University of Nevada, Reno INVESTIGATION of the ROLE for TERMINAL SCHWANN CELLS in NEUROMUSCULAR JUNCTION DEVELOPMENT and DISEAS

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University of Nevada, Reno

INVESTIGATION OF THE ROLE FOR TERMINAL SCHWANN CELLS IN

NEUROMUSCULAR JUNCTION DEVELOPMENT AND DISEASE

A thesis submitted in partial fulfillment of the

requirements for the degree of Master of Science in

Cellular & Molecular Biology

Alexandra Nicole Scurry

Thesis Advisor: Thomas W. Gould, Ph. D.

May, 2016

THE GRADUATE SCHOOL

UNIVERSITY OF NEVADA RENO We recommend that the thesis prepared under our supervision by

ALEXANDRA NICOLE SCURRY

entitled

Investigation of the Role for Terminal Schwan Cells in Neuromuscular Junction Development and Disease

be accepted in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Thomas W. Gould, Ph.D., Advisor

Dennis Mathew, Ph.D., Committee Member

Dean Burkin, Ph.D., Committee Member

Grant Mastick, Ph.D., Committee Member

Normand Leblanc, Ph.D., Graduate School Representative

David W. Zeh, Ph.D., Dean, Graduate School

May, 2016 i

ABSTRACT

The studies performed in this thesis investigate the roles of Schwann cells

(SCs) in the development of the peripheral nervous system and in an inherited peripheral neuropathy, Charcot-Marie-Tooth (CMT) disease. Schwann cells are an integral aspect of the peripheral nervous system and play a large variety of roles to support, maintain, and modulate this system. The experiments performed in this thesis investigated how SCs regulate the development of the neuromuscular junction (NMJ) and maintain early derived NMJs into adulthood.

Differentiation of SCs results in two main subtypes, myelinating or nonmyelinating. The axonal-derived myelinating Schwann cells are essential for the protection, support, and function of motor axons. Axonal-derived nonmyelinating SCs encompass sensory or autonomic nerve bundles and provide structural support to these peripheral nerve subtypes. Nonmyelinating SCs are also found at the motor terminus and are thus known as terminal SCs (TSCs). TSCs are in close proximity with the presynaptic motor nerve and postsynaptic receptor making up the third component of the tripartite synapse. The localization of these cells enables them to directly affect and regulate NMJ survival and function.

Using a murine model deficient in all SC subtypes, the homozygote erbB3

(erbB3-/-) model, we have demonstrated the necessity of SCs for NMJ maintenance ii and viability. However, this model does not allow for the discrimination of roles specific to either axonal SCs or TSCs. We thus applied a unique methodology to isolate the transcriptomes of axonal SCs and TSCs for subsequent RNA sequencing experiments. Analysis of RNA sequencing data revealed 13 and 9 candidate genetic markers specific to TSCs and axonal SCs, respectively.

Finally, we developed novel confocal imaging analysis combined with electron microscopy and electrophysiological methods to examine NMJ deficits in

CMT type 1A (CMT1A). CMT1A is the most common form of CMT and is due to a mutation in the peripheral myelin protein 22 gene, thus affecting SCs. This multifaceted approach allowed us to perform a thorough investigation of the functional and anatomical defects of NMJs in the homozygote Trembler-J mouse.

We found that these mice exhibited symptomology and pathology characteristic of severe hypomyelination despite normal innervation patterns of the NMJ.

Deficits in both structure and function of the NMJ were observed implicating both axonal SCs and TSCs involvement in CMT1A.

iii

TABLE OF CONTENTS

Chapter 1.0

Introduction ...... 1

1.1. Glial cells as major regulator and support cell of the central and

peripheral nervous system ...... 1

1.1.1. Astrocytes...... 2

1.1.2. Oligodendrocytes ...... 7

1.1.3. Microglia ...... 10

1.2. Peripheral glial cells ...... 12

1.2.1. Development of Schwann cells ...... 13

1.2.2. Myelinating axonal Schwann cells ...... 16

1.2.3. Nonmyelinating Remak Schwann cells ...... 20

1.2.4. Nonmyelinating terminal Schwann cells ...... 22

1.3. Neuromuscular junction development ...... 25

1.3.1. Nerve- and muscle-derived factors in NMJ development ..25

1.3.2. Activity-dependent mechanisms of NMJ development ...... 27

1.3.3. Role of Schwann cells in development and maintenance of the

NMJ ...... 29

1.4. Aims of Thesis ...... 31 iv

Chapter 2.0

Characterization of the erbB3-/- Mouse Phenotype During Dramatic

Remodeling in Embryogenesis ...... 44

2.1. Summary ...... 45

2.2. Introduction ...... 46

2.3. Materials and Methods ...... 50

2.4. Results ...... 54

2.5. Discussion ...... 56

Chapter 3.0

Isolation of the Terminal Schwann Cell Transcriptome for the Identification of

Novel Genetic Markers ...... 66

3.1. Summary ...... 67

3.2. Introduction ...... 68

3.3. Materials and Methods ...... 71

3.4. Results ...... 77

3.5. Discussion ...... 79

Chapter 4.0 v

Structural and Functional Abnormalities of the Neuromuscular Junction in the

Trembler-J Homozygote Mouse Model of Congenital Hyopmyelinating

Neuropathy...... 90

4.1. Summary ...... 91

4.2. Introduction ...... 92

4.3. Materials & Methods ...... 96

4.4. Results ...... 104

4.5. Discussion ...... 113

Chapter 5.0

Conclusion ...... 128

References 6.0 ...... 134

LIST OF TABLES

Table 2.1. Top 50 upregulated genes in WT versus erbB3-/- mice ...... 63

Table 3.1. Candidates for TSC genetic markers ...... 87

vii

LIST OF FIGURES

Figure 1.1. Schematic of major astrocyte signaling associated to gliotransmitter release ...... 33

Figure 1.2. Different patterns of myelination on CNS axons ...... 34

Figure 1.3. Polarized microglia play distinct roles in restoration of the neurovascular network after ischemia and other CNS injuries ...... 35

Figure 1.4. Development of myelinating and nonmyelinating Schwann cells

...... 36

Figure 1.5. Control of peripheral nervous system myelination by Schwann cell- axon interactions ...... 37

Figure 1.6. Model of glial-mediated bidirectional modulation of synaptic plasticity ...... 39

Figure 1.7. Synaptogenesis of the neuromuscular junction ...... 40

Figure 1.8. Evoked activity through peripheral nAChRs is required for NMJ degeneration caused by Schwann cell ablation ...... 42

Figure 1.9. Terminal Schwann cells regulate the process of synaptic competition during neuromuscular junction development ...... 43

Figure 2.1. Aberrant developmental patterns followed by complete degeneration of motor nerves in erbB3-/- mice ...... 59 viii

Figure 2.2. Early onset of innervation in erbB3-/- mice...... 60

Figure 2.3. Embryonic erbB3-/- mice exhibit increased widths of endplate band regions in developing diaphragm muscles ...... 61

Figure 2.4. Genes upregulated in WT versus erbB3-/- mice demonstrate a bias toward developmental and cellular processes ...... 62

Figure 3.1. Cre-lox system enables cell-specific transgene expression ...... 82

Figure 3.2. Physical isolation of axonal Schwann cells and terminal Schwann cells enables segregation of ribosomal-tagged Schwann cell subpopulations ...83

Figure 3.3. Specific isolation of SC-labelled ribosomes ...... 84

Figure 3.4. Hierarchical clustering of endplate versus phrenic nerve differential gene expression analysis ...... 85

Figure 3.5. Comparisons of significant changes in gene expression levels between endplate and phrenic nerve samples and between erbB3 wildtype and erbB3-/- samples ...... 86

Figure 4.1. Homozygous Trembler-J mutant (TrJ) mice die early postnatally and exhibit severe hypomyelination without motor pathology at end stage ..119

Figure 4.2. End-stage homozygous Trembler-J mutant (TrJ) mice have fully innervated neuromuscular junctions (NMJs) ...... 120 ix

Figure 4.3. Neuromuscular junctions (NMJs) of end-stage homozygous

Trembler-J mutant (TrJ) mice express normal perisynaptic and postsynaptic markers ...... 121

Figure 4.4. Neuromuscular junctions (NMJs) are reduced in size in end-stage homozygous Trembler-J mutant (TrJ) mice ...... 122

Figure 4.5. Neuromuscular junctions (NMJs) of end-stage homozygous

Trembler-J mutant (TrJ) mice are innervated but contain smaller junctional folds

...... 124

Figure 4.6. Neuromuscular junctions (NMJs) of end-stage homozygous

Trembler-J mutant (TrJ) mice exhibit delayed maturation ...... 125

Figure 4.7. End-stage homozygous Trembler-J mutant (TrJ) mice exhibit physiological deficits at the neuromuscular junctions (NMJs) ...... 126

Figure 4.8. Neuromuscular junctions (NMJs) from end-stage homozygous

Trembler-J mutant (TrJ) mice exhibit greater endplate potentials (EPP) rundown in response to high-frequency stimulation ...... 127 1

Chapter 1.0

INTRODUCTION

1.1. Glial cells as major regulator and support cells of the central and peripheral nervous system

Glial cells are the primary support cell type of the central nervous system

(CNS) and peripheral nervous system (PNS). These cells retain a variety of functions that provide continual support and modulation of neuronal viability and function. The three major types of glial cells in the CNS are astrocytes, oligodendrocytes, and microglia while the major glial cell of the PNS is the

Schwann cell. Each cell type exhibits unique roles that contribute to the overall maintenance and architecture of their respective nervous systems.

1.1.1. Astrocytes

Astrocytes are an essential type of glial cell found in the CNS. These cells are in very close proximity to neurons allowing direct neuronal support and modulation, such as metabolic homeostasis and neurotransmission modulation

(Halassa & Haydon, 2010; Perea, Sur, & Araque, 2014). Additionally, astrocytes are a primary component of the blood brain barrier and directly regulate the structure and function of this barrier in order to maintain the health and viability of the CNS.

While astrocytes cannot elicit action potentials, these glial cells exhibit varying calcium (Ca2+) signals enabling communication between neighboring neurons or other astrocytes. Transmitters released by either neurons or astrocytes activate various receptors expressed by astrocytes, such as NMDA receptors, purinergic receptors, and metabotropic glutamate receptors, leading to increases in intracellular Ca2+ concentration (Figure 1.1). Transient increases of Ca2+ induce propagation of this Ca2+ signal (Ca2+ waves) through other astrocytes via Ca2+ induced Ca2+ release by the endoplasmic reticulum, intracellular signaling molecules like IP3, and Ca2+ induced gliotransmitter release. Propagating Ca2+ signals are essential for efficient communication between astrocytes and continual activation of astrocyte signaling pathways or transmitter release. ATP 3 is a major factor in the activation of these astrocytic Ca2+ waves (Harada, Kamiya,

& Tsuboi, 2015) and enables efficient glial networks.

Astrocyte communication networks function largely through gap junctions. Connexin 43 (Cx43) and connexin 30 (Cx30) are the primary hemichannels expressed by astrocytes that form gap junction channels between these glial cells (Orellana & Stehberg, 2014).These connections between astrocytes allow passage of various signaling molecules, such as Ca2+, glucose, or

IP3, between astrocytes enabling communication (Figure 1.1) (Orellana &

Stehberg, 2014).

Astrocytes modulate neural activity through gliotransmission, or the release of neurotransmitters by astrocytes. The three transmitters classified in astrocytic release are glutamate, D-serine, and ATP. Release of these transmitters induce the activation of neighboring neuronal or astrocyte glutamatergic, purinergic or NMDA receptors. Various mechanisms produce this transmitter release, such as exocytosis, purinergic receptor efflux, or through surface- expressed gap junctions or hemichannels.

Astrocytes express various types of receptors, such as metabotropic glutamate receptors or purinergic receptors (Petzold & Murthy, 2011), allowing for their activation upon neighboring neuronal activity. Changes in astrocytic 4 intracellular calcium stores is observed during this neuronal activity (Perea et al.,

2014) and a significant reduction in this calcium signal occurs upon the antagonism of metabotropic glutamate receptors (Wang et al., 2006).

Furthermore, astrocytes modulate synaptic environments and activity by the uptake of excess neurotransmitters through astrocytic-expressed transporters, such as vesicular glutamate transporters (VGLUTs) (Halassa & Haydon, 2010;

Harada et al., 2015; Murphy-Royal et al., 2015; Rothstein et al., 1994).

Dysregulation of the localization of these glutamate transporters results in aberrant synaptic function and altered time courses of glutamatergic activity

(Murphy-Royal et al., 2015).

A common mechanism of gliotransmission is through exocytosis.

Astrocytes express various SNARE proteins allowing for the generation of the

SNARE complex, a necessary component for exocytosis (Zhang et al., 2004).

These SNARE proteins colocalize with various vesicular transporters, such as

VGLUT1-3, enabling the packaging of astrocytic vesicles with gliotransmitters, such as glutamate and D-serine (Harada et al., 2015; Zhang et al., 2004). Blockade of vesicular ATPase pumps, the pumps that drive vesicular transporters for packaging of synaptic vesicles, reduces secretion of D-serine and glutamate

(Martineau et al., 2013; Parpura & Zorec, 2010) indicating exocytosis in 5 gliotransmitter release. Furthermore, activation of astrocyte-expressed AMPA and metabotropic glutamate receptors induce increases in Ca2+ signals and release of D-serine or glutamate (Halassa & Haydon, 2010; Harada et al., 2015) identifying Ca2+ dependency in this form of gliotransmission. However, astrocyte-mediated neurotransmitter release is still controversial and necessitates more precise and specific methodology to discriminate the exact mechanisms of how and if astrocytes release neurotransmitters in an in vivo system.

Glutamate release can also occur upon low extracellular Ca2+ concentrations and efflux through surface expressed Cx43 hemichannels (Ye,

Wyeth, Baltan-Tekkok, & Ransom, 2003). Activation of P2X7 astrocyte-expressed channels by extracellular ATP results in glutamate efflux through these activated channels (Duan et al., 2003). While extracellular ATP induces Ca2+ waves for signaling within astrocyte communication networks, it is also released by astrocytes through Ca2+ mediated exocytosis (Parpura & Zorec, 2010) or via Cx43

(Torres et al., 2012).

A fundamental component of CNS health and function is the multitude of ways in which astrocytes modulate neuronal and astrocytic communication.

Aberrant gliotransmitter release or hyperactivation of astrocytes (reactive astrocytes) is a key feature of many neurological disorders such as Alzheimer’s 6 disease (Nagele et al., 2004) and epilepsy (Harada et al., 2015). Reactive astrocytes and altered expression of glutamate transporters results in excitoxicity and neuronal damage (Murphy-Royal et al., 2015; Piao et al., 2015). Therefore proper function of astrocytes and their regulatory roles in neural activity is essential.

Astrocytes also maintain the health of the CNS by regulating transport through the blood brain barrier. Astrocytes are a primary component of this barrier and wrap capillaries by their perivascular endfoot processes to provide the CNS with metabolic, structural, and functional support for the exchange of nutrients and oxygen through the bloodstream and the CNS (Abbott,

Patabendige, Dolman, Yusof, & Begley, 2010; Cheslow & Alvarez, 2016).

Astrocytes maintain the homeostasis and structure of the vasculature through release of trophic and structural factors and junctional proteins (Cheslow &

Alvarez, 2016). Astrocytes are an integral component linking the neuronal vasculature to neuronal activity. The neurovascular unit, composed of endothelial cells lining blood vessels and pericytic endfeet of astrocytes, enables complex communication between these various cell types and allows for the dynamic modulation of the blood brain barrier in various physiological settings

(Abbott et al., 2010; Abbott, 2002), including vasodilation or vasoconstriction. 7

1.1.2. Oligodendrocytes

Oligodendrocytes are a subtype of CNS glial cells that ensures myelination of axons. The primary role of these cells is to produce and maintain the myelin sheaths of axons to ensure efficient conductance of the neural signal

(Simons & Nave, 2015). Myelination of axons is a major aspect of proper CNS development and the maintenance of these myelin sheaths throughout a lifetime is required for proper function and axonal viability. To ensure the integrity of myelin, myelin remodeling occurs in the adult human brain by fully differentiated oligodendrocytes, with only 0.3% of oligodendrocyte turnover annually (Yeung et al., 2014).

While the majority of axons in the adult brain are myelinated, stretches of axons are also unmyelinated (Chang, Redmond, & Chan, 2016). Neurons specific to different layers of cortex represent unique patterns of myelination, for instance pyramidal neurons in layers II/III display ~55 µm stretches of unmyelinated axons between internodes resulting in ~35% of myelinated axons in pyramidal neurons from these cortical layers (de Hoz & Simons, 2015). Oligodendrocyte precursor cells (OPCs) are distributed throughout the adult cortex and differentiate into mature oligodendrocytes upon the need of de novo myelination of unmyelinated axonal tracts (Figure 1.2) (Chang et al., 2016). Proliferation and 8 differentiation of OPCs is mediated by neuronal activity (Li, Brus-Ramer, Martin,

& McDonald, 2010) suggesting activity-dependent myelination in the adult.

When neurons in the murine motor cortex were activated, increased rates of OPC maturation and thicker myelin sheaths of the axons from the stimulated neurons were observed (Gibson et al., 2014). Additionally, social isolation of adult mice results in thinner myelin sheaths and reduced expression of myelin genes within the prefrontal cortex (Liu et al., 2012). However, restoration of these deficits occurs after social reintegration (Liu et al., 2012). These various studies provide substantial evidence for activity-dependent mechanisms of oligodendrogenesis and myelin plasticity.

Neurological diseases that affect oligodendrocytes result in dysmyelination and a reduction in white brain matter, such as in multiple sclerosis (Alizadeh, Dyck, & Karimi-Abdolrezaee, 2015). Upon CNS damage or disorders that affect myelin sheaths or axonal integrity, OPCs migrate to the damaged site and elicit a repair response that results in oligodendrogenesis and myelination (Richardson, Young, Tripathi, & McKenzie, 2011). In diseases like multiple sclerosis that exhibit major debilitating effects, the number of migrating

OPCs are not always reduced showing intact translocating ability (Boyd, Zhang,

& Williams, 2013; Chang et al., 2016) however, altered expression of 9 chemorepellent molecules, such as semaphorin 3A, is increased identifying dysregulated mechanisms for remyelination (Boyd et al., 2013). Diseases of this nature demonstrate the necessity of healthy oligodendrocytes in maintaining proper myelin structure and axonal function.

In addition to producing myelin, these cells provide metabolic and trophic support of the axons that they encapsulate (Fünfschilling et al., 2012).

Monocarboxylate transporter 1 is highly expressed in oligodendrocytes and provides a direct means of metabolic support to both myelin and axons by transporting lactate from myelin to its associated axons (Bercury & Macklin,

2015). Oligodendrocytes release exosomes upon calcium entry containing various myelin and cytosolic proteins as well as anti-inflammatory factors (Krämer-

Albers et al., 2007). These oligodendrocyte-derived exosomes are internalized by neurons, presumably enabling the maintenance and support of neuronal and axonal function and integrity (Fünfschilling et al., 2012).

1.1.3. Microglia

Microglial cells are essential to maintaining the health of the CNS through their constant maintenance of a homeostatic CNS environment. Under normal conditions, these cells appear to take on a ‘resting state’ morphology despite their continual scavenging and regulatory activity (Goldmann & Prinz, 2013). These cells play a role in both synaptic and myelin plasticity by supporting the turnover of myelin and remodeling of synapses (Goldmann & Prinz, 2013). They are evenly distributed in the brain parenchyma and establish a continual support system and monitoring system for the brain (Ransohoff & Perry, 2009). Upon injury or highly stressed conditions, microglia play a significant role in modulating an immune and stress response in the CNS (Goldmann & Prinz,

2013).

