentiation of SCs and the expression of several transcription factors closely linked with myelination. However, as described in Chapter 1, mature fully differentiated myelinating SCs are capable of re-entering the cell cycle, de-differentiating, and reverting to an immature state. This occurs in the presence of negative regulators of differentiation such as Sox2, p57kip2 and c-Jun 595. p38 MAPK has been shown to differentially regulate cell cycle progression 330. In some cell systems, p38 MAPK is required for proliferation 6,142, while it inhibits cell cycle progression to consequently promote differentiation in others 419,566. Hence, the role of p38 in cell cycle progression appears to be cell system-dependent. Moreover, transcriptional and post-transcriptional downregulation of cyclin D1 by p38 was cited in other cell types as participating in cell cycle progression at the G1-S checkpoint 375. Notably, in myocytes, p38α was identified as an inhibitor of cyclin D1 expression 375,459. Cyclin D1 gene expression can be regulated via mRNA stability as transcripts contain AREs 529. Moreover, p38 MAPK was shown to regulate the expression of Cyclin D3 36. Thus, while we demonstrate p38 regulates the differentiation of SCs, it may concurrently be modulating other regulators of cell cycle progression, such as Cyclin D.

Another possibility is that p38 MAPK could regulate the expression of negative regulators of myelination, such as Sox2, to promote SC differentiation. As such, we examined the levels of Sox2 in p38-inhibited myelinating SC-DRGN co-cultures. PD169316 did not affect Sox2 expression at the mRNA or protein

227 level. However, PD169316 treatment significantly decreased the number of SCs expressing the cell cycle inhibitor, p27kip1 protein, induced by VC or ECM. p27kip1 expression is associated with a myelinating phenotype 729 and is closely linked with periaxin expression 221,515. Moreover, Krox-20 overexpression has been shown to induce p27kip1 expression in SCs 515. Collectively, these results demonstrate that p38 MAPK modulates the expression of several positive regulators of differentiation, but it does not actively suppress expression of the negative regulator Sox2 to induce myelination. However, SCs express two other negative regulators, p57kip2 and cJun, which could be regulated by p38 during SC differentiation. The cyclin-dependent kinase inhibitor p57kip2 negatively regulates SC differentiation and myelination 514. We are currently exploring the possible regulation of p57kip2 expression by p38 MAPKs at the mRNA and protein level. Moreover, Krox-20, a prime target of p38, actively suppresses c-Jun expression in SCs, and regulates the cJun-JNK pathway through JIP 283. While cJun phosphorylation is linked to some of its inhibitory effects, it was shown that expression by SCs is responsible for abrogating myelination while concurrently promoting proliferation 514. Moreover, it has been postulated that c-Jun may also mediate some of its inhibitory effects indirectly, through Sox2 513. Since activation of p38 and JNK MAPKs yields opposite effects in SCs (differentiation versus proliferation, respectively), it is appealing to explore the effect of p38 inhibition on cJun expression, phosphorylation, and JNK activity. As such, we are currently examining these parameters in SC-DRGN co-cultures.

Thus, we delineate a molecular mechanism by which p38 MAPK regulates SC differentiation and myelination, where p38α regulates p27kip1, Krox- 20, SCIP and Sox10 expression. This is likely effected through two MKs: 1) MK2 which modulates Krox-20 expression, possibly through mRNA transcript stability but does not appear to be directly associated with CREB phosphorylation and 2) p38/MSK-1 to modulate CREB activation and Krox-20 expression. Furthermore, the data presented in Section 4.2 indicate all pro-myelin pathways regulated by p38 ultimately converge on the regulation of the master transcription factor, Krox- 20, required for SCs to myelinate.

228

Rho GTPases regulate SC morphology and Krox-20 expression

The interaction of SCs with components of the extracellular matrix (laminin) is mediated by integrins. As already mentioned, these cell surface receptors lack intrinsic kinase activity. Activation of integrins triggers receptor heterodimerization, and the formation of a focal adhesion complex that includes molecules which direct actin cytoskeleton remodeling such as the Rho family of GTPases. Rho GTPases (RhoA, Rac, Cdc42) act as key molecular switches in various biological contexts, cycling between the GTP-bound (active) and the GDP-bound (inactive) forms. (reviewed in 513). In relation to myelination, various reports denote unique roles for the three key family members, RhoA, Cdc42 and Rac1, in early stages of central and peripheral myelination 191,324,619. As MAPKs (ERKs, JNKs and p38) are among the identified downstream targets of Rho GTPases 45,351,402,493,628,688 and given the central role of p38 MAPK activation in actin-dependent SC elongation and axonal alignment during initiation of myelination 684,685,722, we examined the potential role of Rho GTPases in p38 activation. In Section 4.3, using ectopic overexpression, we confirm and substantiate findings from previous studies implicating RhoGTPases in early events of myelination. Longterm studies of infected SC-DRGNs maintained under myelinating conditions reveal that expression of all constructs (WT, CA, DN) of Rho GTPases (RhoA, Rac1, Cdc42) substantially blocked myelination. Upon further analysis, we discovered, interestingly enough, that some WT-expressing RhoA SCs retain some potential to myelinate, as they stained positively for Krox- 20. Moreover, expression of DN-RhoA adenovirus, revealed a role for RhoA in process extension in SCs. Furthermore, we hereby identify ROCK as a regulator of Krox-20 expression and myelin gene expression. However, our results do not support potential roles of ROCK (consequently RhoA) and Rac1 as activators of p38 MAPK phosphorylation (summarized in Figure 5.1).

As key regulators of actin cytoskeleton remodeling, each RhoGTPase family member was found to carry out specific functions. From well-characterized fibroblast studies, Cdc42 and Rac1 have been identified to orchestrate filopodia and lamellipodia formation, necessary for process extension and cell spreading, whereas RhoA is the primary mediator of cellular contractility, stress fiber formation and establishment of focal adhesions 205,213. In OLGs, RhoA is involved

229 in process extension 274,489,490, membrane sheet formation, endocytic membrane transport 402 and cellular differentiation 351, while Rac1 and Cdc42 modulate proper myelin sheath formation by OLGs 402. On the other hand, in the periphery, Cdc42 is required for normal SC proliferation while Rac1 regulates SC process extension and stabilization, for efficient radial sorting of axon bundles 688. Moreover, Rac1 activation was shown to occur in a β1-integrin dependent manner. In contrast to Rac1 and Cdc42, RhoA has not been extensively studied in relation to SC-mediated myelination. However, in Drosophila melanogaster, RhoA was shown to be involved in glial cell migration and morphogenesis 45,493. Specifically, expression of CA-RhoA prevented peripheral glial cells from migrating peripherally and these mutants were reported to have very long, spike- shaped actin processes. In our hands, expression of either the DN/WT/CA-RhoA construct alone yielded a unique and altered SC morphology. SCs expressing DN-RhoA were found to be bipolar and possessed hyper-elongated processes, contrasting with findings from D. melanogaster 628. This may suggest species- specific roles for RhoGTPases. Also, SCs expressing WT-RhoA exhibited medium to normal process length. We are currently quantifying the lengths of these processes to assess whether there is a significant difference between SCs expressing one RhoA construct or another. Moreover, we found few SCs expressing CA-RhoA to be viable, implicating RhoA in cell survival.

Using SC-DRGN co-cultures, some SCs expressing DN-Cdc42 appear to extend multiple spike-like projections, suggesting a loss of cell polarity, a function ascribed to Cdc42 in other cell systems 628. Also, some SCs expressing DN-Rac1 were bipolar, although each main process may have at least one secondary bifurcation. CA-Rac1-expressing SCs have longer processes, as compared to WT, but the main dysfunction appears to be an agglomeration of cytoplasm at the process tip, having a ‘foot-like’/pseudopodia-like appearance. Notably, a large population of Rac1/Cdc42-infected SCs did not exhibit such distinct morphologies, unlike the phenotypes observed by expressing the various constructs of RhoA, where all SCs displayed one specific phenotype. Instead, multiple phenotypes and aberrations were detected in SCs infected with each construct of Rac1 and Cdc42. Moreover, the mixture of phenotypes exhibited by infected Rac1- and Cdc42-SCs may be due to an overlap in function and

230 signaling pathways between these two GTPases, as has been proposed to exist between them 16,491. As such, the degree of mutant protein expressed by each infected SC may need to pass a specific threshold to yield a distinct phenotype, and unveil its unique roles of Rac1 and Cdc42. We are currently attempting to characterize the frequency of each of these abnormal phenotypes associated with each Rho GTPase construct.

We demonstrate a role for RhoA in early stages of myelination using DN- RhoA constructs. Infection of myelinating cultures early (0 and 1 d after VC addition), abrogated MBP and laminin accumulation, while infection at later days (4 d onwards) had little effect. From these results it may be inferred that RhoA does not participate in myelin maintenance. We also evaluated a principal effector of RhoA, ROCK. It has been shown to modulate actin cytoskeleton rearrangement via myosin light chain phosphatase 144. In our cultures, treatment of SC-DRGNs with Y27632, a selective inhibitor of ROCK, significantly reduced the number of Krox-20 positive SCs. There was also a consequent reduction in the accumulation of myelin proteins MBP and CNP. However, another group of investigators reported no reduction in Krox-20 or myelin proteins with the same inhibitor 576. Melendez-Vasquez reported that pharmacological inhibition of ROCK using Y-27632 resulted in a loss of microvilli and stress fibers in SC cultures, as well as aberrant myelination of DRGNs with an increased number of myelinated internodes and decreased internode length 444. These conflicting results may be explained by the different culture conditions used by the laboratories, in particular the use of serum-containing medium by Melendez-Vasquez. Moreover, they assess changes in Krox-20 expression using Western blotting. As we discovered in Section 4.1.4, in our system, any alterations in protein levels of this master transcription factor can only be detected using immunofluorescence. Moreover, as we demonstrated in Section 4.2, expression of Krox-20 is significantly regulated by p38 MAPK, therefore, suggesting RhoA may partially modulate p38 activity.

To further demarcate the p38 pathway involved in myelination, we sought to determine whether the Rho GTPases are upstream effectors of laminin- or ECM-induced activation of p38 MAPK. However, in our experiments, pharmacological inhibitors of ROCK (Y27632) and Rac1 (NSC 23766) did not

231 inhibit ECM-induced phosphorylation of p38 or of its downstream target, CREB. This suggests that ROCK and Rac1 are not likely upstream activators of p38 MAPK/CREB, however, further studies are required to confirm these results. Thus, we will explore this further by performing GDP/GTP pull-down activation assays (Upstate Biotechnology), following induction with either laminin or ECM in the presence and absence of the p38 inhibitors, PD169316 and SB203580. Additionally, a newly available pharmacological inhibitor of Cdc42 (secramine) will be used to test its relationship to p38 activation. We hope these experiments will help to define roles for Rho family of GTPases in the early stages of SC myelination specifically involving p38 MAPK activation.

p38 MAPKs may also regulate Rho GTPase activity directly or indirectly. For instance, several publications have suggested that p38 MAPK activates Rac1 445. Moreover, in Section 4.2, we demonstrated that p38 MAPK regulates expression of the cip/kip CKI p27kip1 in myelinating SCs. In fact, the three members of the cip/kip family of CKIs have been shown to modulate cytoskeletal dynamics through the inhibition of Rho GTPases. RhoA or ROCK activity is modulated by both p27kip1 801 and p21cip1 50. Furthermore, p57kip2 binds to the LIM- domain containing protein LIMK-1 through its proline-rich domain to direct its intracellular localization in SCs 388,674. LIM domains are cysteine/histidine-rich double zinc fingers that mediate protein-protein interactions 284,721,778. LIMK-1 can phosphorylate cofilin, a modulator of actin filament dynamics.

Several lines of evidence demonstrate a link between SLKs and Rho GTPases. The GEFs are specific to each Rho GTPase, while others are common to Rho, Rac1 and Cdc42 GTPases such as Vav 795. The means by which GEF activation is regulated is not well understood. However, tyrosine phosphorylation by SLKs has been shown to take place. For instance, the Vav family of GEFs can be directly tyrosine phosphorylated and activated by Src 11,406,412,467. Also, GAP activity may be coordinated through tyrosine phosphorylation by SLKs. For instance, Fyn was shown to phosphorylate the p190RhoGAP to modulate RhoA activity in OLGs and direct myelination 406. Indeed, tyrosine phosphorylation of GEFs and GAPs by SLKs represent a potential means to intricately regulate Rho GTPase activity. As we demonstrate in Section 4.1, the involvement of SLKs in peripheral myelination and in p38 MAPK activation induced by laminin, these

232 tyrosine kinases may be also be coordinating Rho GTPases activity in SCs during myelination.

Paxillin, a 68 kDa protein, functions as an adaptor between integrins and the actin cytoskeleton. It possesses multiple domains such as SH2-, SH3-binding domains, five leucine- and aspartate-rich (LD) repeats, four double-zinc finger LIM domains that participate in protein-protein interactions. It localizes to FACs by its carboxy-terminal LIM domains 402,753. It participates in cell adhesion and migration. Numerous extracellular signals converge on this focal adhesion protein. Paxillin is a highly dynamically regulated protein 84. It can be highly phosphorylated at Tyr and Thr residues at its amino-terminal by multiple kinases, including as FAK and SLKs. These sites of Tyr phosphorylation then serve as SH2 binding sites, to recruit proteins to FACs. In SCs, paxillin is a constituent of the actin-associated FAC formed in SCs contacting axons, with FAK, Fyn, and β1 integrin 707,708. Tyrosine phosphorylation of paxillin increased as SCs began differentiating and assembling a basal lamina. Moreover, paxillin has been shown to be phosphorylated at several unique Ser residues in its N-terminal domain by MAPKs p38 during neurite extension in PC-12 cells, 117. It is postulated that this p38-mediated Ser phosphorylation of paxillin modulates focal adhesion organization/assembly. Moreover, paxillin can undergo Ser phosphorylation by JNK during migration 306. Paxillin can interact with many downstream proteins, including Rho GEFs, GAPs and effector proteins to FACs to indirectly coordinate Rho GTPases activity in cells at spatial and temporal levels 307,308. For instance, the p21-activated kinase (PAK), a substrate of Rac1 RhoGTPase, can be recruited by paxillin. Thus, paxillin may be another site of regulation by p38 MAPK following basal lamina stimulation, and may be used to direct Rho GTPases in peripheral myelination. We hope to evaluate its involvement using phospho-specific antibodies to activated paxillin.

Hence, in Section 4.3, we demonstrate a role for RhoA via ROCK in Krox- 20 and myelin proteins gene expression, and it is potentially regulated by SLKs and p38 MAPKs. Further studies are required to tease out any involvement of Cdc42 and Rac1 in myelin gene expression and SC differentiation involving p38 MAPK activation.

233

5.2 Summary of Results and General Discussion In the work presented in this thesis, we have made significant findings regarding SLK, and Rho GTPase involvement in basal lamina-mediated SC differentiation and peripheral myelination, involving the expression and activation of several transcription factors by p38 MAPKs. In Section 4.1, we demonstrate the involvement of Fyn and Lyn in peripheral myelination. Of the few myelinated internodes formed in the presence of the SLK inhibitor, a significantly large number of them were shorter and hypomyelinated. Moreover, early stages of SC differentiation and myelination are directed by SLKs. Using siRNA knockdown, Fyn and Lyn were shown to regulate Krox-20 expression and with a general inhibitor, PP2, SLKs were implicated in the modulation of p38 MAPK, ERK and Akt kinases activation. Next, we provide several lines of evidence (Section 4.2) demarcating p38 MAPK as a direct regulator of SC differentiation and its subsequent myelination of DRGNs through the modulation the expression of transcription factors; Krox-20 is a key target of p38 MAPKα and its downstream effector, MK2, and MSK-1. In addition, the effect of p38 MAPK inhibition of MAG expression is reversed by ectopic expression of Krox-20. Moreover, we found p38 MAPK to regulate SCIP and Sox10 at a transcriptional level, and phosphorylation of CREB. Even though p38 was found to regulate cell cycle exit (p27kip1 expression), we did not however find it negatively regulated Sox2 expression while promoting SC differentiation. Finally, in Section 4.3, using ectopic overexpression studies, we identify roles for RhoA, Rac1 and Cdc42 in modulating process extension, branch stabilization and directionality in SCs, respectively. Moreover, RhoA and ROCK were found to regulate gene expression in SCs through Krox-20. The overall signaling pathway delineated and described in this thesis using this in vitro model system of peripheral myelination involving SLKs, p38 MAPK and Rho GTPases are summarized in the schema below (Figure 5.1).

234

Figure 5. 1 Schema summarizing the signaling pathways activated by ECM, VC in SCDRGN cocultures involving SLK, p38 MAPK and Rho GTPases.

Induction of myelination in SC-DRGN co-cultures with the addition of either ECM or VC, activates SLKs (red), which were found to lie upstream of the p38 MAPK, ERK and Akt phosphorylation. p38 MAPK modulates expression of several transcription factors (Krox-20, SCIP and Sox10) and the activation of CREB. This is mediated by the MKs MSK-1 (Krox-20 expression and CREB activation) or MK2 (Krox-20 expression). Finally, RhoA and its effector, ROCK, modulates Krox-20 expression. The various molecules are colour-coded, as indicated in the figure legend to the right of the schema.

Collectively, through the research presented in this thesis, we have identified important inter-related roles for p38 MAPKs, SLKs and ROCK in directing SC terminal differentiation. In concert, they modulate peripheral myelination. Further studies however, are necessary to determine if indeed p38

235 regulates Rho GTPase activity in early stages of myelination. Moreover, there are some general comments regarding the inferences made and conclusions drawn from the findings presented in this body of work.

Firstly, although the primary upshot for these studies is that the effects of laminin are elicited through integrins to activate Rho GTPases, SLKs and p38 MAPKα, there remains the distinct possibility that some or all of these actions are mediated through the other laminin receptor, DG and its associated DG- dystrophin axis. As described, DG is involved in node formation, organization of myelin and at later stages in myelin maintenance. In order to expel this as a possibility, experiments using β1 integrin and DG-blocking antibodies followed by the assessment of p38 and CREB phosphorylation states should be conducted. Alternatively, studies using β1 integrin conditional knockout SCs and examining p38 activation following addition of laminin will help clarify our findings and further characterize this molecular pathway.

Secondly, the majority of our longterm studies assessing myelination were conducted in SC-DRGN co-cultures under myelinating conditions. NRG-1 has been implicated in multiple stages of SC development, influencing differentiation and myelination (reviewed in 162). It is secreted into medium and is present on DRGN axonal surfaces. As such, it is likely that some of the functions carried out by the SLKs, Rho GTPases and p38 MAPKs, are indeed mediated by NRG-1.

Finally, the majority of the studies presented in this thesis were conducted in SC-DRGN co-culture systems. Our interpretations and evaluation were primarily focused on assessing the involvement for three molecular families SLKs, RhoGTPases and p38 MAPKs in the differentiation of SCs and myelin formation. In these studies, myelin synthesis and SCs have been our areas of interest and end points of assessment. As such, we have used the accumulation of SC-expressed proteins (transcription factors, myelin proteins) as indicators. However, as repeatedly mentioned, the development of peripheral axons, SCs and consequent myelination involves constant interplay between SCs and axons 56,557. In our studies, we only evaluated potential negative secondary effects of pharmacological inhibitors on neurons manifesting as toxicity and axonal degradation, using expression of neurofilaments and ultrastructural axonal

236 integrity as indicators, respectively. However, we did not address the neuronal contribution to SC signaling and development. Thus, we cannot exclude the possibility that changes detected in these defined end points (SC expression of factors and synthesis of myelin) are due to the influence of DRGNs.

Taken together, future studies aimed at clarifying these will help map out a distinct molecular mechanism of myelin regulation attributable to either SC or DRGN.

Implications for Therapy As described in Section 1.9, a number of hereditary demyelinating or dysmyelinating diseases arise due to the abrogation of myelin formation or its maintenance. This indicates that glial transduction of pro-myelinating signals has to be under tight and life-long control to preserve the integrity of the myelinated axon. Understanding cross talk between neurons and SCs will help further define the role of glia in preserving axonal integrity and to develop therapeutic strategies for peripheral neuropathies such as CMT1A.

Moreover, there are several CNS disorders where demyelination occurs, such as multiple sclerosis, viral infections and spinal cord injury, resulting in functional impairments. In most instances, remyelination by OLGs either do not occur or is insufficient 479,557. As a result, there has been a great interest in developing strategies to promote myelin repair in the central nervous system. Oligodendrocyte progenitors, olfactory ensheathing cells, embryonic and neural stem cells are among the various cell types being explored as potential therapeutic options 536. SCs represent another candidate for cellular therapy. The supreme plasticity of SCs has been demonstrated. For instance, SCs cultured in vitro in the presence of the pro-melanocytic factor, endothelin-3 (ET-3), revert to an earlier bipotent glial-melanocytic NC precursor state, and subsequently transdifferentiate to form melanocytes 42,369,536,605. Another study illustrated the ability of SCs to transdifferentiate into myofibroblasts 176,177. Thus, glial cells of the PNS may function as NC stem cells in specific circumstances as repair. Myelin of the central and peripheral nervous systems share both physical and molecular properties. As such, the plastic nature of SCs and its ability to

237 redifferentiate and form myelin has lent itself as a potential therapeutic for spinal cord injuries. However, there are many problems being faced with the success of SC transplantation and myelination in the CNS, primarily arising due to astrocytes 568. Migration of implanted SCs was found to be largely attenuated by astrocyte-produced aggregan and ephrins in a receptor-mediated manner, which disrupted laminin-β1 integrin interaction and VAV2 signaling, respectively 60,321. As such, steps to improve SC migration are being taken. For instance, SCs engineered to express polysialic acid –NCAM and the cell adhesion molecule L1 promoted functional recovery after spinal cord injury 9,10.

