Peripheral Myelin Protein 22 (Pmp22) Is in a Complex with Alpha6 Beta4 Integrin and the Absence of Pmp22 Affects Schwann Cell Adhesion and Migration

Home , Avidin, Myelin protein zero

PERIPHERAL MYELIN PROTEIN 22 (PMP22) IS IN A COMPLEX WITH ALPHA6 BETA4 INTEGRIN AND THE ABSENCE OF PMP22 AFFECTS SCHWANN CELL ADHESION AND MIGRATION

STEPHANIE ANN AMICI

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2006

Stephanie Ann Amici

I dedicate this work to my family for their constant love and support.

ACKNOWLEDGMENTS

Most importantly, I would like to thank my advisor, Dr. Lucia Notterpek, for the inspiration and support she has given to me. She has always had my best interests in mind and has been essential in my growth as a scientist and as a person. I also express my gratitude to my committee members, Dr. Brad Fletcher, Dr. Dena Howland, Dr. Harry

Nick and Dr. Susan Frost, for their insights and suggestions for my project. Similarly, Dr.

William Dunn deserves acknowledgement for his advice and assistance with the electron

microscopy studies. Heartfelt thanks also go to the past and present members of the

Notterpek lab for their amazing friendships over the years. Finally, my deepest gratitude

goes to my family, especially my parents, for their continuous love and encouragement

throughout my life.

TABLE OF CONTENTS

page

ACKNOWLEDGMENTS ...... iv

LIST OF FIGURES ...... vii

LIST OF ABBREVIATIONS...... ix

ABSTRACT...... xi

CHAPTER

1 INTRODUCTION ...... 1

Constituents of Myelin ...... 3 Peripheral Myelin Protein 22-Associated Hereditary Peripheral Neuropathies...... 5 Animal Models of PMP22-Associated Peripheral Neuropathies ...... 8 Potential Disease Mechanisms of PMP22 Neuropathies...... 10 Potential Roles of PMP22 in Cell Biology...... 11

2 PERIPHERAL MYELIN PROTEIN 22 IS IN COMPLEX WITH ALPHA6 BETA4 INTEGRIN AND ITS ABSENCE ALTERS THE SCHWANN CELL BASAL LAMINA ...... 18

Note...... 18 Introduction...... 18 Materials and Methods ...... 20 PMP22-deficient LacZ Mice ...... 20 Morphological Analyses and Teased Nerve Fiber Preparation...... 20 Myelinating Dorsal Root Ganglion (DRG) Neuron-SC Cocultures...... 22 Immunolabeling of Nerves...... 22 Western Blot Analyses ...... 23 Coimmunoprecipitation of Integrins and PMP22...... 24 Cell Surface Labeling...... 25 Protein Interaction Studies in Clone A Cells...... 25 Results...... 26 Phenotype of PMP22-deficient LacZ Mice...... 26 Delayed Myelination and Altered SC-ECM Interactions in the Absence of PMP22...... 31 β4 Integrin Levels Are Reduced in Sciatic Nerves of PMP22 -/- Mice...... 34

v PMP22 Is in a Complex with α6β4 Integrin and Laminin...... 36 Discussion...... 42

3 DELAYED MYELINATION AND ALTERED NODAL ORGANIZATION IN PERIPHERAL MYELIN PROTEIN 22-DEFICIENT MICE...... 48

Introduction...... 48 Materials and Methods ...... 51 Myelinating DRG Explant Cultures ...... 51 Teased Nerve Fiber Preparation ...... 51 Immunolabeling of Nerves and Myelinating DRG Explant Cultures ...... 52 Western Blot Analyses ...... 53 Statistics...... 54 Results...... 54 Altered Myelin Protein Expression and Localization in PMP22 -/- Mice ...... 54 Changes in Integrin Expression Patterns in the Absence of PMP22...... 58 Myelination Is Impaired in DRG Explant Cultures from PMP22 -/- Mice...... 61 Discussion...... 64

4 THE ADHESION AND MIGRATION OF SCHWANN CELLS IS ALTERED IN THE ABSENCE OF PERIPHERAL MYELIN PROTEIN 22 ...... 68

Introduction...... 68 Materials and Methods ...... 70 Mouse Schwann Cell Isolation...... 70 LacZ Activity Assay...... 71 Bromodeoxyuridine Incorporation Assay ...... 71 Immunocytochemistry...... 71 Western Analyses ...... 72 Adhesion Assays ...... 72 Migration Assays...... 73 Results...... 74 PMP22 Is Expressed in Nonmyelinating SCs ...... 74 Increased SC Proliferation in the Absence of PMP22...... 76 PMP22-Deficient Cells Display Impaired Adhesion and Migration...... 77 Lack of PMP22 Results in Altered SC Morphology ...... 79 Discussion...... 82

5 CONCLUSIONS ...... 86

Overview of Findings ...... 86 Unresolved Issues and Future Studies ...... 89

LIST OF REFERENCES...... 93

BIOGRAPHICAL SKETCH ...... 108

LIST OF FIGURES

Figure page

1-1 The myelination program ...... 2

1-2 Putative PMP22 structure...... 5

2-1 Schematic representation of the targeting strategy ...... 27

2-2 Widespread β-gal expression in the E15 mouse embryo ...... 28

2-3 Phenotype of adult PMP22-deficient mice...... 30

2-4 Delayed myelination and ultrastructural alterations in nerves of affected mice ...... 33

2-5 Decreased levels of β4 integrin in nerves of PMP22-deficient mice...... 35

2-6 PMP22 is in a complex with α6β4 integrin and laminin ...... 37

2-7 Coexpression of PMP22 and integrins during myelination ...... 39

2-8 PMP22 and β4 integrin are coimmunoprecipitated from clone A cells...... 41

3-1 The expression of myelin proteins is delayed in nerves from PMP22-deficient mice ...... 55

3-2 Decreased MBP and increased p75 immunostaining in affected nerves...... 57

3-3 Altered integrin expression levels in nerves from PMP22-deficient mice...... 59

3-4 Altered protein localization in teased nerve fibers of PMP22-deficient mice ...... 60

3-5 Shortened myelin internodes in DRG neuron explant cultures from affected mice 62

3-6 Dysmorphic myelin from DRG explant cultures of PMP22 -/- mice ...... 63

4-1 PMP22 expression in cultured mouse SCs...... 75

4-2 SCs from PMP22 -/- mice have an increased rate of proliferation ...... 76

4-3 Adhesion to laminin is reduced in mouse SCs lacking PMP22 ...... 77

vii 4-4 Slowed migration on laminin by PMP22 -/- mouse SCs ...... 79

4-5 Cells at the scratch edge are morphologically different between +/+ and -/- cultures ...... 80

4-6 PMP22 -/- mouse SCs display altered lamellipodial morphology ...... 81

viii

LIST OF ABBREVIATIONS

Arf-6, ADP-ribosylation factor-6

CHN, congenital hypomyelinating neuropathy

CMT, Charcot-Marie-Tooth disease

CNS, central nervous system

Cx32, connexin 32

DRG, dorsal root ganglion

DSS, Dejerine-Sottas Syndrome

E, embryonic day

ECM, extracellular matrix

EMP-2, epithelial membrane protein-2

HNK, human natural killer

HNPP, hereditary neuropathy with liability to pressure palsies

Ig, immunoglobulin kb, kilobase kDa, kilodalton

Kv1.1, voltage-gated potassium channel 1.1

LPA, lysophosphatidic acid

MAG, myelin associated glycoprotein

MBP, myelin basic protein

MDCK, Madin-Darby canine kidney

ix NCV, nerve conduction velocity

OSP, oligodendrocyte specific protein

P, postnatal day

PLP, proteolipid protein

PMP22, peripheral myelin protein 22

PNS, peripheral nervous system

P0, myelin protein zero

SC, Schwann cell

TER, trans-epithelial resistance

TrJ, Trembler J

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

PERIPHERAL MYELIN PROTEIN 22 (PMP22) IS IN A COMPLEX WITH ALPHA6 BETA4 INTEGRIN AND THE ABSENCE OF PMP22 AFFECTS SCHWANN CELL ADHESION AND MIGRATION

Stephanie Ann Amici

May 2006

Chair: Lucia Notterpek Fletcher Major Department: Neuroscience

Myelination in the peripheral nervous system (PNS) is an intricate process that requires precise interactions between Schwann cells (SCs) and their extracellular environment. Peripheral myelin protein 22 (PMP22) is a transmembrane SC glycoprotein associated with a heterogeneous group of inherited demyelinating neuropathies.

However, the exact role of PMP22 in peripheral nerve myelination has not been defined.

To elucidate the potential functions of PMP22 in the cell membrane, we have characterized a novel PMP22-deficient (-/-) mouse model that replaced the second and third coding exons of the pmp22 gene with the lacZ reporter. We utilized complementary in vivo and in vitro approaches to examine the specific purpose of PMP22 in SCs. The

PMP22 -/- mice display strong β-galactosidase reactivity in peripheral nerves, as expected. On morphological examination, the nerves are focally hypermyelinated similar to the phenotypes of neuropathy patients with reduced PMP22 gene dosage. Myelination is delayed during development and loose basal lamina is observed by ultrastructural

xi analysis in sciatic nerves from PMP22 -/- mice, suggesting compromised contacts between SCs and the extracellular matrix (ECM). The linkage between the SC cytoskeleton and the ECM, which is essential for myelin formation in the PNS, is achieved by the integrin family of transmembrane proteins. Therefore, we hypothesized that PMP22 may form a complex with one of the integrins in mediating SC-ECM interactions. Indeed, by immunohistochemical and Western analyses, the levels of β4 integrin are drastically reduced in sciatic nerves from PMP22 -/- mice.

Coimmunoprecipitation experiments from nerve lysates of wild type (+/+) mice and from a cell line overexpressing PMP22 reveal that PMP22 is in a complex with α6β4 integrin and laminin. Additionally, SCs derived from PMP22-null mice exhibit diminished adhesion, slowed motility and altered cell morphology, including reduced lamellipodial formation at the wound edge, as compared to +/+ cells. Together, these data suggest that during development PMP22 is important for mediating the interaction of SCs with the

ECM, likely as a binding partner for the α6β4 integrin and laminin complex. These findings may explain the dysmyelination observed in mouse nerves from PMP22- deficient mice, as well as in pediatric neuropathy patients.

xii CHAPTER 1 INTRODUCTION

The evolution of complex nervous systems in vertebrates has been assisted by the development of the myelin sheath—a multilamellar, lipid-rich structure deposited in segments, termed internodes, along axons of the central and peripheral nervous systems

(CNS and PNS, respectively). The segments are interrupted by bare axonal portions, which are known as nodes of Ranvier. The myelin lamellae are formed by the membrane wrapping and compaction of glial cells (the oligodendrocytes in the CNS and Schwann cells (SCs) in the PNS) around axons. In the CNS, an oligodendrocyte can myelinate many axons whereas, in the PNS, a SC can only myelinate a single axon (Peters et al.,

1991). The myelin sheath insulates axons and decreases the current flow across the internodal axonal membrane. Thus, myelination increases the nerve conduction velocity

(NCV) of signals sent from nerve cell bodies to their target cells.

During PNS development, SC precursors migrate from the neural crest and contact peripheral axons (Kamholz et al., 1999). As SC precursors progress to immature SCs, they begin to ensheath and segregate the axons, termed “radial sorting” (Webster, 1993).

Only axons larger than approximately one micron (µm) in diameter induce SCs to myelinate. It was recently determined that axonal ensheathment and myelin thickness are dependent upon the amount of neuregulin-1 type III (i.e., glial growth factor) expressed on the axon (Michailov et al., 2004; Taveggia et al., 2005). SCs that do not form one-to-

one relationships with axons mature into a non-myelinating type that have multiple axons

1 2 embedded within them (Jessen and Mirsky, 1999). Yet, the reasons that only a subset of axons is myelinated are still not completely understood.

For a SC to initiate myelination, it must first exit the cell cycle. As the SC differentiates, it prompts the synthesis of certain myelin proteins (Jessen and Mirsky,

2005). Once a promyelinating SC associates with an axon (Fig. 1-1A), it secretes a basal lamina and forms connections with the extracellular matrix (ECM) (Fig. 1-1B). This allows it to begin wrapping around the axon (Fig. 1-1C) (Bunge, 1993). As the SC membrane extends, the many layers compress and the majority of the cytoplasm is extruded (Fig. 1-1D). This results in the formation of the compact (myelin lamellae) and non-compact (cytoplasmic regions including the Schmidt-Lantermann incisures, paranodal loops, and the outer and inner mesaxons) compartments of mature PNS myelin

(Peters et al., 1991). The exact mechanisms and molecules mediating myelin wrapping and the establishment of specific domains are unknown at this time.

Figure 1-1. The myelination program. An axon (inner black circle) and a SC (outer white tube encompassing the axon) form a 1:1 relationship (A). The SC secretes a basal lamina (gray ring around the SC) (B). The SC begins to spiral around the SC (C). The myelin wrappings compact to form mature myelin (D).

Constituents of Myelin

The majority of the myelin membrane is comprised of compact myelin. Total

mammalian myelin contains approximately 70-80% lipids by dry mass, including

cholesterol (~23-28%), galactolipids (i.e., Gal-C, SGal-C; ~16-22%) and phospholipids

(i.e., choline, ethanolamine, sphingomyelin; ~50-58%) (Garbay et al., 2000). A lesser

fraction of myelin (~20-30%) consists of proteins. The main proteins in the CNS are

proteolipid protein (PLP), myelin basic protein (MBP) and myelin-associated

glycoprotein (MAG). In the PNS, connexin 32 (Cx32), MAG, myelin protein zero (P0),

MBP and peripheral myelin protein 22 (PMP22) are abundant (Quarles et al., 2006).

Cx32 and MAG are located in non-compact myelin at Schmidt-Lantermann incisures and paranodal loops and make up less than 1% of PNS myelin (Garbay et al.,

2000). Cx32 is a gap junction protein that allows ions and small molecules to move through the myelin membrane (Balice-Gordon et al., 1998). MAG, a member of the immunoglobulin (Ig) superfamily also present in CNS myelin, is believed to participate in axonal recognition and adhesion, intermembrane spacing and maintenance of axon- myelin integrity (Montag et al., 1994). The functions of MAG are due, in part, to the

L2/HNK-1 adhesion/recognition carbohydrate epitope that also is present on P0 and

PMP22 (Kruse et al., 1984; Bollensen and Schachner, 1987; Snipes et al., 1993).

P0 is the most abundant molecule in PNS myelin, comprising ~50% of all proteins of the SC membrane. It is a 28 kilodalton (kDa) member of the Ig superfamily and is found in the compact myelin of SCs (Lemke, 1993). MBP is actually a family of alternatively spliced proteins from the same gene (Garbay et al., 2000). They are cytosolic proteins associated with compact myelin in the CNS and PNS. They are thought to participate with PLP in the CNS and with P0 in the PNS in the maintenance and

compaction of the myelin sheath (Martini et al., 1995). Additionally, tetramers of the

extracellular portion of P0 interact homophilically via the L2/HNK-1 epitope (D’Urso et

al., 1990; Filbin et al., 1990), purportedly to promote and maintain the tight compaction

of myelin. P0 also has been shown to interact with PMP22 (D’Urso et al., 1999),

suggesting these proteins play a part in myelin stability.

The PMP22 gene, located on chromosome 17 in humans, encodes a 160 amino acid

highly hydrophobic protein. PMP22 is a putative four transmembrane domain protein

with two extracellular (EC) domains, a small intracellular (IC) domain and a short

cytoplasmic tail (Fig. 1-2). The L2/HNK-1 adhesion/recognition epitope is present on the

carbohydrate moiety linked to asparagine 41 in the first EC loop of PMP22 (Snipes et al.,

1993). The resulting 22 kDa protein is produced mainly by SCs and comprises 2-5% of

the total protein of PNS myelin (Pareek et al., 1993). PMP22 was first discovered as

PASII, a major glycoprotein in bovine peripheral myelin (Kitamura et al., 1976).

Subsequently, PMP22 was characterized as the growth arrest specific gene, gas-3,

upregulated in serum-starved NIH3T3 fibroblasts (Manfioletti et al., 1990), and as a SC

gene that is down regulated after peripheral nerve crush injury (Spreyer et al., 1991;

Welcher et al., 1991). Based on PMP22 transgenic models and its interaction with P0,

PMP22 is postulated to have roles in myelin formation, compaction and stability, but its

precise function at the molecular level has yet to be delineated (Suter and Scherer, 2003).

PMP22 may have a more general role in cell biology as well, as a homolog of PMP22

was recently discovered in the basal vertebrate, Ciona intestinalis (sea slug) (Gould et al.,

2005), an animal that does not produce myelin.

Figure 1-2. Putative PMP22 structure. EC, extracellular; IC, intracellular; Y, carbohydrate moiety.

Peripheral Myelin Protein 22-Associated Hereditary Peripheral Neuropathies

Misexpression of PMP22 is linked to hereditary peripheral neuropathies, including

Charcot-Marie-Tooth disease type 1A (CMT1A), Dejerine-Sottas Syndrome (DSS), congenital hypomyelinating neuropathy (CHN), and hereditary neuropathy with liability to pressure palsies (HNPP) (Naef and Suter, 1998). Peripheral neuropathies can be associated with problems in neuronal and/or glial cells and result in reduced NCV and axonal degeneration. Causes of peripheral neuropathies include metabolic, inflammatory, infectious or chemical agents. A small but significant fraction of cases are associated with genetic bases (Pearlman and Collins, 1990). CMT, also known as hereditary motor and sensory neuropathy, is the most common of the hereditary peripheral neuropathies,

6 affecting 1 in 2500 individuals (Skre, 1974). It encompasses a wide range of clinically and genetically diverse disorders (CMT1-4) (Suter and Scherer, 2003).

CMT is a neuropathy that progressively worsens as patients age. Muscle atrophy occurs when the signal sent from the nerve soma does not reach the neuromuscular junction and the muscle is not stimulated, either due to abnormalities in the axon (CMT2) or in the glial cells (CMT1, 3, 4) of the myelin sheath (Thomas, 1999). Patients with

CMT1, 3 and 4 have reduced motor NCV, whereas CMT2 patients have relatively preserved signal speeds. Clinical symptoms of CMT1 are distal amyotrophy and weakness in the limbs, tendon areflexia, mild sensory loss and foot deformities such as pes cavus (claw foot) (Thomas, 1999). Initial indications begin as a tingling sensation in the patients’ feet that progresses to muscle weakness and atrophy, sometimes culminating with a loss of ambulatory function (Windebank, 1993).

CMT1A is the most prevalent form of CMT1, with a frequency of 1 in 5,000

(Kuhlenbaumer et al., 2002). It is most commonly associated with the heterozygous inheritance of a 1.5 megabase duplication on chromosome 17p11.2-12 that includes

PMP22 (Vance et al., 1989; Lupski et al., 1991; Valentijn et al., 1992). The two homologous DNA sequences flanking this region have a high degree of transposon-like homology that promotes unequal crossing over during meiosis, resulting in a duplicated

(associated with CMT1A) or a deleted (associated with HNPP) allele (Lupski and Garcia,

2001). Point mutations within the PMP22 gene also account for a small fraction of

CMT1A cases (Roa et al., 1993; Naef and Suter, 1998). Slowed NCV, segmental demyelination and attempted remyelination and axonal loss are observed in the nerves of

CMT1A patients. Nerves also form onion bulbs, which are redundant SC processes

7 around incompletely remyelinated axons (Gabreels-Festen et al., 1995). As the disease progresses, patients develop muscle weakness, atrophy and sensory loss in the lower limbs beginning in their feet and progressing proximally. In addition to foot deformities, slowed or absent reflexes also are present (Suter and Scherer, 2003). While clinical signs usually appear in the second to third decade of life, young individuals carrying the

CMT1A duplication exhibit reduced NCV before clinical signs appear, implying that

PMP22 overexpression is associated with the initial process of myelination, in addition to the more obvious demyelination seen later in life (Berciano et al., 2000).

