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Peripheral Myelin Protein 22 Is a Novel Constituent of Intercellular Junctions


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PERIPHERAL MYELIN PROTEIN 22 IS A NOVEL CONSTITUENT OF INTERCELLULAR JUNCTIONS By KYLE JOSEPH ROUX A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Kyle Joseph Roux

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I dedicate this work to my loving wife and s on; and to my parents, who have steadfastly supported me throughout the years.

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iv ACKNOWLEDGMENTS First and foremost, I would like to tha nk my mentor, Lucia No tterpek. Ever since I started working with Lucia, she has always made my education a priority, and had my best interests in mind. Looking back at my gr aduate career, I can clearly see that from Lucia’s guidance I have gained considerable maturity, and the conf idence necessary to pursue my scientific goals. I would also lik e to thank all of my committee members, whose invaluable insight and positive feedback were grea tly appreciated. Similarly, Bradley Fletcher deserves acknowledgment, for his expertise in molecular biology has been instrumental in promoting my studies. To all of the members of the Notterpek lab, past and present, who have served as perp etual sounding boards for my ideas and endured my endless tirades, I express my gratitude. I cannot thank my parents enough for al l that they have done and endured throughout the years. And most importantly, to my loving wife Amy I offer my deepest appreciation for the sacrifices she has made for our family.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................vi ii CHAPTER 1 INTRODUCTION........................................................................................................1 Introduction................................................................................................................... 1 Peripheral Neuropathies Associated with PMP22........................................................1 Animal Models of PMP22-Associated Neuropathies...................................................4 Disease Mechanism of Altered PMP22 Expression.....................................................5 Genomic Organization of PMP22................................................................................7 Temporospatial Expression of PMP22.........................................................................8 Characteristics of PMP22 Protein.................................................................................9 Role for PMP22 in Cell Proliferation and Cell Morphology......................................11 2 PERIPHERAL MYELIN PROTEIN 22 IS A NOVEL CONSTITUENT OF INTERCELLULAR JUNCTIONS IN EPITHELIA..................................................15 Introduction.................................................................................................................15 Materials and Methods...............................................................................................17 Results........................................................................................................................ .21 Discussion...................................................................................................................31 3 TEMPOROSPATIAL EXPRESSION OF PERIPHERAL MYELIN PROTEIN 22 AT THE DEVELOPING BLOOD-NERV E AND BLOOD-BRAIN BARRIERS....35 Introduction.................................................................................................................35 Materials and Methods...............................................................................................37 Results........................................................................................................................ .41 Discussion...................................................................................................................51 4 MODULATION OF EPITHELIAL MORPHOLOGY, MONOLAYER PERMEABILITY AND CELL MI GRATION BY GAS3/PMP22............................57

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vi Introduction.................................................................................................................57 Materials and Methods...............................................................................................59 Results........................................................................................................................ .65 Discussion...................................................................................................................79 5 CONCLUSIONS........................................................................................................84 Overview of Findings.................................................................................................84 Unresolved Issues.......................................................................................................85 Future Studies.............................................................................................................87 LIST OF REFERENCES...................................................................................................91 BIOGRAPHICAL SKETCH...........................................................................................109

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vii LIST OF FIGURES Figure page 1-1. The secondary structure of PMP22...........................................................................10 2-1. Coexpression of PMP22 with ZO-1 and occludin in colon epithelium.....................22 2-2. Coexpression of PMP22 with ZO-1 and occludin at cell-cell contacts.....................24 2-3. Internalization of PMP22 with o ccludin in EGTA-treated MDCK cells..................26 2.4. Exogenously expressed PMP22-myc is targeted to TJs in MDCK cells...................28 2-5. Colocalization of PM P22-myc with ZO-1 at intercellular junctions.........................29 3-1. Endothelial cell junctions of the BNB in the developing and adult rat sciatic..........41 3-2. Expression of PMP22 mRNA is elev ated in tissues and cells of the........................43 3-3. Endothelial cell contacts of the developing and adult rat BMV are..........................45 3-4. In mouse BECs, PMP22 is a constituent of intercellular junctions...........................47 3-5. In the choroid plexus, PMP22 is a junctional constituent of epithelia......................48 3-6. Neuroepithelial cell junctions of the embryonic ra t brain are immunoreactive........50 4-1. Altered epithelial cell pro liferation and morphology by PMP22..............................66 4-2. Protein polarity and junctiona l constituents in PMP22-MDCK................................68 4-3. Altered paracellular permeability of epithelial monolayers by hPMP22..................70 4-4. Perturbation of epithelial monolayer TER by PMP22 peptides................................72 4-5. An increased paracellular flux of ep ithelial monolayers by PMP22 peptides...........74 4-6. Wound healing is altered by PMP22 in epithelial monolayers.................................75 4-7. Lamellipodial protrusion in migrat ing epithelial monolayers is reduced..................77

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viii Abstract of Dissertation Pres ented 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 IS A NOVEL CONSTITUENT OF INTERCELLULAR JUNCTIONS by Kyle Joseph Roux August 2004 Chair: Lucia Notterpek Fletcher Major Department: Neuroscience Altered expression of peripheral myelin protein 22 (PMP22) is associated with several inherited peripheral neuropathies. Although predominantly expressed in myelinating Schwann cells, PMP 22 is also detected in seve ral cell types outside of the peripheral nervous system. The function of th e protein in myelin or non-neural cells remains unknown; however, PMP22 has been sh own to modulate the proliferation and morphology of Schwann cells and fibroblasts. With homology to the claudin superfamily of tight junction proteins and prominent expre ssion in epithelial cells of the intestine, a role for PMP22 at intercellu lar junctions was hypothesized. The overall aim of this study was to invest igate PMP22 as a cons tituent of apical cell-cell junctions. Initial studies identified that PMP22 is localized to the apical junctional complex in epithelia and endothe lia. Involved in the maintenance of cell polarity and establishment of selective barriers in tissues, these cell junctions play crucial roles in health and disease. Next, the lo calization and expression of the protein at

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ix intercellular junctions of the developi ng and mature rat blood-brain (BBB) and blood-nerve barriers (BNB) was studied. Detected at these cell junctions throughout all developmental stages studied, PMP22 is likely involved in the establishment and maintenance of these barriers. Finally, the role of the protein in multiple aspects of epithelial cell biology was investigated. As reported in other cell types, PMP22 modulated epithelial cell gr owth and morphology. Additionall y, the protein altered the junctional permeability and migration of epithelial monolayers. These results demonstrate that PMP22 is a component of the BNB a nd BBB, and is a functional constituent of apical cell-cell junctions in epithelia. Our studies have laid the foundation for future investigations into the func tion of PMP22 in epithelial cell biology, and provide novel insights into its potential role in peripheral nerve myelin.

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1 CHAPTER 1 INTRODUCTION Introduction First cloned over 15 years ago, periph eral myelin protein-22 (PMP22) was discovered almost simultaneously by thr ee independent laboratories. Described both as growth arrest-specific gene-3 ( gas3 ) in serum-starved or contact-inhibited NIH3T3 fibroblasts (Manfioletti et al ., 1990) and as a myelin gene with altered ex pression after peripheral nerve injury (Spreyer et al., 1991; Snipes et al., 1992), it was determined that PMP22 protein had previously been iden tified as a major glycoprotein of bovine peripheral nerve myelin (Kitamura et al., 1976). Soon after cloning, it became evident that altered expression of PMP22 is associated with several heritable demyelinating disorders. Lacking clear evidence of its stru cture, a role within a signaling pathway, or even an ascribed cellular function, it is know n that PMP22 is critical to the normal function of peripheral nerve myelin. Hypothe ses for the mechanism of disease include dose-dependent loss-of-function and a dom inant negative gain-of-function, ideas supported by the genetics behind altered PMP 22 expression. Despite of extensive genetic characterization of clinical cases, severa l animal models for PMP22 mutations, and intensive analysis of PMP 22 messenger RNA (mRNA) and pr otein, a clear understanding of the basic function and disease mechanism for PMP22 remains unknown Peripheral Neuropathies Associated with PMP22 Predominantly studied for its role in the pathology of the peripheral nervous system (PNS), altered expression of the PMP22 gene is associated with a significant

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2 subset of heritable peripheral neuropathie s in humans, including Charcot-Marie-Tooth disease (CMT), Dejerine-Sotta s syndrome (DSS) and heredita ry neuropathy with liability to pressure palsies (HNPP) (reviewed by Naef and Suter, 1998). These disorders vary in frequency, age of disease onset and functional severity. Accounting for up to 5% of total protein in peripheral nerve myelin (Pareek et al., 1993), PMP22 protein and message levels are highest in myelinating Schwann cells (Welcher et al ., 1991; Snipes et al., 1992). As detected by ultrastruc tural immunocytochemistry, the protein is concentrated in the compact portion of myelin (Haney et al., 1996), the region responsible for maintenance of the ionic resistance that en ables rapid saltatory ne rve impulse conduction. The predominant expression and localization in the PNS, comb ined with its association with demyelinating neuropathies, corresponds with PMP22 as an essential protein constituent of peripheral nerve myelin. Charcot-Marie-Tooth disorders are th e most common heritable peripheral neuropathy with a prevalen ce of 1 in 2,500 (Skre, 1974). Most CMT patients are classified as having CMT type 1 with abnormalities in the PN S myelin that is created by Schwann cells. The most prevalent form (90%) of CMT1, CMT type 1A (CMT1A) (Garcia, 1999) has a prevalence of 1 in 5,000 (Kuhlenbumer et al., 2002), and is predominantly found in patients with a dominant 1.5 Mb duplication of the p11-p12 region of chromosome 17 (Lupski et al., 1991; Raeymaekers et al., 1991), which contains the PMP22 gene. Less frequently, point mutations in the PMP22 gene are associated with CMT1A (Roa et al., 1993). Age of disease on set is variable, typi cally ranging between the 1st and 2nd decade of life. Identified by slowed nerve conduction velocity (NCV), CMT1A can be diagnosed by determination of PMP22 gene duplication or point

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3 mutation (reviewed by Kuhlenbumer et al ., 2002). Peripheral nerve pathology includes demyelination, hypermyelination, and onion bulb formations. Progressi ve distal limb weakness and muscle atrophy, distal and sy mmetrical sensory deficits, and foot deformities are common clinical symptoms of CMT1A that can lead to eventual loss of ambulatory function (reviewed by Kuhlenbumer et al., 2002). Encompassing a genetically diverse gr oup of patients, DSS is a clinical classification for patients with a form of severe peripheral neuropathy. Dominant and recessive mutations have been found in PMP22 myelin protein zero ( MPZ ), early-growth-response-element–2 (EGR ) and periaxin ( PRX) genes. Symptoms arise in infancy or early childhood, and include distal sensory loss and atax ia, motor deficit and palpable nerve hypertrophy (Dejerine and So ttas, 1893). A hallmark diagnostic feature characteristic of DSS is severely reduced NCV. Nerve pathology includes demyelinationremyelination, onion bulb formations, Sc hwann cell hyperprolif eration and nerve hypertrophy (reviewed in Plante-Bodeneauve and Said, 2002). The PMP22-associated DSS usually results from dominant misse nse mutations, although duplication of p11-p12 of chromosome 17 has been reported (Lupski et al., 1991; Mancardi et al., 1994; Silander et al., 1996), illustrating overlapping ge netic bases for DSS and CMT1A. The least severe form of PMP22-as sociated neuropathy is HNPP. Patients predominantly have a dominant 1.5 Mb dele tion of chromosome 17p11-p12; however in rare cases, point mutations have been descri bed, often resulting in premature termination of protein translation (Nicholson et al., 1994). Episodic recurrent motor and sensory peripheral neuropathies, often lasting days to weeks, with onset in childhood or adolescence are typical for HNPP patients (re viewed in Chance et al., 1999). Mildly

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4 slowed symmetrical NCVs are consistent wi th demyelination. Ne rve tomocula, sausage shaped regions of hypermyelination, are a common diagnostic feature found to precede clinical symptoms and hypothesized to be the result of frequent mild injuries (Gabreels-Festen and Wettering, 1999). Segmen tal demyelination and remyelination can also be identified in nerve biopsies. In one study, over 40% of HNPP patients were unaware of their condition and 25% we re symptom free (Pareyson et al., 1996), illustrating the phenotypic variation found in PMP22-associated peripheral neuropathies. Animal Models of PMP22-Associated Neuropathies To determine if altered expression of PMP22 is sufficient to induce heritable peripheral neuropathies, animal models that replicate the deletion, duplication and several point mutations of PMP22 have been genetically engi neered. These animal models recapitulate major aspects of PMP22-related neuropathies in humans and have allowed for a more complete cellular and molecula r analysis of neuropathy nerves than can practically be accomplished with human tissu e samples (Notterpek and Tolwani, 1999). Unlike engineered PMP22-mutant mice, the spontaneously occurring Trembler (Tr) and Trembler-J (TrJ) mice have point mutations in PMP22 resulting in amino-acid substitutions identical to those found in some human CMT1A patie nts (Suter et al., 1992a, 1992b; Valentijn et al., 1992 ; Ionasescu et al., 1997). Frequently used as models for hypertrophic demyelinating ne uropathies similar to CMT1A, the Tr and TrJ mice display phenotypic differences, especially the early postnatal lethality of the homozygous TrJ in comparison to the long-lived homo zygous Tr (Henry and Sidman, 1988). Other neuropathy-associated PMP22 point mutations have since been established in mice (Isaacs et al., 2000; 2002), pr oviding further models for th e study of PMP22-associated neuropathies.

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5 Overexpression of the PMP22 gene in transgenic animals provides a model for CMT1A patients with PMP22 duplication. Rats carrying three copi es of the murine PMP22 gene per allele display slowed NCV and signs of demyelination and dysmyelination (Sereda et al., 1996). Similarly, two mouse models of PMP22 overexpression were designed to recapitula te the CMT1A phenotype (Huxley et al., 1996; Magyar et al., 1996). To study the HNPP phenotype in transgenic mice, expression of the PMP22 gene has been diminished either by antisense technology (Maycox et al., 1997) or by homologous recombinant gene disr uption (Adlkofer et al., 1995). Affected mice display behavioral and pa thological traits found in HNPP patients, including the characteristic tomoculous nerve fibers. The severity of neuropathy found in the homozygous PMP22-null mice as compared to the more mildly affe cted heterozygotes lends support to the principle of PMP22 dose-dependency. In humans, homozygosity for the PMP22 deletion has not been reported, either because of a low frequency of occurrence or an incompatibility with lif e. Crossbreeding between the Tr and a PMP22-null mouse has provided significant ev idence for the dominant negative gain-offunction hypothesis for PMP22 point mutations (Adlkofer et al., 1997). Limitations exist for these animal models of PM P22-neuropathies, including th e brief rodent lifespan that may mask the progressive nature of these disorders. However, their study has led to important discoveries, including the role of protein mistrafficking in disease pathology. Disease Mechanism of Al tered PMP22 Expression The majority of PMP22 protein is locat ed in the plasma membrane of compact myelin in Schwann cells (Pareek et al., 1993; Haney et al., 1996). In rat Schwann cells, most of the newly synthesized PMP22 protei n is rapidly turned over in the endoplasmic reticulum, unable to attain complex glycosyl ation or enter the plas ma membrane (Pareek

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6 et al., 1997). The Tr and Tr-J mutant forms of PMP22 fail to reach the cell surface in myelinating Schwann cells (Colby et al., 2000). In addition to being hemizygous for wt-PMP22 expression, the mutant protein could act to further reduce the surface expression of the wild-type (wt) protein since the mutant protein is capable of associating with the wt form (Tobler et al., 1999 ). However, the duplication of the PMP22 gene leads to disease symptoms similar to many of the point mutants, su ggesting an alternate disease mechanism. Since most of the wt protein neve r reaches the plasma membrane, it has been hypothesized that PMP22 protein processing is difficult for the Schwann cell (Sanders et al., 2001). Either the presence of mutant forms or an increase in the level of wt protein expression may eventually overwhelm the qua lity-control mechanism, causing a negative gain-of-function, perhaps expl aining the progressive natu re of the disease. An upregulation of the lysosomal and ubiquitin-pr oteasomal protein degradation pathways in the Tr-J mouse model lend support to this hypothesis (Notterpek et al., 1997; 1999a; Ryan et al., 2002; Tobler et al., 2002; Fort un et al., 2003), although the actual disease mechanism of the PMP22-associated neuropathies remains uncertain. Current experimental approaches to treating PMP22-associated neuropathies (reviewed in Young and Suter, 2001) incl ude modulation of PMP22 expression and immunosuppression. A progesterone antagonist administered in a transgenic rat model for CMT1A reduces the levels of PM P22 mRNA and improves the CMT1A-like pathology (Sereda et al., 2003). Similarly, ascorbic acid trea tment ameliorates the disease phenotype in a CMT1A mouse model (Passage et al., 2004). Although these studies are promising, no effective treatments are commonly prescribed for clinical use in humans.

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7 Genomic Organization of PMP22 The human PMP22 gene is located at chromosome 17p11.2-p12. The mouse and rat genes are found on chromosome 11 (S uter et al., 1992a) and 10q22 (Liehr and Rautenstrauss, 1995), respectiv ely. In the human genome, PMP22 spans approximately 40kb, with 6 exons coding for the mRNA. The first 2 exons, 1A and 1B are alternatively transcribed under different prom oters, (P1 and P2, respectivel y) resulting in differential 5’ untranslated regions (UTRs), yet maintain ing the same coding region. The existence of dual promoters suggests diversity in regulat ing PMP22 expression. Both transcripts are detected in most tissues; however, the exon 1A containing message is predominant in the peripheral nerve, while the exon 1B form is more common in non-neural tissues (Suter et al., 1994). Exons 2 through 5, code for the PM P22 protein and a large 3’ UTR. This genomic organization is conserved in both the mouse and the rat. Studies of the PMP22 promoters have provided lim ited evidence to suggest how transcription is regulated. A TATA-box-like sequence is pres ent in the P1, but not P2 region, which has a high GC rich sequence, similar to that found in a housekeeping promoter (Suter et al., 1994). Specific tran scription factors know n to regulate PMP22 expression have not been described. Levels of PMP22 message in the sciatic nerve are upregulated during early postn atal development (Bosse al ., 1994). Immediately after nerve injury, PMP22 mRNA levels are reduced followed by upregulation during regeneration (Spreyer et al., 1991; Snipes et al., 1992). In NIH3T3 cells, growth arrest leads to an elevation in PMP22 message (Manfioletti et al., 1990). Similarly, forskolin, an activator of adenylate cyclase that produces cyclic-AMP, results in increased mRNA levels in Schwann cells (Snipe s et al., 1992; Pareek et al ., 1993). Another regulator of PMP22 transcription in Schwann cells is 3 -5 -tetrahydroprogesterone, whose activity is

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8 dependent on the gamma-amino butyric acid (GABAA) receptor (Melcangi et al., 1999; Martini et al., 2003). These findings have provi ded some insight into the expression of PMP22 mRNA; however, a clear understanding of its gene regulation remains elusive. Temporospatial Expression of PMP22 Expressed rather ubiquitously in tissue s outside of the PN S, non-neural PMP22 mRNA is detected by in situ hybridization during murine em bryogenesis in the epithelial ectodermal layer at embryonic day 9.5 (E9.5) (Baechner et al., 1995). In the same study, elevated levels of PMP22 message are enrich ed in the liver and gut during organogenesis (E11.5). By E14.5-16.5 the lung mesenchyme, skin and eye epithelia all contain PMP22 message. As detected by Northern blot anal ysis, tissue-specific expression of PMP22 mRNA in the late embryonic rat heart and kidne y is reduced prior to birth (Rees et al., 1999). Tissues containing signifi cant levels of PMP22 in the adult rat and mouse include the lung, stomach and intestinal tract (Taylo r et al., 1995; Lobsiger et al., 1996). By in situ hybridization, the mature mouse was found to contain signifi cant non-neural PMP22 mRNA in the epithelial villi of the intestine (Baechner et al., 1995). In the central nervous system (CNS), th e highest levels of PMP22 message are detected at E15.5 by Northe rn blot analysis (Wulf and Suter, 1999), and by in situ hybridization at the s ubventricular neuroepithelial laye r of the developing mouse from E11.5 through E17.5 (Baechner et al., 1995; Parmantier et al., 1997). Discrete populations of motor neurons in the developing and adult mouse and rat also contain PMP22 message and protein (Parmantier et al., 1995; 1997). Expr ession of a putative zebrafish orthologue to ma mmalian PMP22 was observed by in situ hybridization in the intestinal and olfactory epithelium and neural crest cells (Wulf et al., 1999), identifying the gene as having both a neural and non-neural expr ession even in nonmammalian

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9 vertebrates. In addition, PMP22 messa ge is found in diverse cell lines in vitro such as differentiated PC12 cells (De Leon et al., 1994) P19-derived neuroepithelial cells (Wulf and Suter, 1999) and shear-stressed endothe lia (Bongrazio et al., 2000). Thus, despite a PNS-specific disease association, the pattern of PMP22 mRNA expression is rather ubiquitous. Characteristics of PMP22 Protein Based on hydropathy plots, PMP22 is a 160 amino-acid hydrophobic protein with a putative four-transmembrane structure, two extracellular loops and intracellular aminoand carboxyl-termini (D’Urso and Muller, 1997, Taylor et al., 2000) (Fig. 1-1). The protein is highly conserved with an 87% am ino-acid identity betw een human and mouse. The 1st transmembrane domain contains a noncleaved signal peptid e sequence, a motif that targets protein insertion into the ER me mbrane (Manfioletti et al., 1990; Welcher et al., 1991; Taylor et al., 1995). The only documented post-translational m odification of PMP22 is the addition of a sugar moiety via N-linked glycosylation of a conserved consensus sequence on the 1st extracellular loop (Pareek et al., 1993). Gl ycosylation of PMP22 gives the core 18 kilodalton (kD) protein its characteristic 22 kD mobility by SDS-PAGE analysis. The sulfated sugar complex is recognized by the L2/HNK-1 antibody, an epitope found on several nervousand immune-system prot eins that function in cell-cell and cell-extracellular matrix in teractions (reviewed in Sc hachner et al., 1995). When glycosylation of the protein is prevented by amino-acid substitution, PMP22 is targeted to the ER and plasma membrane similar to the wt form (Ryan et al., 2000). However, the deglycosylated protein forms less stable homodimers (Ryan et al., 2000) than the wt protein (Tobler et al., 1999).

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10 Figure 1-1. The secondary st ructure of PMP22. Shown above is the putative secondary structure of PMP22. There are four tr ansmembrane regions (grey) and two extracellular loops, the first of which contains an N-glycosylation motif. In addition to forming homotypic interac tions, PMP22 is capable of associating with other transmembrane proteins. PMP22 a ssociates in a glycosylation-independent interaction with the abundant PNS myelin transmembrane protein, myelin protein zero (P0) (D’Urso et al., 1999). It is hypothesized that the inte raction between P0 and PMP22 is required to maintain stable myelin (D’Urso et al., 1999), perhaps by assuring the proper stoichiometry of the two proteins. Anot her protein that interacts with PMP22 is the P2X7 purogenic transmembrane receptor, an ion channel gated by extracellular ATP (Wilson et al., 2002). The P2X7-PMP22 protei n interaction occurs via a unique cytoplasmic domain of the P2X7 receptor. Ther efore, at least in some instances, PMP22’s role in cellular processes may involve the m odulation of other transmembrane proteins.

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11 A member of a family of four tran smembrane proteins, PMP22 has homology to the epithelial membrane protein-1 (EMP-1), -2 and -3 (Taylor et al., 1995; Lobsiger et al., 1996; Taylor and Suter, 1996; Chen et al., 1997). The function of the EMPs remains unclear. However, the most studied of these proteins, EMP-2, associates with 1-integrin and regulates cell-substrate adhesion (Wad ehra et al., 2002), modulates the surface expression of the class I major histocompatabi lity complex (Wadhera et al., 2003), and of caveolins and glycosylphosphatidyl inosito l-linked proteins (W adhera et al., 2004). Studies of EMP-2 further suggest a role for the PMP22-EMP family of proteins in the modulation of other membrane -associated molecules. Role for PMP22 in Cell Proliferation and Cell Morphology Altered PMP22 expression in various in vitro cell lines indicates a role for the protein in regulating both the progressi on of the cell cycle and cell morphology. Upregulated in serum-starved NIH3T3 cells, PMP22 mRNA levels are similarly elevated by contact-inhibited growth arrest (Schne ider et al., 1998; Cic carelli et al., 1990; Manfioletti et al., 1990; Suter et al., 1994). A coincident in crease in PMP22 message and induction of growth arrest is found in Schwann cells (Welch er et al., 1991; Zoidl et al., 1995) and adipoblasts (Shugart et al., 1995). T hus, elevated PMP22 expression appears to be correlated with exit from the cell cycle, at least in a subset of cells. These findings are substantiated by studies that artificially overexpress exogenous PMP22 in Schwann cells, leading to growth arrest (Z oidl et al., 1995). Conversely, Sc hwann cell proliferation is augmented by a reduction of PMP22 message by e xpression of antisense mRNA (Zoidl et al., 1995). Therefore, these studies indicate that PMP22 is capable of both positive and negative regulation of cell-cycle progression. Additionally, nerve growth factor

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12 differentiated PC12, but not C6 glioma cells have an increased level of PMP22 mRNA (DeLeon et al., 1994), suggesting a role for PMP22 in cell differentiation. Elevated levels of exogenous PMP22 can also result in altered cell morphology. In NIH3T3 and HEK-293, but not REF52 ce lls, overexpression of PMP22 results in plasma membrane blebbing and eventual apopt osis (Fabretti et al., 1995; Brancoloni et al., 1999; Wilson et al., 2002), a phenotype inhibited by coexpression of the anti-apoptotic bcl-2 gene (Brancolini et al., 1999). Prolonged activation of the P2X7 purigenic receptor, a PMP22 binding partner, also leads to membrane blebbing and apoptosis (Wilson et al., 2002). The expression of P2X7 in immune and epithelial cells, in addition to Schwann cells (Grafe et al., 1999; Colomar et al., 2001), indicates a potential mechanism for the membrane bl ebbing and apoptosis induced by PMP22 expression. Following PMP22 overexpression, NI H3T3 cells experience RhoA GTPase-dependent altered cell spreading (Brancolini et al., 1999). Conversely, by inhibiting endogenous RhoA GTPase activity, REF52 cells become sensitive to altered cell shape in response to PMP22 overexpr ession (Brancolini et al., 1999). These experiments implicate the Rho GTPase path way in modulating the effects of PMP22 expression. In NIH3T3 cells, PMP22 that is incapable of reaching the plasma membrane, either due to the artificial addition of an ER retrieval signal to th e carboxyl-terminus or the presence of the Tr-J poi nt mutation, is unable to alte r cell spreading or induce apoptosis (Fabretti et al., 1995; Brancolini et al., 2000). While capable of reaching the cell surface and increasing apoptos is, PMP22 protein with a de fective glycosylation motif

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13 fails to affect cell morphology as significantly as the wt protein (Brancolini et al., 2000), suggesting that this motif is cruc ial for certain cellular functions. Prior to the PMP22-induced morphological changes or apoptosis that occur in response to PMP22 overexpression, wt, but not Tr-J, protein is localized to perinuclear endosomes and to large vacuoles near the cell periphery (Chies et al., 2003). These actin/phosphatidyli nositol (4,5)-biphosphate (PIP-2)-pos itive vacuoles ar e part of the ADP-ribosylation factor 6 (Arf-6) plasma -membrane-endosomal recycling pathway involved in cell-cell adhesion and cell migration (reviewed in Donaldson et al., 2003). Thus, PMP22 appears capable of regula ting cell proliferation, morphology and differentiation, all aspects cruc ial to the proper formation of myelin, the structure most obviously affected by altered expression of the gene. In summary, since the original discover y of PMP22 little has been learned about its normal function in the myelinating Schwann cell. This may be due to difficulty in studying the complex and largely unknown process of normal PNS myelination. Advances in dissecting PMP22-related dis ease pathogenesis have focused on protein trafficking and turnover or the characteriza tion of nerve pathology. The function of the protein is largely being examined in cell t ypes unrelated to myelination, an approach justified by extensive non-PNS PMP22 expres sion. However, since message levels are elevated in epithelial cells such as those of the gut, it seems logical to first characterize the localization of PMP22 in these cells. Furthermore, well-characterized cell models of polarized epithelia, amenable to experiment al manipulation, provide certain technical advantages allowing for further investigati on of PMP22’s function. The ultimate goal of these studies lies beyond determining a function for the protein in non-neural cell types,

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14 by providing a foundation of knowledge to be us ed in revealing the role of PMP22 in peripheral nerve myelin in health and diseas e. The purpose of this study was to examine the expression and subcellular localization of PMP22 in non-neural cell types and provide novel insights into the role of the protein in the cell membrane.