Moreover, similar strategies may improve peripheral nerve injury. Although peripheral nerves have the potential to regenerate axons, and reinnervate end organs, total recovery even after repair remains relatively poor 374,511. Patients suffering from such insults have the tendency to suffer from motor and sensory dysfunction, with potential development of chronic pain 297. Electrical stimulation is used clinically to promote repair but success remains minimal 297. Since SC and neuron development are intricately linked, and regional SC dysfunction possibly plays a role 253, cellular therapy is being investigated as an avenue to promote nerve repair 659. Multipotent skin-derived precursors are being studied as potential sources 203,297,728.

5.3 Concluding Remarks Gaining a better understanding of the axo-glial signaling mechanisms and SC biology may help to exploit the intrinsic characteristics of SC and stimulate myelination in demyelinating diseases, injury, and to preserve axonal integrity. Such discoveries may lend themselves to the advancement of oligodendroglial biology and central myelination.

In this thesis, some of the mechanisms of myelination following treatment of SC-DRGN co-cultures with constituents of basal lamina have been deciphered. Moreover, we demonstrate the intertwined signaling pathways of regulation between SLKs, p38 and Rho GTPases signaling that converges on Krox-20. This is key due to the central role of Krox-20 in SC differentiation and

238 peripheral myelination. This contribution may further our knowledge of the complexity of axon-glial biology and interactions mediating peripheral myelination. However, it is evident that further investigation is required to fully comprehend the multiple effectors and their interactions, ultimately influencing the fate of SCs. I hope this work helps set the stage for future studies and forthcoming scientific discoveries in the SC and peripheral myelin field.

239

REFERENCES

1 Myelin, 2nd ed. (Plenum Press, New York, 1984).

2 Myelin: Biology and Chemistry, 1 ed. (CRC Press, USA, 1992).

3 E. Aberg, et al., "Docking of PRAK/MK5 to the atypical MAPKs ERK3 and ERK4 defines a novel MAPK interaction motif," J. Biol. Chem. 284(29), 19392 (2009).

4 C. K. Abrams, et al., "Mutations in connexin 32: the molecular and biophysical bases for the X‐linked form of Charcot‐Marie‐Tooth disease," Brain Res. Brain Res. Rev. 32(1), 203 (2000).

5 R. H. Adams, et al., "Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development," Mol. Cell 6(1), 109 (2000).

6 H. S. Adler, et al., "Activation of MAP kinase p38 is critical for the cell‐cycle‐ controlled suppressor function of regulatory T cells," Blood 109(10), 4351 (2007).

7 K. Adlkofer and C. Lai, "Role of neuregulins in glial cell development," Glia 29(2), 104 (2000).

8 K. Adlkofer, et al., "Hypermyelination and demyelinating peripheral neuropathy in Pmp22‐deficient mice," Nat. Genet. 11(3), 274 (1995).

9 F. T. Afshari, J. C. Kwok, and J. W. Fawcett, "Astrocyte‐produced ephrins inhibit schwann cell migration via VAV2 signaling," J. Neurosci. 30(12), 4246 (2010).

10 F. T. Afshari, et al., "Schwann cell migration is integrin‐dependent and inhibited by astrocyte‐produced aggrecan," Glia 58(7), 857 (2010).

11 B. Aghazadeh, et al., "Structural basis for relief of autoinhibition of the Dbl homology domain of proto‐oncogene Vav by tyrosine phosphorylation," Cell 102(5), 625 (2000).

12 A. Agrawal, et al., "Neurological manifestations of Hansen's disease and their management," Clin. Neurol. Neurosurg. 107(6), 445 (2005).

13 H. C. Agrawal and D. Agrawal, "Proteolipid protein and DM‐20 are synthesized by Schwann cells, present in myelin membrane, but they are not fatty acylated," Neurochem. Res. 16(8), 855 (1991).

14 D. E. Albrecht, et al., "The ABCA1 cholesterol transporter associates with one of two distinct dystrophin‐based scaffolds in Schwann cells," Glia 56(6), 611 (2008).

15 D. Allen, et al., "Antibodies to peripheral nerve myelin proteins in chronic inflammatory demyelinating polyradiculoneuropathy," J. Peripher. Nerv. Syst. 10(2), 174 (2005).

240

16 W. E. Allen, et al., "A role for Cdc42 in macrophage chemotaxis," J. Cell Biol. 141(5), 1147 (1998).

17 B. M. Altevogt, et al., "Connexin29 is uniquely distributed within myelinating glial cells of the central and peripheral nervous systems," J. Neurosci. 22(15), 6458 (2002).

18 S. A. Amici, et al., "Peripheral myelin protein 22 is in complex with alpha6beta4 integrin, and its absence alters the Schwann cell basal lamina," J. Neurosci. 26(4), 1179 (2006).

19 B. Andersen, et al., "Brn‐5 is a divergent POU domain factor highly expressed in layer IV of the neocortex," J. Biol. Chem. 268(31), 23390 (1993).

20 T. J. Anderson, et al., "Modification of Schwann cell phenotype with Plp transgenes: evidence that the PLP and DM20 isoproteins are targeted to different cellular domains," J. Neurosci. Res. 50(1), 13 (1997).

21 R. Anjum and J. Blenis, "The RSK family of kinases: emerging roles in cellular signalling," Nat. Rev. Mol. Cell Biol. 9(10), 747 (2008).

22 P. Anzini, et al., "Structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin 32," J. Neurosci. 17(12), 4545 (1997).

23 M. Aouadi, et al., "Role of MAPKs in development and differentiation: lessons from knockout mice," Biochimie 88(9), 1091 (2006).

24 J. B. Aquino, et al., "In vitro and in vivo differentiation of boundary cap neural crest stem cells into mature Schwann cells," Exp. Neurol. 198(2), 438 (2006).

25 A. Araque, et al., "Tripartite synapses: glia, the unacknowledged partner," Trends Neurosci. 22(5), 208 (1999).

26 E. G. Arias‐Salgado, et al., "Src kinase activation by direct interaction with the integrin beta cytoplasmic domain," Proc. Natl. Acad. Sci. U. S. A 100(23), 13298 (2003).

27 E. J. Arroyo and S. S. Scherer, "On the molecular architecture of myelinated fibers," Histochem. Cell Biol. 113(1), 1 (2000).

28 E. J. Arroyo, et al., "Myelinating Schwann cells determine the internodal localization of Kv1.1, Kv1.2, Kvbeta2, and Caspr," J. Neurocytol. 28(4‐5), 333 (1999).

29 S. Artavanis‐Tsakonas, M. D. Rand, and R. J. Lake, "Notch signaling: cell fate control and signal integration in development," Science 284(5415), 770 (1999).

30 J. S. Arthur, "MSK activation and physiological roles," Front Biosci. 13, 5866 (2008).

31 S. Atanasoski, et al., "Postnatal Schwann cell proliferation but not myelination is strictly and uniquely dependent on cyclin‐dependent kinase 4 (cdk4)," Mol. Cell Neurosci. 37(3), 519 (2008).

241

32 S. Atanasoski, et al., "Cell cycle inhibitors p21 and p16 are required for the regulation of Schwann cell proliferation," Glia 53(2), 147 (2006).

33 S. Atanasoski, et al., "ErbB2 signaling in Schwann cells is mostly dispensable for maintenance of myelinated peripheral nerves and proliferation of adult Schwann cells after injury," J. Neurosci. 26(7), 2124 (2006).

34 A. A. Avilion, et al., "Multipotent cell lineages in early mouse development depend on SOX2 function," Genes Dev. 17(1), 126 (2003).

35 R. Awatramani, et al., "TGFbeta1 modulates the phenotype of Schwann cells at the transcriptional level," Mol. Cell Neurosci. 19(3), 307 (2002).

36 T. Bakheet, et al., "ARED: human AU‐rich element‐containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins," Nucleic Acids Res. 29(1), 246 (2001).

37 R. J. Balice‐Gordon, L. J. Bone, and S. S. Scherer, "Functional gap junctions in the schwann cell myelin sheath," J. Cell Biol. 142(4), 1095 (1998).

38 E. Barbarese, P. E. Braun, and J. H. Carson, "Identification of prelarge and presmall basic proteins in mouse myelin and their structural relationship to large and small basic proteins," Proc. Natl. Acad. Sci. U. S. A 74(8), 3360 (1977).

39 E. Barbarese, J. H. Carson, and P. E. Braun, "Accumulation of the four myelin basic proteins in mouse brain during development," J. Neurochem. 31(4), 779 (1978).

40 D. J. Bare, et al., "p59fyn in rat brain is localized in developing axonal tracts and subpopulations of adult neurons and glia," Oncogene 8(6), 1429 (1993).

41 D. M. Barry, et al., "New movements in neurofilament transport, turnover and disease," Exp. Cell Res. 313(10), 2110 (2007).

42 J. C. Bartolomei and C. A. Greer, "Olfactory ensheathing cells: bridging the gap in spinal cord injury," Neurosurgery 47(5), 1057 (2000).

43 E. Bellone, et al., "A novel mutation (D305V) in the early growth response 2 gene is associated with severe Charcot‐Marie‐Tooth type 1 disease," Hum. Mutat. 14(4), 353 (1999).

44 R. Ben‐Levy, et al., "Nuclear export of the stress‐activated protein kinase p38 mediated by its substrate MAPKAP kinase‐2," Curr. Biol. 8(19), 1049 (1998).

45 Y. Benninger, et al., "Essential and distinct roles for cdc42 and rac1 in the regulation of Schwann cell biology during peripheral nervous system development," J. Cell Biol. 177(6), 1051 (2007).

46 P. Berger, A. Niemann, and U. Suter, "Schwann cells and the pathogenesis of inherited motor and sensory neuropathies (Charcot‐Marie‐Tooth disease)," Glia 54(4), 243 (2006).

242

47 J. Bergoffen, et al., "Connexin mutations in X‐linked Charcot‐Marie‐Tooth disease," Science 262(5142), 2039 (1993).

48 J. R. Bermingham, Jr., et al., "Tst‐1/Oct‐6/SCIP regulates a unique step in peripheral myelination and is required for normal respiration," Genes Dev. 10(14), 1751 (1996).

49 J. R. Bermingham, Jr., et al., "The claw paw mutation reveals a role for Lgi4 in peripheral nerve development," Nat. Neurosci. 9(1), 76 (2006).

50 A. Besson, et al., "p27Kip1 modulates cell migration through the regulation of RhoA activation," Genes Dev. 18(8), 862 (2004).

51 M. A. Bhat, et al., "Discs Lost, a novel multi‐PDZ domain protein, establishes and maintains epithelial polarity," Cell 96(6), 833 (1999).

52 M. A. Bhat, et al., "Axon‐glia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin," Neuron 30(2), 369 (2001).

53 N. R. Bhat, P. Zhang, and S. B. Mohanty, "p38 MAP kinase regulation of oligodendrocyte differentiation with CREB as a potential target," Neurochem. Res. 32(2), 293 (2007).

54 A. Bhattacharyya, et al., "P0 is an early marker of the Schwann cell lineage in chickens," Neuron 7(5), 831 (1991).

55 K. Biffiger, et al., "Severe hypomyelination of the murine CNS in the absence of myelin‐associated glycoprotein and fyn tyrosine kinase," J. Neurosci. 20(19), 7430 (2000).

56 C. Birchmeier and K. A. Nave, "Neuregulin‐1, a key axonal signal that drives Schwann cell growth and differentiation," Glia 56(14), 1491 (2008).

57 M. J. Bitgood and A. P. McMahon, "Hedgehog and Bmp genes are coexpressed at many diverse sites of cell‐cell interaction in the mouse embryo," Dev. Biol. 172(1), 126 (1995).

58 J. A. Black, M. Renganathan, and S. G. Waxman, "Sodium channel Na(v)1.6 is expressed along nonmyelinated axons and it contributes to conduction," Brain Res. Mol. Brain Res. 105(1‐2), 19 (2002).

59 J. A. Black and S. G. Waxman, "The perinodal astrocyte," Glia 1(3), 169 (1988).

60 W. F. Blakemore, P. M. Smith, and R. J. Franklin, "Remyelinating the demyelinated CNS," Novartis. Found. Symp. 231, 289 (2000).

61 A. D. Blanchard, et al., "Oct‐6 (SCIP/Tst‐1) is expressed in Schwann cell precursors, embryonic Schwann cells, and postnatal myelinating Schwann cells: comparison with Oct‐1, Krox‐20, and Pax‐3," J. Neurosci. Res. 46(5), 630 (1996).

62 C. F. Boerkoel, et al., "EGR2 mutation R359W causes a spectrum of Dejerine‐Sottas neuropathy," Neurogenetics. 3(3), 153 (2001).

243

63 C. F. Boerkoel, et al., "Charcot‐Marie‐Tooth disease and related neuropathies: mutation distribution and genotype‐phenotype correlation," Ann. Neurol. 51(2), 190 (2002).

64 C. F. Boerkoel, et al., "Periaxin mutations cause recessive Dejerine‐Sottas neuropathy," Am. J. Hum. Genet. 68(2), 325 (2001).

65 J. M. Boggs, "Myelin basic protein: a multifunctional protein," Cell Mol. Life Sci. 63(17), 1945 (2006).

66 M. A. Bogoyevitch and B. Kobe, "Uses for JNK: the many and varied substrates of the c‐Jun N‐terminal kinases," Microbiol. Mol. Biol. Rev. 70(4), 1061 (2006).

67 D. Bohmann, et al., "Human proto‐oncogene c‐jun encodes a DNA binding protein with structural and functional properties of transcription factor AP‐1," Science 238(4832), 1386 (1987).

68 R. P. Bolande, "Neurocristopathy: its growth and development in 20 years," Pediatr. Pathol. Lab Med. 17(1), 1 (1997).

69 N. Bondurand, et al., "Human Connexin 32, a gap junction protein altered in the X‐ linked form of Charcot‐Marie‐Tooth disease, is directly regulated by the transcription factor SOX10," Hum. Mol. Genet. 10(24), 2783 (2001).

70 L. J. Bone, et al., "New connexin32 mutations associated with X‐linked Charcot‐ Marie‐Tooth disease," Neurology 45(10), 1863 (1995).

71 A. Bosio, E. Binczek, and W. Stoffel, "Functional breakdown of the lipid bilayer of the myelin membrane in central and peripheral nervous system by disrupted galactocerebroside synthesis," Proc. Natl. Acad. Sci. U. S. A 93(23), 13280 (1996).

72 F. Bosse, J. Brodbeck, and H. W. Muller, "Post‐transcriptional regulation of the peripheral myelin protein gene PMP22/gas3," J. Neurosci. Res. 55(2), 164 (1999).

73 F. Bosse, et al., "Differential expression of two mRNA species indicates a dual function of peripheral myelin protein PMP22 in cell growth and myelination," J. Neurosci. Res. 37(4), 529 (1994).

74 H. Bostock, T. A. Sears, and R. M. Sherratt, "The effects of 4‐aminopyridine and tetraethylammonium ions on normal and demyelinated mammalian nerve fibres," J. Physiol 313, 301 (1981).

75 J. M. Boutry, et al., "Establishment and characterization of a mouse Schwann cell line which produces myelin in vivo," J. Neurosci. Res. 32(1), 15 (1992).

76 M. A. Bowe, D. B. Mendis, and J. R. Fallon, "The small leucine‐rich repeat proteoglycan biglycan binds to alpha‐dystroglycan and is upregulated in dystrophic muscle," J. Cell Biol. 148(4), 801 (2000).

77 M. E. Boyle, et al., "Contactin orchestrates assembly of the septate‐like junctions at the paranode in myelinated peripheral nerve," Neuron 30(2), 385 (2001).

244

78 B. Bozon, S. Davis, and S. Laroche, "Regulated transcription of the immediate‐early gene Zif268: mechanisms and gene dosage‐dependent function in synaptic plasticity and memory formation," Hippocampus 12(5), 570 (2002).

79 B. Bozon, S. Davis, and S. Laroche, "A requirement for the immediate early gene zif268 in reconsolidation of recognition memory after retrieval," Neuron 40(4), 695 (2003).

80 B. Bozon, et al., "MAPK, CREB and zif268 are all required for the consolidation of recognition memory," Philos. Trans. R. Soc. Lond B Biol. Sci. 358(1432), 805 (2003).

81 C. Brancolini, et al., "Exposure at the cell surface is required for gas3/PMP22 To regulate both cell death and cell spreading: implication for the Charcot‐ Marie‐Tooth type 1A and Dejerine‐Sottas diseases," Mol. Biol. Cell 11(9), 2901 (2000).

82 C. Brancolini, et al., "Rho‐dependent regulation of cell spreading by the tetraspan membrane protein Gas3/PMP22," Mol. Biol. Cell 10(7), 2441 (1999).

83 S. Britsch, et al., "The transcription factor Sox10 is a key regulator of peripheral glial development," Genes Dev. 15(1), 66 (2001).

84 M. C. Brown, J. A. Perrotta, and C. E. Turner, "Serine and threonine phosphorylation of the paxillin LIM domains regulates paxillin focal adhesion localization and cell adhesion to fibronectin," Mol. Biol. Cell 9(7), 1803 (1998).

85 R. Bruzzone, et al., "Null mutations of connexin32 in patients with X‐linked Charcot‐ Marie‐Tooth disease," Neuron 13(5), 1253 (1994).

86 T. H. Bullock, J. K. Moore, and R. D. Fields, "Evolution of myelin sheaths: both lamprey and hagfish lack myelin," Neurosci. Lett. 48(2), 145 (1984).

87 M. B. Bunge, et al., "A light and electron microscope study of long‐term organized cultures of rat dorsal root ganglia," J. Cell Biol. 32(2), 439 (1967).

88 M. B. Bunge, A. K. Williams, and P. M. Wood, "Neuron‐Schwann cell interaction in basal lamina formation," Dev. Biol. 92(2), 449 (1982).

89 R. P. Bunge, "Expanding roles for the Schwann cell: ensheathment, myelination, trophism and regeneration," Curr. Opin. Neurobiol. 3(5), 805 (1993).

90 R. P. Bunge, "The role of the Schwann cell in trophic support and regeneration," J. Neurol. 242(1 Suppl 1), S19‐S21 (1994).

91 R. P. Bunge, M. B. Bunge, and M. Bates, "Movements of the Schwann cell nucleus implicate progression of the inner (axon‐related) Schwann cell process during myelination," J. Cell Biol. 109(1), 273 (1989).

92 R. P. Bunge, M. B. Bunge, and C. F. Eldridge, "Linkage between axonal ensheathment and basal lamina production by Schwann cells," Annu. Rev. Neurosci. 9, 305 (1986).

245

93 S. Butz, M. Okamoto, and T. C. Sudhof, "A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell adhesion in brain," Cell 94(6), 773 (1998).

94 H. Cai, et al., "The sarcoglycan complex in Schwann cells and its role in myelin stability," Exp. Neurol. 205(1), 257 (2007).

95 K. J. Campbell and N. D. Perkins, "Post‐translational modification of RelA(p65) NF‐ kappaB," Biochem. Soc. Trans. 32(Pt 6), 1087 (2004).

96 K. J. Campbell and N. D. Perkins, "Reprogramming RelA," Cell Cycle 3(7), 869 (2004).

97 L. C. Cantley, "The phosphoinositide 3‐kinase pathway," Science 296(5573), 1655 (2002).

98 D. A. Cantrell, "Phosphoinositide 3‐kinase signalling pathways," J. Cell Sci. 114(Pt 8), 1439 (2001).

99 S. Carenini, et al., "Absence of the myelin‐associated glycoprotein (MAG) and the neural cell adhesion molecule (N‐CAM) interferes with the maintenance, but not with the formation of peripheral myelin," Cell Tissue Res. 287(1), 3 (1997).

100 S. Carenini, et al., "MAG‐deficient Schwann cells myelinate dorsal root ganglion neurons in culture," Glia 22(3), 213 (1998).

101 D. J. Carey, "N‐syndecan: structure and function of a transmembrane heparan sulfate proteoglycan," Perspect. Dev. Neurobiol. 3(4), 331 (1996).

102 D. J. Carey, et al., "Biosynthesis of type IV collagen by cultured rat Schwann cells," J. Cell Biol. 97(2), 473 (1983).

103 D. J. Carey, et al., "Molecular cloning and characterization of N‐syndecan, a novel transmembrane heparan sulfate proteoglycan," J. Cell Biol. 117(1), 191 (1992).

104 D. J. Carey and R. C. Stahl, "Identification of a lipid‐anchored heparan sulfate proteoglycan in Schwann cells," J. Cell Biol. 111(5 Pt 1), 2053 (1990).