Activation of these cells results in a number of consequences, both protective and toxic (Hu et al., 2015; Jha, Lee, & Suk, 2015). Diversity and levels of cytokines and chemokines surrounding and synthesized by microglia determine either the pro-inflammatory (M1) or the anti-inflammatory (M2) state these cells take on during injury (Jha et al., 2015; Perego, Fumagalli, & De Simoni,

2011). Figure 1.3 depicts the multitude of factors produced by the M1 and M2 11 phenotypes of activated microglia and how M1 promotes degradation while M2 promotes repair and restoration.

The M1 phenotype is promoted after stroke, accentuating the neural degeneration seen in patients following ischemia (Cherry, Olschowka, &

O’Banion, 2014). Under the M1 state, microglia become amoeboid-like in shape and boost inflammatory processes including the synthesis and release of cytokines (Goldmann & Prinz, 2013). During repair and healing of neural tissue, the M2 activated microglial cells promotes anti-inflammatory roles and active repair mechanisms. M2 cells secrete proangiogenic factors during repair to promote angiogenesis and vascularization (Hu et al., 2015). Upon increased response of M1 phenotypes and decreased activation of the M2 microglia, murine models of traumatic brain injury display reduced repair mechanisms and increased brain lesions (Kumar et al., 2013).

1.2. Peripheral glial cells.

Glial cells of the peripheral nervous system also play primary roles in the continuous metabolic, structural, and functional support of both motor and sensory nerves as well as neuromuscular junctions (NMJ). These peripheral glial cells express unique functions that separates them from those found in the CNS.

The most well-characterized and understood peripheral glial cell is the myelinating Schwann cell (mSC) found along motor axons and large caliber sensory axons. Schwann cells (SCs) can also be mature and nonmyelinating creating Remak bundles that encompass groups of sensory nerve fibers. Terminal

SCs (TSCs) are a third subtype of SCs found at the synaptic terminus or NMJ and are essential for the development, maintenance and function of the NMJ.

1.2.1. Development of Schwann cells

Schwann cell precursors (SCPs) are derived from ventrally migrating neural crest cells around embryonic day 12 – 13 in mice (E12 -13) (Jessen &

Mirsky, 2008; Monk, Feltri, & Taveggia, 2015). These precursors rely heavily on the axonal-derived survival factor, β-neuregulin-1 type III, for in its absence,

SCPs do not survive past E14 (Jessen & Mirsky, 2008). During this early stage of development, SCPs generate sheet-like processes that separate growing, immature peripheral nerves into distinct regions (Jessen & Mirsky, 2008).

Various molecular signals initiate the differentiation from SCPs into immature

SCs around E15 inducing the abundant expression of glial-specific proteins such as S100 or glial fibrillary acidic protein (GFAP) (Jessen & Mirsky, 2008). Notch-1 is a key factor in the differentiation of SCPs into immature SCs as well as the subsequent proliferative development state of immature SCs (Woodhoo et al.,

2009). By E18, this switch from SCPs to immature SCs is complete and immature

SCs no longer rely on extrinsic survival factors but can support themselves using autocrine signaling mechanisms (Jessen & Mirsky, 2008).

From E18 to birth (postnatal day 0; P0), immature SCs mediate the process of radial sorting which separates larger caliber axons from smaller caliber axons for proper SC association and eventual myelination or grouping of smaller 14 caliber axons into Remak bundles (Jessen & Mirsky, 2008; Monk et al., 2015).

This process begins with the translocation of immature SCs into nerve regions, followed by the reorganization of peripheral axons into nerve bundles that associate with 3-8 immature SCs (Monk et al., 2015). Connective tissue surrounding peripheral nerves begins to appear, vascularization of nerves occurs and an immature basal lamina is generated by the surrounding SCs (Jessen &

Mirsky, 2008; Monk et al., 2015). To segregate larger caliber axons from these nerve bundles, immature SCs generate lamellipodia-like processes that attach to single large axons and move them to the periphery of the nerve bundle (Monk et al., 2015). By postnatal day 7 (P7), these bundles of axons dramatically reduce in the total number of axons due to this radial sorting and segregation of large caliber axons (Webster, Martin, & O’Connell, 1973). As SC differentiation is continuing during this sorting process, immature SCs continue to proliferate and daughter SCs develop 1:1 relationships with these separated large caliber axons

(Monk et al., 2015). This 1:1 relationship distinguishes promyelinating daughter

SCs from immature SCs and is dependent upon the balance of proliferative and cell death pathways (Mirsky & Jessen, 2009). Once this 1:1 ratio is established,

Krox20 is upregulated stimulating promyelin pathways and promyelinating daughter SCs begin to generate compact myelin, ensheath these individual 15 axons, and mature into fully differentiated axonal myelinating SCs (Jessen &

Mirsky, 2008; Monk et al., 2015). Upon completion of radial sorting, the remaining small caliber axons stay grouped in compact bundles and their associated SCs differentiate into nonmyelinating Remak SCs that encompass these Remak nerve bundles (Jessen & Mirsky, 2008; Monk et al., 2015). This entire process is highly dependent on the proper ratio of axonal and SC number.

Between birth and P3, axon and SC proliferation hugely increases leading to a

50% and 90% increase, respectively (Webster et al., 1973). Dysregulation of this proliferative phase leads to delays or prevention in radial sorting and ultimate dysmyelination (Monk et al., 2015; Porrello et al., 2014). Figure 1.4 illustrates this process of SC differentiation from SCPs to immature SCs to either fully matured myelinating SCs or nonmyelinating SCs.

1.2.2. Myelinating axonal Schwann cells

The proper myelination of peripheral axons is essential for structural support, trophic support, and efficient neural signal transduction by saltatory propagation. This mechanism of conduction uses the insulating myelin, which increases the membrane resistance, to immediately propagate the neural signal along the axon through dense expression of voltage-gated sodium channels at the Nodes of Ranvier, unmyelinated regions interspersed along an axon.

Various factors and pathways tightly control the myelination of axons by mSCs throughout development as well as the maintenance of myelin sheaths under normal and stressed/injured conditions in fully developed individuals.

Furthermore, mSCs exhibit mechanisms to communicate with their ensheathed axons and provide trophic support. This communication is established by direct connections to motor axons through paranodal junctions whereby chemical or electrical signals are transferred (Court, Hendriks, MacGillavry, Alvarez, & van

Minnen, 2008; Samara, Poirot, Domènech-Estévez, & Chrast, 2013).

β-neuregulin-1 (NRGI) is essential for regulating SC myelination. β- neuregulin-1 type III (NRGI-III) is expressed by the surface of axons and thus acts as an extrinsic signal guiding either the promyelinating state or the nonmyelinating state of differentiating SCs. Heavily myelinated dorsal root 17 ganglia neurons express very high levels NRGI-III whereas minimally myelinated peripheral nerves of the autonomic nervous system have very limited expression of NRGI-III (Quintes, Goebbels, Saher, Schwab, & Nave,

2010). NRGI and NRGI-III bind to erbB receptors to initiate various signaling pathways for the promotion of myelination, such as the phosphoinositide 3- kinase/AKT pathway (Taveggia et al., 2005a). The MEK/Erk signaling pathway induces phosphorylation of Krox20 (an upregulator of myelination) by Yy1 activation (Pereira, Lebrun-Julien, & Suter, 2012). Binding of erbB receptors by

NRGI and NRGI-III also activates the phospholipase C-γ/calcineurin pathway which stimulates the translocation of NFATc4 to the nucleus where it interacts with Sox10 for the ultimate upregulation of Krox20 (Pereira et al., 2012). Figure

1.5 demonstrates the interaction between NRGI-III and erbB receptors and the multitude of downstream cascades initiated by activated erbB receptors.

These promyelinating pathways involve the activation of the transcription factors, SRY-related HMG-box 10 (Sox10) and octamer-binding transcription factor 6 (Oct6), which induce the expression of the transcription factor, KROX20

(Jessen & Mirsky, 2008). KROX20 promotes the transcription of various myelin proteins as well as downregulates inhibitory myelinating pathways (Jessen &

Mirsky, 2008; Pereira et al., 2012). Timing of myelination is essential, for 18 myelination prior to completion of radial sorting results in improper differentiation of SCs and unsorted axons leading to debilitating peripheral neuropathies (Monk et al., 2015). The inhibitory signaling pathway that helps control the timing of myelination is the inhibitory c-Jun N-terminal kinase (JNK) pathway which is downregulated by KROX20 expression (Rhona Mirsky et al.,

2008; Parkinson et al., 2008).

About 70% of myelin is composed of lipids while the remaining 30% is composed of proteins. Cholesterol makes up ~28% of the lipid composition and is an essential element of myelin. Through their intrinsic ability to synthesize cholesterol, mSCs promote their own survival and function. Mice lacking squalene synthase (an enzyme necessary for cholesterol biosynthesis) show deficits in myelination and reduced expression of myelin proteins (Saher et al.,

2009). Furthermore, erbB receptors respond to depleted cholesterol levels and upregulate HMGR expression, a rate-limiting enzyme in cholesterol biosynthesis

(Pertusa, Morenilla-Palao, Carteron, Viana, & Cabedo, 2007). The ability of the

SC-expressed erbB receptors to respond to levels of nerve-derived NRGI allows continual regulation of adequate myelination in fully developed, fully myelinated peripheral nerves (Chang et al., 2016). 19

Axonal myelinating SCs also retain the unique capacity for dedifferentiation under stressful states, such as peripheral nerve damage. Upon this dedifferentiation, essential intracellular pathways result in altered gene expression that promotes macrophage invasion, axonal growth, and other functions for healing (Arthur-Farraj et al., 2012; Z.-L. Chen, Yu, & Strickland,

2007; Jessen & Mirsky, 2016; Woodhoo et al., 2009). mSCs are able to revert to an immature state and clear myelin and other cellular debris until proper regrowth has occurred and remyelination begins (Chen et al., 2007; Jessen & Mirsky, 2016).

SC-derived exosomes have also proven extremely important in the proper repair and guidance of axonal regeneration after nerve injury (Lopez-Leal & Court,

2016; Lopez-Verrilli, Picou, & Court, 2013). SC-derived exosomes containing various survival factors and mRNAs are taken up by their neighboring axons

(Court et al., 2008; Lopez-Verrilli et al., 2013) allowing for cross-talk between mSCs and their ensheathed axons.

1.2.3. Nonmyelinating Remak Schwann cells

Mature axonal SCs that do not produce myelin ensheath multiple sensory and sympathetic nerve fibers into bundles, termed Remak bundles. Therefore, this type of SC is commonly referred to as a Remak SC. Remak bundles conduct sensory signals via cable propagation, a more passive process when compared to saltatory conduction due to the lack of insulation or control of axonal membrane resistance resulting in reduced conduction velocities (Salzer, 2012).

The literature suggests that axonal signals determine the phenotype of maturing Schwann cells in their vicinity (Salzer, 2012) for instance sympathetic nerve fibers do not express significant amounts of β-neuregulin-1 like motor fibers do thus resulting in limited myelination (Quintes et al., 2010). A major player in the proper development of Remak bundles is the LDL receptor-related protein 1 (LRP1). In mice deficient for this protein, Remak bundles contain larger caliber axons, increased total number of axons, and abnormal separation of axons by SC cytoplasm as compared to Remak bundles of WT mice (Orita et al., 2013).

This also leads to increased activation of M1 microglia in the spinal cord after nerve injury as well as significant allodynia, or hyper-sensitization to pain (Orita et al., 2013). Proper development of Remak SCs and their engulfed C fibers is necessary for normal sensory perception and transduction. 21

Remak SCs and fibers are often considered the ‘first-responders’ to injury.

Due to their unmyelinated state, these fibers and glial cells retain a higher degree of plasticity than do mSCs. Therefore, upon nerve injury and denervation, these cells are able to sprout and direct Remak fibers to these injured regions for immediate relay of damage to the CNS and initiation of the repair and reinnervation process (Griffin & Thompson, 2008). Key events initiating this response is reduced expression of basic myelin genes, such as Krox20 or myelin basic protein, as well as upregulation of neurotrophic factors, such as nerve growth factor and vascular endothelial growth factor (Jessen & Mirsky, 2016).

A major regulator controlling the altered genetic and functional profile of activated Remak Schwan cells is the transcription factor, c-Jun. Murine models lacking c-Jun do not express similar genetic profiles of activated Remak SCs and do not successfully repair injured nerves like those of normal mice (Arthur-Farraj et al., 2012). Interestingly, Krox20, a major regulator of myelin genes, suppresses expression of c-Jun in healthy mSCs and unactivated Remak bundles (Arthur-

Farraj et al., 2012). Thus, properly functioning Remak SCs are not only necessary for proper structural and functional integrity of C-fibers, but also for the repair response found in the PNS after nerve injury.

1.2.4. Nonmyelinating terminal Schwann cells.

Terminal Schwann cells are nonmyelinating periphal glia that surround the terminus of a motor axon and therefore are referred to as terminal Schwann cells (TSCs). Due to the direct localization of TSCs to motor synapses, or neuromuscular junctions (NMJs), these cells provide numerous modulatory and survival functions for the NMJ. During developmental ages and into adulthood,

TSCs are necessary for the maintenance and support of NMJs (Chen, Sugiura,

Myers, Liu, & Lin, 2010; Darabid, Perez-Gonzalez, & Robitaille, 2014; Feng & Ko,

2009; Reddy, Koirala, Sugiura, Herrera, & Ko, 2003b). They can sense and modulate neuronal activity by calcium signaling mechanisms and a variety of receptors including purinergic receptors (P2Y and P2X), muscarinic acetylcholine receptors (mAChRs), and adenosine receptors (A2A and A1) (Bélair & Robitaille,

2009; Darabid, Arbour, & Robitaille, 2013a; Rousse & Robitaille, 2006; Todd,

Darabid, & Robitaille, 2010a). Furthermore, these cells serve a protective and regenerative role under stressful conditions, such as axonal damage or denervation.

The numerous types of receptors found on TSCs allow for sensing and responding to synaptic activity (Bélair & Robitaille, 2009). Activation of P2Y receptors on TSCs results in increases in intracellular Ca2+ by the activation of 23 pertussis-toxin sensitive G-proteins while activation of P2X receptors leads to increased Ca2+ by activation of L-type Ca2+ channels (Bélair & Robitaille, 2009).

Activation of muscarinic and purinergic receptors by acetylcholine (ACh) or ATP bath application, respectively, results in increased Ca2+ via intracellular Ca2+ stores (Rousse & Robitaille, 2006).

TSCs can also regulate synaptic activity and plasticity by altering the chemical environment at the synapse through SC release of ATP (Todd, Darabid,

& Robitaille, 2010b). Transient increases of intracellular Ca2+ in TSCs leads to minimal amounts glial-derived ATP which is broken down into ADP, AMP, and adenosine (Todd et al., 2010a). Low adenosine levels leads to the activation of A1 receptors on TSCs or presynaptic nerve terminals and block presynaptic Ca2+ entry via P/Q-type Ca2+ channels resulting in glia-mediated synaptic depression

(Figure 1.6) (Todd et al., 2010a). Conversely, a sustained Ca2+ response by TSCs results in increased release of ATP, greater amounts of adenosine, and activation of A2A receptors causing opening of L-type Ca2+ channels in presynaptic nerve terminals leading to potentiation (Figure 1.6) (Todd et al., 2010a).

TSCs are extremely plastic cells with regenerative capabilities for the restoration of structure and function after injury. During peripheral nerve injury and denervation, TSCs upregulate various molecules, such as the pro- 24 inflammatory cytokine interleukin-1, to promote the immune response and transfer neurotrophic and growth factors to damaged axons (Armati & Mathey,

2013; Kang, Tian, & Thompson, 2003; Lopez-Verrilli et al., 2013; Webber et al.,

2011; Webber & Zochodne, 2010). TSCs form growth sprouts creating bridges between adjacent innervated, functional endplates and the damaged sites allowing for ultimate repair and re-innervation (Griffin & Thompson, 2008; Kang et al., 2003). Certain genes, such as Sox2, Oct6, or glial-derived neurotrophic factor, are activated in TSCs during these stages of repair to switch TSCs into a synaptogenesis state (Ellerton, Thompson, & Rimer, 2008; Jessen & Mirsky, 2016).

These various studies on regeneration and nerve repair by TSCs have been conducted in adult models.

TSCs perform multiple functions, each critical to the neural-glia-muscle tripartite synapse. These include sensing and modulation of NMJ activity

(Ansselin, Davey, & Allen, 1996; Rousse & Robitaille, 2006; Todd et al., 2010a), development and maintenance of the NMJ (Darabid et al., 2013a; Sugiura & Lin,

2011), and regenerative and immunological roles (Armati & Mathey, 2013; Son &

Thompson, 1995; Webber et al., 2011). 25

1.3. Neuromuscular junction development

1.3.1. Nerve- and muscle-derived factors in NMJ development

The synaptogenesis of the neuromuscular junction is a complex process that involves various factors and signals from the innervating motor neuron, the postsynaptic muscle fiber, and the surrounding terminal Schwann cells.

Postsynaptic nicotinic acetylcholine receptor (nAChR) clustering occurs in an organized fashion in the center of muscle fibers independent of motor nerve signals (Noakes, Phillips, Hanley, Sanes, & Merlie, 1993; Yang et al., 2001). This prepatterning of nAChRs appears as early as embryonic day 13 (E13) (Noakes et al., 1993) and creates the appearance of a centralized endplate band comprised of these postsynaptic nAChRs (Figure 1.7A).

In the absence of proper factors derived from both muscle and nerve, such as MuSK and agrin, respectively, these nAChRs eventually disperse and degenerate. The activation of the muscle specific kinase, MuSK, is critical for the integrity of nAChR during synaptic maturation (DeChiara et al., 1996) as well as for the maintenance of synaptic integrity postnatally (Hesser, Henschel, &

Witzemann, 2006). Wnt ligands interact with MuSK for additional stabilization of

NMJs (Darabid et al., 2014). The nerve-derived factor agrin is essential for the activation of MuSK and in its absence, the nAChR clustering becomes highly 26 disorganized despite postsynaptic structure development. Rather than an organized, densely innervated endplate band region localized within the center of the muscle fiber, agrin mutant mice display aberrant nAChR localization, extensive nerve growth, and insufficient innervation patterns (Gautam et al.,

1996). The agrin receptor, the muscle-derived LRP4, is essential for precise localization of NMJs and nAChR aggregates (Wu et al., 2012). While LRP4 knockout mice are not embryonically lethal, diaphragm innervation patterns show major discrepancies in both pre- and post-synaptic formation and structure as well as in immense reductions in spontaneous activity of these NMJs (Wu et al., 2012). LRP4 binds agrin allowing for the proper signaling between nerve and muscle, however LRP4 is also a major activating and stabilizing factor for MuSK

(Kim et al., 2008). Therefore, the LRP4-agrin-MuSK complex is necessary for proper synaptogenesis, NMJ maintenance, and neuromuscular activity. The variety of signaling pathways involved in NMJ formation and maturation is a tightly controlled, coordinated system as visualized in Figure 1.7B.

1.3.2. Activity-dependent mechanisms of NMJ development

The various factors described above are also influenced by the level of neural activity occurring at these developing synapses. Mice lacking the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), display a major block of neurotransmission, and a multitude of effects demonstrating impaired synaptic formation and activity. ChAT knockout (KO) mice are embryonically lethal and demonstrate increased branching of motor nerves, hyperinnervation of myofibers, immature appearance of NMJs, and widened endplate regions (Figure 1.8) (Brandon et al., 2003; Misgeld et al., 2002). ChAT

KO mice show various aspects of NMJ development that are activity-dependent.

Interestingly, the roles of agrin and ACh seem to counterbalance each other, for in double knockout, ChAT and agrin deficient mice, there is a rescue effect of

NMJ formation and integrity (Misgeld, Kummer, Lichtman, & Sanes, 2005).