DSS and CHN are associated with dominant mutations in PMP22 and have the most severe phenotype of the neuropathies linked with PMP22 misexpression (Suter and

Scherer, 2003). Disease onset begins earlier than in CMT1A, usually in infancy or early childhood, with delayed motor development before three years of age. If clinically recognized at birth, the neuropathy is identified as CHN, whereas it is termed DSS if it is detected later in infancy. Patients typically have dysmyelinated axons, where the myelin sheaths have not formed properly. A hallmark diagnostic feature of DSS and CHN is a severely reduced NCV, typically less than 10 meters per second (normal NCV is ~38 m/s) (Suter and Scherer, 2003). Symptoms include distal sensory loss, ataxia and motor deficits. Similar to CMT1A, affected nerves show demyelination and attempts of remyelination, onion bulbs, supernumerary SCs, axonal degeneration and nerve hypertrophy (Plante-Bordeneuve and Said, 2002).

HNPP, the mildest form of hereditary peripheral neuropathy, occurs when one copy of PMP22 is deleted (Chance et al., 1993), or rarely when the gene is truncated or mutated (Nicholson et al., 1994). As with CMT1A, the onset is usually in the second to

8 third decade of life. Initial symptoms of HNPP may be related to mild nerve trauma, such as sleeping on a limb that results in palsy (i.e., a feeling of “pins and needles”), which lasts for days to weeks instead of seconds to minutes (Windebank, 1993). Patients exhibit a phenotype that includes slowed axonal conduction resulting from focal hypermyelination, “sausage-like” myelin thickenings known as tomacula. These tomacula form along portions of the internodes of nerve fibers (Verhagen et al., 1993), presumably from minor nerve traumas. Nerve biopsies also show segmental demyelination and attempts of remyelination (Suter and Scherer, 2003). Affected individuals can develop muscle weakness and pes cavus, similar to CMT1A patients, but the disease progression is generally slower in HNPP.

Animal Models of PMP22-Associated Peripheral Neuropathies

Animal models of PMP22 neuropathies have helped to establish a causal relationship between PMP22 misexpression and resulting peripheral neuropathies.

Rodent lines with overexpression, underexpression or point mutations in PMP22 have been engineered, in addition to lines with spontaneously occurring point mutations

(Martini and Schachner, 1997). These models parallel human diseases in that they exhibit demyelination over time. However, early postnatal studies also detect alterations in axo- glial relationships, indicating PMP22 is necessary during myelin initiation as well

(Notterpek et al., 1997; Robertson et al., 1997; Robaglia-Schlupp et al., 2002).

Trembler J (TrJ) mice, a model of CMT1A, have a dominantly inherited demyelinating neuropathy, associated with a leucine to proline amino acid substitution at position 16 in the first transmembrane domain of PMP22 (Suter et al., 1992). Upon morphological examination, these mice exhibit primary dysmyelination and myelin instability. Frequently, their SCs are not well adhered to their axons and they produce

9 excess basal lamina (Notterpek et al., 1997; Robertson et al., 1997). Additionally, in some instances the SC terminal loops of myelin turn out toward the ECM instead of adhering to the axon (Robertson et al., 1997). PMP22 overexpressor (C22) mice, a model of CMT1A expressing seven copies of the human PMP22 gene, have abnormal myelin, excess basal lamina (Huxley et al., 1998) and delayed myelination (Robaglia-Schlupp et al., 2002). The C22 mice and a rat model overexpressing one copy of PMP22, display SC hypertrophy, muscle weakness and reduced NCV, similar to CMT1A patients (Huxley et al., 1996; Sereda et al., 1996).

Reduced or absent PMP22 also is associated with neuropathies, but the diseases are usually less disabling than those incurred by overexpression or point mutations of

PMP22. Homozygous PMP22-deficient mice have dysmyelination and tomacula at a young age, and develop severe demyelination, axonal loss and slowed NCV later in life

(Adlkofer et al, 1995). The tomacula observed early in life are replaced by thin or absent myelin sheaths with time. Most of the studies have been performed in heterozygous

PMP22-deficient animals because these mice resemble human HNPP. Heterozygous mice eventually share characteristics similar to the homozygous PMP22 knockouts, but symptoms occur on a delayed time course. Additionally, their nerves have a smaller decrease in NCV and display less obvious demyelination (Adlkofer et al., 1997a). A study of the fate of tomacula during aging reveals that they are intrinsically unstable structures, but the severity of these abnormalities is variable in heterozygous mice, perhaps explaining the multifarious phenotypes of patients presenting with HNPP

(Adlkofer et al., 1997a). Transgenic mice expressing antisense PMP22 mRNA have reduced levels of PMP22, and display traits comparable to PMP22 knockout mice,

including movement disorder, slowed NCV over time, focal myelin thickenings and

progressive demyelination (Maycox et al., 1997). While these studies have been helpful

to characterize the neuropathies, they have not been able to elucidate the role of PMP22

in normal myelination.

Potential Disease Mechanisms of PMP22 Neuropathies

The fact that both overexpression and underexpression of PMP22 is associated with a host of demyelinating peripheral neuropathies suggests that the levels of PMP22 must be tightly regulated for normal SC cell function. The newly synthesized PMP22 is a short-lived protein. Most PMP22 is rapidly degraded in the endoplasmic reticulum, with only ~20% of the de novo protein acquiring complex glycosylation in the Golgi and

trafficking to the SC membrane (Pareek et al., 1997). The rapid turnover rate is likely due

to the inability of this hydrophobic protein to fold properly, but it could be necessary for

an intracellular function of PMP22 (Naef and Suter, 1998).

Various point mutants of PMP22 are not incorporated into the plasma membrane,

but are instead retained inside the cell, as indicated by in vitro studies (D’Urso et al.,

1998; Tobler et al., 1999; Naef and Suter, 1999; Brancolini et al., 2000; Colby et al.,

2000; Johnson et al., 2005). These mutant forms of PMP22 can heterooligomerize with

the normal protein, and possibly impair its progress to the cell membrane and its

incorporation into myelin (Tobler et al, 1999; 2002; Johnson et al., 2005). Therefore,

although PMP22 is overexpressed, its incorporation into myelin is reduced. Indeed, most

patients with PMP22 point mutations have more severe neuropathies than those with a

single duplication or deletion of the PMP22 gene (Lupski and Garcia, 2001).

Mutant and/or misfolded PMP22 (TrJ and C22) can accumulate inside the cell and

overload the endoplasmic reticulum-associated degradation pathway and the proteasome,

11 leading to aggregate formation (Notterpek et al., 1999a; Ryan et al., 2002; Fortun et al.,

2003). In TrJ neuropathy nerves, PMP22 has an extended half-life and forms aggresome- like structures that are surrounded by molecular chaperones and lysosomes (Fortun et al.,

2003). The aggregates also recruit MBP (Fortun et al., 2005), another protein known to be degraded by the proteasome, suggesting an additional reason for myelin instability in

PMP22 mutants. SCs have the ability to eliminate aggresomes by a mechanism, which is enhanced when autophagy is activated and is primarily prevented when autophagy is inhibited, a function that may be impaired in PMP22 mutants (Fortun et al., 2003; 2005).

Studies in mice expressing one copy of the Trembler allele on a PMP22-null background show increased myelin deficiencies compared to heterozygous and homozygous PMP22-null animals (Adlkofer et al., 1997b). Additionally, mice lacking a copy of PMP22 do not have aggregates or an extended protein half-life. In cells from heterozygous TrJ mice (TrJ/+), the mutant protein is intracellularly retained in the endoplasmic reticulum and/or Golgi apparatus, often forming aggregates (Fortun et al.,

2003). Mice expressing one copy of PMP22 display a less severe neuropathy than TrJ/+ mice, suggesting a reduced amount of PMP22 at the cell surface is less toxic than the accumulation of misfolded PMP22 inside the cell. Therefore, it appears that the pathology of these neuropathies results from a reduced amount of normal PMP22 incorporation into myelin, and in some cases a toxic gain-of-function from the mutated copy of PMP22, which accrues along the secretory pathway.

Potential Roles of PMP22 in Cell Biology

Diseases associated with PMP22 are only observed in myelinating Schwann cells of the PNS, but PMP22 mRNA is found at lower levels in many other tissues, including the lung, heart and intestine (Baechner et al., 1995; Parmantier et al., 1995; 1997; Taylor

12 et al., 1995). The broad expression pattern suggests that PMP22 has a role in general cell biology in addition to a specialized function in myelin. PMP22 has been associated with changes in proliferation, cell shape and motility (Zoidl et al., 1995; Brancolini et al.,

1999; Roux et al., 2005, respectively).

As stated earlier, PMP22 also was discovered as a gene upregulated in serum- starved fibroblasts (gas-3) (Manfioletti et al., 1990). The level of PMP22 modulates the rate of cell division in many cell lines, with increased PMP22 expression resulting in a slowed transition from the G0/G1 to the S phase of the cell cycle (Zoidl et al., 1995;

1997; Roux et al., 2005), which may lead to apoptosis (Fabretti et al., 1995; Zoidl et al.,

1997). Reduced PMP22 mRNA levels have the opposite effect, enhancing DNA synthesis (Zoidl et al., 1995). PMP22 also induces morphological changes in multiple cell types. In NIH3T3 fibroblasts, PMP22 overexpression alters cell shape (Brancolini et al.,

1999). These morphological changes are seen with wild-type PMP22, but not with

PMP22 point mutants (Brancolini et al., 1999, 2000). These mutants do not traffic to the cell membrane, suggesting PMP22 must be at the cell surface to perform its function.

Stable PMP22-expressing Madin Darby canine kidney (MDCK) subclones have larger cell circumferences, increased nuclear dimensions and are flatter than control MDCK cells (Roux et al., 2005). The morphological changes brought about by PMP22 appear to be dependent on RhoA (Brancolini et al., 1999; Roux et al., 2005), a GTPase belonging to a family of molecular switches that are involved in regulating the actin cytoskeleton, cell polarity and membrane transport pathways (Etienne-Manneville and Hall, 2002).

In addition, in PMP22-overexpressing fibroblasts there are alterations in the ADP- ribosylation factor 6 (Arf6)-regulated plasma membrane endosomal-recycling pathway

(Chies et al., 2003). PMP22 accumulates in late endosomes close to the juxtanuclear

region similar to other myelin proteins. It induces the formation of vacuoles that are

positive for actin and (4, 5)-bisphosphate (PIP2), which indicates that it is specifically related to the Arf-6 pathway. PMP22 may induce vacuole formation by increasing the

GTP-bound state of Arf6 at the plasma membrane, or the overexpressed protein may

interfere with the recycling of vesicles back to the plasma membrane (Chies et al., 2003).

PMP22 also interacts with the P2X7 receptor in vitro (Wilson et al., 2002), an ATP-gated

ion channel that associates with integrins and cytoskeletal proteins (Kim et al., 2001).

The binding of ATP for short periods leads to the formation of a cation permeable

channel, but prolonged activation can induce cell blebbing and eventual death (Virginio

et al., 1999), a phenotype also seen in PMP22-overexpressing fibroblasts (Fabretti et al.,

1995; Brancolini et al., 1999).

Similar to epithelia and endothelia, SCs are polarized and have specialized

membrane domains (Bunge and Bunge, 1983). Epithelial cells are compartmentalized

into apical and basolateral cell membranes, whereas SCs are partitioned into compact

(apical) and non-compact (basolateral) myelin membranes. Studies in MDCK cells show

that specific CNS myelin proteins trafficked to either the apical (PLP) or basolateral

(myelin-oligodendrocyte glycoprotein) cell membranes, depending on distinct sorting

signals (Kroepfl and Gardinier, 2001).

PMP22 localizes to apical intercellular junctions in epithelia and endothelia

(Notterpek et al., 2001, Roux et al., 2004). A PMP22 homolog in Caenorhabditis

elegans, VAB-9, also has been described at adherens junctions (Simske et al., 2003).

Apical junctions are involved in cell polarity and paracellular permeability (Mitic et al.,

2000). The integral membrane junction molecules, such as claudins and occludin, form homophilic and heterophilic contacts via their EC domains in opposing cell membranes

(Furuse et al., 1999, Van Itallie et al., 1997; Fujita et al., 2000). PMP22 shares ~54% amino acid similarity and ~ 30% identity with the claudin family of tight junction- associated proteins (Notterpek et al., 2001). While it is tempting to speculate that PMP22 is a claudin family member, overexpression of PMP22 in L fibroblasts (Notterpek et al.,

2001) and in C6 glioma cells (Takeda et al., 2001) failed to induce tight junction strands, a characteristic of the claudin family (Tsukita and Furuse, 2000). However, PMP22 does modulate transepithelial resistance (TER) and paracellular flux in MDCK cells (Roux et al., 2005), two assays that test the functionality of apical junctional complexes (Wong and Gumbiner, 1997). Specifically, overexpression of human wild type PMP22 leads to an increase in TER and paracellular flux, indicating a tighter epithelial cell monolayer.

Function-blocking peptides composed of the second EC domain of PMP22 lower monolayer TER, and interfere with tight junction formation by binding to homophilic or heterophilic binding partners of PMP22 (Roux et al., 2005).

In addition to potentially adhesive peptide motifs in the EC domains, PMP22 contains the L2/HNK-1 adhesion/recognition carbohydrate epitope in its first EC loop

(Snipes et al., 1993). This glycosylation site is conserved among members of the PMP22 gene family (Taylor et al., 1995), and on PMP22 homologs throughout evolution (Gould et al., 2005). L2/HNK-1 was first discovered using a monoclonal antibody that stained an epitope on the surface of human natural killer (HNK) cells (Abo and Balch, 1981).

Further studies have shown that it is present on a variety of cells in the nervous system

(Kruse et al., 1984; Schachner et al., 1995) and is highly conserved among many species,

15 including human, mouse, rat, chicken and insect (Dennis et al., 1988). The L2/HNK-1 epitope modulates interactions within the PNS (Schachner et al., 1995), such as the homophilic binding of P0 molecules (D’Urso et al., 1990; Filbin et al., 1990) and the heterophilic linkage of certain adhesive molecules to laminin, including α1β1 integrin

(Pesheva et al., 1987; Lallier and Bronner-Fraser, 1992) and L1 (Hall et al., 1997).

The function of the L2/HNK-1 epitope on PMP22 is not fully understood. Recent in vitro assays suggest that PMP22 interacts homophilically via its first EC loop but the sugar moiety is not necessary for this interaction (Hasse et al., 2004). Additionally, the

L2/HNK-1 epitope is not involved in expression or trafficking of the protein to the SC membrane (Ryan et al., 2000). However, in vitro deglycosylation studies indicate that the

L2/HNK-1 epitope stabilizes PMP22 homodimers (Tobler et al., 1999; Ryan et al., 2000) and is linked to cell spreading effects (Brancolini et al., 2000). Also, migration is slowed in response to wounding in an MDCK epithelial cell monolayer when PMP22 is overexpressed (Roux et al., 2005), suggesting altered cell-substrate interactions. In vivo, the epitope may interact with other PMP22 molecules or with proteins such as laminin, playing a structural role in myelin stability and/or SC adhesion to the basal lamina. Yet, as the vast majority of PMP22 is thought to be embedded in the membrane, its EC domains (including the L2/HNK-1 epitope) may not be large enough to interact directly with the basal lamina. Thus, PMP22 may be part of a signaling complex in the membrane that links the basal lamina and the SC cytoskeleton.

Laminins are fundamental constituents of the SC basal lamina that contain a binding site for the L2/HNK-1 epitope (Hall et al., 1993; 1997). They are known to mediate SC proliferation, differentiation, cytoskeletal reorganization and myelination in

the PNS (Chen and Strickland, 2003). When specific laminin chains are absent, SC

proliferation is altered (Stirling, 1975; Jaros and Bradley, 1979), which renders SCs

incapable of extending their processes, and this in turn prevents the defasciculation of

axons and myelination of peripheral nerves (Colognato et al., 2005). These impairments

can lead to muscular dystrophy and peripheral neuropathy (Feltri and Wrabetz, 2005).

Laminins in the basal lamina are ligands for the integrins in the SC plasma membrane.

The integrins are a heterodimeric family of key integral membrane receptors,

important in cell adhesion, migration and myelination (Hynes, 1992). There are 24 α- and

nine β-subunits in humans that form 25 known integrin heterodimers (Clegg et al., 2003).

Three main dimers are present in the PNS–α1β1, α6β1, and α6β4 (Previtali et al., 2001).

The importance of integrins in the PNS is supported by the phenotypes of integrin

mutants. The SC-specific conditional disruption of β1 integrin leads to impaired axonal

sorting and loose basal lamina. However, some axons are sorted and myelinated,

suggesting partial redundancy of function with other laminin receptors, such as

dystroglycan or α6β4 integrin (Feltri et al., 2002). α6β4 knockout mice die around birth

due to their inability to form proper hemidesmosomes, yet; myelination appears to initiate

and progress properly (Previtali et al., 2003a). α7β1, an integrin present in mature SCs,

also has been shown to be non-essential for myelination (Previtali et al., 2003b). As with

β1 integrin knockout mice, these studies imply integrins have overlapping functions in

the process of myelination. Integrins are known to interact with many tetraspan proteins

(Berditchevski and Odintsova, 1999), including oligodendrocyte specific protein (OSP),

also known as claudin-11 (Tiwari-Woodruff et al., 2001), epithelial membrane protein-2

(EMP-2), a PMP22 family member (Wadehra et al., 2002), and PLP, a tetraspan protein

17 in CNS myelin (Gudz et al., 2002). An interaction between PMP22 and an integrin would provide PMP22 with the ability to modulate the observed cell-ECM communications, as well as intracellular events.

Based on the phenotypes of PMP22 mutant mice, PMP22 is a key molecule in peripheral nerve development. To better understand the pathogenesis behind PMP22- associated disorders, it is necessary to ascertain the normal function of the protein in SC development and myelin formation and stability. In vitro studies in various nonneural cell types imply that PMP22 also has a more general role in cell biology uncoupled to myelin structure. The studies described here characterize a novel PMP22-deficient mouse model to elucidate the function of PMP22 in the cell membrane through a variety of in vivo and in vitro approaches.

CHAPTER 2 PERIPHERAL MYELIN PROTEIN 22 IS IN COMPLEX WITH ALPHA6 BETA4 INTEGRIN AND ITS ABSENCE ALTERS THE SCHWANN CELL BASAL LAMINA

Note

The work presented in this chapter was published in The Journal of Neuroscience

26(4) 1179-89 (2006). William A. Dunn, Jr. assisted with the electron microscopy studies. Andrew J. Murphy, Niels C. Adams, Nicholas W. Gale, David M. Valenzuela and George D. Yancopoulos engineered the PMP22-LacZ transgenic mouse line.