105 D. J. Carey, et al., "Processing and subcellular distribution of the Schwann cell lipid‐ anchored heparan sulfate proteoglycan and identification as glypican," Exp. Cell Res. 208(1), 10 (1993).

106 D. J. Carey, et al., "Syndecan‐1 expressed in Schwann cells causes morphological transformation and cytoskeletal reorganization and associates with actin during cell spreading," J. Cell Biol. 124(1‐2), 161 (1994).

107 K. L. Carraway, III, et al., "Neuregulin‐2, a new ligand of ErbB3/ErbB4‐receptor tyrosine kinases," Nature 387(6632), 512 (1997).

108 L. A. Cary, et al., "SRC catalytic but not scaffolding function is needed for integrin‐ regulated tyrosine phosphorylation, cell migration, and cell spreading," Mol. Cell Biol. 22(8), 2427 (2002).

246

109 E. Casanova, et al., "Construction of a conditional allele of RSK‐B/MSK2 in the mouse," Genesis. 32(2), 158 (2002).

110 F. Cavalieri, et al., "Isolation of three new avian sarcoma viruses: ASV 9, ASV 17, and ASV 25," Virology 143(2), 680 (1985).

111 M. E. Cavet, S. Lehoux, and B. C. Berk, "14‐3‐3beta is a p90 ribosomal S6 kinase (RSK) isoform 1‐binding protein that negatively regulates RSK kinase activity," J. Biol. Chem. 278(20), 18376 (2003).

112 J. R. Chan, et al., "Neurotrophins are key mediators of the myelination program in the peripheral nervous system," Proc. Natl. Acad. Sci. U. S. A 98(25), 14661 (2001).

113 P. F. Chance, et al., "DNA deletion associated with hereditary neuropathy with liability to pressure palsies," Cell 72(1), 143 (1993).

114 K. J. Chandross, et al., "TNF alpha inhibits Schwann cell proliferation, connexin46 expression, and gap junctional communication," Mol. Cell Neurosci. 7(6), 479 (1996).

115 P. Chavrier, et al., "Characterization of a mouse multigene family that encodes zinc finger structures," Mol. Cell Biol. 8(3), 1319 (1988).

116 C. Y. Chen and A. B. Shyu, "AU‐rich elements: characterization and importance in mRNA degradation," Trends Biochem. Sci. 20(11), 465 (1995).

117 L. M. Chen, D. Bailey, and C. Fernandez‐Valle, "Association of beta 1 integrin with focal adhesion kinase and paxillin in differentiating Schwann cells," J. Neurosci. 20(10), 3776 (2000).

118 M. Chen, S. C. Chen, and C. J. Pallen, "Integrin‐induced tyrosine phosphorylation of protein‐tyrosine phosphatase‐alpha is required for cytoskeletal reorganization and cell migration," J. Biol. Chem. 281(17), 11972 (2006).

119 S. Chen, et al., "Disruption of ErbB receptor signaling in adult non‐myelinating Schwann cells causes progressive sensory loss," Nat. Neurosci. 6(11), 1186 (2003).

120 S. Chen, et al., "Neuregulin 1‐erbB signaling is necessary for normal myelination and sensory function," J. Neurosci. 26(12), 3079 (2006).

121 Z. Chen, et al., "MAP kinases," Chem. Rev. 101(8), 2449 (2001).

122 Z. L. Chen and S. Strickland, "Laminin gamma1 is critical for Schwann cell differentiation, axon myelination, and regeneration in the peripheral nerve," J. Cell Biol. 163(4), 889 (2003).

123 H. L. Cheng, et al., "Insulin‐like growth factor‐I and Bcl‐X(L) inhibit c‐jun N‐terminal kinase activation and rescue Schwann cells from apoptosis," J. Neurochem. 76(3), 935 (2001).

124 L. Cheng, et al., "Control of Schwann cell survival and proliferation: autocrine factors and neuregulins," Mol. Cell Neurosci. 12(3), 141 (1998).

247

125 G. F. Chernoff, "Shiverer: an autosomal recessive mutant mouse with myelin deficiency," J. Hered. 72(2), 128 (1981).

126 M. A. Chernousov, et al., "Glypican‐1 and alpha4(V) collagen are required for Schwann cell myelination," J. Neurosci. 26(2), 508 (2006).

127 M. A. Chernousov, et al., "Schwann cells synthesize type V collagen that contains a novel alpha 4 chain. Molecular cloning, biochemical characterization, and high affinity heparin binding of alpha 4(V) collagen," J. Biol. Chem. 275(36), 28208 (2000).

128 M. A. Chernousov, et al., "p200, a collagen secreted by Schwann cells, is expressed in developing nerves and in adult nerves following axotomy," J. Neurosci. Res. 56(3), 284 (1999).

129 M. A. Chernousov, R. C. Stahl, and D. J. Carey, "Schwann cells use a novel collagen‐ dependent mechanism for fibronectin fibril assembly," J. Cell Sci. 111 ( Pt 18), 2763 (1998).

130 M. A. Chernousov, et al., "Regulation of Schwann cell function by the extracellular matrix," Glia 56(14), 1498 (2008).

131 S. Y. Chiu and W. Schwarz, "Sodium and potassium currents in acutely demyelinated internodes of rabbit sciatic nerves," J. Physiol 391, 631 (1987).

132 M. B. Clark and M. B. Bunge, "Cultured Schwann cells assemble normal‐appearing basal lamina only when they ensheathe axons," Dev. Biol. 133(2), 393 (1989).

133 T. Coetzee, et al., "Myelination in the absence of galactocerebroside and sulfatide: normal structure with abnormal function and regional instability," Cell 86(2), 209 (1996).

134 P. Cohen and S. Frame, "The renaissance of GSK3," Nat. Rev. Mol. Cell Biol. 2(10), 769 (2001).

135 R. D. Cohn, "Dystroglycan: important player in skeletal muscle and beyond," Neuromuscul. Disord. 15(3), 207 (2005).

136 E. J. Collarini, et al., "Down‐regulation of the POU transcription factor SCIP is an early event in oligodendrocyte differentiation in vitro," Development 116(1), 193 (1992).

137 H. Colognato, et al., "CNS integrins switch growth factor signalling to promote target‐dependent survival," Nat. Cell Biol. 4(11), 833 (2002).

138 H. Colognato, et al., "Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development," J. Cell Biol. 167(2), 365 (2004).

139 O. Coqueret, "New roles for p21 and p27 cell‐cycle inhibitors: a function for each cell compartment?," Trends Cell Biol. 13(2), 65 (2003).

140 G. Corfas, et al., "Mechanisms and roles of axon‐Schwann cell interactions," J. Neurosci. 24(42), 9250 (2004).

248

141 C. J. Cornbrooks, et al., "In vivo and in vitro observations on laminin production by Schwann cells," Proc. Natl. Acad. Sci. U. S. A 80(12), 3850 (1983).

142 C. Correze, J. P. Blondeau, and M. Pomerance, "p38 mitogen‐activated protein kinase contributes to cell cycle regulation by cAMP in FRTL‐5 thyroid cells," Eur. J. Endocrinol. 153(1), 123 (2005).

143 J. M. Cosgaya, J. R. Chan, and E. M. Shooter, "The neurotrophin receptor p75NTR as a positive modulator of myelination," Science 298(5596), 1245 (2002).

144 S. Cotteret and J. Chernoff, "The evolutionary history of effectors downstream of Cdc42 and Rac," Genome Biol. 3(2), REVIEWS0002 (2002).

145 Court FA, et al., "A laminin‐2, dystroglycan, utrophin axis is required for compartmentalization and elongation of myelin segments," J. Neurosci. 29(12), 3908 (2009).

146 Court FA, et al., "Restricted growth of Schwann cells lacking Cajal bands slows conduction in myelinated nerves," Nature 431(7005), 191 (2004).

147 Court FA, L. Wrabetz, and M. L. Feltri, "Basal lamina: Schwann cells wrap to the rhythm of space‐time," Curr. Opin. Neurobiol. 16(5), 501 (2006).

148 G. R. Crabtree and E. N. Olson, "NFAT signaling: choreographing the social lives of cells," Cell 109 Suppl, S67‐S79 (2002).

149 A. Cuenda, et al., "SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin‐1," FEBS Lett. 364(2), 229 (1995).

150 Q. L. Cui and G. Almazan, "IGF‐I‐induced oligodendrocyte progenitor proliferation requires PI3K/Akt, MEK/ERK, and Src‐like tyrosine kinases," J. Neurochem. 100(6), 1480 (2007).

151 Q. L. Cui, et al., "Inhibition of Src‐like kinases reveals Akt‐dependent and ‐ independent pathways in insulin‐like growth factor I‐mediated oligodendrocyte progenitor survival," J. Biol. Chem. 280(10), 8918 (2005).

152 D. D'Urso, P. Ehrhardt, and H. W. Muller, "Peripheral myelin protein 22 and protein zero: a novel association in peripheral nervous system myelin," J. Neurosci. 19(9), 3396 (1999).

153 D. D'Urso, et al., "Overloaded endoplasmic reticulum‐Golgi compartments, a possible pathomechanism of peripheral neuropathies caused by mutations of the peripheral myelin protein PMP22," J. Neurosci. 18(2), 731 (1998).

154 M. Dahme, et al., "Disruption of the mouse L1 gene leads to malformations of the nervous system," Nat. Genet. 17(3), 346 (1997).

155 S. R. Datta, A. Brunet, and M. E. Greenberg, "Cellular survival: a play in three Akts," Genes Dev. 13(22), 2905 (1999).

156 W. Davidson, et al., "Discovery and characterization of a substrate selective p38alpha inhibitor," Biochemistry 43(37), 11658 (2004).

249

157 S. Davis, B. Bozon, and S. Laroche, "How necessary is the activation of the immediate early gene zif268 in synaptic plasticity and learning?," Behav. Brain Res. 142(1‐2), 17 (2003).

158 S. M. de Waegh, V. M. Lee, and S. T. Brady, "Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells," Cell 68(3), 451 (1992).

159 Felipe C. De and S. P. Hunt, "The differential control of c‐jun expression in regenerating sensory neurons and their associated glial cells," J. Neurosci. 14(5 Pt 1), 2911 (1994).

160 Ferra F. de, et al., "Alternative splicing accounts for the four forms of myelin basic protein," Cell 43(3 Pt 2), 721 (1985).

161 M. Deak, et al., "Mitogen‐ and stress‐activated protein kinase‐1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB," EMBO J. 17(15), 4426 (1998).

162 N. O. Deakin and C. E. Turner, "Paxillin comes of age," J. Cell Sci. 121(Pt 15), 2435 (2008).

163 J. L. Dean, et al., "The involvement of AU‐rich element‐binding proteins in p38 mitogen‐activated protein kinase pathway‐mediated mRNA stabilisation," Cell Signal. 16(10), 1113 (2004).

164 L. S. DeBruin, et al., "Developmental partitioning of myelin basic protein into membrane microdomains," J. Neurosci. Res. 80(2), 211 (2005).

165 G. Dechant, A. Rodriguez‐Tebar, and Y. A. Barde, "Neurotrophin receptors," Prog. Neurobiol. 42(2), 347 (1994).

166 L. Decker, et al., "Peripheral myelin maintenance is a dynamic process requiring constant Krox20 expression," J. Neurosci. 26(38), 9771 (2006).

167 M. A. Del Pozo, "Integrin signaling and lipid rafts," Cell Cycle 3(6), 725 (2004).

168 Hertog J. den, et al., "Receptor protein tyrosine phosphatase alpha activates pp60c‐ src and is involved in neuronal differentiation," EMBO J. 12(10), 3789 (1993).

169 E. Denarier, et al., "Functional organization of a Schwann cell enhancer," J. Neurosci. 25(48), 11210 (2005).

170 N. Denisenko‐Nehrbass, et al., "A molecular view on paranodal junctions of myelinated fibers," J. Physiol Paris 96(1‐2), 99 (2002).

171 L. M. Donahue and A. J. Reinhart, "POU domain genes are differentially expressed in the early stages after lineage commitment of the PNS‐derived stem cell line, RT4‐AC," Brain Res. Dev. Brain Res. 106(1‐2), 1 (1998).

172 Z. Dong, et al., "Neu differentiation factor is a neuron‐glia signal and regulates survival, proliferation, and maturation of rat Schwann cell precursors," Neuron 15(3), 585 (1995).

250

173 A. J. Douglas, et al., "Structure and chromosomal localization of the human 2',3'‐ cyclic nucleotide 3'‐phosphodiesterase gene," Ann. Hum. Genet. 56(Pt 3), 243 (1992).

174 A. J. Douglas and R. J. Thompson, "Structure of the myelin membrane enzyme 2',3'‐ cyclic nucleotide 3'‐phosphodiesterase: evidence for two human mRNAs," Biochem. Soc. Trans. 21(2), 295 (1993).

175 H. Duclohier, "Structure‐function studies on the voltage‐gated sodium channel," Biochim. Biophys. Acta 1788(11), 2374 (2009).

176 E. Dupin, et al., "Endothelin 3 induces the reversion of melanocytes to glia through a neural crest‐derived glial‐melanocytic progenitor," Proc. Natl. Acad. Sci. U. S. A 97(14), 7882 (2000).

177 E. Dupin, et al., "Reversal of developmental restrictions in neural crest lineages: transition from Schwann cells to glial‐melanocytic precursors in vitro," Proc. Natl. Acad. Sci. U. S. A 100(9), 5229 (2003).

178 L. Dytrych, et al., "Two PDZ domain proteins encoded by the murine periaxin gene are the result of alternative intron retention and are differentially targeted in Schwann cells," J. Biol. Chem. 273(10), 5794 (1998).

179 J. M. Edgar and J. Garbern, "The myelinated axon is dependent on the myelinating cell for support and maintenance: molecules involved," J. Neurosci. Res. 76(5), 593 (2004).

180 S. Einheber, M. A. Bhat, and J. L. Salzer, "Disrupted Axo‐Glial Junctions Result in Accumulation of Abnormal Mitochondria at Nodes of Ranvier," Neuron Glia Biol. 2(3), 165 (2006).

181 S. Einheber, et al., "Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination," J. Cell Biol. 123(5), 1223 (1993).

182 S. Einheber, et al., "The axonal membrane protein Caspr, a homologue of neurexin IV, is a component of the septate‐like paranodal junctions that assemble during myelination," J. Cell Biol. 139(6), 1495 (1997).

183 C. F. Eldridge, M. B. Bunge, and R. P. Bunge, "Differentiation of axon‐related Schwann cells in vitro: II. Control of myelin formation by basal lamina," J. Neurosci. 9(2), 625 (1989).

184 C. F. Eldridge, et al., "Differentiation of axon‐related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation," J. Cell Biol. 105(2), 1023 (1987).

185 D. J. Epstein, M. Vekemans, and P. Gros, "Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax‐3," Cell 67(4), 767 (1991).

186 R. Erdman, et al., "Schwann cell adhesion to a novel heparan sulfate binding site in the N‐terminal domain of alpha 4 type V collagen is mediated by syndecan‐ 3," J. Biol. Chem. 277(9), 7619 (2002).

251

187 J. M. Ervasti, et al., "Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle," Nature 345(6273), 315 (1990).

188 Y. Eshed, et al., "Gliomedin mediates Schwann cell‐axon interaction and the molecular assembly of the nodes of Ranvier," Neuron 47(2), 215 (2005).

189 R. M. Esper and J. A. Loeb, "Rapid axoglial signaling mediated by neuregulin and neurotrophic factors," J. Neurosci. 24(27), 6218 (2004).

190 R. M. Esper and J. A. Loeb, "Neurotrophins induce neuregulin release through protein kinase Cdelta activation," J. Biol. Chem. 284(39), 26251 (2009).

191 S. Etienne‐Manneville and A. Hall, "Rho GTPases in cell biology," Nature 420(6916), 629 (2002).

192 E. Fabbretti, et al., "Apoptotic phenotype induced by overexpression of wild‐type gas3/PMP22: its relation to the demyelinating peripheral neuropathy CMT1A," Genes Dev. 9(15), 1846 (1995).

193 A. Faissner, et al., "Expression of neural cell adhesion molecule L1 during development, in neurological mutants and in the peripheral nervous system," Brain Res. 317(1), 69 (1984).

194 C. Faivre‐Sarrailh, et al., "The glycosylphosphatidyl inositol‐anchored adhesion molecule F3/contactin is required for surface transport of paranodin/contactin‐associated protein (caspr)," J. Cell Biol. 149(2), 491 (2000).

195 D. L. Falls, "Neuregulins: functions, forms, and signaling strategies," Exp. Cell Res. 284(1), 14 (2003).

196 D. L. Falls, et al., "ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family," Cell 72(5), 801 (1993).

197 A. M. Fannon, et al., "Novel E‐cadherin‐mediated adhesion in peripheral nerve: Schwann cell architecture is stabilized by autotypic adherens junctions," J. Cell Biol. 129(1), 189 (1995).

198 K. Feinberg, et al., "A glial signal consisting of gliomedin and NrCAM clusters axonal Na+ channels during the formation of nodes of Ranvier," Neuron 65(4), 490 (2010).

199 M. L. Feltri, et al., "Conditional disruption of beta 1 integrin in Schwann cells impedes interactions with axons," J. Cell Biol. 156(1), 199 (2002).

200 M. L. Feltri, et al., "Beta 4 integrin expression in myelinating Schwann cells is polarized, developmentally regulated and axonally dependent," Development 120(5), 1287 (1994).

201 M. L. Feltri and L. Wrabetz, "Laminins and their receptors in Schwann cells and hereditary neuropathies," J. Peripher. Nerv. Syst. 10(2), 128 (2005).

202 Z. Feng and C. P. Ko, "Neuronal glia interactions at the vertebrate neuromuscular junction," Curr. Opin. Pharmacol. 7(3), 316 (2007).

252

203 K. J. Fernandes, J. G. Toma, and F. D. Miller, "Multipotent skin‐derived precursors: adult neural crest‐related precursors with therapeutic potential," Philos. Trans. R. Soc. Lond B Biol. Sci. 363(1489), 185 (2008).

204 C. Fernandez‐Valle, et al., "Expression of the protein zero myelin gene in axon‐ related Schwann cells is linked to basal lamina formation," Development 119(3), 867 (1993).

205 C. Fernandez‐Valle, et al., "Actin plays a role in both changes in cell shape and gene‐ expression associated with Schwann cell myelination," J. Neurosci. 17(1), 241 (1997).

206 C. Fernandez‐Valle, et al., "Anti‐beta 1 integrin antibody inhibits Schwann cell myelination," J. Neurobiol. 25(10), 1207 (1994).

207 C. Fernandez‐Valle, P. M. Wood, and M. B. Bunge, "Localization of focal adhesion kinase in differentiating Schwann cell/neuron cultures," Microsc. Res. Tech. 41(5), 416 (1998).

208 S. N. Fewou, et al., "Myelin protein composition is altered in mice lacking either sulfated or both sulfated and non‐sulfated galactolipids," J. Neurochem. 112(3), 599 (2010).

209 M. T. Filbin and G. I. Tennekoon, "Homophilic adhesion of the myelin P0 protein requires glycosylation of both molecules in the homophilic pair," J. Cell Biol. 122(2), 451 (1993).

210 M. T. Filbin, et al., "Role of myelin P0 protein as a homophilic adhesion molecule," Nature 344(6269), 871 (1990).

211 A. D. Forrest, et al., "Focal adhesion kinase (FAK): A regulator of CNS myelination," J. Neurosci. Res. 87(15), 3456 (2009).

212 G. Fragoso, et al., "p38 mitogen‐activated protein kinase is required for central nervous system myelination," Glia 55(15), 1531 (2007).

213 G. Fragoso, et al., "Inhibition of p38 mitogen‐activated protein kinase interferes with cell shape changes and gene expression associated with Schwann cell myelination," Exp. Neurol. 183(1), 34 (2003).

214 T. Franz and R. Kothary, "Characterization of the neural crest defect in Splotch (Sp1H) mutant mice using a lacZ transgene," Brain Res. Dev. Brain Res. 72(1), 99 (1993).

215 R. Frei, et al., "Myelin formation by Schwann cells in the absence of beta4 integrin," Glia 27(3), 269 (1999).

216 M. Freidin, et al., "Connexin 32 increases the proliferative response of Schwann cells to neuregulin‐1 (Nrg1)," Proc. Natl. Acad. Sci. U. S. A 106(9), 3567 (2009).

217 M. A. Frevel, et al., "p38 Mitogen‐activated protein kinase‐dependent and ‐ independent signaling of mRNA stability of AU‐rich element‐containing transcripts," Mol. Cell Biol. 23(2), 425 (2003).

253

218 F. R. Fricker, et al., "Sensory axon‐derived neuregulin‐1 is required for axoglial signaling and normal sensory function but not for long‐term axon maintenance," J. Neurosci. 29(24), 7667 (2009).

219 H. C. Friedman, et al., "A distinct pattern of trophic factor expression in myelin‐ deficient nerves of Trembler mice: implications for trophic support by Schwann cells," J. Neurosci. 16(17), 5344 (1996).

220 R. P. Friedrich, et al., "The class III POU domain protein Brn‐1 can fully replace the related Oct‐6 during schwann cell development and myelination," Mol. Cell Biol. 25(5), 1821 (2005).