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15 CHAPTER 2 PERIPHERAL MYELIN PROTEIN 22 IS A NOVEL CONSTITUENT OF INTERCELLULAR JUNCTIONS IN EPITHELIA Note The work presented in this chapter was published in Proceedings of the National Academy of Sciences USA 98(25) 14404-14409 (2001) Amy Yazdanpour and Christoph Rahner assisted with the cryosectioning and immunostaining, Stepha nie Amici assisted with the RT-PCR and Western blots, and Bradle y Fletcher assisted with the retroviral infections. Introduction Peripheral myelin protein 22 (PMP22), also known as gas3 is a tetraspan glycoprotein with proposed roles in peri pheral nerve myelin formation, cell-cell interactions, and cell prolifer ation (Suter and Snipes, 1995). PMP22 expression is highest in myelin-forming Schwann cells; however, PM P22 mRNA can be detected in a variety of non-neural tissues. Epithelial cells of the lungs and intestin es are known to express the highest levels of PMP22 mRNA outside of the peripheral nervous system (Baechner et al., 1995; Taylor et al., 1995; Wulf et al., 1999) yet the localization or the role of the protein in these tissues has not been de termined. Although the function of PMP22 in Schwann cells and non-neural cells is larg ely undefined, it is we ll established that deletions, duplications, or mutations in PM P22 account for the majority of heritable demyelinating peripheral neuropathies, includi ng Charcot-Marie-Tooth disease type IA. Myelin-forming Schwann cells and epithe lial cells, two cell types with high levels

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16 of PMP22 mRNA expression, share similarities in that they are both polarized and maintain compositionally unique membrane dom ains. In addition, sim ilar to the barrier function of epithelia, Schwann cells sepa rate intramyelinic and extramyelinic extracellular space (Mugnaini and Schnapp, 1974) The molecular bases of how Schwann cells attain these functions are not yet unde rstood, although they ar e likely to involve specialized intercellular junctions, such as a dherens and/or tight j unctions (TJs). Freeze fracture studies of PNS myelin detected rows of TJ-like fibrils within the Schwann cell membrane (Shinowara et al., 1980); neverthele ss the identities of the proteins forming these structures are unknown. Recent studies re vealed the presence of TJ strands in CNS myelin (Morita et al., 1999a), which is deposited by oligodendrocytes. A protein component of TJ strands in CNS myelin is oligodendrocytespecific protein/claudin-11, a PMP22-related, tetraspan membrane protein (M orita et al., 1999a; Bronstein et al., 1996). In addition to oligodendrocyte-speci fic protein/claudin-11, PMP22 shares significant sequence identity and structural simi larity with other claudins, including the first discovered claudin in liver, claudin-1 (Furuse et al., 1998a). The claudin protein family now includes more than 20 members with unique, as well as overlapping, tissue distribution (Mitic et al., 2000, Rahner et al., 2001; Tsukita et al., 2001). Claudins appear to have roles in the formation of TJ stra nds and in the establishment of the ionic selectivity of the junctional barrier (Ts ukita et al., 2001). The essential function of claudins at TJs is supported by recent reports on claudin misexpression and disease-causing alteration in epithelial phys iology (Simon et al., 1999; Wilcox et al., 2001). Occludin, also a tetraspan pr otein of TJs, is an adhesive molecule that may have roles in the barrier function of TJs (Furuse et al., 1996; Wong and Gumbiner, 1997; Van

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17 Itallie and Anderson, 1997). These transmembr ane junctional protei ns form complexes with cytoplasmic molecules, such as zonula occludens-1 and -2 (ZO-1, ZO-2), which link the membrane proteins to cytoskeletal elements (Fanning et al., 1998). As the molecular architecture of intercellular junctions is bei ng uncovered, studies show that in addition to ionic barrier and fence functions, TJs are invo lved in intracellular vesicle targeting and signaling (Zahraoui et al., 2000). Based on the mRNA expression pattern, a nd the primary and secondary structure of PMP22, we hypothesized that PMP22 might be a component of intercellu lar junctions in epithelia. Therefore, we examined th e expression and localiz ation of PMP22 in cultured epithelia and a variety of tissues with ZO. Using immunochemical, biochemical, and molecular approaches, we found that in epithelial cells PMP22 is coexpressed with occludin and ZO-1 at or near TJs and that overexpression of PMP22 in L cell fibroblasts mediated the formation of ZO-1-positive inte rcellular junctions. These studies suggest that the plasma membrane-associated biologi cal function of PMP22 might involve a role in the establishment and/or maintenance of in tercellular junctions a nd possibly of TJs. Materials and Methods Cell Culture Primary Schwann cell cultures were established from newborn rat pups (Notterpek et al., 1999b). L cells (American Type Culture Coll ection) were maintained in 10% horse serum containing DMEM. MadinDarby canine kidney (MDCK) cells were cultured in 10% FBS containing DMEM on 0.4-m pore size Transwell filters (Costar), or glass coverslips, with or without type I collagen coat ing. Highly polarized, confluent MDCK cell monolayers were in cubated with 4 mM EGTA for 1-4 h to chelate the calcium from the culture medium (Gumbine r and Simons, 1986; Kartenbeck et al., 1991).

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18 EGTA treatment results in th e rounding up of the cells and di sassembly of intercellular contacts. Retroviral Overexpression of PMP22-myc in MDCK and L Cells The mouse PMP22 ORF with a myc epitope in the 2nd extracellular loop (Tobler et al., 1999) was directionally inserted into the retroviral plasmid pBMN (H itoshi et al., 1998). The resulting pBMN-PMP22myc, or a control pBMN-GFP (green fluorescent protein) plasmid, was transien tly transfected into the amphot ropic retroviral packaging cell line Phoenix A (obtained from Garry Nola n, Stanford University, CA). Retroviral supernatants were collected after 30 h incuba tion at 32C and directly applied to 1 106 MDCK or L cells (~40% confluency). Retrovi ral transductions were performed at 32C for 24 h in the presence of 5 g/ml polybrene. Forty-eight hours pos tinfection, cells were replated and allowed to form confluent monolayers. Estimated from the number of pBMN-GFP-expressing cells, the infection rate in the L cells was ~99% and ~15% in MDCK cells. Immunostaining Procedures MDCK cells and 2or 8-m thick cryosect ions of adult rat li ver and colon were double immunostained with pol yclonal anti-PMP22 (Notterpek et al., 1999b) and monoclonal anti-tight junction protein anti bodies, according to published procedures (Itoh et al., 1997). Primary antibodies include d monoclonal anti-occl udin and anti-ZO-1 (Zymed), and polyclonal anti-claudin-1 (Z ymed) and anti-PMP22 (Notterpek et al., 1999b). Twelve distinct polycl onal antibodies made against 16-aa peptides of the 1st (amino acids 27-42) or 2nd (amino acids 117-133) extracellular loops of the mouse, rat, or human PMP22 were used to localize PMP 22 in the studied samples. Preimmune and

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19 peptide preadsorbed (0.1 mg/ml) rabbit se rum and nonspecific mouse IgGs served as controls of antibody binding. Bound primary antibodies were detected with Alexa fluorochrome-conjugated sec ondary antibodies, including FITC-conjugated anti-mouse IgG and Texas red-conjugated anti-rabbit IgG (Molecular Probes). Nuclei were stained with Hoechst dye. Coverslips were mounted by using a ProLong Antifade kit (Molecular Probes), and images were acquired with a Spot camera attached to a Nikon Eclipse 1000 or an Olympus MRC-1024 confocal microscope Images were processed for printing by using Adobe PHOTOSHOP 5.0. To increase the resolution of the i mmunoreactivity in filter-grown MDCK cells, filters with confluent monolayers were sectioned after freezing and processed for immunostaining (Itoh et al., 1997). For optim al detection of the myc epitope-tagged PMP22, retrovirally infected MDCK and L cel ls were fixed in 4% paraformaldehyde, followed by permeabilization and immunolab eling with polyclonal or monoclonal anti-myc antibodies (Ryan et al., 2000). These fixation conditions are suboptimal for the detection of endogenous TJ prot eins, which is reflected by reduced levels of claudin, ZO-1, and occludin-like immunoreactivities. RNA isolation, Northern analysis, and Reverse Transcriptase-PCR (RT-PCR) Total RNA was isolated from rat liver and rat Schwann cells by using the TRIzol reagent (GIBCO Life Technologies). The Titan One Tube RT-PCR System (Roche Diagnostic) was used to generate and am plify a 425-bp PMP22 cDNA fragment by using 1 g of total RNA. Specific primers were synthesized according to the nucleotide sequence of rat PMP22 (sense primer 5'-ACACTTGACCCTGAAGG C-3' and reverse primer 5'-AGCATCAGAAGGACA CCG-3'). Half of each RT-PCR product was digested

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20 with a PMP22 sequence-specific restriction enzyme ( Bsa I), and samples were analyzed on acrylamide gels. Negative controls includ ed samples without the RT enzyme and samples that were RNase-treated. Biochemical Procedures Bile canaliculi-enriched fractions from P 70 rat livers were processed according to established procedures (Song et al., 1969; Tsukita and Tsukita, 1989). Three different membrane fractions were collected (Song et al., 1969) and analyzed by Western blotting with anti-PMP22 antiserum. To confirm that our anti-human, antirat, or anti-mouse PMP22 antibodies can detect canine PMP22 in MDCK cells we purch ased frozen dog sciatic nerves (Pel-Freez Biologicals). Adult rat, mouse, and canine sciatic nerve lysates were analyzed on 12.5% SDS gels as described (N otterpek et al., 1999b). Control and retrovirally infected MDCK cell monolayers were extracted with 0.5% TX-100-containing buffer, and detergen t soluble (S) and inso luble (I) fractions were collected (Jou et al., 1998). Control and retrovirally infected L cells were directly lysed in SDS gel sample buffer, and protein concentrations were determined. Endoglycosidase H and N-glycosidase digestions were performed as described (Pareek et al., 1997). To prevent the aggreg ation of PMP22, protein samples were heated to 80C before loading of the gels. Gels were tr ansferred to nitrocellulose membranes and processed for immunoblotting with monoclona l anti-occludin and anti-ZO-1 (Zymed), and polyclonal anti-claudin-1 (Zymed) a nd anti-PMP22 (Notterpek et al., 1999b) antibodies. Bound antibodies were detected with horseradish peroxidase-conjugated

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21 anti-mouse, or anti-rabbit, secondary antibodies (Sigma) by using ECL chemiluminescent reagents (Amersham Pharmacia). Results PMP22 Localizes to Cell-Cell Junctions in the Rat Liver and Colon Previous studies have shown high levels of PMP22 mRNA in various non-neural tissues (Baechner et al., 1995; Taylor et al., 1995; Wulf et al., 1999); however, to date the localization and the expression of protein at these sites has not been determined. Using a TX-100 pre-extraction immunostaining procedure (Itoh et al., 1997), we detected bright PMP22-like immunoreactivity at the surface ep ithelium of the mucosa in colon (Fig. 2-1A) and liver bile canaliculi (Fig. 2-1 C, E, and F). In colon, PMP22 and ZO-1 are found at apical junctions of epithelial cells and in small blood vessels transversing the submucosa (Fig. 2-1 A and B, arrowheads). In liver, PMP22 and ZO-1 are colocalized to bile canaliculi (Fig. 2-1 C and D, respectiv ely); however, only ZO-1, but not PMP22, is present at endothelial cell junctions of the portal vein (Fig. 2-1E, arrows). Nerve terminals show bright PMP22 and no ZO-1 im munoreactivity (Fig. 2-1E). In addition to ZO-1, PMP22 is colocalized with occludin at bile canaliculi (Fig. 2-1 F and G, respectively). The lack of PMP22-like immunor eactivities in liver sections incubated with preimmune (Fig. 2-1A inset) or anti genic peptide preincuba ted serum (Fig. 2-1F, inset) support the specificity of the PMP22-like immunostain ing at these novel locations. The localization of PMP22 in colon epithelium and bile canaliculi was verified by eight distinct antibodies, including antisera ra ised against the 1st rather than the 2nd extracellular loop of the pr otein (data not shown). The expression of PMP22 in liver wa s confirmed by RT-PCR (Fig. 2-1H) and Western analysis (Fig. 2-1I). Using specific primers to th e rat PMP22 cDNA we detected

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22 Figure 2-1. Coexpression of PMP22 with ZO-1 and occludin in colon epithelium and bile canaliculi. Frozen sections of nor mal adult rat colon (A and B) and liver (C-G) were coimmunostained with poly clonal anti-PMP22 (A, C, E, and F) and monoclonal anti-ZO-1 (B, D, and E) or occludin (G) antibodies. (A and B) Confocal images showing the presence of PMP22 at the surface epithelium of the mucosa and in submucosal vasculature (arrowheads). (E) A highresolution thin section of adult rat liver stained with anti-PMP22 (red), anti-ZO-1 (green) antibodies and nuclear dye (blue). PV, porta l vein; N, nerve terminal; BD, bile duct; HC, hepatocyte. Liver sections incubated with preimmune serum (A Inset) or peptide preadsorbed antiserum (F Inset) do not result in TJ-like immunostaining. Magni fications: 40 (A-E) and 60 (F and G). (H) The expression of PMP22 mRNA in liver was verified by RT-PCR. BsaI undigested (-) and digested (+) PCR-amplified fragments are shown for each sample. The numbers on the left i ndicate bp. (I) Membrane pellets (P) (75 g) from adult rat liver homogena tes were fractiona ted and proteins isolated at sucrose densities 1.22 (D 3) and 1.18 (D2) and 1.16 (D1) were analyzed (75 g/lane) for the presence of PMP22. Rat sciatic nerve (N) lysate (4 g) was used as a positive control for the anti-PMP22 antibody immunoreactivity. S, total liver supernatan t (75 g). Molecular mass, in kDa. the identical 425-bp fragment in liver and Sc hwann cell RNA (Fig. 2-1H). The identities of the PCR fragments were verified by Bsa I restriction enzyme digests. To further

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23 corroborate the expression of PMP22 in liv er, crude liver membrane pellets were subfractionated by discontinuous sucrose density-gradient ultracentrifugation (Fig. 2-1I) (Song et al., 1969; Tsukita and Tsukita, 1989). Although PMP22 is difficult to detect in total liver membrane preparations, in bile ca naliculi-enriched fractions (sucrose density fractions 1 (D1) and 2 (D2)) (Ryan et al., 2000 ), we observed a significant enrichment for PMP22 (Fig. 2-1I). The majority of PMP22 wa s concentrated at the interface of sucrose densities 1.22 and 1.18 (D2) and was absent from the highest sucrose density fraction (D3), which contains nuclei, mitochodria, and erythrocyte gho sts (Song et al., 1969). Parallel blots incubated with preimmune or antigenic peptide preadsorbed serum were completely blank at the 21-to 35-kDa range (data not shown). The slower migration of PMP22 in bile canaliculi compared with sc iatic nerve is likely caused by differential posttranslational modification of PMP22 in myelin and non-neural tissue. PMP22 is Localized to Epithelial Apical Cell Junctions The in vivo tissue localization studies suggest that PMP22 is a component of intercellular junctions in epithelia, therefor e we examined the distribution of PMP22 in MDCK cell monolayers. In subconfluent MD CK cell cultures, PMP22 (Fig. 2-2A) is found at anti-ZO-1 antibody (Fig. 2-2B) imm unoreactive intercellu lar junctions. The nuclear staining observed with the anti-PM P22 antibody has been described before (Pareek et al., 1997) and, in part, is caus ed by nonspecific imm unoreactivity of the antiserum (Fig. 2-2 A and C, insets). The dist ribution of PMP22 in c onfluent filter-grown MDCK monolayers also was determined (F ig. 2-2 C-G). Similar to the subconfluent cultures (Fig. 2-2 A and B), PMP22-like immunoreactivity (Fig. 2-2 C and E) is colocalized with ZO-1 (not shown) and occludin (Fig. 22 D and F) at intercellular

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24 Figure 2-2. Coexpression of PMP22 with ZO -1 and occludin at ce ll-cell contacts in MDCK cells. Subconfluent (A and B) and filter-grown (C-G) MDCK cells were immunostained with polyclonal anti-PMP22 (A, C, E, and G) and monoclonal anti-ZO-1 (B), or anti-occludi n (D and F) antibodies. (A Inset) Cells stained with preimmune rabbit se rum. In filter-grown MDCK cultures PMP22 (C) is codistributed with occludi n (D) at apical cell contacts. PMP22 antigenic peptide preadsorbed antiserum does not stain intercellular contacts of MDCK cells (C Inset). On sectioned (8 m) filt ers (Z plane) PMP22-like immunoreactivity (E and G) is associat ed with the apical border of the monolayer, which is also reactive w ith the anti-occludi n (F) antibodies (arrows in E and F). (G) Anti-PMP22 (re d) and Hoechst nuclear dye (blue) stained MDCK cell monolayer is shown. Magnifications: 60 (A, B, and E-G) and 40 (C and D). (H) Protein blots of (18 g/lane) normal adult rat, canine, and mouse sciatic nerves were reacte d with anti-PMP22 antiserum. The upper arrow on the right indicate s the glycosylated 22-kDa PMP22, while the lower arrow points to the newly synthesize d 18-kDa, endoglycosidase-H sensitive protein. Molecular mass, in kDa. junctions. Sectioned filters, double-stai ned with anti-occludin and anti-PMP22 antibodies, demonstrate that PMP22 (Fig. 2-2E ) is present at api cal cell-cell contacts, similar to occludin (Fig. 2-2F). The colocal ization of PMP22 and occludin at apical

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25 intercellular contacts was confirmed by confocal microscopy and rotated three-dimensional images of deconvoluti on microscopy (not shown). Because MDCK cells are of canine origin, we verified by Western analysis that our anti-PMP22 antibodies raised against human, rat, or mouse peptid es can recognize the dog PMP22 (Fig. 2-2H). As the anti-PMP22 immunoblot shows, we pos itively identified the dog PMP22 in total sciatic nerve lysates, using an anti-human PMP22 antibody that stains intercellular junctions in MDCK cells (Fig. 2-2H). On SDS gels, the canine nerve PMP22 has a similar mobility as the rat or the mouse protein (~22 kDa) and shifts ~4 kDa upon N-glycosidase treatment (not shown) (Pareek et al., 1997). To begin to elucidate the relations hip of PMP22 to known tight junctional proteins, filter-grown MDCK cell monolayer s were treated with EGTA to disrupt intercellular contacts (Fig. 2-3) Previous studies showed that such treatment leads to the internalization of proteins found at adherens and TJs (Cereijido et al., 2000). After a 1-h EGTA treatment of the cultures, the major ity of anti-ZO-1 and anti-occludin antibody immunoreactive intercellular co ntacts disappeared, and both occludin (Fig. 2-3B) and ZO-1 (not shown) were in ternalized in vesicles. Us ing double immunolabeling, PMP22 and occludin were detected together in a subpopulation of vesicles (arrows in Fig. 2-3 A and B). These results strongly support that PMP22 is a protein component of apical intercellular junctions in epithelial cells. Tight junctional proteins are insoluble in TX-100 (Jou et al., 1998); therefore we compared the solubility properties of PMP 22 to ZO-1, occludin, and claudin-1 (Fig. 2-3C). Confluent MDCK monolayers were incubated with 0.5% TX-100 containing

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26 Figure 2-3. Internalization of PMP22 with occludin in EGTA-treated MDCK cells. Confluent MDCK monolayers were cultu red in the presence of 4 mM EGTA for 1 h followed by immunostaining w ith polyclonal anti-PMP22 (A) and monoclonal anti-occludin (B) antibodies. Arrows point to vesicles that contain both PMP22 (A) and occludin (B). (M agnification: 60.) (C) Confluent MDCK cell monolayers were extracted with 0.5% TX-100 containing buffer and detergent soluble (S), and detergent-insoluble (I) fractions were immunoblotted with antibodies ag ainst ZO-1, occludin, PMP22, and claudin-1. Molecular mass, in kDa. buffer, and detergent soluble and insoluble fr actions (Jou et al., 1998) were analyzed for the four antigens (Fig. 2-3C). In agreement with previous studies, we found that the greater portion of ZO-1 and o ccludin remain in the TX-100 insoluble fraction (Jou et al., 1998), whereas the majority of claudin-1 is ex tracted in the detergent (S). The greater solubility of claudin-1 correlates with its high intracellular levels in MDCK cells (Fig. 2-4B). In contrast to claudin-1, PMP22 is largely insoluble in 0.5% TX-100 containing buffer (I) (Fig. 2-3C). Epitope Tagged PMP22 is Targeted to Epithelial Cell Junctions To further validate our findings on the ap ical junctional localization of PMP22 in MDCK cells, we studied the targeting of myc-tagged mouse PMP22 (Fig. 2-4). A myc

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27 epitope tag in the 2nd extrac ellular loop of PMP22 does not interfere with the normal processing and trafficking of the protein (Tobler et al., 1999, Ryan et al., 2000). MDCK cells infected with the pBMN-PMP22-myc cons truct were plated on coverslips or filters and allowed to proliferate. In subconfluent cultures we detected PMP22-myc (Fig. 2-4 A and C) and claudin-1 (Fig. 2-4 B and C) at intercellular junctions. Cells that are overexpressing the mouse PMP22-myc are able to integrate and establish intercellular contacts with parental cells (Fig. 2-4 A, C, and D). Furthermore, in filter-grown MDCK cell monolayers, the exogenous PMP22-myc protei n is correctly targeted to apical cell contacts (Fig. 2-4E). The apical junctional targeting of PM P22-myc also was established by Western analysis (Fig. 2-4F). Anti-myc immunoblot s of control, pBMN-GFP, and PMP22-myc infected MDCK cells specifi cally detects an ~27-kDa a nd a less abundant ~33-kDa band in the PMP22-myc sample (Fig. 2-4F, lane 3). Both ~27-kDa and ~33-kDa bands shift upon deglycosylation with N-glycosidase (d ata not shown) (Pareek et al., 1997). The arrowheads at ~34 kDa indicate a nonspecifi c protein that is immunoreactive with the myc antibody in control (Fig. 2-4F, lane 1) and GFP-infected (Fig. 2-4F, lane 2) cells. Significantly, similarly to the endogenous canin e PMP22 (Fig. 2-3C), the majority of PMP22-myc is also insoluble in 0.5% TX-100 (F ig. 2-4F, lane I). In addition to the ~27 kDa and ~33 kDa bands, the detergent-inso luble fraction contai ns a range of high molecular mass anti-myc antibody immunoreac tive proteins, which likely represent aggregates of PMP22 multimers (Tobler et al., 1999). Together, these overexpression experiments strongly support that PMP22 is a component of the ap ical intercellular junctional complex in epithelia.

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28 Figure 2.4. Exogenously expressed PMP22-myc is targeted to TJs in MDCK cells. pBMN-PMP22-myc-infected cells were cultured on coverslips (A-D) or Transwell filters (E) and immunostained with monoclonal anti-myc (A and C-E), and polyclonal anti-claudin-1 (B and C) antibodies. Note, only ~15% of the cells (green cells) were infected with the PMP22-myc construct (A and C-E). PMP22-myc is targeted to anti -claudin-1 immunoreac tive intercellular contacts (arrows in A-C) and PMP22-my c-expressing cells fo rm contacts with noninfected cells (D). Nuclei were stai ned with Hoechst dye (C and D Inset). [Magnifications: 60 (A-C), and 40 (D and E).] (F) The expression of PMP22-myc was verified by anti-myc Western analysis. Lysates of PMP22-myc-infected cells (lane 3) show expression of a ~27-kDa and a ~33-kDa PMP22-myc protein. A nonspecifi c ~34-kDa band is present in all samples, including uninfected contro l (lane 1) and pBMN-GFP (lane 2)infected cell lysates (arrowheads). Th e majority of PMP22-myc protein is insoluble in TX-100 (I). S, TX-100 so luble. Molecular mass, in kDa. Exogenous PMP22 Alters Cell-Cell Contacts in L-Fibroblasts Overexpression of claudins or occludin in nonadherent L fibroblasts has been shown to induce the formation of intercellu lar contacts, including well-organized TJs

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29 Figure 2-5. Colocalization of PMP22-myc with ZO-1 at intercellular junctions of L fibroblasts. Uninfected control L cells (A and B) show diffuse ZO-1-like membrane staining with focal concentr ation at cell processes (A) and low levels of nonspecific immunoreactivity with polyclonal anti-myc (B) antibodies. In PMP22-myc-infected cells PMP22-myc is detected at cell-cell contacts (arrows in C and F), which ar e costained with the anti-ZO-1 antibody (arrows in D and G). PMP22-myc is detect ed in aggresome-like structures in some cells (* in F). Nuclei are staine d with Hoechst dye (H). (Magnification: 60.) (E) The expression and proces sing of PMP22-myc was studied in lysates of control, uninfected (lane 1), and PMP22-myc-infected L cells (lanes 2-5) by anti-myc Western blotting. Un treated (lane 2), endoglycosidase H (lane 3), N-glycosidase (lane 4), and no enzyme (lane 5) PMP22-myc samples are shown. N-glycosidase (N) treatment of PMP22-myc cell lysates results in a characteristic shift of PMP22, fr om a mature high molecular mass, endoglycosidase H-resistant form (upper arrow) to a deglycosylated, lower molecular mass core protein (lower arrow). The N-glycosidase resistant, anti-myc immunoreactive ~29-kDa band likely represents a mono-ubiquitinylated PMP22-myc (lane 4). Molecular mass, in kDa. (Furuse et al., 1998b). Therefore, we examin ed the targeting of mouse PMP22-myc in mouse L fibroblasts (Fig. 2-5). Parental L cells express low levels of PMP22 mRNA and undetectable PMP22 protein (data not shown). In agreement with previous studies in parental L cells, ZO-1 is diffu sely distributed over cell bodies and concentrated in puncta at processes (Fig. 2-5A). In cells overexpressing PMP22-myc, we detected anti-myc (Fig.