Introduction

Myelination in the peripheral nervous system (PNS) is dependent on the secretion of a basal lamina by Schwann cells (SCs) (Bunge, 1993). This linkage with the extracellular matrix (ECM) stabilizes axon-SC contacts and permits the initiation of myelination (Eldridge et al., 1987; 1989). Integrins, specifically α6β1 and α6β4, play key roles in mediating the interaction between SCs and basal lamina constituents, such as laminin (Previtali et al., 2001). Besides integrins, SCs express a number of adhesive proteins, including myelin associated glycoprotein, myelin protein zero (P0) and peripheral myelin protein 22 (PMP22), which play critical roles at various stages of myelination (Schachner and Martini, 1995). These molecules contain the L2/HNK-1 adhesion/recognition carbohydrate epitope that can modulate cell-cell and cell-matrix interactions.

PMP22, a putative tetraspan glycoprotein, is thought to be a key molecule in PNS myelin, as its misexpression is associated with hereditary demyelinating neuropathies

18 19

(Naef and Suter, 1998). While the exact function of PMP22 in the SC membrane is still unclear, the phenotypes of mice deficient in PMP22, or expressing mutated copies, suggest an involvement in myelin formation and maintenance (Adlkofer et al., 1995;

Maycox et al., 1997; Robertson et al., 1997). One mechanism by which PMP22 may influence myelin stability is by interacting with the extracellular domain of P0 (D’Urso et al., 1999; Hasse et al., 2004). An additional role in embryonic and early postnatal development is supported by the expression of PMP22 by migratory neural crest cells

(Hagedorn et al., 1999), and the dysmyelinating phenotypes of PMP22 mutant rodents

(Martini and Schachner, 1997; Notterpek and Tolwani, 1999). In agreement, certain mutations in PMP22 are associated with childhood onset dysmyelinating neuropathies in humans (Roa et al., 1993; Garcia et al., 1998).

The localization of PMP22 at cell-cell junctions of various epithelia and endothelia, suggests a possible role in mediating myelin wrapping and intercellular adhesion

(Notterpek et al., 2001; Roux et al., 2004; 2005). Overexpression of PMP22 in epithelial cells alters their migratory behavior and changes the functional properties of cell-cell contacts (Roux et al., 2005). In addition, in NIH3T3 cells (Brancolini et al., 1999) and rat

SCs (Nobbio et al., 2004), the overexpression of PMP22 mediates membrane expansion.

These findings, together with the phenotypes of PMP22 mutant mice, indicate that

PMP22 is capable of modulating intercellular and cell-matrix communications in a variety of cell types. How PMP22 attains these functions in the plasma membrane is still unknown, but could involve direct or indirect links with constituents or binding partners of the ECM.

To elucidate the potential roles of PMP22 in the cell membrane, we engineered a

novel PMP22-deficient mouse line. Affected mice broadly express the LacZ reporter in a

variety of neural and nonneural tissues and display tomaculous neuropathy.

Ultrastructural analyses of sciatic nerves revealed alterations in the SC basal lamina,

which led us to examine the potential interaction of PMP22 with integrins. By a variety

of approaches, we show that PMP22 is a binding partner for the α6β4 integrin complex.

Materials and Methods

PMP22-deficient LacZ Mice

This PMP22-deficient LacZ mouse line was engineered using the VelociGene

targeting technology (Valenzuela et al., 2003). A breeding colony is maintained under

specific pathogen-free housing. The Institutional Animal Care and Use Committee have

approved the use of animals for these studies. For genotyping, DNA was isolated from

tail biopsies of pups under 10-days old, and digested with BamHI. Genotypes were determined by Southern blot analysis using a 1.4 kb probe, which recognizes a portion of the murine pmp22 upstream of the start codon (5’ probe) and a portion of the neomycin resistance gene (neo probe). The lacZ reporter activity was detected histochemically on embryonic day (E) 15 and adult heterozygous tissue samples by X-gal staining

(Valenzuela et al., 2003). Sections from the embryos were counterstained with neutral red. Wholemount preparations were imaged using a Leica MZ12.5 dissecting microscope with a Soft Imaging Systems ColorView II camera. Cryostat sections were scanned using an Aperio ScanScope and all images were formatted for printing with PHOTOSHOP CS.

Morphological Analyses and Teased Nerve Fiber Preparation

All reagents used for these studies were obtained from Electron Microscopy

Sciences (Fort Washington, PA). Sciatic nerves were collected from genotyped postnatal

(P) day 3, P10, P13 and P18 mice. Samples were fixed by immersion in 1%

glutaraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4,

overnight at 4°C, followed by osmication in 2% OsO4 in 0.1 M sodium cacodylate buffer

for 1 hr at room temperature (RT), dehydrated in an ascending ethanol and acetone series,

and embedded in Spurr’s medium. Thick sections of the samples were stained with

toluidine blue and surveyed by light microscopy using a SPOT camera (Diagnostic

Instruments, Inc., Sterling Heights, MI) attached to a Nikon Eclipse E800 microscope.

Measurements of axon and fiber (axon with myelin) diameters on light level images were

obtained using Advanced SPOT RT Software (Diagnostic Instruments, Inc.). G ratios

were determined by dividing the axon diameter by the fiber diameter. To quantify

potential differences in g ratios, more than 100 individual fibers per animal were

analyzed, using 4-5 mice per genotype. Statistical analysis was performed using

Microsoft Excel 2000 software. Thin sections of the samples were poststained with lead citrate and uranyl acetate, and examined on a Jeol 100CX transmission electron

microscope (Peabody, MA). Images were formatted for printing by using Adobe

PHOTOSHOP 5.5.

Details of the teased nerve fiber procedure were described by Martini et al. (1995).

Briefly, sciatic nerves from genotyped P21 mice were collected and preteased into small

bundles, followed by osmification and dehydration, as above. Single fibers were obtained

by teasing the nerves in embedding medium and slides were baked overnight at 60ºC.

Light microscopy was performed using a Nikon T1-SM inverted microscope equipped

with a Nikon DS-L1 camera.

Myelinating Dorsal Root Ganglion (DRG) Neuron-SC Cocultures

Rat DRG-SC cocultures were established as described (Notterpek et al., 1999b).

DRGs were collected from embryonic day 15 rodents, digested with 0.25% trypsin

(Gibco, Rockville, MD), dissociated and plated on rat-tail collagen (Biomedical

Technologies, Inc., Stoughton, MA) coated glass coverslips. Explants were maintained in

Minimum Essential Medium (Gibco) supplemented with 10% fetal calf serum (Hyclone,

Logan, UT), 0.3% glucose (Sigma-Aldrich, St. Louis, MO), 10 mM HEPES (Cellgro,

Mediatech, Inc., Herndon, VA) and 100 ng/ml nerve growth factor (Harlan Bioproducts for Science, Madison, WI) for 7 days. Ascorbic acid (50 µg/ml) (Sigma-Aldrich) was then added for an additional 15 days, at which point the cultures were used for biochemical studies.

Immunolabeling of Nerves

Sciatic nerves were dissected from genotyped P10 and 3-month old +/+ and PMP22

-/- littermates. Samples were frozen by immersion in liquid nitrogen-cooled N-methyl butane. Frozen sections (5 µm thickness) were dried for 1 hr on Superfrost/Plus microslides (Fisher, Pittsburgh, PA), followed by fixation with 4% paraformaldehyde in

PBS for 10 min at RT, and permeabilization with 100% acetone for 2 min at -20°C

(Melendez-Vasquez et al., 2004). Samples were blocked in PBS containing 20% normal goat serum for 1 hr. Primary antibodies, including polyclonal rabbit anti-laminin,

1:10,000 (Sigma-Aldrich), anti-PMP22 (Notterpek et al., 1999b); and monoclonal rat anti-β4, 1:4000; anti-β1, 1:4000 and anti-α6 integrin, 1:4000 (all from BD Biosciences

PharMingen, San Diego, CA), were added in the blocking solution overnight at 4°C. For the PMP22 and integrin double labeling studies, nerve sections were fixed in 4% paraformaldehyde (10 min, RT) and permeabilized with 100% methanol (10 min, -20°C)

(Einheber et al., 1993). These fixation conditions are optimal for the detection of β4 integrin; however the PMP22-like staining is weaker than usual (Notterpek et al., 1999b).

The samples were then incubated with Alexa Fluor 594 goat anti-rabbit IgG (red) and/or

Alexa Fluor 488 goat anti-rat IgG (green) (both from Molecular Probes, Inc., Eugene,

OR) for 1-2 hr. Hoechst dye #33342 (Molecular Probes, Inc.) was included in the secondary antibody solution at 10 µg/ml to visualize nuclei. Coverslips were mounted by using the ProLong Antifade kit (Molecular Probes, Inc.). Samples were imaged with a

Spot camera attached to a Nikon Eclipse E800 microscope or an Olympus Optical

(Tokyo, Japan) MRC-1024 confocal microscope and were formatted for printing by using

Adobe PHOTOSHOP 5.5.

Western Blot Analyses

Sciatic nerves collected from genotyped mice were frozen immediately in liquid nitrogen. For total protein analyses, nerves from three to four mice were crushed under liquid nitrogen, and solubilized in SDS gel sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 3% SDS). Protein concentrations were determined using BCA reagents (Pierce

Chemicals, Rockford, IL). Samples (20 µg/lane) were separated on 7.5% or 12.5% acrylamide gels under reducing conditions, and transferred to nitrocellulose membranes

(Bio-Rad Laboratories, Inc., Hercules, CA). Polyclonal anti-PMP22, 1:2000; anti-β- galactosidase, 1:1000 (Molecular Probes, Inc.); anti-laminin, 1:10,000 (Sigma-Aldrich); anti-β4 integrin, 1:1000; monoclonal rat anti-β1 integrin, 1:1000 (both from Chemicon,

Temecula, CA) and monoclonal mouse anti-glyceraldehyde phosphate dehydrogenase

(GAPDH, a kind gift from Dr. Gerry Shaw, University of Florida, Gainesville, FL) were used. Bound antibodies were visualized using an enhanced chemiluminescence kit

(PerkinElmer Life Sciences, Boston, MA). Films were digitally imaged using a GS-710 densitometer (Bio-Rad) and were formatted for printing by using Adobe PHOTOSHOP

5.5.

Coimmunoprecipitation of Integrins and PMP22

For the coimmunoprecipitation experiments, a previously established procedure was used (Gudz et al., 2002). Briefly, sciatic nerves from P21 +/+ mice were lysed in immunoprecipitation (IP) buffer (0.15 M NaCl, 0.05 M Tris, 0.5 mM EDTA, pH 7.5, 1%

Triton X-100, 0.05% SDS, and 0.2% bovine serum albumin), supplemented with

complete protease inhibitor (Roche, Indianapolis, IN) and 500 µM phenylmethylsulfonyl

fluoride (Sigma-Aldrich). Lysates from PMP22 -/- mice were analyzed in parallel as a

negative control. After centrifugation, pelleted material was solubilized in IP buffer with

2.5% SDS and combined with the supernatant (final SDS volume, 0.3%). Following a

second centrifugation, protein lysate supernatants (1 mg/ml) were precleared by

incubation with non-specific rabbit or rat serum, followed by incubation with protein A

Sepharose (Amersham Biosciences, Piscataway, NJ) or protein G agarose (Santa Cruz

Biotechnology, Santa Cruz, CA), respectively. The supernatants were then incubated with rabbit polyclonal anti-PMP22, anti-β4 integrin, anti-laminin or rat monoclonal anti-α6

integrin antibodies, and the antibody-antigen complexes were captured with either protein

A Sepharose or protein G agarose. Immunoprecipitates were eluted by boiling in SDS gel

sample buffer with 2% β-mercaptoethanol (Bio-Rad). Total protein lysates and

immunoprecipitates were separated on 10% acrylamide gels, blotted to polyvinylidene

difluoride membrane (Bio-Rad), blocked with 5% nonfat dry milk in PBS-T buffer (10

mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.4), and subsequently probed with the

appropriate antibody (polyclonal anti-PMP22, 1:1000; anti-β4 integrin, 1:1000; anti-

laminin, 1:10,000; and mouse monoclonal β-dystroglycan, 1:1000 (NovoCastra,

Newcastle, UK)). Bound antibodies were visualized using enhanced chemiluminescence

kit, as above.

Cell Surface Labeling

Myelinating rat DRG-SC cocultures were rinsed with PBS containing 0.1 mM

CaCl2 and 1 mM MgCl2, supplemented with complete protease inhibitor (Roche,

Indianapolis, IN) and 500 µM phenylmethylsulfonyl fluoride (Sigma-Aldrich) (PBS-CM)

(Gudz et al., 2002). After incubation with 2 mM Sulfo-NHS-LC-biotin (Pierce Chemical

Co., Rockford, IL) in PBS-CM for 1 hour at 4°C the samples were washed with PBS and

0.1 M Tris-HCl, and solubilized in IP buffer, as above. Following centrifugation,

Strepavidin-agarose beads (Sigma-Aldrich) were added to the supernatant for 3 h at 4˚C.

Beads were collected by brief centrifugation and washed in IP buffer. The biotin-avidin

protein complexes were dissociated from the beads by heating in SDS and ß-

mercaptoethanol-containing gel sample buffer. The eluted proteins were analyzed on

12.5% acrylamide gels, and blotted with polyclonal rabbit anti-PMP22 and anti-β4

integrin antibodies and monoclonal mouse anti-actin (Sigma-Adlrich).

Protein Interaction Studies in Clone A Cells

Clone A cells, originally isolated from a poorly differentiated colon

adenocarcinoma (Dexter et al. 1979), were a kind gift from Dr. Arthur Mercurio (Beth

Israel Medical Center, Boston, MA). Cells were maintained in RPMI medium

supplemented with 10% fetal calf serum, 50 µg/ml penicillin, 50 units streptomycin

(Sigma-Aldrich), 5 mM HEPES, 200 µM L-glutamine (Gibco). The human PMP22 (kind

gift of Dr. Clare Huxley, Imperial College School of Science, Technology and Medicine)

open reading frame, tagged with a myc-epitope in the second extracellular loop, was

inserted into the pLNCX2 retroviral vector (Clontech, Palo Alto, CA), under the control

of the cytomegalovirus (CMV) promoter. Forty eight hours following transfection with

LipofectamineTM and PLUSTM reagent (Invitrogen, Carlsbad, CA) samples were

processed for double immunolabeling, as described above except that cells were

permeabilized with 0.2% Triton-X 100/PBS, 15 min, RT and incubated with polyclonal

rabbit anti-myc (Santa Cruz Biotechnology) and monoclonal mouse anti-β4 integrin

(Ancell Corporation, Bayport, MN) antibodies. For the coimmunoprecipitation

experiments, monoclonal mouse anti-myc (Santa Cruz Biotechnology) and polyclonal

rabbit anti-β4 integrin antibodies were used.

Results

Phenotype of PMP22-deficient LacZ Mice

Targeted deletion of the pmp22 gene was accomplished using the VelociGene

technology (Valenzuela et al., 2003). This approach circumvents potential complications

of previous gene silencing technologies, such as targeted disruption, which may allow for

alternative splicing and some translation of PMP22. For this mouse model, the first two coding exons of pmp22, exons 2 and 3 (Fig. 2-1A), encoding the first transmembrane segment, were replaced with the lacZ reporter in tandem with the neo selection gene (Fig.

2-1B). The entire pmp22 coding sequence was not deleted because of concerns that removal of the large intron between exons 3 and 4 (~17,000 base pairs) may include regulatory elements, which could affect the expression pattern of the lacZ reporter. In this

design, the lacZ gene is driven by the pmp22 promoters, allowing for high-resolution

analysis of the β-galactosidase (β-gal) protein throughout the organism.

Figure 2-1. Schematic representation of the targeting strategy. Genomic structure of the mouse pmp22, showing the alternatively utilized exons 1A and 1B, which are preceded by promoters 1 and 2, respectively (A). The double bars between exons 3 and 4, and 4 and 5, indicate 16.6 kilobase (kb) and 7.0 kb intronic sequences, respectively. Diagram of the pmp22-lacZ construct (B). The first two coding exons of pmp22 (exons 2 and 3) were replaced with the lacZ reporter followed by a strong transcription termination signal, the PGK-neo gene and a second stop signal. BamHI restriction sites (B) and locations of the PMP22 (5’ probe) and neo probes are shown (A, B). Southern blot analysis of genomic DNA digested with BamHI from wild type (+/+), heterozygous (+/-), and homozygous PMP22 knockout (-/-) littermates (C). The endogenous allele is detected as a 2.2 kb fragment while the mutant allele yields a 7.6 kb fragment. Western blot analyses of sciatic nerve lysates (7.5 µg/lane) from adult +/+, +/-, -/- mice with anti-PMP22 and anti-β-galactosidase antibodies (D). Anti-GAPDH is shown as a loading control. Molecular mass in kb (C) and kDa (D) is shown on the left.

To determine the genotypes of the mice, genomic DNA is blotted with a probe that contains a portion of the mouse pmp22 and the neomycin resistance gene (Fig. 2-1C). In wild type (+/+) and heterozygote (+/-) samples, a 2.2 kilobase (kb) fragment, indicative of the endogenous pmp22 gene, is detected. The mutant allele is identified as a 7.6 kb

28 band in +/- and homozygous knockout (-/-) samples. In accordance, Western blot analysis on whole sciatic nerve lysates confirms the expression of PMP22 in adult +/+ mice, with lower and undetectable levels in +/- and -/-, respectively (Fig. 2-1D). β-gal reporter expression is reciprocal of PMP22, with the highest levels in the -/- nerves. To monitor equal protein loading, the blot was reprobed with an anti-GAPDH antibody.

Figure 2-2. Widespread β-gal expression in the E15 mouse embryo. The β-gal reporter is expressed in a variety of neural and nonneural tissues on a cryosection of an E15 embryo (A). Higher magnification images show reporter expression in the ventricular zones of the rhombencephalon (R) and mesencephalon (M) (10X B), dorsal root ganglion (8X C, arrow), skeletal muscle (C, asterisk) and cartilage of the vertebrae (C, arrowhead). β-gal activity is prominent in the aortic arch of the heart (7X D, arrow). High levels of the reporter are also present in the lungs (18X E) and the intestines (9X F). The sections are counterstained with neutral red.

Expression of the β-gal reporter was first examined on sections of heterozygote

embryos at E15 (Fig. 2-2). Detected as a blue reaction product, β-gal is expressed

broadly, with robust staining in the lung and intestines (Fig. 2-2A). At higher

magnification, β-gal is evident in the rhombencephalic and mesencephalic neuroepithelium (Fig. 2-2B), the dorsal root ganglia (Fig. 2-2C, arrow), the skeletal muscle (Fig. 2-2C, asterisk) and the cartilage of the vertebrae (Fig. 2-2C, arrowhead).

The reporter is also prominent at the aortic arch of the heart, a previously unrecognized

area for the expression of PMP22 (Fig. 2-2D, arrow). Panels E and F show a higher

magnification of the lungs and the intestines, respectively. These results are in agreement

with previous reports on the broad tissue distribution of the PMP22 mRNA during

murine development (Baechner et al., 1995; Parmantier et al., 1995; 1997; Taylor et al.,

1995), and validate the specificity of our targeting design.

Next, we investigated the expression of β-gal in adult heterozygous transgenic mice

(Fig. 2-3). Many nonneural tissues remain positive for the reporter, including the lungs,

intestines, gallbladder, kidneys, urinary bladder, reproductive organs and stomach (data

not shown). Particularly, the cartilage of the ribs (Fig. 2-3A) and the aortic arch (Fig. 2-

3B) display high levels of β-gal expression. On whole-mounts of the brain, the deep

nuclei of the brainstem (Fig. 2-3C, arrows) and the cranial nerves (Fig. 2-3C,

arrowheads) are reactive. In addition, the high abundance of PMP22 in the PNS is

confirmed on spinal cord preparations, which display robust β-gal reactivity in the

peripheral nerves (Fig. 2-3D).