221 A. J. Friessen, W. K. Miskimins, and R. Miskimins, "Cyclin‐dependent kinase inhibitor p27kip1 is expressed at high levels in cells that express a myelinating phenotype," J. Neurosci. Res. 50(3), 373 (1997).

222 Q. Fu, et al., "Control of cholesterol biosynthesis in Schwann cells," J. Neurochem. 71(2), 549 (1998).

223 N. Fujita, et al., "The cytoplasmic domain of the large myelin‐associated glycoprotein isoform is needed for proper CNS but not peripheral nervous system myelination," J. Neurosci. 18(6), 1970 (1998).

224 R. Fukunaga and T. Hunter, "MNK1, a new MAP kinase‐activated protein kinase, isolated by a novel expression screening method for identifying protein kinase substrates," EMBO J. 16(8), 1921 (1997).

225 H. Funakoshi, et al., "Differential expression of mRNAs for neurotrophins and their receptors after axotomy of the sciatic nerve," J. Cell Biol. 123(2), 455 (1993).

226 C. M. Gabriel, N. A. Gregson, and R. A. Hughes, "Anti‐PMP22 antibodies in patients with inflammatory neuropathy," J. Neuroimmunol. 104(2), 139 (2000).

227 M. Gaestel, "MAPKAP kinases ‐ MKs ‐ two's company, three's a crowd," Nat. Rev. Mol. Cell Biol. 7(2), 120 (2006).

228 B. Garbay, et al., "Myelin synthesis in the peripheral nervous system," Prog. Neurobiol. 61(3), 267 (2000).

229 J. Y. Garbern, et al., "Peripheral neuropathy caused by proteolipid protein gene mutations," Ann. N. Y. Acad. Sci. 883, 351 (1999).

230 J. Y. Garbern, et al., "Proteolipid protein is necessary in peripheral as well as central myelin," Neuron 19(1), 205 (1997).

231 M. L. Garcia, et al., "Phosphorylation of highly conserved neurofilament medium KSP repeats is not required for myelin‐dependent radial axonal growth," J. Neurosci. 29(5), 1277 (2009).

232 A. N. Garratt, S. Britsch, and C. Birchmeier, "Neuregulin, a factor with many functions in the life of a schwann cell," Bioessays 22(11), 987 (2000).

254

233 A. N. Garratt, et al., "A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool," J. Cell Biol. 148(5), 1035 (2000).

234 C. L. Gatto, B. J. Walker, and S. Lambert, "Local ERM activation and dynamic growth cones at Schwann cell tips implicated in efficient formation of nodes of Ranvier," J. Cell Biol. 162(3), 489 (2003).

235 J. Georgiou, et al., "Synaptic regulation of glial protein expression in vivo," Neuron 12(2), 443 (1994).

236 N. Gerits, S. Kostenko, and U. Moens, "In vivo functions of mitogen‐activated protein kinases: conclusions from knock‐in and knock‐out mice," Transgenic Res. 16(3), 281 (2007).

237 M. D. Gershon, A. Chalazonitis, and T. P. Rothman, "From neural crest to bowel: development of the enteric nervous system," J. Neurobiol. 24(2), 199 (1993).

238 J. Ghislain and P. Charnay, "Control of myelination in Schwann cells: a Krox20 cis‐ regulatory element integrates Oct6, Brn2 and Sox10 activities," EMBO Rep. 7(1), 52 (2006).

239 J. Ghislain, et al., "Characterisation of cis‐acting sequences reveals a biphasic, axon‐ dependent regulation of Krox20 during Schwann cell development," Development 129(1), 155 (2002).

240 S. Ghosh, M. J. May, and E. B. Kopp, "NF‐kappa B and Rel proteins: evolutionarily conserved mediators of immune responses," Annu. Rev. Immunol. 16, 225 (1998).

241 F. G. Giancotti, "Complexity and specificity of integrin signalling," Nat. Cell Biol. 2(1), E13‐E14 (2000).

242 F. G. Giancotti and E. Ruoslahti, "Integrin signaling," Science 285(5430), 1028 (1999).

243 B. I. Giasson and W. E. Mushynski, "Aberrant stress‐induced phosphorylation of perikaryal neurofilaments," J. Biol. Chem. 271(48), 30404 (1996).

244 K. Giese, J. Pagel, and R. Grosschedl, "Distinct DNA‐binding properties of the high mobility group domain of murine and human SRY sex‐determining factors," Proc. Natl. Acad. Sci. U. S. A 91(8), 3368 (1994).

245 K. P. Giese, et al., "Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons," Cell 71(4), 565 (1992).

246 W. R. Gilbert, et al., "Immunoblot identification of phosphorylated basic proteins of rat and rabbit CNS and PNS myelin: evidence for four phosphorylated basic proteins and P2 in rat PNS myelin," Neurochem. Res. 7(12), 1495 (1982).

247 C. S. Gillespie, et al., "Periaxin, a novel protein of myelinating Schwann cells with a possible role in axonal ensheathment," Neuron 12(3), 497 (1994).

255

248 C. S. Gillespie, et al., "Peripheral demyelination and neuropathic pain behavior in periaxin‐deficient mice," Neuron 26(2), 523 (2000).

249 J. A. Girault and E. Peles, "Development of nodes of Ranvier," Curr. Opin. Neurobiol. 12(5), 476 (2002).

250 M. I. Givogri, et al., "Expression and regulation of golli products of myelin basic protein gene during in vitro development of oligodendrocytes," J. Neurosci. Res. 66(4), 679 (2001).

251 X. Gong, et al., "Mechanisms regulating the nuclear translocation of p38 MAP kinase," J. Cell Biochem. (2010).

252 A. D. Goodearl, et al., "Purification of multiple forms of glial growth factor," J. Biol. Chem. 268(24), 18095 (1993).

253 T. Gordon, T. M. Brushart, and K. M. Chan, "Augmenting nerve regeneration with electrical stimulation," Neurol. Res. 30(10), 1012 (2008).

254 R. M. Gould, A. L. Byrd, and E. Barbarese, "The number of Schmidt‐Lanterman incisures is more than doubled in shiverer PNS myelin sheaths," J. Neurocytol. 24(2), 85 (1995).

255 M. D. Goulding, et al., "Pax‐3, a novel murine DNA binding protein expressed during early neurogenesis," EMBO J. 10(5), 1135 (1991).

256 L. Goutebroze, et al., "Syndecan‐3 and syndecan‐4 are enriched in Schwann cell perinodal processes," BMC. Neurosci. 4, 29 (2003).

257 A. Gow, V. L. Friedrich, Jr., and R. A. Lazzarini, "Myelin basic protein gene contains separate enhancers for oligodendrocyte and Schwann cell expression," J. Cell Biol. 119(3), 605 (1992).

258 I. A. Graef, F. Chen, and G. R. Crabtree, "NFAT signaling in vertebrate development," Curr. Opin. Genet. Dev. 11(5), 505 (2001).

259 S. Greenfield, et al., "Protein composition of myelin of the peripheral nervous system," J. Neurochem. 20(4), 1207 (1973).

260 S. Greenfield, S. W. Brostoff, and E. L. Hogan, "Characterization of the basic proteins from rodent peripheral nervous system myelin," J. Neurochem. 34(2), 453 (1980).

261 S. Greenfield, et al., "Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins," J. Neurochem. 39(5), 1278 (1982).

262 O. Griesbeck, et al., "Expression of neurotrophins in skeletal muscle: quantitative comparison and significance for motoneuron survival and maintenance of function," J. Neurosci. Res. 42(1), 21 (1995).

263 I. R. Griffiths, P. Dickinson, and P. Montague, "Expression of the proteolipid protein gene in glial cells of the post‐natal peripheral nervous system of rodents," Neuropathol. Appl. Neurobiol. 21(2), 97 (1995).

256

264 I. R. Griffiths, P. Dickinson, and P. Montague, "Expression of the proteolipid protein gene in glial cells of the post‐natal peripheral nervous system of rodents," Neuropathol. Appl. Neurobiol. 21(2), 97 (1995).

265 C. A. Grimes and R. S. Jope, "CREB DNA binding activity is inhibited by glycogen synthase kinase‐3 beta and facilitated by lithium," J. Neurochem. 78(6), 1219 (2001).

266 R. Grosschedl, K. Giese, and J. Pagel, "HMG domain proteins: architectural elements in the assembly of nucleoprotein structures," Trends Genet. 10(3), 94 (1994).

267 K. S. Grossmann, et al., "The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin‐1/ErbB signaling throughout Schwann cell development," Proc. Natl. Acad. Sci. U. S. A 106(39), 16704 (2009).

268 M. Grove, et al., "FAK is required for axonal sorting by Schwann cells," J. Cell Biol. 176(3), 277 (2007).

269 R. Guennoun, et al., "Progesterone stimulates Krox‐20 gene expression in Schwann cells," Brain Res. Mol. Brain Res. 90(1), 75 (2001).

270 A. Guilbot, et al., "A mutation in periaxin is responsible for CMT4F, an autosomal recessive form of Charcot‐Marie‐Tooth disease," Hum. Mol. Genet. 10(4), 415 (2001).

271 J. Gunther, "Impulse conduction in the myelinated giant fibers of the earthworm. Structure and function of the dorsal nodes in the median giant fiber," J. Comp Neurol. 168(4), 505 (1976).

272 J. D. Haines, et al., "Mitogen‐Activated Protein Kinase Activated Protein Kinase 2 (MK2) Participates in P38 MAPK Regulated Control of Oligodendrocyte Differentiation,"in 2010).

273 J. D. Haines, et al., "p38 Mitogen‐activated protein kinase regulates myelination," J. Mol. Neurosci. 35(1), 23 (2008).

274 A. Hall, "Rho GTPases and the actin cytoskeleton," Science 279(5350), 509 (1998).

275 K. HAMA, "Some observations on the fine structure of the giant nerve fibers of the earthworm, Eisenia foetida," J. Biophys. Biochem. Cytol. 6(1), 61 (1959).

276 K. HAMA, "The fine structure of the Schwann cell sheath of the nerve fiber in the shrimp (Penaeus japonicus)," J. Cell Biol. 31(3), 624 (1966).

277 C. A. Haney, et al., "Heterophilic binding of L1 on unmyelinated sensory axons mediates Schwann cell adhesion and is required for axonal survival," J. Cell Biol. 146(5), 1173 (1999).

278 D. Harari, et al., "Neuregulin‐4: a novel growth factor that acts through the ErbB‐4 receptor tyrosine kinase," Oncogene 18(17), 2681 (1999).

257

279 G. Harauz, et al., "Myelin basic protein‐diverse conformational states of an intrinsically unstructured protein and its roles in myelin assembly and multiple sclerosis," Micron. 35(7), 503 (2004).

280 K. W. Harder, et al., "Protein‐tyrosine phosphatase alpha regulates Src family kinases and alters cell‐substratum adhesion," J. Biol. Chem. 273(48), 31890 (1998).

281 M. C. Harrisingh, et al., "The Ras/Raf/ERK signalling pathway drives Schwann cell dedifferentiation," EMBO J. 23(15), 3061 (2004).

282 S. Harroch, et al., "No obvious abnormality in mice deficient in receptor protein tyrosine phosphatase beta," Mol. Cell Biol. 20(20), 7706 (2000).

283 A. Heinen, et al., "The cyclin‐dependent kinase inhibitor p57kip2 is a negative regulator of Schwann cell differentiation and in vitro myelination," Proc. Natl. Acad. Sci. U. S. A 105(25), 8748 (2008).

284 A. Heinen, et al., "p57 kip2's role beyond Schwann cell cycle control," Cell Cycle 7(18), 2781 (2008).

285 C. E. Henderson, et al., "GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle," Science 266(5187), 1062 (1994).

286 J. E. Heuser and C. F. Doggenweiler, "The fine structural organization of nerve fibers, sheaths, and glial cells in the prawn, Palaemonetes vulgaris," J. Cell Biol. 30(2), 381 (1966).

287 E. Hitti, et al., "Mitogen‐activated protein kinase‐activated protein kinase 2 regulates tumor necrosis factor mRNA stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/uridine‐rich element," Mol. Cell Biol. 26(6), 2399 (2006).

288 M. E. Hodes and S. R. Dlouhy, "The proteolipid protein gene: double, double, ... and trouble," Am. J. Hum. Genet. 59(1), 12 (1996).

289 M. E. Hodes, V. M. Pratt, and S. R. Dlouhy, "Genetics of Pelizaeus‐Merzbacher disease," Dev. Neurosci. 15(6), 383 (1993).

290 P. N. Hoffman, et al., "Neurofilament gene expression: a major determinant of axonal caliber," Proc. Natl. Acad. Sci. U. S. A 84(10), 3472 (1987).

291 P. N. Hoffman and R. J. Lasek, "The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons," J. Cell Biol. 66(2), 351 (1975).

292 P. G. Hogan, et al., "Transcriptional regulation by calcium, calcineurin, and NFAT," Genes Dev. 17(18), 2205 (2003).

293 A. Hoke, et al., "Glial cell line‐derived neurotrophic factor alters axon schwann cell units and promotes myelination in unmyelinated nerve fibers," J. Neurosci. 23(2), 561 (2003).

258

294 W. E. Holmes, et al., "Identification of heregulin, a specific activator of p185erbB2," Science 256(5060), 1205 (1992).

295 H. M. Hong, et al., "A novel mutation in the connexin 29 gene may contribute to nonsyndromic hearing loss," Hum. Genet. 127(2), 191 (2010).

296 K. Honke, et al., "Paranodal junction formation and spermatogenesis require sulfoglycolipids," Proc. Natl. Acad. Sci. U. S. A 99(7), 4227 (2002).

297 B. Hood, H. B. Levene, and A. D. Levi, "Transplantation of autologous Schwann cells for the repair of segmental peripheral nerve defects," Neurosurg. Focus. 26(2), E4 (2009).

298 I. Horresh, et al., "Organization of myelinated axons by Caspr and Caspr2 requires the cytoskeletal adapter protein 4.1B," J. Neurosci. 30(7), 2480 (2010).

299 I. Horresh, et al., "Multiple molecular interactions determine the clustering of Caspr2 and Kv1 channels in myelinated axons," J. Neurosci. 28(52), 14213 (2008).

300 T. Hoshi, et al., "Nodal protrusions, increased Schmidt‐Lanterman incisures, and paranodal disorganization are characteristic features of sulfatide‐deficient peripheral nerves," Glia 55(6), 584 (2007).

301 H. Houlden and M. M. Reilly, "Molecular genetics of autosomal‐dominant demyelinating Charcot‐Marie‐Tooth disease," Neuromolecular. Med. 8(1‐2), 43 (2006).

302 S. T. Hsieh, T. O. Crawford, and J. W. Griffin, "Neurofilament distribution and organization in the myelinated axons of the peripheral nervous system," Brain Res. 642(1‐2), 316 (1994).

303 S. T. Hsieh, et al., "Regional modulation of neurofilament organization by myelination in normal axons," J. Neurosci. 14(11 Pt 1), 6392 (1994).

304 Q. D. Hu, et al., "F3/contactin acts as a functional ligand for Notch during oligodendrocyte maturation," Cell 115(2), 163 (2003).

305 Y. Hu, et al., "Synergistic interactions of lipids and myelin basic protein," Proc. Natl. Acad. Sci. U. S. A 101(37), 13466 (2004).

306 C. Huang, et al., "Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC‐12 cells," J. Cell Biol. 164(4), 593 (2004).

307 C. Huang, K. Jacobson, and M. D. Schaller, "MAP kinases and cell migration," J. Cell Sci. 117(Pt 20), 4619 (2004).

308 C. Huang, et al., "JNK phosphorylates paxillin and regulates cell migration," Nature 424(6945), 219 (2003).

309 T. Hubert, et al., "Collagens in the developing and diseased nervous system," Cell Mol. Life Sci. 66(7), 1223 (2009).

259

310 L. D. Hudson, et al., "Aberrant splicing of proteolipid protein mRNA in the dysmyelinating jimpy mutant mouse," Proc. Natl. Acad. Sci. U. S. A 84(5), 1454 (1987).

311 R. A. Hughes and D. R. Cornblath, "Guillain‐Barre syndrome," Lancet 366(9497), 1653 (2005).

312 R. A. Hughes, et al., "Pathogenesis of Guillain‐Barre syndrome," J. Neuroimmunol. 100(1‐2), 74 (1999).

313 R. A. Hughes and H. C. Powell, "Experimental allergic neuritis: demyelination induced by P2 alone and non‐specific enhancement by cerebroside," J. Neuropathol. Exp. Neurol. 43(2), 154 (1984).

314 D. J. Hunter, et al., "Structure of myelin P2 protein from equine spinal cord," Acta Crystallogr. D. Biol. Crystallogr. 61(Pt 8), 1067 (2005).

315 O. Ibraghimov‐Beskrovnaya, et al., "Primary structure of dystrophin‐associated glycoproteins linking dystrophin to the extracellular matrix," Nature 355(6362), 696 (1992).

316 M. Imamura, et al., "A sarcoglycan‐dystroglycan complex anchors Dp116 and utrophin in the peripheral nervous system," Hum. Mol. Genet. 9(20), 3091 (2000).

317 Y. Imamura, I. C. Scott, and D. S. Greenspan, "The pro‐alpha3(V) collagen chain. Complete primary structure, expression domains in adult and developing tissues, and comparison to the structures and expression domains of the other types V and XI procollagen chains," J. Biol. Chem. 275(12), 8749 (2000).

318 K. Inoue, et al., "Congenital hypomyelinating neuropathy, central dysmyelination, and Waardenburg‐Hirschsprung disease: phenotypes linked by SOX10 mutation," Ann. Neurol. 52(6), 836 (2002).

319 K. Inoue, Y. Tanabe, and J. R. Lupski, "Myelin deficiencies in both the central and the peripheral nervous systems associated with a SOX10 mutation," Ann. Neurol. 46(3), 313 (1999).

320 K. Itoh, et al., "Disrupted Schwann cell‐axon interactions in peripheral nerves of mice with altered L1‐integrin interactions," Mol. Cell Neurosci. 30(4), 624 (2005).

321 Y. Iwashita, et al., "Schwann cells transplanted into normal and X‐irradiated adult white matter do not migrate extensively and show poor long‐term survival," Exp. Neurol. 164(2), 292 (2000).

322 M. Jaegle, et al., "The POU proteins Brn‐2 and Oct‐6 share important functions in Schwann cell development," Genes Dev. 17(11), 1380 (2003).

323 M. Jaegle, et al., "The POU factor Oct‐6 and Schwann cell differentiation," Science 273(5274), 507 (1996).

260

324 A. B. Jaffe and A. Hall, "Rho GTPases: biochemistry and biology," Annu. Rev. Cell Dev. Biol. 21, 247 (2005).

325 S. W. Jang, et al., "In vivo detection of Egr2 binding to target genes during peripheral nerve myelination," J. Neurochem. 98(5), 1678 (2006).

326 M. L. Jaramillo, et al., "Identification of tyrosine 620 as the major phosphorylation site of myelin‐associated glycoprotein and its implication in interacting with signaling molecules," J. Biol. Chem. 269(44), 27240 (1994).

327 C. J. Jensen, et al., "90‐kDa ribosomal S6 kinase is phosphorylated and activated by 3‐ phosphoinositide‐dependent protein kinase‐1," J. Biol. Chem. 274(38), 27168 (1999).

328 K. R. Jessen and R. Mirsky, "Schwann cells and their precursors emerge as major regulators of nerve development," Trends Neurosci. 22(9), 402 (1999).

329 K. R. Jessen and R. Mirsky, "The origin and development of glial cells in peripheral nerves," Nat. Rev. Neurosci. 6(9), 671 (2005).

330 K. R. Jessen and R. Mirsky, "Negative regulation of myelination: relevance for development, injury, and demyelinating disease," Glia 56(14), 1552 (2008).

331 H. Jiang, et al., "Proteolipid protein mRNA stability is regulated by axonal contact in the rodent peripheral nervous system," J. Neurobiol. 44(1), 7 (2000).

332 K. Jirsova, et al., "Cold jet: a method to obtain pure Schwann cell cultures without the need for cytotoxic, apoptosis‐inducing drug treatment," J. Neurosci. Methods 78(1‐2), 133 (1997).

333 M. Johannessen, M. P. Delghandi, and U. Moens, "What turns CREB on?," Cell Signal. 16(11), 1211 (2004).

334 E. A. Jones, et al., "Interactions of Sox10 and Egr2 in myelin gene regulation," Neuron Glia Biol. 3(4), 377 (2007).

335 M. W. Jones, et al., "A requirement for the immediate early gene Zif268 in the expression of late LTP and long‐term memories," Nat. Neurosci. 4(3), 289 (2001).

336 J. P. Julien and W. E. Mushynski, "Multiple phosphorylation sites in mammalian neurofilament polypeptides," J. Biol. Chem. 257(17), 10467 (1982).

337 J. P. Julien and W. E. Mushynski, "Neurofilaments in health and disease," Prog. Nucleic Acid Res. Mol. Biol. 61, 1 (1998).

338 D. Jung, et al., "Identification and characterization of the dystrophin anchoring site on beta‐dystroglycan," J. Biol. Chem. 270(45), 27305 (1995).