Furthermore, mice lacking Schwann cells that result in complete loss of synapses and motor nerves demonstrate a rescue effect upon blocking activity by genetic ablation of ChAT (Figure 1.8). Normal synaptic activity during development thus exhibits significant negative effects and must be antagonized by signals from nerve-derived agrin as well as Schwann cells. 28

Around postnatal day 7 (P7) in mice, over 60% of NMJs have multiple- innervating nerves and are undergoing activity-dependent competition and pruning (Kopp, Perkel, & Balice-Gordon, 2000). During synaptic competition, neural inputs with weak signals likely demonstrate limited ability to maintain consistent neurotransmitter release making them unlikely to be selected for strengthening and stabilization (Kopp et al., 2000). Axons selected for single- innervation from poly-innervated sites display similar quantal content to that from the strongest inputs in multiple-innervated fibers (Colman, 1997).

Furthermore, activity-dependent synaptic pruning helps redistribute axonal resources, for axons without ChAT have preferential loss than compared to those that have normal levels of ChAT (Buffelli et al., 2003).

The complex interplay between motor nerve-derived factors, muscle- derived factors, and synaptic activity demonstrates a system that heavily relies on the proper coordination of this multitude of signals. Synaptic activity is an important contributor to the strengthening and functioning of these developing

NMJs, however ACh also disperses nAChR clusters. Therefore, the expression of agrin and the LRP4-agrin-MuSK complex is important to maintain the integrity and localization of these early innervations that are being negatively regulated by early neural activity. 29

1.3.3. Role of Schwann cells in development and maintenance of the NMJ

The erbB receptor family comprises a distinct group of tyrosine-kinase receptors with altered catalytic domains eliminating their tyrosine kinase activity

(Riethmacher et al., 1997). erbB receptors bind neuregulins 1-4 and are expressed by a few different cell types including SCs (Buonanno & Fischbach, 2001). In mice with genetic deletions of the erbB2 or erbB3 receptors, SCs do not proliferate or differentiate resulting in embryonic lethality (Buonanno &

Fischbach, 2001). While cardiac deficits result in erbB2 KO mice and cause lethality around E11 (Lee et al., 1995; Woldeyesus et al., 1999), erbB3 KO mice evade this effect and display a lack of SCs and SCPs (Riethmacher et al., 1997).

This typically results in embryonic lethality around E15 but some embryos can survive to term (Riethmacher et al., 1997), allowing for the characterization and study of developmental effects resultant from an absence of SCs.

At early postnatal ages, TSCs modulate synaptic pruning through competition with motor axons for nAChR contact and continual removal of axonal boutons for single rather than poly-innervated sites (Smith et al., 2013).

TSCs are able to decipher competing information from the poly-innervated synapses through the varying strengths of the TSC Ca2+ response (Figure 1.9)

(Darabid et al., 2013a). The Ca2+ response to neurotransmitter release by 30 developing motor nerves is not the only factor modulating synaptic pruning, intrinsic properties of TSCs, such as their receptor expression, also plays a role.

During this time of synaptic pruning, TSCs densely express P2Y receptors in areas of heightened neural activity (Figure 1.9) (Darabid et al., 2013a). Upon completion of synaptic pruning and full development of NMJs (around P21),

TSCs revert to expressing both P2Y and muscarinic receptors near the synapse

(Figure 1.9) (Darabid et al., 2013a). This unique ability of TSC responsiveness to neural signal strength and activity-dependent receptor expression is a major player in the postnatal developmental stage of synaptic pruning.

Motor axons and SCs have a symbiotic relationship, neither can develop, survive, nor function without the other. In cell culture, SC medium showed an alteration between an initial growth state of motor axons and then a synaptogenic state as the axon became in close proximity to its target muscle fiber (Peng et al., 2003). SCs mediate synaptogenesis through the colocalization of motor axon growth and developing motor synapses (Reddy, Koirala, Sugiura,

Herrera, & Ko, 2003a). In these various studies, either cell culture was used or cell ablation techniques were used. In either case, it is obvious that SCs are needed for proper and efficient synaptogenesis to occur.

1.4. Aims of Thesis

The aims of this thesis are to examine the effects of Schwann cells during the development of the neuromuscular junction. Specifically, this thesis sought to distinguish axonal Schwann cells from terminal Schwann cells for the future characterization of both Schwann cell subtypes in NMJ development.

Additionally, this thesis investigated the role SCs played in the early lethality characterized by a homozygote Trembler-J model, a murine model that typifies

Charcot-Marie-Tooth disease, a disease that targets the peripheral myelin protein

22 gene and thus directly affects SCs. The aims by chapter are the following:

Chapter 2: Characterization of the erbB3-/- mouse phenotype during dramatic remodeling in embryogenesis. This chapter will characterize structural and genetic alterations in the diaphragm and hindlimb muscles of erbB3-/- mice as compared to WT mice. These mutant models exhibit a complete loss of all SC subtypes and exhibit dramatic changes in the developing diaphragm muscle between embryonic day 14 and 15.5.

Chapter 3. Isolation of the terminal Schwann cell transcriptome for the identification of novel genetic markers. Assess the qualitative and quantitative differences in mRNA transcript expression between myelinating Schwann cells 32 and terminal Schwann cells using a novel combination of a genetic and dissection approach followed by extensive bioinformatics analysis.

Chapter 4. Structural and Functional Abnormalities of the Neuromuscular

Junction in the Trembler-J Homozygote Mouse Model of Congenital

Hypomyelinating Neuropathy. Investigate the alterations in NMJ structure and function in Charcot-Marie Tooth disease using Trembler-J homozygote mice, a murine model that capitulates the Schwann cell directed CMT disease and does not survive long postnatally.

Figure 1.1. Schematic of major astrocyte signaling associated to gliotransmitter release. Increased intracellular free Ca2+ concentration ([Ca2+]i) allows the release of gliotransmitters into the synaptic cleft through vesicles and hemichannels (HCs) made up of Connexin proteins. D-serine and glutamate released by astrocytes can activate NMDA receptors at the post-synaptic neuron and modulate neuronal plasticity. Astroglial glutamate also binds to mGluR at the presynaptic neuron increasing neuronal release of glutamate into the synapse. In astrocytes, increasing ([Ca2+]i) allows Ca2+ wave propagation between astrocytes, mediated by gap junction channels (GJCs) and by release of glutamate and ATP, resulting in further activation of NMDA and P2YR receptors at neighboring astrocytes. (Modified from Orellana & Stehberg, 2014).

Figure 1.2. Different patterns of myelination on CNS axons. This scheme shows a partially myelinated axon with a ‘patchy’ myelination pattern (upper image) and a fully myelinated axon (lower image). It also depicts how myelin may integrate through the de novo myelination of previously unmyelinated axons or by myelin replacement and remodeling. OPC, oligodendrocyte progenitor cell; OL, oligodendrocyte. (de Hoz & Simons, 2014).

Figure 1.3. Polarized microglia play distinct roles in restoration of the neurovascular network after ischemia and other CNS injuries. The activated M1 phenotype upregulates pro-inflamatory factors (listed on figure) to promote oxidative stress and degenerative pathways upon injury. The activated M2 phenotype upregulates anti-inflammatory molecules (listed on figure) to promote healing mechanisms like neuronal repair and remyelination (modified from Hu et al., 2015).

Figure 1.4. Development of myelinating and nonmyelinating Schwann cells. Schematic depicting Schwann cell (SC) development. SCs are orange and axons are blue. From left, SC precursors (SCPs), depicted longitudinally, are proliferative and migrate with growing axons. The remaining stages are depicted in cross-section. SCPs become immature SCs, which are associated with many axons. Immature SCs have ceased migration, remain proliferative, and form an immature basal lamina (BL, purple). The BL persists and matures through subsequent stages of SC development. During radial sorting, immature SCs interdigitate their cytoplasmic processes into the axon bundle, and promyelinating SCs are associated with a single axonal segment. Myelinating SCs spiral their membrane many times around the axonal segment to form the myelin sheath. Immature SCs can also develop into nonmyelinating SCs. A nonmyelinating SC, entheathing multiple small caliber axons is depicted (modified from Monk et al. 2015).

2 3

Figure 1.5. Control of peripheral nervous system myelination by Schwann cell- axon interactions. SC myelination is a process strongly dependent on instructive signals provided by the axons. Neuregulin 1 (NRG1) and its membrane-bound isoform, neuregulin 1 type III (NRG1-III) bind to erbB receptors on SCs to activate downstream signaling pathways for the upregulation of myelination. Upon NRG1/NRG1-III binding to erbB receptors, multiple downstream signaling cascades are activated: 1) Phoshoplipase C-γ mediates calcium (Ca2+) release and activation of calcineurin. Calcineurin activation dephosphorylates nuclear factor of activated T-cells 4 (NFATc4) for its translocation into the nucleus where it forms a complex with the transcription factor, Sox10. This NFATc4-Sox10 38 complex binds to and induces transcription of the myelin protein zero (P0) gene and Krox20 (a major upregulator of promyelinating pathways) 2) The MEK pathway activates ERK1/2 for upregulation of myelination or phosphorylates Yy1 which then binds to and activates transcription of Krox20 and 3) the PI3K/AKT signaling pathway is activated leading to upregulation of myelination mechanisms (modified from Pereira et al., 2011).

Figure 1.6. Model of glial- mediated bidirectional modulation of synaptic plasticity. A) Bursts of activity (1) induce the release of neurotransmitter to activate terminal Schwann cells (TSCs) (2) and the postsynaptic terminal. Receptor activation on TSCs leads to oscillatory Ca2+ elevations (3) and the release of a lesser amount of glial-derived ATP (4) that is degraded to adenosine. Relatively low levels of synaptic adenosine lead to synaptic depression through activation of A1 receptors and a decrease in presynaptic Ca2+ entry through P/Q type Ca2+ channels (5 and 6). B) Continuous presynaptic activity (1) induces the release of neurotransmitter to activate TSCs (2) and the postsynaptic terminal. Receptor activation on TSCs leads to a single Ca2+ elevation (3) and the release of a larger amount of glial-derived ATP (4) that is degraded to adenosine. Relatively high levels of synaptic adenosine lead to activation of A2A receptors, activation of L-types Ca2+ channels (5) and synaptic potentiation (6) (Todd, et al. 2010). 40

Figure 1.7. Synaptogenesis of the neuromuscular junction. A) A developing embryonic day 15.5 (E15.5) wild-type (WT) murine diaphragm muscle has clustering of post-synaptic nicotinic acetylcholine receptors (nAChRs), stained with α-bungarotoxin (BTX), generating a centralized endplate band (middle panel). Schwann cells are labelled with an antibody against S100 (green; left panel) and follow the migration of developing motor nerves that synapse onto the postsynaptic nAChRs. B) Parallel signaling pathways at the neuromuscular junction (NMJ) are involved in the coordinated maturation of the presynaptic terminal, the postsynaptic muscle fiber and the terminal Schwann cells (TSCs); dotted lines indicate pathways that are still being debated. Agrin, which is released by the nerve terminal, acts on the LRP4-MuSK complex, which comprises low-density lipoprotein receptor-related protein 4 (LRP4) and muscle- specific tyrosine kinase receptor (MuSK). The phosphorylatin of MusK leads to rapsyn-mediated clustering of ionotropic AChRs and postsynaptic maturation. 41

AChR clustering can also be enhanced by WNT ligands (associated with MuSK), whereas release of ACh inhibits AChR clustering. Neuregulin 1 (NRGI) can be released by the nerve terminal and/or surround TSCs, and binds to TSC expressed erbB receptors (erbB2 or erbB3). NRGI binds to erbB receptors on TSCs and promotes TSC survival and maturation. Postsynaptically, laminins interact with integrin β1, which increases AChR clustering. TSC-derived transforming growth factor-β (TGFβ) induces presynaptic maturation and postsynaptic differentiation by upregulating the expression of agrin. Synaptically released ATP is detected by TSC-expressed purinergic type 2Y receptors (P2YRs) and triggers increases in intra-TSC calcium concentrations. TSCs also express muscarinic AChRs (mAChRs), and their activation by the local application of Ach triggers increases in intra-TSC calcium concentrations. However, mAChRs are not activated by endogenous Ach release during NMJ development (modified from Darabid et al., 2014).

Figure 1.8. Evoked activity through peripheral nAChRs is required for NMJ degeneration caused by Schwann cell ablation. Diaphragms from embryonic day 18 (E18) mice lacking the acetylcholine synthesizing enzyme (top right panel), choline acetyltransferase (ChAT), result in excessive axonal branching, increased innervated sites, and widened endplate regions as compared to diaphragms from E18 wildtype (WT) mice (top left panel). Neural and NMJ degeneration occurs by E18 in mice deficient in Schwann cells (erbB3 knockout [KO] mice; bottom left panel). In double KO mice, a rescue effect is observed (bottom right panel). Note the defasciculation phenotype of double KOs vs. ChAT single KO mice. 43

Figure 1.9. Terminal Schwann cells regulate the process of synaptic competition during neuromuscular junction development. Upper panel. Early in synaptogenesis (from embryonic day 18 [E18] to ~ postnatal day 10 [P10]) there is immense synaptic competition at poly-innervated sites. As terminal Schwann cells (TSCs) proceed in their differentiation during this same time period, they are able to detect and decode signals from the multiple nerve fibers located at singular sites. As synaptic pruning progresses, TSCs differentiate and multiple TSCs surround single innervated sites in the mature NMJ. Lower panel. As synaptic competition proceeds, surrounding TSCs express P2YRs that are activated by ATP released by the developing motor fiber. Only after full maturation of the NMJ and TSC, the TSC begins to express muscarinic acetylcholine receptors (mAChRs) through which they can sense neural activity via ACh release (modified from Darabid et al., 2013a). 44

CHAPTER 2

CHARACTERIZATION OF THE ERBB3-/- MOUSE PHENOTYPE DURING

DRAMATIC REMODELING IN EMBRYOGENESIS.

2.1. SUMMARY

Axonal Schwann cells (SCs) either ensheath bundles of sensory or autonomic nerves and are nonmyelinating, or myelinate peripheral motor nerves for proper nerve conduction. A second subtype of peripheral glial cell, are nonmyelinating, terminal Schwann Cells (TSCs) that are localized to the neuromuscular synapse or junction (NMJ) between the motor axon and muscle. TSCs sense and modulate neural activity at the NMJ and have been implicated in regulating the development and maintenance of this synapse. In embryonic mice where both subtypes of SCs are absent, motor neurons initially form synaptic connections with the target muscle but subsequently retract leading to denervation, paralysis and eventual fatality. This early innervation occurs around embryonic day 14

(E14) and only 36 hours later this degeneration has fully transpired. Using confocal imaging and analysis and RNA sequencing, we characterize this 36 hour time period in order to more fully understand how SCs normally maintain immature NMJs.

2.2. INTRODUCTION

Schwann cells (SCs), a subtype of peripheral glia, serve major supportive and modulatory roles in the peripheral nervous system. Axonal, myelinating SCs

(mSCs) are characterized by their ability to myelinate peripheral axons enabling efficient motor nerve conduction velocity while terminal SCs (TSCs) are nonmyelinating and surround the neuromuscular junction (NMJ). This close association of the TSC with the NMJ allows TSCs to provide an array of roles for the maintenance and proper function of the NMJ.

Glia are known as neural support cells involved in the protection and regeneration of nerves after axonal damage. Recent studies have provided additional insight into the significant roles glia play in development, neurotransmission, and initiating repair and regeneration after denervated or damaged conditions. Upon nerve injury, TSCs upregulate pro-inflammatory cytokines, transfer neurotrophic and growth factors to damaged axons, and form sprouts that guide axons for proper regeneration and reinnervation (Armati &

Mathey, 2013; Kang et al., 2003; Lopez-Verrilli et al., 2013; Son & Thompson,

1995; Webber et al., 2011; Webber & Zochodne, 2010). Throughout development and continuing into adulthood, Schwann cells are necessary for the maintenance and support of NMJs, for ablation of TSCs or blockade of the SC-derived growth factor, transforming growth factor-β1, results in reduced rates of synaptogenesis 47 and eventual degeneration of previously formed synapses (Darabid et al., 2013a;

Z. Feng & Ko, 2009; Reddy et al., 2003b; Sugiura & Lin, 2011). TSCs are involved in the sensing and modulation of neuronal activity using a variety of receptors and calcium signaling mechanisms (Ansselin et al., 1996; Castonguay &

Robitaille, 2001; Zhihua Feng & Ko, 2008; Rousse & Robitaille, 2006; Todd et al.,

2010a). Not only can TSCs respond to synaptic activity by changes in their intracellular calcium levels, they can also regulate this activity by altering the biochemical environment at the synapse through release of ATP or uptake of acetylcholine (Ansselin et al., 1996; Caillol et al., 2012; Musarella et al., 2006;

Todd et al., 2010a).

Schwann cell proliferation and migration is dependent on the erbB/neuregulin signaling pathways (Jessen & Mirsky, 2008; Pereira et al., 2012;

Taveggia et al., 2005b). Neuregulin 1 is part of the epidermal growth-factor like molecular family and signals through erbB receptor tyrosine kinases throughout

SC differentiation and maintenance (Garratt, Britsch, & Birchmeier, 2000; Perlin,

Lush, Stephens, Piotrowski, & Talbot, 2011). Proper expression and signaling between neuregulin 1 and the erbB2 and erbB3 receptors are necessary for the differentiation and appropriate localization of neural crest cells into SC precursor cells (Garratt et al., 2000). Neuregulin type III expression is critical for immature 48

SC survival and provides migration signals decoding the ultimate fate of axons becoming myelinated or not (Taveggia et al., 2005b). In the absence of the family of epidermal growth-factor like molecules, specifically neuregulin 1 and 2, their receptors, the erbB family, or regulators of these receptors, Sox10; the proliferation and maturation of Schwann cells does not occur and these glia are absent (Britsch, 2001; Wolpowitz et al., 2000).

In studies where the neuregulin receptor erbB3, a receptor critical for the survival and differentiation of SCs, has been genetically ablated (erbB3-/- mice), initial NMJs are formed but motor axons quickly retract and synapses do not stabilize nor survive (Britsch, 2001; Lin et al., 2000; Riethmacher et al., 1997;

Wolpowitz et al., 2000). When the neural activity generated by these early synaptic connections is blocked, motor neuron degeneration and NMJ denervation is simultaneously recovered leading to the implication that SCs normally inhibit activity in order to maintain NMJs and regulate their development (Brandon et al., 2003; Misgeld et al., 2002).

In the present study, we characterized and quantified the structural differences in the developing diaphragm muscle from erbB3-/- mice as compared to erbB3 WT mice. Initially formed synapses and peripheral nerve growth and branching occurs by embryonic day 14 (E14) however dramatic alterations occur 49 leading to denervation and degeneration of motor axons and postsynaptic receptors by E15.5 in erbB3-/- mice (Lin et al., 2000). Therefore, we used time points within this 36 hour developmental period to examine the phenotype of embryonic erbB3-/- mice using confocal imaging and analysis as well as RNA- sequencing analysis.

2.3. MATERIALS & METHODS

Mice

erbB3 heterozygote mice (Jackson Laboratory, Sacramento, CA, USA) aged 8 weeks were backcrossed into a Balb/C background for three generations to generate homozygous erbB3 knockout mice (erbB3-/-). With a strong Balb/C background, females carried more homozygote knockout mice to late embryonic ages (embryonic day 15; E15) versus early fatality (E13 or earlier) prior to initial stages of NMJ development. At designated stages between E14 and E15 development, pregnant homozygote mothers were euthanized by inhalation of

5% isofluorane followed by cervical dislocation. Embryos were carefully removed from the uterus and kept alive in oxygenated Krebs’ solution

(containing (mM): NaCl, 120.35; KCl, 5.9; NaHCO3, 15.5; NaH2PO4, 1.2; MgCl2,

1.2; CaCl2, 2.5; glucose, 11.5 (continuously gassed with 5% CO2–95% O2, pH 7.3–

7.4), until the time of experiment. Wild-type (WT), heterozygote (erbB3+/-), and homozygote (erbB3-/-) embryonic mice were genotyped for mutated erbB3 alleles.