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30 2-5 C and F) and anti-ZO-1 (Fig. 2-5 D a nd G) immunoreactivity at cell-cell contacts. Well-defined intercellular junctions can be seen, which are immunoreactive with both anti-myc and anti-ZO-1 antibodies (arrows in Fig. 2-5 C, D, and F-H). Significantly, overexpression of PMP22 alters the distribution of ZO-1 and appears to cause the recruitment of ZO-1 to inte rcellular contacts (compare Fig. 2-5A and D). Although the formation of intermittent intercellular contac ts is consistently observed in PMP22-myc infected L cells, overexpression of PMP22 does not appear to induce long strands involving multiple cells, such as described for claudins (Furuse et al., 1998b). Cells that have integrated multiple copies of PM P22-myc often contain intracellular PMP22 aggregates, termed aggresomes (Fig. 2-5F *) (Notterpek et al., 1999a). Uninfected (Fig. 2-5B) or pBMN-GFP-infected ( not shown) L cells do not adhe re together and exhibit low levels of nonspecific immunoreac tivity with the myc antibody. The expression and processing of PMP22myc in L cells was studied by anti-myc Western analysis of endoglycosidase H-trea ted and N-glycosidase-treated cell lysates (Fig. 2-5E). Overexpression of PMP22-myc in L cells yields several bands with apparent mobilities ~30 kDa. A portion of the overexpressed protein is resistant to endoglycosidase H treatment (Fig. 2-5E, lane 3) and likely represents the membrane fraction of the protein (Pareek et al., 1997). N-glycosidase treatment shifts the majority of these bands to ~22 kDa, which corresponds to the peptide backbone of PMP22-myc (Fig. 2-5E, lane 4). The anti-myc immunoreactive ~29-kDa band in the N-glycosidase-treated sample might represent ubiquitinylated PMP22myc, which is suggested by the presence of PMP22-myc aggregates in PMP22-myc infected L cells (Fig. 2-5F *) (Notterpek et al., 1999a). These L cell overexpression studies sugg est that PMP22 might serve an adhesive

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31 role at intercellular juncti ons and through indirect protei n interactions it affects the localization of ZO-1. Discussion Because of its well-established disease association, PMP22 has received considerable attention during the last decade (Suter et al., 1995; Naef and Suter, 1998). Although we have gained significant insight into the genetics of PMP22-associated peripheral neuropathies, as well as the intrace llular turnover and proc essing of PMP22 in normal and neuropathy Schwann cells, we st ill do not understand the function of the protein. Here, we present data on interce llular junctional localization of PMP22 in epithelial cells, suggesting th at PMP22 plays a role in cell-cell inte ractions. Given that PMP22 is primarily known as a peripheral nervous system myelin component, the expression of PMP22 at epit helial cell junctions may seem unexpected. Nonetheless, our results are in complete ag reement with previous studies on the tissue distribution of PMP22 mRNA (B aechner et al., 1995; Taylor et al., 1995; Wulf et al., 1999). One of the tissues with reported highest levels of PMP22 mRNA is the gastrointestinal tract (Baec hner et al., 1995), where we detected bright PMP22-like immunoreactivity at intercellula r junctions of absorptive colonic epithelium. In addition to the epithelial cells of the gastrointestinal tract, PMP22 is present at TJs of the liver. Previously, PMP22 mRNA expression was s hown to be high in embryonic liver; however, message levels decreased significan tly during postnatal development (Baechner et al., 1995). These findings ar e in agreement with reports describing that the turnover rate of junctional proteins at established memb rane contacts is fairly slow (48 h) (Pasdar and Nelson, 1989; McCarthy et al., 2000), th erefore the rate of PMP22 mRNA and protein synthesis in postembryonic liver is ex pected to be low. Nonetheless, by RT-PCR

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32 we detected PMP22 mRNA in the adult rat li ver; and by using an established membrane subfractionation procedure (Song et al., 1969), we identified PMP22 in bile canaliculi-enriched liver membrane preparations. Although PMP22 is expressed in a variet y of tissues, the gross pathological findings in PMP22 mutant animals are limite d to myelinated peripheral nerves. These data may indicate that Schwann cells are pa rticularly sensitive to PMP22 missexpression and/or that PMP22-related pr oteins compensate for the normal function of PMP22 in other tissues. A similar compensatory mechan ism might operate in occludin-deficient mice, which form morphologically and functi onally intact TJs (Sa itou et al., 2000). Our results on the epithelial localization of PMP 22 warrant a closer examination of PMP22 neuropathy animals, as it is known that certain PMP22 mutant mice display nonglial abnormalities, which are difficult to explain by myelination defects alone. For example, during early postnatal development homozygous Tr-J animals exhibit ~35% reduction in weight compared with wild-type litter mates (unpublished data) and die at around postnatal day 18 (Henry et al., 1983). The homozygous Tr-J condition is the only known lethal phenotype associated with PMP22 missexpression and it ca nnot be explained by peripheral myelination defects al one, as several other periphe ral myelin-deficient animals live normal life spans (Martini and Schac hner, 1997). These paradoxes regarding the phenotypes of PMP22 neuropathy animals have puzzled investigators of the field for many years; however, to date possible explanatio ns have not previously been put forth. The in vivo protein expression studi es suggest that PMP22 is a constituent of membrane junctions in epithelia; however, they provide limited information on the relationship of PMP22 to esta blished TJ membrane protei ns. The internalization of

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33 PMP22 with occludin, in EGTA -treated MDCK cells, support s the notion that PMP22 is a constituent of the apical junctional comple x. The detergent solubility properties of PMP22 imply that PMP22 might be associated with occludin and/or ZO-1, or other TX-100-insoluble proteins, rather than with claudin-1. Because PMP22 shares 28% amino acid sequence identity with claudin-1 (Takeda et al., 2001), it is important to note that in this assay the two proteins segreg ate differently. These results strongly argue against any cross-reac tivity of our anti-PMP22 antibodi es with claudin-1, or other claudins, as the two proteins are detected in opposite fractions of the cell lysates. Nonetheless, because PMP22 shares significa nt sequence identity with several members of the claudin gene family (Takeda et al., 2001), our studies raise the question of whether PMP22 may be another claudin. It has recently been established that all well-characterized claudins are ab le to reconstitute long TJ st rands in fibroblasts (Tsukita et al., 2001). Previous studies performed in HeLa cells (D’Urso et al., 1999) and our results in L cells suggest that PMP22 ha s adhesive properties, as it can mediate intercellular contacts between nonadherent cells and is able to recruit ZO-1 to newly formed cell junctions (Fig. 2-5). In comp arison to L cells, PMP22 overexpression does not appear to induce intercellular adhesion in C6 glioma cells (Takeda et al., 2001), a central nervous system-derived tumor cell line. These differences in response to PMP22 overexpression are likely the results of cell sp ecificities in en dogenous junctional molecules and/or differences in the pro cessing and trafficking of the overexpressed PMP22. Nonetheless, further studies will be ne cessary to examine the ultrastructure of these newly formed membrane junctions, as cl audins are known to mediate the assembly

PAGE 43

34 of long fibrils, in comparison to short strands that are formed by occludin overexpression (Van Itallie and Anderson, 1997; Furuse et al., 1998b). Besides structural similarities, PMP22 sh ares functional proper ties with some of the claudins. Recent reports revealed that single point mutations in claudin-16 and claudin-14 cause kidney and hearing abnor malities, respectively (Simon et al., 1999; Wilcox et al., 2001). Of the known claudins claudin-15 is the most homologous to PMP22, sharing 30% identity and 54% sim ilarity in their amino acid sequences (unpublished data). Although the total molecu lar mass of the claudins and PMP22 is identical (22 kDa), 4 kDa of the total mo lecular mass of PMP22 is comprised of carbohydrate that is attached to Asn-41 in the 1st extra cellular loop. This carbohydrate motif has a role in the homodimerization of PMP22 (Tobler et al ., 1999; Ryan et al., 2000) and is required for the cell spreadi ng effect observed in PMP22-overexpressing fibroblasts (Brancoloini et al., 2000). The carbohydrate modification of PMP22 in epithelial cells is unknown, but it could have a role in me diating homophilic interaction between neighboring cells. The studies described here provide insights into the potential function of PMP22 in membrane physiology. Our results demonstr ate that PMP22 is a protein component of intercellular junctions, where it might medi ate the formation of cell-to-cell contacts and/or stabilize membrane contacts. A sim ilar role for PMP22 in the Schwann cells membrane could explain the demyelinating phe notypes associated with various forms of PMP22 misexpression.

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35 CHAPTER 3 TEMPOROSPATIAL EXPRESSION OF PE RIPHERAL MYELIN PROTEIN 22 AT THE DEVELOPING BLOOD-NERVE AND BLOOD-BRAIN BARRIERS Note The work presented in this chapter was published in The Journal of Comparative Neurology 474(4) 578-588 (2004). Julie Oakley and Shale Joy assisted with the cryosectioning and Stephanie Amici assisted wi th the establishment of choroid epithelial cultures and Northern blot analysis. Introduction The peripheral (PNS) and central ne rvous systems (CNS) are privileged environments, selectively restri ctive to molecules found in th e general circulatory system. Enclosing the peripheral nerve endoneurium is the blood-nerve barrier (BNB), established and maintained largely by inter cellular junctions of the perineurium and endothelial cells of endoneur ial vasculature (Allt and Lawrenson, 2000; Smith et al., 2001). The blood-brain barrier ( BBB) similarly provides a rest rictive environment for the CNS parenchyma, relying on cell-cell juncti ons of the brain va sculature, choroid epithelium and arachnoid (Saunders et al., 2000). In the mature BBB, tight junctions are t hought to regulate the pa racellular flow of molecules (Kniesel and Wolburg, 2000; H uber et al., 2001). During development, interendothelial junctions of th e brain microvasculature displa y increasing ultrastructural complexity (from ~E11 to early postnatal) with a loss of expanded para cellular clefts and more strand continuity (Schulze and Firth, 1992; Stewart and Hayakawa, 1994; Kniesel et

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36 al., 1996). Nonetheless, throughout developm ent, functional studies detected low transendothelial permeab ility to serum proteins (Knies el and Wolburg, 2000; Saunders et al., 2000). Thus, changes in ultrastructural j unction complexity may not be necessarily concurrent with physiologic alterati ons. The choroid epithelium regulates the composition of the cerebrospinal fluid (CSF), which in the mature mammalian brain has a low protein concentration as compared to se rum (Dziegielewska et al., 2000). The CSF of the immature brain contains high levels of protein (Dziegielews ka et al., 2000) that bypass restrictive tight junctions (Ek et al ., 2003) and penetrate th e choroid epithelium through an intracellular route (Balslev et al., 1997; Knott et al., 1997). In the embryonic mammalian brain, specialized neuroe pithelial junctions are thought to enable the brain parenchyma to exclude CSF proteins (Mollgard et al., 1987), thus maintaining a restrictive environment for the immature CNS. Several proteins involved in the formation and maintenance of the BBB intercellular junctions have been identified. Such proteins include cadherins and -catenin at adherens junc tions; tight junction-associat ed occludin, claudins, and junctional adhesion molecules (JAMs), and ZO-1 which can be detected at both tight and adherens junctions (Kniesel and Wolburg, 2000; Lippoldt et al., 2000; Huber et al., 2001; Wolburg et al., 2001; Vorbr odt and Dobrogowska, 2003). Pe ripheral myelin protein 22 (PMP22) is a recently described constituent of interepithelial junctions in the rat colon and Madin-Darby canine kidney cells (C hapter 1, Notterpek et al., 2001). Also known as growth arrest specific gene 3 ( gas3 ), PMP22 is a 22kD tetraspan glycoprotein with proposed roles in peripheral nerve myelination, cell-cell interactions and cell proliferation (Jetten and Suter, 2000). While the function of PMP22 in Schwann cells and non-neural

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37 cells is largely undefined, it is well establis hed that deletions, duplic ations or mutations of PMP22 account for the majority of heritable demyelinating peripheral neuropathies, including Charcot-Marie-Tooth disease type IA (Gabreels-Festen and Wetering, 1999). Indeed, PMP22 expression is highest in my elin-forming Schwann cells; however, PMP22 mRNA is readily detected outside of the PNS (Bosse et al., 1994; Taylor et al., 1995; Baechner et al., 1995; Parmantier et al., 1995; 1997; Lobsiger et al ., 1996; Wulf et al., 1999). In the rodent CNS, PMP22 message is found in a subset of motoneurons, and during embryogenesis, at the neuroepithelial ce ll layer of the ventri cular zone (Baechner et al., 1995; Parmantier et al., 1995; 1997; Wulf and Suter, 1999). Based on the mRNA expression pattern a nd epithelial distribution of PMP22, we investigated the presence of the protein at epithelial and e ndothelial cell contacts of the rodent BNB and BBB. Utilizing an antig enic PMP22 peptide-purified antibody, we detected PMP22 at occludin, claudin-5 and ZO-1 immunoreactive endothelial and choroid epithelial cell juncti ons. Furthermore, PMP22 is also present at unique neuroepithelial junctions of the subventricu lar zone in the embr yonic rat brain. These studies suggest a ubiquitous role fo r PMP22 at intercellular junctions. Materials and Methods Northern Blot Analysis For the RNA isolation, rats of the sp ecified ages were euthanized by CO2 asphyxiation followed by decapitation, or by de capitation alone (postnatal day 1 (P1) pups), and freshly collected tissues were im mediately frozen in liquid nitrogen. The use of animals for these studies has been approved by the Univ ersity of Florida IACUC. Total RNA was isolated using TriZol LS r eagent (Gibco BRL) from the following P1 and P70 rat tissues and cells: cortices without pi al surfaces or choroid plexuses, cortical

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38 microvessels, choroid plexuses, primary cultur es of choroid epithelia (see below) and Schwann cells (Notterpek et al., 2001) Total RNA from Schwann cells (4 g) and CNS derived tissues and cells (10 g) were electrophoretically separated on a formaldehyde agarose gel and transferred to a nylon me mbrane (Hybond, Amersham International). A 32P-labeled probe corresponding to the entire rat PMP22 open reading frame was used to detect PMP22 mRNA expression. An 18S ribos omal RNA probe (gift of Dr. Sue SempleRowland, University of Florida) served as an internal loading control. Message levels were quantified using the Scion Image dens itometry program (Scion Corporation). Morphological Studies of Rat Sciatic Nerve and Brain Embryonic day 15 (E15), P1 and P70 brains and P10, P20 and P70 sciatic nerves were removed from rats euthanized by CO2 asphyxiation followed by decapitation, and were immersed in liquid nitrogen cooled nmethylbutane. Nerve and brain samples were cut on a cryostat at 8 # m thickness. To increase the resolution of the studied molecules at endothelial junctions of brain microvessels, one mm3 isolated rat cortices were pressed between glass slides (Utsumi et al., 2000) and allowed to dry prior to fixation and immunostaining (Itoh et al., 1997; Notterpek et al., 2001). Primar y antibodies included monoclonal anti-occludin, an ti-claudin-5, anti-ZO-1 (Z ymed Laboratories), anti! -catenin (BD Transduction Labs), and polyclonal anti-occludin (Zymed) and anti-PMP22 (Notterpek et al., 2001). Pr eimmune rabbit serum, antigen ic PMP22 peptide-adsorbed (0.1 mg/ml) immune serum and secondary anti body alone served as controls of antibody binding specificity. Bound primary antibodies were detected with Alexa fluorochromeconjugated secondary antibodies, including FITC-conjugated anti-mouse IgG and Texas red-conjugated anti-rabbit IgG (Molecular Pr obes, Inc.). Nuclei were stained with

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39 Hoechst dye (Molecular Probes) Parallel samples were fixed as described above and stained with cresyl violet (Nissl stain). Coverslips were mounted by using the ProLong Antifade kit (Molecular Probes). Samples were imaged with a Spot camera attached to a Nikon Eclipse 1000 microscope and were formatted for printing by using Adobe PHOTOSHOP 5.0. Isolation and Culture of Brain Endothelia Primary cultures of brain endotheli al cells (BECs) from rat and mouse microvasculature were established following a published protocol (Tontsch and Bauer, 1989). Briefly, brain cortices were isolated from decapitated P1 rat or P4 mouse pups, followed by the removal of pial surfaces and choroid plexuses. Cortices were minced and enriched for microvessels by processing with a loose fitting Dounce homogenizer and centrifugation in a sucrose buffer. Isolated mi crovessels were disso ciated with type I collagenase (Sigma) and cultured on fibronectin -coated glass coverslips in 30% S180 cell (American Type Culture Collection) conditione d media, 10% heat-inactivated fetal calf serum, endothelial cell growth supplement (ECGS: 20 g/ml) (Becton Dickinson), and heparin (100 g/ml) (Sigma) in medium 199. Cells were fixed 48 hours after plating according to the protocol outlined above for the tissue samples (Itoh et al., 1997). To immunostain the BECs, polyclonal anti-PMP22 antibodies from whole rabbit serum were antigenic peptide-purified us ing a cyanogen bromide-activated sepharose 4B (Sigma) column. Rabbit IgGs bound to peptide, corr esponding to the 2nd loop (amino acids 117133) of the rat PMP22, were isolated and used for double immunolabeling with monoclonal anti-ZO-1 antibodies. Specificity of the antigenic peptide-purified antibodies

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40 for PMP22 was determined by Western blotti ng rat nerve lysates (Notterpek et al., 1999b). Isolation and Culture of Choroid Plexus Epithelia Primary cultures of rat and mouse choroi d plexus epithelia we re established with minor modifications of a publis hed protocol (Strazeille a nd Ghersi-Egea, 1999). Briefly, choroid plexus tissue was isolated from P1 rat or P4 mouse brains, rinsed in calciumand Mg2+-free Hank’s balanced salt solution (HBSS) and incubated in Pronase E (Sigma) for 25 minutes at 37C. After rinsing in HB SS, the tissue was incubated in 0.025% trypsin/HBSS (Ce llGro) with 12.5 # g/ml DNase I (Sigma), and following sedimentation, the supernatant was collected into chilled feta l bovine serum. This was repeated 5 times, followed by a 5 minute centrifugation of the pooled supernatants to collect the cells for resuspension in culture medium (DMEM w ith 10% fetal calf serum) and plating on a laminin-coated dish for two hours. Unatta ched cells, enriched for choroid plexus epithelia, were transferred to a laminin-co ated coverslip or dish and cultured until confluent (~ 6 days). Immunostaining for junc tional proteins and PMP22, or cell lysis for RNA isolation was performed, as described above. Calcium Switch Assay Primary cultures of mouse BECs were in cubated with 4 mM EGTA for 4 hours to chelate the calcium in the culture medium (Notterpek et al., 2 001). EGTA treatment results in disassembly and internalizati on of intercellular c ontacts (Gumbiner and Simons, 1986). Subsequently, cells were fixed and immunostained for PMP22 and ZO-1, as above.

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41 Results PMP22 is Detected at the BNB Compared to other tissues, peripheral nerv e myelin contains the highest level of PMP22 (Snipes et al., 1992; Lobsiger et al., 1996). It is not establishe d, however, if other Figure 3-1. Endothelial cell j unctions of the BNB in the de veloping and adult rat sciatic nerve are PMP22 immunoreactive. Rat sciatic nerve sections were stained with Nissl (A, F and K, arrows), or coimmunostained for ZO-1 (B, G and L) and PMP22 (C, H and M). On parallel sections, the localiz ation of occludin (D, I and N) and claudin-5 (E, J and O) was examined. In P10 (A-E), P20 (F-J) and P70 (K-O) rat sciatic nerves PMP22 colocalizes with ZO-1 at interendothelial cell junctions of nerve vessels. Unlike PMP22 (C, H and M), junctional occludin-like immunoreactivity (D, I and N) is less intense at P10 than at P20, or P70. No primary antib ody control (C') and peptide-adsorbed anti-PMP22 antibodies (M') fail to imm unolabel endothelial intercellular junctions. Scale bar = 30 # m (O). cell types in the PNS besides Schwann cells express detectable levels of the protein. Since PMP22 is present at ep ithelial and endothelial cell ju nctions in various non-neural

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42 tissues (Notterpek et al., 2001), we examined endoneurial blood vessels of the rat sciatic nerve for the expression of the protein duri ng the development of the BNB (Fig. 3-1). Longitudinal cryosections of rat sciatic nerv es were fixed and stained with Nissl, or double immunolabeled utilizing a procedure op timized to detect junctional molecules (Itoh et al., 1997). This detergent permeabiliz ation method does not permit the detection of PMP22 in peripheral myelin, which is th e most recognized staining pattern for the protein (Notterpek et al., 1997). At all studied ages, elongated nuclei of the nerve vasculature are readily visible by the Nissl stain (Fig. 3-1A, F, K). In the P10 rat nerve (Fig. 3-1A-E), ZO-1 (Fig. 3-1B) and PMP22 (Fig. 3-1C) are colocalized at interendothelial cell junc tions. Immunolabeling of parallel nerve sections reveal that occludin is barely detectable at these cellcell contacts in the P 10 nerve (Fig. 3-1D). Claudin-5, a tight juncti on protein predominantly expresse d in endothelia (Morita et al., 1999b), exhibits a primarily diffuse, likely intracellular pattern (Fig. 3-1E). With maturation of the nerve (at P20 and P70), both ZO-1 and PMP22 remain at endothelial junctions (Fig. 3-1G, L, and 3-1H, M, respectively), while occludin-like immunoreactivity gradually increases between P20 (Fig. 3-1I) and P70 (Fig. 3-1N). During the same developmental period, the s ubcellular distribution of claudin-5 becomes more distinct at cell-cell junctions (Fig. 3-1J, O). Secondary antibody alone (Fig. 3-1C') and antigenic PMP22 peptideadsorbed antibody (Fig. 3-1M') controls fail to label endothelial cell contacts. Thus, the temporal expression of PMP22 at interendothelial junctions of the developing rat nerve parallels that of ZO-1 and claudin-5, rather than of occludin.

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43 PMP22 in the Developing and Adult Rat Brain Microvasculature Since PMP22 is present in endothelia and epithelia of various tissues (Notterpek et al., 2001) and mRNA expression is re ported in the CNS (Baechner et al., 1995; Parmantier et al., 1995; 1997, Wulf and Suter, 1999), we investigated whether PMP22 Figure 3-2. Expression of PM P22 mRNA is elevated in tissues and cells of the developing rat BBB. (A) PMP22 expression in various rat tissues and cell lines was investigated by Northern blot analysis. Total RNA from P1 rat cortex (lane 1), BMV-enriched fraction (l ane 2), choroid plexus tissue (lane 3) and cultured choroid ep ithelia (lane 4) (10 # g/lane) were probed for PMP22 and 18S ribosomal RNA (loading control) (B) Densitometric analysis of the blot was performed after correction for RNA loading. The level of PMP22 mRNA in the P1 cortex was arbitrar ily set to a value of 1, allowing for comparison of the samples. is expressed in endothelial and/ or epithelial cells of the br ain (Fig. 3-2). Northern blot analysis of rat brain cortex, isolated cort ical microvessels and choroid plexuses, and primary cultures of choroid epithelia was performed to compare the levels of PMP22

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44 mRNA in the rat brain microvascul ature (BMV) and choroid epithelia to that of total cortex (Fig. 3-2A). Densitometric analysis of the blot, af ter correction for loading, is shown in Figure 2B. Cultured rat Schwann ce ll RNA was used as a positive control for the PMP22 hybridization (data no t shown). In the P1 cortex, without meninges or choroid plexuses, the level of PMP22 mRNA is very low (Fig. 3-2, lane 1). Compared to total cortical RNA, isolated rat BMV (lane 2) a nd choroid plexus tissue (lane 3) from early postnatal rats have an approximately 8-fold enrichment in PMP22 message (Fig. 3-2B). The expression of PMP22 observed for chor oid plexuses was confirmed in cultured choroid epithelia (lane 4), wh ich contains PMP22 message le vels nearly 17-fold higher than total cortex. Low levels of PMP22 mRNA are detected in the total cortex, BMV and choroid plexus from the P70 rat brain (data not shown). These resu lts demonstrate that during early postnatal development, PMP22 message levels are elevated in BMV and choroid plexus epithelia, as compared to total cortex. The localization of PMP22 at intercellular junctions of the BMV was investigated by double immunolabeling pressed preparations of E15, P1 and P70 rat brain cortices (Fig. 3-3). As the Nissl stained samples reve al (Fig. 3-3A, F, K), this method of tissue preparation preserves vessel co ntinuity and optimizes the visualization of cell-cell contacts (Utsumi et al., 2000). At the earliest age examined (E15), ZO-1 (Fig. 3-3B) and PMP22 (Fig. 3-3C) are already present at interendothelial contacts and pers ist throughout development without obvious changes in levels or distribution (Fi g. 3-3G and L; 3-3H and M). On parallel sections, the localizations of the tight junction proteins occludin (Fig. 3-3D, I, N) and claudin-5 (Fig. 3-3E, J, O) were also examined. In the E15 BMV, occludin-like immunostaining is ba rely detectable (Fig. 3-3D), with a gradual increase in

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45 Figure 3-3. Endothelial ce ll contacts of the devel oping and adult rat BMV are immunoreactive for PMP22. Rat brain cortic es (1 mm3) were pressed, stained with Nissl (A, F, and K), or coimmu nostained for ZO-1 (B, G and L) and PMP22 (C, H and M). Occludin (D, I a nd N) and claudin-5 (E, J and O) immunoreactivities were examined in para llel. In the E15 (A-E), P1 (F-J) and P70 (K-O) rat cortices, PMP22 colocali zes with ZO-1 at endothelial cell contacts of microvessels. During early development (E15 and P1), junctional occludin-like immunoreactivity is less pronounced (D and I), as compared to PMP22 (C and H). Endothelial claudin5 expression is observed for all ages studied (E, J and O). No primary an tibody control (C'), preimmune rabbit serum (H') and peptide-adsorbed anti-PM P22 antibodies (M') fail to label the cell contacts. Scale bar = 20 # m (O). signal between P1 and P70 (Fig. 3-3I, N). In comparison, similar to PMP22 and ZO-1, claudin-5-like immunoreactivity (Fig. 3-3E, J, O) is present throughout development. The subcellular localization of claudin-5, in addition to be ing found at junctions, includes

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46 significant amounts of diffuse staining, likel y representing intracellular protein. At all ages studied, the anti-rabbit secondary an tibodies (Fig. 3-3C'), the preimmune rabbit serum (Fig. 3-3H') and antigenic PMP22 peptid e-adsorbed antibodies (Fig. 3-3M') fail to label cell-cell contacts of the rat BMV. To improve the subcellula r resolution of PMP22-like immunoreactivity in brain endothelia, microvessels isolat ed from P4 mouse cortices we re dissociated and cultured on fibronectin-coated glass coverslips. Semi-confluent BEC monolayers were double immunostained for PMP22 and ZO-1 (Fig. 3-4). Whole anti-PMP 22 rabbit serum (Fig. 3-4A') not only detects cell junctions (arrow), but also labels BEC nuclei (asterisk). In order to establish the specificity of th e PMP22 immunoreactivity pattern, antigenic PMP22 peptide-purified antibod ies were prepared and used to immunolabel the BEC cultures (Fig. 3-4A, D). In agreement with the in vivo studies (Fig. 3-3), PMP22-like immunoreactivity, detected with the peptidepurified antibody (Fig. 3-4A) colocalizes with ZO-1 (Fig. 3-4B, C, arrows) at endotheli al cell junctions. Low levels of PMP22 are also seen in the cytoplasm of the cells, po ssibly reflecting the ER-G olgi fraction of the protein. Significantly, the nuclear immunoreactivity of the whole PMP22 antiserum is absent with the antigenic peptide-purified an tibody. To corroborate the colocalization of PMP22 with ZO-1 at cell contacts, cultured BECs were treated with 4 mM EGTA for 4 hours to induce the internaliz ation and disassembly of e ndothelial junctions (Gumbiner and Simons, 1986; Notterpek et al., 2001) (Fig. 3-4D-F). After calcium depletion, intracellular vesicular structures are coi mmunoreactive for PMP22 (Fig. 3-4D) and ZO-1 (Fig. 3-4E), likely representing internalized junctional complexes. The merged image

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47 reveals PMP22and ZO-1-containing vesicles (Fig. 3-4F, arrows) and perinuclear, ERGolgi PMP22-like immunoreactivity (Fig. 3-4F, arrowhead). These subcellular studies in Figure 3-4. In mouse BECs, PMP22 is a const ituent of intercellular junctions. Affinity purified anti-PMP22 antibodies were used to detect PMP22 (A) and ZO-1 (B) in primary cultures of mouse BECs. Colocalization of PMP22 and ZO-1 at intercellular junctions (arrows) is seen as yellow in the merged image (C). Whole anti-PMP22 rabbit serum (A') labels intercellular junc tions (arrow) and cell nuclei (asterisk). Perturbation of endothelial junctions with EGTA causes the internalization of PMP22 (D ) and ZO-1 (E), resulting in coimmunoreactive vesicular structures (yellow in F, arrows). Perinuclear PMP22 immunoreactive regions that do not colocalize with ZO-1 likely represent the ER-Golgi pr otein fraction (F, arrowhead). Hoechst dye was used to label nuclei (blue in C and F). Scale bar = 20 # m (B). cultured BECs further support the notion that PMP22 is a constituent of intercellular junctions in the rodent BMV. Epithelial Junctions of Choroid Plexus are PMP22 Immunoreactive Epithelial cells of the ch oroid plexus are crucial to the establishment and maintenance of the blood-CSF barrier (Segal, 2000). In the adult m ouse and rat brain,

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48 Figure 3-5. In the choroid plexus, PMP22 is a junctional constituent of epithelia. Cryosections from E15 (A-C) and P70 (D-F) rat brains were fixed and coimmunolabeled for occludin (A and D) and PMP22 (B and E). Parallel sections were immunostained for ZO-1 (C and F). In the E15 rat brain, PMP22 (B) colocalizes with occludin (A) at the ZO-1 immunoreactive (C) junctions of the budding choroid plexus (A-C, arrows). However, occludin is absent from the adjacent PMP22 a nd ZO-1 immunoreactive ventricular surface (A-C, arrowheads). In the P70 choroid plexus, occludin, PMP22, and ZO-1 remain at the choroid epithelial junctions (D-F, respectively, arrows). Preimmune serum (B') and peptide-adsorbed anti-PMP22 antibody (E') controls fail to label cell-cell contacts Confluent primary cultures of choroid epithelia were immunostained with affinity purified anti-PMP22 (G) and anti-ZO-1 (H) antibodies. Colocalization at intercellular junctions was detected (G and H, arrows). Whole anti-PMP22 rabbit serum (G') labels intercellular junctions (arr ow) and cell nuclei (asteris k). Nuclei were stained with Hoechst dye (blue in A, C, D, F, and H). Scale bar = 50 # m (F). occludin, ZO-1 and various claudins are present at interepithelial junctions of the choroid (Lippoldt et al., 2000; Wolburg et al., 2001). As PMP22 is detected at epithelial cell contacts of the rat colon (N otterpek et al., 2001), and PM P22 message is elevated in choroid epithelia (Fig. 3-2), we also investigated the e xpression and localization of