Figure 2-3. Phenotype of adult PMP22-deficient mice. Distinct β-gal expression is evident in the ribs (A), aortic arch of the heart (B), brainstem deep nuclei (C, arrows), cranial nerve roots (C, arrowheads) and in the peripheral roots of a spinal cord preparation (D). Behavioral phenotypes of three-month old littermates (E). Wild type (+/+) mice have splayed hind limbs and relaxed forelimbs; whereas PMP22-deficient littermates (-/-) clasp their hind and forelimbs close to their body (top right) and display gait abnormalities (bottom). Teased nerve fibers from genotyped P21 littermates (F). Nerve fibers of PMP22 -/- mice exhibit frequent tomacula (arrowhead). Magnification, X40.

Phenotypically, heterozygous PMP22-deficient mice should model hereditary neuropathy with liability to pressure palsies (HNPP), which is commonly associated with deletion of one copy of the PMP22 gene (Chance et al., 1993). Homozygous PMP22- deficient mice are predicted to have a more severe neuropathy similar to CMT1A

(Adlkofer et al., 1995). Indeed, when the mice are held by the base of the tail, wild type adults splay their hind limbs and relax their forelimbs (Fig. 2-3E, +/+). In comparison, homozygous PMP22-deficient mice manifest visible signs of motor deficits, including clasped hind limbs, tremor and hindered gait (Fig. 2-3E, -/-), which become noticeable by two weeks of age. As the animals age, distal muscle weakness advances to hind limb paralysis. Heterozygous mice are less affected during the first two months of postnatal

31 development, but display various severities of visible motor deficits by five months of age. PMP22 -/- mice are less capable of mating, and show postnatal growth retardation, as compared to +/+ littermates. By three weeks of age, PMP22 -/- mice weigh ~2 grams less than their +/+ littermates (+/+: 9.7 grams, n=7; -/-: 7.7 grams, n=8; p<0.005). On morphological examination, the sciatic nerves from P21 -/- mice reveal tomacula, sausage-like thickenings of myelin (Fig. 2-3F, arrowhead). Paranodal tomacula are the most common, however internodal myelin thickenings were also noted (data not shown).

At three weeks of age, tomacula are less common in the sciatic nerves of heterozygous mice, but increase in frequency as the animals grow older (our unpublished data;

Adlkofer et al., 1995). Compared to young animals (Fig. 2-3F), our preliminary studies show fewer tomacula and an increase in demyelinating profiles in nerve samples from adult -/- mice (data not shown). As tomacula are the neuropathological hallmarks of

HNPP (Meier and Moll, 1982), their presence in the nerves of affected mice further validates our model.

Delayed Myelination and Altered SC-ECM Interactions in the Absence of PMP22

During myelination, PMP22 is thought to play a role in the interactions between

SCs and axons, possibly mediating myelin wrapping (Martini and Schachner, 1997).

Therefore, sciatic nerves from genotyped +/+ and -/- littermates between the ages of P3 and P18, within the period of active myelination, were fixed and processed for morphological examination (Fig. 2-4). Thick sections (1 µm) of nerves from P10 +/+ mice show the proper one-to-one axon-SC relationships and many well-myelinated fibers

(Fig. 2-4A). In comparison, in the absence of PMP22, there is a notable increase in the number of SCs that are halted in the promyelinating stage, where they have surrounded axons but have not begun wrapping them (Fig. 2-4B and D, arrowheads). At P10, 95±3%

of defasciculated axons from +/+ mice are myelinated, whereas only 56±13% of axons from -/- mice are (n=3 per genotype, p<0.0005). In agreement with the detection of tomacula in teased nerve fibers (Fig. 2-3F), some axons are hypermyelinated (Fig. 2-4B, asterisks). After the exclusion of promyelinated and hypermyelinated profiles, we quantified axon and fiber diameters in multiple sections from P10, P13 and P18 littermates. At the ages examined, measurements of over five hundred axon-SC profiles per genotype reveal an ~19% reduction in axon (+/+: 3.37 µm, -/-: 2.68 µm) and a 23% reduction in fiber (+/+: 5.04 µm, -/-: 3.84 µm) diameters in nerves of -/- mice (n=5) as compared to +/+ littermates (n=4) (p<0.0005 for both). As the result of these changes, the g ratios in the affected samples are increased by ~5% (+/+: 0.668, -/-: 0.701; p<0.005), indicating dysmyelination.

Ultrathin sections of the same samples from P10 +/+ mice show tightly adhered

SCs against their axons and each axon-SC unit is surrounded by a basal lamina (Fig. 2-

4C, arrow in inset). In comparison, pockets of loose basal lamina are prominent in cross- sections of nerves from P10 -/- mice (Fig. 2-4D, arrows). The boxed area in 2-4D is magnified to illustrate the excess, loose basal lamina around a particular SC-axon profile

(Fig. 2-4F, arrow). In some instances, bare axons with pockets of basal lamina are observed (Fig. 2-4D, a), suggesting that the SCs have retracted their processes. SC profiles, as judged by the presence of a surrounding basal lamina, with irregular cytoplasmic processes are also present (Fig. 2-4D, open arrowhead). The described morphological alterations persist throughout the first three weeks of postnatal development (Fig. 2-4E-G, arrows), and can also be seen in samples from adult mice (not shown).

Figure 2-4. Delayed myelination and ultrastructural alterations in nerves of affected mice. Semithin (1 µm thickness), toluidine blue stained cross-sections from P10 wild type (A, +/+) and PMP22-deficient (B, -/-) littermates are shown. In contrast to the normal nerve (A) there is a delay in myelination in PMP22- deficient samples, as many promyelinating fibers are observed (B, arrowheads). Thickened myelin profiles are also present (B, asterisks). On ultrastuctural examination, nerves of P10 +/+ mice show tight basal lamina (C, arrow in inset, 3X magnification) around the SCs. In comparison, promyelinating SCs (arrowhead), loose basal lamina (arrows), bare axons (a) and irregular, hypertrophic SC processes (open arrowhead) are evident in the P10 -/- sample (D-G). The boxed area from D is magnified in F (3X). SC profiles with pockets of loose basal lamina are also seen in nerves from P3 (E) and P18 PMP22 -/- mice (G). Scale bar, 10 µm (A-B), 2 µm (C-D), 0.67 µm (inset in C, and F), 0.5 µm (E, G).

β4 Integrin Levels Are Reduced in Sciatic Nerves of PMP22 -/- Mice

To investigate the molecular changes associated with the altered basal lamina profiles, nerves from P10 genotyped littermates were processed for analyses with anti- basal lamina and SC outer surface (abaxonal) protein antibodies, including laminin and integrins (Fig. 2-5). As previously described (Einheber et al., 1993), β4 integrin is mainly localized to the outer membrane of SCs in nerves of P10 +/+ mice (Fig. 2-5A, arrow). In comparison, β4-like staining intensity is notably reduced in the homozygous PMP22 knockout sample and the basal lamina-like pattern is discernable only occasionally (Fig.

2-5B, arrow). Likewise, in nerves of +/+ mice, β1 integrin antibodies label the outer SC membrane (Fig. 2-5C, arrow), whereas in the absence of PMP22, β1-like immunoreactivity is patchy and less distinct (Fig. 2-5D). Laminin, a major protein component of the SC basal lamina, is detected around each axon-SC unit, adjacent to the abaxonal SC membrane in normal nerves (Fig. 2-5E). Similar to the integrins (Fig. 2-5B,

D), in sections from PMP22 -/- mice, laminin-like immunoreactivity appears less organized and thickenings of the basal lamina are seen (Fig. 2-5F, arrowheads).

Nonspecific rat or rabbit sera do not label the nerve sections (Fig. 2-5A, B, E, F, insets).

The nerve cross sections shown in Figure 2-5 are costained with the Hoechst nuclear dye, which reveals elevated cell density in the absence of PMP22. Quantification of independent samples from three mice per genotype shows an ~1.5-fold increase in nuclei in affected nerves (p<0.05). This finding agrees with a previous report on increased cell density at P10 in nerves of homozygous PMP22-deficient mice (Sancho et al., 2001).

Figure 2-5. Decreased levels of β4 integrin in nerves of PMP22-deficient mice. Cryosections of sciatic nerves from +/+ (A, C, E) and PMP22 -/- (B, D, F) mice were immunostained with monoclonal rat anti-β4 (A, B), anti-β1 integrin (C, D), or polyclonal rabbit anti-laminin (E, F) antibodies. In nerves of P10 +/+ mice, β4 and β1 integrin are detected at the abaxonal SC surface (A, C, arrows). In comparison, abaxonal integrin-like staining is only discernable around a fraction of the fibers in the -/- samples (B, D arrows). In addition, when the images are collected at the same exposure times, the level of β4-like immunoreactivity is reduced (compare A and B). Laminin is detected at the SC basal lamina in +/+ (E, arrow) and -/- nerves (F), with thickened basal lamina (arrowheads), and a tomaculum (open arrowhead) marked in the affected sample (F). Nonspecific rat (inset in A, B) and rabbit (inset in E, F) sera serve as controls for staining specificity. Scale bar, 5 µm (A-F). Sciatic nerve lysates (20 µg/lane) from P10 +/+, +/- and -/- mice were analyzed with polyclonal rabbit anti-β4 integrin and anti-laminin, and monoclonal rat anti-β1 integrin antibodies (G). The blots were reprobed with monoclonal mouse anti-GAPDH antibody as a protein loading control. Molecular mass is in kDa.

To substantiate the reduction in β4-like immunoreactivity in affected samples, whole sciatic nerve lysates from P10 littermates were blotted with the indicated antibodies (Fig. 2-5G). In agreement with the immunolocalization studies (Fig. 2-5A-F), the levels of β4 integrin are reduced in nerves of PMP22 -/- mice, while β1 integrin remains relatively constant. GAPDH is shown as a protein loading control. Since the mobility of laminin is similar to β4 on SDS gels, a parallel blot was probed with anti- laminin antibody. As predicted from the immunostaining (Fig. 2-5E and F), the level of laminin is unaltered in nerves of PMP22 -/- mice. The reduction in the levels of β4 integrin is consistent among several independent samples and at all ages tested, including

P18 and adult (data not shown).

PMP22 Is in a Complex with α6β4 Integrin and Laminin

The reduction in β4 integrin levels in the absence of PMP22 could suggest that the two proteins are in a complex. To investigate the potential interaction of β4 integrin and

PMP22, sciatic nerve lysates of 3-week old +/+ mice were immunoprecipitated (IP) with antibodies against candidate proteins. Subsequently, the immunoprecipitates were analyzed by Western blots for the indicated binding partners, as shown at the right of each Western blot. First, total nerve lysates (T) were precleared (PC lanes) with nonspecific rabbit IgGs and then incubated with a rabbit anti-β4 integrin antibody (Fig. 2-6A, left). Probing of the immunoprecipitate with the indicated antibodies; after electrophoretic separation, identifies not only β4 integrin but also PMP22 in the precipitate. In a reciprocal experiment, PMP22 was immunoprecipitated and analyzed for

β4 integrin (Fig. 2-6A, right). As before, the blot was reprobed with anti-PMP22

37 antibody to verify the precipitation of PMP22. These coimmunoprecipitation experiments indicate that PMP22 is in a complex with β4 integrin.

Figure 2-6. PMP22 is in a complex with α6β4 integrin and laminin. Sciatic nerve lysates (T lanes) from P21 +/+ mice were processed for immunoprecipitation (IP), after preclearing (PC lanes) with nonspecific immunoglobulins of the appropriate isotype. Lysates were incubated with polyclonal rabbit anti-β4 integrin (A, left, and C), anti-PMP22 (A, right), monoclonal rat anti-α6 integrin (B, left) or polyclonal rabbit anti-laminin (B, right) antibodies and captured immunoprecipitates were probed for the indicated proteins (A-C) as designated at the right of each blot. On reprobes (marked with asterisks), after stripping the membranes, the anti-β4 integrin and anti-laminin antibodies do not work efficiently when 1 µg/lane total (T) nerve protein is analyzed. Immunoprecipitation with anti-β4 integrin on nerve lysates of homozygous PMP22-deficient mice is shown as a negative control (C). Molecular mass is in kDa.

In myelinating peripheral nerves, β4 is known to interact with α6 integrin and laminin (Lee et al., 1992; Previtali et al., 2001); therefore, we investigated whether laminin could be detected in the immunoprecipitate (Fig. 2-6A, right). Probing with anti- laminin reveals that it coprecipitates with PMP22, whereas β-dystroglycan, another

38 laminin receptor in peripheral nerves (Scherer and Arroyo, 2002), does not (Fig. 2-6A, right). The two bands on the anti-laminin Western blot of the PMP22 immunoprecipitate may represent differential mobilities of the ~200 kDa β and γ chains that do not always resolve in the total or on the reprobes.

To substantiate the interaction of PMP22 with the integrin protein complex, the nerve lysates were incubated with anti-α6 integrin or anti-laminin antibodies (Fig. 2-6B).

Subsequent Western blot analyses of the immunoprecipitates identify PMP22 in the pull- down fractions. As the anti-α6 antibody does not work on blots of denatured samples, the

α6 precipitate was reprobed for β4, the binding partner of α6 in peripheral nerves (Fig. 2-

6B, left). Compared to the integrins and laminin, PMP22 is more abundant in whole nerve lysates, therefore when only 1 µg of total protein is analyzed (T lanes) PMP22 is readily detected, while laminin and β4 are barely visible on the reprobes (Fig. 2-6A-C, asterisks). However, laminin and β4 are prominent when 20 µg of whole nerve lysates are analyzed (Fig. 2-5G). As a control for the specificity of the coimmunoprecipitation experiments, lysates from PMP22 -/- mouse nerves were incubated with β4 integrin antibodies (Fig. 2-6C). In agreement with the reduced levels of β4 integrin in affected nerves, there is a faint enrichment in β4 in the pull-down fraction, yet PMP22 is not detected. Together, these studies indicate that a fraction of PMP22 is in complex with

α6β4 integrin and laminin in nerves of +/+ mice.

In myelinating peripheral nerves, the expression levels of PMP22 and α6β4 integrin substantially increase during the first two weeks of postnatal development

(Notterpek et al., 1999b; Previtali et al., 2001); however, the potential colocalization of these molecules has not been examined. To corroborate the interaction of PMP22 with

39 the integrin complex, nerve samples from +/+ mice and myelinating DRG neuron-SC cocultures were studied (Fig. 2-7). While the fixation conditions are not optimal for the codetection of the two antigens, on cross sections of sciatic nerves from 3-mo old mice, a portion of PMP22-like staining overlaps with β4 integrin (Fig. 2-7A). On the merged single plane confocal image, the yellow color indicates the colocalization of PMP22 with

β4 integrin at the abaxonal SC membrane (Fig. 2-7A, arrows). Normal rat or rabbit serum, used as controls of specificity, do not stain the nerve sections (Fig. 2-7A, insets).

Figure 2-7. Coexpression of PMP22 and integrins during myelination. Sciatic nerve cryosections from 3-mo old +/+ mice were labeled with monoclonal rat anti- β4 and polyclonal rabbit anti-PMP22 antibodies and examined by confocal microscopy (A). The merged single plane image reveals the partial colocalization (merge, yellow) of β4 integrin (green) and PMP22 (red). Scale bar, 5 µm. Nonspecific rat and rabbit sera (bottom insets) serve as controls for staining specificity. Whole protein lysates of P1, P3, P8 and P21 sciatic nerves (10 µg/lane) from +/+ mice were analyzed with anti-PMP22 and anti-β4 integrin antibodies (B). The arrows indicate the migration of β4 integrin at ~200 kDa (top) and of PMP22 at ~22 kDa (bottom), while the arrowhead marks a non-specific immunoreactive band. Cell surface biotinylation of myelinating DRG-SC cocultures identifies β4 integrin and PMP22 in the avidin pull-down (AP), from which actin is excluded (C). Total lysate (T) and agarose bead preclear (PC) fractions are also shown. Molecular mass in kDa.

Additionally, the steady-state levels of β4 integrin and PMP22 increase concomitantly during the first 3-weeks of postnatal development (Fig. 2-7B, arrows). The arrowhead on the PMP22 Western blot identifies a non-specific band that is reactive with our polyclonal antibody. As integrins are established cell surface receptors of SCs

(Previtali et al., 2001), the fraction of PMP22 that is in complex with the integrins should also be accessible to cell surface labeling. Therefore, myelinating DRG-SC cocultures were incubated with Sulfo-NHS-LC-biotin and subsequently precipitated with strepavidin

(Fig. 2-7C). β4 integrin, as expected, and PMP22 are detected in the pull-down fraction, which represents the cell surface pool of these molecules. Actin, a known intracellular protein, served as a negative control. Together, these studies indicate that PMP22 is coexpressed in time and locale with β4 integrin in myelinating nerves and DRG-SC cocultures.

Next, we asked whether PMP22 may form a complex with β4 integrin in nonneural samples as well (Fig. 2-8). A human colon adenocarcinoma cell line, termed clone A, is known to express high levels of α6β4 integrin, and is amenable to transfection (Dexter et al., 1979; Lotz et al., 1990). Clone A cells were transfected with epitope (myc)-tagged human PMP22 (hPMP-myc) and analyzed with anti-myc and anti-β4 integrin antibodies

(Fig. 2-8). In a cluster of transfected cells, hPMP-myc is targeted to the β4 integrin- positive cell membrane, resulting in a yellow color on the merged image (Fig. 2-8A-C, arrows). Non-transfected cells in the periphery of the image act as an internal control for the anti-myc antibody (Fig. 2-8B, C).

Figure 2-8. PMP22 and β4 integrin are coimmunoprecipitated from clone A cells. Epitope (myc)-tagged hPMP22 was expressed in clone A cells and samples were double immunolabeled with anti-β4 (A) and anti-myc (B) antibodies. As the merged image (C) reveals, hPMP22 is targeted to the β4 integrin-positive plasma membrane of the cells (A-C, arrows). Scale bar, 10 µm. Vector control and hPMP-myc expressing clone A cells were lysed and processed for immunoprecipitation (IP) with the indicated antibodies (D). The precipitates were subsequently probed for the marked proteins by Western blot. The arrows on the right indicate the overexpressed glycosylated PMP22 (~22 kDa) and the endogenous β4 integrin (~200 kDa). The arrowheads point at the position of the deglycosylated ~18 kDa and ~190 kDa forms. Molecular mass is in kDa.

In parallel, clone A cells transfected with either vector alone or hPMP-myc were lysed in immunoprecipitation buffer and incubated with the indicated antibodies (Fig. 2-

8D). When lysates of vector control cultures are immunoprecipitated with the β4 integrin antibody and blotted with an anti-myc, no bands are visible. In comparison, when the same experiment is performed in PMP-myc transfected cells, hPMP-myc is coimmunoprecipitated with β4 integrin (Fig. 2-8D, WB with myc, arrow). To confirm the identity of the myc-immunoreactive band as PMP22, one half of the immunoprecipitate was treated with PNGase F to remove the carbohydrate moiety of PMP22. As previously

42 described (Pareek et al., 1997), upon PNGase F treatment the ~22 kDa band shifts to 18 kDa, which represent the core PMP22 peptide (Fig. 2-8D, WB with myc, arrowhead).

The blots were reprobed with β4 integrin to show that β4 integrin is efficiently immunoprecipitated from both vector control and hPMP-myc expressing cultures. Upon

PNGase F treatment the mobility of β4 is also changes from about 200 kDa to 190 kDa

(Fig. 2-8D, WB with β4, arrow and arrowhead, respectively) (Sonnenberg et al., 1988).