Upon confirmation of genotyping, WT and erbB3-/- mice were used for subsequent experiments and were sacrificed by decapitation. Diaphragms and hindlimbs from erbB3-/- or WT mice were used for immunohistochemistry and flash frozen in liquid nitrogen and stored at -80°C for subsequent RNA isolation, 51 respectively. These procedures were in accordance with National Institutes of

Health guidelines for the care and use of laboratory animals and approved by the

Animal Ethics Committee at the University of Nevada, Reno.

Immunohistochemistry

Diaphragm muscles from embryonic erbB3-/- and littermate control animals were fixed in 4% paraformaldehyde overnight at 4°C. 24 hours later, diaphragms were rinsed in phosphate-buffered saline (PBS), blocked in 0.1M glycine for 1 hour at room temperature (RT), rinsed again, and then incubated as whole mounts in primary antibody solution overnight at 4°C in PBS containing

10% fetal bovine serum and 1% triton-X. Primary antibodies were rabbit-anti- synaptophysin ([Syp] Santa Cruz Biotechnology, Santa Cruz, CA, USA), or rabbit-anti-S100 (Dako, Carpinteria, CA, USA) diluted to 1:500. 18 hours later, muscles were rinsed and incubated for 1 hour at RT in the dark with AlexaFluor

488-conjugated donkey-anti-rabbit antibodies (Jackson ImmunoResearch, West

Grove, PA, USA) together with AlexaFluor 594-conjugated α-bungarotoxin

([BTX] Biotium, Hayward, CA, USA) diluted to 1:1000.

Confocal Image Analysis

Images of developing presynaptic motor neurons labeled with antibodies against synaptophysin or S100 (a SC marker) and postsynaptic α-BTX-labeled 52 nicotinic acetylcholine receptor (nAChR) clusters were acquired with the

Olympus Ix81 confocal microscope (Olympus Scientific Solutions Americas

Corp., Waltham, MA, USA) and Fluoview 1000 software using a 10x or 20x objective. Images were analyzed using an in house developed software,

Volumetry G8c (GWH). Maximum fluorescence-intensity maps of either Syp or

α-BTX were created from confocal stacks after normalization of signal to noise ratios. A Euclidian distance based algorithm generated maps outlining the boundary of pixels containing fluorescent signal from anti-Syp labelled nerves.

Binary images were then generated from maximum fluorescence intensity maps of α-BTX-labelled pixels. These binary images were overlayed onto Euclidian distance maps (EDM) and the total area of α-BTX-labelled particles within the

EDM was taken and compared between erbB3-/- and littermate control diaphragms.

RNA Sequencing

Using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara,

CA, USA), the quantity and quality of RNA samples was assessed. Only samples with RNA integrity numbers greater than 7 were used for library preparation.

Due to the low quantity of RNA from this isolation method, the SMARTer Ultra

Low Input RNA Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) was 53 used to generate cDNA libraries for high-throughput sequencing by Illumina

NextSeq 500 (Illumina, Inc., San Diego, CA, USA).

RNA Sequencing Analysis

RNA sequencing reads were initially trimmed to remove barcodes and adenosine stretches from the 3’ end. Reads were aligned to the mouse transcriptome and then to the mouse genome (GRCm38/mm10, retrieved from

UCSC Genome Bioinformatics) using TopHat2 (Kim et al., 2013). Transcript sets were assembled using Cufflinks (Trapnell et al., 2012) and then merged with

Cuffmerge (Trapnell et al., 2012). Statistical differences between RNA-sequencing data sets (erbB3-/- versus WT) were generated using Cufflinks. The PANTHER overrepresentation test (version 10.0) was used to perform gene ontology enrichment analysis using the GO Ontology database (released 03-25-2016) (Gene

Ontology Consortium).

Statistics

Statistical comparisons of data were performed using unpaired Student's t tests, and P < 0.05 was considered as statistically significant. n refers to the number of animals from which tissues were taken. All data are presented as means ± SEM. In the figures, *P < 0.05, **P < 0.01 and ***P < 0.001.

2.4. RESULTS

At E14.5, a WT diaphragm exhibits organized branching of developing motor nerves and localized regions of synaptic innervation along the midline of the diaphragm, a region termed the endplate band (Figure 2.1A). Conversely, in the diaphragm of E14.5 erbB3-/- mice, motor nerves exhibit aberrant branching patterns, a loss of a focalized endplate band, and massive defasciculation (Figure

2.1B). By E15.5, erbB3-/- mice display complete loss of motor nerves and denervation (Figure 2.1D) as compared to the organized, condensed appearance of innervating motor nerves in E15.5 WT mice (Figure 2.1C).

In E14.25 erbB3-/- mice, innervation of muscle fibers is still apparent despite abnormal structure of axons as compared to E14.25 WT mice (Figure

2.2A.i-ii; n=3). Disorganization of motor axons and degenerating nerve terminals are apparent as early as E14.25 in erbB3-/- mice when compared to the organized, innervated NMJs of immature synapses in E14.25 WT mice (Figure 2.2A.iii-iv; n=3). We then characterized the innervation patterns observed in the defasciculated, degenerating diaphragms of erbB3-/- mice. While developing motor axons of E14.5 WT mice present with early branching near postsynaptic nAChR clusters within the endplate band, minimal innervation of these nAChRs is apparent (Figure 2.2B.i). Contrary, the aberrant branching and defasciculation 55 of motor axons in E14.5 erbB3-/- mice show increased numbers of innervated nAChRs (Figure 2.2B.ii).

α-BTX labelled structures extend beyond the area of synaptophysin- labelled nerve structures (Euclidean distance maps) in E14.25 erbB3-/- mice as compared to WT mice (Figure 2.3A). α-BTX labelled postsynaptic nAChR clusters at the center of the EDM are only 8.5% of the total α-BTX labelled area in erbB3-/- mice as compared to 72.5% of nAChR clusters in WT mice (Figure 2B).

RNA sequencing of E14.5 erbB3-/- and WT hindlimb muscle quantified transcriptional changes. Table 2.1 shows the 50 most upregulated genes in E14.5

WT versus erbB3-/-. The high percentage of SC specific genes (Sox10, Fabp7, Mpz,

Sox5, Mal) in WT datasets as compared to erbB3-/- provides confidence to the

RNA sequencing analysis. Gene ontology analysis of the top 300 upregulated genes in WT versus erbB3-/- datasets reveals a significant number of genes categorized in cellular, processes, developmental processes, metabolic processes, and biological regulation (Figure 2.4A; 85, 43, 71, and 44 genes, respectively; p <

0.001). Gene ontology classification of the 50 highest upregulated genes in WT versus erbB3-/- datasets revealed the same pattern of categorization (Figure 2.4B;

Cellular processes = 16 genes, developmental processes = 8 genes, metabolic processes = 10 genes, biological regulation = 10 genes, p < 0.001). 56

2.5. DISCUSSION

TSCs make up the tripartite synapse at the NMJ and have direct association with the presynaptic motor nerve and postsynaptic receptor. Rather than their previously assumed role of passive support cells, TSCs exhibit extensive roles in the regulation and maintenance of mature NMJs (Darabid et al., 2013a; Todd et al., 2010a). The role of TSCs in development is less clear, however their necessity for the stabilization of the developing NMJ is evident.

Axonal SCs are also necessary for the complete myelination of developing motor nerves, and absence of these cells results in diminished structural axon integrity and reduced efficiency in neural transduction (Jessen & Mirsky, 2008; Pereira et al., 2012). Utilizing a murine model exhibiting a severe, dennervating phenotype in the absence of both TSCs and mSCs, we characterized the embryonic time points when these fatal changes occur.

While erbB3-/- mice show innervation of postsynaptic nAChRs by E14 (as do WT mice), the complete degeneration and loss of motor axons seen in erbB3-/- mice by E15.5 suggests immense alterations in normal developmental processes.

Despite this initial innervation, motor axons in diaphragm muscles of erbB3-/- mice had a disorganized, defasciculated appearance, indicating a role of SCs for the proper organization and pattern of motor axon migration and branching. 57

Although erbB3-/- mice show nAChR innervation, this pattern appears premature as compared to WT mice. Without the modulatory roles of SCs in this innervating process, motor axons synapse onto muscle fibers prematurely, leading to early neural activity. The endplate band widths are also significantly larger in erbB3-/- mice than in WT suggesting a role of SCs in the maintenance and integrity of the condensed nAChRs making up the endplate band. The role of acetylcholine (ACh) in development is known to be a negative regulator of the synaptogenesis process (Brandon et al., 2003; Misgeld et al., 2002), therefore it is possible that SCs counterbalance this negative effect and regulate the proper timing of innervation. In mice deficient for both erbB3 and the enzyme necessary for ACh production, choline acetyltransferase (ChAT), a rescue effect is observed

(Figure 1.8). Further studies are necessary to reveal how the interactions of SCs and nascent NMJ activity is involved in normal NMJ development.

The two subtypes of SCs, axonal SCs and TSCs, serve varying purposes in the fully developed PNS. Nevertheless, the differentiation processes for SCs only begins ~E18 (Kristján R Jessen & Mirsky, 2008; Monk et al., 2015) and myelination doesn’t fully initiate until P0 (Jessen & Mirsky, 2008; Mirsky et al., 2008). The specific signaling processes regulating the development of these two SC subtypes, remains unclear, therefore the precise form of SCs and the subsequent 58 mediating effects they play during development remains elusive. The strength of the erbB3-/- model is total ablation of SCs, however owing to the fact that SCs along the nerve have yet to make myelin, the difference between these SCs and nonmyelinating TSCs at these early ages is unclear. It is possible that mSCs serve a different role than TSCs during development and the maturation of these two

SC subtypes coincides with the proper NMJ development in a reciprocal model.

The absence of multiple SC specific genes, such as Mpz, and of genes required for essential developmental and biological processes in erbB3-/- mice, such as Sox10, implicates these cells as major mediators of PNS development. A precise time course experiment is necessary to analyze genetic changes in WT mice throughout the E14 to E15 period to characterize the genetic mechanisms responsible for this drastic period of embryogenesis. Our RNA sequencing analysis reveals possible candidates, such as Gpr17 which inhibits myelination until maturation of oligodendrocytes (Chen et al., 2009) or Fabp7 which is integral to the proper differentiation of oligodendrocytes from oligodendrocyte progenitor cells (Sharifi et al., 2013). The identification of specific genetic markers for both mSCs and TSCs will enable an extremely thorough analysis of the precise pathways that mediate normal synaptogenesis in the PNS and the identification of cell types and factors that are required at each stage. 59

Figure 2.1. Aberrant developmental patterns followed by complete degeneration of motor nerves in erbB3-/- mice. 10x images of diaphragm muscles from E14.5 WT (A) and E14.5 erbB3-/- mice (B) stained with anti- synaptophysin. E14.5 erbB3-/- mice exhibit immense disorganization of developing motor nerves characterized by increased width of an endplate band and defasciculation of nerves. 10x images of diaphragm muscles from E15.5 WT (C) and E15.5 erbB3-/- mice (D) stained with anti-synaptophysin. E15.5 WT diaphragm muscle shows increased organization and condensation of nerve terminals within a centralized endplate band (A,C) as opposed to the complete degeneration of synapses and almost complete loss of motor nerves in E15.5 erbB3-/- mice (B,D). Scalebar = 200 µm

Figure 2.2. Early onset of innervation in erbB3-/- mice. A) 10x mages of E14.25 WT (i) and E14.25 erbB3-/- mice (ii) stained with anti-synaptophysin. Diaphragms of E14.25 erbB3-/- mice exhibit early degeneration and disorganization as compared to WT mice. 63x images of endplate regions in these diaphragms (red boxes in i and ii) show excessive branching of degenerating nerve terminals in erbB3-/- mice (iv) as compared to WT mice (iii). Scalebar in i = 100 µm; scalebar in iii = 10 µm. B) 63x images of E14.5 WT (i) and erbB3-/- diaphragms (ii) stained with antibodies against synaptophysin (Syp; green) and 594-conjugated-α-BTX (BTX; red). Defasciculated motor axons of E14.5 erbB3-/- diaphragms depict early innervation of nAChRs in erbB3-/- mice (ii) as compared to WT mice (i). Scalebar = 10 µm 61

Figure 2.3. Embryonic erbB3-/- mice exhibit increased widths of endplate band regions in developing diaphragm muscles. A) 20x images of E14.25 erbB3 WT diaphragm muscles (left panels) and E14.25 erbB3-/- diaphragm muscles (right panels) stained with anti-synaptophysin (Syp) and α-BTX (BTX). Binary images created from pixels labelled with 594-conjugated-α-BTX were created to depict the area occupied by nAChRs (top right panels) while Euclidian distance maps (EDMs) were generated to show the boundary of Syp-labled particles (bottom right panels). Binary images were overlayed onto EDMs to quantify the localization of nAChRs within the Syp-labelled areas in order to determine endplate band width. B) In WT mice, the total area of α-BTX labelled particles within EDMs drastically reduces as the distance from the midline of the EDM increases. This reduction is less apparent in E14.25 erbB3-/- mice. 62

Figure 2.4. Genes upregulated in WT versus erbB3-/- mice demonstrate a bias toward developmental and cellular processes. A) The top 300 upregulated genes in WT versus erbB3-/- mice show preference for biological regulation, cellular processes, developmental processes, and metabolic processes. B) The top 50 upregulated genes in WT versus erbB3-/- mice show a similar preference as in (A), biological regulation, cellular processes, developmental processes, and metabolic processes. 63

Gene Description Fold P WT KO Change adjusted Average Average Expression Expression Gpr17 -G protein—coupled receptor 17 [Source:MGI 2.334 4.76E-175 578.58 11.38 Symbol;Acc:MGI:3584514]- Col20a1 collagen, type XX, alpha 1 [Source:MGI 2.334 2.97E-127 970.61 177.27 Symbol;Acc:MGI:1920618] Cdh19 cadherin 19, type 2 [Source:MGI 2.334 1.14E-59 198.14 14.32 Symbol;Acc:MGI:3588198] Plxnb3 plexin B3 [Source:MGI 2.334 1.76E-55 304.11 32.65 Symbol;Acc:MGI:2154240] Sox10 -SRY (sex determining region Y)—box 10 2.334 8.82E-55 839.68 124.78 [Source:MGI Symbol;Acc:MGI:98358]- Ednrb endothelin receptor type B [Source:MGI 2.334 3.97E-64 2739.75 774.84 Symbol;Acc:MGI:102720] Plekhb pleckstrin homology domain containing, family 2.334 7.95E-38 214.67 34.29 1 B (evectins) member 1 [Source:MGI Symbol;Acc:MGI:1351469] L1cam L1 cell adhesion molecule [Source:MGI 2.334 1.04E-25 349.12 59.86 Symbol;Acc:MGI:96721] Fabp7 fatty acid binding protein 7, brain [Source:MGI 2.334 4.23E-24 168.29 41.59 Symbol;Acc:MGI:101916] Gjc3 gap junction protein, gamma 3 [Source:MGI 2.334 8.62E-25 135.89 22.36 Symbol;Acc:MGI:2153041] Egfl8 -EGF—like domain 8 [Source:MGI 2.334 2.33E-22 221.58 52.26 Symbol;Acc:MGI:1932094]- Ptprz1 protein tyrosine phosphatase, receptor type Z, 2.334 3.62E-21 773.38 282.26 polypeptide 1 [Source:MGI Symbol;Acc:MGI:97816] Pou3f1 POU domain, class 3, transcription factor 1 2.334 NA 142.98 22.95 [Source:MGI Symbol;Acc:MGI:101896] Afap1l2 -actin filament associated protein 1—like 2 2.334 6.44E-25 869.82 395.73 [Source:MGI Symbol;Acc:MGI:2147658]- Ttyh1 tweety homolog 1 (Drosophila) [Source:MGI 2.334 7.53E-21 287.76 66.75 Symbol;Acc:MGI:1889007] Sfrp5 -secreted frizzled—related sequence protein 5 2.334 9.80E-24 170.04 9.89 [Source:MGI Symbol;Acc:MGI:1860298]- Atp10b ATPase, class V, type 10B [Source:MGI 2.334 3.99E-21 160.75 17.00 Symbol;Acc:MGI:2442688] Plekha4 pleckstrin homology domain containing, family 2.334 6.68E-14 603.09 237.72 A (phosphoinositide binding specific) member 4 [Source:MGI Symbol;Acc:MGI:1916467] Mpz myelin protein zero [Source:MGI 2.334 7.26E-21 733.51 6.78 Symbol;Acc:MGI:103177] Fam210 family with sequence similarity 210, member B 2.334 1.29E-18 534.94 303.63 b [Source:MGI Symbol;Acc:MGI:1914267] Plp1 proteolipid protein (myelin) 1 [Source:MGI 2.334 7.49E-12 591.63 264.80 Symbol;Acc:MGI:97623] Cnp -2prime,3prime—cyclic nucleotide 3prime 2.334 1.72E-17 901.39 519.69 phosphodiesterase [Source:MGI Symbol;Acc:MGI:88437]- Lgi4 -leucine—rich repeat LGI family, member 4 2.334 1.71E-14 96.84 13.25 [Source:MGI Symbol;Acc:MGI:2180197]- 64

Myl3 myosin, light polypeptide 3 [Source:MGI 2.334 1.16E-10 915.58 479.51 Symbol;Acc:MGI:97268] Slitrk2 -SLIT and NTRK—like family, member 2 2.334 1.23E-09 377.91 197.93 [Source:MGI Symbol;Acc:MGI:2679449]- Dhh desert hedgehog [Source:MGI 2.334 3.08E-10 135.67 36.53 Symbol;Acc:MGI:94891] Insc inscuteable homolog (Drosophila) [Source:MGI 2.334 3.28E-08 169.99 79.48 Symbol;Acc:MGI:1917942] Sema3g sema domain, immunoglobulin domain (Ig), 2.334 1.65E-09 769.35 461.61 short basic domain, secreted, (semaphorin) 3G [Source:MGI Symbol;Acc:MGI:3041242] Foxd3 forkhead box D3 [Source:MGI 2.334 1.07E-07 376.80 201.71 Symbol;Acc:MGI:1347473] Olfml2 -olfactomedin—like 2A [Source:MGI 2.334 2.14E-07 1171.75 606.75 a Symbol;Acc:MGI:2444741]- Sh3tc2 SH3 domain and tetratricopeptide repeats 2 2.334 7.55E-07 199.79 104.72 [Source:MGI Symbol;Acc:MGI:2444417] Gal3st1 -galactose—3—O—sulfotransferase 1 2.334 6.89E-10 68.99 12.54 [Source:MGI Symbol;Acc:MGI:1858277]- Igsf11 immunoglobulin superfamily, member 11 2.334 1.32E-07 132.31 46.46 [Source:MGI Symbol;Acc:MGI:2388477] Aatk -apoptosis—associated tyrosine kinase 2.334 7.33E-10 1236.02 797.01 [Source:MGI Symbol;Acc:MGI:1197518]- Spon1 -spondin 1, (f—spondin) extracellular matrix 2.334 3.13E-12 1836.39 1219.71 protein [Source:MGI Symbol;Acc:MGI:2385287]- Kcna2 -potassium voltage—gated channel, shaker— 2.334 2.48E-08 176.29 48.52 related subfamily, member 2 [Source:MGI Symbol;Acc:MGI:96659]- Tgfa transforming growth factor alpha [Source:MGI 2.334 5.71E-07 116.11 45.28 Symbol;Acc:MGI:98724] Sox5 -SRY (sex determining region Y)—box 5 2.334 1.51E-06 278.79 153.38 [Source:MGI Symbol;Acc:MGI:98367]- Srcin1 SRC kinase signaling inhibitor 1 [Source:MGI 2.334 1.84E-06 166.08 79.54 Symbol;Acc:MGI:1933179] Kcna1 -potassium voltage—gated channel, shaker— 2.334 6.51E-07 100.30 37.25 related subfamily, member 1 [Source:MGI Symbol;Acc:MGI:96654]- Bche butyrylcholinesterase [Source:MGI 2.334 5.44E-06 892.75 470.93 Symbol;Acc:MGI:894278] Gfra3 glial cell line derived neurotrophic factor 2.334 4.50E-06 468.12 267.98 family receptor alpha 3 [Source:MGI Symbol;Acc:MGI:1201403] Kcna6 -potassium voltage—gated channel, shaker— 2.334 9.90E-10 742.98 514.89 related, subfamily, member 6 [Source:MGI Symbol;Acc:MGI:96663]- Gm975 predicted pseudogene 9755 [Source:MGI 2.334 1.03E-05 185.06 73.01 5 Symbol;Acc:MGI:3642279] Fam198 family with sequence similarity 198, member B 2.334 2.56E-09 1645.72 1150.75 b [Source:MGI Symbol;Acc:MGI:1915909] Chadl -chondroadherin—like [Source:MGI 2.334 5.03E-05 231.48 144.68 Symbol;Acc:MGI:3036284]- 65

Camk2 -calcium/calmodulin—dependent protein 2.334 9.51E-05 544.77 315.29 b kinase II, beta [Source:MGI Symbol;Acc:MGI:88257]- Cmtm5 -CKLF—like MARVEL transmembrane domain 2.334 2.76E-06 67.85 20.66 containing 5 [Source:MGI Symbol;Acc:MGI:2447164]- Moxd1 -monooxygenase, DBH—like 1 [Source:MGI 2.334 6.83E-06 2114.99 1409.45 Symbol;Acc:MGI:1921582]- Mal myelin and lymphocyte protein, T cell 2.334 3.33E-10 200.48 10.58 differentiation protein [Source:MGI Symbol;Acc:MGI:892970]

Table 2.1. Top 50 upregulated genes in WT versus erbB3-/- mice. This table shows the top 50 genes with a fold change greater than 2.3 expressed in log2 terms. These genes represent candidates that are highly expressed in Schwann cells under normal conditions. Gene names, description, fold change (log2(WT/KO)), p adjusted, WT average expression levels, and KO average expression levels are presented.