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49 PMP22 in choroid plexus tissue (Fig. 3-5). Cryosections of E15 (Fig. 3-5A-C) and P70 (Fig. 3-5D-F) rat brains were double immunolabeled with m onoclonal anti-occludin (Fig. 3-5A and D) and polyclonal anti-PMP22 (Fi g. 3-5B and E) antibodies. In the E15 rat brain, occludin (Fig. 3-5A, arrows) and PMP22 (Fig. 3-5B, arrows), as well as ZO-1 (Fig. 3-5C, arrows), are readily detected at ce ll junctions of the budding choroid plexus. In contrast, occludin is absent from th e anti-PMP22 and anti -ZO-1 immunoreactive neuroepithelial layer of the periventricula r region (Fig. 3-5A, B, C, arrowheads) (see below). In the P70 rat brain, occludin (Fig. 35D), as well as PMP22 (Fig. 3-5E), remain at interepithelial cell contacts of the mature choroid plexus (Fig. 3-5D, E, arrows). Immunolabeling of parallel sections reveals that the expression of ZO-1 (Fig. 3-5F, arrow) persists at these junctions. Se condary antibody alone (data not shown), preimmune serum (Fig. 3-5B') and antigenic PMP22 peptide-adsorbed antiserum (Fig. 3-5E') controls do not label cell contacts. In parallel with the in vivo studies, we investigated th e subcellular localization of PMP22 in purified cultures of choroid plexus epithelia (F ig. 3-5G and H). Confluent monolayers were coimmunolabeled with pe ptide-purified polycl onal anti-PMP22 (Fig. 3-5G) and monoclonal anti-ZO-1 (Fig. 3-5H) antibodies. As observed in the mouse BECs (Fig. 3-4A'), the anti-PMP22 whole rabbit serum labels the cell junctions, as well as the nuclei of choroid epithelia (Fig. 3-5G', arro w and asterisk, respectively). However, the nuclear staining is largely abolished by using the antigen ic peptide-purified PMP22 antibody (Fig. 3-5G). Similar to the rat brain tissue (Fig. 3-5A-F), PMP22 and ZO-1 are present at cell-cell contacts of cultured mouse choroid epithelia (Fig. 3-5G and H,

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50 arrows). Together, these results identify PMP22 as a constituent of intercellular junctions in the developing and adult rodent choroid epithelia. PMP22 is Detected at Neuroepithelial Junctions Neuroepithelial cell junctions lose ex pression of occludin during neural tube invagination (Aaku-Saraste et al., 1996) but continue to express ZO-1 and -catenin Figure 3-6. Neuroepithelial cell junctions of the embryoni c rat brain are immunoreactive for PMP22. Cryosections of E15 rat brain were fixed and coimmunostained for ZO-1 (A) and PMP22 (B), o ccludin (C) and PMP22 (D), or -catenin (E) and PMP22 (F). At the apical surface of the ventricular zone, ZO-1 (A) and -catenin (E), but not occludin (C) co localize with PMP22 (B, D, and F). Nuclei were stained with Hoechst dye (blue in A, C and E). V: Ventricular space. Scale bar = 17 # m (A). (Chenn et al., 1998; Manabe et al., 2002). In ag reement, in the E15 rat brain, we did not detect occludin at the ventricular zone, while PMP22-like immunoreactivity is readily

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51 visible (Fig. 3-5A, B, arrowheads). To furthe r investigate the dist ribution of PMP22 at the neuroepithelial cell layer, E15 rat brain cryosections were processed for double immunolabeling with ZO-1 (Fig. 3-6A), occludin (Fig. 3-6C), or -catenin (Fig. 3-6E) and PMP22 (Fig. 3-6B, D, F) antibodies. As the higher magnification view of this region reveals, PMP22 is colocalized with ZO-1 (Fig. 3-6A) and -catenin (Fig. 3-6E) at the apical surface of the neuroepithelial cell layer. In agreement with previous investigations in the chicken (Aaku-Saraste et al., 1996), occl udin (Fig. 3-6C) is not detected at these cell junctions. In comparis on to ZO-1 and PMP22, the -catenin-like i mmunoreactivity, although concentrated at the apical surface of the ventricle, extends along the lateral contacts of the neuroepithe lial cell layer (Fig. 3-6E). Th e observed distribution patterns for ZO-1 and -catenin are in agreement with pr evious studies in the developing mammalian brain (Chenn et al., 199 8; Manabe et al., 2002). Thes e data illustrate that, at the subventricular region, PMP22 is present at unique neuroepithelia l cell-cell contacts, which lack classical tight junctions (Mollg ard et al., 1987; Aaku-Sa raste et al., 1996). Discussion This study identifies PMP22 as a constituen t of intercellular contacts of the BNB and BBB. The colocalization with known juncti onal proteins in the developing and adult rat sciatic nerve, BMV and choroid epithe lia, suggests a role for PMP22 in the establishment of the BNB and BBB. Additiona lly, the presence of PMP22 at occludinnegative, specialized adherens-like junctions of the embryonic rat neuroepithelia may indicate a ubiquitous role at intercellular contacts. PMP22 is a broadly distributed membrane protein with documented expression in a variety of developing and mature tissues, including epithelial cells of the lung and

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52 intestine (Taylor et al., 1995; Baechner et al ., 1995; Wulf et al., 1999). The detection of PMP22 at the paraventricular re gion of the E15 rat brain is c onsistent with prior reports of neuroepithelial mRNA expression (Baec hner et al., 1995; Parmantier et al., 1997). Additionally, we observed both PMP22 message and protein in endothelial cells of the developing BMV and choroid epithelia. Whereas PMP22 remains localized to intercellular junctions of the adult rat BBB, message levels are reduced, as compared to early postnatal ages. This discrepancy between mRNA levels and detection of protein in the adult rodent brain could result from a lo w turnover rate of PMP22 at stable cell contacts and/or an increased half-life for the mRNA. The elevated expression of PMP22 in the developing rodent brain, prior to the maturation of the BBB, might reflect de novo junction formation or remodeling. Endotheli al cell-cell junctions undergo a similar structural remodeling following shear stress (Ogunrinade, 2002). Supporting a role for PMP22 in the assembly of intercellula r junctions, PMP22 mR NA is significantly upregulated after prolonged (24-48 hours) laminar shear stress in human umbilical vein and cardiac microvascular endotheli al cells (Bongrazio et al., 2000). The molecular composition of tight j unctions at the BBB is rapidly being elucidated (Wolburg and Li ppoldt, 2002). For all ages and tissues examined, we consistently detected PMP22 together with ZO-1, a broadly dist ributed structural constituent of cell junctions. Previous studi es show ZO-1 in the developing mouse BMV as early as E15, but not at E9 (Nico et al., 1999). It remain s to be determined whether prior to E15 in the rat brain or P10 in th e nerve, PMP22 and ZO-1 are targeted to junctions simultaneously or sequentially. Noneth eless, at both locations their junctional localization precedes that of occludin. Our fi ndings are in agreement with earlier reports

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53 describing junctional occludin in the rat BMV at P1 (Utsumi et al ., 2000) and elevated occludin immunoreactivity at P70, as compared to P8 (Hirase et al., 1997). Similar to the BBB, the expression of occludi n at endothelial cell juncti ons of the BNB lags behind PMP22 and ZO-1. It has been reported that the rat peripheral nerve vasculature becomes more restrictive to Evans Blue albumin a nd horseradish peroxidase, between P13-18 in the rat (Smith et al., 2001), coinciding w ith our observation of increased occludin expression. Occludin is thought to regulat e tight junction physiology; however, the precise function of occludin at intercellular juncti ons has not been definitively established (Saitou et al., 2000). The expression pattern of PMP22 was al so compared to claudin-5; a tight junction-associated integral membrane pr otein present in embryonic mouse BMV (Nitta et al., 2003) and cultured BECs (Chen et al., 199 8). In all of the studied nerve and brain samples, both PMP22 and claudin-5 are pres ent at endothelial junctions; however, a notable fraction of claudin-5 is intracellular at the younger ages. Thus, in the rodent CNS and PNS, we identified PMP22 as an early c onstituent of intercellular contacts, prior to structural maturation of the BNB and BBB. The temporospatial expression pattern of PMP22 is similar to ZO-1 and claudin-5, but not occludin. Although homologous to the claudin family (Notterpek et al., 2001; Takeda et al., 2001) and present at apical intercellular j unctions (Notterpek et al., 2001), functional studies to date suggest a distin ct role for PMP22 at these loca tions. In C6 glioma cells or L fibroblasts, unlike the claudins (Furuse et al., 1998b), PMP22 overexpression does not induce the formation of tight junction-like strands (Takeda et al., 2001; Notterpek et al., 2001). The detection of PMP22 in immature rat nerve and BMV suggests involvement

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54 early in the formation of cellcell contacts. The presence of PMP22 at the apical surface of the neuroepithelial cell la yer of the embryonic rat brain, which lack classical tight junctions (Mollgard et al., 1987; Aaku-Saraste et al., 1996), s upport a role for the protein either at adherens junctions or at the unique apical ‘strap -junctions’ (Mollgard et al., 1987). Together, these findings imply that, un like the claudins, PMP22 may not directly take part in the formation of paracellular resistance at the BNB a nd BBB; but instead may participate in the establishment and/or mainte nance of cell polarity and cell-cell adhesion. In agreement with this notion, VAB-9, a recen tly identified PMP22/epithelial membrane protein (EMP)/claudin family member, is an adherens junction protein crucial for the proper development of C. elegans (Simske et al., 2003). If PMP22 is indeed a crucial constitu ent of epithelial and endothelial cell junctions, then one might expect to see wi despread pathology when the protein is misexpressed. Nonetheless, the well-documented phenotype of PMP22 mutations, deletion or duplication is the demyelination of peripheral nerves (Gabreels-Festen and Wetering, 1999). Similar to the occludin-null mice (Saitou et al., 2000), PMP22-deficient mice are viable with no overt morphologi cal CNS pathology (Adlkofer et al., 1995), suggestive of some functional re dundancy for this protein at in tercellular junc tions of the BBB. However, mice homozygous for PMP22 mutations often display seemingly nonPNS related pathologies such as seizure and reduced growth rate (Henry et al., 1983; Suter et al., 1992a; Notterpek et al., 1997, Isaacs et al., 2000). A less understood phenotype associated with PMP22 misexpression is CNS demyelination in a subset of hereditary neuropathy with liability to pressu re palsies (HNPP) patients (Amato et al., 1996; Schneider et al., 2000; Dackovic et al ., 2001). Since PMP22 is not expressed in

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55 oligodendrocytes (Baechner et al., 1995: Parmantier et al., 19 95; 1997), it is unlikely that the protein has a direct role in CNS myelin. Based on our finding of PMP22 at intercellular junctions of BMV and choroi d epithelium, it is possible that the CNS pathology reported in some HNPP patients is the result of a compromised BBB. How might the localization of PMP22 at the BNB relate to the demyelinating phenotype of PMP22 neuropathies? Myelinated Sc hwann cells of the PNS share several characteristics with endothel ia and epithelia, including pol arization, distinct membrane domains (Bunge and Bunge, 1983) and close me mbrane apposition to create discrete compartments to restrict ion flow, all lik ely established by intermembranous junctions (Poliak et al., 2002; Scherer and Arroyo, 2002) It is conceivable that the myelin pathology observed in PMP22-associated neur opathies (Gabreels-Fe sten and Wetering, 1999), in part, is a result of disrupted PMP22 function at j unction-like structures within peripheral myelin or at the BN B. Perturbation of the BNB is a feasible hypothesis for the etiology of at least some of the nerve pathology observed in patients with PMP22associated neuropathies. Elevated levels of endoneurial macrophages are found in the PMP22-mutant TrJ mice (Misko et al., 2002) and macrophage-associated demyelination is detected in a Charcot-Marie-Toot h disease type IA patient with a PMP22 duplication (Vital et al., 2003). However, similar observa tions in P0 and connexin-32 mutant mice suggest macrophage infiltration may be co mmon to several here ditary peripheral neuropathies (Carenini et al ., 2001; Kobsar et al., 2002). The results described here support the no tion that PMP22 is a constituent of epithelial and endothelial interc ellular junctions. Fu rthermore, our findings suggest a role for PMP22 early in the establishment a nd/or maintenance of cell-cell contacts. Future

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56 investigations will attempt to establish the function of PMP22 at these locations, which may also help to clarify the role of the protein in peripheral nerves.

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57 CHAPTER 4 MODULATION OF EPITHELIAL MORPHOLOGY, MONOLAYER PERMEABILITY AND CELL MI GRATION BY GAS3/PMP22 Introduction The tetraspan glycoprotein peripheral myelin protein 22 (PMP22), also known as growth arrest-specific gene-3 ( gas-3 ), has proposed roles in peripheral nerve myelin formation, cell-cell interactions and cell pr oliferation (Suter a nd Snipes, 1995). Although the highest expression levels are found in myelin-forming Schwann cells, PMP22 mRNA can be detected in a multitude of devel oping and mature non-neural tissues; including epithelia of the intes tine (Taylor et al., 1995; Baechner et al., 1995; Wulf et al., 1999) and the choroid plexus (Roux et al., 2004). The specific role of PMP22 in Schwann cell biology remains undefined; although, it is known that altered expression is associated with heritable demyelinating peripheral neur opathies (reviewed by Naef and Suter, 1998). Similarly, the function of the protein at th ese non-neural locations remains undetermined. To date, in vitro studies have identified a role fo r PMP22 in the regulation of cell proliferation and morphology. In Schwann cells elevated expression delays the transition from G0/G1 to the S phase of the cell cycle (Z oidl et al., 1995), and can lead to apoptosis in some instances (Fabretti et al., 1995, Zo idl et al., 1997). Conversely, reduced PMP22 mRNA levels are associated with enhanced DNA synthesis and entr y into the S+G2/M phases (Zoidl et al. 1995). In NIH3T3 fibr oblasts, PMP22 overexpression regulates cell spreading, an effect that is dependent on the Rho-GTPase pathway (Brancolini et al., 1999). Recent studies have detected exoge nous PMP22 in ADP-ribosylation factor 6

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58 (Arf-6) positive plasma membrane-endosomal r ecycling vacuoles prio r to apoptosis or changes in cell shape (Chies et al., 2003). This pathway is known to be involved in modulating the actin cytoskeleton, cell pol arity, adhesion and migration (Donaldson, 2003). Together, these findings s upport the notion that PMP22 has a significant role in basic cellular processes, extending beyond an involvement in Schwann cell myelination. We previously described PMP22 as a consti tuent of apical intercellular junctions in epithelial and endothelial cells (Notte rpek et al., 2001; Roux et al., 2004). While PMP22 shares significant amino acid ho mology with members of the claudin superfamily, overexpression of the protein in Lfibroblasts (Notterpek et al., 2001) or C6 glioma cells (Takeda et al., 2001) did not indu ce the formation of tight junction strands. Nonetheless, PMP22 might function in the establishment and maintenance of ion-selective paracellular barriers. Transmem brane proteins of th e apical junctional complex such as the claudins, occludin a nd the junctional adhesion molecules (JAMs) (reviewed by Gonzlez-Mariscal et al., 2003) all participate in the regulation of junctional permeabililty. In addition, based on the findings of Brancolini and co lleagues (Brancolini et al., 1999; Brancolini et al ., 2000; Chies et al., 2003), PMP 22 might be involved in the regulation of epithelial pro liferation and/or cell migra tion, dynamic processes that involve changes in cell adhe sion and morphology. In support of this possibility, PMP22 contains the carbohydrate L2/HNK1 adhesion/r ecognition epitope in the 1st extracellular loop (Snipes et al., 1993; Sc hachner et al., 1995). In this report, we examined the role of PMP22 in several facet s of epithelial cell biology, including proliferation, cell shape a nd migration. An elevated level of PMP22 alters the migration of epithelia and reduces the formation of lamellipodial protrusions.

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59 The transepithelial electrical resistance (TER) and paracellular flux are also affected in these cultures. Application of PMP22 peptides has a similar effect on the TER and the permeability of MDCK monolayers. Together, th ese results indicate that PMP22 plays a role in modulating growth, morphology, migr ation and paracellular permeability in epithelial cells. Materials and Methods Cell Culture MDCK type I (high resistance) and type II (low resistance) cells were grown in Eagle's minimum essential medium supplemen ted with 5% FCS or Dulbecco's modified Eagle's medium with 10% FCS, respectively. Cells were cultured on 6.5 or 12 mm Costar Transwell filters (0.4 # m pores) (Corning Incorporated) or tissue culture dishes, and maintained at 37C and 5% CO2. For transepithelial electrical resistance (TER) or paracellular flux studies (see below), MDCK II monolayers were grown on filters (3x105 cells/cm2) in low calcium media (see below) fo r 48 h to ensure confluency prior to calcium addition. TER measurements were r ecorded every 24 h after calcium addition until 6 days post-plating at which time TER levels had reached a steady-state. After plating MDCK I cells on filters (3x105 cells/cm2), the medium was replaced every 24 h until the 6th day when TER levels had reached a steady-state. For the calcium-switch assay (Gumbiner and Simons, 1986), cells were treated with EDTA (4 mM) containing media for 4 h (Notterpek et al., 2001), or for 18 h in calciumand magnesium-free media with 5% Chelex (Sigma) treated FCS (Suzuki et al., 2001).

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60 Establishment of Stable Transgene Expressing Cells The human PMP22 (kind gift of Dr. Clar e Huxley, Imperial College School of Science, Technology and Medicine) and hu man occludin (Van Itallie and Anderson, 1997) ORFs were inserted into the pLNCX -II retroviral vector (Clontech). Transgene expression is regulated by the cytomegal ovirus (CMV) promoter. Following transient transfection with LipofectamineTM and PLUSTM reagent (Invitrogen), MDCK cells were treated with 1.1 mg/ml Genetici n (G418 sulfate) (Gibco) for four weeks to establish a population of stably expressing cells. Subclones of the stab ly expressing MDCK II cells were monitored for transgene expression by immunoblotting as desc ribed below. Three subclones were established for each construc t. Where indicated, 2.5 mM sodium butyrate was added for 20 h to induce the transgene expression under the CMV promoter (Gorman et al., 1983). Primary Antibodies Monoclonal mouse anti-o ccludin, anti-ZO-1 (Zymed Laboratories, Inc.), anti! -catenin (BD Transduction Labs), anti-E-c adherin clone rrl (Dev elopmental Studies Hybridoma Bank), anti" -tubulin, anti-actin (Sigma), an ti-GP-135 (kind gift from Dr. George Ojakian, SUNY Do wnstate Medical Center), rat anti-E-cadherin (Zymed), and rabbit polyclonal anti-occludin (Zymed), an ti-PMP22 and affinity purified anti-PMP22 antibodies (Notterpek et al., 2001; Roux et al., 2004) were used. Immunofluorescent Labeling MDCK cells plated on glass coverslips or 12 mm Transwells were grown to confluency and fixed with either 3% PFA followed by a 1 min incubation in 100% -20C acetone (for PMP22 detection) or 1% PFA followed by permeabilization with 0.2%

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61 Triton X-100 (TX-100) (juncti ons staining). For z-plan e imaging of confluent monolayers, filters were fixed with 4% PFA followed by 5 min 100% methanol at -20C and then frozen in liquid nitrogen-cooled Nmethylbutane prior to cryosectioning filters along the z-plane. Samples were blocked in 10% normal goat serum in PBS. After incubation with primary antibodies, Alex a FITC-conjugated anti-mouse IgG and Texas red-conjugated anti-rabbit IgG (Molecular Pr obes) antibodies were added. Nuclei were stained with Hoechst dye (10 # g/ml) (Molecular Probes). Actin filaments were visualized with FITC-conjugated phalloidin (Molecular Probes). Coverslips were mounted by using the ProLong Antifade kit (Mol ecular Probes). Samples were imaged with a Spot camera attached to a Nikon Eclipse 800 microscope and formatted for printing by using Adobe PHOTOSHOP 5.0. Images were measured using Spot Advanced 3.5. BrdU Incorporation The DNA synthesis rate of subconfluent MD CK II cells plated on glass coverslips was analyzed using a BrdU labeling a nd detection kit (Roc he) optimized for immunofluorescence of adherent cells following the manufacturer's recommended protocol. The percentage of BrdU positive ce lls was determined by counting in 4 random fields (0.8 mm2)and comparing to the total number of cells visualized by Hoechst staining. More than 500 cells were counted for each condition. The percentage was calculated from the ratio between BrdU positive and total cells. Epithelial Cyst Formation To generate MDCK II cysts, we followed an established protocol (Pollack et al., 1998). Briefly, MDCK II cells (5X104 cells/ml) were suspended in 2 mg/ml rat tail collagen type I (Sigma) on 6.5 mm Transwells (0.4 # m). After the collagen gel solidified

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62 at 37C, medium was added to the apical and basal chambers and was replaced every 48 h. Eight days after plating, the collagen gels were removed from the filters and fixed in 4% PFA prior to freezing in liquid-nitrogen cooled N-methylbutane. Cryosections (7 # m) were fixed on glass slides in 100% methanol for 5 min at -20C prior to blocking and immunolabeling as above. Cell Surface Biotinylation and Western Blotting For Western blotting, monolayers of confluent MDCK cells were lysed (Notterpek et al., 2001) and where indicated, treated with N-glycosidase (PNGase F) (Pareek et al., 1997). Samples were separa ted by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad) pr ior to immunoblotting. Bound HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Sigma) were detected using ECL reagents (Perkin Elmer). For the detection of PMP22 at the cell surface, confluent monolayers of stably expressing MDCK II cells (6 cm dish) were bi otinylated with biocyt in hydrazide (Lisanti et al., 1989; Prince et al., 1993). Monolayers were rinsed with PBS containing 10 mM CaCl2 and 1 mM MgCl2 (PBS-CM) and incubated with 10 mM NaIO4 in PBS-CM for 30 min at 4C in the dark while rocking. Afte r rinsing with PBS-CM, cell monolayers were kept in the dark for 1 h at 23C with 2 mM biocytin hydrazide (Pierce). Following extensive rinsing in PBS-CM, cells were ly sed for affinity precipitation in 3.2 ml 4C NP-40 buffer (25 mM Hepes/NaOH pH 7.4, 150 mM NaCl, 4 mM EDTA, 25 mM, NaF, 1% NP-40, 1 mM Na3VO4, 1X Complete protease i nhibitor (Roche Diagnostics)) (modified from Sakakibara et al., 1997), scrape d from the culture dish and gently rocked for 30 min at 4C. Cell lysates were centrifuged 10,000 g for 30 min at 4C and the

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63 supernatants reserved. The pe llet was solubilized in 320 # l SDS buffer (25 mM Hepes, pH 7.4, 4 mM EDTA, 25 mM NaF, 1% SDS, 1 mM Na3VO4) by sonication on ice. To the solubilized pellet, 9 volumes of NP-40 buffer was added, passed through a 27-gauge needle 10 times on ice, and incubated for 30 min at 4C. The lysates were centrifuged 10,000 g for 30 min at 4C and the supernatants reserved. ImmunoPure immobilized streptavadin beads (Pierce) suspended in lysi s buffer, were added to the cell lysate and gently rocked for 2 h at 4C. Streptavadin beads were washed 4 times with NP-40 buffer and boiled in 30 # l SDS gel sample buffer (Notterpek et al., 1997). After brief centrifugation, the 30 # l of sample buffer was removed a nd treated with PNGase F, as above. Samples were processed for immunobl otting with anti-PMP22 antibodies, as above. Measurement of Junctional Permeability TER was measured in 37 $ C culture media using an EVOM Epithelial voltohmmeter with an STX-2 electrode (Wor ld Precision Instruments, Inc.). The TER values were calculated by subtracting the background TER of blank filters and normalized by the area of the monolayer. Steady state TER measurements (N=9 wells per construct) were detected 6 days after cell plating under th e described culture conditions. To measure nonionic paracellular flux, FITC-dextran of 3 and 40 kD (Molecular Probes) was dissolved in P-buffer (Balda et al. 1996) at a concentration of 10 mg/ml. Apical and basolateral compartments of cells cultured on Transwell filters (N=3-4 wells per construct) were rinsed with P-buffer and a llowed to equilibrate for 10 min. The 3 and 40 kD FITC-dextran stock solutions (25 and 50 # g/ # l, respectively) was added to the apical chamber and the cells were incubated at 37C for 30 min. By sampling the basal media,

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64 the amount of FITC-dextran diffusion from the apical to the basal chamber was measured in a VersaFluor fluorometer (Bio-Rad). A st andard curve was used to convert relative fluorescent units to the concentr ation of dextran in solution. Peptide Perturbation HPLC-purified peptides were purchased from United Biochemical Research, Inc. Peptides corresponding to a portion of the 1st (aa 45-63) (NH2-SALGAVQHCYSSSVSEWLQ-COOH) (PMP22-1st) and the entire 2nd loop (aa 117-132) (NH2-YTVRHEWHVNTDYSY-COOH) (PMP22-2nd) of murine PMP22 were chosen. A scrambled peptide using the same amino acids as the 2nd loop of PMP22 (NH2-HDEYVSNTHWYRSYTV-COOH) (scrambled-2nd) served as a control. A 44 aa peptide corresponding to the 2nd extracellular lo op of the chicken occludin (Occ-2nd) was used as a positive control (Wong and Gumbiner, 1997). Peptides were dissolved in DMSO (10 mM) and added to calcium-containing media at the indicated concentrations. Monolayers (N=3-4 wells per condition) were fed every 24 h with fresh peptide-containing medium. Wound Migration Studies Wound assays using MDCK cells have previously been reported (Fenteany et al. 2000; Sabo et al., 2001). Highly confluen t MDCK II monolayers on either glass coverslips or tissue culture plas tic wells were wounded with a 200 # l pipette tip (Sabo et al., 2001). Long scratches and short wounds were made prior to rinsing the monlayer in fresh media to remove detached cells. At various time point s after wounding, wound areas were imaged with a Nikon DS camera a ttached to a Nikon Eclipse TS100 inverted microscope. In order to ensure that identic al areas were imaged between time points,

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65 multiple positioning-marks were made at the center of the denuded surface with a small needle. Relative wound areas (N=3 per constr uct) were measured with the NIH image analysis program. Alternatively, the mean distance migrated along the wound edge (N=6, measurements in 2 separate fields) was determined using Adobe PHOTOSHOP 5.0. The Rho-kinase inhibitor Y-27632 (10 # M) (Calbiochem) was applied to MDCK monolayers 2 h after wounding and monolayers were fixe d for staining after an additional 3 h (Omelchenko et al., 2003). Scatter factor ( SF) was obtained by cultu ring highly confluent NIH3T3 fibroblasts in DMEM with 1% FCS for 72 hr, followed by 0.45 # m filtration. Prior to use, SF was tested confirming its ability to induce the dispersion of small colonies of neo-MDCK cells (Stoker et al., 1987). Statistical Analysis Where indicated, means and standard de viations (SD) were calculated and statistical significance was de termined by unpaired 2-tail t test using GraphPad Prism 4.0. Results PMP22 Overexpression Alters Epithelial Cell Proliferation and Morphology To investigate the role of PMP22 in epithelial cells, human PMP22 (hPMP22) was overexpressed in the pLNCX-2 vector und er the control of the CMV promoter in MDCK II cells (PMP22-MDCK). These low resistance kidney-derived cells are frequently used for studies of polarized ep ithelia (Stevenson et al ., 1988). In total cell lysates (T), using an antibody optimized to detect the human protein, PMP22 is faintly observed at ~25kD (Fig. 4-1A, arrowhead). Upon PNGase F treatment, the protein is detected more prominently at 18kD (Fig. 41A, arrow). Enhanced immunoreactivity of PMP22 after endoglycosidase treatment has been previously observed (Fabretti et al.,

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66 1995). When the cell lysate is fractionated into detergent solubl e (S) and detergent insoluble fractions (I), the overexpressed human PMP22 protein is highly enriched in the detergent-insoluble pool. A similar detergen t solubility profiles has been observed Figure 4-1. Altered epithelia l cell proliferati on and morphology by PMP22. (A) Stable, neo and human PMP22 (hPMP22) expre ssing MDCK II cells were lysed in 3% SDS or 0.5% TX-100 buffer, and total lysates (T ), TX-100 soluble (S) and insoluble (I) fractions (30 # g/lane) were analyzed w ith (+) and without (-) PNGase F digestion. The glycosylated hP MP22 protein is detected at ~26 kD (arrowhead), and following PNGase F treatment becomes readily visible, migrating at ~18 kD (arrow). Compared to total lysates, human PMP22 is enriched in the detergent insoluble pelle t. (B) Plasma memb rane targeting of hPMP22 was determined by cell surface biotinylation, followed by PNGase F treatment and immunoblotting with anti-hP MP22 antibodies. The majority of biotinylated hPMP22 is detected in the 1% NP-40 insoluble (I) fraction (arrow). Molecular mass, in kDs. (C) As measured by BrdU incorporation in subconfluent cultures, compared to neo cells, hPMP22 expression reduces DNA synthesis by 32.99.5%, (*, P<0.004). (D) In confluent PMP22-MDCK monolayers, the cell density is 51.78.4% of the neo cultures (*, P<0.004). (E) The reduced cell density in confluent PM P22 monolayers is in agreement with an increase in nuclear area. Hoechst st aining of representative cultures is shown (Bar, 15 m). Quantification of nuclei re veals ~1.5-fold increase in nuclear dimension of PMP22-MDCK cells as compared to neo controls (*, P <0.0001). (F) The apical area of the PM P22-MDCK cells, outlined by ZO-1 immunostaining, is significantly larger than in neo cells (*, P<0.0004). Error bars in C, D, E and F, show means SD. P-values were determined by t test.