These results indicate that PMP22 and β4 integrin are in a complex in a variety of cell types.

Discussion

Understanding the role of PMP22 in the cell membrane has been hindered by lack of knowledge on interacting proteins, particularly molecules with signaling potential. In the current study we characterized a novel PMP22-deficient mouse model that led us to identify PMP22 as a binding partner for the α6β4 integrin and laminin complex.

Analyses of heterozygous PMP22-deficient mice revealed broad tissue distribution of the

β-gal reporter and an HNPP-like neuropathic phenotype. In nerves of homozygous knockout mice we detected loose SC basal lamina and reduced levels of β4 integrin, suggesting a role for PMP22 in the stabilization of integrins in the cell membrane.

Indeed, coimmunoprecipitation experiments from nerves of +/+ mice and clone A human colonic carcinoma cells show that PMP22 is in a complex with α6β4 integrin and laminin.

The VelociGene technology employed in this study uses targeting vectors based on bacterial artificial chromosomes that can precisely replace the gene of interest with a reporter, allowing for high-resolution localization of target gene expression (Valenzuela

et al., 2003). The β-gal reporter studies described here confirm and extend previous studies on the expansive tissue distribution of PMP22 mRNA during embryonic development and in the adult rodent (Baechner et al., 1995; Parmantier et al., 1995;

1997). The detection of β-gal in the aortic arch of the heart (Fig. 2-2D) agrees with our findings on the expression of PMP22 in a variety of vascular tissue (Notterpek et al.,

2001; Roux et al., 2004). Other nonneural tissues that show robust reporter expression

include the lungs, intestines and cartilage. The function of PMP22 at these locations is

not yet known, although frequent amplification and overexpression of PMP22 in high-

grade osteosarcoma has recently been reported (Huhne et al., 1999; van Dartel and

Hulsebos, 2004). In contrast, the levels of PMP22 transcripts were reduced by about 20-

fold in urethane-induced mouse lung tumors (Re et al., 1992). These reports indicate that

PMP22 may play crucial roles in a variety of tissues, likely regulating cell proliferation

and differentiation. Nonetheless, while homozygous PMP22 -/- consistently show

postnatal growth retardation, the gross pathological alterations are limited to myelinated

peripheral nerves. These data may indicate a particular sensitivity of SCs to PMP22

expression and/or the existence of compensatory mechanisms in other tissues.

Phenotypically, PMP22 -/- mice display a severe neuropathy that is apparent by the

third week after birth. The peripheral nerves contain an excess number of SCs, and show

delayed myelination, as well as hypermyelination of individual axons (Figs. 2-3 and 2-4).

These abnormalities are in agreement with data from young PMP22-deficient (Adlkofer

et al., 1995) and PMP22 overexpressor mice (Magyar et al., 1996; Rogablia-Schlupp et

al., 2002). In nerves of PMP22-deficient and overproducer mice, prominent pockets of

basal laminae surrounding hypomyelinated axons were also described (Adlkofer et al.,

1997a; Magyar et al., 1996), but the molecular mechanisms underlying these changes were not addressed. Similarly, inaccurate SC-axon interactions and redundant basal lamina were noted in Trembler J mice (Robertson et al., 1997; 1999), which carry a

Leu16Pro mutation in the first transmembrane domain of PMP22 (Suter et al., 1992). The described abnormalities in the nerves of -/- mice (Figs. 2-4 and 2-5), together with our findings in an epithelial wound healing model (Roux et al., 2005) suggest a direct role for

PMP22 in mediating the interactions between cells and the ECM. Cell-matrix interactions can influence cell shape, which is profoundly modulated by PMP22 (Brancolini et al.,

1999; Roux et al., 2005). In fibroblasts and SCs, the overexpression of PMP22 induces membrane blebbing and regulates cell spreading (Brancolini et al., 2000), while in

MDCK epithelial monolayers it leads to a flattened morphology (Roux et al., 2005).

Changes in cells shape, as well as actin remodeling, are prerequisites for myelinogenesis in the PNS (Bunge et al., 1989; Fernandez-Valle et al., 1997). Therefore, based on the described biological effects of PMP22, it is expected that in its absence, SC differentiation and myelination are delayed (Fig. 2-4).

Laminins are major components of the basal lamina and known mediators of SC differentiation, axon myelination and regeneration in the PNS (Chen and Strickland,

2003). In agreement, the ablation of specific laminin chains alters SC proliferation, which in turn prevents the defasciculation and myelination of peripheral nerves (Colognato et al., 2005). In addition to integrins, laminins bind a variety of adhesive molecules that carry the L2/HNK1 carbohydrate adhesion recognition epitope (Schmidt and Schachner,

1998). PMP22 contains this epitope (Snipes et al., 1993) therefore it may directly bind laminin. The glycosylation consensus sequence at asparagine 41, which bears this

45 epitope, is conserved among members of the PMP22 gene family and across species as distant as zebrafish (Taylor et al., 1995; Wulf et al., 1999). This carbohydrate modification of PMP22 is involved in the stabilization of PMP22 homodimers (Tobler et al., 1999) and in the cell spreading effects (Brancolini et al., 1999; 2000), but not in the trafficking of the protein to the SC membrane (Ryan et al., 2000), or in the interaction with P0 (Hasse et al., 2004). The L2/HNK-1 epitope can also modulate the heterophilic interaction of some nervous system molecules, including α1β1 integrin and laminin

(Pesheva et al., 1987; Lallier and Bronner-Fraser, 1992). Further studies will determine if

PMP22 has the ability to directly bind laminin and if this interaction depends on its carbohydrate moiety.

Similar to laminins, abnormal expression of integrins is associated with degenerative, inflammatory and malignant disorders of the PNS, suggesting a key role for these molecules in nerve biology (Previtali et al., 2001). The α6β1 integrin dimer is prominent during the promyelinating stage and its expression influences the migration of mouse SCs in vitro (Milner et al., 1997). The conditional disruption of β1 integrin in SCs is associated with dysmyelination, loose, undulating basal lamina and aberrant SC morphology (Feltri et al., 2002). The observed defects in the basal lamina of PMP22 -/- nerves are comparable to those seen in this β1 integrin-deficient model (Feltri et al.,

2002). During early postnatal development, the α6β4 dimer becomes prevalent, and is thought to be involved in myelinogenesis (Previtali et al., 2001). Since mice lacking β4 integrin die shortly after birth, developmental studies of myelination are not feasible.

However, DRG neuron cultures established from these mice can be induced to form myelin and have a continuous basal lamina, suggesting that β4 integrin is not essential for

46 myelination (Frei et al., 1999). As α6 integrin can dimerize with β1, in the absence of β4, an α6β1 dimer may compensate for the α6β4 complex. Alternatively, β4 integrin may have effects on myelination later in development that cannot be evaluated in the currently available β4 -/- mice. In a recent report, α7β1 integrin was shown to be dispensable for peripheral nerve development and myelination (Previtali et al., 2003b). Together, these findings indicate a redundancy in the integrin pathways and suggest that under certain conditions they may compensate for one another.

The recognition of PMP22 as a binding partner for the integrin/laminin complex is not unexpected. Previous studies have identified three PMP22-like tetraspan proteins, oligodendrocyte specific protein (OSP), epithelial membrane protein 2 (EMP-2) and proteolipid protein (PLP) that interact with integrins. OSP and OSP-associated protein form a complex with β1 integrin and regulate the migration of oligodendrocytes (Tiwari-

Woodruff et al., 2001). EMP-2, a PMP22 family member, interacts with α6β1 integrin and mediates the adhesion of 3T3 fibroblasts to ECM proteins (Wadehra et al., 2002).

PLP, a constituent of CNS myelin, forms a complex with αv integrin, which appears to be important for the binding of fibronectin to oligodendrocytes (Gudz et al., 2002). The interaction with the integrins provides PMP22 with the ability to modulate the cell-ECM communications, as well as intracellular events. Signaling between the ECM and the intracellular compartment is essential for SC myelination, as well as cellular differentiation and motility, in general. Independently, both integrins and PMP22 have been shown to modulate these physiologic functions (Murgia et al., 1998; Rabinovitz and

Mercurio, 1997; Danen and Sonnenberg, 2003; Roux et al., 2005); however, in the case of PMP22, the underlying molecular events are yet to be elucidated.

The results described here place PMP22 in a complex with α6β4 integrin and laminin in SCs and in a colonic carcinoma cell line. The identification of PMP22 as a binding partner for an integrin signaling complex provides a major step towards understanding the role of this disease-linked molecule in the nervous system and in nonneural cell types.

CHAPTER 3 DELAYED MYELINATION AND ALTERED NODAL ORGANIZATION IN PERIPHERAL MYELIN PROTEIN 22-DEFICIENT MICE

Introduction

The process of myelination in the peripheral nervous system (PNS) begins at birth and is completed by postnatal day (P) 28 of murine development. However, the preparations for myelination commence soon after neural tube formation (Jessen and

Mirsky, 2005). A subset of neural crest cells gives rise to the dorsal root ganglion (DRG) sensory neurons and glia. SC precursors are the initial glial cells (generated at embryonic day (E) 12-13 in mice) that provide essential trophic support to neurons. They progress to immature SCs (E13-16), which defasciculate the axons. Neural crest cells, SC precursors and immature SCs all proliferate rapidly until they begin to differentiate, at which point the SCs must exit the cell cycle (Jessen and Mirsky, 2005).

Depending on the size of the axons that the immature SCs randomly associate with, they will evolve into nonmyelinating or myelinating SCs. A single nonmyelinating SC will ensheath multiple axons in troughs on its surface. In contrast, the immature SCs that come in contact with large caliber axons (>1 µm in diameter) progress through a promyelinating stage to become myelinating SCs (Jessen and Mirsky, 1999). Myelinated fibers are organized into distinct domains, including internodes, juxtaparanodes, paranodes and nodes. This composition is necessary for the proper conduction of nerve impulses along an axon and results from associations between SCs and axons, which are poorly understood at the present time (Peles and Salzer, 2000).

48 49

The stages of myelination are marked by the modulation of certain proteins expressed throughout this time. The p75 low affinity neurotrophin receptor (p75) is a marker for immature and nonmyelinating Schwann cells (Jessen and Mirsky, 2005).

When a SC commits to myelination, p75 levels decrease, while the myelinating proteins increase exponentially. Myelin associated glycoprotein (MAG) is one of the first markers of myelinating SCs (Martini and Schachner, 1986). MAG is expressed in non-compact myelin and is postulated to have a role in transmitting signals from SCs to axons that are necessary to stabilize myelin (Garbay et al., 2000).

Myelin protein zero (P0), myelin basic protein (MBP) and peripheral myelin protein 22 (PMP22) are upregulated as myelination progresses. While these proteins are typically thought of as compact myelin proteins, PMP22 and P0 also are expressed at low levels in immature and nonmyelinating SCs (Jessen and Mirsky, 2005). P0 is a single transmembrane spanning glycoprotein that represents 50-70% of total myelin protein

(Garbay et al., 2000). Its structure suggests that it is necessary for myelin compaction both extracellularly, binding homophilically via its extracellular domains (Shapiro et al.,

1996), and intracellularly, where it most likely interacts with MBP in the formation of the major dense line (Garbay et al., 2000). MBP, the second most abundant protein in PNS myelin (5-15%), is actually a family of proteins encoded by one gene. Alternative splicing results in the expression of four major proteins, with molecular masses of 14, 17,

18.5 and 21 kDa. In adult rodents, the 14 and 18.5 kDa bands are the most prominent

(Quarles et al., 2006). The MBPs participate in myelin sheath compaction and maintenance (Martini et al., 1995). PMP22, as discussed in earlier chapters, is a four transmembrane spanning glycoprotein that makes up 2-5% of peripheral myelin protein,

50 with proposed roles in myelin formation and maintenance (Suter and Scherer, 2003). It may achieve a part of its function by interacting with P0 to stabilize compact myelin

(D’Urso et al., 1999; Hasse et al., 2004). However, PMP22 also is expressed in nonneural tissues (Baechner et al., 1995; Notterpek et al., 2001), indicating it has another function uncoupled to its structural role in myelin, potentially in SC differentiation or membrane expansion.

In addition to myelin proteins, specific integrin heterodimers, including α6β1 and

α6β4, also are modulated during PNS development. β1 integrin is expressed in all stages of SC development, but its levels decrease with myelination (Feltri et al., 2002). β1 integrin has proposed roles in axonal segregation and myelin initiation (Fernandez-Valle et al., 1994; Feltri et al., 2002). Conversely, another integrin subunit, β4, increases concomitantly with myelination (Feltri et al., 1994), having a pattern similar to PMP22

(Notterpek et al., 1999b). The function of β4 integrin in myelin is not fully understood because β4 integrin-null mice die shortly after birth, as a result of severe epidermolysis bullosa with detachment of the epidermis and esophageal epithelia (van der Neut et al.,

1996). Since myelination progresses normally in DRG explant cultures collected from

E14 null animals (Frei et al., 1999) and β4 integrin mRNA levels continue to increase throughout development (Feltri et al., 1994), β4 is postulated to have a role in myelin stability (Feltri and Wrabetz, 2005).

Previous studies suggest PMP22 is important during myelin initiation (Adlkofer et al., 1995; Robaglia-Schlupp et al., 2002). To examine the early nerve development in the absence of PMP22, the quantity and the quality of the myelin constituents were evaluated. The absence of PMP22 leads to a delay in the expression of myelin proteins,

51 both in vivo and in vitro. Additionally, the organization of certain proteins, including β4 integrin and the potassium channel, Kv1.1, are altered at the nodal regions in affected nerves.

Materials and Methods

Myelinating DRG Explant Cultures

Mouse DRG explant cultures were established as described (Cosgaya et al., 2002).

Pregnant PMP22 heterozygote females were sacrificed according to University of Florida

Institutional Animal Care and Use Committee guidelines to obtain wild type (+/+), heterozygous PMP22-deficient (+/-) and homozygous PMP22-deficient (-/-) cultures.

DRG neurons were collected from E12-14 mice, digested with 0.25% trypsin (Gibco,

Rockville, MD), dissociated and plated on rat-tail collagen-coated (Biomedical

Technologies, Inc., Stoughton, MA) glass coverslips. DNA was isolated from each embryo for genotyping by Southern blot analyses (See Ch. 2 for detailed protocol).

Explants were maintained in Minimum Essential Medium (MEM) (Gibco) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 0.3% glucose (Sigma-Aldrich, St.

Louis, MO), 10 mM HEPES (Cellgro, Mediatech, Inc., Herndon, VA) and 100 ng/ml nerve growth factor (Harlan Bioproducts for Science, Madison, WI) for 7 days. Ascorbic acid (50 µg/ml) (Sigma-Aldrich) was then added for 7-11 days, and the cultures were processed for immunolabeling studies.

Teased Nerve Fiber Preparation

Teased nerve fibers from the sciatic nerves of P21 +/+ and PMP22 -/- mice were prepared as described by Devaux and Scherer (2005). Briefly, nerves were fixed in 4% paraformaldehyde/PBS, pH 7.4 for 30 min at RT. The epineurium was removed and the

nerves were teased into single fibers. The fibers were dried on glass slides overnight at

RT and processed for immunohistochemistry or stored at -20°C.

Immunolabeling of Nerves and Myelinating DRG Explant Cultures

Sciatic nerves were dissected from genotyped P11 +/+ and PMP22 -/- littermates.

Samples were frozen by immersion in liquid nitrogen-cooled N-methyl butane. Frozen

sections (5 µm thickness) were dried for 1 hr on Superfrost/Plus microslides (Fisher,

Pittsburgh, PA), followed by fixation with 4% paraformaldehyde in PBS for 10 min at

RT, and permeabilization for 2 min with 100% acetone chilled to -20°C (Melendez-

Vasquez et al., 2004). DRG explant cultures were fixed in 4% paraformaldehyde/MEM

for 10 min at RT and permeabilized with 100% methanol for 10 min at -20°C (Einheber

et al., 1993). Both nerve sections and DRG samples were blocked in PBS containing 20%

normal goat serum (NGS). Teased nerve fibers were permeabilized by immersion in

acetone for 10 min at -20°C and blocked with 5% NGS containing 0.1% Triton X-100 in

PBS (Devaux and Scherer, 2005). All samples were blocked for 1 hr at RT.

Primary antibodies, including polyclonal rabbit anti-laminin, 1:10,000 (Sigma-

Aldrich); anti-phosphorylated myosin light chain, 1:500 (Cell Signaling Technologies,

Danvers, MA); chicken anti-neurofilament (a kind gift from Dr. G. Shaw, University of

Florida, Gainesville, FL); and monoclonal rat anti-β4 integrin, 1:4000 (BD Biosciences

PharMingen, San Diego, CA); and anti-myelin basic protein, 1:500 (Chemicon,

Temecula, CA) antibodies were added in the blocking solution overnight at 4°C. The

samples were then incubated with Alexa Fluor 594 goat anti-rabbit IgG (red) and/or

Alexa Fluor 488 goat anti-rat IgG (green) (both from Molecular Probes, Inc., Eugene,

OR) for 1-2 hr. Hoechst dye (Molecular Probes, Inc.) was included in the secondary

antibody solution at 10 µg/ml to visualize nuclei. Coverslips were mounted by using the

ProLong Antifade kit (Molecular Probes, Inc.). Samples were imaged with a Spot camera

attached to a Nikon Eclipse E800 microscope or an Olympus Optical (Tokyo, Japan)

MRC-1024 confocal microscope and were formatted for printing by using Adobe

PHOTOSHOP 5.5.

Western Blot Analyses

Sciatic nerves collected from genotyped P4, P11, P23 and 3-month old mice were

frozen immediately in liquid nitrogen. For total protein analyses, nerves from three to four mice were crushed under liquid nitrogen, and tissue lysates were prepared in SDS gel

sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 3% SDS) supplemented with

complete protease inhibitor (Roche, Indianapolis, IN) and 500 µM phenylmethylsulfonyl

fluoride (Sigma-Aldrich). Protein concentrations were determined using the BCA

reagents (Pierce Chemicals, Rockford, IL). Protein samples (10 µg/lane) were separated

on 7.5% or 12.5% SDS-PAGE under reducing or non-reducing conditions, and proteins

were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA).

Polyclonal rabbit anti-β4 integrin, 1:500; anti-p75, 1:1000; and monoclonal rat anti-β1

integrin (non-reduced), 1:500; and anti-MBP, 1:1000 (all from Chemicon, Temecula,

CA); monoclonal mouse anti-alpha-tubulin, 1:5000 (Sigma-Aldrich); anti-GAPDH (a

kind gift from Dr. Gerry Shaw, University of Florida, Gainesville, FL); and anti-P0 (Dr.

J. Archelos, Karl-Franzens University, Graz, Austria) antibodies were used. Bound

antibodies were visualized using an enhanced chemiluminescence kit (PerkinElmer Life

Sciences, Boston, MA). Films were digitally imaged using a GS-710 densitometer (Bio-

Rad) and were formatted for printing by using Adobe PHOTOSHOP 5.5.

Statistics

Myelinated DRG explant cultures were maintained for 7 or 11 days in ascorbate

and immunostained with an anti-MBP antibody to label myelin internodal segments. The

internode lengths from +/+, +/- and -/- cultures were measured with SPOT RT software

(Diagnostic Instruments, Inc., Sterling Heights, MI). Measurements were collected from

three coverslips per genotype at each time point. Significance was determined using an

unpaired two-tailed Student’s t-test.