CHAPTER 3

ISOLATION OF THE TERMINAL SCHWANN CELL TRANSCRIPTOME

FOR THE IDENTIFICATION OF NOVEL GENETIC MARKERS.

3.1. SUMMARY

Schwann cells (SCs) are a type of peripheral glia cell that perform essential functions for the biological and functional integrity of motor axons and their synapses. Axonal SCs provide structural and functional support to peripheral nerves while nonmyelinating terminal Schwann cells (TSCs) surround, protect, and modulate the neuromuscular junction (NMJ). The close association that axonal SCs and TSCs have with motor axons and NMJs, respectively, provide these glial cells the ability to directly affect the structural and functional integrity of these peripheral structures under developed conditions and throughout development. Current models available to study SCs do not differentiate between these two subtypes, therefore it remains unknown how axonal SCs and

TSCs modulate development in their individual ways. We sought to identify genetic markers unique to each subtype of SCs for the application in future studies regarding the distinct roles of axonal SCs and TSCs in NMJ development.

To identify these genetic components, the whole transcriptomes of axonal and synaptic regions isolated by a genetic ribosomal tagging method were compared and analyzed. Rigid statistical standards and comparison of axonal vs synaptic

RNA sequencing data with RNA sequencing data from WT vs erbB3-/- tissue revealed 13 and 19 possible genes specific to TSCs and axonal SCs, respectively.

3.2. INTRODUCTION

The role of SCs, a type of peripheral glial cell, in synaptogenesis and peripheral nervous system (PNS) development remains unclear. The axonal- derived myelinating Schwann cell is necessary to promote myelination and provides structural and trophic support to motor axons (Rhona Mirsky et al.,

2008; Samara et al., 2013) while axonal nonmyelinating SCs provide this support to sensory and autonomic nerves. However, the possible roles axonal SCs could have in the proper migration and organization of motor axons is unknown. The fully developed neuromuscular junction (NMJ) is comprised of three major components: the presynaptic motor nerve, the postsynaptic muscle-derived receptor, and the terminal Schwann cell (TSC). The close proximity of the TSC to the NMJ allows the TSC to sense and respond to synaptic activity (Darabid,

Arbour, & Robitaille, 2013b; Rousse & Robitaille, 2006; Todd et al., 2010b) and provide continual support and maintenance to the synapse (Griffin & Thompson,

2008; Webber et al., 2011). However, the ways in which TSCs direct NMJ maturation during development is unclear.

For proper maturation and function of SCs, the activation of erbB receptors by neuregulin ligand isoforms (mainly neuregulin 1 and neuregulin 1 type III) is required (Pereira et al., 2012; Quintes et al., 2010; Taveggia et al.,

2005b). Murine models that lack the neuregulin receptor, erbB3 (erbB3-/-), present 69 with a severe phenotype characterized by motor neuron retraction, loss, and denervation resulting in embryonic lethality (Riethmacher et al., 1997). The erbB3-/- model is not specific to either SC subtype and leads to a loss of all SCs, making the discrimination of axonal SC-specific or TSC-specific roles in PNS development irresolvable.

In order to understand and manipulate various phases of NMJ synaptogenesis, it is necessary to identify the mediating effects induced by either axonal SCs or TSCs. Applying a novel method will allow us to detect genetic markers unique to either axonal SCs or TSCs. By establishing markers for these two subtypes of SCs, future studies can investigate the roles of axonal SCs and

TSCs in development.

In order to identify genetic markers of axonal SCs and TSCs, we applied a genetic approach developed by Sanz et al. in 2009 that enables the isolation of polysomes from Cre-expressing cells. A genetically engineered mouse was created in which a modified exon 4 of the Rpl22 gene (a core ribosomal protein) with a hemagglutinin (HA) epitope tag was inserted downstream of a wildtype

(WT) exon 4 and flanked by loxP sites (Sanz et al., 2009). Upon expression of the

Cre recombinase enzyme (through genetically modified cell-specific Cre expression), loxP sites are targeted enabling the removal of the WT exon 4 and 70 expression of the HA-labelled exon 4 (Figure 3.1). This successfully labels ribosomes in Cre-expressing cells allowing for subsequent cell-specific ribosomal isolation by means of immunoprecipitation methodology. Using a combined genetic, physical dissection, and RNA sequencing approach, we sought to identify candidates for TSC-specific genetic markers.

3.3. MATERIALS & METHODS

Mice

erbB3 heterozygote mice (Jackson Laboratory, Sacramento, CA, USA) aged 8 weeks were backcrossed into a Balb/C background for three generations to generate homozygous erbB3 knockout mice (erbB3-/-). With a strong Balb/C background, females carried more homozygote knockout mice to late embryonic ages (embryonic day 15; E15) versus early fatality (E13 or earlier) prior to initial stages of NMJ development. At E14.5, pregnant homozygote mothers were euthanized by inhalation of 5% isofluorane followed by cervical dislocation.

Embryos were carefully removed from the uterus and kept alive in oxygenated

Krebs’ solution (containing (mM): NaCl, 120.35; KCl, 5.9; NaHCO3, 15.5;

NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.5; glucose, 11.5 (continuously gassed with 5%

CO2–95% O2, pH 7.3–7.4), until the time of experiment. Wild-type (WT), heterozygote (erbB3+/-), and homozygote (erbB3-/-) embryonic mice were genotyped for mutated erbB3 alleles. Upon confirmation of genotyping, WT and erbB3-/- mice were used for subsequent experiments and were sacrificed by decapitation. Hindlimbs from erbB3-/- or WT mice were flash frozen in liquid nitrogen and stored at -80°C for subsequent RNA isolation. 72

Rpl22 hemagglutinin (HA) tagged (RiboTag) mice (Jackson Laboratory,

Sacramento, CA, USA) and Wnt-1Cre (neural crest promoter) mice (Jackson

Laboratory, Sacramento, CA, USA) were bred with each other to generate SC- specific ribosome labeled mice (RiboTag:Wnt-1Cre) (Figure 3.1) as described by

Sanz et al. (2009). At postnatal day 0 (P0), the time point when myelination has fully begun (Rhona Mirsky et al., 2008) and SCs have differentiated into myelinating or nonmyelinating subtypes, mice from RiboTag:Wnt-1Cre breeding were genotyped using Ribotag loxP primers (Sanz et al., 2009). P0 RiboTag:Wnt-

1Cre mice were sacrificed by decapitation. Diaphragm muscles and hindlimb muscle were physically dissected for subsequent immunohistochemistry and

RNA isolation experiments, respectively. These procedures were in accordance with National Institutes of Health guidelines for the care and use of laboratory animals and approved by the Animal Ethics Committee at the University of

Nevada, Reno.

Immunohistochemistry

Diaphragm muscles from P0 RiboTag:Wnt-1Cre and littermate controls were fixed in 4% paraformaldehyde overnight at 4°C. 24 hours later, diaphragms were rinsed in phosphate-buffered saline (PBS), blocked in 0.1M glycine for 1 hour at room temperature (RT), rinsed again, and then incubated as whole 73 mounts in primary antibody solution overnight at 4°C in PBS containing 10% fetal bovine serum and 1% triton-X. Primary antibodies were goat-anti- hemagglutinin ([HA] Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit-anti-

S100 (Dako, Carpinteria, CA) diluted 1:500. 18-24 hours after primary antibody incubation, muscles were rinsed and incubated for 1 hour at RT in the dark with

AlexaFluor 488-conjugated donkey-anti-goat antibodies (Jackson

ImmunoResearch, West Grove, PA) or 488-conjugated donkey-anti-goat antibodies (Jackson ImmunoResearch, West Grove, PA) diluted 1:1000.

Confocal Imaging

Images of Schwann cells lining motor axons and terminals labeled with antibodies against S100 (a SC marker) or HA were acquired with the Olympus

Ix81 confocal microscope (Olympus Scientific Solutions Americas Corp.,

Waltham, MA, USA) and Fluoview 1000 software using a 10x or 20x objective.

Panoramas of the Wnt-1 labeled nerve trunk and endplate regions were constructed using a Nikon FN-PT upright fluorescent microscope (Nikon USA,

Melville, NY, USA) by taking a montage of high-resolution images (10X), which were combined using the Photomerge function in Adobe Photoshop CS6 (Adobe

Systems Inc., San Jose, CA, USA).

Ribosome Immunoprecipitation 74

To segregate axonal SCs from terminal SCs, we physically dissected the phrenic nerve from isolated P0 RiboTag:Wnt-1Cre mice or age-matched WT littermate diaphragm muscles to obtain an axonal SC sample (Figure 3.2, red outline). We then dissected the entire endplate band from these diaphragm muscles to isolate the terminal SCs (Figure 3.2, white outline) (while some axonal

SCs will be found in this population, the majority of SCs will be TSCs). In order to isolate HA-tagged ribosomes from each sample, we used the method described by Sanz et al. (2009). Briefly, tissue samples were placed in immunoprecipitation (IP) buffer (50 mM Tris [pH 7.5], 100 mM KCl, 12 mM

MgCl2, 1% Nonidet P-40) and homogenized using the Bullet Blender tissue homogenizer (Next Advance, Averill Park, NY, USA). Tissue lysates were then added to citrate phosphate buffer (24 mM citric acid, 52 mM dibasic sodium phosphaste; pH 5.0) containing magnetic Protein G Dynabeads (Thermo Fisher

Scientific, Waltham, MA, USA) conjugated to an antibody against hemagglutinin

(HA) and rotated overnight at 4°C. 24 hours later, samples were rinsed once in citrate phosphate buffer and 3 times in high salt buffer (50 mM Tris, pH 7.5, 300 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 100 µg/mL cycloheximide) while supernatants were retrieved using a magnetic plate. RNA was isolated from supernatants by the direct addition of Qiagen RLT buffer (Qiagen Inc., 75

Valencia, CA, USA). Total RNA was extracted using the RNeasy mini kit (Qiagen

Inc., Valencia, CA, USA).

RNA Sequencing

Using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA,

USA), the quantity and quality of RNA samples was assessed. Only samples with

RNA integrity numbers greater than 7 were used for library preparation. Due to the low quantity of RNA from this isolation method, the SMARTer Ultra Low

Input RNA Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate cDNA libraries for high-throughput sequencing by Illumina NextSeq

500 (Illumina, Inc., San Diego, CA, USA).

RNA Sequencing Analysis

UCSC Genome Bioinformatics) using TopHat2 (Kim et al., 2013). Transcript sets were assembled using Cufflinks (Trapnell et al., 2012) and then merged with

Cuffmerge (Trapnell et al., 2012). Statistical differences between RNA-sequencing data sets (erbB3-/- versus WT) were generated using Cufflinks. Differential expression analysis of FPKM values generated pure expression values for RNA 76 sequencing reads. Expression values were used to compare fold changes between datasets, expressed as log2 values.

Hierarchical clustering was performed on RiboTag:Wnt-1Cre samples derived from either the endplate region (EP) or the phrenic nerve (PN). Filtering of datasets was performed with Gene Cluster 3.0 software (Open Source

Clustering Software, University of Tokyo, Tokyo, Japan). First, datasets were filtered to eliminate genes that had less than 25 normalized reads in each sample and less than a 2-fold difference (log2 = 1.0 for a 2-fold expression level difference) between EP and PN conditions. Filtered data was then clustered based on expression level differences compared to the mean expression level for each gene. Cluster analyses were visualized using Java TreeView software

(Saldanha, 2004).

Statistics

Statistical comparisons of RNA sequencing data were performed using p values generated from DESeq software after differential expression analysis. P values were then adjusted based on a normalized histogram of all p values for each dataset to limit the rate of false positives. Fold changes of expression levels with P < 0.05 and Padj < 0.001 were considered statistically significant.

3.4. RESULTS

Schwann cell specific expression of the epitope labelled (HA) Rpl22 gene

(Figure 3.3) confirmed the specificity of this genetic approach. In order to visualize the integrity of our RNA sequencing data and establish consistent patterns between datasets from similar conditions, we performed hierarchical clustering analysis of gene expression data. The dendrogram displayed in Figure

3.4 depicts filtered data from RiboTag:Wnt-1Cre phrenic nerve (PN) and

RiboTag:Wnt-1Cre endplate (EP) samples. The similarity in expression patterns between phrenic nerve samples and between endplate samples provides confidence to successful isolation of these two regions and valid sequencing results without any major outliers.

Table 3.1 depicts the top 50 genes that were upregulated in the endplate samples as compared to the phrenic nerve samples. After application of strict statistical filtering, 13 genes were upregulated in both the endplate region and in

WT samples as compared to the phrenic nerve and erbB3-/- samples, respectively

(Figure 3.4A). These genes showed high statistical significance in either the endplate versus phrenic nerve differential expression analysis (Padj < 0.04) or in the WT versus erbB3-/- differential expression analysis (Padj < 0.004). These genes also showed a 1.25 fold-change as compared to phrenic nerve samples. 78

Additionally, 9 genes were found to be downregulated in the endplate region as compared to the phrenic nerve and upregulated in WT samples as compared to erbB3-/- samples (Figure 3.4B). These genes were downregulated by a factor of 1.25 in the endplate versus phrenic nerve differential expression analysis and upregulated by a factor of 1.25 in the WT versus erbB3-/- differential expression analysis (Padj < 0.05 in EP versus PN; Padj < 0.0006 in WT versus KO).

One gene, Sorbs1, was found to be statistically significant in both differential expression analyses (Padj = 0.00594 in EP versus PN; Padj = 0.0104 in WT versus

KO) with only a fold-change greater than a factor of 1.25 in the WT versus KO analysis (log2 = 0.30 for WT expression levels compared to KO expression levels; data not shown). Four genes, Cp, Ptch2, Myl9, Kif1a, were found to be statistically significant in both differential expression analyses comparing PN to

EP and WT to KO (PN vs EP Padj values: Cp = 0.0366, Ptch2 = 0.0013, Myl9 =

0.00014, Kif1a = 0.0192; WT vs KO Padj values: Cp = 0.0004, Ptch2 = 0.00117, Myl9

= 0.00137, Kif1a = 0.0022).

3.5. DISCUSSION

Development of the NMJ is a tightly regulated process, coordinating a vast array of signaling pathways in both the presynaptic motor nerve and the postsynaptic muscle fiber. In the absence of SCs, the synaptic connection is initially made and then quickly deteriorates leading to synapse and motor nerve degeneration and embryonic fatality. While SCs play a crucial part in the maintenance and maturation of the NMJ, it is difficult to distinguish how axonal

SCs and TSCs each regulate this process. In the absence of genetic markers specific to each SC subtype, transgenic cell ablation methodologies result in the loss of the entire population of SCs. It is imperative to identify markers specific to axonal SCs and TSCs in order to develop a more detailed understanding of

PNS development and NMJ synaptogenesis.

The Ribotag approach enables cell-specific epitope tagging of ribosomes.

RiboTag:Wnt-1Cre mice display SC specific expression of the hemagglutinin tag, allowing subsequent isolation of SC-derived ribosomes. This genetic approach combined with the manual isolation of TSCs from axonal SCs reflects a unique methodology for obtaining the TSC and axonal SC transcriptome. Comparison of gene expression levels in the EP and PN populations to those from erbB3-/- and

WT RNA sequencing datasets provided further validity to this approach. 80

Analysis of RNA sequencing data generated from low RNA input can cause extensive outliers and false positive results. Applying stringent levels for significance and using adjusted p values based on a normalized threshold for each dataset can help diminish this problem. Clustering analysis of Ribotag RNA sequencing results also provided a method for ensuring the quality of our differential expression analysis. Comparison of filtered gene expression levels from the Ribotag RNA sequencing study to upregulated expression levels of genes in WT RNA sequencing datasets as compared to erbB3-/- datasets provided assurance for SC derived genes. After subjecting RNA sequencing data to strict filters for both fold-change and statistical significance, only 13 and 9 genes remained as candidates for TSC or mSCs, respectively.

Genes that passed statistical tests and showed upregulation in both the EP samples as compared to PN and WT samples as compared to KO samples necessitate further investigation for their specific expression in TSCs during early developmental ages. Those genes that met the same statistical standard and demonstrated downregulation in the EP as compared to PN and upregulation in

WT as compared to KO are likely candidates for axonal SC specific expression.

The present study utilized early developmental ages, P0, for Ribotag RNA sequencing and E14.5 for erbB3 RNA sequencing, since P0 initiates myelination 81 in development and SC differentiation has occurred (Jessen & Mirsky, 2008;

Rhona Mirsky et al., 2008). Nonetheless, these various candidates should also be tested in adult models to ensure continued expression of TSC or axonal SC specific markers.

Subsequent studies from this approach will ascertain the specificity of these various genes for SC subtypes. Identification of these markers will be crucial for understanding various stages in development as well as in disease.

Multiple peripheral neuropathies present with damaging effects to SCs as well as motor neurons and synapses. The ability to apply SC-subtype specific markers to disease investigations will allow us to dissect the contributing effect TSCs may have at the NMJ or axonal SCs may have on motor axons. Amyotrophic lateral sclerosis is one neurodegenerative disease that affects upper and lower motor neurons. However, recent studies provide evidence for presymptomatic dysfunction at the NMJ by TSCs as well as in axonal SCs (Arbour, Tremblay,

Martineau, Julien, & Robitaille, 2015; De Winter et al., 2006; Keller, Gravel, &

Kriz, 2009; Verheijen et al., 2014). The ability to investigate one subtype of SCs through the use of subtype-specific markers would enable the more precise discrimination of toxic effects exerted by TSCs or axonal SCs in these disease models. 82

Figure 3.1. Cre-lox system enables cell-specific transgene expression. The cre recombinase enzyme (Cre) is expressed downstream of a cell-specific or tissue- specific promoter (Wnt-1: SC specific). The gene of interest (GeneX) is flanked by loxP sites, sites that are recognized by Cre in a homozygous loxP ‘floxed’ mouse. Breeding of the SC-specific Cre mouse with the homozygous loxP ‘floxed’ mouse derives first generation Cre-lox mice that are heterozygous for the conditional knockout of GeneX. Following this genetic cross, cells expressing Cre recognize the loxP sites on the gene of interest and excise that gene so it is no longer flanked and can be expressed (modified from Jackson Laboratory; jax.org/news- and-insights/jax-blog/2011/september/cre-lox-breeding-for-dummies).