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67 previously for both the endogenous canine and exogenous myc-tagged rat PMP22 in MDCK cells (Notterpek et al., 2001). Vector-only cells (neo) are not immunoreactive with the anti-human PMP22 antibody (Fig. 4-1A). We next examined the targeting of hu man PMP22 to the cell surface, utilizing biotinylation and subsequent precipitation (R yan et al., 2002) (Fig. 4-1B). Endoglysidase treatment of the precipitated protein reveal ed hPMP22 at the plasma membrane. Since the majority of hPMP22 is found in the detergen t insoluble fraction (a rrow), the cell-surface protein is likely accumulated at apical interc ellular junctions and/ or, as reported in Schwann cells, possibly in lipid rafts (E rne et al., 2002; Hasse et al., 2002). Since PMP22 is known to modulate cell cy cle progression (Schneider et al., 1988; Manfioletti et al., 1990; Zoidl et al., 1995; 1997; Karl sson et al., 1999), we next examined how hPMP22 might affect epithelial prolifer ation. Similar to previous reports, in subconfluent cultures, elevated levels of hP MP22 resulted in a 33% reduction of BrdU incorporation, as compared to neo cells (F ig. 4-1C). At confluency, 51.78.4% fewer cells are in the PMP22-MDCK cultures, compar ed to controls (Fig. 4-1D). The lower cell density of PMP22 monolayers is readily vi sible by Hoechst imaging of nuclei from confluent filter-grown cultures (Fig. 4-1E). On images taken at the same magnification, the PMP22 cell nuclei appear larger and, when quantified, reveal an approximately 1.5-fold increase in area (graph, Fig. 4-1E). As predicted from a confluent monolayer with reduced cell density, the apical surface area of the PMP22 cells, as determined by ZO-1 immunostaining (see below) is ~1.9-fold larger than neo controls (Fig. 4-1F). These morphological characteri stics of PMP22-MDCK cultu res suggest the monolayer consists of fewer cells with a flattened morphology.

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68 PMP22 Does Not Alter Epithelial Polarity or the Localization of Junctional Proteins To further evaluate the altered epithelial morphology induced by hPMP22 overexpression, confluent filte r-grown monolayers were double immunostained with Figure 4-2. Protein polar ity and junctional constitue nts in PMP22-MDCK. (A) Confluent, filter-grown neoa nd PMP22-MDCK monolayers were immunostained and examined by from the z-plane by cryosectioning. In neo and PMP22-expressing monolayers, the protein polarity of GP-135 (green) and E-cadherin (red) are apical and ba solateral, respectively. The flattened morphology of the PMP22 cells is appa rent (Bar, 10 m). (B) In a 3-D collagen matrix polarization model, both cultures form multicellular cysts with apical GP-135 (green) oriented towa rds the center. Nuclei are visualized by Hoechst dye (blue) (Bar, 15 # m). (C) Compared to neo cultures, an increased level of PMP22 immunoreactiv ity is detected at intercellular junctions of PMP22-MDCK cells, when the images are taken at a constant exposure time. Parallel monolayers were also immunostained for a representative tight and adherens junc tion constituent, ZO-1 and -catenin, respectively. Both junction proteins a ppear similarly localized in neo and PMP22 cells. Bar, 20 # m. (D) Immunoblotting of 0.5% TX-100 soluble (S) and insoluble (I) fractions (20 # g/lane) reveals comparable levels and detergent solubilities for occludin, -catenin, as well as actin, between confluent neoand PMP22-MDCK monol ayers. Occludin appears slightly elevated in the detergent insoluble fraction of the PMP22-MDCK sample.

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69 anti-E-cadherin and anti-GP135 antibodi es following z-plane cryosectioning. As expected, in neo-MDCK cultures, GP135 is apical (Ojakian and Schwimmer, 1988) and E-cadherin labels the latera l borders (Fig. 4-2A). While the PMP22-MDCK cells are flattened, the protein polarity is similar to that of the neo controls. We also examined protein polarity in a three-dimensional (3 -D) model of epithelia l cysts (Ojakia and Schwimmer, 1994). In this model, MDCK cells form a polarized multicellular structure in which the apical surface faces a lumen. After sectioning and immunostaining for GP135, similar to neo cells, PMP22 cells formed cysts with normal polarity (Fig. 4-2B). As PMP22 is a protein constituent of apical intercellular junctions (Notterpek et al., 2001; Roux et al., 2004), we next inves tigated how PMP22 overexpression affects the localization and detergent sol ubility properties of tight a nd adherens-junction molecules (Fig. 4-2 C and D). As compared to neo cells, an elevated level of PMP22-like immunoreactivity is associated with the cel l-cell contacts of PM P22-MDCK monolayers (Fig. 4-2C). The expression and localizati on of tight junction-a ssociated occludin, as well as the adherens junction-associated -catenin, appear similar between the control neo and PMP22-MDCK samples (Fig. 4-2C). Similarly, the localiza tion of claudin-1, ZO-1 and E-cadherin was unaltered by PMP 22 (data not shown). As described above (Fig. 4-1F), the larger api cal dimensions of the PMP22 cells are apparent by the junctional immunostaining. To further examine the levels and detergent-solubility characteristics of junction-associated proteins, parallel samples were proce ssed for immunoblotting with the indicated antibodies (Fig. 4-2D). The three examined proteins, occludin, -catenin and actin, show similar expression levels and detergent-solubility properties between the

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70 neo and PMP22-MDCK cells, with a slight in crease for occludin in the PMP22 sample. Thus, the overexpression of PMP22 does not drastically alter epithelial protein polarity, nor the levels or localization of representative tight and a dherens junction molecules. Paracellular Permeability is Altered by PMP22 Expression Overexpression of the tight junction-associ ated occludin in MDCK cells revealed a role for this protein in regulating par acellular resistance (Bal da et al., 1996; 2000; Figure 4-3. Altered paracellular permeab ility of epithelial monolayers by hPMP22 expression. (A) TER recordings beginni ng two days after the addition of calcium, from newly-confluent MDCK monol ayers are shown. Three different clones of neo, PMP22 and Occ cells (N=3 filters per clone) were used for the quantification. Compared to neo cultures, the TER is elevated in PMP22 and Occ cells. (B) At steady-state, six days after plating, PMP22and Occ-MDCK monolayers have increased TER, comp ared to neo cells (*, P<0.0001). (C) Treatment with sodium butyrate (20 h), further enhances the differences in TER values between control and PMP 22, as well as Occ monolayers (*, P<0.0001). (D) The paracellular flux of 3kD nonionic FITC-dextran by PMP22and Occ-MDCK monolayers is significantly elevated (*, P<0.0001), as compared to neo cells. Error bars show means SD. P-values were determined by t-test.

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71 McCarthy et al., 1996; 2000). Since PMP22 is similarly localized to intercellular junctions of MDCK cells (No tterpek et al., 2001), we inve stigated whether the TER of the hPMP22-expressing epithelia l monolayers is altered comp ared to neo controls. Two days after the addition of calcium, PMP22and occludin-expressing cells have a high TER level indicative of newlyconfluent monolayers, after which the TER decreases to steady-state by day four. (F ig. 4-3A). Occludin expre ssing monolayers (Occ-MDCK), used as a positive control, have TERs sligh tly more elevated than the hPMP22 cells. By four days after calcium add ition, the monolayers reach a stea dy-state level of confluency and the TER of the hPMP22 and occludin expr essing cells is ~1.6-fo ld higher than the neo control (Fig. 4-3B). To further induce transgene expression, confluent monolayers were treated with sodium butyrate for 20 h (Gorman et al., 1983). As indicated in Fig. 4-3C, both the PMP22and Occ-MDCK monolay ers exhibit a 3.2and 4.1-fold increase in TER, respectively, as compared to butyrat e-treated neo cells. A similar phenomenon has been observed previously in epithelial monolayers overe xpressing occludin (Balda et al., 1996; 2000; McCarthy et al., 1996; 2000). As overexpression of junctional proteins ha s been shown to alte r paracellular flow of nonionic molecules (Balda et al., 1996; 2000; McCart hy et al., 1996; 2000), we performed a dextran flux assay on confluent PMP22-MDCK monolayers. As compared to neo controls, the flux of a 3kD nonionic FITC-l abeled dextran is elevated ~17-fold in both the PMP22and Occ-MDCK monolayers (Fig. 4-3D). Th ese results i ndicate that elevated expression of PMP22 results in altered permeability of MDCK monolayers.

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72 Epithelial Monolayer Permeability is Perturbed by PMP22 Peptides An alternative approach to elucidate the role of junctional pr oteins is to apply peptides that correspond to extracellular domains of endogenous proteins (Wong, 1997; Figure 4-4. Perturbation of epithelial monol ayer TER by PMP22 peptides. (A) Peptides corresponding to a portion of the 1st (1) and the entire 2nd (2) extracellular loops of murine PMP22, a scrambled 2nd loop and the 2nd extracellular loop of chicken occludin were applied to confluent MDCK m onolayers after a calcium-switch. (B) Twenty hours after th e addition of the peptides, the TER of PMP22-2nd peptide treated monolayers (32 # M) remains low compared to naive, DMSO, PMP22-1st or scrambled-2nd treated cells (*, P<0.0001). TER reformation by monolayers exposed to Occ-2nd peptide (16 # M) is also significantly perturbed (**, P<0.0006). (C) A dosage curve for PMP22-2nd peptide reveals an effective concentra tion range for TER disruption between 8 to 32 M. Error bars show means SD. P-values were determined by t test. (D) Following a 20h PMP22-2nd peptide treatment, and subsequent washout, the monolayers regain a TER similar to untreated samples.

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73 Wong and Gumbiner, 1997; Lacaz-Vieira et al ., 1999; Chung et al., 2001; Vietor et al., 2001; Tavelin et al., 2003; Lee et al., 2004). As the canine PMP22 has not been cloned, we designed peptides representing portions of the 1st and 2nd extr acellular domains of the mouse protein (Fig. 4-4A). While PM P22 has homology to some members of the claudin family, the proteins are less similar in the extracellular domains (Align software, data not shown). Confluent MDCK I monol ayers were treated with the indicated peptides, following a calcium-switch. Twenty hour s after the re-additi on of calcium with the corresponding peptides (32 M PMP22-1st, -2nd, PMP22-scrambled-2nd, or 16 M Occ-2nd), the TER of the monolayers was recorded (Fig. 4-4B). As expected based on the literature (Wong, 1997; Wong a nd Gumbiner, 1997; Vietor et al., 2001), the Occ-2nd loop peptide inhibited TER recovery by 50.48.7%. Similarly, the PMP22-2nd loop peptide diminished TER recovery by 91.30.4%. Appl ication of vehicle (DMSO), PMP22-1st or scrambled-2nd peptides had no significant effect on the TER compared to nave cells (Fig. 4-4B). A concentration curve for PMP22-2nd peptide identified an effective range of TER disruption between 8 and 32 # M (Fig. 4-4C). The washout of PMP22-2nd peptide from MDCK monolayers results in the restoration of the TER to leve ls similar to controls (Fig. 4-4D). Therefore, the disrup tive effect of the PMP22-2nd loop peptide on the monolayer TER is reversible. Next, we examined the paracellular fl ux and the morphology of peptide-treated monolayers (Fig. 4-5). The flux of the 3 kD, but not the 40 kD, FITC-dextran is significantly elevated in both the PMP22-2ndand Occ-2nd-loop treated monolayers, indicating a size selective dist urbance of paracellular permeability (Fig. 4-5A). The observed 3-fold increase in the fl ow of the 3kD dextran in Occ-2nd treated cultures is in

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74 Figure 4-5. An increased paracellular flux of epithelial monolayers by PMP22 peptides. (A) MDCK monolayers were treated with the indicated peptides and the paracellular flux of 3 kD, and 40 kD FITC-labeled nonionic dextrans were determined. In PMP22-2nd (*, P<0.0001) and Occ-2nd treated monolayers (**, P<0.002) the paracellular flow of th e 3kD, but not the 40kD, dextran is significantly elevated. Error bars show means SD. P-values were determined by t test. (B) In PMP22-2nd and scrambled-2nd peptide-treated monolayers (32 M), the localization of ZO-1 and E-ca dherin remain comparable. Bar, 20 # m. agreement with previous reports (Wong and Gu mbiner, 1997). In order to determine if the PMP22-2nd peptide treatment alters the distribution of junctional constituents, the localization of ZO-1 and E-ca dherin was examined in parall el peptide-treated monolayers (Fig. 4-5B). Both ZO-1 and E-cadherin a ppear unaltered following treatment with PMP22-2nd or scrambled-2nd peptides. These findings indicate that the PMP22-2nd

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75 peptide reduces monolayer permeability withou t radically altering major constituents of apical intercellular junctions. PMP22 Expression Slows the Migration of Epithelial Monolayers Injured epithelial monolayers down-regulat e ZO-1 and occludin mRNAs (Cao et al., 2002), suggestive of junctional remodeling. After observing that the expression of Figure 4-6. Wound healing is a ltered by PMP22 in epithelial monolayers. (A) Confluent neoand PMP22-MDCK monolayers were wounded with a pipette tip and the migration of the cells into the wound area (borders outlined in black) was evaluated at 2 and 24h. Neo-MDCK m onolayers nearly close the wound by 24 h, while PMP22-MDCK cells are unable to similarly reduce the wound area. Bar, 800 # m. (B) Quantification of woundi ng experiments reveal that compared to nave and neo cells, PMP 22-MDCK cells are significantly less competent to migrate (*, P < 0.0003). Error bars show means SD. P-values determined by t test. (C) Hoechst staining of parallel samples shows dispersed nuclei along the wound edge in the neo cultures. In comparison, the nuclei of the PMP22-MDCK cells appear densel y packed and uniform throughout the image. Bar, 150 # m.

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76 hPMP22 alters the morphology and permeability of epithelial monolayers, we hypothesized that the protein might modul ate the dynamic processes involved in epithelial migration. To test this idea, hPMP22-overexpre ssing epithelial cells were observed in a two-dimensional wound-migra tion assay (Fig. 4-6A) (Fenteany et al., 2000). By phase microscopy 24 hours after wou nding, the neo cultures nearly close the denuded area (Fig. 4-6A, top panels). In co mparison, the rate of monolayer closure is visibly reduced in PMP22-MCDK cells (Fig. 4-6A, bottom panels). Indeed, within a 24h period, PMP22 monolayers exhibit a 60.36% reduction in wound closure as compared to neo cells (Fig. 4-6 B). At higher magnification of Hoechst stained samples, the neo monolayers display a typical wave of migra ting cells at the wound edge that appear flattened and spread out (Sheffers et al., 2003; Matsubayashi et al., 2004) (Fig. 4-6C). In contrast, the nuclei of PMP22-expressing cells are more compact at the wound edge and are more uniformly spaced throughout the monolayer. In response to monolayer wounding, migr ating MDCK cells maintain cell-cell contacts, form an actin purse-string along the wound edge and pu ll multiple cell rows forward in by Rac-dependent lamellipodi al crawling (Fenteany et al., 2000). Lamellipodial protrusion by leader cells, but not the formation of an ac tin purse-string, is required by MDCK monolayers to close a wound (Fenteany et al., 2000). Therefore, utilizing immunolabeling we examined these two structures in PMP22-MDCK cells (Fig. 4-7A). As expected, 24 h after monolayer w ounding, neo cells (top ro w) have an actinbelt along the wound edge, with periodic breaks (arrowhead on the right) representative of migrating cells. Leader cell lamellipodia are observed in the neo monolayers by

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77 Figure 4-7. Lamellipodial protru sion in migrating epithelia l monolayers is reduced by PMP22. (A) The distribution of actin, -tubulin and E-cadherin was examined by fluorescence microscopy along the wound edge at 24 h post-wounding. In neo cultures, an actin pur se-string and lamellipodial protrusions (arrowheads) are detected (top row). In wounded PMP22 samples, the actin purse-string is uninterrupted and -tubulin and E-cadherin appe ar concentrated along the wound edge. Lamellipodial protrusions into the wound space are largely absent in the PMP22-MDCK monolayers. Bar, 40 # m. (B) A 3 h treatment of wounded monolayers with Y-27632, a Rho ki nase inhibitor, induces extensive lamellipodial protrusions (arrows) al ong the wound edge of neo cells, visualized by fluorescent labeling of actin. In PMP22-MDCK monolayers, Y-27632 is unable to bring about a simila r response, as cells with lamellipodia are sparse (arrow). Bar, 50 # m. (C) Quantification of epithelial migration after wounding (5 h), in the absence and presen ce of scatter factor (SF). In normal culture medium, neo cells migrate fa ster than PMP22-MDCK monolayers. The addition of SF to the medium signif icantly increases the migration of neo and PMP22-MDCK cells (*, P<0.0003; **, P<0.0001, respectively). Error bars show means SD. P-values were determined by t test.

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78 tubulin and E-cadherin labeling (arrowheads). An actin purse-string is continuous along the migrating edge of PMP22 monolayers (Fi g. 4-7A, bottom panel); however, breaks or perturbations in this actinbelt are largely absent. A dditionally, the tubulin and Ecadherin immunoreactivities appear concentrat ed along the leading edge in PMP22 cells of the wound, and the monolayers have fewer cells extending lamellipodia into the wound area. Since lamellipodial protrusion is cruc ial for MDCK monolayer migration, we examined if inhibition of Rho-kinase, known to induce lamellipodial expansion in wounded epithelial monolayers (Omelchenko et al., 2003), could overcome the effects of PMP22 overexpression (Fig. 4-7B). As expected, a three hour treatment of neo-MDCK cells with a Rho kinase inhibitor (Y-27632) leads to increased lamellipodial-like cell protrusion, visualized by actinphalloidin fluorescent imaging (Fig. 4-7B, arrows). In comparison, PMP22-MDCK cells appear resistan t to the formation of lamellipodia, with few cell protrusions apparent along the wound edge (arrow). Thus, the expression of PMP22 results in the reduced migr ation of MDCK cells after wounding. Fibroblast-derived scatter-fact or (SF), has been shown to induce an epithelial to mesenchymal transition (EMT) in MDCK ce lls (Stoker et al. 1987). Therefore, we investigated whether SF is capable of ove rriding the inhibitory effect of PMP22 expression on MDCK monolayer migration. Wh en cultured in SF, neo and PMP22 cells migrate a similar distance five hours after a scratch wound (Fig. 4-7C, gray bars). Compared to monolayers in normal medium (F ig. 4-7C, white bars), wound closure in the presence of SF increases by 1.8and 4.7fold for the neo and PMP22-MDCK cells,

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79 respectively. These results indicate that wh ile PMP22-MDCK cells are refractory to the effects of Rho kinase inhibi tion, they are competent to migrate after SF-induced EMT. Discussion The described results indicate that PMP 22 plays a role in several aspects of epithelial biology. The overexpression of PMP 22 reduces the prolifer ation and final cell density of epithelial monolay ers, and induces flattened cell morphology. Monolayers of such cultures have increased TER and paracellular flux of nonionic dextrans. In agreement, a PMP22 peptide disrupts the refo rmation of paracellula r resistance following calcium-switch. The migration of epithelial monolayers is also reduced by PMP22 overexpression, possibly due to a deficiency in lamellipodial forming leader cells. These results suggest that PMP22 takes part in a pa thway by which apical cell junctions regulate the proliferation and morphology of epithe lial cells, and modulate paracellular permeability and cell motility. Cell junction-associated pr oteins have previously been shown to influence cell proliferation. For example, in addition to regulating paracellular permeability (Balda and Matter, 2000; Reichert et al., 2000), elevated levels of ZO-1 reduce proliferation and cell density in MDCK cells (Balda et al., 2003). This effect is thought to result from sequestration of the transcription factor Z ONAB, a ZO-1 binding partner, from the cell nucleus (Balda et al., 2003). Currentl y, it is unknown through which pathway the transmembrane protein PMP22 elicits a si milar response in the MDCK model. In Schwann cells and fibroblasts, in addition to growth arrest, overexpression of PMP22 by retroviral and transien t transfection has been shown to induce apoptosis (Fabretti et al., 1995; Zoidl et al., 1997), a pr ocess counteracted by exogenous Bcl-2 (Brancolini et al.,

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80 1999). In our stably trans duced cell populations, increased apoptosis as judged by Hoechst staining was not observed, possibly due to a lower level of PMP22 expression. It has been reported in several cell types that overe xpression of PMP22 affects cellular morphology (Brancolini et al., 1999; 2000; Chies et al., 2003). The altered cell shape observed in PMP22-MDCK monolayers is likely the consequence of reduced cell density at confluency, in which a flattene d morphology is necessary for maintaining functional cell-cell junctions. The elevated TER might be the result of this phenomenon, as confluent monolayers with reduced cell de nsity have less total tight junctional space (Marcial et al., 1984). Since para cellular junctions are more pe rmeable than th e cell itself (Stefani and Cereijido, 1983), an elevated TER would be expected. Yet, the reduced cell density reported after ZO-1 overexpression did not signifi cantly alter the TER of the MDCK monolayers (Balda et al., 2000). Therefore, PMP22’s effect on monolayer resistance is not entirely based on altered morphology. In a ccordance, as shown here and by others (Balda et al., 1 996; 2000; McCarthy et al., 1996; 2000), elevated levels of occludin increased the TER, but did not signi ficantly alter the ce ll morphology or density of confluent monolayers. A role for PMP22 in modulating paracel lular flow is supported by the increased ionic and nonionic permeability fo llowing exposure to the PMP22-2nd loop peptide. The reduced TER and increased flux of small dextrans may indicate that the 2nd loop peptide disrupts homotypic interactions of PMP22. I ndeed, PMP22 is known to form dimers and larger oligomers in vivo and in vitro (Tobler et al., 1999; 2002) As the extracellular domains of PMP22 share no significant homol ogy with the claudins, a potential direct effect on claudins is unlikely. However, th e peptides may be perturbing the function of

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81 other, as of yet undetermined, epithelial junc tion-associated protei ns that are binding partners for PMP22. The localization of exogenous and e ndogenous PMP22 at apical intercellular junctions (Notterpek et al., 2001; Roux et al., 2004), combined w ith the effects of elevated PMP22 expression or PMP22 peptides on paracellular perm eability, supports the notion that PMP22 is a functional constituent of the apical junctional complex. Although it has not been determined ul trastructurally whether PMP22 is at tight or adherens junctions, the protein is capable of alteri ng both the ionic and nonionic permeability of epithelial monolayers. Similar effects have b een attained in previous studies when the expression of the tight junc tion protein occludin (Balda et al., 1996; 2000; McCarthy et al., 1996; 2000) was modulated. As PMP22 is de tected at intercellu lar contacts of rat neuroepithelia (Roux et al., 2004), cells devoid of classical tight junctions (Mollgard et al., 1987; Aaku-Saraste et al., 1996 ), the role of the protein might not be exclusive to tight junctions. In this respect, PMP22 is si milar to ZO-1, a junctional protein that in some cell types exists at site s other than the tight junctiona l complex (Itoh et al., 1993). Epithelial cells maintain physical co ntacts during wound closure, while they extend Rac-GTPase-dependent lamellipodia (Fen teany et al., 2000). In normal epithelia, lamellipodial protrusion is promoted by Y27632, likely by disrupting the actin marginal bundles along the wound edge (Omelchenko et al., 2003). PMP22 expression however, even after Y-27632 treatment, prevents the la mellipodial formation. This suggests that an elevated level of the protein interferes w ith the signaling for lamellipodial protrusion, possibly by acting via the actin cytoskeleton di rectly or indirectly by modulating the Rac GTPase pathway. In comparison, the same MDCK cells are capable of migrating similar

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82 to controls following the application of SF. Th is result suggests that the inhibitory action of PMP22 on wound-induced migration is likely dependent upon epithelial cell-cell contacts, as SF induces a j unction-disrupting EMT (Nusrat et al., 1994, Gris endi et al., 1998). Since PMP22 is predominantly expressed in myelinating Schwann cells, what relevance do our studies in ep ithelia have to our understand ing of the protein’s function in the PNS? The growth arrest properties of PMP22 appear to occur independent of cell type, but have yet to be directly linked to PMP22-associated disease pathology. Cell morphology is drastically altered during mye lination, an event that involves extensive membrane expansion; however, PMP22 is not required for myelin wrapping (Adlkofer et al., 1995). Cell migration is also crucial to proper nerve development (reviewed in Lobsiger et al., 2002). Nonetheless, our resu lts show that when epithelia undergo EMT and migrate as individual cells, the overexpr ession of PMP22 has no inhibitory effect. Typically thought of as a component of comp act myelin (Haney et al., 1996), PMP22 has not yet been localized to tight junctions of PNS myeli n. Based on the effects of PMP22 on epithelial paracellular permeability, the protein could have a similar role in PNS myelin at claudin-1 and -5-positive autot ypic tight junctions (Poliak et al., 2002). Therefore, modulating PMP22 in an epithelia l model may provide some clues as to the function of the protein in PNS myelin. In addition to identifying the participati on of PMP22 in epithelial cell biology, we established an in vitro model that is amenable to furt her experimentation. Utilizing this system, the specific activity of various PM P22 domains can be dissected by examining their effects on epithelial pe rmeability and migration. It will be equally important to

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83 identify binding partners of PMP22 at interc ellular junctions in order to fully understand how the protein signals such globa l changes in epith elial cell biology.