Results

Altered Myelin Protein Expression and Localization in PMP22 -/- Mice

PMP22 is implicated in the initiation of myelination during murine development

(Adlkofer et al., 1995; Notterpek et al., 1997; Robaglia-Schlupp et al., 2002; Amici et al.,

2006). To examine the temporo-spatial expression of myelin proteins during postnatal growth, whole sciatic nerve lysates from +/+, +/- and -/- mice were analyzed at P4, P11,

P23 and 3-months (Fig. 3-1). As previously described (Notterpek et al., 1999b), the expression of structural myelin components, such as P0 and MBP, begins by P4 in +/+ nerves (Fig. 3-1A). The abundance of P0 increases in +/+ and +/- samples as myelination progresses, continuing through 3-months of age. In addition to the 30 kDa band indicative of the P0 glycoprotein, a band also is present at 28 kDa in the 3-month +/+ and

+/- lysates, which may represent the unglycosylated form of the protein. The levels of P0 in the nerves from +/- mice do not appear noticeably different from +/+ samples.

However, P0 is reduced in the nerves of PMP22 -/- mice at P4, P11 and 3-months (Fig. 3-

1A). Additionally, the unglycosylated form of P0 is not present at the 3-month time point, suggesting a reduction in the synthesis of the core protein (Fig. 3-1A). At P23, P0

55 expression is similar to +/+ levels, which may be due to the prominence of tomacula

(focal hypermyelination) in the nerves from PMP22 -/- mice at this age (Fig. 2-3F).

Figure 3-1. The expression of myelin proteins is delayed in nerves from PMP22-deficient mice. Sciatic nerve lysates (10 μg/lane) from +/+, +/- and -/- mice (3-4 mice per genotype, ages P4, P11, P23 and 3-months) were analyzed by Western blots (A-B). The blots were probed with monoclonal mouse anti-P0 and rat anti-MBP (A) and polyclonal rabbit anti-p75 (B) antibodies. The reprobed blots were incubated with monoclonal mouse anti-α-tubulin antibody as a loading control (A, B). Molecular mass in kDa.

The temporal expression pattern of MBP is similar to P0 in the nerves of +/+ mice.

At P4, low levels of the 14 and 18.5 kDa forms of MBP are detected in +/+ samples (Fig.

3-1A, +/+), which is the expected pattern for rodent nerve development (Quarles et al.,

2006). By P11, these MBP isoforms are strongly expressed in addition to the 17 kDa protein. While the 21 kDa MBP is present in low abundance by P23 (Fig. 3-1A, +/+). The nerves from +/- mice have a comparable pattern of expression to wild type (Fig. 3-1A,

+/-). However, at P4 in the -/- samples, no MBP isoforms can be detected (Fig. 3-1A. -/-), even on longer exposures (data not shown). The 14 and 18.5 kDa MBPs are the only

56 isoforms expressed at P11. A fraction of the 21 kDa form is not present until P23 (Fig. 3-

1A. -/-). As with P0, the 18.5 and 14 kDa proteins do not exhibit noticeable decreases at

P23 compared to +/+ samples.

The p75 low affinity neurotrophin receptor, a protein expressed in immature and nonmyelinating SCs, declines with the differentiation of SCs into a myelinating phenotype (Jessen and Mirsky, 2005). The nerves from +/+ and +/- mice both adhere to this expected progression. Conversely, p75 remains elevated at all ages studies in PMP22

-/- samples, potentially indicating a reduction in the number of SCs differentiating into myelinating SCs (Fig. 3-1B). Alpha tubulin is shown as a protein loading control (Fig. 3-

1A-B).

To complement biochemistry, nerves from P11 and adult animals were processed for immunohistochemistry and analyzed with anti-MBP and anti-p75 antibodies. At P11, the majority of large caliber axons in nerves from +/+ animals (Fig. 3-2A, red, anti- neurofilament antibody) are surrounded by MBP immunolabeling (Fig. 3-2A, green), characteristic of ongoing myelination. In contrast, there is a reduction in the number of

MBP-positive fibers in affected nerves (Fig. 3-2B) and abundant p75 staining (data not shown). This is in agreement with the findings shown by electron microscopy in Chapter

2. The number of nuclei in the nerves from PMP22 -/- mice is increased by ~1.5-fold at

P11, which was quantified in Chapter 2 and agrees with earlier studies in a different

PMP22-deficient line (Sancho et al., 2001). To examine demyelination, longitudinal sciatic nerve sections from 3-month old animals were costained with anti-MBP antibody and Hoechst dye (Fig. 3-2C-D). Wild type samples depict normal myelination, with MBP labeling compact myelin (Fig. 3-2C, green, arrows) surrounding the axons. Nerves from

-/- animals appear less structured, with increased space between the individual fibers

(Fig. 3-2D, arrowheads) and fewer properly myelinated axons (Fig. 3-2D, arrows).

Additionally, the MBP-like staining intensity is reduced and there is an increased amount of nuclei in the affected samples (Fig. 3-2D).

Figure 3-2. Decreased MBP and increased p75 immunostaining in affected nerves. Cross- sections of cryopreserved sciatic nerves from P11 +/+ (A) and -/- (B) littermates are labeled with a monoclonal rat anti-MBP antibody to indicate myelin (A-B, green). Sections are coimmunostained with polyclonal chicken anti-neurofilament light chain antibody (red) to visualize axons. Hoechst dye (blue) denotes nuclei (A-F). Scale bar, A-B, 10 μm. Sciatic nerves from adult +/+ (C, E) and -/- (D, F) animals were longitudinally sectioned and immunostained with monoclonal rat anti-MBP (green, C-D) or polyclonal rabbit anti-p75 (red, E-F) antibodies. Arrows indicate myelin. Arrowheads denote extracellular spaces. Asterisks mark nonmyelinating SC bundles. Open arrowheads designate thin nonmyelinating SC processes that do not appear to be included within bundles. Scale bar, C-F, 10 μm.

The distribution of nonmyelinating SCs in sciatic nerves from 3-month +/+ and -/- mice was evaluated by the immunolabeling the tissues with anti-p75 antibody and

Hoechst (Fig. 3-2E-F). In normal nerves, nonmyelinating SCs, as indicated by anti-p75 reactivity, are localized to distinct bundles, encompassing small caliber axons (Fig. 3-2E, asterisk). However, in nerves from affected animals, p75 immunostaining still labels bundles (Fig. 3-2F, asterisk), but nonmyelinating SCs also intrude throughout the entire nerve (Fig. 3-2F, open arrowheads). This suggests that, either SCs are dedifferentiating due to demyelination, followed by subsequent attempts to remyelinate and repair the nerve fibers (Stoll and Muller, 1999), or that the p75-positive SCs have not differentiated from an immature state (Jessen and Mirsky, 2005).

Changes in Integrin Expression Patterns in the Absence of PMP22

Several integrin heterodimers are involved with PNS myelination. During embryonic development, α6β1 integrin is robustly expressed on SC precursors and immature SCs. As myelination progresses, α6β1 levels decline and α6β4 becomes the prominent integrin heterodimer in peripheral nerves (Previtali et al., 2001). In agreement with these studies, β1 integrin diminishes with age in the +/+ lysates (Fig. 3-3, +/+, A). In contrast, the amount of β1 integrin remains elevated in the nerves from affected mice at

3-months when compared with +/+ and +/- counterparts (Fig. 3-3A). As expected, β4 integrin is upregulated with myelination in both +/+ and +/- samples (Fig. 3-3B).

However, when PMP22 is absent, β4 integrin expression is dramatically reduced, never reaching +/+ or +/- levels. As α6 integrin is known to dimerize with both the β1 and β4 integrin subunits, the sustained expression of β1 integrin in the P23 and 3-month -/-

samples may compensate for the reduced levels of β4 integrin at these time points.

GAPDH is shown as a loading control (Fig. 3-3A-B).

Figure 3-3. Altered integrin expression levels in nerves from PMP22-deficient mice. Whole sciatic nerve lysates (10 μg/lane) from +/+, +/- and -/- mice (ages P4, P11, P23 and 3-months) were analyzed on non-reduced (A) and reduced (B) Western blots. The blots were probed with monoclonal rat anti-β1 integrin (A), and polyclonal rabbit anti-β4 integrin (B) antibodies. Monoclonal mouse anti-GAPDH antibody served as a protein loading control (A, B). Molecular mass in kDa.

Teased nerve fiber analyses of P21 sciatic nerves from +/+ and PMP22 -/- mice confirm the altered localization of β4 integrin at the abaxonal SC membrane (Amici et al., 2006). In +/+ nerves, anti-β4 integrin antibody localizes to the Schwann cell outer membrane (Figs. 2-5A; 3-4A) and displays slightly stronger staining at the nodes of

Ranvier (Fig. 3-4A, asterisk), in agreement with previous studies (Feltri and Wrabetz,

2005). In affected nerves, β4 integrin exhibits focally intense immunostaining at the nodes of Ranvier (Fig. 3-4A, asterisk), with a notably weaker signal along the internodes.

Figure 3-4. Altered protein localization in teased nerve fibers of PMP22-deficient mice. Teased nerve fibers from P21 +/+ and -/- littermates (A-C) were stained with monoclonal rat anti-β4 integrin (A), polyclonal rabbit anti-Kv 1.1 (B) and monoclonal rat anti-E-cadherin (C) antibodies. Hoechst dye labels nuclei (C). Asterisks denote nodes of Ranvier (A-C). T indicates tomacula (A-B). Arrowheads point to Schmidt-Lantermann incisures (C). Arrows mark punctate e-cadherin staining (C). Scale bar, 10 μm (A-C).

As proper SC-axon interactions are necessary for the efficient conduction of nerve impulses, proteins at the nodes of Ranvier were examined (Peles and Salzer, 2000).

Sodium channels are localized properly to the nodes in +/+ and -/- mice (data not shown).

The voltage-gated potassium channel antibody, anti-Kv1.1, labels the juxtaparanodal region of the axon between the internodes and paranodes in the fibers from +/+ mice

(Peles and Salzer, 2000; Fig. 3-4B). While Kv1.1 is present in the affected nerves, it is not localized correctly. The juxtaparanodal region is farther within the internode, due to a paranodal tomacula in the affected fiber (Fig. 3-4B, T). This alters the nodal spacing and agrees with previous studies from another PMP22-deficient mouse line (Neuberg et al.,

1999). The proper compartmentalization of non-compact myelin proteins also is different

in PMP22 -/- samples. E-cadherin is an adherens junction protein that is believed to

stabilize proper myelin formation (Fannon et al., 1995; Hasegawa et al., 1996). In normal

nerve fibers, e-cadherin is localized to the Schmidt-Lantermann incisures and paranodes

(Fig. 3-4C, arrowheads). However, it is distributed in punctate patches along the fibers

from -/- nerves (Fig. 3-4C, arrows), suggesting myelin instability in these nerves.

Myelination Is Impaired in DRG Explant Cultures from PMP22 -/- Mice

To specifically study the delay in the induction of myelination observed in PMP22

-/- mice, an in vitro DRG neuron explant culture model was utilized (Wood et al., 1990).

As opposed to the 1-2 week period of myelin initiation that occurs in vivo (Jessen and

Mirsky, 1999), this system provides a way to synchronize the entrance of SCs into the myelin program. Therefore, this method can accurately measure the delays in SC wrapping and internode elongation. To induce SC myelination of the DRG axons, cultures were treated with ascorbic acid. This facilitates SC basal lamina formation, an essential step in PNS myelination (Eldridge et al., 1987). After the cultures were treated with ascorbic acid for seven or eleven days, they were fixed and immunolabeled with anti-MBP antibody to visualize myelin internodes (Fig. 3-5A-C, green). Cultures were costained with phosphorylated-myosin light chain (MLC-P) to label the nodes of Ranvier

(Fig. 3-5A-C, red) and Hoechst dye to denote nuclei (Fig. 3-5A-C, blue). At seven days post ascorbic acid addition, cultures from +/+ mice contain abundant myelin, as do +/- cultures (Fig. 3-5A-B). However, MBP-positive myelinated segments are rare in -/- cultures (Fig. 3-5C).

Figure 3-5. Shortened myelin internodes in DRG neuron explant cultures from affected mice. DRG explant cultures isolated from +/+, +/- and -/- embryos were stained with monoclonal rat anti-MBP (green) and polyclonal rabbit anti- MLC-P (red) antibodies and examined by confocal microscopy (A-C). Cultures were labeled with Hoechst dye to visualize nuclei. Scale bar, 40 μm. The lengths of the myelin internodes from +/+, +/- and -/- cultures treated for 7 days (D7) or 11 days (D11) with ascorbic acid were measured using Spot Advanced software (in μm) (D).

The amount of myelin in the cultures appears comparable between the +/+ and +/- explants, but the internode lengths in the +/- cultures are significantly shortened compared to control samples (Fig. 3-5A-B). Subsequent quantification of internode length at this time reveals that internodes from +/- cultures are approximately half as long as internodes from +/+ cultures (68.8±11.4 μm and 149.5±10.6 μm, respectively) (Fig. 3-

5D, D7). Cultures from PMP22 -/- mice also have shortened myelin segments, with an average internode length of 14.6±3.2 μm, approximately one tenth the length of +/+

cultures. By day 11 with ascorbic acid treatment, internodes from wild type (140.1±7.3

μm) and heterozygous (81.5±6.3 μm) cultures maintain lengths measured at day 7 with no significant changes, however the +/- show a trend toward elongation. In -/- cultures, internodes extend to 32.5±4.1 μm (Fig. 3-5D, D11), approximately doubling in length from day 7. Potentially, the internodes from the PMP22 +/- and -/- cultures will reach the same length as their +/+ counterparts if allowed a long enough period of time to myelinate.

Figure 3-6. Dysmorphic myelin from DRG explant cultures of PMP22 -/- mice. DRG neuron explant cultures from +/+ and -/- mice are stained with monoclonal rat anti-MBP (green) and polyclonal rabbit anti-MLC-P (red, asterisks) antibodies (A-B) and visualized by confocal microscopy. Cultures are labeled with Hoechst dye to denote nuclei. Scale bar, 8 μm. The insets in A and B are single plane, confocal images (2X). Arrows indicate compact myelin internodes. Arrowheads signify altered myelin architecture in -/- cultures. Asterisks specify nodal MLC-P localization.

Upon higher power examination of the +/+ DRG explant cultures, the SCs form tightly compacted myelin internodes, indicated by the MBP-positive fibers (Fig. 3-6A,

green). The intense lines of myelin staining (Fig. 3-6A, arrows) surround a dimmer line,

denoting the axon. However, the myelin architecture is altered in the -/- cultures (Fig. 3-

6B, arrowheads). Single plane confocal images reveal tightly compacted myelin

internodes in +/+ cultures (Fig. 3-6, inset in A, arrow), but loose, dysmorphic myelin in

-/- cultures (Fig. 3-6, inset in B, arrowhead).

SC processes must extend along the length of the axon while simultaneously

wrapping around it, a process that requires precise communication between the axon and

the SC. MLC-P is upregulated prior to myelination in rat sciatic nerves, and its levels

sharply decline as the nerves initiate myelination (Melendez-Vasquez et al., 2004). MLC

is phosphorylated by Rho kinase (Amano et al., 1996). Previous studies connected

cellular alterations in morphology with RhoA in PMP22 overexpression models

(Brancolini et al., 1999; Roux et al., 2005). Therefore, DRG cultures were labeled with

MLC-P to assess the changes of it during myelination. MLC-P staining is detected at

nodal areas in +/+ cultures (Fig. 3-6A, red, asterisks), but it is undetectable in -/- cultures

(Fig. 3-6B), suggesting that there may be altered axo-glial interactions in PMP22 -/-

cultures.

Discussion

PMP22 misexpression is associated with a family of demyelinating neuropathies

(Suter and Scherer, 2003). In addition to its role in compact myelin stability, there is abundant evidence that PMP22 has a function in myelinogenesis (Adlkofer et al., 1995;

Notterpek et al., 1997; Robertson et al., 1997; Robaglia-Schlupp et al., 2002; Amici et al.,

2006). In this chapter, internodal and nodal proteins were characterized during active myelination in the absence of PMP22. The normal localization of these proteins was disturbed in PMP22 -/- animals, as evidenced by both in vivo and in vitro methods.

Additionally, myelin internodal lengths were shortened in DRG explants from +/- and

PMP22 -/- mice.

The decreased levels of myelin proteins at P4 and P11 in the absence of PMP22 are indicative of delayed myelination; however, at P23 overall myelin protein levels are comparable between the three genotypes. This suggests that myelination recovers over time in the affected nerves, yet, by electron microscopy, ~40% of the fibers are still promyelinated at P18 (Chapter 2, data not shown), and p75 levels remain elevated (Fig.

3-1). However, nerves from PMP22 -/- mice have many fibers with tomacula at this age

(Adlkofer et al., 1995; Amici et al., 2006), which may be responsible for the increased amounts of myelin proteins in the nerve lysates from the PMP22 -/- mice. These hypermyelinated fibers subsequently become unstable and demyelinate as the animals age, leading to the diminished amount of myelinated fibers observed by immunostaining and biochemistry in the nerves of older PMP22 -/- mice.

As p75 is normally downregulated at the start of myelination (Jessen and Mirsky,

2005), the persistently elevated p75 expression in the absence of PMP22 implies that the

SCs do not undergo differentiation in a timely manner, in agreement with the EM studies from Chapter 2. Additionally, the increased p75 levels at the 3-month time point in

PMP22 -/- samples may indicate a reversion of the myelinating SCs to an earlier immature phenotype (Jessen and Mirsky, 2005). Nerve injury models of Wallerian degeneration (Stoll and Muller, 1999), in which SCs dedifferentiate, proliferate and then attempt to remyelinate the demyelinated nerves, support this idea. Alternatively, some

SCs may remain in the immature state, never entering into the myelin program (Jessen and Mirsky, 2005).

As myelination progresses in normal nerves, β4 integrin is robustly expressed

(Previtali et al., 2001). However, in PMP22-deficient mice, β4 integrin levels fail to rise after P11, and the protein is localized focally to the nodal/paranodal regions of nerve fibers. β1 integrin levels remain elevated, possibly to compensate for the lower β4 integrin levels in the -/- nerves. As β4 integrin heterodimerizes with α6 integrin (Hynes,

1992; Previtali et al., 2001), when β4 is decreased, α6 may be free to partner with β1 integrin. There seems to be functional redundancy between the integrin heterodimers present in peripheral nerves (Previtali et al., 2001). For example, conditional β1 integrin- null animals, in which the protein is specifically absent from their SCs, still have some fibers that undergo myelination (Feltri et al., 2002).

PMP22 -/- mice display altered potassium channel (Kv1.1) localization by teased nerve fiber analysis. Kv1.1 is restricted to the juxtaparanodal region, a specialized portion of the internode, just adjacent to the innermost paranodal loop (Peles and Salzer, 2000).

In the affected nerves, the axo-glial interactions may be perturbed, due to paranodal tomacula formation. Additionally, e-cadherin, a non-compact myelin protein, should be confined to the Schmidt-Lantermann incisures and paranodes (Fannon et al., 1995), but is present in punctate patches along the entire internode. This suggests the myelin of

PMP22 -/- animals is intrinsically unstable, regardless of tomacula formation.