Figure 3.2. Physical isolation of axonal Schwann cells and terminal Schwann cells enables segregation of ribosomal-tagged Schwann cell subpopulations. A montage image comprised of 10x images from the diaphragm of a postnatal day 4 (P4) RiboTag:Wnt-1Cre mouse. The region encompassed by the white oval represents the endplate area that was dissected for physical isolation of TSCs. The innervating phrenic nerve, circled in red, contains mSCs that were physically isolated by dissection. Scalebar = 200 µm.

Figure 3.3. Specific isolation of SC-labelled ribosomes. 20x image of a P0 diaphragm labelled with an antibody against hemagglutinin (HA) shows HA expression along the nerve, location of myelinating Schwann cells (mSCs), and at the nerve terminals, location of terminal Schwann cells (TSCs). Scalebar = 50 µm

Figure 3.4. Hierarchical clustering of endplate versus phrenic nerve differential gene expression analysis. The dendrogram shows relative changes in gene expression levels as compared to the mean expression level for each gene. Genes are depicted across each level of the dendrogram and variations in expression level are expressed by a factor of log2, represented by changes in color. 86

Figure 3.5. Comparisons of significant changes in gene expression levels between endplate and phrenic nerve samples and between erbB3 wildtype and erbB3-/- samples. A) 13 genes were found to be upregulated (log2(fold change) > 1.25) in both the endplate versus phrenic nerve RNA sequencing analysis (white bars) and in the WT versus KO RNA sequencing analysis (black bars). The fold change in expression levels of these genes (depicted as a log2 value) was significant in either the EP versus PN analysis (green) or in the WT versus KO analysis (tan). B) 9 genes were found to be downregulated (log2(fold change) > 1.25) in the endplate versus phrenic nerve RNA sequencing analysis (white bars) and upregulated in the WT versus KO RNA sequencing analysis (black bars). The fold change in expression levels of these genes (depicted as a log2 value) was significant in either the EP versus PN analysis (green), in the WT versus KO analysis (tan), or in both analyses (grey). 87

PN Fold P EP Average Gene Description Average Change adjusted Expression Expression Myh13 myosin, heavy polypeptide 13, skeletal muscle 12.395 1.09E-33 6301.79 0.00 [Source:MGI Symbol;Acc:MGI:1339967] Myh4 myosin, heavy polypeptide 4, skeletal muscle 11.018 1.28E-70 48938.54 16.71 [Source:MGI Symbol;Acc:MGI:1339713] Myh2 myosin, heavy polypeptide 2, skeletal muscle, 10.957 5.60E-53 19150.27 5.65 adult [Source:MGI Symbol;Acc:MGI:1339710] Myh8 myosin, heavy polypeptide 8, skeletal muscle, 10.954 1.10E-68 2908969.93 1035.19 perinatal [Source:MGI Symbol;Acc:MGI:1339712] Myh1 myosin, heavy polypeptide 1, skeletal muscle, 10.517 2.89E-50 45661.96 20.39 adult [Source:MGI Symbol;Acc:MGI:1339711] Apobec apolipoprotein B mRNA editing enzyme, 10.168 2.67E-30 5186.66 1.87 2 catalytic polypeptide 2 [Source:MGI Symbol;Acc:MGI:1343178] Sptb spectrin beta, erythrocytic [Source:MGI 10.106 6.04E-46 236033.69 140.93 Symbol;Acc:MGI:98387] Rpl3l -ribosomal protein L3--like [Source:MGI 10.026 1.83E-27 2272.73 0.97 Symbol;Acc:MGI:1913461]- Neb nebulin [Source:MGI Symbol;Acc:MGI:97292] 9.720 8.62E-61 159318.44 144.01 Smtnl1 -smoothelin--like 1 [Source:MGI 9.668 1.50E-21 1243.82 0.49 Symbol;Acc:MGI:1915928]- Ampd1 adenosine monophosphate deaminase 1 9.662 2.23E-21 1252.90 0.49 [Source:MGI Symbol;Acc:MGI:88015] Atp1b4 ATPase, (Na+)/K+ transporting, beta 4 9.563 1.41E-36 5484.81 4.23 polypeptide [Source:MGI Symbol;Acc:MGI:1915071] Myo18 myosin XVIIIb [Source:MGI 9.526 9.57E-23 1580.20 0.80 b Symbol;Acc:MGI:1921626] Lmod3 leiomodin 3 (fetal) [Source:MGI 9.493 9.91E-20 1180.48 0.49 Symbol;Acc:MGI:2444169] Ckmt2 creatine kinase, mitochondrial 2 [Source:MGI 9.459 5.58E-42 7803.44 7.66 Symbol;Acc:MGI:1923972] Gm286 predicted gene 28653 [Source:MGI 9.325 1.14E-28 2477.05 1.87 53 Symbol;Acc:MGI:5579359] Cacng6 -calcium channel, voltage--dependent, gamma 9.275 3.35E-15 553.94 0.00 subunit 6 [Source:MGI Symbol;Acc:MGI:1859168]- Atp2a1 ATPase, Ca++ transporting, cardiac muscle, 9.230 4.21E-47 18315.08 22.33 fast twitch 1 [Source:MGI Symbol;Acc:MGI:105058] Tmod4 tropomodulin 4 [Source:MGI 9.230 1.20E-27 1981.76 1.56 Symbol;Acc:MGI:1355285] Tnnt3 troponin T3, skeletal, fast [Source:MGI 9.215 3.39E-33 269988.24 289.78 Symbol;Acc:MGI:109550] Myot myotilin [Source:MGI 9.206 1.47E-32 4073.43 4.51 Symbol;Acc:MGI:1889800] Tnnc2 troponin C2, fast [Source:MGI 9.120 3.02E-33 35285.07 41.37 Symbol;Acc:MGI:98780] Nctc1 -non--coding transcript 1 [Source:MGI 8.964 3.45E-17 699.94 0.31 Symbol;Acc:MGI:1306816]- 88

Tcap -titin--cap [Source:MGI 8.930 1.15E-27 4416.28 5.65 Symbol;Acc:MGI:1330233]- Obscn -obscurin, cytoskeletal calmodulin and titin-- 8.879 3.84E-44 8413.44 13.35 interacting RhoGEF [Source:MGI Symbol;Acc:MGI:2681862]- Tnni2 troponin I, skeletal, fast 2 [Source:MGI 8.846 4.08E-40 73276.04 116.34 Symbol;Acc:MGI:105070] Nrap -nebulin--related anchoring protein 8.840 2.79E-17 649.01 0.49 [Source:MGI Symbol;Acc:MGI:1098765]- Mylk4 myosin light chain kinase family, member 4 8.806 3.95E-13 392.86 0.00 [Source:MGI Symbol;Acc:MGI:3643758] Hhatl -hedgehog acyltransferase--like [Source:MGI 8.770 2.09E-18 835.26 0.62 Symbol;Acc:MGI:1922020]- Myh3 myosin, heavy polypeptide 3, skeletal muscle, 8.679 1.14E-28 9595.46 15.22 embryonic [Source:MGI Symbol;Acc:MGI:1339709] Dusp27 dual specificity phosphatase 27 (putative) 8.677 1.34E-12 357.27 0.00 [Source:MGI Symbol;Acc:MGI:2685055] Ttn titin [Source:MGI Symbol;Acc:MGI:98864] 8.656 3.98E-42 267418.51 501.75 Tmem1 transmembrane protein 182 [Source:MGI 8.604 2.96E-18 753.57 0.80 82 Symbol;Acc:MGI:1923725] Scn4a -sodium channel, voltage--gated, type IV, 8.530 6.03E-12 328.97 0.00 alpha [Source:MGI Symbol;Acc:MGI:98250]- Smyd1 SET and MYND domain containing 1 8.526 3.15E-18 742.56 0.97 [Source:MGI Symbol;Acc:MGI:104790] 4632404 RIKEN cDNA 4632404M16 gene [Source:MGI 8.494 5.49E-15 489.34 0.31 M16Rik Symbol;Acc:MGI:1921598] Myf6 myogenic factor 6 [Source:MGI 8.425 1.64E-11 308.72 0.00 Symbol;Acc:MGI:97253] Ryr1 ryanodine receptor 1, skeletal muscle 8.305 2.67E-25 14454.46 29.13 [Source:MGI Symbol;Acc:MGI:99659] BC0021 cDNA sequence BC002189 [Source:MGI 8.188 1.11E-10 254.58 0.00 89 Symbol;Acc:MGI:3642300] Capn3 calpain 3 [Source:MGI 8.180 7.05E-14 409.81 0.49 Symbol;Acc:MGI:107437] Actn2 actinin alpha 2 [Source:MGI 8.139 1.15E-27 20708.81 51.12 Symbol;Acc:MGI:109192] Scn4b sodium channel, type IV, beta [Source:MGI 8.083 2.75E-10 237.08 0.00 Symbol;Acc:MGI:2687406] Dok7 docking protein 7 [Source:MGI 8.043 1.38E-12 412.61 0.49 Symbol;Acc:MGI:3584043] Acta1 actin, alpha 1, skeletal muscle [Source:MGI 8.013 7.30E-32 1340295.18 3857.33 Symbol;Acc:MGI:87902] - -microRNA 133a--1 [Source:MGI 7.967 7.17E-10 219.42 0.00 Mir133 Symbol;Acc:MGI:2676818]- a--1- Ckm creatine kinase, muscle [Source:MGI 7.959 8.46E-30 126763.25 372.96 Symbol;Acc:MGI:88413] Fsd2 fibronectin type III and SPRY domain 7.931 4.52E-14 452.27 0.62 containing 2 [Source:MGI Symbol;Acc:MGI:2444310] 89

Fitm1 -fat storage--inducing transmembrane protein 7.866 5.65E-17 976.80 2.43 1 [Source:MGI Symbol;Acc:MGI:1915930]- Itgb6 integrin beta 6 [Source:MGI 7.850 1.91E-09 204.88 0.00 Symbol;Acc:MGI:96615] - -microRNA 133a--2 [Source:MGI 7.846 2.65E-09 219.07 0.00 Mir133 Symbol;Acc:MGI:3618718]- a--2-

Table 3.1. Candidates for TSC genetic markers. This table depicts the 50 most upregulated genes, determined by the fold change of gene expression level, in the endplate RNA sequencing dataset as compared to the dataset of the phrenic nerve.

CHAPTER 4

STRUCTURAL AND FUNCTIONAL ABNORMALITIES OF THE

NEUROMUSCULAR JUNCTION IN THE TREMBLER-J HOMOZYGOTE

MOUSE MODEL OF CONGENITAL HYPOMYELINATING NEUROPATHY.

Alexandra N. Scurry, Dante J. Heredia, Cheng-Yuan Feng, Gregory B. Gephart, Grant W. Hennig, & Thomas W. Gould

Published in final edited form:

Journal of Neuropathology & Experimental Neurology. 2016 April; 75(4): 334- 346. doi: 10.1093/jnen/nlw004 91

4.1. SUMMARY

Mutations in peripheral myelin protein 22 (PMP22) result in the most common form of Charcot-Marie-Tooth (CMT) disease, CMT1A. This hereditary peripheral neuropathy is characterized by dysmyelination of peripheral nerves, reduced nerve conduction velocity, and muscle weakness. A PMP22 point mutation in

L16P (leucine 16 to proline) underlies a form of human CMT1A as well as the

Trembler-J mouse model of CMT1A. Homozygote Trembler-J mice (TrJ ) die early postnatally, fail to make peripheral myelin, and are thus more similar to patients with congenital hypomyelinating neuropathy than those with CMT1A.

Because recent studies of inherited neuropathies in humans and mice have demonstrated that dysfunction and degeneration of neuromuscular synapses or junctions (NMJs) often precede impairments in axonal 25 conduction, we examined the structure and function of NMJs in TrJ mice. Although synapses appeared to be normally innervated even in end-stage TrJ mice, the growth and maturation of the NMJs were altered. In addition, the amplitudes of nerve- evoked muscle endplate potentials were reduced and there was transmission failure during sustained nerve stimulation. These results suggest that the severe congenital hypomyelinating neuropathy that characterizes TrJ mice results in structural and functional deficits of the developing NMJ. 92

4.2. INTRODUCTION

Charcot Marie Tooth disease (CMT) is the most frequently inherited peripheral neuropathy, occurring in 1 of 2500 people in the United States

(Tanaka & Hirokawa, 2002). Mutations underlying CMT directly affect motor and sensory neurons (Type 2) or indirectly impair their function by causing damage to Schwann cells (Type 1) (Shy & Rose, 2005; Ueli Suter & Scherer, 2003).

The principal clinical feature of CMT is muscle weakness and atrophy which begins in the distal limb and spreads proximally (Sereda & Nave, 2006).

Physiologically, patients with CMTI but not those with CMT2 exhibits a reduction of motor nerve conduction velocity (MCV), consistent with a myelin disruption; however, the clinical manifestations of CMT1 more closely correlate with functional evidence of muscle denervation than with reduced MCVs

(Krajewski, 2000). Consistent with this idea, animal models of CMT1 exhibit a loss of large-diameter axons and impaired axonal transport even before impairment of MCV (Vavlitou et al., 2010). In addition to these signs of axonal dysfunction, neuromuscular junctions (NMJs) show evidence of denervation in

CMT1 mutant mice (Ang et al., 2010; Yin et al., 2004). Therefore, peripheral dysfunction of motor nerve terminals may represent an important early step in the pathophysiology of CMT1. 93

CMT Type 1A (CMT1A) is the most common form of all CMTs and is characterized by mutations or other genetic disruptions of peripheral myelin protein 22 (PMP22), a 22kd tetraspan transmembrane glycoprotein located within compact myelin. Histologically, peripheral nerves exhibit signs of demyelination and/or dysmyelination, depending on the PMP22 genotype. For example, replacement of glycine at 150 by aspartic acid (G150D) in the Trembler mouse (Tr) is associated with demyelinated axons with a compensatory, ongoing increase in Schwann cell proliferation; by contrast, substitution of leucine 16 with proline (L16P) in the Trembler-J mouse (TrJ) is associated with dysmyelination with only a transient increase in dividing Schwann cells (Robertson, Huxley,

King, & Thomas, 1999; U. Suter et al., 1992; Ueli Suter et al., 1992). The L16P

PMP22 allele, which also underlies a form of human CMT1A (Valentijn et al.,

1992), is semidominant in TrJ mice and triggers moderate dysmyelination in heterozygotes and severe hypomyelination and early postnatal lethality

(~postnatal day 15; P15) in homozygotes (Henry & Sidman, 1998). This mutation disrupts the formation and stability of myelin in part by increasing the lysosomal degradation of PMP22 protein (Notterpek, Shooter, & Snipes, 1997).

The difference in myelination (hypomelination vs. dysmyelination) and prognosis (shortened vs. normal longevity) between homozygotic and 94 heterozygotic TrJ mice, respectively, suggests that homozygotes (TrJ mice) may more closely represent a model of congenital hypomyelinating neuropathy, which also exhibits severe hypomyelination and a more severe neurological prognosis than that occurs in patients with CMT1A (Harati & Butler, 1985;

Warner et al., 1998). In particular, some cases of congenital hypomyelination present with defects in respiration, swallowing and early death (Hahn, Henry,

Hudgins, & Madan, 2001). Respiratory failure may be caused by central or peripheral mechanisms, such as impaired respiratory drive (Amano et al., 2009), or peripheral dysfunction or degeneration of NMJs (Vanoli, Rinchetti, Porro,

Parente, & Corti, 2015). Recently, it was reported that TrJ heterozygote mice, similar to other animal models of CMT1, show axonal degeneration and denervated NMJs (Meekins, Carter, Emery, & Weiss, 2007; Nicks et al., 2013).

However, whether the more severely affected TrJ homozygotes exhibit degeneration of motor axons or NMJs has not been determined. Additionally, although PMP22 is expressed by non-myelinating Schwann cells of the nerve

(Snipes, 1992), whether this pattern includes the nonmyelinating subtype at the

NMJ (terminal/perisynaptic Schwann cells; T/PSCs) has not been examined.

Because T/PSCs are required to maintain the NMJ (Reddy et al., 2003b), toxicity 95 of the TrJ PMP22 mutation within these cells could lead to non-cell autonomous dysfunction of the NMJ.

Together with previous reports demonstrating that in some animal models of motor neuron disease, peripheral denervation of NMJs occurs before or in the absence of motor neuron death (F. Chen et al., 2010; Fischer et al., 2004;

Gould et al., 2006), these observations raise the possibility that homozygote TrJ mice die early postnatally because of defects in neuromuscular synaptic function or maintenance. In the present study, we examined the anatomical and physiological characteristics of the NMJ in TrJ mice and showed that although neuromuscular innervation is maintained in multiple muscle subtypes, some abnormal structural and functional features of the NMJ are observed. We discuss these deficits in the context of human congenital hypomyelinating neuropathies.

4.3. MATERIALS & METHODS

Mice

Trembler-J heterozygote mice were purchased from The Jackson

Laboratory (Bar Harbor, ME) and then backcrossed 3 times into a Balb/C background before mating to themselves to generate homozygous TrJ mice. This approach was taken because heterozygote females in this strain were better mothers to homozygote pups (personal communication, L. Notterpek). Wild- type (WT), heterozygote and homozygote mice were genotyped for the L16P point mutation by a combined PCR and BanI restriction digest protocol as previously described (Notterpek et al., 1997).

Homozygote mice become easily identifiable between P8-P10, growing less quickly than WT and never beyond 17 days of age. In contrast, heterozygote mice begin to shake near P20-P25. For these studies, we exclusively characterized

WT and homozygote TrJ mice. TrJ and WT mice were killed at “end-stage,” or when they were no longer able to right themselves (between P13-P16). Motor performance was measured by noting the day at which TrJ mice could be overtly distinguished by virtue of impaired coordination and shakiness. Because we failed to note any difference between male and female mice in our analyses, they were pooled. Animals were either cervically dislocated (for electrophysiological 97 studies) or anaesthetized with a mixture of ketamine and xylazine and then transcardially perfused with 4% paraformaldehyde (immunohistochemistry) or with a mixture of glutaraldehyde and paraformaldehyde in sodium cacodylate buffer (electron microscopy). The University of Nevada, Reno Institutional

Animal Care and Use Committee approved the experimental use of these animals.

Immunohistochemistry

Diaphragm muscles from paraformaldehyde-perfused animals were dissected, rinsed in phosphate-buffered saline (PBS), blocked in 0.1M glycine for

1 hour at room temperature (RT), rinsed again, and then incubated as whole mounts in primary antibody solution overnight at 4°C in PBS containing 10% fetal bovine serum and 1% triton-X. Primary antibodies were rabbit-anti- synaptophysin ([Syp] Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit-anti-

S100 (Dako, Carpinteria, CA). The next day, muscles were rinsed and incubated for 1 hour at RT in the dark with AlexaFluor 488-conjugated donkey-anti-rabbit antibodies (Jackson ImmunoResearch, West Grove, PA) together with

AlexaFluor594-conjugated α-bungarotoxin ([BTX] Biotium, Hayward, CA). The extensor digitorum longus (EDL), soleus, tibialis anterior (TA) and erector spinae muscles were individually dissected from perfused animals, postfixed in 4% 98 paraformaldehyde at 4°C overnight, immersed in 30% sucrose, embedded in 3:2 sucrose:OCT cryoprotectant (Tissue-Tek, Sakura, Torrance, CA) and then snap- frozen in isomethylbutane. Sections were cut at 20- or 40-μm thickness and mounted on Superfrost slides pretreated with 0.01% poly-D-lysine and stained with the following antibodies: Syp, S100, goat-anti-vesicular acetylcholine transporter (VAChT) (EMD Millipore, Temecula, CA), rabbit-anti-muscle-specific kinase (kindly provided by L. Mei, University of Georgia). Some slides were incubated at the time of secondary antibody application with AlexaFluor 647- conjugated FasII (kindly provided by W. Thompson, Texas A&M), which binds and labels acetylcholinesterase.