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84 CHAPTER 5 CONCLUSIONS Overview of Findings At the time that these studies began, PM P22 was viewed primarily as a myelin protein involved in peripheral nerve pa thology. With little known about its function, especially in myelinating Schwann cells, the majority of research has focused on characterizing nerve pathology and the mechanism of disease with the goal to ameliorate or prevent neuropathy. The research presente d here has sought to examine fundamental properties of non-neural PMP22 complemen ting other efforts by providing knowledge about the function of PMP22. With its wide spread and extensive expression pattern throughout development and maturity, an unders tanding of the role for PMP22 in basic cell biology is a justified end eavor that may lead to nove l discoveries about undefined cellular processes. By following clues such as homology to th e claudin superfamily of tight junction proteins (Chapter 2, Notterp ek et al., 2001; Takeda et al., 2001) and expression in epithelial (Baechner et al., 1995; Wulf et al ., 1999) and endothelial cells (Bongrazio et al., 2000), we correctly hypothesized that PMP22 is a constituent of ce ll-cell contacts in epithelia and endothelia (C hapter 2, Notterpek et al ., 2001). Subsequently, we characterized the expression and localizati on of PMP22 in the developing and adult blood-nerve and blood-brain barr iers (BBB) (Chapter 3, Roux et al., 2004), The BBB is a system extensively researched for its involve ment in several CNS disorders (reviewed by

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85 Neuwelt, 2004). As a result of those studies, PMP22 was detected early in development at junctions of the brain vasc ulature and choroidal epithelia. Additionally, the protein was found at neuroepithelial interc ellular junctions, substantiati ng previous reports of PMP22 mRNA expression in the neuroepithelium (Baec hner et al., 1995; Parmantier et al., 1995; 1997). These cell-cell junctions are thought to be requi site for neurogenesis (Chenn and McConnell, 1995; Manabe et al., 2002). Finally, we began to investigate the function of the protein in epithelia, where we found evid ence for its involvement in regulating the cell cycle and cell morphology. However, perhap s most importantly, these studies led to the discovery of a novel role for PMP22 in the modulation of junction permeability and cell migration, and identified an in vitro model in which to further investigate the function of the protein. Unresolved Issues As with most scientific research, thes e studies have led to many as of yet unanswered questions. With regards to specific subcellular localizat ion, it is clear that PMP22 does not display a pattern of immunolab eling similar to E-cadherin or -catenin, typical adherens junction proteins detected at lateral cell contacts. Instead, the protein colocalizes with ZO-1, occludin and claudin-1 at apical inte rcellular junctions, suggestive of a tight junction protein. However, this i ssue is complicated by the detection of the protein at apical neuroepith elial cell junctions, sites whic h are thought to be devoid of classical tight junctions (Mo llgard et al., 1987; Aaku-Saraste et al., 1996). These results suggest that PMP22, like ZO-1 (Itoh et al., 1993), might be ca pable of existing at either type of junction depending upon the cell type. This hypothesis may explain its expression in cells, such as fibroblasts (Manfioletti et al., 1990), which lack tight junctions, but contain ZO-1 (Itoh et al., 1993; Chapter 2, No tterpek et al., 2001). Nonetheless, we still

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86 do not know at the ultrastructural level, whet her PMP22 resides primar ily at the tight or adherens junctions of epithelia or endothel ia. The most direct way to resolve this uncertainty is to attempt ultrastructura l immunolabeling, ideally on freeze-fractured samples. Another unresolved topic concerns non-PN S related consequences to peripheral neuropathy-associated PMP22 misexpression. In humans, the only reported clinical pathology seemingly unrelated to periphera l neuropathy is the CNS demyelination reported in a small subset of patients defici ent in PMP22 expression (Amato et al., 1996; Schneider et al., 2000; Dackovic et al., 2001) This phenotype remains infrequently reported in humans; however, a seizure-like behavior, suggestive of CNS pathology, is also observed in homozygous PMP22-defici ent and Tr-J mouse models. Additionally, other organ systems, besides the PNS, have not been extensively examined for pathology in mouse models for PMP22-misexpression. A lik ely candidate for such studies would be the homozygous Tr-J mouse that is not viable beyond three weeks postn atal (Henry et al., 1983). Of course, the possibility exists that there is a redundancy of function for PMP22 outside of the Schwann cell, with the family of epithelial membrane proteins being the most likely candidates based on homology (Taylo r et al., 1995; 1996; Lobsiger et al., 1996). A similar redundancy likely occurs in mice deficient in claudin-14, a protein expressed in several tissues, but with pathology detected in only cochlear hair cells (BenYoseph et al., 2003). If any redundancy can be identified in vitro the establishment of double knockout transgenic animals may allow for in vivo analysis. Since PMP22 is localized to apical in tercellular junctions it was perhaps not unexpected that junctional permeability w ould be affected by overexpression of the

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87 protein (Chapter 4). Nonetheless, it s till remains unknown how PMP22 induces this effect. Based on its homology to the claudins, it is temping to assume that PMP22 itself acts to modulate the barrier properties of the epithelial m onolayer. However, it may be more likely that PMP22 modulates, as of yet unknown, binding partners that themselves function to maintain paracellu lar resistance. Possible candidates for this role would certainly include the claudins or occludin. To date, attemp ts to co-immunoprecipitate proteins with PMP22 have proven difficult, la rgely due to the prot ein’s insolubility. Future Studies The studies described in Chapter 4 iden tified that an elevated PMP22 level increases both the TER and pa racellular flux and inhibits th e proper migration of MDCK monolayers; however, it remains unknown how a reduction in PMP22 expression will affect these processes. Future studies may ta ke advantage of an i nducible antisense or RNA-silencing technology to cr eate a transient decrease in the level of the protein. Hopefully, if a functional redundancy for PM P22 exists, it will not occur rapidly, but instead require a lack of PMP22 expr ession during cellular development and differentiation. It is hoped that the studies described he re will lead to a clearer understanding of the mechanisms by which PMP22 modulates such seemingly diverse cel lular processes as proliferation, morphology, junctional pe rmeability and epithelial migration. One approach to this issue is to find binding pa rtners for PMP22, either by hypothesis driven testing or using a more comprehensive ‘shot gun’ technique, such as a yeast two hybrid assay. Myelin protein zero (D’Urso et al., 1999) and P2X7 (Wilson et al., 2002), were identified as proteins that interact with PMP22 by these approaches, respectively. Once

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88 binding partners are discovered, it is possibl e that the modulation of their function, if known, by PMP22 could account for the effect s of altered PMP22 expression. Based on its extensive expression pattern and ability to associate w ith two seemingly unrelated proteins, it is conceivable that PMP22 may act as a transmembrane protein-chaperone, possibly involved in the targeting or stoich eometry of other proteins within specific membrane domains. An alternative approach to investigate the function of PMP22 is to dissect the signaling mechanisms by which the protein modu lates cellular behaviors. For example, it may be informative to evaluate the activation or inactivation of the major GTPase pathways, Rho, Rac and Cdc42, following an in crease or decrease in PMP22 expression. A role for the RhoA-GTPase pathway in PM P22-induced altered cellular morphology has already been identified in fibroblasts (Branc olini et al., 1999; Chies et al., 2003). In these studies, it appears that active RhoA, either ar tificially induced or naturally occuring, is able to counteract the e ffects of PMP22 overexpression on cell morphology. In addition to cell morphology, the GTPase pathways are crucial to maintena nce of paracellular permeability (reviewed in Hopkins et al., 2000) and wound closure (Fenteany et al., 2000) in epithelial ce lls. Overexpression of PMP22 pr oduces a flattened morphology and inhibits lamellipodial formattion required for wound closure (Chapter 4), phenotypes similar to that found in MDCK cells e xpressing a dominant negative Rac-GTPase (Takaishi et al., 1997, Fenteany et al., 2000, re spectively). These same GTPases are likely involved in the migration and immense memb rane expansion required for PNS myelin formation (Melendez-Vasquez et al., 2004).

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89 In parallel to inve stigating binding partners and signaling pathways involved in the PMP22 modulation of epithelial biology, the MDCK cell model can be used to identify protein domains or specific amino aci ds crucial to its normal functions. Based on the peptide perturbation experiments in Chapter 4, the 2nd loop of PMP22 would be a prime candidate for site directed mutagenesis, a technique to create proteins with altered amino acids. The charged amino acids of both the 1st and 2nd loop should be analyzed as these have been shown to mediate the speci fic ionic selectivity of several claudins (Colegio et al., 2002; 2003; Va n Itallie et al., 2003; Yu et al., 2003). As glycosylated PMP22 is found at the cell surf ace in epithelia (Chapter 4) it may be insightful to compare the effects of the wild type protein to a mutant lacking the proper glycosylation motif. Previous studies in Schwann cells have shown that the non-glycosylated protein is capable of proper targeting (Ryan et al., 2002) ; however, in Cos7 cells the protein is unable to induce as signifi cant a change in cell morphol ogy (Brancolini et al., 2000). Another unique domain of PMP22 is the short carboxyl tail of charged amino acids (Fig 1-1). Previous studies have s hown that this domain does not ac t as an ER retrieval motif (Brancolini et al., 2000 ), but no functional significance has yet to be ascribed to this domain. It is unlikely that this carboxyl region would se rve as a PDZ binding domain (Gonzalez-Mariscal et al., 2003), a feature common to many junc tional proteins, as it is not similar to previously identified sequences and is rather short and likely very close to the membrane. Perhaps with the exception of proliferation, a quantifiable functional analysis of PMP22 in in vitro Schwann cells remains elusive. Thus, it may be tempting to study neuropathy-associated PMP22 point mutants in epithelia since junctional permeability and wound migration are quantifiable characteristics. Unfortunately, many

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90 of the diseased-linked point mutant proteins a ppear to have defects in trafficking and are unable to reach the cell surface. In such situations, it is unclear if the results are due to disrupted function or targeting. Only afte r a non-neural clinical pathology can be identified in PMP22 mutant mice, or humans would it seem worthwhile to examine the effects of the altered prot ein on epithelial biology. In summary, the studies described in th is dissertation have identified PMP22, for the first time, as a constituent of apical intercellu lar junctions in epit helia and endothelia, and have provided novel evidence of a functiona l role in epithelial biology. The findings of this work will affect future efforts of scientists investigating the role for PMP22 in hereditary peripheral neuropathies, as well as those seeking to unders tand basic epithelial or endothelial cell biology.

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109 BIOGRAPHICAL SKETCH Born outside of Chicago, Illinois, Kyle Joseph Roux is the son of Kenneth and Shirley Roux. Raised in Tallahassee, Flor ida from early childhood, he graduated high school from Alfred B. Maclay College Prep aratory School. For undergraduate studies he attended Emory University where he received a Bachelor of Science degree in biological anthropology/human biology and met his wi fe, Amy. After graduating, he moved to Gainesville, Florida and worked for a year in the laboratory of Dr. Margaret Wallace before beginning graduate studies in the Interdisciplinary Program for Biomedical Research at the University of Florida in th e fall of 1999. In the summer of 2000 he began to pursue his doctoral degree in the departme nt of Neuroscience unde r the supervision of Dr. Lucia Notterpek. During his graduate studies Kyle and Amy became the proud parents of their son Aidan.


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PERIPHERAL MYELIN PROTEIN 22 IS A NOVEL CONSTITUENT OF
INTERCELLULAR JUNCTIONS














By

KYLE JOSEPH ROUX


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


2004

































Copyright 2004

by

Kyle Joseph Roux



























I dedicate this work to my loving wife and son; and to my parents, who have steadfastly
supported me throughout the years.















ACKNOWLEDGMENTS

First and foremost, I would like to thank my mentor, Lucia Notterpek. Ever since

I started working with Lucia, she has always made my education a priority, and had my

best interests in mind. Looking back at my graduate career, I can clearly see that from

Lucia's guidance I have gained considerable maturity, and the confidence necessary to

pursue my scientific goals. I would also like to thank all of my committee members,

whose invaluable insight and positive feedback were greatly appreciated. Similarly,

Bradley Fletcher deserves acknowledgment, for his expertise in molecular biology has

been instrumental in promoting my studies. To all of the members of the Notterpek lab,

past and present, who have served as perpetual sounding boards for my ideas and endured

my endless tirades, I express my gratitude.

I cannot thank my parents enough for all that they have done and endured

throughout the years. And most importantly, to my loving wife Amy I offer my deepest

appreciation for the sacrifices she has made for our family.
















TABLE OF CONTENTS



A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF FIGURES .. ................... ............ ........ .............. vii

ABSTRACT ................................................... ................. viii

CHAPTER

1 IN TR O D U C T IO N ........ .. ......................................... ..........................................1.

Introduction .................. ............ ...... ..... .......... ................................................ 1
Peripheral Neuropathies Associated with PMP22..................................................1...
Animal Models of PMP22-Associated Neuropathies.............................................4...
Disease Mechanism of Altered PMP22 Expression...............................................5...
G enom ic O organization of PM P22 ........................................................... ...............7...
Tem porospatial Expression of PM P22 .................................................... ............... 8
C characteristics of PM P22 Protein............................................................ ...............9...
Role for PMP22 in Cell Proliferation and Cell Morphology................................11

2 PERIPHERAL MYELIN PROTEIN 22 IS A NOVEL CONSTITUENT OF
INTERCELLULAR JUNCTIONS IN EPITHELIA.............................................15

In tro d u ctio n ............................................................................................................... .. 1 5
M materials and M ethods .. ..................................................................... ............... 17
R e su lts....................................................................................................... ....... .. 2 1
D isc u ssio n ............................................................................................................... ... 3 1

3 TEMPOROSPATIAL EXPRESSION OF PERIPHERAL MYELIN PROTEIN 22
AT THE DEVELOPING BLOOD-NERVE AND BLOOD-BRAIN BARRIERS.... 35

In tro d u ctio n ................................................................................................................ 3 5
M materials and M ethods .. ..................................................................... ................ 37
R e su lts....................................................................................................... ....... .. 4 1
D isc u ssio n ............................................................................................................... ... 5 1

4 MODULATION OF EPITHELIAL MORPHOLOGY, MONOLAYER
PERMEABILITY AND CELL MIGRATION BY GAS3/PMP22......................... 57









Introduction .................................................................................. ...................... 57
M materials and M ethods ...................... ................................................................ 59
Results ............................................... ........................ 65
Discussion ............................................. ............................. 79

5 C O N C L U S IO N S ........................................................................................................84

O v erview of F finding s ............................................................................. ...............84
U resolved Issues ................................................................................................. 85
F u tu re S tu d ie s ............................................................................................................. 8 7

LIST O F R EFEREN CE S .................................................................................................91

B IO G R A PH IC A L SK E TCH ................ .. ........................ .................... ...............109















LIST OF FIGURES

Figure page

1-1. The secondary structure of PM P22 ...................................................... ................ 10

2-1. Coexpression of PMP22 with ZO-1 and occludin in colon epithelium..................22

2-2. Coexpression of PMP22 with ZO-1 and occludin at cell-cell contacts..................24

2-3. Internalization of PMP22 with occludin in EGTA-treated MDCK cells ...............26

2.4. Exogenously expressed PMP22-myc is targeted to TJs in MDCK cells................28

2-5. Colocalization of PMP22-myc with ZO-1 at intercellular junctions......................29

3-1. Endothelial cell junctions of the BNB in the developing and adult rat sciatic..........41

3-2. Expression of PMP22 mRNA is elevated in tissues and cells of the .....................43

3-3. Endothelial cell contacts of the developing and adult rat BMV are.......................45

3-4. In mouse BECs, PMP22 is a constituent of intercellular junctions........................47

3-5. In the choroid plexus, PMP22 is a junctional constituent of epithelia ...................48

3-6. Neuroepithelial cell junctions of the embryonic rat brain are immunoreactive ........50

4-1. Altered epithelial cell proliferation and morphology by PMP22 .............................66

4-2. Protein polarity and junctional constituents in PMP22-MDCK...............................68

4-3. Altered paracellular permeability of epithelial monolayers by hPMP22 ...............70

4-4. Perturbation of epithelial monolayer TER by PMP22 peptides ............................. 72

4-5. An increased paracellular flux of epithelial monolayers by PMP22 peptides...........74

4-6. Wound healing is altered by PMP22 in epithelial monolayers ...............................75

4-7. Lamellipodial protrusion in migrating epithelial monolayers is reduced...............77















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 IS A NOVEL CONSTITUENT OF
INTERCELLULAR JUNCTIONS

by

Kyle Joseph Roux

August 2004


Chair: Lucia Notterpek Fletcher
Major Department: Neuroscience

Altered expression of peripheral myelin protein 22 (PMP22) is associated with

several inherited peripheral neuropathies. Although predominantly expressed in

myelinating Schwann cells, PMP22 is also detected in several cell types outside of the

peripheral nervous system. The function of the protein in myelin or non-neural cells

remains unknown; however, PMP22 has been shown to modulate the proliferation and

morphology of Schwann cells and fibroblasts. With homology to the claudin superfamily

of tight junction proteins and prominent expression in epithelial cells of the intestine, a

role for PMP22 at intercellular junctions was hypothesized.

The overall aim of this study was to investigate PMP22 as a constituent of apical

cell-cell junctions. Initial studies identified that PMP22 is localized to the apical

junctional complex in epithelia and endothelia. Involved in the maintenance of cell

polarity and establishment of selective barriers in tissues, these cell junctions play crucial

roles in health and disease. Next, the localization and expression of the protein at









intercellular junctions of the developing and mature rat blood-brain (BBB) and

blood-nerve barriers (BNB) was studied. Detected at these cell junctions throughout all

developmental stages studied, PMP22 is likely involved in the establishment and

maintenance of these barriers. Finally, the role of the protein in multiple aspects of

epithelial cell biology was investigated. As reported in other cell types, PMP22

modulated epithelial cell growth and morphology. Additionally, the protein altered the

junctional permeability and migration of epithelial monolayers. These results demonstrate

that PMP22 is a component of the BNB and BBB, and is a functional constituent of

apical cell-cell junctions in epithelia. Our studies have laid the foundation for future

investigations into the function of PMP22 in epithelial cell biology, and provide novel

insights into its potential role in peripheral nerve myelin.














CHAPTER 1
INTRODUCTION

Introduction

First cloned over 15 years ago, peripheral myelin protein-22 (PMP22) was

discovered almost simultaneously by three independent laboratories. Described both as

gin ,i i/ arrest-specific gene-3 (gas3) in serum-starved or contact-inhibited NIH3T3

fibroblasts (Manfioletti et al., 1990) and as a myelin gene with altered expression after

peripheral nerve injury (Spreyer et al., 1991; Snipes et al., 1992), it was determined that

PMP22 protein had previously been identified as a major glycoprotein of bovine

peripheral nerve myelin (Kitamura et al., 1976). Soon after cloning, it became evident

that altered expression of PMP22 is associated with several heritable demyelinating

disorders. Lacking clear evidence of its structure, a role within a signaling pathway, or

even an ascribed cellular function, it is known that PMP22 is critical to the normal

function of peripheral nerve myelin. Hypotheses for the mechanism of disease include

dose-dependent loss-of-function and a dominant negative gain-of-function, ideas

supported by the genetics behind altered PMP22 expression. Despite of extensive genetic

characterization of clinical cases, several animal models for PMP22 mutations, and

intensive analysis of PMP22 messenger RNA (mRNA) and protein, a clear understanding

of the basic function and disease mechanism for PMP22 remains unknown

Peripheral Neuropathies Associated with PMP22

Predominantly studied for its role in the pathology of the peripheral nervous

system (PNS), altered expression of the PMP22 gene is associated with a significant









subset of heritable peripheral neuropathies in humans, including Charcot-Marie-Tooth

disease (CMT), Dejerine-Sottas syndrome (DSS) and hereditary neuropathy with liability

to pressure palsies (HNPP) (reviewed by Naef and Suter, 1998). These disorders vary in

frequency, age of disease onset and functional severity. Accounting for up to 5% of total

protein in peripheral nerve myelin (Pareek et al., 1993), PMP22 protein and message

levels are highest in myelinating Schwann cells (Welcher et al., 1991; Snipes et al.,

1992). As detected by ultrastructural immunocytochemistry, the protein is concentrated in

the compact portion of myelin (Haney et al., 1996), the region responsible for

maintenance of the ionic resistance that enables rapid saltatory nerve impulse conduction.

The predominant expression and localization in the PNS, combined with its association

with demyelinating neuropathies, corresponds with PMP22 as an essential protein

constituent of peripheral nerve myelin.

Charcot-Marie-Tooth disorders are the most common heritable peripheral

neuropathy with a prevalence of 1 in 2,500 (Skre, 1974). Most CMT patients are

classified as having CMT type 1 with abnormalities in the PNS myelin that is created by

Schwann cells. The most prevalent form (90%) of CMT1, CMT type lA (CMT1A)

(Garcia, 1999) has a prevalence of 1 in 5,000 (Kuhlenbaumer et al., 2002), and is

predominantly found in patients with a dominant 1.5 Mb duplication of the pI 1-p12

region of chromosome 17 (Lupski et al., 1991; Raeymaekers et al., 1991), which contains

the PMP22 gene. Less frequently, point mutations in the PMP22 gene are associated with

CMT1A (Roa et al., 1993). Age of disease onset is variable, typically ranging between

the 1st and 2nd decade of life. Identified by slowed nerve conduction velocity (NCV),

CMT1A can be diagnosed by determination of PMP22 gene duplication or point









mutation (reviewed by Kuhlenbaumer et al., 2002). Peripheral nerve pathology includes

demyelination, hypermyelination, and onion bulb formations. Progressive distal limb

weakness and muscle atrophy, distal and symmetrical sensory deficits, and foot

deformities are common clinical symptoms of CMTIA that can lead to eventual loss of

ambulatory function (reviewed by Kuhlenbaumer et al., 2002).

Encompassing a genetically diverse group of patients, DSS is a clinical

classification for patients with a form of severe peripheral neuropathy. Dominant and

recessive mutations have been found in PMP22, myelin protein zero (MPZ),

early-growth-response-element-2 (EGR) and periaxin (PRX) genes. Symptoms arise in

infancy or early childhood, and include distal sensory loss and ataxia, motor deficit and

palpable nerve hypertrophy (Dejerine and Sottas, 1893). A hallmark diagnostic feature

characteristic of DSS is severely reduced NCV. Nerve pathology includes demyelination-

remyelination, onion bulb formations, Schwann cell hyperproliferation and nerve

hypertrophy (reviewed in Plante-Bodeneauve and Said, 2002). The PMP22-associated

DSS usually results from dominant missense mutations, although duplication of p 11-pl2

of chromosome 17 has been reported (Lupski et al., 1991; Mancardi et al., 1994; Silander

et al., 1996), illustrating overlapping genetic bases for DSS and CMT1A.

The least severe form of PMP22-associated neuropathy is HNPP. Patients

predominantly have a dominant 1.5 Mb deletion of chromosome 17pl -p12; however in

rare cases, point mutations have been described, often resulting in premature termination

of protein translation (Nicholson et al., 1994). Episodic recurrent motor and sensory

peripheral neuropathies, often lasting days to weeks, with onset in childhood or

adolescence are typical for HNPP patients (reviewed in Chance et al., 1999). Mildly









slowed symmetrical NCVs are consistent with demyelination. Nerve tomocula, sausage

shaped regions of hypermyelination, are a common diagnostic feature found to precede

clinical symptoms and hypothesized to be the result of frequent mild injuries

(Gabreels-Festen and Wettering, 1999). Segmental demyelination and remyelination can

also be identified in nerve biopsies. In one study, over 40% of HNPP patients were

unaware of their condition and 25% were symptom free (Pareyson et al., 1996),

illustrating the phenotypic variation found in PMP22-associated peripheral neuropathies.

Animal Models of PMP22-Associated Neuropathies

To determine if altered expression of PMP22 is sufficient to induce heritable

peripheral neuropathies, animal models that replicate the deletion, duplication and several

point mutations of PMP22 have been genetically engineered. These animal models

recapitulate major aspects of PMP22-related neuropathies in humans and have allowed

for a more complete cellular and molecular analysis of neuropathy nerves than can

practically be accomplished with human tissue samples (Notterpek and Tolwani, 1999).

Unlike engineered PMP22-mutant mice, the spontaneously occurring Trembler (Tr) and

Trembler-J (TrJ) mice have point mutations in PMP22 resulting in amino-acid

substitutions identical to those found in some human CMT1A patients (Suter et al.,

1992a, 1992b; Valentijn et al., 1992; Ionasescu et al., 1997). Frequently used as models

for hypertrophic demyelinating neuropathies similar to CMT1A, the Tr and TrJ mice

display phenotypic differences, especially the early postnatal lethality of the homozygous

TrJ in comparison to the long-lived homozygous Tr (Henry and Sidman, 1988). Other

neuropathy-associated PMP22 point mutations have since been established in mice

(Isaacs et al., 2000; 2002), providing further models for the study of PMP22-associated

neuropathies.









Overexpression of the PMP22 gene in transgenic animals provides a model for

CMT1A patients with PMP22 duplication. Rats carrying three copies of the murine

PMP22 gene per allele display slowed NCV and signs of demyelination and

dysmyelination (Sereda et al., 1996). Similarly, two mouse models of PMP22

overexpression were designed to recapitulate the CMT1A phenotype (Huxley et al.,

1996; Magyar et al., 1996). To study the HNPP phenotype in transgenic mice, expression

of the PMP22 gene has been diminished either by antisense technology (Maycox et al.,

1997) or by homologous recombinant gene disruption (Adlkofer et al., 1995). Affected

mice display behavioral and pathological traits found in HNPP patients, including the

characteristic tomoculous nerve fibers. The severity of neuropathy found in the

homozygous PMP22-null mice as compared to the more mildly affected heterozygotes

lends support to the principle of PMP22 dose-dependency. In humans, homozygosity for

the PMP22 deletion has not been reported, either because of a low frequency of

occurrence or an incompatibility with life. Crossbreeding between the Tr and a

PMP22-null mouse has provided significant evidence for the dominant negative gain-of-

function hypothesis for PMP22 point mutations (Adlkofer et al., 1997). Limitations exist

for these animal models of PMP22-neuropathies, including the brief rodent lifespan that

may mask the progressive nature of these disorders. However, their study has led to

important discoveries, including the role of protein mistrafficking in disease pathology.

Disease Mechanism of Altered PMP22 Expression

The majority of PMP22 protein is located in the plasma membrane of compact

myelin in Schwann cells (Pareek et al., 1993; Haney et al., 1996). In rat Schwann cells,

most of the newly synthesized PMP22 protein is rapidly turned over in the endoplasmic

reticulum, unable to attain complex glycosylation or enter the plasma membrane (Pareek









et al., 1997). The Tr and Tr-J mutant forms of PMP22 fail to reach the cell surface in

myelinating Schwann cells (Colby et al., 2000). In addition to being hemizygous for

wt-PMP22 expression, the mutant protein could act to further reduce the surface

expression of the wild-type (wt) protein since the mutant protein is capable of associating

with the wt form (Tobler et al., 1999). However, the duplication of the PMP22 gene leads

to disease symptoms similar to many of the point mutants, suggesting an alternate disease

mechanism. Since most of the wt protein never reaches the plasma membrane, it has been

hypothesized that PMP22 protein processing is difficult for the Schwann cell (Sanders et

al., 2001). Either the presence of mutant forms or an increase in the level of wt protein

expression may eventually overwhelm the quality-control mechanism, causing a negative

gain-of-function, perhaps explaining the progressive nature of the disease. An

upregulation of the lysosomal and ubiquitin-proteasomal protein degradation pathways in

the Tr-J mouse model lend support to this hypothesis (Notterpek et al., 1997; 1999a;

Ryan et al., 2002; Tobler et al., 2002; Fortun et al., 2003), although the actual disease

mechanism of the PMP22-associated neuropathies remains uncertain.

Current experimental approaches to treating PMP22-associated neuropathies

(reviewed in Young and Suter, 2001) include modulation of PMP22 expression and

immunosuppression. A progesterone antagonist administered in a transgenic rat model

for CMT1A reduces the levels of PMP22 mRNA and improves the CMTIA-like

pathology (Sereda et al., 2003). Similarly, ascorbic acid treatment ameliorates the disease

phenotype in a CMT1A mouse model (Passage et al., 2004). Although these studies are

promising, no effective treatments are commonly prescribed for clinical use in humans.









Genomic Organization of PMP22

The human PMP22 gene is located at chromosome 17p 11.2-pl2. The mouse and

rat genes are found on chromosome 11 (Suter et al., 1992a) and 10q22 (Liehr and

Rautenstrauss, 1995), respectively. In the human genome, PMP22 spans approximately

40kb, with 6 exons coding for the mRNA. The first 2 exons, lA and IB are alternatively

transcribed under different promoters, (PI and P2, respectively) resulting in differential

5' untranslated regions (UTRs), yet maintaining the same coding region. The existence of

dual promoters suggests diversity in regulating PMP22 expression. Both transcripts are

detected in most tissues; however, the exon lA containing message is predominant in the

peripheral nerve, while the exon IB form is more common in non-neural tissues (Suter et

al., 1994). Exons 2 through 5, code for the PMP22 protein and a large 3' UTR. This

genomic organization is conserved in both the mouse and the rat.

Studies of the PMP22 promoters have provided limited evidence to suggest how

transcription is regulated. A TATA-box-like sequence is present in the P1, but not P2

region, which has a high GC rich sequence, similar to that found in a housekeeping

promoter (Suter et al., 1994). Specific transcription factors known to regulate PMP22

expression have not been described. Levels of PMP22 message in the sciatic nerve are

upregulated during early postnatal development (Bosse al., 1994). Immediately after

nerve injury, PMP22 mRNA levels are reduced followed by upregulation during

regeneration (Spreyer et al., 1991; Snipes et al., 1992). In NIH3T3 cells, growth arrest

leads to an elevation in PMP22 message (Manfioletti et al., 1990). Similarly, forskolin, an

activator of adenylate cyclase that produces cyclic-AMP, results in increased mRNA

levels in Schwann cells (Snipes et al., 1992; Pareek et al., 1993). Another regulator of

PMP22 transcription in Schwann cells is 3ca-5u.-tetrahydroprogesterone, whose activity is









dependent on the gamma-amino butyric acid (GABAA) receptor (Melcangi et al., 1999;

Martini et al., 2003). These findings have provided some insight into the expression of

PMP22 mRNA; however, a clear understanding of its gene regulation remains elusive.