339 M. Jurynczyk and K. Selmaj, "Notch: a new player in MS mechanisms," J. Neuroimmunol. 218(1‐2), 3 (2010).

340 D. Kabzinska, et al., "Charcot‐Marie‐Tooth type 4F disease caused by S399fsx410 mutation in the PRX gene," Neurology 66(5), 745 (2006).

261

341 L. V. Kalia, J. R. Gingrich, and M. W. Salter, "Src in synaptic transmission and plasticity," Oncogene 23(48), 8007 (2004).

342 S. Kalwy, et al., "Myelin basic protein‐related proteins in mouse brain and immune tissues," J. Neurochem. 70(1), 435 (1998).

343 Y. Kamachi, M. Uchikawa, and H. Kondoh, "Pairing SOX off: with partners in the regulation of embryonic development," Trends Genet. 16(4), 182 (2000).

344 J. Kamholz, J. Toffenetti, and R. A. Lazzarini, "Organization and expression of the human myelin basic protein gene," J. Neurosci. Res. 21(1), 62 (1988).

345 Y. J. Kang, et al., "Multiple activation mechanisms of p38alpha mitogen‐activated protein kinase," J. Biol. Chem. 281(36), 26225 (2006).

346 S. C. Kao, et al., "Calcineurin/NFAT signaling is required for neuregulin‐regulated Schwann cell differentiation," Science 323(5914), 651 (2009).

347 A. Keren, Y. Tamir, and E. Bengal, "The p38 MAPK signaling pathway: a major regulator of skeletal muscle development," Mol. Cell Endocrinol. 252(1‐2), 224 (2006).

348 H. A. Kim, et al., "A developmentally regulated switch directs regenerative growth of Schwann cells through cyclin D1," Neuron 26(2), 405 (2000).

349 J. Kim, et al., "SOX10 maintains multipotency and inhibits neuronal differentiation of neural crest stem cells," Neuron 38(1), 17 (2003).

350 C. Kioussi, M. K. Gross, and P. Gruss, "Pax3: a paired domain gene as a regulator in PNS myelination," Neuron 15(3), 553 (1995).

351 A. Kippert, et al., "Rho regulates membrane transport in the endocytic pathway to control plasma membrane specialization in oligodendroglial cells," J. Neurosci. 27(13), 3560 (2007).

352 D. A. Kirschner, et al., "Myelin membrane structure and composition correlated: a phylogenetic study," J. Neurochem. 53(5), 1599 (1989).

353 D. Kiryushko, V. Berezin, and E. Bock, "Regulators of neurite outgrowth: role of cell adhesion molecules," Ann. N. Y. Acad. Sci. 1014, 140 (2004).

354 M. Kishi, et al., "Requirement of Sox2‐mediated signaling for differentiation of early Xenopus neuroectoderm," Development 127(4), 791 (2000).

355 C. Klein, et al., "Process outgrowth of oligodendrocytes is promoted by interaction of fyn kinase with the cytoskeletal protein tau," J. Neurosci. 22(3), 698 (2002).

356 E. Knapska and L. Kaczmarek, "A gene for neuronal plasticity in the mammalian brain: Zif268/Egr‐1/NGFI‐A/Krox‐24/TIS8/ZENK?," Prog. Neurobiol. 74(4), 183 (2004).

357 C. G. Koh, "Rho GTPases and their regulators in neuronal functions and development," Neurosignals. 15(5), 228 (2006).

262

358 M. Komada and P. Soriano, "[Beta]IV‐spectrin regulates sodium channel clustering through ankyrin‐G at axon initial segments and nodes of Ranvier," J. Cell Biol. 156(2), 337 (2002).

359 H. Kondoh and Y. Kamachi, "SOX‐partner code for cell specification: Regulatory target selection and underlying molecular mechanisms," Int. J. Biochem. Cell Biol. 42(3), 391 (2010).

360 G. Koopmans, B. Hasse, and N. Sinis, "Chapter 19: The role of collagen in peripheral nerve repair," Int. Rev. Neurobiol. 87, 363 (2009).

361 E. Kordeli, S. Lambert, and V. Bennett, "AnkyrinG. A new ankyrin gene with neural‐ specific isoforms localized at the axonal initial segment and node of Ranvier," J. Biol. Chem. 270(5), 2352 (1995).

362 A. Kotlyarov and M. Gaestel, "Is MK2 (mitogen‐activated protein kinase‐activated protein kinase 2) the key for understanding post‐transcriptional regulation of gene expression?," Biochem. Soc. Trans. 30(Pt 6), 959 (2002).

363 S. Krause, et al., "Small Rho GTPases are key regulators of peripheral nerve biology in health and disease," J. Peripher. Nerv. Syst. 13(3), 188 (2008).

364 D. Kremer, et al., "p57kip2 is dynamically regulated in experimental autoimmune encephalomyelitis and interferes with oligodendroglial maturation," Proc. Natl. Acad. Sci. U. S. A 106(22), 9087 (2009).

365 V. Krutovskikh and H. Yamasaki, "Connexin gene mutations in human genetic diseases," Mutat. Res. 462(2‐3), 197 (2000).

366 K. Kuhlbrodt, et al., "Cooperative function of POU proteins and SOX proteins in glial cells," J. Biol. Chem. 273(26), 16050 (1998).

367 K. Kuhlbrodt, et al., "Sox10, a novel transcriptional modulator in glial cells," J. Neurosci. 18(1), 237 (1998).

368 G. Kuhn, et al., "Coexpression of PMP22 gene with MBP and P0 during de novo myelination and nerve repair," Glia 8(4), 256 (1993).

369 I. Kulbatski, et al., "Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis," Prog. Histochem. Cytochem. 43(3), 123 (2008).

370 M. S. Kwa, et al., "Investigation of serum response to PMP22, connexin 32 and P(0) in inflammatory neuropathies," J. Neuroimmunol. 116(2), 220 (2001).

371 S. Lambert, J. Q. Davis, and V. Bennett, "Morphogenesis of the node of Ranvier: co‐ clusters of ankyrin and ankyrin‐binding integral proteins define early developmental intermediates," J. Neurosci. 17(18), 7025 (1997).

372 P. D. Lampe and A. F. Lau, "Regulation of gap junctions by phosphorylation of connexins," Arch. Biochem. Biophys. 384(2), 205 (2000).

373 J. Landry and J. Huot, "Modulation of actin dynamics during stress and physiological stimulation by a signaling pathway involving p38 MAP kinase and heat‐ shock protein 27," Biochem. Cell Biol. 73(9‐10), 703 (1995).

263

374 A. A. Lavdas, et al., "Schwann cells engineered to express the cell adhesion molecule L1 accelerate myelination and motor recovery after spinal cord injury," Exp. Neurol. 221(1), 206 (2010).

375 J. N. Lavoie, et al., "Cyclin D1 expression is regulated positively by the p42/p44MAPK and negatively by the p38/HOGMAPK pathway," J. Biol. Chem. 271(34), 20608 (1996).

376 N. M. Le Douarin and C Kalcheim, The Neural Crest, 2nd ed. (Cambridge University Press, Cambridge, United Kingdom, 1999).

377 Douarin N. Le, et al., "Glial cell lineages in the neural crest," Glia 4(2), 175 (1991).

378 N. Le, et al., "Analysis of congenital hypomyelinating Egr2Lo/Lo nerves identifies Sox2 as an inhibitor of Schwann cell differentiation and myelination," Proc. Natl. Acad. Sci. U. S. A 102(7), 2596 (2005).

379 A. C. LeBlanc and J. F. Poduslo, "Axonal modulation of myelin gene expression in the peripheral nerve," J. Neurosci. Res. 26(3), 317 (1990).

380 S. E. LeBlanc, et al., "Direct regulation of myelin protein zero expression by the Egr2 transactivator," J. Biol. Chem. 281(9), 5453 (2006).

381 S. E. LeBlanc, et al., "Regulation of cholesterol/lipid biosynthetic genes by Egr2/Krox20 during peripheral nerve myelination," J. Neurochem. 93(3), 737 (2005).

382 S. E. LeBlanc, R. M. Ward, and J. Svaren, "Neuropathy‐associated Egr2 mutants disrupt cooperative activation of myelin protein zero by Egr2 and Sox10," Mol. Cell Biol. 27(9), 3521 (2007).

383 K. K. Lee, et al., "Dominant‐negative beta1 integrin mice have region‐specific myelin defects accompanied by alterations in MAPK activity," Glia 53(8), 836 (2006).

384 M. Lee, et al., "P0 is constitutively expressed in the rat neural crest and embryonic nerves and is negatively and positively regulated by axons to generate non‐ myelin‐forming and myelin‐forming Schwann cells, respectively," Mol. Cell Neurosci. 8(5), 336 (1997).

385 M. J. Lee, et al., "In early development of the rat mRNA for the major myelin protein P(0) is expressed in nonsensory areas of the embryonic inner ear, notochord, enteric nervous system, and olfactory ensheathing cells," Dev. Dyn. 222(1), 40 (2001).

386 M. M. Lee, A. Badache, and G. H. DeVries, "Phosphorylation of CREB in axon‐induced Schwann cell proliferation," J. Neurosci. Res. 55(6), 702 (1999).

387 M. M. Lee, C. Sato‐Bigbee, and G. H. De Vries, "Schwann cells stimulated by axolemma‐enriched fractions express cyclic AMP responsive element binding protein," J. Neurosci. Res. 46(2), 204 (1996).

264

388 S. Lee and D. M. Helfman, "Cytoplasmic p21Cip1 is involved in Ras‐induced inhibition of the ROCK/LIMK/cofilin pathway," J. Biol. Chem. 279(3), 1885 (2004).

389 S. L. Lee, et al., "Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI‐A (Egr‐1)," Science 273(5279), 1219 (1996).

390 K. R. Legate, et al., "ILK, PINCH and parvin: the tIPP of integrin signalling," Nat. Rev. Mol. Cell Biol. 7(1), 20 (2006).

391 G. Lemke and R. Axel, "Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin," Cell 40(3), 501 (1985).

392 G. Lemke and M. Chao, "Axons regulate Schwann cell expression of the major myelin and NGF receptor genes," Development 102(3), 499 (1988).

393 G. Lemke, et al., "Transcriptional controls underlying Schwann cell differentiation and myelination," Ann. N. Y. Acad. Sci. 605, 248 (1990).

394 G. Levi, et al., "Defective bone formation in Krox‐20 mutant mice," Development 122(1), 113 (1996).

395 G. Levi, et al., "Role of Krox‐20 in endochondral bone formation," Ann. N. Y. Acad. Sci. 785, 288 (1996).

396 J. U. Levi, R. R. Cowden, and G. H. Collins, "The microscopic anatomy and ultrastructure of the nervous system in the earthworm (Lumbricus sp.) with emphasis on the relationship between glial cells and neurons," J. Comp Neurol. 127(4), 489 (1966).

397 C. Li, et al., "Myelin associated glycoprotein modulates glia‐axon contact in vivo," J. Neurosci. Res. 51(2), 210 (1998).

398 C. Li, et al., "Myelination in the absence of myelin‐associated glycoprotein," Nature 369(6483), 747 (1994).

399 J. Li, et al., "Analysis of connexin expression during mouse Schwann cell development identifies connexin29 as a novel marker for the transition of neural crest to precursor cells," Glia 55(1), 93 (2007).

400 L. Li, et al., "Egr3, a synaptic activity regulated transcription factor that is essential for learning and memory," Mol. Cell Neurosci. 35(1), 76 (2007).

401 S. Li, et al., "Laminin‐sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts," J. Cell Biol. 169(1), 179 (2005).

402 X. Liang, N. A. Draghi, and M. D. Resh, "Signaling from integrins to Fyn to Rho family GTPases regulates morphologic differentiation of oligodendrocytes," J. Neurosci. 24(32), 7140 (2004).

403 A. S. Limpert and B. D. Carter, "Axonal neuregulin 1 type III activates NF‐{kappa}B in schwann cells during myelin formation," J. Biol. Chem. (2010).

265

404 J. T. Littleton, M. A. Bhat, and H. J. Bellen, "Deciphering the function of neurexins at cellular junctions," J. Cell Biol. 137(4), 793 (1997).

405 A. Lonigro and J. J. Devaux, "Disruption of neurofascin and gliomedin at nodes of Ranvier precedes demyelination in experimental allergic neuritis," Brain 132(Pt 1), 260 (2009).

406 M. Lopez‐Lago, et al., "Tyrosine phosphorylation mediates both activation and downmodulation of the biological activity of Vav," Mol. Cell Biol. 20(5), 1678 (2000).

407 Z. Lu, et al., "Developmental abnormalities of myelin basic protein expression in fyn knock‐out brain reveal a role of Fyn in posttranscriptional regulation," J. Biol. Chem. 280(1), 389 (2005).

408 Z. Lu, et al., "The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2," Mol. Cell 9(5), 945 (2002).

409 L. Luo, "Rho GTPases in neuronal morphogenesis," Nat. Rev. Neurosci. 1(3), 173 (2000).

410 J. R. Lupski, et al., "DNA duplication associated with Charcot‐Marie‐Tooth disease type 1A," Cell 66(2), 219 (1991).

411 A. Ma, et al., "Serine phosphorylation of focal adhesion kinase in interphase and mitosis: a possible role in modulating binding to p130(Cas)," Mol. Biol. Cell 12(1), 1 (2001).

412 A. D. Ma, et al., "Cytoskeletal reorganization by G protein‐coupled receptors is dependent on phosphoinositide 3‐kinase gamma, a Rac guanosine exchange factor, and Rac," Mol. Cell Biol. 18(8), 4744 (1998).

413 K. Mackay and D. Mochly‐Rosen, "An inhibitor of p38 mitogen‐activated protein kinase protects neonatal cardiac myocytes from ischemia," J. Biol. Chem. 274(10), 6272 (1999).

414 S. Maddika, et al., "Cell survival, cell death and cell cycle pathways are interconnected: implications for cancer therapy," Drug Resist. Updat. 10(1‐ 2), 13 (2007).

415 G. M. Mager, et al., "Active gene repression by the Egr2.NAB complex during peripheral nerve myelination," J. Biol. Chem. 283(26), 18187 (2008).

416 V. Magnaghi, et al., "Progesterone derivatives increase expression of Krox‐20 and Sox‐10 in rat Schwann cells," J. Mol. Neurosci. 31(2), 149 (2007).

417 M. Mahalingam and J. A. Cooper, "Phosphorylation of mammalian eIF4E by Mnk1 and Mnk2: tantalizing prospects for a role in translation," Prog. Mol. Subcell. Biol. 27, 132 (2001).

418 N. K. Mahanthappa, E. S. Anton, and W. D. Matthew, "Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth," J. Neurosci. 16(15), 4673 (1996).

266

419 P. Maher, "p38 mitogen‐activated protein kinase activation is required for fibroblast growth factor‐2‐stimulated cell proliferation but not differentiation," J. Biol. Chem. 274(25), 17491 (1999).

420 S. Maitra, et al., "The AU‐rich element mRNA decay‐promoting activity of BRF1 is regulated by mitogen‐activated protein kinase‐activated protein kinase 2," RNA. 14(5), 950 (2008).

421 E. T. Mambetisaeva, V. Gire, and W. H. Evans, "Multiple connexin expression in peripheral nerve, Schwann cells, and Schwannoma cells," J. Neurosci. Res. 57(2), 166 (1999).

422 M. A. Marchionni, et al., "Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system," Nature 362(6418), 312 (1993).

423 G. S. Maro, et al., "Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS," Nat. Neurosci. 7(9), 930 (2004).

424 C. J. Marshall, "Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal‐regulated kinase activation," Cell 80(2), 179 (1995).

425 R. Martini, "The effect of myelinating Schwann cells on axons," Muscle Nerve 24(4), 456 (2001).

426 R. Martini and S. Carenini, "Formation and maintenance of the myelin sheath in the peripheral nerve: roles of cell adhesion molecules and the gap junction protein connexin 32," Microsc. Res. Tech. 41(5), 403 (1998).

427 R. Martini, et al., "Mice doubly deficient in the genes for P0 and myelin basic protein show that both proteins contribute to the formation of the major dense line in peripheral nerve myelin," J. Neurosci. 15(6), 4488 (1995).

428 R. Martini and M. Schachner, "Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N‐CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve," J. Cell Biol. 103(6 Pt 1), 2439 (1986).

429 R. Martini and M. Schachner, "Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N‐CAM, and myelin‐associated glycoprotein) in regenerating adult mouse sciatic nerve," J. Cell Biol. 106(5), 1735 (1988).

430 R. Martini and M. Schachner, "Molecular bases of myelin formation as revealed by investigations on mice deficient in glial cell surface molecules," Glia 19(4), 298 (1997).

431 R. Martini and K. V. Toyka, "Immune‐mediated components of hereditary demyelinating neuropathies: lessons from animal models and patients," Lancet Neurol. 3(8), 457 (2004).

432 M. C. Marty, et al., "The myelin basic protein gene is expressed in differentiated blood cell lineages and in hemopoietic progenitors," Proc. Natl. Acad. Sci. U. S. A 99(13), 8856 (2002).

267

433 T. Masaki, et al., "Expression of dystroglycan and laminin‐2 in peripheral nerve under axonal degeneration and regeneration," Acta Neuropathol. 99(3), 289 (2000).

434 S. Masui, et al., "Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells," Nat. Cell Biol. 9(6), 625 (2007).

435 M. Mata, D. Alessi, and D. J. Fink, "S100 is preferentially distributed in myelin‐ forming Schwann cells," J. Neurocytol. 19(3), 432 (1990).

436 N. Matsunami, et al., "Peripheral myelin protein‐22 gene maps in the duplication in chromosome 17p11.2 associated with Charcot‐Marie‐Tooth 1A," Nat. Genet. 1(3), 176 (1992).

437 P. Maurel, et al., "Nectin‐like proteins mediate axon Schwann cell interactions along the internode and are essential for myelination," J. Cell Biol. 178(5), 861 (2007).

438 P. Maurel and J. L. Salzer, "Axonal regulation of Schwann cell proliferation and survival and the initial events of myelination requires PI 3‐kinase activity," J. Neurosci. 20(12), 4635 (2000).

439 J. H. McALEAR, N. S. MILBURN, and G. B. CHAPMAN, "The fine structure of Schwann cells, nodes of Ranvier and Schmidt‐Lanterman incisures in the central nervous system of the crab, Cancer irroratus," J. Ultrastruct. Res. 2(2), 171 (1958).

440 M. L. McGarvey, et al., "Synthesis and effects of basement membrane components in cultured rat Schwann cells," Dev. Biol. 105(1), 18 (1984).

441 F. Mechta‐Grigoriou, D. Gerald, and M. Yaniv, "The mammalian Jun proteins: redundancy and specificity," Oncogene 20(19), 2378 (2001).

442 C. Meier, et al., "Developing Schwann cells acquire the ability to survive without axons by establishing an autocrine circuit involving insulin‐like growth factor, neurotrophin‐3, and platelet‐derived growth factor‐BB," J. Neurosci. 19(10), 3847 (1999).

443 C. Melendez‐Vasquez, et al., "Differential expression of proteoglycans at central and peripheral nodes of Ranvier," Glia 52(4), 301 (2005).

444 C. V. Melendez‐Vasquez, S. Einheber, and J. L. Salzer, "Rho kinase regulates schwann cell myelination and formation of associated axonal domains," J. Neurosci. 24(16), 3953 (2004).

445 C. V. Melendez‐Vasquez, et al., "Nodes of Ranvier form in association with ezrin‐ radixin‐moesin (ERM)‐positive Schwann cell processes," Proc. Natl. Acad. Sci. U. S. A 98(3), 1235 (2001).

446 I. N. Melnikova, et al., "Synergistic transcriptional activation by Sox10 and Sp1 family members," Neuropharmacology 39(13), 2615 (2000).

447 M. Menegoz, et al., "Paranodin, a glycoprotein of neuronal paranodal membranes," Neuron 19(2), 319 (1997).

268

448 D. Meyer, et al., "Isoform‐specific expression and function of neuregulin," Development 124(18), 3575 (1997).

449 H. Mi, et al., "Differential distribution of closely related potassium channels in rat Schwann cells," J. Neurosci. 15(5 Pt 2), 3761 (1995).

450 H. Mi, et al., "Inwardly rectifying K+ channels that may participate in K+ buffering are localized in microvilli of Schwann cells," J. Neurosci. 16(8), 2421 (1996).

451 G. V. Michailov, et al., "Axonal neuregulin‐1 regulates myelin sheath thickness," Science 304(5671), 700 (2004).

452 E. Mikesova, et al., "Novel EGR2 mutation R359Q is associated with CMT type 1 and progressive scoliosis," Neuromuscul. Disord. 15(11), 764 (2005).

453 J. Milbrandt, "A nerve growth factor‐induced gene encodes a possible transcriptional regulatory factor," Science 238(4828), 797 (1987).

454 R. J. Milner, et al., "Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein," Cell 42(3), 931 (1985).

455 Y. Min, et al., "Interaction forces and adhesion of supported myelin lipid bilayers modulated by myelin basic protein," Proc. Natl. Acad. Sci. U. S. A 106(9), 3154 (2009).