Confocal Image Analysis

Images of postsynaptic α-BTX-labeled nicotinic acetylcholine receptor

(nAChR) clusters (referred to as NMJ) were acquired with the Olympus Ix81 confocal microscope and Fluoview 1000 software using a 63x objective. Using

Volumetry G8c, a custom software program developed by author GWH, confocal stacks were rotated for alignment to an orthogonal axis and individual NMJs were isolated from the stack. A normalized threshold, based on a signal-to-noise ratio, was applied to each endplate stack. Maximum fluorescence-intensity maps were created and depth coloring was used to assess surface curvature and depth. 99

A bounding cube around each NMJ was used to calculate overall NMJ dimensions, and a subvoxel volume routine was used to calculate NMJ volume.

Individual NMJ depth maps were further processed for perforation analysis.

Only NMJs located primarily on the top and bottom surface of muscle fibers was analyzed (depth < 25 µm) to minimize the effect of reduced z-axis resolution in any NMJ located on the side of muscle fibers. Perforations present in isolated

NMJs were excluded from analysis if they were less than 0.22 µm2 (10 pixels).

The size and volume of each perforation were measured.

Muscle Fiber Analysis

Semithin sections (1 µm) of end-stage TrJ and control diaphragm muscles were cut using an UltraCut ultramicrotome (Type 706201, Leica Microsystems,

Vienna, Austria) and stained with toluidine blue. These muscle sections were imaged with a Zeiss Axioskop 2 plus light microscope using a 100x objective (NA

1.25) and a Zeiss Axiocam 105 color camera. Diameters of individual diaphragm muscle fibers were measured utilizing the Feret’s diameter measurement tool in

ImageJ (http://rsb.info.nih.gov/ij/index.html), which computes the longest distance between any 2 points along a region of interest’s boundary, and is the most sensitive measure to differences in sectioning angle. Myofiber area measurements were calculated using the Area measurement tool in ImageJ, 100 which uses values and coordinates of neighboring pixels within each selected object or muscle fiber.

Electrophysiology

Diaphragm muscles from end-stage TrJ and WT mice were dissected and pinned on a Sylgard-coated dish containing oxygenated (95% O2 and 5% CO2)

Krebs-Ringer’s solution (121 mM NaCl, 5 mM KCl, 2mM CaCl2, 1 mM MgCl2, 1 mM NaH2P04, 12 mM NaHCO3, and 11 mM glucose), pH 7.3, at RT. After 30 minutes of perfusion, the phrenic nerve was drawn into a suction electrode and stimulated for 0.1 ms with supramaximal square waves from an S88 stimulator together with a stimulation isolation unit (Grass Technologies, Middleton, WI).

Sharp intracellular recording electrodes were made from borosilicate (1mm OD,

0.5mm ID; Sutter, CA) on a P-97 Flaming-Brown puller of approximately 60 MΩ, which were backfilled with 3M KCl. Signals were amplified and digitized at

2KHz by an AxoClamp 900A amplifier and Digidata 1550 and recorded by pClamp 10 software (Molecular Devices, Sunnyvale, CA). Correct positioning of micro-electrodes at the motor endplate of the costal diaphragm was confirmed at the beginning of an experiment by electrophysiological measures (i.e., rise-to- peak or 10-90% rise times of miniature endplate potentials [mEPPs] less than 2 ms) as well as by post-hoc BTX labeling. mEPP decay times were calculated by 101 measuring the descent time from peak to half amplitude. Endplate potentials

(EPPs) were measured after treatment with 2.5 µM Nav1.4 antagonist µ- conotoxin GIIIb ((Kaja et al., 2010); Peptides International, Louisville, KY). EPPs were normalized to a resting membrane potential of -70 mV and the acetylcholine (Ach) reversal potential was assumed to be 0 mV (Magleby &

Stevens, 1972). EPPs were then corrected for the effects of non-linear summation

(McLachlan & Martin, 1981) using the formula: corrected EPP = EPP/(1-f [EPP/E]) where f=0.8 and E=the difference between the resting and ACh reversal potentials. The number of quanta released in response to a nerve impulse was measured by the direct method by dividing the mean amplitude of normalized and corrected EPPs by the mean amplitude of mEPPs. Only muscle cells with resting membrane potentials between -60 and -75 mV were included for analysis.

For 40 Hz trains, each value represents the average of 3 EPPs taken at a time after the onset of stimulation. Stimulation episodes were separated by 20 minutes to allow recovery.

Electron Microscopy

End-stage TrJ and WT mice were transcardially perfused after making a small incision in the diaphragm (precordial region), with rinse buffer (0.1M sodium cacodylate) followed by fix buffer (1.5% glutaraldehyde, 2% 102 paraformaldehyde in 0.1M sodium cacodylate). The intact and straight segment of the phrenic nerve (for proximal axon area and axon/Schwann cell counts), as well as the endplate-enriched medial portion of the costal diaphragm (for distal axon area and diameter as well as NMJ structural analysis) were each dissected and incubated in fixative at 4°C overnight and then in rinse for several hours at

4°C. Nerve and muscle samples were then post-fixed in 2% osmium tetroxide for

30 minutes, dehydrated in a graded series of ethanol dehydrations, incubated in propylene oxide, embedded in Spurr’s resin, oriented to generate transverse sections of axons or myofibers, respectively, and polymerized at 60°C overnight.

Ultrathin sections were cut at 90 µm and stained with uranyl acetate followed by lead citrate. Sections were photographed or digitized using a Phillips CM 10 transmission electron microscope equipped with a Gatan BioScan digital imaging system. For intramuscular phrenic nerve branch axon analysis, we cut the central portion of the NMJ enriched strip to focus on the central-most branch (i.e., most central branch along the dorsoventral axis). For axon/Schwann cell counts, the number of axons and the number of Schwann cells with nuclei in the plane of the section were quantified in proximal phrenic nerve transverse sections.

Statistics 103

Differences between TrJ and WT mice were assessed by unpaired Student t-tests assuming equal variance. For physiological studies, data were generated from 3 or more cells per animal, n=3 or greater. For studies of NMJ volume and maturation in which the total number of examined NMJs was different in each genotype, no less than 10 NMJs per diaphragm (volume) or 4 NMJs per diaphragm (maturation) and 3 diaphragms per genotype were included for each comparison. A P < 0.05 was considered significant.

104

4.4. RESULTS

We first examined motor incoordination and lifespan of homozygotic TrJ mice backcrossed at least 3 times into the Balb/C locus. TrJ mice were indistinguishable from heterozygote and wild-type controls until ~P7. At this age, TrJ mice began to appear uncoordinated and moved unsteadily and shakily, reflected as poor motor performance (Figure 4.1A). Next, we examined lifespan in TrJ mice. We defined end-stage as the day at which animals could not right themselves in less than 30 seconds after being placed on their backs, although mice sometimes died even before this end-stage diagnostic. TrJ mice began to die as early as P11, most reaching endstage between P13 and P15; no mice lived beyond P17 (Figure 4.1B). Near the end of their lifespans, TrJ mice occasionally exhibited seizure-like episodes, after which they appeared lifeless for up to several minutes, before finally regaining vigor. At end-stage, TrJ mice weighed less than their WT littermates (8.8 + 0.31 vs. 7.8 + 0.47 g, p < 0.05, WT vs. TrJ mice, n=4). When we examined the wet weight of the rectus femoris, we also observed a small but significant difference between genotypes (6.32 + 0.23 vs. 5.58 + 0.33 mg, p < 0.05, WT vs. TrJ mice, n=5).

In order to determine whether TrJ mice die early postnatally because of motor axon or nerve terminal degeneration, we examined the area and 105 ultrastructure of axons in the phrenic nerve, which is almost exclusively composed of motor axons innervating the diaphragm muscle. We found that in the phrenic nerve of end-stage TrJ mice (although motor axons were severely hypomyelinated), there were no overt signs of Schwann cell pathology or axonal degeneration, such as debris or axon swellings (Figure 4.1C). The area of these proximal phrenic motor axons was also unaffected (4.66 + 1.53 vs. 4.29 + 2.13

µm2, p = 0.41, WT vs. TrJ mice, at least 10 axons per nerve, n=3). However, more distal, intramuscular phrenic nerve branches were smaller in TrJ than in WT mice

(area, 6.42 + 3.74 vs. 3.96 + 2.26 µm2, p < 0.01; diameter, 2.16 + 0.3 vs. 2.76 + 0.4

µm, p < 0.005, WT vs. TrJ mice, 70 axons from n=3 (WT) and 46 axons from n=3

(TrJ) mice). When we quantified the ratio of Schwann cell nuclei per axon, we observed an increase (36.7 + 5.25 vs. 49.8 + 6.8 glial cells /100 phrenic nerve axons, p < 0.05, WT vs. TrJ mice, n=4; Figure 4.1C), similar to a previous report of sciatic axons (Robertson et al., 1999). Together, these data suggest that while phrenic motor axons fail to exhibit overt pathological signs of degeneration in end-stage

TrJ mice, the distal segments of these axons are atrophic.

In order to evaluate the most distal component of the motor neuron, the

NMJ, we wholemount immunostained the diaphragm muscle with antibodies against the presynaptic protein synaptophysin and the postsynaptic marker BTX, 106 which labels nAChR clusters at the NMJ. Similar to control, diaphragm from end-stage TrJ mice exhibited a normal gross pattern of innervation, as indicated by the number of BTX-labeled nAChR clusters exhibiting apposed, synaptophysin-immunoreactive nerve terminals (Figure 4.2A). We failed to detect denervated nAChRs, terminal sprouts, or other markers of denervation.

We did, however, note that the size of BTX-labeled nAChR clusters appeared smaller in TrJ than in WT mice (see below). Since motor innervation of specific muscle subtypes often develops differently and responds differentially to disease and injury (Pun et al., 2002), we also examined NMJs in cryosections of muscles containing distinct fiber subtypes or originating from distinct embryological masses, even though overt differentiation of fiber subtypes on the basis of myosin heavy-chain expression does not reportedly occur until the third postnatal week, well after the death of TrJ mice (Sieck & Prakash, 1997).

Innervation of the soleus (Type I or oxidative fiber), the EDL (Type IIB or fast glycolytic fiber), and the TA (mixed) muscles, similar to the diaphragm (mixed), was unaffected in TrJ mice (Figure 4.2A). Innervation of the erector spinae muscles (epaxial muscle group), similar to the diaphragm (hypaxial group) and leg muscles (appendicular group), was unaffected (data not shown). In order to ensure that synaptophysin antibody labeling accurately represented presynaptic 107 nerve terminals, we immunostained sections of these muscles with an antibody against another presynaptic marker, the vesicular acetylcholine transporter

(VAChT). Similar to results with synaptophysin antibodies, VAChT antibodies labeled the presynaptic terminals of all NMJs in each of the muscles examined

(Figure 4.2B).

We then examined whether T/PSCs at the NMJ were affected in end-stage

TrJ mice. S100-immunoreactive Schwann cells (Ueli Suter & Scherer, 2003;

Tanaka & Hirokawa, 2002) were localized to the NMJ in both genotypes (Figure

4.3A,B), consistent with previous studies (Love & Thompson, 1998). We failed to observe S100-positive bridges between NMJs (Son & Thompson, 1995), further indicating a lack of peripheral degenerative changes in these mice. Finally, we examined postsynaptic markers such as muscle-specific kinase (MuSK) and acetylcholinesterase (AChE), and, similar to pre- and perisynaptic markers, were unable to observe a difference between TrJ and WT mice (Figure 4.3B). There was no fragmentation in the pattern of BTX labeling, providing further evidence that these synapses are not in the process of degeneration. Together, these studies show that neuromuscular synapses of end-stage TrJ mice are innervated and contain a normal tripartite complement of cell-specific markers. 108

We next analyzed the size of individual postsynaptic nAChR clusters because they appeared smaller in end-stage TrJ mice. The height (parallel to long axis of muscle) and volume from single NMJs were extracted and compared between muscles of TrJ and control mice (Figure 4.4A). We observed a significant reduction in the volume of BTX-labeled nAChR clusters in the diaphragm and in the soleus and TA but not the EDL (soleus volume, 329.27 ± 87.61 vs. 265.41 ±

67.40 μm3, *p < 0.005, WT vs. TrJ mice, 65 NMJs from n=3 (WT) and 80 NMJs from n=3 (TrJ); EDL volume, 330.83 ± 86.49 vs. 314.91 ± 93.70 μm3, p = 0.56, WT vs. TrJ mice, 46 NMJs from n=3 (WT) and 31 NMJs from n=3 (TrJ); TA volume, 553.31 ±

103.21 vs. 315.80 ± 66.69 μm3, **p < 0.001; WT vs. TrJ mice, 46 NMJs from n=3

(WT) and 60 NMJs from n=3 (TrJ); diaphragm volume, 1265.72 ± 265.11 vs. 605.58

± 87.70 μm3, **p < 0.001, WT vs. TrJ mice, 55 NMJs from n=3 (WT) and 101 NMJs from n=3 (TrJ); Figure 4.4B-D). In order to determine whether this reduction in

NMJ size was due to a concomitant decrease of muscle size, we stained semithin sections of transverse sections of diaphragm with toluidine blue (Figure 4.4E).

Both the average cross-sectional area and Feret’s diameter were significantly reduced in TrJ vs. WT mice (area, 272.7 + 78.6 vs. 244.7 + 69.8 µm2, p < 0.05;

Ferret’s, 24.66 + 5.2 vs. 22.12 + 3.7 μm, p < 0.005, WT vs. TrJ mice; 70 cells per animal, n=3). The relative increase in NMJ volume between WT vs. TrJ mice was 109

2.1-fold, whereas the corresponding increases in overall weight, muscle wet weight, muscle fiber diameter and muscle fiber area were all between 1.1-1.3- fold, suggesting that the reduction of NMJ size was largely not due to these factors.

Next we performed an ultrastructural analysis of diaphragm NMJs.

T/PSCs were present at the synapse in both TrJ and WT mice, and in some cases the cytoplasm of these cells could be seen to terminate partially but not totally into the synaptic gutter (Figure 4.5). When we measured the depth of junctional folds in single cross sections of NMJs, we observed a statistically significant reduction in TrJ vs. WT mice (individual fold depth, 1.43 + 0.18 vs. 1.03 + 0.32 µm, p < 0.05, WT vs. TrJ mice, n=5 folds per NMJ, 3 NMJs per animal, 3 animals), consistent with the results obtained above by BTX volume analysis.

The reduction of NMJ size may result from atrophy or delayed maturation. The

NMJ undergoes a defined change in structural complexity during the first several weeks of postnatal development (Shi, Fu, & Ip, 2012). For example, during postnatal week 1, NMJs exhibit a plaque-like morphology, characterized by an oval shape, minimal folds, and little or no perforations. During the second and third postnatal weeks, NMJs adopt a more pretzel-like structure, displaying a greater number of invaginations and perforations (Marques, Conchello, & 110

Lichtman, 2000). We could also identify an intermediate morphology where some but not all of the plaques disappeared and were replaced by folds and nascent perforations. Hence, we divided the NMJs of the diaphragm into 3 categories and observed that while the number of NMJs exhibiting intermediate maturation was approximately even between genotypes, the number of immature, plaque-like NMJs was significantly higher in TrJ than in WT mice, and the number of more mature, pretzel-like NMJs was greater in WT than in TrJ mice (Figure 4.6B). Next, we performed a detailed analysis that measured the total number of perforations per NMJ, the average area of individual perforations, and the percentage of NMJ area that were occupied by perforations.

NMJs in control mice exhibited larger perforations than those in TrJ mice

(perforation hole number, 2.2 + 1.08 vs. 1.7 + 0.95 holes/NMJ, p = 0.25; perforation size, 3.66 + 3.46 vs. 1.57 + 1.45 µm2, *p < 0.05; perforation area, 5.03 + 3.87 vs. 2.35

+ 2.14 % of total NMJ area, *p < 0.05, WT vs. TrJ mice, 33 perforations from 15

NMJs from n=3 (WT) and 17 perforations from 13 NMJs from n=3 (TrJ) diaphragms; Figure 4.6C). The difference in size and maturation of NMJs between genotypes could also be observed when they were labeled with S100 antibodies marking T/PSCs (Figure 4.3B). These results, therefore, favor the idea 111 that the reduced volume of NMJs observed in end-stage TrJ mice results at least in part because of a delay in maturation.

To determine whether mutant NMJs exhibited a difference in neuromuscular function in addition to the delay in motor endplate maturation, we performed standard electrophysiological analysis of the diaphragm from end-stage TrJ and control mice. Although we failed to detect a difference in the resting membrane potential between genotypes (-70.2 + 4.0 mV vs. -68.8 + 3.0 mV, p=0.621, WT vs. TrJ mice), when we evoked endplate potentials by stimulating the phrenic nerve in the presence of the Nav1.4 antagonist, µ-conotoxin

(conotoxin GIIIb), we observed a significant decrease in the size of the EPP

(amplitude, 25.2 + 0.93 vs. 22.9 + 1.1 mV, *p < 0.05, WT vs. TrJ mice; Figure

4.7A,B). To determine if this reduction was caused by changes in quantal size, we examined the amplitudes and durations of mEPPs, which reflect the postsynaptic response to individual quanta (Figure 4.7A-B). Neither of these features were affected (mEPP amplitude, 1.54 + 0.16 vs. 1.56 + 0.10 mV, p=0.862; mEPP rise-to- peak, 1.38 + 0.62 vs. 1.06 + 0.11 ms, p=0.294, mEPP decay time, 2.72 + 0.44 vs. 2.74

+ 0.89 ms, p=0.970, WT vs. TrJ mice). Quantal content was therefore decreased in

TrJ vs. WT mice (12.77 + 0.37 vs. 11.70 + 0.45 quanta/EPP, *p < 0.005, WT vs. TrJ mice). Next, we examined the frequency of mEPPs and found that it was 112 significantly reduced in TrJ vs. WT mice (Figure 7C; 1.38 + 0.19 vs. 0.38 + 0.18 events/s, **p < 0.01, WT vs. TrJ mice), suggesting that the lower EPP amplitude obtained from recordings in these mutants reflects a presynaptic deficit (Figure

4.7C, D). Next, we examined the response of NMJs to high-frequency nerve stimulation, which induces the depression of ACh release at neuromuscular synapses via presynaptic mechanisms. Moreover, this form of short-term plasticity is also modulated by T/PSCs (Darabid et al., 2013a). While NMJs in both genotypes exhibited a quick initial reduction of EPP amplitude, TrJ mice exhibited an exaggerated rundown in synaptic transmission as the period of high-frequency stimulation persisted (Figure 4.8A, B). Additionally, high- frequency stimulation failed to trigger EPPs occasionally after ~30s in TrJ mice

(Figure 4.8C, D). This complete lack of nerve-evoked ACh release was never observed in control mice. Therefore, these data indicate that NMJs in TrJ mice exhibit a presynaptic deficit that causes an enhanced rundown in neuromuscular transmission.

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4.5. DISCUSSION

Because homozygous TrJ mice exhibit a congenital lack of myelination and die early in the postnatal period, we tested the hypothesis that this severely shortened lifespan was caused by a failure to maintain neuromuscular synapses, leading to muscle atrophy and paralysis. We failed to find evidence supporting this idea. Across multiple muscle subtypes examined within 1-2 days prior to death, NMJs of TrJ mice contained qualitatively normal expression of markers associated with pre-, peri- and post-synaptic elements. These findings are in contrast to congenital models of motor neuron disease, such as spinal muscular atrophy, which exhibit anatomical as well as functional evidence of synaptic degeneration at the NMJ (Goulet, Kothary, & Parks, 2013). They are also different than results obtained from adult, heterozygote TrJ mice as well as other CMT1 mouse models, which exhibit a modest but significant loss of neuromuscular synaptic maintenance (Ang et al., 2010; Nicks et al., 2013; Yin et al., 2004).