Temporospatial Expression of PMP22

Expressed rather ubiquitously in tissues outside of the PNS, non-neural PMP22

mRNA is detected by in situ hybridization during murine embryogenesis in the epithelial

ectodermal layer at embryonic day 9.5 (E9.5) (Baechner et al., 1995). In the same study,

elevated levels of PMP22 message are enriched in the liver and gut during organogenesis

(El 1.5). By E14.5-16.5 the lung mesenchyme, skin and eye epithelia all contain PMP22

message. As detected by Northern blot analysis, tissue-specific expression of PMP22

mRNA in the late embryonic rat heart and kidney is reduced prior to birth (Rees et al.,

1999). Tissues containing significant levels of PMP22 in the adult rat and mouse include

the lung, stomach and intestinal tract (Taylor et al., 1995; Lobsiger et al., 1996). By in

situ hybridization, the mature mouse was found to contain significant non-neural PMP22

mRNA in the epithelial villi of the intestine (Baechner et al., 1995).

In the central nervous system (CNS), the highest levels of PMP22 message are

detected at E15.5 by Northern blot analysis (Wulf and Suter, 1999), and by in situ

hybridization at the subventricular neuroepithelial layer of the developing mouse from

E11.5 through E17.5 (Baechner et al., 1995; Parmantier et al., 1997). Discrete

populations of motor neurons in the developing and adult mouse and rat also contain

PMP22 message and protein (Parmantier et al., 1995; 1997). Expression of a putative

zebrafish orthologue to mammalian PMP22 was observed by in situ hybridization in the

intestinal and olfactory epithelium and neural crest cells (Wulf et al., 1999), identifying

the gene as having both a neural and non-neural expression even in nonmammalian









vertebrates. In addition, PMP22 message is found in diverse cell lines in vitro, such as

differentiated PC12 cells (De Leon et al., 1994), P19-derived neuroepithelial cells (Wulf

and Suter, 1999) and shear-stressed endothelia (Bongrazio et al., 2000). Thus, despite a

PNS-specific disease association, the pattern of PMP22 mRNA expression is rather

ubiquitous.

Characteristics of PMP22 Protein

Based on hydropathy plots, PMP22 is a 160 amino-acid hydrophobic protein with

a putative four-transmembrane structure, two extracellular loops and intracellular amino-

and carboxyl-termini (D'Urso and Muller, 1997, Taylor et al., 2000) (Fig. 1-1). The

protein is highly conserved with an 87% amino-acid identity between human and mouse.

The 1st transmembrane domain contains a non-cleaved signal peptide sequence, a motif

that targets protein insertion into the ER membrane (Manfioletti et al., 1990; Welcher et

al., 1991; Taylor et al., 1995).

The only documented post-translational modification of PMP22 is the addition of

a sugar moiety via N-linked glycosylation of a conserved consensus sequence on the 1st

extracellular loop (Pareek et al., 1993). Glycosylation of PMP22 gives the core 18

kilodalton (kD) protein its characteristic 22 kD mobility by SDS-PAGE analysis. The

sulfated sugar complex is recognized by the L2/HNK-1 antibody, an epitope found on

several nervous- and immune-system proteins that function in cell-cell and

cell-extracellular matrix interactions (reviewed in Schachner et al., 1995). When

glycosylation of the protein is prevented by amino-acid substitution, PMP22 is targeted to

the ER and plasma membrane similar to the wt form (Ryan et al., 2000). However, the

deglycosylated protein forms less stable homodimers (Ryan et al., 2000) than the wt

protein (Tobler et al., 1999).





























N2HJ \ IICOOH


Figure 1-1. The secondary structure of PMP22. Shown above is the putative secondary
structure of PMP22. There are four transmembrane regions (grey) and two
extracellular loops, the first of which contains an N-glycosylation motif.

In addition to forming homotypic interactions, PMP22 is capable of associating

with other transmembrane proteins. PMP22 associates in a glycosylation-independent

interaction with the abundant PNS myelin transmembrane protein, myelin protein zero

(PO) (D'Urso et al., 1999). It is hypothesized that the interaction between PO and PMP22

is required to maintain stable myelin (D'Urso et al., 1999), perhaps by assuring the

proper stoichiometry of the two proteins. Another protein that interacts with PMP22 is

the P2X7 purogenic transmembrane receptor, an ion channel gated by extracellular ATP

(Wilson et al., 2002). The P2X7-PMP22 protein interaction occurs via a unique

cytoplasmic domain of the P2X7 receptor. Therefore, at least in some instances, PMP22's

role in cellular processes may involve the modulation of other transmembrane proteins.









A member of a family of four transmembrane proteins, PMP22 has homology to

the epithelial membrane protein-1 (EMP-1), -2 and -3 (Taylor et al., 1995; Lobsiger et al.,

1996; Taylor and Suter, 1996; Chen et al., 1997). The function of the EMPs remains

unclear. However, the most studied of these proteins, EMP-2, associates with pl3-integrin

and regulates cell-substrate adhesion (Wadehra et al., 2002), modulates the surface

expression of the class I major histocompatability complex (Wadhera et al., 2003), and of

caveolins and glycosylphosphatidyl inositol-linked proteins (Wadhera et al., 2004).

Studies of EMP-2 further suggest a role for the PMP22-EMP family of proteins in the

modulation of other membrane-associated molecules.

Role for PMP22 in Cell Proliferation and Cell Morphology

Altered PMP22 expression in various in vitro cell lines indicates a role for the

protein in regulating both the progression of the cell cycle and cell morphology.

Upregulated in serum-starved NIH3T3 cells, PMP22 mRNA levels are similarly elevated

by contact-inhibited growth arrest (Schneider et al., 1998; Ciccarelli et al., 1990;

Manfioletti et al., 1990; Suter et al., 1994). A coincident increase in PMP22 message and

induction of growth arrest is found in Schwann cells (Welcher et al., 1991; Zoidl et al.,

1995) and adipoblasts (Shugart et al., 1995). Thus, elevated PMP22 expression appears to

be correlated with exit from the cell cycle, at least in a subset of cells. These findings are

substantiated by studies that artificially overexpress exogenous PMP22 in Schwann cells,

leading to growth arrest (Zoidl et al., 1995). Conversely, Schwann cell proliferation is

augmented by a reduction of PMP22 message by expression of antisense mRNA (Zoidl et

al., 1995). Therefore, these studies indicate that PMP22 is capable of both positive and

negative regulation of cell-cycle progression. Additionally, nerve growth factor









differentiated PC12, but not C6 glioma cells, have an increased level of PMP22 mRNA

(DeLeon et al., 1994), suggesting a role for PMP22 in cell differentiation.

Elevated levels of exogenous PMP22 can also result in altered cell morphology.

In NIH3T3 and HEK-293, but not REF52 cells, overexpression of PMP22 results in

plasma membrane blebbing and eventual apoptosis (Fabretti et al., 1995; Brancoloni et

al., 1999; Wilson et al., 2002), a phenotype inhibited by coexpression of the

anti-apoptotic bcl-2 gene (Brancolini et al., 1999). Prolonged activation of the P2X7

purigenic receptor, a PMP22 binding partner, also leads to membrane blebbing and

apoptosis (Wilson et al., 2002). The expression of P2X7 in immune and epithelial cells,

in addition to Schwann cells (Grafe et al., 1999; Colomar et al., 2001), indicates a

potential mechanism for the membrane blebbing and apoptosis induced by PMP22

expression.

Following PMP22 overexpression, NIH3T3 cells experience RhoA

GTPase-dependent altered cell spreading (Brancolini et al., 1999). Conversely, by

inhibiting endogenous RhoA GTPase activity, REF52 cells become sensitive to altered

cell shape in response to PMP22 overexpression (Brancolini et al., 1999). These

experiments implicate the Rho GTPase pathway in modulating the effects of PMP22

expression. In NIH3T3 cells, PMP22 that is incapable of reaching the plasma membrane,

either due to the artificial addition of an ER retrieval signal to the carboxyl-terminus or

the presence of the Tr-J point mutation, is unable to alter cell spreading or induce

apoptosis (Fabretti et al., 1995; Brancolini et al., 2000). While capable of reaching the

cell surface and increasing apoptosis, PMP22 protein with a defective glycosylation motif









fails to affect cell morphology as significantly as the wt protein (Brancolini et al., 2000),

suggesting that this motif is crucial for certain cellular functions.

Prior to the PMP22-induced morphological changes or apoptosis that occur in

response to PMP22 overexpression, wt, but not Tr-J, protein is localized to perinuclear

endosomes and to large vacuoles near the cell periphery (Chies et al., 2003). These

actin/phosphatidylinositol (4,5)-biphosphate (PIP-2)-positive vacuoles are part of the

ADP-ribosylation factor 6 (Arf-6) plasma-membrane-endosomal recycling pathway

involved in cell-cell adhesion and cell migration (reviewed in Donaldson et al., 2003).

Thus, PMP22 appears capable of regulating cell proliferation, morphology and

differentiation, all aspects crucial to the proper formation of myelin, the structure most

obviously affected by altered expression of the gene.

In summary, since the original discovery of PMP22 little has been learned about

its normal function in the myelinating Schwann cell. This may be due to difficulty in

studying the complex and largely unknown process of normal PNS myelination.

Advances in dissecting PMP22-related disease pathogenesis have focused on protein

trafficking and turnover or the characterization of nerve pathology. The function of the

protein is largely being examined in cell types unrelated to myelination, an approach

justified by extensive non-PNS PMP22 expression. However, since message levels are

elevated in epithelial cells such as those of the gut, it seems logical to first characterize

the localization of PMP22 in these cells. Furthermore, well-characterized cell models of

polarized epithelia, amenable to experimental manipulation, provide certain technical

advantages allowing for further investigation of PMP22's function. The ultimate goal of

these studies lies beyond determining a function for the protein in non-neural cell types,






14


by providing a foundation of knowledge to be used in revealing the role of PMP22 in

peripheral nerve myelin in health and disease. The purpose of this study was to examine

the expression and subcellular localization of PMP22 in non-neural cell types and provide

novel insights into the role of the protein in the cell membrane.














CHAPTER 2
PERIPHERAL MYELIN PROTEIN 22 IS A NOVEL CONSTITUENT OF
INTERCELLULAR JUNCTIONS IN EPITHELIA

Note

The work presented in this chapter was published in Proceedings of the National

Academy of Sciences USA 98(25) 14404-14409 (2001) Amy Yazdanpour and Christoph

Rahner assisted with the cryosectioning and immunostaining, Stephanie Amici assisted

with the RT-PCR and Western blots, and Bradley Fletcher assisted with the retroviral

infections.

Introduction

Peripheral myelin protein 22 (PMP22), also known as gas3, is a tetraspan

glycoprotein with proposed roles in peripheral nerve myelin formation, cell-cell

interactions, and cell proliferation (Suter and Snipes, 1995). PMP22 expression is highest

in myelin-forming Schwann cells; however, PMP22 mRNA can be detected in a variety

of non-neural tissues. Epithelial cells of the lungs and intestines are known to express the

highest levels of PMP22 mRNA outside of the peripheral nervous system (Baechner et

al., 1995; Taylor et al., 1995; Wulf et al., 1999), yet the localization or the role of the

protein in these tissues has not been determined. Although the function of PMP22 in

Schwann cells and non-neural cells is largely undefined, it is well established that

deletions, duplications, or mutations in PMP22 account for the majority of heritable

demyelinating peripheral neuropathies, including Charcot-Marie-Tooth disease type IA.

Myelin-forming Schwann cells and epithelial cells, two cell types with high levels









of PMP22 mRNA expression, share similarities in that they are both polarized and

maintain compositionally unique membrane domains. In addition, similar to the barrier

function of epithelia, Schwann cells separate intramyelinic and extramyelinic

extracellular space (Mugnaini and Schnapp, 1974). The molecular bases of how Schwann

cells attain these functions are not yet understood, although they are likely to involve

specialized intercellular junctions, such as adherens and/or tight junctions (TJs). Freeze

fracture studies of PNS myelin detected rows of TJ-like fibrils within the Schwann cell

membrane (Shinowara et al., 1980); nevertheless the identities of the proteins forming

these structures are unknown. Recent studies revealed the presence of TJ strands in CNS

myelin (Morita et al., 1999a), which is deposited by oligodendrocytes. A protein

component of TJ strands in CNS myelin is oligodendrocyte-specific protein/claudin-11, a

PMP22-related, tetraspan membrane protein (Morita et al., 1999a; Bronstein et al., 1996).

In addition to oligodendrocyte-specific protein/claudin-11, PMP22 shares

significant sequence identity and structural similarity with other claudins, including the

first discovered claudin in liver, claudin-1 (Furuse et al., 1998a). The claudin protein

family now includes more than 20 members with unique, as well as overlapping, tissue

distribution (Mitic et al., 2000, Rahner et al., 2001; Tsukita et al., 2001). Claudins appear

to have roles in the formation of TJ strands and in the establishment of the ionic

selectivity of the junctional barrier (Tsukita et al., 2001). The essential function of

claudins at TJs is supported by recent reports on claudin misexpression and

disease-causing alteration in epithelial physiology (Simon et al., 1999; Wilcox et al.,

2001). Occludin, also a tetraspan protein of TJs, is an adhesive molecule that may have

roles in the barrier function of TJs (Furuse et al., 1996; Wong and Gumbiner, 1997; Van









Itallie and Anderson, 1997). These transmembrane junctional proteins form complexes

with cytoplasmic molecules, such as zonula occludens-1 and -2 (ZO-1, ZO-2), which link

the membrane proteins to cytoskeletal elements (Fanning et al., 1998). As the molecular

architecture of intercellular junctions is being uncovered, studies show that in addition to

ionic barrier and fence functions, TJs are involved in intracellular vesicle targeting and

signaling (Zahraoui et al., 2000).

Based on the mRNA expression pattern, and the primary and secondary structure

of PMP22, we hypothesized that PMP22 might be a component of intercellular junctions

in epithelia. Therefore, we examined the expression and localization of PMP22 in

cultured epithelia and a variety of tissues with ZO. Using immunochemical, biochemical,

and molecular approaches, we found that in epithelial cells PMP22 is coexpressed with

occludin and ZO-1 at or near TJs and that overexpression of PMP22 in L cell fibroblasts

mediated the formation of ZO-1-positive intercellular junctions. These studies suggest

that the plasma membrane-associated biological function of PMP22 might involve a role

in the establishment and/or maintenance of intercellular junctions and possibly of TJs.

Materials and Methods

Cell Culture

Primary Schwann cell cultures were established from newborn rat pups

(Notterpek et al., 1999b). L cells (American Type Culture Collection) were maintained in

10% horse serum containing DMEM. Madin-Darby canine kidney (MDCK) cells were

cultured in 10% FBS containing DMEM on 0.4-pm pore size Transwell filters (Costar),

or glass coverslips, with or without type I collagen coating. Highly polarized, confluent

MDCK cell monolayers were incubated with 4 mM EGTA for 1-4 h to chelate the

calcium from the culture medium (Gumbiner and Simons, 1986; Kartenbeck et al., 1991).









EGTA treatment results in the rounding up of the cells and disassembly of intercellular

contacts.

Retroviral Overexpression of PMP22-myc in MDCK and L Cells

The mouse PMP22 ORF with a myc epitope in the 2nd extracellular loop (Tobler

et al., 1999) was directionally inserted into the retroviral plasmid pBMN (Hitoshi et al.,

1998). The resulting pBMN-PMP22myc, or a control pBMN-GFP (green fluorescent

protein) plasmid, was transiently transfected into the amphotropic retroviral packaging

cell line Phoenix A (obtained from Garry Nolan, Stanford University, CA). Retroviral

supernatants were collected after 30 h incubation at 320C and directly applied to 1 x 106

MDCK or L cells (-40% confluency). Retroviral transductions were performed at 32C

for 24 h in the presence of 5 [g/ml polybrene. Forty-eight hours postinfection, cells were

related and allowed to form confluent monolayers. Estimated from the number of

pBMN-GFP-expressing cells, the infection rate in the L cells was -99% and -15% in

MDCK cells.

Immunostaining Procedures

MDCK cells and 2- or 8-[tm thick cryosections of adult rat liver and colon were

double immunostained with polyclonal anti-PMP22 (Notterpek et al., 1999b) and

monoclonal anti-tight junction protein antibodies, according to published procedures

(Itoh et al., 1997). Primary antibodies included monoclonal anti-occludin and anti-ZO-1

(Zymed), and polyclonal anti-claudin-1 (Zymed) and anti-PMP22 (Notterpek et al.,

1999b). Twelve distinct polyclonal antibodies made against 16-aa peptides of the 1st

(amino acids 27-42) or 2nd (amino acids 117-133) extracellular loops of the mouse, rat,

or human PMP22 were used to localize PMP22 in the studied samples. Preimmune and









peptide preadsorbed (0.1 mg/ml) rabbit serum and nonspecific mouse IgGs served as

controls of antibody binding. Bound primary antibodies were detected with Alexa

fluorochrome-conjugated secondary antibodies, including FITC-conjugated anti-mouse

IgG and Texas red-conjugated anti-rabbit IgG (Molecular Probes). Nuclei were stained

with Hoechst dye. Coverslips were mounted by using a ProLong Antifade kit (Molecular

Probes), and images were acquired with a Spot camera attached to a Nikon Eclipse 1000

or an Olympus MRC-1024 confocal microscope. Images were processed for printing by

using Adobe PHOTOSHOP 5.0.

To increase the resolution of the immunoreactivity in filter-grown MDCK cells,

filters with confluent monolayers were sectioned after freezing and processed for

immunostaining (Itoh et al., 1997). For optimal detection of the myc epitope-tagged

PMP22, retrovirally infected MDCK and L cells were fixed in 4% paraformaldehyde,

followed by permeabilization and immunolabeling with polyclonal or monoclonal

anti-myc antibodies (Ryan et al., 2000). These fixation conditions are suboptimal for the

detection of endogenous TJ proteins, which is reflected by reduced levels of claudin,

ZO-1, and occludin-like immunoreactivities.

RNA isolation, Northern analysis, and Reverse Transcriptase-PCR (RT-PCR)

Total RNA was isolated from rat liver and rat Schwann cells by using the TRIzol

reagent (GIBCO Life Technologies). The Titan One Tube RT-PCR System (Roche

Diagnostic) was used to generate and amplify a 425-bp PMP22 cDNA fragment by using

1 [g of total RNA. Specific primers were synthesized according to the nucleotide

sequence of rat PMP22 (sense primer 5'-ACACTTGACCCTGAAGGC-3' and reverse

primer 5'-AGCATCAGAAGGACACCG-3'). Half of each RT-PCR product was digested









with a PMP22 sequence-specific restriction enzyme (Bsal), and samples were analyzed

on acrylamide gels. Negative controls included samples without the RT enzyme and

samples that were RNase-treated.

Biochemical Procedures

Bile canaliculi-enriched fractions from P70 rat livers were processed according to

established procedures (Song et al., 1969; Tsukita and Tsukita, 1989). Three different

membrane fractions were collected (Song et al., 1969) and analyzed by Western blotting

with anti-PMP22 antiserum.

To confirm that our anti-human, anti-rat, or anti-mouse PMP22 antibodies can

detect canine PMP22 in MDCK cells we purchased frozen dog sciatic nerves (Pel-Freez

Biologicals). Adult rat, mouse, and canine sciatic nerve lysates were analyzed on 12.5%

SDS gels as described (Notterpek et al., 1999b).

Control and retrovirally infected MDCK cell monolayers were extracted with

0.5% TX-100-containing buffer, and detergent soluble (S) and insoluble (I) fractions

were collected (Jou et al., 1998). Control and retrovirally infected L cells were directly

lysed in SDS gel sample buffer, and protein concentrations were determined.

Endoglycosidase H and N-glycosidase digestions were performed as described (Pareek et

al., 1997). To prevent the aggregation of PMP22, protein samples were heated to 80C

before loading of the gels. Gels were transferred to nitrocellulose membranes and

processed for immunoblotting with monoclonal anti-occludin and anti-ZO-1 (Zymed),

and polyclonal anti-claudin-1 (Zymed) and anti-PMP22 (Notterpek et al., 1999b)

antibodies. Bound antibodies were detected with horseradish peroxidase-conjugated









anti-mouse, or anti-rabbit, secondary antibodies (Sigma) by using ECL chemiluminescent

reagents (Amersham Pharmacia).

Results

PMP22 Localizes to Cell-Cell Junctions in the Rat Liver and Colon

Previous studies have shown high levels of PMP22 mRNA in various non-neural

tissues (Baechner et al., 1995; Taylor et al., 1995; Wulf et al., 1999); however, to date the

localization and the expression of protein at these sites has not been determined. Using a

TX-100 pre-extraction immunostaining procedure (Itoh et al., 1997), we detected bright

PMP22-like immunoreactivity at the surface epithelium of the mucosa in colon (Fig.

2-1A) and liver bile canaliculi (Fig. 2-1 C, E, and F). In colon, PMP22 and ZO-1 are

found at apical junctions of epithelial cells and in small blood vessels transversing the

submucosa (Fig. 2-1 A and B, arrowheads). In liver, PMP22 and ZO-1 are colocalized to

bile canaliculi (Fig. 2-1 C and D, respectively); however, only ZO-1, but not PMP22, is

present at endothelial cell junctions of the portal vein (Fig. 2-1E, arrows). Nerve

terminals show bright PMP22 and no ZO-1 immunoreactivity (Fig. 2-1E). In addition to

ZO-1, PMP22 is colocalized with occludin at bile canaliculi (Fig. 2-1 F and G,

respectively). The lack of PMP22-like immunoreactivities in liver sections incubated

with preimmune (Fig. 2-1A inset) or antigenic peptide preincubated serum (Fig. 2-1F,

inset) support the specificity of the PMP22-like immunostaining at these novel locations.

The localization of PMP22 in colon epithelium and bile canaliculi was verified by eight

distinct antibodies, including antisera raised against the 1st rather than the 2nd

extracellular loop of the protein (data not shown).

The expression of PMP22 in liver was confirmed by RT-PCR (Fig. 2-1H) and

Western analysis (Fig. 2-11). Using specific primers to the rat PMP22 cDNA we detected


























H rat liver RSC N Total Sucrose
42+^ N S P D3 D2 D1
425 35-
263 29-
172 21U



Figure 2-1. Coexpression of PMP22 with ZO-1 and occludin in colon epithelium and
bile canaliculi. Frozen sections of normal adult rat colon (A and B) and liver
(C-G) were coimmunostained with polyclonal anti-PMP22 (A, C, E, and F)
and monoclonal anti-ZO-1 (B, D, and E) or occludin (G) antibodies. (A and
B) Confocal images showing the presence of PMP22 at the surface epithelium
of the mucosa and in submucosal vasculature (arrowheads). (E) A high-
resolution thin section of adult rat liver stained with anti-PMP22 (red),
anti-ZO-1 (green) antibodies and nuclear dye (blue). PV, portal vein; N, nerve
terminal; BD, bile duct; HC, hepatocyte. Liver sections incubated with
preimmune serum (A Inset) or peptide preadsorbed antiserum (F Inset) do not
result in TJ-like immunostaining. Magnifications: x40 (A-E) and x60 (F and
G). (H) The expression ofPMP22 mRNA in liver was verified by RT-PCR.
Bsal undigested (-) and digested (+) PCR-amplified fragments are shown for
each sample. The numbers on the left indicate bp. (I) Membrane pellets (P)
(75 gg) from adult rat liver homogenates were fractionated and proteins
isolated at sucrose densities 1.22 (D3) and 1.18 (D2) and 1.16 (D1) were
analyzed (75 gg/lane) for the presence of PMP22. Rat sciatic nerve (N) lysate
(4 gg) was used as a positive control for the anti-PMP22 antibody
immunoreactivity. S, total liver supernatant (75 gg). Molecular mass, in kDa.

the identical 425-bp fragment in liver and Schwann cell RNA (Fig. 2-1H). The identities

of the PCR fragments were verified by Bsal restriction enzyme digests. To further









corroborate the expression of PMP22 in liver, crude liver membrane pellets were

subfractionated by discontinuous sucrose density-gradient ultracentrifugation (Fig. 2-11)

(Song et al., 1969; Tsukita and Tsukita, 1989). Although PMP22 is difficult to detect in

total liver membrane preparations, in bile canaliculi-enriched fractions (sucrose density

fractions 1 (Dl) and 2 (D2)) (Ryan et al., 2000), we observed a significant enrichment for

PMP22 (Fig. 2-11). The majority of PMP22 was concentrated at the interface of sucrose

densities 1.22 and 1.18 (D2) and was absent from the highest sucrose density fraction

(D3), which contains nuclei, mitochodria, and erythrocyte ghosts (Song et al., 1969).

Parallel blots incubated with preimmune or antigenic peptide preadsorbed serum were

completely blank at the 21-to 35-kDa range (data not shown). The slower migration of

PMP22 in bile canaliculi compared with sciatic nerve is likely caused by differential

posttranslational modification of PMP22 in myelin and non-neural tissue.

PMP22 is Localized to Epithelial Apical Cell Junctions

The in vivo tissue localization studies suggest that PMP22 is a component of

intercellular junctions in epithelia, therefore we examined the distribution of PMP22 in

MDCK cell monolayers. In subconfluent MDCK cell cultures, PMP22 (Fig. 2-2A) is

found at anti-ZO-1 antibody (Fig. 2-2B) immunoreactive intercellular junctions. The

nuclear staining observed with the anti-PMP22 antibody has been described before

(Pareek et al., 1997) and, in part, is caused by nonspecific immunoreactivity of the

antiserum (Fig. 2-2 A and C, insets). The distribution of PMP22 in confluent filter-grown

MDCK monolayers also was determined (Fig. 2-2 C-G). Similar to the subconfluent

cultures (Fig. 2-2 A and B), PMP22-like immunoreactivity (Fig. 2-2 C and E) is

colocalized with ZO-1 (not shown) and occludin (Fig. 2-2 D and F) at intercellular






















U
^^^^^^^^^^ PMP22~T*-C



PMP/flalffHoehs


H Rat Dog Mouse

21- m
1 2 3


Figure 2-2. Coexpression of PMP22 with ZO-1 and occludin at cell-cell contacts in
MDCK cells. Subconfluent (A and B) and filter-grown (C-G) MDCK cells
were immunostained with polyclonal anti-PMP22 (A, C, E, and G) and
monoclonal anti-ZO-1 (B), or anti-occludin (D and F) antibodies. (A Inset)
Cells stained with preimmune rabbit serum. In filter-grown MDCK cultures
PMP22 (C) is codistributed with occludin (D) at apical cell contacts. PMP22
antigenic peptide preadsorbed antiserum does not stain intercellular contacts
of MDCK cells (C Inset). On sectioned (8 gm) filters (Z plane) PMP22-like
immunoreactivity (E and G) is associated with the apical border of the
monolayer, which is also reactive with the anti-occludin (F) antibodies
(arrows in E and F). (G) Anti-PMP22 (red) and Hoechst nuclear dye (blue)
stained MDCK cell monolayer is shown. Magnifications: x60 (A, B, and E-G)
and x40 (C and D). (H) Protein blots of (18 gg/lane) normal adult rat, canine,
and mouse sciatic nerves were reacted with anti-PMP22 antiserum. The upper
arrow on the right indicates the glycosylated 22-kDa PMP22, while the lower
arrow points to the newly synthesized 18-kDa, endoglycosidase-H sensitive
protein. Molecular mass, in kDa.
junctions. Sectioned filters, double-stained with anti-occludin and anti-PMP22
antibodies, demonstrate that PMP22 (Fig. 2-2E) is present at apical cell-cell contacts,
similar to occludin (Fig. 2-2F). The colocalization of PMP22 and occludin at apical









intercellular contacts was confirmed by confocal microscopy and rotated

three-dimensional images of deconvolution microscopy (not shown). Because MDCK

cells are of canine origin, we verified by Western analysis that our anti-PMP22 antibodies

raised against human, rat, or mouse peptides can recognize the dog PMP22 (Fig. 2-2H).

As the anti-PMP22 immunoblot shows, we positively identified the dog PMP22 in total

sciatic nerve lysates, using an anti-human PMP22 antibody that stains intercellular

junctions in MDCK cells (Fig. 2-2H). On SDS gels, the canine nerve PMP22 has a

similar mobility as the rat or the mouse protein (-22 kDa) and shifts ~4 kDa upon

N-glycosidase treatment (not shown) (Pareek et al., 1997).