456 J. H. Miner and J. R. Sanes, "Collagen IV alpha 3, alpha 4, and alpha 5 chains in rodent basal laminae: sequence, distribution, association with laminins, and developmental switches," J. Cell Biol. 127(3), 879 (1994).

457 R. Miskimins, et al., "p27(Kip1) enhances myelin basic protein gene promoter activity," J. Neurosci. Res. 67(1), 100 (2002).

458 R. Mollaaghababa and W. J. Pavan, "The importance of having your SOX on: role of SOX10 in the development of neural crest‐derived melanocytes and glia," Oncogene 22(20), 3024 (2003).

459 A. Molnar, et al., "Cdc42Hs, but not Rac1, inhibits serum‐stimulated cell cycle progression at G1/S through a mechanism requiring p38/RK," J. Biol. Chem. 272(20), 13229 (1997).

460 P. V. Monje, G. Athauda, and P. M. Wood, "Protein kinase A‐mediated gating of neuregulin‐dependent ErbB2‐ErbB3 activation underlies the synergistic action of cAMP on Schwann cell proliferation," J. Biol. Chem. 283(49), 34087 (2008).

461 P. V. Monje, Bunge M. Bartlett, and P. M. Wood, "Cyclic AMP synergistically enhances neuregulin‐dependent ERK and Akt activation and cell cycle progression in Schwann cells," Glia 53(6), 649 (2006).

462 K. R. Monk, et al., "A G protein‐coupled receptor is essential for Schwann cells to initiate myelination," Science 325(5946), 1402 (2009).

269

463 E. S. Monuki, R. Kuhn, and G. Lemke, "Cell‐specific action and mutable structure of a transcription factor effector domain," Proc. Natl. Acad. Sci. U. S. A 90(21), 9978 (1993).

464 E. S. Monuki, et al., "SCIP: a glial POU domain gene regulated by cyclic AMP," Neuron 3(6), 783 (1989).

465 P. Morell and H. Jurevics, "Origin of cholesterol in myelin," Neurochem. Res. 21(4), 463 (1996).

466 L. Morgan, K. R. Jessen, and R. Mirsky, "The effects of cAMP on differentiation of cultured Schwann cells: progression from an early phenotype (04+) to a myelin phenotype (P0+, GFAP‐, N‐CAM‐, NGF‐receptor‐) depends on growth inhibition," J. Cell Biol. 112(3), 457 (1991).

467 N. Movilla and X. R. Bustelo, "Biological and regulatory properties of Vav‐3, a new member of the Vav family of oncoproteins," Mol. Cell Biol. 19(11), 7870 (1999).

468 F. Moya, M. B. Bunge, and R. P. Bunge, "Schwann cells proliferate but fail to differentiate in defined medium," Proc. Natl. Acad. Sci. U. S. A 77(11), 6902 (1980).

469 P. Murphy, et al., "The regulation of Krox‐20 expression reveals important steps in the control of peripheral glial cell development," Development 122(9), 2847 (1996).

470 M. R. Murru, et al., "A novel Cx32 mutation causes X‐linked Charcot‐Marie‐Tooth disease with brainstem involvement and brain magnetic resonance spectroscopy abnormalities," Neurol. Sci. 27(1), 18 (2006).

471 M. Musso, et al., "The D355V mutation decreases EGR2 binding to an element within the Cx32 promoter," Neurobiol. Dis. 8(4), 700 (2001).

472 R. Naef, et al., "Aberrant protein trafficking in Trembler suggests a disease mechanism for hereditary human peripheral neuropathies," Mol. Cell Neurosci. 9(1), 13 (1997).

473 R. Naef and U. Suter, "Many facets of the peripheral myelin protein PMP22 in myelination and disease," Microsc. Res. Tech. 41(5), 359 (1998).

474 R. Nagarajan, et al., "EGR2 mutations in inherited neuropathies dominant‐negatively inhibit myelin gene expression," Neuron 30(2), 355 (2001).

475 R. Nagarajan, et al., "EGR2 mutations in inherited neuropathies dominant‐negatively inhibit myelin gene expression," Neuron 30(2), 355 (2001).

476 V. Narayanan, K. H. Kaestner, and G. I. Tennekoon, "Structure of the mouse myelin P2 protein gene," J. Neurochem. 57(1), 75 (1991).

477 R. Nashmi, O. T. Jones, and M. G. Fehlings, "Abnormal axonal physiology is associated with altered expression and distribution of Kv1.1 and Kv1.2 K+ channels after chronic spinal cord injury," Eur. J. Neurosci. 12(2), 491 (2000).

270

478 K. A. Nave and J. L. Salzer, "Axonal regulation of myelination by neuregulin 1," Curr. Opin. Neurobiol. 16(5), 492 (2006).

479 K. A. Nave and B. D. Trapp, "Axon‐glial signaling and the glial support of axon function," Annu. Rev. Neurosci. 31, 535 (2008).

480 A. R. Nebreda and A. Porras, "p38 MAP kinases: beyond the stress response," Trends Biochem. Sci. 25(6), 257 (2000).

481 S. Nemoto, Z. Sheng, and A. Lin, "Opposing effects of Jun kinase and p38 mitogen‐ activated protein kinases on cardiomyocyte hypertrophy," Mol. Cell Biol. 18(6), 3518 (1998).

482 L. New, et al., "PRAK, a novel protein kinase regulated by the p38 MAP kinase," EMBO J. 17(12), 3372 (1998).

483 V. Ng, et al., "Role of the cell wall phenolic glycolipid‐1 in the peripheral nerve predilection of Mycobacterium leprae," Cell 103(3), 511 (2000).

484 S. M. Nicholson, et al., "Altered gene expression in Schwann cells of connexin32 knockout animals," J. Neurosci. Res. 66(1), 23 (2001).

485 J. C. Nickols, et al., "Activation of the transcription factor NF‐kappaB in Schwann cells is required for peripheral myelin formation," Nat. Neurosci. 6(2), 161 (2003).

486 A. Niemann, P. Berger, and U. Suter, "Pathomechanisms of mutant proteins in Charcot‐Marie‐Tooth disease," Neuromolecular. Med. 8(1‐2), 217 (2006).

487 S. S. Nikam, et al., "The zinc finger transcription factor Zif268/Egr‐1 is essential for Schwann cell expression of the p75 NGF receptor," Mol. Cell Neurosci. 6(4), 337 (1995).

488 R. A. Nixon and S. E. Lewis, "Differential turnover of phosphate groups on neurofilament subunits in mammalian neurons in vivo," J. Biol. Chem. 261(35), 16298 (1986).

489 C. D. Nobes and A. Hall, "Rho, rac and cdc42 GTPases: regulators of actin structures, cell adhesion and motility," Biochem. Soc. Trans. 23(3), 456 (1995).

490 C. D. Nobes and A. Hall, "Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia," Cell 81(1), 53 (1995).

491 C. D. Nobes and A. Hall, "Rho GTPases control polarity, protrusion, and adhesion during cell movement," J. Cell Biol. 144(6), 1235 (1999).

492 A. Nodari, et al., "Alpha6beta4 integrin and dystroglycan cooperate to stabilize the myelin sheath," J. Neurosci. 28(26), 6714 (2008).

493 A. Nodari, et al., "Beta1 integrin activates Rac1 in Schwann cells to generate radial lamellae during axonal sorting and myelination," J. Cell Biol. 177(6), 1063 (2007).

271

494 L. A. Noon and A. C. Lloyd, "Treating leprosy: an Erb‐al remedy?," Trends Pharmacol. Sci. 28(3), 103 (2007).

495 L. Notterpek, et al., "Peripheral myelin protein 22 is a constituent of intercellular junctions in epithelia," Proc. Natl. Acad. Sci. U. S. A 98(25), 14404 (2001).

496 S. D. Novakovic, et al., "Disruption and reorganization of sodium channels in experimental allergic neuritis," Muscle Nerve 21(8), 1019 (1998).

497 K. J. O'Donovan and J. M. Baraban, "Major Egr3 isoforms are generated via alternate translation start sites and differ in their abilities to activate transcription," Mol. Cell Biol. 19(7), 4711 (1999).

498 V. J. Obremski, A. M. Hall, and C. Fernandez‐Valle, "Merlin, the neurofibromatosis type 2 gene product, and beta1 integrin associate in isolated and differentiating Schwann cells," J. Neurobiol. 37(4), 487 (1998).

499 S. Occhi, et al., "Both laminin and Schwann cell dystroglycan are necessary for proper clustering of sodium channels at nodes of Ranvier," J. Neurosci. 25(41), 9418 (2005).

500 T. Ogata, et al., "Opposing extracellular signal‐regulated kinase and Akt pathways control Schwann cell myelination," J. Neurosci. 24(30), 6724 (2004).

501 Y. Ogawa, et al., "Spectrins and ankyrinB constitute a specialized paranodal cytoskeleton," J. Neurosci. 26(19), 5230 (2006).

502 S. Oh, et al., "Changes in permeability caused by connexin 32 mutations underlie X‐ linked Charcot‐Marie‐Tooth disease," Neuron 19(4), 927 (1997).

503 F. X. Omlin, et al., "Immunocytochemical localization of basic protein in major dense line regions of central and peripheral myelin," J. Cell Biol. 95(1), 242 (1982).

504 K. Ono and J. Han, "The p38 signal transduction pathway: activation and function," Cell Signal. 12(1), 1 (2000).

505 K. W. Orford and D. T. Scadden, "Deconstructing stem cell self‐renewal: genetic insights into cell‐cycle regulation," Nat. Rev. Genet. 9(2), 115 (2008).

506 S. Ortega, M. Malumbres, and M. Barbacid, "Cyclin D‐dependent kinases, INK4 inhibitors and cancer," Biochim. Biophys. Acta 1602(1), 73 (2002).

507 T. Osawa and C. Ide, "Changes in thickness of collagen fibrils in the endo‐ and epineurium of the mouse sciatic nerve during development," Acta Anat. (Basel) 125(4), 245 (1986).

508 D. J. Osterhout, et al., "Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase," J. Cell Biol. 145(6), 1209 (1999).

509 H. L. Pahl, "Activators and target genes of Rel/NF‐kappaB transcription factors," Oncogene 18(49), 6853 (1999).

272

510 E. Pannese, "The satellite cells of the sensory ganglia," Adv. Anat. Embryol. Cell Biol. 65, 1 (1981).

511 F. Papastefanaki, et al., "Grafts of Schwann cells engineered to express PSA‐NCAM promote functional recovery after spinal cord injury," Brain 130(Pt 8), 2159 (2007).

512 D. Pareyson, et al., "Cranial nerve involvement in CMT disease type 1 due to early growth response 2 gene mutation," Neurology 54(8), 1696 (2000).

513 D. B. Parkinson, et al., "c‐Jun is a negative regulator of myelination," J. Cell Biol. 181(4), 625 (2008).

514 D. B. Parkinson, et al., "Krox‐20 inhibits Jun‐NH2‐terminal kinase/c‐Jun to control Schwann cell proliferation and death," J. Cell Biol. 164(3), 385 (2004).

515 D. B. Parkinson, et al., "Regulation of the myelin gene periaxin provides evidence for Krox‐20‐independent myelin‐related signalling in Schwann cells," Mol. Cell Neurosci. 23(1), 13 (2003).

516 D. B. Parkinson, et al., "Transforming growth factor beta (TGFbeta) mediates Schwann cell death in vitro and in vivo: examination of c‐Jun activation, interactions with survival signals, and the relationship of TGFbeta‐mediated death to Schwann cell differentiation," J. Neurosci. 21(21), 8572 (2001).

517 D. B. Parkinson, et al., "beta‐Neuregulin and autocrine mediated survival of Schwann cells requires activity of Ets family transcription factors," Mol. Cell Neurosci. 20(1), 154 (2002).

518 E. Parmantier, et al., "Peripheral myelin protein‐22 is expressed in rat and mouse brain and spinal cord motoneurons," Eur. J. Neurosci. 7(5), 1080 (1995).

519 E. Parmantier, et al., "Schwann cell‐derived Desert hedgehog controls the development of peripheral nerve sheaths," Neuron 23(4), 713 (1999).

520 J. T. Parsons, "Focal adhesion kinase: the first ten years," J. Cell Sci. 116(Pt 8), 1409 (2003).

521 J. T. Parsons, et al., "Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement," Oncogene 19(49), 5606 (2000).

522 P. I. Patel, et al., "The gene for the peripheral myelin protein PMP‐22 is a candidate for Charcot‐Marie‐Tooth disease type 1A," Nat. Genet. 1(3), 159 (1992).

523 D. L. Paul, "New functions for gap junctions," Curr. Opin. Cell Biol. 7(5), 665 (1995).

524 R. I. Peirano, et al., "Protein zero gene expression is regulated by the glial transcription factor Sox10," Mol. Cell Biol. 20(9), 3198 (2000).

525 R. I. Peirano and M. Wegner, "The glial transcription factor Sox10 binds to DNA both as monomer and dimer with different functional consequences," Nucleic Acids Res. 28(16), 3047 (2000).

273

526 E. Peles, et al., "Isolation of the neu/HER‐2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells," Cell 69(1), 205 (1992).

527 E. Peles, et al., "Identification of a novel contactin‐associated transmembrane receptor with multiple domains implicated in protein‐protein interactions," EMBO J. 16(5), 978 (1997).

528 E. Peles and J. L. Salzer, "Molecular domains of myelinated axons," Curr. Opin. Neurobiol. 10(5), 558 (2000).

529 E. Perdiguero, et al., "Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38alpha in abrogating myoblast proliferation," EMBO J. 26(5), 1245 (2007).

530 J. A. Pereira, et al., "Integrin‐linked kinase is required for radial sorting of axons and Schwann cell remyelination in the peripheral nervous system," J. Cell Biol. 185(1), 147 (2009).

531 A. Peretz, et al., "Hypomyelination and increased activity of voltage‐gated K(+) channels in mice lacking protein tyrosine phosphatase epsilon," EMBO J. 19(15), 4036 (2000).

532 C. Perrin‐Tricaud, U. Rutishauser, and N. Tricaud, "P120 catenin is required for thickening of Schwann cell myelin," Mol. Cell Neurosci. 35(1), 120 (2007).

533 M. Pertusa, et al., "Transcriptional control of cholesterol biosynthesis in Schwann cells by axonal neuregulin 1," J. Biol. Chem. 282(39), 28768 (2007).

534 A. C. Peterson and G. M. Bray, "Hypomyelination in the peripheral nervous system of shiverer mice and in shiverer in equilibrium normal chimaera," J. Comp Neurol. 227(3), 348 (1984).

535 D. Pham‐Dinh, et al., "Proteolipid DM‐20 predominates over PLP in peripheral nervous system," Neuroreport 2(2), 89 (1991).

536 G. Piaton, et al., "Remyelination in multiple sclerosis," Prog. Brain Res. 175, 453 (2009).

537 B. Pierrat, et al., "RSK‐B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38alpha mitogen‐activated protein kinase (p38alphaMAPK)," J. Biol. Chem. 273(45), 29661 (1998).

538 G. Pimienta and J. Pascual, "Canonical and alternative MAPK signaling," Cell Cycle 6(21), 2628 (2007).

539 V. Pingault, et al., "SOX10 mutations in patients with Waardenburg‐Hirschsprung disease," Nat. Genet. 18(2), 171 (1998).

540 V. Pingault, et al., "The SOX10 transcription factor: evaluation as a candidate gene for central and peripheral hereditary myelin disorders," J. Neurol. 248(6), 496 (2001).

274

541 V. Pingault, et al., "Peripheral neuropathy with hypomyelination, chronic intestinal pseudo‐obstruction and deafness: a developmental "neural crest syndrome" related to a SOX10 mutation," Ann. Neurol. 48(4), 671 (2000).

542 J. L. Podratz, E. Rodriguez, and A. J. Windebank, "Role of the extracellular matrix in myelination of peripheral nerve," Glia 35(1), 35 (2001).

543 R. Poirier, et al., "Distinct functions of egr gene family members in cognitive processes," Front Neurosci. 2(1), 47 (2008).

544 S. Poliak, et al., "Caspr2, a new member of the neurexin superfamily, is localized at the juxtaparanodes of myelinated axons and associates with K+ channels," Neuron 24(4), 1037 (1999).

545 S. Poliak, et al., "Localization of Caspr2 in myelinated nerves depends on axon‐glia interactions and the generation of barriers along the axon," J. Neurosci. 21(19), 7568 (2001).

546 S. Poliak and E. Peles, "The local differentiation of myelinated axons at nodes of Ranvier," Nat. Rev. Neurosci. 4(12), 968 (2003).

547 S. Poliak, et al., "Juxtaparanodal clustering of Shaker‐like K+ channels in myelinated axons depends on Caspr2 and TAG‐1," J. Cell Biol. 162(6), 1149 (2003).

548 S. C. Previtali, et al., "Schwann cells synthesize alpha7beta1 integrin which is dispensable for peripheral nerve development and myelination," Mol. Cell Neurosci. 23(2), 210 (2003).

549 S. C. Previtali, et al., "Role of integrins in the peripheral nervous system," Prog. Neurobiol. 64(1), 35 (2001).

550 S. C. Previtali, et al., "Expression of laminin receptors in schwann cell differentiation: evidence for distinct roles," J. Neurosci. 23(13), 5520 (2003).

551 C. Puckett, et al., "Myelin‐specific proteolipid protein is expressed in myelinating Schwann cells but is not incorporated into myelin sheaths," J. Neurosci. Res. 18(4), 511 (1987).

552 B. J. Pulverer, et al., "Phosphorylation of c‐jun mediated by MAP kinases," Nature 353(6345), 670 (1991).

553 S. Pyronnet, "Phosphorylation of the cap‐binding protein eIF4E by the MAPK‐ activated protein kinase Mnk1," Biochem. Pharmacol. 60(8), 1237 (2000).

554 R. H. Quarles, "Myelin‐associated glycoprotein in development and disease," Dev. Neurosci. 6(6), 285 (1983).

555 R. H. Quarles, "Myelin‐associated glycoprotein (MAG): past, present and beyond," J. Neurochem. 100(6), 1431 (2007).

556 A. Quattrini, et al., "Peripheral nerve dysmyelination due to P0 glycoprotein overexpression is dose‐dependent," Ann. N. Y. Acad. Sci. 883, 294 (1999).

275

557 S. Quintes, et al., "Neuron‐glia signaling and the protection of axon function by Schwann cells," J. Peripher. Nerv. Syst. 15(1), 10 (2010).

558 M. Rahmatullah, et al., "Synergistic regulation of Schwann cell proliferation by heregulin and forskolin," Mol. Cell Biol. 18(11), 6245 (1998).

559 M. Raman, W. Chen, and M. H. Cobb, "Differential regulation and properties of MAPKs," Oncogene 26(22), 3100 (2007).

560 A. Rambukkana, et al., "Role of alpha‐dystroglycan as a Schwann cell receptor for Mycobacterium leprae," Science 282(5396), 2076 (1998).

561 Santiago Ramon y Cajal, The Neuron and The Glial Cell (Charles C.Thomas Books, Springfield, Illinois, U.S.A., 1984).

562 M. V. Rao, et al., "The neurofilament middle molecular mass subunit carboxyl‐ terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate," J. Cell Biol. 163(5), 1021 (2003).

563 M. N. Rasband, "It's "juxta" potassium channel!," J. Neurosci. Res. 76(6), 749 (2004).

564 M. N. Rasband, et al., "CNP is required for maintenance of axon‐glia interactions at nodes of Ranvier in the CNS," Glia 50(1), 86 (2005).

565 M. N. Rasband, et al., "Potassium channel distribution, clustering, and function in remyelinating rat axons," J. Neurosci. 18(1), 36 (1998).

566 O. Rausch and C. J. Marshall, "Cooperation of p38 and extracellular signal‐regulated kinase mitogen‐activated protein kinase pathways during granulocyte colony‐stimulating factor‐induced hemopoietic cell proliferation," J. Biol. Chem. 274(7), 4096 (1999).

567 C. Readhead and L. Hood, "The dysmyelinating mouse mutations shiverer (shi) and myelin deficient (shimld)," Behav. Genet. 20(2), 213 (1990).

568 C. Real, et al., "The instability of the neural crest phenotypes: Schwann cells can differentiate into myofibroblasts," Int. J. Dev. Biol. 49(2‐3), 151 (2005).

569 S. Rehberg, et al., "Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10‐mediated transactivation," Mol. Cell Biol. 22(16), 5826 (2002).

570 S. Reiprich, et al., "Activation of Krox20 gene expression by Sox10 in myelinating Schwann cells," J. Neurochem. (2009).

571 S. Reiprich, et al., "Activation of Krox20 gene expression by Sox10 in myelinating Schwann cells," J. Neurochem. 112(3), 744 (2010).

572 C. Ressot and R. Bruzzone, "Connexin channels in Schwann cells and the development of the X‐linked form of Charcot‐Marie‐Tooth disease," Brain Res. Brain Res. Rev. 32(1), 192 (2000).

276

573 S. F. Retta, et al., "Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity," Exp. Cell Res. 229(2), 307 (1996).

574 S. A. Richards, et al., "Characterization of regulatory events associated with membrane targeting of p90 ribosomal S6 kinase 1," Mol. Cell Biol. 21(21), 7470 (2001).