In the absence of PMP22, the signal for myelin initiation is not induced in the vast majority of SCs associated with DRG axons, and DRG neuron explant cultures from both

+/- and -/- animals have significantly shorter myelinated internodes than +/+ cultures

(Fig. 3-5, 3-6). We have shown that PMP22 interacts with β4 integrin, which links the basal lamina with the SC cytoskeleton. In rat DRG neuron-Schwann cell cocultures

67 treated with a Rho-kinase inhibitor (Y-27632), myelin segments are shorter (Melendez-

Vasquez et al., 2004). As Rho-kinase is known to phosphorylate MLC, I examined

MLC-P in PMP22 -/- cultures and found that it is not present at the nodes of Ranvier, in contrast to +/+ cultures. Potentially, MLC-P is important for segment elongation, and the actin cytoskeleton of the SC may be altered when it is abnormally expressed.

Alternatively, since internodes do not form at close intervals along a single axon, the proper signals might not be received by the axon for nodal formation.

These studies characterize myelin proteins during nerve development in the absence of PMP22. The delay in the initiation of myelination, the altered nodal organization and the shortened myelinating internodes observed in PMP22-deficient mice suggest a role for PMP22 in axo-glial communications.

CHAPTER 4 THE ADHESION AND MIGRATION OF SCHWANN CELLS IS ALTERED IN THE ABSENCE OF PERIPHERAL MYELIN PROTEIN 22

Introduction

Peripheral myelin protein 22 (PMP22) is thought to play roles in Schwann cell (SC) differentiation, the initiation of myelination and the structure and stability of myelin

(Adlkofer et al., 1995; Zoidl et al., 1995; Notterpek et al., 1997; Carenini et al., 1999), processes in which cytoskeletal reorganization is essential. Previous studies from our lab place PMP22 at apical intercellular junctions, where it influences epithelial cell morphology and motility (Roux et al., 2005). Madin Darby canine kidney (MDCK) epithelial cells stably expressing PMP22 have a flattened morphology, exhibiting larger nuclear and apical border areas. These monolayers fail to close a wound as rapidly as control epithelia and do not produce the lamellipodia observed in the control cultures

(Roux et al., 2005). PMP22 also mediates membrane expansion in NIH3T3 fibroblasts and rat SCs (Brancolini et al., 1999; Nobbio et al., 2004).

Normal adhesion and migration in SCs, fibroblasts and epithelia is a dynamic process, whereby the actin cytoskeleton regulates the clustering of integrins into focal adhesions and focal complexes. In turn, actin dynamics are governed by the Rho family of GTPases. Integrin-mediated adhesion activates these GTPases, which triggers the assembly of filopodia (associated with Cdc42), lamellipodia (linked to Rac) and stress fibers (affiliated with Rho), membrane structures involved in cell adhesion and migration

(Schoenwaelder and Burridge, 1999).

68 69

PMP22 has been placed in a complex with α6β4 integrin, a molecule that links the actin cytoskeleton to laminin (Amici et al., 2006). Integrins are heterodimeric molecules, which have roles in cell proliferation, adhesion, migration and differentiation (Danen and

Sonnenberg, 2003). The association of PMP22 with an integrin provides a potential mechanism for the links reported between PMP22 and the cell cycle, cell shape and motility (Zoidl et al., 1995; Brancolini et al., 1999; Roux et al., 2005). In addition to interacting with α6β4 integrin, PMP22 contains the L2/HNK-1 epitope on the asparagine-linked carbohydrate moiety of its first extracellular loop (Snipes et al., 1993), which is involved in cell adhesion and recognition of neural and immune cells

(Schachner and Martini, 1995).

PMP22 is alternatively named growth arrest specific gene 3 (gas-3) because

PMP22 mRNA levels were found to be upregulated in serum-starved NIH3T3 fibroblasts

(Schneider et al., 1988; Manfioletti et al., 1990). The growth regulatory properties of

PMP22 expression have been observed in rat Schwann cells (SCs), as well. When it is overexpressed, PMP22 inhibits SC proliferation (Welcher et al., 1991; Zoidl et al., 1995;

Johnson et al., 2005). Conversely, expression of antisense PMP22 mRNA increased the proliferation rate of SCs (Zoidl et al., 1995).

In vivo analyses of specific molecules associated with essential processes, such as myelination, are difficult because other molecules often compensate for the missing or mutant protein that is being studied. This redundancy masks the true function of the protein, as is the case with the analyses of several integrin (Frei et al., 1999; Feltri et al.,

2002; Previtali et al., 2003b) and PMP22 (Adlkofer et al., 1995; Martini and Schachner,

1997; Carenini et al., 1999; Notterpek and Tolwani, 1999) transgenic animals. In vitro

70 models remove some of the factors that are present in vivo, so fewer compensatory mechanisms are present. Therefore, to attempt to comprehend the role of PMP22 in nerve biology, mouse Schwann cells (SCs) isolated from wild type and homozygous PMP22- deficient mice (Amici et al., 2006) will be utilized to investigate the growth-modulatory, adhesive and migratory properties of PMP22.

Materials and Methods

Mouse Schwann Cell Isolation

Wild type, +/- and -/- mice from P4 to P10 were sacrificed according to proper animal protocols and their sciatic nerves were collected in a sterile hood. Nerves were cut into ~5 mm pieces and allowed to adapt to culture conditions for 1-4 days depending on their age at the time of collection. This step mimics Wallerian degeneration seen after nerve injury in vivo and causes the SCs to hyperproliferate (Stoll and Muller, 1999). The nerves were digested with 0.25% trypsin (Gibco) and 0.03% collagenase (Worthington

Biochemical Corp., Lakewood, NJ) in Hank’s buffered salt solution (HBSS), for two cycles, 10 min each. They were triturated in serum-containing media with 18, 21 and 23 gauge needles to separate the SCs from the axons and the ECM and these suspensions were filtered through sterile Nitex fabric by centrifugation at 1200 rpm for 5 minutes.

The cells were plated and allowed to adhere for 30 min. Then, the supernatants were transferred to new poly-L lysine coated dishes to reduce fibroblast contamination. The

SC cultures were maintained in DMEM (Gibco) supplemented with 10% FCS (Hyclone), gentamycin (20 µg/ml, Gibco), glial growth factor (10-20 µg/ml, BTI) and forskolin (2

µM, Calbiochem) (mouse SC medium).

LacZ Activity Assay

Schwann cells from +/+ and PMP22 -/- mice were plated on poly-L-lysine coated

60 mm2 dishes and maintained in mouse SC medium for 24 hr. The lacZ reporter activity

was detected histochemically by X-Gal (5-bromo-4-chloro-3-inodyl-β-D-galactoside) staining, according to the procedure of Valenzuela et al., 2003. Images were collected using a Nikon T1-SM inverted microscope equipped with a Nikon DS-L1 camera.

Bromodeoxyuridine Incorporation Assay

The DNA synthesis rate of subconfluent mouse SCs isolated from +/+ and -/- mice was analyzed using a bromodeoxyuridine (BrdU) labeling and detection kit (Roche,

Indianapolis, IN) optimized for immunofluorescence of adherent cells. After an 8 hr labeling period with BrdU, cells were processed following the manufacturer's recommended protocol. BrdU-positive cells were counted in 4 random fields of view (0.8 mm2) and divided by the total number of cells visualized by Hoechst staining. More than

200 cells were counted for each condition. The number of PMP22 -/- cells was calculated

as a percentage of +/+ cells (arbitrarily assigned to 100%). Statistical significance was

determined using a two-tailed Student’s t-test for unpaired samples (Microsoft Excel).

Error bars indicate the standard error of mean.

Immunocytochemistry

Mouse SCs were fixed with 4% paraformaldehyde in PBS for 10 min at RT, and

permeabilized with 0.2% Triton X-100 for 15 min at RT (Notterpek et al., 2001). Samples were blocked in PBS containing 20% normal goat serum for 1 hr. Primary antibodies,

including monoclonal mouse anti-vinculin, 1:500; anti-actin 1:1000, (both from Sigma-

Aldrich); and polyclonal rabbit anti-p75 (Chemicon) antibodies were added in the

blocking solution overnight at 4°C. The samples were then incubated for 2 hrs with Alexa

Fluor 594 goat anti-rabbit IgG (red) and/or Alexa Fluor 488 goat anti-rat IgG (green) and

Hoechst dye at 10 µg/ml to visualize nuclei (all from Molecular Probes, Inc., Eugene,

OR). Coverslips were mounted by using the ProLong Antifade kit (Molecular Probes,

Inc.). Samples were imaged with a Spot camera attached to a Nikon Eclipse E800

microscope and were formatted for printing by using Adobe PHOTOSHOP 5.5.

Western Analyses

For total protein analyses, mouse SCs isolated from +/+ and -/- sciatic nerves were

solubilized in SDS gel sample buffer (62.5 mM Tris, pH 6.8, 10% glycerol, 3% SDS)

supplemented with complete protease inhibitor (Roche) and protein concentrations were

determined using BCA reagents (Pierce Chemicals, Rockford, IL). Deglycosylation procedures were done according to the manufacturer’s instructions (New England

Biolabs). Samples (20 µg/lane) were separated on 12.5% acrylamide gels under reducing conditions and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.,

Hercules, CA). Polyclonal rabbit anti-PMP22, 1:500 (Notterpek et al., 1999b);

monoclonal mouse anti-vinculin, 1:500; anti-actin, 1:1000, (both from Sigma-Aldrich);

and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 1:4000, (a kind gift from

Dr. G. Shaw, University of Florida, Gainesville, FL) antibodies were used. Bound

antibodies were visualized with an enhanced chemiluminescence kit (PerkinElmer Life

Sciences, Boston, MA). Films were digitally imaged using a GS-710 densitometer (Bio-

Rad) and were formatted for printing by using Adobe PHOTOSHOP 5.5.

Adhesion Assays

96-well microtiter plates were coated for 2 hrs at 37°C with 10 µg/ml laminin

(Promega, Madison, WI). Plates were then blocked with 1% bovine serum albumin in

DMEM for 1 hr at 37°C. Mouse SCs were dissociated from tissue culture plates with

mild trypsinization and 10,000 cells/well were plated in 10% FCS/DMEM for 30 min.

Cell numbers were determined by counting cells with a hemocytometer. After a 30 min

incubation, the cells were fixed in 1% glutaraldehyde/DMEM for 15 min at RT, stained

in 0.1% crystal violet/dH2O for 30 min at RT, dried, and subsequently permeabilized in

0.2% TritonX-100/PBS for 30 min at RT. The absorbance was measured in a microplate reader at 550 nm, and the data were graphed and analyzed using Microsoft Excel. Assays were repeated at least three times to verify the results, and each assay contained 4 wells per condition. A positive correlation between the amount of cells adhered to the plate and the absorbance value obtained has already been established (Gillies et al., 1986;

Aumailley et al., 1989). Statistical significance was determined using an unpaired, two- tailed Student’s t-test (Microsoft Excel). Error bars indicate the standard deviation.

Migration Assays

SCs were plated in 40 µl drops on poly-L-lysine or laminin-coated glass coverslips

at 5x104 cells/coverslip in mouse SC medium. After 3 hr, an additional 200 µl of medium

was added. Twenty-four hours later, the medium was changed to 2% FCS/DMEM and

the cells were incubated for 12 hrs. Next, the cell monolayer was gently scratched with a

sterile yellow pipette tip to generate an ~1 mm wide gap. Immediately after scratching

(t=0 hrs), 10%FCS/DMEM was added and migration of the cells within the gap was

monitored microscopically at t=0 and t=8 hr using a Nikon T1-SM inverted microscope

equipped with a Nikon DS-L1 camera. The gaps were asymmetrically marked with a

needle at points along the scratches and the images at t=0 and t=8 hr were overlapped and

aligned in Photoshop 5.5. The area migrated over 8 hr by the cells was measured with the

ImageJ software program (National Institute of Health, Bethesda, MD). The SC front was

traced on each side of the gap and the area within the gap at t=0 and t=8 hr was estimated

74 directly by the computer. The difference between the areas at the two times was then calculated ((area at t=0 hr)-(area at t=8 hr)) and the area migrated by the +/+ cells was set to 100%. The motility of the PMP22 -/- SCs was compared to this baseline. Assays were repeated at least three times to verify the results. Statistical significance was determined using an unpaired, two-tailed Student’s t-test (Microsoft Excel). Error bars indicate the standard deviation. SC migration was observed for 8 hr to ensure that the presence of cells within the gap is due to movement, not proliferation (Meintainis et al., 2001).

Parallel cultures were processed for immunostaining, as described above.

Results

PMP22 Is Expressed in Nonmyelinating SCs

PMP22 is linked to many cellular processes in vivo, including Schwann cell differentiation and initiation of myelination (Adlkofer et al., 1995; Magyar et al., 1996).

To attempt to understand the function of PMP22, mouse Schwann cells from +/+ and -/- littermates were isolated from sciatic nerves (Fig. 4-1A-B) as a model to examine cell proliferation, adhesion, migration and morphology.

The transgenic mouse line that is used in these studies is missing the first two coding exons of pmp22, exons 2 and 3 (Fig. 2-1A), which have been replaced with the lacZ reporter in the PMP22-deficient mice (Fig. 2-1B). When a lacZ activity assay is performed, the expression of the β-gal reporter is detected as a blue reaction product in homozygous PMP22 -/- SC cultures (Fig. 4-1B), indicating the PMP22 promoter is active in these SCs.

Figure 4-1. PMP22 expression in cultured mouse SCs. Mouse SCs isolated from sciatic nerves of wild type (A) and PMP22-deficient mice (B) were treated with X- gal to visualize β-gal expression in -/- SCs (A-B, magnification, X40). Wild type mouse SC lysates (c) were treated with N-glycosidase (n) or endoH (h) and probed with a polyclonal rabbit anti-PMP22 antibody (C). Arrow, glycosylated PMP22. Arrowhead, unglycosylated PMP22. Molecular mass in kDa.

In parallel, SC lysates from wild type mice were analyzed biochemically and immunoblotted with anti-PMP22 antibody (Fig. 4-1C). Bands at 22 (Fig. 4-1C, arrow) and 18 (Fig. 4-1C, arrowhead) kDa are present in control lysates (Fig. 4-1C, lane c). In comparison, treatment with N-glycosidase, an enzyme that cleaves all asparagine-linked sugars regardless of their position in the secretory pathway, removes the carbohydrate moiety on PMP22. As previously described (Pareek et al., 1997), the 22 kDa band shifts to 18 kDa representing the core PMP22 protein (Fig. 4-1C, lane n). Upon enzymatic digestion with endoglycosidase H (endoH), an enzyme that removes the high mannose sugars acquired by proteins in the ER, a fraction of PMP22 is resistant to digestion (Fig.

4-1C, lane h). This indicates that a portion of newly synthesized PMP22 has exited the

ER, acquired complex glycosylation in the medial Golgi (Kornfeld and Kornfeld, 1985) and has most likely been stably incorporated into the cell membrane.

Increased SC Proliferation in the Absence of PMP22

PMP22 also was discovered as growth arrest specific gene-3 (gas-3), a gene upregulated in serum-starved fibroblasts (Schneider et al., 1988; Manfioletti et al., 1990) and its growth-altering properties are well characterized in multiple cell types (Zoidl et al., 1995; 1997; Roux et al., 2005). Additionally, increased SC density has been noted in nerves from PMP22-deficient animals (Sancho et al., 2001; Amici et al., 2006).

Therefore, isolated SCs from the nerves of +/+ and -/- littermates were examined to determine if there are differences in the rates of proliferation (Fig. 4-2). Cells were plated at equal densities, grown for 24 hr and labeled with BrdU for 8 hr. Next, they were processed and evaluated for BrdU incorporation. Subconfluent cultures lacking of

PMP22 have an ~20% increase in their rate of proliferation, compared with +/+ cells

(Fig. 4-2, p<0.05).

Figure 4-2. SCs from PMP22 -/- mice have an increased rate of proliferation. BrdU incorporation analyses for +/+, +/- and -/- mouse SCs. Control SCs incorporation rate over a 8 hr period is arbitrarily set to 100±15%, compared with 123±16% of -/- SCs. P<0.05.

PMP22-Deficient Cells Display Impaired Adhesion and Migration

In addition to proliferation studies, functional assays have been performed linking

PMP22 with cell shape and motility (Brancolini et al., 1999; Roux et al., 2005). Rat SCs that are overexpressing PMP22 have hindered motility and cell spreading (Nobbio et al.,

2004), and epithelial monolayers stably expressing PMP22 have slowed migration (Roux et al., 2005). In addition, reduced PMP22 expression is associated with dysmyelination and loose basal lamina in vivo (Adlkofer et al., 1995; Magyar et al., 1996; Amici et al.,

2006). To determine if there are intrinsic deficits in motility, in vitro adhesion and migration assays were performed with SCs isolated from +/+ and -/- littermates.

Figure 4-3. Adhesion to laminin is reduced in mouse SCs lacking PMP22. Control and -/- SCs were plated at 10,000 cells/well in a 96-well plate coated with BSA alone or with 10 µg/ml laminin and allowed to adhere for 30 minutes. Cells were fixed, stained with crystal violet and lysed in 0.2% TX-100 in PBS. The absorbance was read at 550 nm. *=p<0.0001.

As PMP22 interacts with β4 integrin and contains the L2/HNK-1 epitope in its first extracellular loop, we hypothesized that SCs deficient in PMP22 would be impaired in their adhesion to laminin, an extracellular matrix substrate. The L2/HNK-1 epitope

78 mediates adhesion between neural cells and laminin (Hall et al., 1993), and α6β4 specifically interacts with laminin (Lee et al., 1992). Wild type and PMP22 -/- SCs were plated in wells coated with BSA alone or in wells coated with laminin and subsequently blocked with BSA to prevent non-specific adhesion. Cells were allowed to adhere for 30 minutes, and were then fixed and processed for a crystal violet assay. This adhesion assay reveals that PMP22 -/- SCs adhere ~60% less to laminin than control cells (Fig. 4-3A p<0.0001). While -/- SCs have a lower affinity to laminin than +/+ SCs, they appear to adhere more strongly to BSA-coated wells, attaching only slightly more to laminin than to BSA alone (Fig. 4-3).

Integrins regulate the migration of cells as well as their adhesive properties (Hynes,

1992). Similarly, PMP22 has been linked to the regulation of cell spreading and migration in fibroblast and epithelial cell models (Brancolini et al., 1999; Roux et al.,

2005). Therefore, to study the potential differences in the motility of +/+ and PMP22 -/-

SCs, a scratch migration assay was performed (Meintainis et al., 2001). Cells were plated at high density (50,000 cells/40 µl drop) and allowed to adhere to poly-l-lysine (not shown) or laminin (Fig. 4-4A-D) for 12 hr. To synchronize the cells, serum was removed for 12 hours. Following this starvation period, cells were scratched through the middle of the culture with a pipette tip, creating an approximately 1 mm wide gap. At this point, serum-containing media was provided to the cells (Fig.4-4A, C; t=0 hr), and they migrated into the scratch over an 8 hr time frame (Fig. 4-4B, D; t=8 hr). The areas migrated by the +/+ and -/- cultures were calculated and converted to percent area migrated. Wild type cultures were set to 100% (Fig. 4-4E). SCs from +/+ and -/- mice cover comparable areas on poly-L-lysine (Fig. 4-4E). Wild type cells advance at a faster

79 pace when they are plated on laminin as opposed to poly-L-lysine. However, PMP22 -/-

SCs migrate significantly slower than +/+ SCs on laminin (an ~70% reduction in wound closure) (Fig. 4-4E, p<0.002).