Together with the absence of severe muscle atrophy, the lack of peripheral denervation in TrJ mice suggests that the early lethality in this mouse is not caused by a failure to maintain neuromuscular synaptic connections.

The NMJ is abnormal in several muscles of end-stage TrJ mice, however, indicating that the development of this synapse is affected in the absence of 114 myelination. Specifically, the size and complexity of BTX-labeled postsynaptic endplates as well as the depth of postsynaptic junctional folds were reduced in the diaphragm, relative to WT mice. There was no loss of postsynaptic MuSK or

AChE, suggesting that key functional elements of the postsynaptic apparatus were intact. Similarly, the expression of S100 and the number of nuclei affiliated with this protein were similar in WT and TrJ NMJs, indicating that nonmyelinating T/PSCs were not overtly affected. These results instead suggest that the maturation of the NMJ is impaired in TrJ mice. The significance of impaired

NMJ maturation is unclear, but a recent report showed that a mouse model of

CMT2 exhibits similarly altered NMJ maturation in a subset of muscles that precedes subsequent degeneration (Sleigh, Grice, Burgess, Talbot, & Cader,

2014). Interestingly, similar to NMJs of the diaphragm, NMJs of the soleus and

TA, but not EDL muscles, were smaller in TrJ than in WT mice. Although these muscles in the adult express different complements of myosin isoforms, with the

EDL expressing the highest percentage of fast-fatigable Type IIB fibers, the development of these isoform expression subtypes typically occurs later than the time of lethality of homozygous TrJ mice (Sieck & Prakash, 1997), suggesting that other features underlie the differential sensitivity of these muscle subtypes to

NMJ maturation. In addition to the decreased size of NMJs as deduced by 115 quantitative BTX analysis, the distal but not proximal phrenic nerve is reduced in size in TrJ mice. We attribute these changes to impaired growth rather than to atrophy, based on the assessment of NMJ size and maturation discussed above, as well as the lack of overt axonal pathology in either proximal or distal phrenic nerves.

Synaptic function is also abnormal in the diaphragm of end-stage TrJ mice.

Individual EPPs were smaller and the frequency but not amplitude or duration of mEPPs was reduced in these mice relative to control, suggesting a presynaptic deficit. Whether this deficit precedes the structural postsynaptic alteration is unclear but could be tested by performing a time course of functional and anatomical studies. A more severe deficit in synaptic transmission was observed in response to high-frequency (40 Hz) phrenic nerve stimulation. Although the initial reduction to 80% of transmitter release within the first second of such stimulation was similar between genotypes and to previous studies (Kaja et al.,

2010; Moyer & van Lunteren, 1999), NMJs in TrJ mice exhibited a more pronounced reduction in release as the stimulation continued. Together with the reduced amplitude of the initial EPP, and based on a safety factor at the vertebrate NMJ between 2 and 4 (Gertler & Robbins, 1978), this reduction is predicted to cause neuromuscular transmission failure when the initial control 116

EPP of ~26mV reaches 6.5-13 mV, or within 10 seconds (vs. 45 seconds in control), after the onset of stimulation. Even more strikingly, after a period of 30-

35 seconds of high-frequency stimulation, NMJs in TrJ mice exhibit a complete lack of transmitter release. In contrast, such total failure was never observed in control mice even after a minute of 60-Hz stimulation (data not shown).

Together, these results suggest that NMJs in TrJ mice are unable to maintain synaptic transmission in response to high-frequency stimulation. Future studies will be aimed at elucidating the mechanisms of this effect, such as impairments of synaptic vesicle mobilization or synaptic currents. For example, it has been reported that conotoxin GIIIb reduces the potassium current (and thus quantal content) at motor nerve terminals through an action on presynaptic sodium channels (Braga, Anderson, Harvey, & Rowan, 1992), an effect that could account for the reduced EPP in TrJ mice.

Although it is tempting to speculate that the failure to release neurotransmitter in response to 40-Hz stimulation contributes to respiratory failure, the present data are too preliminary to support this conclusion. First, while 40-Hz stimulation is well within the phrenic nerve firing rates obtained from ventilated adult rats (Kong & Berger, 1986), these frequencies are typically maintained for 1 second during an inspiration, followed by an intercycle rest 117 period, resulting in a use- or duty-cycle of ~0.35 (St John & Bartlett, 1979). This difference may affect the interpretation of our results because less EPP rundown was observed in response to phasic vs. continuous stimulation (Moyer & van

Lunteren, 1999). Second, it would be useful to determine whether the differences observed at the endplate translate into functional deficits by measuring respiratory and diaphragm function via plethysmography and isometric tension measurements, respectively. Third, whether failed EPPs result from deficits in

ACh release or from impaired nerve conduction remains unclear, but could be addressed by studies of nerve conduction velocity and distal nerve propagation via compound muscle action potentials or extracellular axon segment recording, respectively. Evidence in support of impaired conduction comes from studies of heterozygote TrJ mice, which exhibit significant deficits in motor nerve velocity

(Meekins, Emery, & Weiss, 2004). Together, the results from these studies would provide insight into whether the deficits reported here at the NMJ contribute to the early lethality of homozygous TrJ mice. Alternatively, alterations in central white matter in TrJ mice may contribute to early lethality because PMP22 is expressed, albeit at lower levels, in the CNS (Ohsawa, Murakami, Miyazaki,

Shirabe, & Sunada, 2006). Consistent with this idea, reductions in the volume of 118

CNS white matter as well as cognitive impairment have been reported in humans with CMT1A (Chanson et al., 2013).

Together, our studies describe a set of specific structural and functional deficits in the neuromuscular system of TrJ mice, which we propose as an excellent animal model of congenital hypomyelinating neuropathy. Surprisingly, these mice exhibit only modest structural alterations at the NMJ in contrast to the evidence of synaptic degeneration seen in other models of CMT1. Rather, the principal neuromuscular deficit in TrJ mice is functional. Future studies will examine motor nerve conduction velocities in these mice in order to determine if they are impaired and underlie the peripheral transmission deficits we observed in this study, as would be expected based on the severe hypomyelination of peripheral nerves in these mice. In summary, our data suggest that neurological deficits associated with congenital hypomyelinating neuropathy are likely caused by a functional impairment of synaptic transmission at the NMJ.

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Figure 4.1. Homozygous Trembler-J mutant (TrJ) mice die early postnatally and exhibit severe hypomyelination without motor pathology at end stage. (A) Kaplan-Meier curve illustrates the onset of motor incoordination. (B) Kaplan- Meier curve shows the onset of death. (C) Toluidine blue-stained semithin sections of phrenic nerve obtained from end-stage TrJ and wild-type (WT) mice exhibit a complete absence of myelination but fail to show evidence of motor axon pathology. Arrowheads depict axons surrounded by myelin sheaths (left panel) or by Schwann cell cytoplasm (right panel); Schwann cells are indicated by arrows. Scale bar in C = 10 µm.

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Figure 4.2. End-stage homozygous Trembler-J mutant (TrJ) mice have fully innervated neuromuscular junctions (NMJs). (A) NMJs from diaphragm, extensor digitorum longus (EDL), and soleus muscles of end-stage TrJ or wild- type (WT) mice are stained immunohistochemically with an antibody directed against a presynaptic protein (synaptophysin; Syp; green) and with a fluorescently labeled toxin that binds postsynaptic acetylcholine receptor (AChR) clusters (Alexa-Fluor-594-conjugated α-bungarotoxin; 594-α-BTX; red). Merged images show complete apposition or overlap of these markers. (B) NMJs of the diaphragm were also colabeled with an antibody against presynaptic vesicular ACh transporter (VAChT; blue) and 594-α-BTX (red). (C) High-power images of Syp immunoreactivity in NMJs from the tibialis anterior (TA) muscle illustrate the increased size and elaboration of NMJs in WT vs TrJ mice. Scale bar in A (same as in B), 50 µm; C, 10 µm. 121

Figure 4.3. Neuromuscular junctions (NMJs) of end-stage homozygous Trembler-J mutant (TrJ) mice express normal perisynaptic and postsynaptic markers. (A) Terminal/perisynaptic Schwann cells (T/PSCs) at the NMJ of the tibialis anterior of wild-type (WT) or TrJ mice were stained with an antibody directed against S100 (S100; green); NMJs are colabeled with bungarotoxin (BTX) (red). (B) Merged high-power images show 1–2 Hoechst-labeled nuclei present at each NMJ (arrows, right panels) as well as the increased size and elaboration of NMJs in WT vs TrJ mice. (C, D) 594-α-BTX-labeled postsynaptic nicotinic acetylcholine receptor (nAChR) clusters (red) were colabeled with an antibody against postsynaptic muscle-specific kinase (MuSK; green) (C) and a fluorescently labeled toxin that binds acetylcholinesterase (AChE; Alexa-Fluor- 647-conjugated fasciculin II; 647-FasII; pseudocolored green) (D). Scale bars: A, C, 20 µm; B, 10 µm; D, 50µm.

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Figure 4.4. Neuromuscular junctions (NMJs) are reduced in size in end-stage homozygous Trembler-J mutant (TrJ) mice. (A) Maximum fluorescence-intensity maps of individual 594-α-bungarotoxin (BTX)-labeled nicotinic acetylcholine receptor (nAChR) clusters were created (Max); depth coloring was used to assess surface curvature and depth (Depth); and a subvoxel volume routine was used to calculate volume (Volume). The scale bar indicates varying depths relative to the surface. (B) Reduced volumes of individual BTX-labeled NMJs in diaphragm, 123 tibialis anterior (TA), and soleus (but not extensor digitorum longus [EDL]) of TrJ (black bars), relative to WT mice (white bars). (C, D) Frequency distribution (C) and cumulative frequency distribution (D) of 594-α-BTX-labeled NMJ volumes in the diaphragm shows a large increase in the number of smaller NMJs in end- stage TrJ mice. (E) Toluidine blue-stained semithin sections of the diaphragm show muscle cells with similar diameter. Scale bar in E = 10 µm.

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Figure 4.5. Neuromuscular junctions (NMJs) of end-stage homozygous Trembler-J mutant (TrJ) mice are innervated but contain smaller junctional folds. Low-power (left panels) and high-power (right panels) ultrastructural images of NMJs taken from the diaphragm of wild-type (WT) (upper panels) and TrJ mice (lower panels). Terminal/perisynaptic Schwann cells (SCs) are correctly positioned at the motor endplate (arrow in upper right panel denotes SC cytoplasm extending partially around nerve terminal (NT). Postsynaptic junctional folds (JF) and myofilaments (MF), as well as mitochondria (M) and synaptic vesicles (SV) in the presynaptic nerve terminal, are shown. Arrowheads in right panels mark individual junctional folds. Scale bar = 2 µm.

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Figure 4.6. Neuromuscular junctions (NMJs) of end-stage homozygous Trembler-J mutant (TrJ) mice exhibit delayed maturation. (A) Representative examples of sequential stages of postnatal NMJ development including plaque, intermediate, and near-pretzel. (B) The relative percentages of diaphragm NMJs in these different stages of maturation are shown for control (wild-type [WT], white bars) and TrJ (black bars) mice. (C) The area of an NMJ encompassed by perforations and the size of individual perforations were significantly reduced in TrJ vs WT mice. Scale bar =10 µm.

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Figure 4.7. End-stage homozygous Trembler-J mutant (TrJ) mice exhibit physiological deficits at the neuromuscular junctions (NMJs). (A, B) Intracellular microelectrode recording measurements of endplate potentials (EPPs) and miniature EPPs (mEPPs) taken at the motor endplate of diaphragm muscle from P13 wild-type (WT) and TrJ mice in the presence of the Nav1.4 channel blocker µ-conotoxin GIIIb. The amplitude of EPPs but not mEPPs is significantly reduced in TrJ vs wild-type (WT) mice. (C, D) Examples and quantification of 1.5-second recording traces show that the frequency of mEPPs is significantly reduced in TrJ vs WT mice.

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Figure 4.8. Neuromuscular junctions (NMJs) from end-stage homozygous Trembler-J mutant (TrJ) mice exhibit greater endplate potentials (EPP) rundown in response to high-frequency stimulation. (A, B) Sixty-second recording traces (A) and enlargements of the beginning, middle, and end of these traces in response to continuous 40-Hz stimulation of the phrenic nerve (B). (C) Examples of 200-ms recording traces after 35 seconds of 40-Hz stimulation of the phrenic nerve. Note the examples of transmission failure in TrJ mice (asterisks), which was never observed in wild-type (WT) mice. Vertical lines next to potentials indicate stimulation artifacts. (D) EPP rundown in response to 40-Hz stimulation is similar between genotypes for 15 seconds but becomes significantly greater over time in NMJs of TrJ vs WT mice (*p < 0.05, ** p< 0.001).

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Chapter 5.0

CONCLUSION

In chapter 2, we examined the 36 hour period between embryonic day 14.0

(E14) and E15.5 in erbB3-/- mice as compared to littermate WT control mice.

Applying quantification methods to confocal images, we observed significant defasciculation of developing motor nerves in E14.25 and E14.5 mice followed by complete axonal and NMJ degeneration by E15.5. We identified premature innervation of NMJs in E14.5 erbB3-/- mice as compared to WT mice coinciding with disorganization and scattering of postsynaptic nAChR clusters in erbB3-/- mice as early as E14.25. The generation of Euclidian distance maps from synaptophysin-labelled particles enabled the quantification of the aberrant localization of nAChRs. In normal development, postsynaptic nAChR clusters condense along the midline of the diaphragm muscle creating a centralized endplate band (Pun et al., 2002; Shi et al., 2012). In erbB3-/- mice, the endplate band is significantly wider and diminishes by E15.5. Using RNA isolated from

E14.5 erbB3-/- mice and age-matched WT mice, we also performed RNA sequencing and subsequent analysis. Differential gene expression analysis of

RNA sequencing datasets revealed significant numbers of SC specific genes and 129 genes necessary for biological regulation and nervous system development upregulated in WT versus erbB3-/- samples.

In chapter 3, we developed a novel approach to analyze the TSC and axonal SC transcriptome. Combining the genetic epitope-tagging of ribosomes in

Cre-expressing SCs with physical segregation of axonal SCs from TSCs, we were able to successfully isolate these two SC subtype populations. Axonal SCs were isolated from phrenic nerve tissue (PN) while TSCs from tissue dissected from the endplate band (EP), a region enriched in TSCs over axonal SCs. RNA extraction from these samples yielded high quality RNA for RNA sequencing analysis. Differential gene expression of the PN and EP RNA sequencing datasets was filtered with stringent statistical conditions and compared to genes upregulated in WT samples discussed in chapter 2. These results revealed 13 possible candidates for genetic markers specific to TSCs as they were upregulated in both EP versus PN differential expression analyses and in WT versus erbB3-/- differential expression analyses. Conversely, 9 genes were identified as possible axonal SC genetic markers for these genes were significantly downregulated in EP versus PN differential expression analyses and upregualted in WT versus erbB3-/- differential expression analyses. 130

In chapter 4, we examined a homozygote model of the most common inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1A

(CMT1A). This model, the Trembler-J homozygote mouse (TrJ), presents with hypomyelination and early postnatal lethality despite evidence of motor axon pathology. NMJs in oxidative, fast-fatigable, and mixed muscle fiber types exhibit normal innervation patterns and normal expression of perisynaptic (S100) and postsynaptic (MuSK and FasII) markers. However, the neural activity at

NMJs in TrJ mice is impaired with decreased amplitudes of EPPs and decreased frequency of mEPPs. We generated novel analytical methods to quantify the total volume of NMJs extracted from confocal stacks from TrJ mice as compared to age-matched WT mice. Despite normal composition and innervation of the tripartite synapse, maturation of NMJs is delayed and the volume of NMJs were significantly reduced in mixed muscle fibers (diaphragm and tibialis anterior) and oxidative fibers (soleus) but not in fast-fatigable fibers (extensor digitorum longus). The size of junctional folds was also significantly reduced in TrJ mice as compared to WT.

In chapter 2, we showed that E14.5 erbB3-/- mice exhibit premature innervation of nAChRs that comprise a characteristic disorganized, widened endplate region. When neural activity is blocked by genetic deletion of ChAT in 131 erbB3-/- mice, motor neurons and synaptic innervation is maintained, albeit with a defasciculated phenotype (Figure 1.8). Presumably under normal conditions,

SCs exhibit antagonistic effects on the negative, nAChR dispersal activity of ACh

(Brandon et al., 2003; Misgeld et al., 2002). Observations made in the erbB3-/- murine model show a critical role for SCs in the development of the NMJ.

However, the erbB3-/- mouse exhibits a loss of all SCs, therefore it remains unclear whether axonal SCs or TSCs exert this modulating effect in NMJ development.

Additionally, SCs are differentiating from SC precursors into immature

SCs at ~E15 (Jessen & Mirsky, 2008; Rhona Mirsky et al., 2008) and full maturation of SCs does not conclude until ~E18. While various factors, such as

Oct6, Sox10, and Krox20, are known to mediate various stages of SC development, the mechanisms responsible for TSC differentiation are unknown

(Mirsky & Jessen, 2009; Mirsky et al., 2008). The ability to elucidate the regulatory mechanisms induced by axonal SCs or TSCs throughout embryogenesis would significantly improve our understanding of this process.

Conditional ablation of individual SC subtypes, axonal SCs or TSCs, would allow the investigation of each subtype at specific time points in both embryological and early postnatal development. Further studies are imperative to validate the 132 specificity of the 13 candidate genes and 9 candidate genes found through our

RNA sequencing analysis in chapter 3. Upon confirmation of these genetic markers, we can begin to create novel murine models to investigate the roles of axonal SCs and TSCs in development.

The benefits of identifying genetic markers specific to either axonal SCs or

TSCs extends beyond application to developmental studies. A substitution of leucine 16 with proline in the peripheral myelin protein gene is the underlying genetic cause in CMT1A and leads to hypomyelination and fatality characteristic of the TrJ mouse (Suter et al., 1992a; Suter et al., 1992b; Valentijn et al., 1992). In peripheral neuropathies directly affecting Schwann cells, such as CMT1A, axonal

SCs and TSCs could have differential susceptibilities to toxicity. Directing this mutation to either TSCs or axonal SCs would enable the characterization of pathology due to dysfunction of only one SC subtype.

The involvement of glia in neuropathies is becoming more prevalent.

Rather than glia being a secondary contributor to dysfunction; mounting evidence is suggesting a primary role. Amyotrophic lateral sclerosis is classically considered a motor neuron disease affecting both upper and lower motor neurons. Nonetheless, growing evidence suggests dysfunction at the neuromuscular junction (NMJ) as well as deficits in retrograde axonal transport 133 prior to any manifestation of symptoms suggesting a ‘dying back’ progression of disease (Bilsland et al., 2010; K. Chen, Northington, & Martin, 2010; Dadon-

Nachum, Melamed, & Offen, 2011; De Winter et al., 2006; Fischer et al., 2004; Frey et al., 2000; Gould & Oppenheim, 2007; Moloney et al., 2014; Verheijen et al.,

2014). These asymptomatic disease indicators are found in axonal SCs in some models of familial ALS (K. Chen et al., 2010; Verheijen et al., 2014) and more predominantly in TSCs (Arbour et al., 2015; De Winter et al., 2006; Keller et al.,

2009).

In order to extend our understanding of the function of axonal SCs and

TSCs in development and disease, future studies to assess the explicit expression of the specific TSC genetic markers and possible axonal SC genetic markers identified in chapter 3 is needed. This will provide us with the novel ability to examine intrinsic processes of SC differentiation as well as the direct ways in which axonal SCs and TSCs modulate motor nerve and synapse development.

Utilization of these markers will also help us assess the ways TSCs and axonal

SCs contribute or protect against diseases targeting SCs, motor synapses or motor axons, respectively.

134

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