575 S. A. Richards, et al., "Ribosomal S6 kinase 1 (RSK1) activation requires signals dependent on and independent of the MAP kinase ERK," Curr. Biol. 9(15), 810 (1999).

576 K. Riento and A. J. Ridley, "Rocks: multifunctional kinases in cell behaviour," Nat. Rev. Mol. Cell Biol. 4(6), 446 (2003).

577 J. C. Rios, et al., "Paranodal interactions regulate expression of sodium channel subtypes and provide a diffusion barrier for the node of Ranvier," J. Neurosci. 23(18), 7001 (2003).

578 P. Rispoli, et al., "A thermodynamic and structural study of myelin basic protein in lipid membrane models," Biophys. J. 93(6), 1999 (2007).

579 J. M. Ritchie, J. A. Black, and S. G. Waxman, "Sodium channels in the cytoplasm of Schwann cells. Proc. Natl. Acad. Sci. 87 (1990) 9290‐9294," Brain Res. 761(1), 3 (1997).

580 B. B. Roa, C. A. Garcia, and J. R. Lupski, "Charcot‐Marie‐Tooth disease type 1A: molecular mechanisms of gene dosage and point mutation underlying a common inherited peripheral neuropathy," Int. J. Neurol. 2526, 97 (1991).

581 A. Roach, et al., "Chromosomal mapping of mouse myelin basic protein gene and structure and transcription of the partially deleted gene in shiverer mutant mice," Cell 42(1), 149 (1985).

582 F. Robert, et al., "Synthesis of progesterone in Schwann cells: regulation by sensory neurons," Eur. J. Neurosci. 13(5), 916 (2001).

583 P. M. Rodriguez‐Waitkus, et al., "Steroid hormone signaling between Schwann cells and neurons regulates the rate of myelin synthesis," Ann. N. Y. Acad. Sci. 1007, 340 (2003).

584 N. Ronkina, A. Kotlyarov, and M. Gaestel, "MK2 and MK3‐‐a pair of isoenzymes?," Front Biosci. 13, 5511 (2008).

585 J. Rosenbluth, "Intramembranous particle distribution at the node of Ranvier and adjacent axolemma in myelinated axons of the frog brain," J. Neurocytol. 5(6), 731 (1976).

586 J. Rosenbluth, "Central myelin in the mouse mutant shiverer," J. Comp Neurol. 194(3), 639 (1980).

587 J. Rosenbluth, "Peripheral myelin in the mouse mutant Shiverer," J. Comp Neurol. 193(3), 729 (1980).

277

588 K. Rothblum, R. C. Stahl, and D. J. Carey, "Constitutive release of alpha4 type V collagen N‐terminal domain by Schwann cells and binding to cell surface and extracellular matrix heparan sulfate proteoglycans," J. Biol. Chem. 279(49), 51282 (2004).

589 K. J. Roux, et al., "Modulation of epithelial morphology, monolayer permeability, and cell migration by growth arrest specific 3/peripheral myelin protein 22," Mol. Biol. Cell 16(3), 1142 (2005).

590 K. J. Roux, S. A. Amici, and L. Notterpek, "The temporospatial expression of peripheral myelin protein 22 at the developing blood‐nerve and blood‐ brain barriers," J. Comp Neurol. 474(4), 578 (2004).

591 P. P. Roux and J. Blenis, "ERK and p38 MAPK‐activated protein kinases: a family of protein kinases with diverse biological functions," Microbiol. Mol. Biol. Rev. 68(2), 320 (2004).

592 A. K. Ryan and M. G. Rosenfeld, "POU domain family values: flexibility, partnerships, and developmental codes," Genes Dev. 11(10), 1207 (1997).

593 R. A. Saavedra, et al., "The myelin proteins of the shark brain are similar to the myelin proteins of the mammalian peripheral nervous system," J. Mol. Evol. 29(2), 149 (1989).

594 K. Sagane, et al., "Ataxia and peripheral nerve hypomyelination in ADAM22‐deficient mice," BMC. Neurosci. 6, 33 (2005).

595 R. N. Saha, M. Jana, and K. Pahan, "MAPK p38 regulates transcriptional activity of NF‐ kappaB in primary human astrocytes via acetylation of p65," J. Immunol. 179(10), 7101 (2007).

596 G. Saher, et al., "Cholesterol regulates the endoplasmic reticulum exit of the major membrane protein P0 required for peripheral myelin compaction," J. Neurosci. 29(19), 6094 (2009).

597 F. Saito, et al., "Characterization of the transmembrane molecular architecture of the dystroglycan complex in schwann cells," J. Biol. Chem. 274(12), 8240 (1999).

598 F. Saito, et al., "Unique role of dystroglycan in peripheral nerve myelination, nodal structure, and sodium channel stabilization," Neuron 38(5), 747 (2003).

599 R. Saito, N. Fujita, and S. Nagata, "Overexpression of Fyn tyrosine kinase causes abnormal development of primary sensory neurons in Xenopus laevis embryos," Dev. Growth Differ. 43(3), 229 (2001).

600 J. L. Salzer, "Polarized domains of myelinated axons," Neuron 40(2), 297 (2003).

601 J. L. Salzer, P. J. Brophy, and E. Peles, "Molecular domains of myelinated axons in the peripheral nervous system," Glia 56(14), 1532 (2008).

602 T. Samorajski and R. L. Friede, "Size‐dependent distribution of axoplasm, Schwann cell cytoplasm, and mitochondri in the peripheral nerve fibers of mouse," Anat. Rec. 161(3), 281 (1968).

278

603 S. Sancho, P. Young, and U. Suter, "Regulation of Schwann cell proliferation and apoptosis in PMP22‐deficient mice and mouse models of Charcot‐Marie‐ Tooth disease type 1A," Brain 124(Pt 11), 2177 (2001).

604 D. Santamaria and S. Ortega, "Cyclins and CDKS in development and cancer: lessons from genetically modified mice," Front Biosci. 11, 1164 (2006).

605 M. Sasaki, et al., "Remyelination of the injured spinal cord," Prog. Brain Res. 161, 419 (2007).

606 M. D. Schaller, et al., "Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2‐dependent binding of pp60src," Mol. Cell Biol. 14(3), 1680 (1994).

607 G. E. Schepers, R. D. Teasdale, and P. Koopman, "Twenty pairs of sox: extent, homology, and nomenclature of the mouse and human sox transcription factor gene families," Dev. Cell 3(2), 167 (2002).

608 S. S. Scherer, "Nodes, paranodes, and incisures: from form to function," Ann. N. Y. Acad. Sci. 883, 131 (1999).

609 S. S. Scherer, et al., "Connexin32 is a myelin‐related protein in the PNS and CNS," J. Neurosci. 15(12), 8281 (1995).

610 S. S. Scherer, et al., "Axons regulate Schwann cell expression of the POU transcription factor SCIP," J. Neurosci. 14(4), 1930 (1994).

611 S. S. Scherer, et al., "Ezrin, radixin, and moesin are components of Schwann cell microvilli," J. Neurosci. Res. 65(2), 150 (2001).

612 S. S. Scherer, et al., "Periaxin expression in myelinating Schwann cells: modulation by axon‐glial interactions and polarized localization during development," Development 121(12), 4265 (1995).

613 S. S. Scherer, et al., "Transgenic expression of human connexin32 in myelinating Schwann cells prevents demyelination in connexin32‐null mice," J. Neurosci. 25(6), 1550 (2005).

614 S. S. Scherer, et al., "Connexin32‐null mice develop demyelinating peripheral neuropathy," Glia 24(1), 8 (1998).

615 S. S. Scherer, et al., "Expression of growth‐associated protein‐43 kD in Schwann cells is regulated by axon‐Schwann cell interactions and cAMP," J. Neurosci. Res. 38(5), 575 (1994).

616 M. R. Schiller, "Coupling receptor tyrosine kinases to Rho GTPases‐‐GEFs what's the link," Cell Signal. 18(11), 1834 (2006).

617 P. Schindler, et al., "Developmental study of proteolipids in bovine brain: a novel proteolipid and DM‐20 appear before proteolipid protein (PLP) during myelination," J. Neurochem. 55(6), 2079 (1990).

279

618 L. S. Schlesinger and M. A. Horwitz, "Phenolic glycolipid‐1 of Mycobacterium leprae binds complement component C3 in serum and mediates phagocytosis by human monocytes," J. Exp. Med. 174(5), 1031 (1991).

619 A. Schmidt and A. Hall, "Guanine nucleotide exchange factors for Rho GTPases: turning on the switch," Genes Dev. 16(13), 1587 (2002).

620 S. Schneider‐Maunoury, et al., "Disruption of Krox‐20 results in alteration of rhombomeres 3 and 5 in the developing hindbrain," Cell 75(6), 1199 (1993).

621 J. Schneider‐Schaulies, Brunn A. von, and M. Schachner, "Recombinant peripheral myelin protein P0 confers both adhesion and neurite outgrowth‐promoting properties," J. Neurosci. Res. 27(3), 286 (1990).

622 J. Schreiber, et al., "Redundancy of class III POU proteins in the oligodendrocyte lineage," J. Biol. Chem. 272(51), 32286 (1997).

623 M. Schumacher, et al., "Progesterone synthesis and myelin formation in peripheral nerves," Brain Res. Brain Res. Rev. 37(1‐3), 343 (2001).

624 M. A. Schwartz, M. D. Schaller, and M. H. Ginsberg, "Integrins: emerging paradigms of signal transduction," Annu. Rev. Cell Dev. Biol. 11, 549 (1995).

625 K. B. Seamon, W. Padgett, and J. W. Daly, "Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells," Proc. Natl. Acad. Sci. U. S. A 78(6), 3363 (1981).

626 J. Sedzik, A. E. Blaurock, and M. Hoechli, "Reconstituted P2/myelin‐lipid multilayers," J. Neurochem. 45(3), 844 (1985).

627 C. Seiwa, et al., "Fyn tyrosine kinase participates in the compact myelin sheath formation in the central nervous system," Neurosci. Res. 37(1), 21 (2000).

628 K. J. Sepp and V. J. Auld, "RhoA and Rac1 GTPases mediate the dynamic rearrangement of actin in peripheral glia," Development 130(9), 1825 (2003).

629 G. Sessa, et al., "Circulating fragments of myelin‐associated alpha 6 beta 4 integrin in Guillain‐Barre syndrome," J. Neuroimmunol. 80(1‐2), 115 (1997).

630 A. Sgambato, et al., "Multiple functions of p27(Kip1) and its alterations in tumor cells: a review," J. Cell Physiol 183(1), 18 (2000).

631 N. M. Shah, et al., "Glial growth factor restricts mammalian neural crest stem cells to a glial fate," Cell 77(3), 349 (1994).

632 D. L. Sherman and P. J. Brophy, "A tripartite nuclear localization signal in the PDZ‐ domain protein L‐periaxin," J. Biol. Chem. 275(7), 4537 (2000).

633 D. L. Sherman, et al., "Specific disruption of a schwann cell dystrophin‐related protein complex in a demyelinating neuropathy," Neuron 30(3), 677 (2001).

280

634 D. L. Sherman, et al., "Neurofascins are required to establish axonal domains for saltatory conduction," Neuron 48(5), 737 (2005).

635 C. J. Sherr and J. M. Roberts, "CDK inhibitors: positive and negative regulators of G1‐ phase progression," Genes Dev. 13(12), 1501 (1999).

636 Y. Shimoji, et al., "A 21‐kDa surface protein of Mycobacterium leprae binds peripheral nerve laminin‐2 and mediates Schwann cell invasion," Proc. Natl. Acad. Sci. U. S. A 96(17), 9857 (1999).

637 A. Shiryaev and U. Moens, "Mitogen‐activated protein kinase p38 and MK2, MK3 and MK5: Menage a trois or menage a quatre?," Cell Signal. (2010).

638 P. Shrager, "Ionic channels and signal conduction in single remyelinating frog nerve fibres," J. Physiol 404, 695 (1988).

639 S. Shuman, M. Hardy, and D. Pleasure, "Peripheral nervous system myelin and Schwann cell glycoproteins: identification by lectin binding and partial purification of a peripheral nervous system myelin‐specific 170,000 molecular weight glycoprotein," J. Neurochem. 41(5), 1277 (1983).

640 M. E. Shy, et al., "Schwann cell expression of PLP1 but not DM20 is necessary to prevent neuropathy," Ann. Neurol. 53(3), 354 (2003).

641 M. E. Shy, et al., "Axon‐Schwann cell interactions regulate the expression of c‐jun in Schwann cells," J. Neurosci. Res. 43(5), 511 (1996).

642 K. Simons and D. Toomre, "Lipid rafts and signal transduction," Nat. Rev. Mol. Cell Biol. 1(1), 31 (2000).

643 H. Skre, "Genetic and clinical aspects of Charcot‐Marie‐Tooth's disease," Clin. Genet. 6(2), 98 (1974).

644 K. Slentz‐Kesler, et al., "Identification of the human Mnk2 gene (MKNK2) through protein interaction with estrogen receptor beta," Genomics 69(1), 63 (2000).

645 M. X. Sliwkowski, et al., "Coexpression of erbB2 and erbB3 proteins reconstitutes a high affinity receptor for heregulin," J. Biol. Chem. 269(20), 14661 (1994).

646 T. Smeal, et al., "Oncogenic and transcriptional cooperation with Ha‐Ras requires phosphorylation of c‐Jun on serines 63 and 73," Nature 354(6353), 494 (1991).

647 C. Smith‐Slatas and E. Barbarese, "Myelin basic protein gene dosage effects in the PNS," Mol. Cell Neurosci. 15(4), 343 (2000).

648 G. J. Snipes, et al., "Characterization of a novel peripheral nervous system myelin protein (PMP‐22/SR13)," J. Cell Biol. 117(1), 225 (1992).

649 A. Sobko, A. Peretz, and B. Attali, "Constitutive activation of delayed‐rectifier potassium channels by a src family tyrosine kinase in Schwann cells," EMBO J. 17(16), 4723 (1998).

281

650 Y. H. Soung, J. L. Clifford, and J. Chung, "Crosstalk between integrin and receptor tyrosine kinase signaling in breast carcinoma progression," BMB. Rep. 43(5), 311 (2010).

651 B. R. Sperber, et al., "A unique role for Fyn in CNS myelination," J. Neurosci. 21(6), 2039 (2001).

652 B. R. Sperber and F. A. McMorris, "Fyn tyrosine kinase regulates oligodendroglial cell development but is not required for morphological differentiation of oligodendrocytes," J. Neurosci. Res. 63(4), 303 (2001).

653 I. Spiegel, et al., "A central role for Necl4 (SynCAM4) in Schwann cell‐axon interaction and myelination," Nat. Neurosci. 10(7), 861 (2007).

654 N. Stahl, J. Harry, and B. Popko, "Quantitative analysis of myelin protein gene expression during development in the rat sciatic nerve," Brain Res. Mol. Brain Res. 8(3), 209 (1990).

655 H. J. Stewart, "Expression of c‐Jun, Jun B, Jun D and cAMP response element binding protein by Schwann cells and their precursors in vivo and in vitro," Eur. J. Neurosci. 7(6), 1366 (1995).

656 H. J. Stewart, et al., "Changes in DNA synthesis rate in the Schwann cell lineage in vivo are correlated with the precursor‐‐Schwann cell transition and myelination," Eur. J. Neurosci. 5(9), 1136 (1993).

657 D. Stokoe, et al., "Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins," FEBS Lett. 313(3), 307 (1992).

658 S. Sugita, et al., "A stoichiometric complex of neurexins and dystroglycan in brain," J. Cell Biol. 154(2), 435 (2001).

659 O. A. Sulaiman and T. Gordon, "Role of chronic Schwann cell denervation in poor functional recovery after nerve injuries and experimental strategies to combat it," Neurosurgery 65(4 Suppl), A105‐A114 (2009).

660 S. Suresh, et al., "Myelin basic protein and myelin protein 2 act synergistically to cause stacking of lipid bilayers," Biochemistry (2010).

661 K. Susuki, et al., "Gangliosides contribute to stability of paranodal junctions and ion channel clusters in myelinated nerve fibers," Glia 55(7), 746 (2007).

662 K. Susuki, et al., "Anti‐GM1 antibodies cause complement‐mediated disruption of sodium channel clusters in peripheral motor nerve fibers," J. Neurosci. 27(15), 3956 (2007).

663 U. Suter and S. S. Scherer, "Disease mechanisms in inherited neuropathies," Nat. Rev. Neurosci. 4(9), 714 (2003).

664 U. Suter, et al., "Regulation of tissue‐specific expression of alternative peripheral myelin protein‐22 (PMP22) gene transcripts by two promoters," J. Biol. Chem. 269(41), 25795 (1994).

282

665 N. Suzuki, et al., "Oct‐6: a POU transcription factor expressed in embryonal stem cells and in the developing brain," EMBO J. 9(11), 3723 (1990).

666 J. Svaren and D. Meijer, "The molecular machinery of myelin gene transcription in Schwann cells," Glia 56(14), 1541 (2008).

667 D. E. Syroid, et al., "Cell death in the Schwann cell lineage and its regulation by neuregulin," Proc. Natl. Acad. Sci. U. S. A 93(17), 9229 (1996).

668 A. Tabernero, et al., "The Neuron‐Glia Signal beta Neuregulin Induces Sustained CREB Phosphorylation on Ser‐133 in Cultured Rat Schwann Cells," Mol. Cell Neurosci. 10(5/6), 309 (1998).

669 S. Tait, et al., "An oligodendrocyte cell adhesion molecule at the site of assembly of the paranodal axo‐glial junction," J. Cell Biol. 150(3), 657 (2000).

670 Y. Takeda, et al., "The roles of cell adhesion molecules on the formation of peripheral myelin," Keio J. Med. 50(4), 240 (2001).

671 J. F. Talts, et al., "Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha‐dystroglycan and several extracellular matrix proteins," EMBO J. 18(4), 863 (1999).

672 K. Tamura, et al., "Requirement for p38alpha in erythropoietin expression: a role for stress kinases in erythropoiesis," Cell 102(2), 221 (2000).

673 Y. Tan, et al., "FGF and stress regulate CREB and ATF‐1 via a pathway involving p38 MAP kinase and MAPKAP kinase‐2," EMBO J. 15(17), 4629 (1996).

674 H. Tanaka, et al., "Cytoplasmic p21(Cip1/WAF1) regulates neurite remodeling by inhibiting Rho‐kinase activity," J. Cell Biol. 158(2), 321 (2002).

675 W. Tang, et al., "Connexin29 is highly expressed in cochlear Schwann cells, and it is required for the normal development and function of the auditory nerve of mice," J. Neurosci. 26(7), 1991 (2006).

676 Y. Tao, et al., "Erbin regulates NRG1 signaling and myelination," Proc. Natl. Acad. Sci. U. S. A 106(23), 9477 (2009).

677 N. Tapinos, M. Ohnishi, and A. Rambukkana, "ErbB2 receptor tyrosine kinase signaling mediates early demyelination induced by leprosy bacilli," Nat. Med. 12(8), 961 (2006).

678 N. Tapinos and A. Rambukkana, "Insights into regulation of human Schwann cell proliferation by Erk1/2 via a MEK‐independent and p56Lck‐dependent pathway from leprosy bacilli," Proc. Natl. Acad. Sci. U. S. A 102(26), 9188 (2005).

679 E. F. Targ and J. D. Kocsis, "4‐Aminopyridine leads to restoration of conduction in demyelinated rat sciatic nerve," Brain Res. 328(2), 358 (1985).

680 E. F. Targ and J. D. Kocsis, "Action potential characteristics of demyelinated rat sciatic nerve following application of 4‐aminopyridine," Brain Res. 363(1), 1 (1986).

283

681 C. Taveggia, et al., "Neuregulin‐1 type III determines the ensheathment fate of axons," Neuron 47(5), 681 (2005).

682 A. R. Taylor, S. E. Geden, and C. Fernandez‐Valle, "Formation of a beta1 integrin signaling complex in Schwann cells is independent of rho," Glia 41(1), 94 (2003).

683 V. Taylor, et al., "Epithelial membrane protein‐1, peripheral myelin protein 22, and lens membrane protein 20 define a novel gene family," J. Biol. Chem. 270(48), 28824 (1995).

684 H. Teramoto, et al., "Signaling from the small GTP‐binding proteins Rac1 and Cdc42 to the c‐Jun N‐terminal kinase/stress‐activated protein kinase pathway. A role for mixed lineage kinase 3/protein‐tyrosine kinase 1, a novel member of the mixed lineage kinase family," J. Biol. Chem. 271(44), 27225 (1996).

685 H. Teramoto, et al., "The small GTP‐binding protein rho activates c‐Jun N‐terminal kinases/stress‐activated protein kinases in human kidney 293T cells. Evidence for a Pak‐independent signaling pathway," J. Biol. Chem. 271(42), 25731 (1996).

686 S. R. Tew and T. E. Hardingham, "Regulation of SOX9 mRNA in human articular chondrocytes involving p38 MAPK activation and mRNA stabilization," J. Biol. Chem. 281(51), 39471 (2006).

687 S. M. Thomas and J. S. Brugge, "Cellular functions regulated by Src family kinases," Annu. Rev. Cell Dev. Biol. 13, 513 (1997).