Figure 4-4. Slowed migration on laminin by PMP22 -/- mouse SCs. SCs from +/+ and -/- mice were plated on poly-L-lysine (not shown) or laminin-coated 24-well dishes, scratched with a pipette tip and allowed to migrate for 8 hr (A-D). Images from +/+ (A, B) and -/- (C, D) were collected at t=0 hr (A, C) and t=8 hr (B, D) post-scratch (magnification, X10) and overlaid in Photoshop 5.5. The needle mark used to align the images can be seen at the top of panels A and B. The area migrated was calculated using ImageJ software. Percent area migrated on poly-L-lysine and laminin are shown (E). *=p<0.002.

Lack of PMP22 Results in Altered SC Morphology

To examine the purity of the cultures, SCs were plated on laminin-coated glass coverslips in parallel with the cultures in the migration assay described in Fig. 4-4. After

identical treatments of serum-starvation and migration, the cells were processed for

immunostaining. The cultures were immunolabeled with an antibody to the SC surface

marker, p75 (Fig. 4-5, t=8 hr), which demonstrated that the cultures have a high level of

SC purity with few fibroblasts (Fig. 4-5A-B, asterisks).

Figure 4-5. Cells at the scratch edge are morphologically different between +/+ and -/- cultures. Eight hours after the induction of migration, cells from +/+ (A) and -/- (B) mice were immunolabeled with a polyclonal rabbit anti-p75 (A-B) antibody. Cultures were labeled with Hoechst to mark nuclei. s, scratch area. Scale bar, 20 µm. Arrows indicate lamellipodia. Arrowheads denote filopodia. Asterisks signify fibroblasts. The quantification of lamellipodia migrating into the scratch area reveals a decreased number of lamellipodia in the -/- compared to control cultures (C).

Since p75 specifically labels the SC membrane, cell morphology also can be evaluated. Wild type cells have many broad well-formed lamellipodia (Fig. 4-5A, arrows), and fewer filopodia-like protrusions (Fig. 4-5A, arrowheads). Conversely,

PMP22-deficient SCs have a reduced number of properly formed lamellipodia at the scratch edge than +/+ cells (Fig. 4-5B, arrows), with a greater percentage of filopodia- like projections into the scratch zone (Fig. 4-5A, arrowheads). Quantification reveals that

PMP22 -/- SCs have an ~20% reduction in the number of well-formed lamellipodia migrating into the wound (Fig. 4-5C).

Figure 4-6. PMP22 -/- mouse SCs display altered lamellipodial morphology. Immunostaining of +/+ and -/- SCs plated on laminin at a scratch edge (A). Cells are immunostained with anti-vinculin antibody (red) and actin phalloidin (green). Scale bar, 5 µm. Western blots of +/+ and -/- total SC lysates immunoblotted with anti-vinculin, anti-actin and anti-GAPDH antibodies (B). Molecular mass in kDa.

The migration of SCs requires certain morphological alterations in the cells.

Integrin-mediated adhesion activates the Rho family of GTPases, which in turn trigger the assembly of filopodia, lamellipodia and stress fibers (Schoenwaelder and Burridge,

1999). SCs form lamellipodia at their leading edges, which guide the cells forward

(Meintainis et al., 2001). After migration, cultures were immunolabeled with anti- vinculin (red), an antibody that recognizes focal adhesions, and actin phalloidin (green) to label the actin cytoskeleton (Fig. 4-6A) and to visualize the organization of the lamellipodia. The cytoskeleton appears to be collapsed in the lamellipodia of -/- SCs.

Westerns indicate that vinculin levels are slightly decreased in -/- cultures (Fig. 4-6B).

Total β-actin expression remains constant. GAPDH serves as a protein loading control.

Discussion

In this chapter, the consequences associated with a lack of PMP22 expression were examined at the cellular level. As PMP22-associated neuropathies specifically manifest

PNS disease phenotypes, studies were performed on mouse SCs isolated from +/+ and

PMP22 -/- sciatic nerves to attempt to understand the biological function of PMP22.

Differences in proliferation, adhesion and migration were detected between the genotypes. In addition, the cytoskeletal architecture of the PMP22 -/- cells appeared to be altered.

In myelinating rat SCs, PMP22 is robustly expressed (Pareek et al., 1997), however, PMP22 is produced at much lower levels in nonmyelinating SCs (Notterpek et al., 1999b). A β-gal activity assay (Valenzuela et al., 2003) verified that the PMP22 promoter elements were active in the PMP22 -/- SCs, as β-gal expression is under the control of the PMP22 promoter elements (Amici et al., 2006). PMP22 expression was

83 confirmed in nonmyelinating mouse SC cultures from +/+ animals by biochemical analyses. A fraction of PMP22 is resistant to digestion with endoH, an enzyme that cleaves high mannose sugars present on proteins that have not yet reached the medial

Golgi of the secretory pathway (Kornfeld and Kornfeld, 1985). This suggests that the protein proceeds through the secretory pathway and is stably incorporated into the SC plasma membrane.

PMP22-deficient SCs proliferate faster than control SCs, agreeing with other studies of the growth-modulatory behavior of PMP22 in cell cycle regulation (Manfioletti et al., 1990; Zoidl et al., 1995). In vivo studies of PMP22-deficient mice showed an increase in SC density at P10 (Sancho et al., 2001, Amici et al., 2006). Overexpression of human PMP22 leads to slowed SC propagation in epithelia and rat SCs (Johnson et al.,

2005; Roux et al., 2005). In rat SCs, the localization of the PMP22 protein constructs within the cell may be important for proliferation, as different exogenous human PMP22 constructs have different proliferation rates (Johnson et al., 2005). In support of this idea, zonula occludens-1, a molecule that colocalizes with PMP22 at adherens and tight junctions (Notterpek et al., 2001; Roux et al., 2004), has recently been implicated in cell cycle regulation depending on its location in the cell (Balda et al., 2003). The interaction of PMP22 and β4 integrin also may explain the growth-altering properties, as integrins are linked to cell proliferation modulation (Danen and Sonnenberg, 2003).

An important question that arises from these studies is how does the loss of PMP22 expression lead to reduced adhesion and slowed migration? PMP22 contains the

L2/HNK-1 adhesion/recognition carbohydrate epitope on its first EC loop (Snipes et al.,

1993). Therefore, adhesion and migration may be directly affected in PMP22-deficient

SCs by the lack of this epitope, which has been shown to bind laminin (Hall et al., 1993;

1997). PMP22 might act as an anchoring molecule in the membrane for proteins with

adhesive and signal transduction mechanisms. PMP22 is a highly hydrophobic protein,

the majority of which is embedded in the membrane. It also is present in lipid rafts

(Hasse et al., 2002), which are dynamic membrane patches that are enriched in signaling

molecules, including integrins. Indeed, we have shown that PMP22 interacts with β4 integrin (Amici et al., 2006), a molecule known to link laminin to the cytoskeleton (Lee et al., 1992). If the absence of PMP22 destabilizes the expression of β4 integrin at the cell

surface, alternate integrin subunits with different binding properties to ECM substrates

might be expressed to compensate for its absence (Milner et al., 1997), and therefore,

may be responsible for the altered adhesion and migration to laminin.

The impaired migration exhibited by SCs deficient in PMP22 also may be a

consequence of improper activation of Rho GTPases, which are necessary for the

formation of membrane protrusions including filopodia and lamellipodia (Etienne-

Manneville and Hall, 2002). MDCK cells expressing PMP22 also are hindered in their

migratory abilities (Roux et al., 2005). If PMP22 is involved in the linkage between the

ECM and the actin cytoskeleton, then either the overexpression or the underexpression of

PMP22 could cause slowed migration. Activated RhoA induces increased adhesion to the

substrate, and MDCK cells display a strengthened actin cytoskeleton. Therefore, the

slowed migration in PMP22 overexpressing epithelia may be due to an increased amount

of active RhoA. In contrast, mouse SCs lacking PMP22 may not receive the proper

signals from the ECM for Rho GTPase activation. Altered activation of Rho GTPases

85 also would explain the changes observed in cell morphology and reduced lamellipodial formation in SCs lacking PMP22 (Etienne-Manneville and Hall, 2002).

Our studies suggest that reduced levels of PMP22 are linked to observed changes in cell proliferation, morphology, migration and adhesion. However, future studies are still necessary to determine the specific niche of this versatile protein in cell biology.

CHAPTER 5 CONCLUSIONS

Overview of Findings

The misexpression of peripheral myelin protein 22 (PMP22) has been extensively studied, as it is linked to a family of demyelinating hereditary neuropathies (Suter and

Scherer, 2003). However, the specific function of PMP22 remains unclear. By the characterization of a newly established PMP22-deficient mouse model and the identification of a binding partner for PMP22, the studies described here provide novel information toward elucidating the role of PMP22 in peripheral nerve biology. The results indicate that, at the cell membrane, PMP22 is in a complex with α6β4 integrin and is a participant in the linkage of the Schwann cell (SC) cytoskeleton with the extracellular matrix (ECM). Moreover, the absence of PMP22 results in impaired SC adhesion and motility, two functions that are required for myelination.

While PMP22 has previously been linked to myelin initiation (Adlkofer et al.,

1995; Robertson et al., 1997; Robaglia-Schlupp et al., 2002), this dissertation characterizes these developmental delays in greater detail (Chapters 2 and 3).

Heterozygous PMP22-deficient mice presented a broad pattern of tissue distribution of the β-gal reporter protein in embryonic and adult animals, and a neuropathic phenotype comparable to HNPP. The sciatic nerves of PMP22 -/- mice have an increased number of

SCs and exhibit delayed myelination, as well as focal hypermyelinated areas along internodes.

86 87

Loose basal lamina is another characteristic of mice lacking PMP22 (Fig. 2-4), which has been noted in previous PMP22 animal models (Magyar et al., 1996; Adlkofer et al., 1997a). Yet, the reasons for its presence were not examined until the current studies. As SCs must secrete a basal lamina to initiate the myelination process, an altered interaction between the basal lamina and the SC abaxonal membrane may be the reason for the excess basal lamina in nerves from PMP22 -/- mice. Integrins are a family of plasma membrane receptors that link the cytoskeleton of a cell with the basal lamina

(Hynes et al., 1992). When β4 integrin is not expressed, affected mice die shortly after birth due to epidermal detachment (Georges-Labouesse et al., 1996; van der Neut et al.,

1996). Moreover, loose basal lamina has been observed in mice with a SC-specific disruption of β1 integrin (Feltri et al., 2002).

In the nerves of PMP22 -/- mice, the levels of the β4 integrin subunit are severely reduced (Figs. 2-5 and 3-3). Based on the loose basal lamina and reduced β4 expression in these nerves, I hypothesized that PMP22 and β4 integrin may interact in the sciatic nerves of +/+ animals. Indeed, coimmunoprecipitation experiments revealed that β4 integrin and PMP22 are in a complex with α6 integrin and laminin (Fig. 2-6).

Additionally, PMP22 interacts with β4 integrin in a human colon adenocarcinoma cell line (Fig. 2-8; Dexter et al., 1979), indicating that this complex is not restricted to the nervous system.

To further analyze the myelination defects in PMP22 -/- mice, developmental time course studies were performed on sciatic nerves and dorsal root ganglion (DRG) explant cultures from +/+, +/- and -/- mice (Chapter 3). Myelin protein expression is delayed in the nerves of affected mice, as expected. Additionally, p75, a marker of non-myelinating

SCs remains elevated throughout development, indicating delayed differentiation of the

SCs. Moreover, the expression of β4 integrin is decreased, while β1 integrin remains elevated in the PMP22 -/- samples (Fig. 3-3), indicative of dysmyelination (Previtali et al., 2001). Some SCs are able to initiate myelination in the absence of PMP22 expression; however, the fibers can exhibit focal hypermyelination, which results in the altered localization of nodal proteins (Fig. 3-4A-B). The myelin in PMP22 -/- mice appears intrinsically unstable, as e-cadherin, a marker of non-compact myelin compartments, is localized in punctate patches along the internode.

In DRG explant cultures established from PMP22 -/- embryos, myelination also is impaired. PMP22 -/- explants do not produce as many myelinated fibers as +/+ cultures, and the lengths of the myelinated segments are dramatically reduced. The altered nodal structure suggests that PMP22 is involved in interactions between the SC and the axon.

However, their internodes extend with prolonged exposure to myelin-inducing medium, which may be due to compensatory mechanisms observed in other myelin protein knockouts (Martini and Schachner, 1997).

As PMP22-associated neuropathies are linked to intrinsic SC deficits as opposed to axonal defects (Aguayo et al., 1977), mouse SC cultures from +/+ and -/- littermates were analyzed for alterations in cell proliferation, adhesion, migration and morphology

(Chapter 4). As expected, SCs proliferate at a faster rate when PMP22 is absent. PMP22

-/- SCs have reduced adhesion and hindered motility, compared to control cultures.

Additionally, cells isolated from -/- mice have fewer morphologically correct lamellipodia. The findings described here reveal that the lack of PMP22 results in phenotypes similar to those observed in PMP22 overexpression paradigms (Brancolini et

89 al., 1999; Nobbio et al., 2004; Roux et al., 2005). Thus, a specific level of PMP22 expression is necessary in Schwann cells, as either too much or too little PMP22 leads to deficits in proper functioning.

Unresolved Issues and Future Studies

The developmental studies in Chapters 2 and 3 provide novel insights into the role of PMP22 in PNS myelination and suggest changes in axo-glial communications in the absence of PMP22. This could be further analyzed, in part, by electron microscopy examination of sciatic nerve longitudinal sections; comparing the morphologies of the nodal regions and the distances between the nodes of Ranvier in the +/+ and PMP22 -/- samples. If PMP22 is important in these interactions, then the paranodal loops of the -/-

SCs should not adhere correctly to the axon and the lengths of the nodes and internodes may be affected. Along with the morphological studies, the expression of certain molecules, including myelin-related transcription factors can be analyzed. SCIP/Oct6 decreases as myelination is initiated, and conversely Krox20 is more robustly expressed

(Zorick et al., 1996). Therefore, as myelin protein levels are delayed in the nerves from

PMP22 -/- mice, it is expected that they will have a slower downregulation of SCIP and upregulation of Krox20 (Fig. 3-1). Based on the altered nodal formation in PMP22 -/- samples observed by immunohistochemistry (Fig. 3-4), biochemical analyses of the nodal proteins, including the sodium and potassium channels, may reveal changes to their overall levels during development. Alterations in nodal protein expression provide further evidence for impaired axo-glial interactions (Peles and Salzer, 2000).

In addition to axo-glial communications, PMP22 affects SC adhesion and migration to the ECM substrate, laminin (Chapter 4). As PMP22 contains the L2/HNK-1 adhesion/recognition epitope, it may interact directly with the ECM, specifically laminin

(Hall et al., 1993; 1997). To test this possibility, adhesion and migration assays should be

performed in cells stably expressing a deglycosylated form of PMP22. If PMP22 is

directly binding to the substrate, then these cultures will exhibit reduced attachment and

hindered migration, compared with control cells.

Motility deficits suggest changes in the actin cytoskeleton (Etienne-Manneville and

Hall, 2002). Previous studies in epithelia and fibroblasts link PMP22 to the Rho GTPase

pathway (Brancolini et al., 1999; Roux et al., 2005).Phosphorylated-myosin light chain

(MLC-P) is a downstream target of Rho kinase (Amano et al., 1996), and is undetectable

from the nodal regions of -/- DRG explant cultures (Fig. 3-6). As MLC-P is severely

reduced from PMP -/- DRG explant cultures, its expression pattern in vivo also may be delayed. It has been shown that MLC-P is robustly expressed in a transient fashion at the initiation of myelination in rat sciatic nerves (Melendez-Vasquez et al., 2004). An investigation of the levels of phosphorylated, compared with total MLC at the onset of myelination in +/+ and PMP22 -/- nerves will provide insights into the regulation of

MLC-P by Rho kinase in murine nerve development. This initial peak of MLC-P may be delayed in the nerves of -/- animals.

Based on the reduction in MLC-P immunoreactivity in DRG explants cultures established from PMP22 -/- mice, Rho appears to be affected. Adhesion and migration deficits in SCs lacking PMP22 were described in Chapter 4, so the signaling pathways involved with these cellular processes may now be examined. Specifically, Rho GTPase

GST pull-down assays can measure the amount of activated GTPases in the cells. If

PMP22 is necessary for activation of Rho, then I would expect that the levels of active

Rho are reduced in the SC cultures from PMP22-deficient mice.

The levels of Rho GTPases in cells are modulated by the integrins (Schoenwaelder and Burridge, 1999). The studies described in Chapters 2 and 3 reveal decreased levels of

β4 integrin in the nerves of PMP22 -/- mice. This reduction in steady-state β4 integrin protein may be the result of transcriptional or post-translational factors. To rule out transcription, I analyzed mRNA levels from +/+, +/- and -/- nerves by Northern blots, and found no major differences in β4 mRNA levels (data not shown). This suggests that the diminished steady-state expression of β4 integrin is due to post-transcriptional effects.

Pulse-chase studies can be used to elucidate differences between the genotypes in the stability of the newly-synthesized β4 integrin protein. Based on reduced levels of the steady-state pool of β4 integrin in affected nerves, it may have a shortened half-life, compared with wild type. Additionally, endoH analysis of de novo and steady-state protein levels will track the progress of the protein in the secretory pathway. These studies also will help to determine where β4 and PMP22 interact. If PMP22 and β4 integrin interact after both proteins are targeted separately to the plasma membrane, then

PMP22 may stabilize the localization of this integrin. It also is possible that PMP22 and

β4 integrin traffic together through the secretory pathway to the cell membrane. In this situation, in the absence of PMP22, β4 integrin will not be incorporated into the membrane. These studies should be expanded to the other PMP22 mouse models (C22 and TrJ) to determine if integrin functions play a role in other PMP22-associated neuropathies as well.

In regards to the interaction between PMP22 and β4 integrin, the clone A cells can be further utilized in PMP22 domain-mapping studies. By the same techniques used in

Fig. 2-8, PMP22 mutant molecules that are incorporated into the plasma membrane–

including point, truncation and glycosylation–mutants can be used to analyze the

interaction between β4 integrin and PMP22. Mutations in the transmembrane domains, as with the spontaneously occurring Trembler (G150D) and Trembler J (L16P) point mutants, may change the structure of PMP22 in the membrane and prevent it from interacting with the integrin complex. The truncated form of PMP22 is lacking the second extracellular loop, the third and fourth transmembrane domains and the C-terminal intracellular tail, which could reveal which portion of PMP22 interacts with the integrin complex. However, many of these mutants are intracellularly retained and form cytosolic aggregates. Therefore, if β4 interacts with them, it is most likely retained within the cell, as occurs with the heterodimeric interaction between the wild-type and mutant PMP22 proteins (Tobler et al., 1999; Johnson et al., 2005).

In summary, the work described here places PMP22 in a complex with α6β4 integrin and laminin. Additionally, the adhesion and migration of SCs is affected in the absence of PMP22. Therefore, this dissertation provides the groundwork for future studies of PMP22 in SC-ECM interactions and in PMP22-related signaling pathways.

Ultimately, these findings add to the knowledge on the function of PMP22 and will contribute to the development of therapies for PMP22-associated inherited peripheral neuropathies.

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BIOGRAPHICAL SKETCH

Stephanie Amici was born in Columbus, Ohio, in 1977 to Richard and Sandra

Amici. After graduating from Westerville South High School in 1995, she attended The

Ohio State University for her undergraduate studies. She graduated with a bachelor’s degree in zoology in 1999. She then worked at the Ohio State University Veterinary

Hospital for a year before moving to Gainesville, Florida, and beginning her graduate studies in the Interdisciplinary Program for Biomedical Research at the University of

Florida in 2000. She joined the laboratory of Dr. Lucia Notterpek in the summer of 2001.

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