688 T. Thurnherr, et al., "Cdc42 and Rac1 signaling are both required for and act synergistically in the correct formation of myelin sheaths in the CNS," J. Neurosci. 26(40), 10110 (2006).

689 V. Timmerman, et al., "Novel missense mutation in the early growth response 2 gene associated with Dejerine‐Sottas syndrome phenotype," Neurology 52(9), 1827 (1999).

690 S. G. Timsit, et al., "DM‐20 mRNA is expressed during the embryonic development of the nervous system of the mouse," J. Neurochem. 58(3), 1172 (1992).

691 Z. Tiran, et al., "Tyrosine phosphatases epsilon and alpha perform specific and overlapping functions in regulation of voltage‐gated potassium channels in Schwann cells," Mol. Biol. Cell 17(10), 4330 (2006).

692 A. R. Tobler, et al., "Transport of Trembler‐J mutant peripheral myelin protein 22 is blocked in the intermediate compartment and affects the transport of the wild‐type protein by direct interaction," J. Neurosci. 19(6), 2027 (1999).

693 A. Tomar and D. D. Schlaepfer, "Focal adhesion kinase: switching between GAPs and GEFs in the regulation of cell motility," Curr. Opin. Cell Biol. 21(5), 676 (2009).

694 P. Topilko, et al., "Differential regulation of the zinc finger genes Krox‐20 and Krox‐ 24 (Egr‐1) suggests antagonistic roles in Schwann cells," J. Neurosci. Res. 50(5), 702 (1997).

284

695 P. Topilko, et al., "Krox‐20 controls myelination in the peripheral nervous system," Nature 371(6500), 796 (1994).

696 R. L. Touraine, et al., "Neurological phenotype in Waardenburg syndrome type 4 correlates with novel SOX10 truncating mutations and expression in developing brain," Am. J. Hum. Genet. 66(5), 1496 (2000).

697 M. Traka, et al., "The neuronal adhesion protein TAG‐1 is expressed by Schwann cells and oligodendrocytes and is localized to the juxtaparanodal region of myelinated fibers," J. Neurosci. 22(8), 3016 (2002).

698 M. Traka, et al., "Association of TAG‐1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers," J. Cell Biol. 162(6), 1161 (2003).

699 B. D. Trapp, et al., "Co‐localization of the myelin‐associated glycoprotein and the microfilament components, F‐actin and spectrin, in Schwann cells of myelinated nerve fibres," J. Neurocytol. 18(1), 47 (1989).

700 B. D. Trapp, M. Dubois‐Dalcq, and R. H. Quarles, "Ultrastructural localization of P2 protein in actively myelinating rat Schwann cells," J. Neurochem. 43(4), 944 (1984).

701 B. D. Trapp, et al., "P2 protein in oligodendrocytes and myelin of the rabbit central nervous system," J. Neurochem. 40(1), 47 (1983).

702 B. D. Trapp and R. H. Quarles, "Presence of the myelin‐associated glycoprotein correlates with alterations in the periodicity of peripheral myelin," J. Cell Biol. 92(3), 877 (1982).

703 B. D. Trapp, R. H. Quarles, and K. Suzuki, "Immunocytochemical studies of quaking mice support a role for the myelin‐associated glycoprotein in forming and maintaining the periaxonal space and periaxonal cytoplasmic collar of myelinating Schwann cells," J. Cell Biol. 99(2), 594 (1984).

704 N. Tricaud, et al., "Adherens junctions in myelinating Schwann cells stabilize Schmidt‐Lanterman incisures via recruitment of p120 catenin to E‐ cadherin," J. Neurosci. 25(13), 3259 (2005).

705 M. V. Tsiper and P. D. Yurchenco, "Laminin assembles into separate basement membrane and fibrillar matrices in Schwann cells," J. Cell Sci. 115(Pt 5), 1005 (2002).

706 A. G. Turjanski, J. P. Vaque, and J. S. Gutkind, "MAP kinases and the control of nuclear events," Oncogene 26(22), 3240 (2007).

707 C. E. Turner, "Paxillin and focal adhesion signalling," Nat. Cell Biol. 2(12), E231‐E236 (2000).

708 C. E. Turner, "Paxillin interactions," J. Cell Sci. 113 Pt 23, 4139 (2000).

709 J. C. Ulzheimer, et al., "Altered expression of ion channel isoforms at the node of Ranvier in P0‐deficient myelin mutants," Mol. Cell Neurosci. 25(1), 83 (2004).

285

710 H. Umemori, et al., "Stimulation of myelin basic protein gene transcription by Fyn tyrosine kinase for myelination," J. Neurosci. 19(4), 1393 (1999).

711 H. Umemori, et al., "Initial events of myelination involve Fyn tyrosine kinase signalling," Nature 367(6463), 572 (1994).

712 K. Uyemura, et al., "Comparative studies on the myelin proteins of bovine peripheral nerve and spinal cord," J. Neurochem. 19(11), 2607 (1972).

713 Y. Uziyel, S. Hall, and J. Cohen, "Influence of laminin‐2 on Schwann cell‐axon interactions," Glia 32(2), 109 (2000).

714 I. Vabnick, et al., "Dynamic potassium channel distributions during axonal development prevent aberrant firing patterns," J. Neurosci. 19(2), 747 (1999).

715 B. Vanhaesebroeck and D. R. Alessi, "The PI3K‐PDK1 connection: more than just a road to PKB," Biochem. J. 346 Pt 3, 561 (2000).

716 T. Vartanian, et al., "Axonal neuregulin signals cells of the oligodendrocyte lineage through activation of HER4 and Schwann cells through HER2 and HER3," J. Cell Biol. 137(1), 211 (1997).

717 K. Verhoeven, et al., "Slowed conduction and thin myelination of peripheral nerves associated with mutant rho Guanine‐nucleotide exchange factor 10," Am. J. Hum. Genet. 73(4), 926 (2003).

718 L. Vermeulen, et al., "The versatile role of MSKs in transcriptional regulation," Trends Biochem. Sci. 34(6), 311 (2009).

719 J. D. Verrier, et al., "Peripheral myelin protein 22 is regulated post‐transcriptionally by miRNA‐29a," Glia 57(12), 1265 (2009).

720 P. W. Vesely, et al., "Translational regulation mechanisms of AP‐1 proteins," Mutat. Res. 682(1), 7 (2009).

721 P. Vlachos and B. Joseph, "The Cdk inhibitor p57(Kip2) controls LIM‐kinase 1 activity and regulates actin cytoskeleton dynamics," Oncogene 28(47), 4175 (2009).

722 A. B. Vojtek and J. A. Cooper, "Rho family members: activators of MAP kinase cascades," Cell 82(4), 527 (1995).

723 Wichert G. von, et al., "Force‐dependent integrin‐cytoskeleton linkage formation requires downregulation of focal complex dynamics by Shp2," EMBO J. 22(19), 5023 (2003).

724 S. Vucic, M. C. Kiernan, and D. R. Cornblath, "Guillain‐Barre syndrome: an update," J. Clin. Neurosci. 16(6), 733 (2009).

725 T. V. Waehneldt, "Phylogeny of myelin proteins," Ann. N. Y. Acad. Sci. 605, 15 (1990).

726 Y. Wakamatsu, et al., "Multiple roles of Sox2, an HMG‐box transcription factor in avian neural crest development," Dev. Dyn. 229(1), 74 (2004).

286

727 Y. Wakamatsu, T. M. Maynard, and J. A. Weston, "Fate determination of neural crest cells by NOTCH‐mediated lateral inhibition and asymmetrical cell division during gangliogenesis," Development 127(13), 2811 (2000).

728 S. Walsh and R. Midha, "Use of stem cells to augment nerve injury repair," Neurosurgery 65(4 Suppl), A80‐A86 (2009).

729 H. Wang, et al., "Involvement of the p38 mitogen‐activated protein kinase alpha, beta, and gamma isoforms in myogenic differentiation," Mol. Biol. Cell 19(4), 1519 (2008).

730 P. S. Wang, et al., "Protein‐tyrosine phosphatase alpha acts as an upstream regulator of Fyn signaling to promote oligodendrocyte differentiation and myelination," J. Biol. Chem. 284(48), 33692 (2009).

731 X. Wang and X. Jiang, "PTEN: a default gate‐keeping tumor suppressor with a versatile tail," Cell Res. 18(8), 807 (2008).

732 Y. Wang, et al., "Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen‐activated protein kinase family," J. Biol. Chem. 273(4), 2161 (1998).

733 I. B. Wanner, et al., "Role of N‐cadherin in Schwann cell precursors of growing nerves," Glia 54(5), 439 (2006).

734 L. E. Warner, et al., "Mutations in the early growth response 2 (EGR2) gene are associated with hereditary myelinopathies," Nat. Genet. 18(4), 382 (1998).

735 A. J. Waskiewicz, et al., "Mitogen‐activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2," EMBO J. 16(8), 1909 (1997).

736 S. G. Waxman, "Determinants of conduction velocity in myelinated nerve fibers," Muscle Nerve 3(2), 141 (1980).

737 S. G. Waxman, et al., "Sodium channels and their genes: dynamic expression in the normal nervous system, dysregulation in disease states(1)," Brain Res. 886(1‐2), 5 (2000).

738 S. G. Waxman and J. M. Ritchie, "Molecular dissection of the myelinated axon," Ann. Neurol. 33(2), 121 (1993).

739 D. J. Webb, J. T. Parsons, and A. F. Horwitz, "Adhesion assembly, disassembly and turnover in migrating cells ‐‐ over and over and over again," Nat. Cell Biol. 4(4), E97 (2002).

740 M. Wegner and C. C. Stolt, "From stem cells to neurons and glia: a Soxist's view of neural development," Trends Neurosci. 28(11), 583 (2005).

741 Q. Wei, W. K. Miskimins, and R. Miskimins, "Sox10 acts as a tissue‐specific transcription factor enhancing activation of the myelin basic protein gene promoter by p27Kip1 and Sp1," J. Neurosci. Res. 78(6), 796 (2004).

287

742 J. A. Weiner and J. Chun, "Schwann cell survival mediated by the signaling phospholipid lysophosphatidic acid," Proc. Natl. Acad. Sci. U. S. A 96(9), 5233 (1999).

743 M. J. Weise, et al., "Protein composition of PNS myelin: developmental comparison of control and quaking mice," J. Neurochem. 41(2), 448 (1983).

744 D. Wen, et al., "Neu differentiation factor: a transmembrane glycoprotein containing an EGF domain and an immunoglobulin homology unit," Cell 69(3), 559 (1992).

745 D. Wen, et al., "Structural and functional aspects of the multiplicity of Neu differentiation factors," Mol. Cell Biol. 14(3), 1909 (1994).

746 J. M. White, "ADAMs: modulators of cell‐cell and cell‐matrix interactions," Curr. Opin. Cell Biol. 15(5), 598 (2003).

747 G. R. Wiggin, et al., "MSK1 and MSK2 are required for the mitogen‐ and stress‐ induced phosphorylation of CREB and ATF1 in fibroblasts," Mol. Cell Biol. 22(8), 2871 (2002).

748 G. Wildhardt, et al., "Two different PAX3 gene mutations causing Waardenburg syndrome type I," Mol. Cell Probes 10(3), 229 (1996).

749 C. Williams, S. Aston, and T. D. Rees, "The effect of hematoma on the thickness of pseudosheaths around silicone implants," Plast. Reconstr. Surg. 56(2), 194 (1975).

750 R. A. Williamson, et al., "Dystroglycan is essential for early embryonic development: disruption of Reichert's membrane in Dag1‐null mice," Hum. Mol. Genet. 6(6), 831 (1997).

751 A. J. Windebank, et al., "Myelination determines the caliber of dorsal root ganglion neurons in culture," J. Neurosci. 5(6), 1563 (1985).

752 R. Winzen, et al., "The p38 MAP kinase pathway signals for cytokine‐induced mRNA stabilization via MAP kinase‐activated protein kinase 2 and an AU‐rich region‐targeted mechanism," EMBO J. 18(18), 4969 (1999).

753 R. M. Wolf, et al., "Tyrosine phosphorylation of p190 RhoGAP by Fyn regulates oligodendrocyte differentiation," J. Neurobiol. 49(1), 62 (2001).

754 P. Wood, et al., "Studies of the initiation of myelination by Schwann cells," Ann. N. Y. Acad. Sci. 605, 1 (1990).

755 P. Wood, E. Okada, and R. Bunge, "The use of networks of dissociated rat dorsal root ganglion neurons to induce myelination by oligodencrocytes in culture," Brain Res. 196(1), 247 (1980).

756 P. M. Wood, M. Schachner, and R. P. Bunge, "Inhibition of Schwann cell myelination in vitro by antibody to the L1 adhesion molecule," J. Neurosci. 10(11), 3635 (1990).

288

757 A. Woodhoo, et al., "Notch controls embryonic Schwann cell differentiation, postnatal myelination and adult plasticity," Nat. Neurosci. 12(7), 839 (2009).

758 M. A. Wozniak, et al., "Focal adhesion regulation of cell behavior," Biochim. Biophys. Acta 1692(2‐3), 103 (2004).

759 L. Wrabetz, et al., "P(0) glycoprotein overexpression causes congenital hypomyelination of peripheral nerves," J. Cell Biol. 148(5), 1021 (2000).

760 R. Wu, et al., "The POU gene Brn‐5 is induced by neuregulin and is restricted to myelinating Schwann cells," Mol. Cell Neurosci. 17(4), 683 (2001).

761 Z. Xing, et al., "Direct interaction of v‐Src with the focal adhesion kinase mediated by the Src SH2 domain," Mol. Biol. Cell 5(4), 413 (1994).

762 J. Xu, et al., "A three‐dimensional collagen lattice activates NF‐kappaB in human fibroblasts: role in integrin alpha2 gene expression and tissue remodeling," J. Cell Biol. 140(3), 709 (1998).

763 W. Xu, et al., "Absence of P0 leads to the dysregulation of myelin gene expression and myelin morphogenesis," J. Neurosci. Res. 60(6), 714 (2000).

764 H. Yamada, et al., "Dystroglycan is a dual receptor for agrin and laminin‐2 in Schwann cell membrane," J. Biol. Chem. 271(38), 23418 (1996).

765 H. Yamada, et al., "Dystroglycan is a binding protein of laminin and merosin in peripheral nerve," FEBS Lett. 352(1), 49 (1994).

766 J. Yamauchi, et al., "The neurotrophin‐3 receptor TrkC directly phosphorylates and activates the nucleotide exchange factor Dbs to enhance Schwann cell migration," Proc. Natl. Acad. Sci. U. S. A 102(14), 5198 (2005).

767 J. Yamauchi, J. R. Chan, and E. M. Shooter, "Neurotrophin 3 activation of TrkC induces Schwann cell migration through the c‐Jun N‐terminal kinase pathway," Proc. Natl. Acad. Sci. U. S. A 100(24), 14421 (2003).

768 J. Yamauchi, J. R. Chan, and E. M. Shooter, "Neurotrophins regulate Schwann cell migration by activating divergent signaling pathways dependent on Rho GTPases," Proc. Natl. Acad. Sci. U. S. A 101(23), 8774 (2004).

769 J. Yamauchi, et al., "Ras activation of a Rac1 exchange factor, Tiam1, mediates neurotrophin‐3‐induced Schwann cell migration," Proc. Natl. Acad. Sci. U. S. A 102(41), 14889 (2005).

770 B. D. Yandava, L. L. Billinghurst, and E. Y. Snyder, ""Global" cell replacement is feasible via neural stem cell transplantation: evidence from the dysmyelinated shiverer mouse brain," Proc. Natl. Acad. Sci. U. S. A 96(12), 7029 (1999).

771 D. Yang, et al., "Coordinate control of axon defasciculation and myelination by laminin‐2 and ‐8," J. Cell Biol. 168(4), 655 (2005).

289

772 S. H. Yang, A. D. Sharrocks, and A. J. Whitmarsh, "Transcriptional regulation by the MAP kinase signaling cascades," Gene 320, 3 (2003).

773 A. S. Yap, W. M. Brieher, and B. M. Gumbiner, "Molecular and functional analysis of cadherin‐based adherens junctions," Annu. Rev. Cell Dev. Biol. 13, 119 (1997).

774 X. Yin, et al., "Evolution of a neuroprotective function of central nervous system myelin," J. Cell Biol. 172(3), 469 (2006).

775 X. Yin, et al., "Myelin‐associated glycoprotein is a myelin signal that modulates the caliber of myelinated axons," J. Neurosci. 18(6), 1953 (1998).

776 X. Yin, et al., "P0 protein is required for and can induce formation of schmidt‐ lantermann incisures in myelin internodes," J. Neurosci. 28(28), 7068 (2008).

777 X. Yin, et al., "Schwann cell myelination requires timely and precise targeting of P(0) protein," J. Cell Biol. 148(5), 1009 (2000).

778 T. Yokoo, et al., "p57Kip2 regulates actin dynamics by binding and translocating LIM‐kinase 1 to the nucleus," J. Biol. Chem. 278(52), 52919 (2003).

779 C. Yoon, Z. Korade, and B. D. Carter, "Protein kinase A‐induced phosphorylation of the p65 subunit of nuclear factor‐kappaB promotes Schwann cell differentiation into a myelinating phenotype," J. Neurosci. 28(14), 3738 (2008).

780 M. Yoshida and D. R. Colman, "Parallel evolution and coexpression of the proteolipid proteins and protein zero in vertebrate myelin," Neuron 16(6), 1115 (1996).

781 T. Yoshihara, et al., "A novel missense mutation in the early growth response 2 gene associated with late‐onset Charcot‐‐Marie‐‐Tooth disease type 1," J. Neurol. Sci. 184(2), 149 (2001).

782 T. Yoshimura, M. Satake, and T. Kobayashi, "Connexin43 is another gap junction protein in the peripheral nervous system," J. Neurochem. 67(3), 1252 (1996).

783 J. E. Yoshino, et al., "Localization of 2',3'‐cyclic nucleotide 3'‐phosphodiesterase on cultured Schwann cells," Brain Res. 325(1‐2), 199 (1985).

784 J. E. Yoshino, J. W. Griffin, and G. H. DeVries, "Identification of an axolemma‐enriched fraction from peripheral nerve," J. Neurochem. 41(4), 1126 (1983).

785 W. M. Yu, et al., "Laminin is required for Schwann cell morphogenesis," J. Cell Sci. 122(Pt 7), 929 (2009).

786 W. M. Yu, et al., "Schwann cell‐specific ablation of laminin gamma1 causes apoptosis and prevents proliferation," J. Neurosci. 25(18), 4463 (2005).

290

787 W. M. Yu, et al., "Disruption of laminin in the peripheral nervous system impedes nonmyelinating Schwann cell development and impairs nociceptive sensory function," Glia 57(8), 850 (2009).

788 G. Zanazzi, et al., "Glial growth factor/neuregulin inhibits Schwann cell myelination and induces demyelination," J. Cell Biol. 152(6), 1289 (2001).

789 T. Zarubin and J. Han, "Activation and signaling of the p38 MAP kinase pathway," Cell Res. 15(1), 11 (2005).

790 L. Zeng, et al., "PTP alpha regulates integrin‐stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration," J. Cell Biol. 160(1), 137 (2003).

791 D. Zhang, et al., "Neuregulin‐3 (NRG3): a novel neural tissue‐enriched protein that binds and activates ErbB4," Proc. Natl. Acad. Sci. U. S. A 94(18), 9562 (1997).

792 S. M. Zhang, et al., "Myelin glycoprotein P0 is expressed at early stages of chicken and rat embryogenesis," J. Neurosci. Res. 40(2), 241 (1995).

793 S. Q. Zhang, et al., "Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment," Mol. Cell 13(3), 341 (2004).

794 S. Zhao, A. Fort, and D. C. Spray, "Characteristics of gap junction channels in Schwann cells from wild‐type and connexin‐null mice," Ann. N. Y. Acad. Sci. 883, 533 (1999).

795 Q. Zheng and Y. Zhao, "The diverse biofunctions of LIM domain proteins: determined by subcellular localization and protein‐protein interaction," Biol. Cell 99(9), 489 (2007).

796 X. M. Zheng, Y. Wang, and C. J. Pallen, "Cell transformation and activation of pp60c‐ src by overexpression of a protein tyrosine phosphatase," Nature 359(6393), 336 (1992).

797 G. Zoidl, et al., "Retroviral‐mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth," EMBO J. 14(6), 1122 (1995).

798 G. Zoidl, et al., "Influence of elevated expression of rat wild‐type PMP22 and its mutant PMP22Trembler on cell growth of NIH3T3 fibroblasts," Cell Tissue Res. 287(3), 459 (1997).

799 T. S. Zorick, et al., "The Transcription Factors SCIP and Krox‐20 Mark Distinct Stages and Cell Fates in Schwann Cell Differentiation," Mol. Cell Neurosci. 8(2/3), 129 (1996).

800 S. Zuchner and J. M. Vance, "Molecular genetics of autosomal‐dominant axonal Charcot‐Marie‐Tooth disease," Neuromolecular. Med. 8(1‐2), 63 (2006).

801 S. Zuluaga, et al., "p38alpha MAPK can positively or negatively regulate Rac‐1 activity depending on the presence of serum," FEBS Lett. 581(20), 3819 (2007).

291