To begin to elucidate the relationship of PMP22 to known tight junctional

proteins, filter-grown MDCK cell monolayers were treated with EGTA to disrupt

intercellular contacts (Fig. 2-3). Previous studies showed that such treatment leads to the

internalization of proteins found at adherens and TJs (Cereijido et al., 2000). After a 1-h

EGTA treatment of the cultures, the majority of anti-ZO-1 and anti-occludin antibody

immunoreactive intercellular contacts disappeared, and both occludin (Fig. 2-3B) and

ZO-1 (not shown) were internalized in vesicles. Using double immunolabeling, PMP22

and occludin were detected together in a subpopulation of vesicles (arrows in Fig. 2-3 A

and B). These results strongly support that PMP22 is a protein component of apical

intercellular junctions in epithelial cells.

Tight junctional proteins are insoluble in TX-100 (Jou et al., 1998); therefore we

compared the solubility properties of PMP22 to ZO-1, occludin, and claudin-1 (Fig.

2-3C). Confluent MDCK monolayers were incubated with 0.5% TX-100 containing




















C Z01 Occludin PMP22 Claudini

225- a 21-

S I S I S I S I

Figure 2-3. Internalization of PMP22 with occludin in EGTA-treated MDCK cells.
Confluent MDCK monolayers were cultured in the presence of 4 mM EGTA
for 1 h followed by immunostaining with polyclonal anti-PMP22 (A) and
monoclonal anti-occludin (B) antibodies. Arrows point to vesicles that contain
both PMP22 (A) and occludin (B). (Magnification: x60.) (C) Confluent
MDCK cell monolayers were extracted with 0.5% TX-100 containing buffer
and detergent soluble (S), and detergent-insoluble (I) fractions were
immunoblotted with antibodies against ZO-1, occludin, PMP22, and
claudin-1. Molecular mass, in kDa.

buffer, and detergent soluble and insoluble fractions (Jou et al., 1998) were analyzed for

the four antigens (Fig. 2-3C). In agreement with previous studies, we found that the

greater portion of ZO-1 and occludin remain in the TX-100 insoluble fraction (Jou et al.,

1998), whereas the majority of claudin-1 is extracted in the detergent (S). The greater

solubility of claudin-1 correlates with its high intracellular levels in MDCK cells (Fig.

2-4B). In contrast to claudin-1, PMP22 is largely insoluble in 0.5% TX-100 containing

buffer (I) (Fig. 2-3C).

Epitope Tagged PMP22 is Targeted to Epithelial Cell Junctions

To further validate our findings on the apical junctional localization of PMP22 in

MDCK cells, we studied the targeting of myc-tagged mouse PMP22 (Fig. 2-4). A myc









epitope tag in the 2nd extracellular loop of PMP22 does not interfere with the normal

processing and trafficking of the protein (Tobler et al., 1999, Ryan et al., 2000). MDCK

cells infected with the pBMN-PMP22-myc construct were plated on coverslips or filters

and allowed to proliferate. In subconfluent cultures we detected PMP22-myc (Fig. 2-4 A

and C) and claudin-1 (Fig. 2-4 B and C) at intercellular junctions. Cells that are

overexpressing the mouse PMP22-myc are able to integrate and establish intercellular

contacts with parental cells (Fig. 2-4 A, C, and D). Furthermore, in filter-grown MDCK

cell monolayers, the exogenous PMP22-myc protein is correctly targeted to apical cell

contacts (Fig. 2-4E).

The apical junctional targeting of PMP22-myc also was established by Western

analysis (Fig. 2-4F). Anti-myc immunoblots of control, pBMN-GFP, and PMP22-myc

infected MDCK cells specifically detects an -27-kDa and a less abundant -33-kDa band

in the PMP22-myc sample (Fig. 2-4F, lane 3). Both -27-kDa and -33-kDa bands shift

upon deglycosylation with N-glycosidase (data not shown) (Pareek et al., 1997). The

arrowheads at -34 kDa indicate a nonspecific protein that is immunoreactive with the

myc antibody in control (Fig. 2-4F, lane 1) and GFP-infected (Fig. 2-4F, lane 2) cells.

Significantly, similarly to the endogenous canine PMP22 (Fig. 2-3 C), the majority of

PMP22-myc is also insoluble in 0.5% TX-100 (Fig. 2-4F, lane I). In addition to the -27

kDa and -33 kDa bands, the detergent-insoluble fraction contains a range of high

molecular mass anti-myc antibody immunoreactive proteins, which likely represent

aggregates of PMP22 multimers (Tobler et al., 1999). Together, these overexpression

experiments strongly support that PMP22 is a component of the apical intercellular

junctional complex in epithelia.






























35-123 S I-



Figure 2.4. Exogenously expressed PMP22-myc is targeted to TJs in MDCK cells.
pBMN-PMP22-myc-infected cells were cultured on coverslips (A-D) or
Transwell filters (E) and immunostained with monoclonal anti-myc (A and
C-E), and polyclonal anti-claudin-1 (B and C) antibodies. Note, only -15% of
the cells (green cells) were infected with the PMP22-myc construct (A and
C-E). PMP22-myc is targeted to anti-claudin-1 immunoreactive intercellular
contacts (arrows in A-C) and PMP22-myc-expressing cells form contacts with
noninfected cells (D). Nuclei were stained with Hoechst dye (C and D Inset).
[Magnifications: x60 (A-C), and x40 (D and E).] (F) The expression of
PMP22-myc was verified by anti-myc Western analysis. Lysates of
PMP22-myc-infected cells (lane 3) show expression of a -27-kDa and a
-33-kDa PMP22-myc protein. A nonspecific -34-kDa band is present in all
samples, including uninfected control (lane 1) and pBMN-GFP (lane 2)-
infected cell lysates (arrowheads). The majority of PMP22-myc protein is
insoluble in TX-100 (I). S, TX-100 soluble. Molecular mass, in kDa.

Exogenous PMP22 Alters Cell-Cell Contacts in L-Fibroblasts

Overexpression of claudins or occludin in nonadherent L fibroblasts has been

shown to induce the formation of intercellular contacts, including well-organized TJs


myc/Claudin




















21- 4 4
Ii 4


E1


Figure 2-5. Colocalization of PMP22-myc with ZO-1 at intercellular junctions of L
fibroblasts. Uninfected control L cells (A and B) show diffuse ZO-1-like
membrane staining with focal concentration at cell processes (A) and low
levels of nonspecific immunoreactivity with polyclonal anti-myc (B)
antibodies. In PMP22-myc-infected cells, PMP22-myc is detected at cell-cell
contacts (arrows in C and F), which are costained with the anti-ZO-1 antibody
(arrows in D and G). PMP22-myc is detected in aggresome-like structures in
some cells (* in F). Nuclei are stained with Hoechst dye (H). (Magnification:
x60.) (E) The expression and processing of PMP22-myc was studied in
lysates of control, uninfected (lane 1), and PMP22-myc-infected L cells (lanes
2-5) by anti-myc Western blotting. Untreated (lane 2), endoglycosidase H
(lane 3), N-glycosidase (lane 4), and no enzyme (lane 5) PMP22-myc samples
are shown. N-glycosidase (N) treatment of PMP22-myc cell lysates results in
a characteristic shift of PMP22, from a mature high molecular mass,
endoglycosidase H-resistant form (upper arrow) to a deglycosylated, lower
molecular mass core protein (lower arrow). The N-glycosidase resistant,
anti-myc immunoreactive -29-kDa band likely represents a
mono-ubiquitinylated PMP22-myc (lane 4). Molecular mass, in kDa.
(Furuse et al., 1998b). Therefore, we examined the targeting of mouse PMP22-myc in

mouse L fibroblasts (Fig. 2-5). Parental L cells express low levels ofPMP22 mRNA and

undetectable PMP22 protein (data not shown). In agreement with previous studies in

parental L cells, ZO-1 is diffusely distributed over cell bodies and concentrated in puncta

at processes (Fig. 2-5A). In cells overexpressing PMP22-myc, we detected anti-myc (Fig.









2-5 C and F) and anti-ZO-1 (Fig. 2-5 D and G) immunoreactivity at cell-cell contacts.

Well-defined intercellular junctions can be seen, which are immunoreactive with both

anti-myc and anti-ZO-1 antibodies (arrows in Fig. 2-5 C, D, and F-H). Significantly,

overexpression of PMP22 alters the distribution of ZO-1 and appears to cause the

recruitment of ZO-1 to intercellular contacts (compare Fig. 2-5A and D). Although the

formation of intermittent intercellular contacts is consistently observed in PMP22-myc

infected L cells, overexpression of PMP22 does not appear to induce long strands

involving multiple cells, such as described for claudins (Furuse et al., 1998b). Cells that

have integrated multiple copies of PMP22-myc often contain intracellular PMP22

aggregates, termed aggresomes (Fig. 2-5F *) (Notterpek et al., 1999a). Uninfected (Fig.

2-5B) or pBMN-GFP-infected (not shown) L cells do not adhere together and exhibit low

levels of nonspecific immunoreactivity with the myc antibody.

The expression and processing of PMP22-myc in L cells was studied by anti-myc

Western analysis of endoglycosidase H-treated and N-glycosidase-treated cell lysates

(Fig. 2-5E). Overexpression of PMP22-myc in L cells yields several bands with apparent

mobilities -30 kDa. A portion of the overexpressed protein is resistant to

endoglycosidase H treatment (Fig. 2-5E, lane 3) and likely represents the membrane

fraction of the protein (Pareek et al., 1997). N-glycosidase treatment shifts the majority of

these bands to -22 kDa, which corresponds to the peptide backbone of PMP22-myc (Fig.

2-5E, lane 4). The anti-myc immunoreactive -29-kDa band in the N-glycosidase-treated

sample might represent ubiquitinylated PMP22-myc, which is suggested by the presence

of PMP22-myc aggregates in PMP22-myc infected L cells (Fig. 2-5F *) (Notterpek et al.,

1999a). These L cell overexpression studies suggest that PMP22 might serve an adhesive









role at intercellular junctions and through indirect protein interactions it affects the

localization of ZO-1.

Discussion

Because of its well-established disease association, PMP22 has received

considerable attention during the last decade (Suter et al., 1995; Naef and Suter, 1998).

Although we have gained significant insight into the genetics of PMP22-associated

peripheral neuropathies, as well as the intracellular turnover and processing of PMP22 in

normal and neuropathy Schwann cells, we still do not understand the function of the

protein. Here, we present data on intercellular junctional localization of PMP22 in

epithelial cells, suggesting that PMP22 plays a role in cell-cell interactions.

Given that PMP22 is primarily known as a peripheral nervous system myelin

component, the expression of PMP22 at epithelial cell junctions may seem unexpected.

Nonetheless, our results are in complete agreement with previous studies on the tissue

distribution of PMP22 mRNA (Baechner et al., 1995; Taylor et al., 1995; Wulf et al.,

1999). One of the tissues with reported highest levels of PMP22 mRNA is the

gastrointestinal tract (Baechner et al., 1995), where we detected bright PMP22-like

immunoreactivity at intercellular junctions of absorptive colonic epithelium. In addition

to the epithelial cells of the gastrointestinal tract, PMP22 is present at TJs of the liver.

Previously, PMP22 mRNA expression was shown to be high in embryonic liver;

however, message levels decreased significantly during postnatal development (Baechner

et al., 1995). These findings are in agreement with reports describing that the turnover

rate of junctional proteins at established membrane contacts is fairly slow (48 h) (Pasdar

and Nelson, 1989; McCarthy et al., 2000), therefore the rate of PMP22 mRNA and

protein synthesis in postembryonic liver is expected to be low. Nonetheless, by RT-PCR









we detected PMP22 mRNA in the adult rat liver; and by using an established membrane

subfractionation procedure (Song et al., 1969), we identified PMP22 in bile

canaliculi-enriched liver membrane preparations.

Although PMP22 is expressed in a variety of tissues, the gross pathological

findings in PMP22 mutant animals are limited to myelinated peripheral nerves. These

data may indicate that Schwann cells are particularly sensitive to PMP22 missexpression

and/or that PMP22-related proteins compensate for the normal function of PMP22 in

other tissues. A similar compensatory mechanism might operate in occludin-deficient

mice, which form morphologically and functionally intact TJs (Saitou et al., 2000). Our

results on the epithelial localization of PMP22 warrant a closer examination of PMP22

neuropathy animals, as it is known that certain PMP22 mutant mice display nonglial

abnormalities, which are difficult to explain by myelination defects alone. For example,

during early postnatal development homozygous Tr-J animals exhibit -35% reduction in

weight compared with wild-type littermates (unpublished data) and die at around

postnatal day 18 (Henry et al., 1983). The homozygous Tr-J condition is the only known

lethal phenotype associated with PMP22 missexpression and it cannot be explained by

peripheral myelination defects alone, as several other peripheral myelin-deficient animals

live normal life spans (Martini and Schachner, 1997). These paradoxes regarding the

phenotypes of PMP22 neuropathy animals have puzzled investigators of the field for

many years; however, to date possible explanations have not previously been put forth.

The in vivo protein expression studies suggest that PMP22 is a constituent of

membrane junctions in epithelia; however, they provide limited information on the

relationship of PMP22 to established TJ membrane proteins. The internalization of









PMP22 with occludin, in EGTA-treated MDCK cells, supports the notion that PMP22 is

a constituent of the apical junctional complex. The detergent solubility properties of

PMP22 imply that PMP22 might be associated with occludin and/or ZO-1, or other

TX-100-insoluble proteins, rather than with claudin-1. Because PMP22 shares 28%

amino acid sequence identity with claudin-1 (Takeda et al., 2001), it is important to note

that in this assay the two proteins segregate differently. These results strongly argue

against any cross-reactivity of our anti-PMP22 antibodies with claudin-1, or other

claudins, as the two proteins are detected in opposite fractions of the cell lysates.

Nonetheless, because PMP22 shares significant sequence identity with several members

of the claudin gene family (Takeda et al., 2001), our studies raise the question of whether

PMP22 may be another claudin. It has recently been established that all

well-characterized claudins are able to reconstitute long TJ strands in fibroblasts (Tsukita

et al., 2001). Previous studies performed in HeLa cells (D'Urso et al., 1999) and our

results in L cells suggest that PMP22 has adhesive properties, as it can mediate

intercellular contacts between nonadherent cells and is able to recruit ZO-1 to newly

formed cell junctions (Fig. 2-5). In comparison to L cells, PMP22 overexpression does

not appear to induce intercellular adhesion in C6 glioma cells (Takeda et al., 2001), a

central nervous system-derived tumor cell line. These differences in response to PMP22

overexpression are likely the results of cell specificities in endogenous junctional

molecules and/or differences in the processing and trafficking of the overexpressed

PMP22. Nonetheless, further studies will be necessary to examine the ultrastructure of

these newly formed membrane junctions, as claudins are known to mediate the assembly









of long fibrils, in comparison to short strands that are formed by occludin overexpression

(Van Itallie and Anderson, 1997; Furuse et al., 1998b).

Besides structural similarities, PMP22 shares functional properties with some of

the claudins. Recent reports revealed that single point mutations in claudin-16 and

claudin-14 cause kidney and hearing abnormalities, respectively (Simon et al., 1999;

Wilcox et al., 2001). Of the known claudins, claudin-15 is the most homologous to

PMP22, sharing 30% identity and 54% similarity in their amino acid sequences

(unpublished data). Although the total molecular mass of the claudins and PMP22 is

identical (22 kDa), 4 kDa of the total molecular mass of PMP22 is comprised of

carbohydrate that is attached to Asn-41 in the 1st extracellular loop. This carbohydrate

motif has a role in the homodimerization of PMP22 (Tobler et al., 1999; Ryan et al.,

2000) and is required for the cell spreading effect observed in PMP22-overexpressing

fibroblasts (Brancoloini et al., 2000). The carbohydrate modification of PMP22 in

epithelial cells is unknown, but it could have a role in mediating homophilic interaction

between neighboring cells.

The studies described here provide insights into the potential function of PMP22

in membrane physiology. Our results demonstrate that PMP22 is a protein component of

intercellular junctions, where it might mediate the formation of cell-to-cell contacts

and/or stabilize membrane contacts. A similar role for PMP22 in the Schwann cells

membrane could explain the demyelinating phenotypes associated with various forms of

PMP22 misexpression.














CHAPTER 3
TEMPOROSPATIAL EXPRESSION OF PERIPHERAL MYELIN PROTEIN 22 AT
THE DEVELOPING BLOOD-NERVE AND BLOOD-BRAIN BARRIERS

Note
The work presented in this chapter was published in The Journal of Comparative

Neurology 474(4) 578-588 (2004). Julie Oakley and Shale Joy assisted with the

cryosectioning and Stephanie Amici assisted with the establishment of choroid epithelial

cultures and Northern blot analysis.

Introduction

The peripheral (PNS) and central nervous systems (CNS) are privileged

environments, selectively restrictive to molecules found in the general circulatory system.

Enclosing the peripheral nerve endoneurium is the blood-nerve barrier (BNB),

established and maintained largely by intercellular junctions of the perineurium and

endothelial cells of endoneurial vasculature (Allt and Lawrenson, 2000; Smith et al.,

2001). The blood-brain barrier (BBB) similarly provides a restrictive environment for the

CNS parenchyma, relying on cell-cell junctions of the brain vasculature, choroid

epithelium and arachnoid (Saunders et al., 2000).

In the mature BBB, tight junctions are thought to regulate the paracellular flow of

molecules (Kniesel and Wolburg, 2000; Huber et al., 2001). During development,

interendothelial junctions of the brain microvasculature display increasing ultrastructural

complexity (from ~E11 to early postnatal) with a loss of expanded paracellular clefts and

more strand continuity (Schulze and Firth, 1992; Stewart and Hayakawa, 1994; Kniesel et









al., 1996). Nonetheless, throughout development, functional studies detected low

transendothelial permeability to serum proteins (Kniesel and Wolburg, 2000; Saunders et

al., 2000). Thus, changes in ultrastructural junction complexity may not be necessarily

concurrent with physiologic alterations. The choroid epithelium regulates the

composition of the cerebrospinal fluid (CSF), which in the mature mammalian brain has a

low protein concentration as compared to serum (Dziegielewska et al., 2000). The CSF of

the immature brain contains high levels of protein (Dziegielewska et al., 2000) that

bypass restrictive tight junctions (Ek et al., 2003) and penetrate the choroid epithelium

through an intracellular route (Balslev et al., 1997; Knott et al., 1997). In the embryonic

mammalian brain, specialized neuroepithelial junctions are thought to enable the brain

parenchyma to exclude CSF proteins (Mollgard et al., 1987), thus maintaining a

restrictive environment for the immature CNS.

Several proteins involved in the formation and maintenance of the BBB

intercellular junctions have been identified. Such proteins include cadherins and

P-catenin at adherens junctions; tight junction-associated occludin, claudins, and

junctional adhesion molecules (JAMs), and ZO-1 which can be detected at both tight and

adherens junctions (Kniesel and Wolburg, 2000; Lippoldt et al., 2000; Huber et al., 2001;

Wolburg et al., 2001; Vorbrodt and Dobrogowska, 2003). Peripheral myelin protein 22

(PMP22) is a recently described constituent of interepithelial junctions in the rat colon

and Madin-Darby canine kidney cells (Chapter 1, Notterpek et al., 2001). Also known as

gi ,i i/i arrest specific gene 3 (gas3), PMP22 is a 22kD tetraspan glycoprotein with

proposed roles in peripheral nerve myelination, cell-cell interactions and cell proliferation

(Jetten and Suter, 2000). While the function of PMP22 in Schwann cells and non-neural









cells is largely undefined, it is well established that deletions, duplications or mutations

of PMP22 account for the majority of heritable demyelinating peripheral neuropathies,

including Charcot-Marie-Tooth disease type IA (Gabreels-Festen and Wetering, 1999).

Indeed, PMP22 expression is highest in myelin-forming Schwann cells; however, PMP22

mRNA is readily detected outside of the PNS (Bosse et al., 1994; Taylor et al., 1995;

Baechner et al., 1995; Parmantier et al., 1995; 1997; Lobsiger et al., 1996; Wulf et al.,

1999). In the rodent CNS, PMP22 message is found in a subset of motoneurons, and

during embryogenesis, at the neuroepithelial cell layer of the ventricular zone (Baechner

et al., 1995; Parmantier et al., 1995; 1997; Wulf and Suter, 1999).

Based on the mRNA expression pattern and epithelial distribution of PMP22, we

investigated the presence of the protein at epithelial and endothelial cell contacts of the

rodent BNB and BBB. Utilizing an antigenic PMP22 peptide-purified antibody, we

detected PMP22 at occludin, claudin-5 and ZO-1 immunoreactive endothelial and

choroid epithelial cell junctions. Furthermore, PMP22 is also present at unique

neuroepithelial junctions of the subventricular zone in the embryonic rat brain. These

studies suggest a ubiquitous role for PMP22 at intercellular junctions.

Materials and Methods

Northern Blot Analysis

For the RNA isolation, rats of the specified ages were euthanized by CO2

asphyxiation followed by decapitation, or by decapitation alone (postnatal day 1 (P1)

pups), and freshly collected tissues were immediately frozen in liquid nitrogen. The use

of animals for these studies has been approved by the University of Florida IACUC.

Total RNA was isolated using TriZol LS reagent (Gibco BRL) from the following P1 and

P70 rat tissues and cells: cortices without pial surfaces or choroid plexuses, cortical









microvessels, choroid plexuses, primary cultures of choroid epithelia (see below) and

Schwann cells (Notterpek et al., 2001). Total RNA from Schwann cells (4 [g) and CNS

derived tissues and cells (10 [g) were electrophoretically separated on a formaldehyde

agarose gel and transferred to a nylon membrane (Hybond, Amersham International). A

32P-labeled probe corresponding to the entire rat PMP22 open reading frame was used to

detect PMP22 mRNA expression. An 18S ribosomal RNA probe (gift of Dr. Sue Semple-

Rowland, University of Florida) served as an internal loading control. Message levels

were quantified using the Scion Image densitometry program (Scion Corporation).

Morphological Studies of Rat Sciatic Nerve and Brain

Embryonic day 15 (E15), P1 and P70 brains and P10, P20 and P70 sciatic nerves

were removed from rats euthanized by CO2 asphyxiation followed by decapitation, and

were immersed in liquid nitrogen cooled n-methylbutane. Nerve and brain samples were

cut on a cryostat at 8 |tm thickness. To increase the resolution of the studied molecules at

endothelial junctions of brain microvessels, one mm3 isolated rat cortices were pressed

between glass slides (Utsumi et al., 2000) and allowed to dry prior to fixation and

immunostaining (Itoh et al., 1997; Notterpek et al., 2001). Primary antibodies included

monoclonal anti-occludin, anti-claudin-5, anti-ZO-1 (Zymed Laboratories), anti-P-catenin

(BD Transduction Labs), and polyclonal anti-occludin (Zymed) and anti-PMP22

(Notterpek et al., 2001). Preimmune rabbit serum, antigenic PMP22 peptide-adsorbed

(0.1 mg/ml) immune serum and secondary antibody alone served as controls of antibody

binding specificity. Bound primary antibodies were detected with Alexa fluorochrome-

conjugated secondary antibodies, including FITC-conjugated anti-mouse IgG and Texas

red-conjugated anti-rabbit IgG (Molecular Probes, Inc.). Nuclei were stained with









Hoechst dye (Molecular Probes). Parallel samples were fixed as described above and

stained with cresyl violet (Nissl stain). Coverslips were mounted by using the ProLong

Antifade kit (Molecular Probes). Samples were imaged with a Spot camera attached to a

Nikon Eclipse 1000 microscope and were formatted for printing by using Adobe

PHOTOSHOP 5.0.

Isolation and Culture of Brain Endothelia

Primary cultures of brain endothelial cells (BECs) from rat and mouse

microvasculature were established following a published protocol (Tontsch and Bauer,

1989). Briefly, brain cortices were isolated from decapitated P1 rat or P4 mouse pups,

followed by the removal of pial surfaces and choroid plexuses. Cortices were minced and

enriched for microvessels by processing with a loose fitting Dounce homogenizer and

centrifugation in a sucrose buffer. Isolated microvessels were dissociated with type I

collagenase (Sigma) and cultured on fibronectin-coated glass coverslips in 30% S180 cell

(American Type Culture Collection) conditioned media, 10% heat-inactivated fetal calf

serum, endothelial cell growth supplement (ECGS: 20 tg/ml) (Becton Dickinson), and

heparin (100 tg/ml) (Sigma) in medium 199. Cells were fixed 48 hours after plating

according to the protocol outlined above for the tissue samples (Itoh et al., 1997). To

immunostain the BECs, polyclonal anti-PMP22 antibodies from whole rabbit serum were

antigenic peptide-purified using a cyanogen bromide-activated sepharose 4B (Sigma)

column. Rabbit IgGs bound to peptide, corresponding to the 2nd loop (amino acids 117-

133) of the rat PMP22, were isolated and used for double immunolabeling with

monoclonal anti-ZO-1 antibodies. Specificity of the antigenic peptide-purified antibodies









for PMP22 was determined by Western blotting rat nerve lysates (Notterpek et al.,

1999b).

Isolation and Culture of Choroid Plexus Epithelia

Primary cultures of rat and mouse choroid plexus epithelia were established with

minor modifications of a published protocol (Strazeille and Ghersi-Egea, 1999). Briefly,

choroid plexus tissue was isolated from P1 rat or P4 mouse brains, rinsed in calcium- and

Mg2+-free Hank's balanced salt solution (HBSS) and incubated in Pronase E (Sigma) for

25 minutes at 37C. After rinsing in HBSS, the tissue was incubated in 0.025%

trypsin/HBSS (CellGro) with 12.5 [tg/ml DNase I (Sigma), and following sedimentation,

the supernatant was collected into chilled fetal bovine serum. This was repeated 5 times,

followed by a 5 minute centrifugation of the pooled supernatants to collect the cells for

resuspension in culture medium (DMEM with 10% fetal calf serum) and plating on a

laminin-coated dish for two hours. Unattached cells, enriched for choroid plexus

epithelia, were transferred to a laminin-coated coverslip or dish and cultured until

confluent (~ 6 days). Immunostaining for junctional proteins and PMP22, or cell lysis for

RNA isolation was performed, as described above.

Calcium Switch Assay

Primary cultures of mouse BECs were incubated with 4 mM EGTA for 4 hours to

chelate the calcium in the culture medium (Notterpek et al., 2001). EGTA treatment

results in disassembly and internalization of intercellular contacts (Gumbiner and

Simons, 1986). Subsequently, cells were fixed and immunostained for PMP22 and ZO-1,

as above.








Results

PMP22 is Detected at the BNB

Compared to other tissues, peripheral nerve myelin contains the highest level of

PMP22 (Snipes et al., 1992; Lobsiger et al., 1996). It is not established, however, if other


Nissi


ZO-.1


PMP22 occludin claudin-5
- Umm -


Kyj


Figure 3-1. Endothelial cell junctions of the BNB in the developing and adult rat sciatic
nerve are PMP22 immunoreactive. Rat sciatic nerve sections were stained
with Nissl (A, F and K, arrows), or coimmunostained for ZO-1 (B, G and L)
and PMP22 (C, H and M). On parallel sections, the localization of occludin
(D, I and N) and claudin-5 (E, J and 0) was examined. In P10 (A-E), P20
(F-J) and P70 (K-O) rat sciatic nerves, PMP22 colocalizes with ZO-1 at
interendothelial cell junctions of nerve vessels. Unlike PMP22 (C, H and M),
junctional occludin-like immunoreactivity (D, I and N) is less intense at P10
than at P20, or P70. No primary antibody control (C') and peptide-adsorbed
anti-PMP22 antibodies (M') fail to immunolabel endothelial intercellular
junctions. Scale bar = 30[tm (0).

cell types in the PNS besides Schwann cells express detectable levels of the protein.

Since PMP22 is present at epithelial and endothelial cell junctions in various non-neural


P10





P20





P70









tissues (Notterpek et al., 2001), we examined endoneurial blood vessels of the rat sciatic

nerve for the expression of the protein during the development of the BNB (Fig. 3-1).

Longitudinal cryosections of rat sciatic nerves were fixed and stained with Nissl, or

double immunolabeled utilizing a procedure optimized to detect junctional molecules

(Itoh et al., 1997). This detergent permeabilization method does not permit the detection

of PMP22 in peripheral myelin, which is the most recognized staining pattern for the

protein (Notterpek et al., 1997). At all studied ages, elongated nuclei of the nerve

vasculature are readily visible by the Nissl stain (Fig. 3-1A, F, K). In the P10 rat nerve

(Fig. 3-1A-E), ZO-1 (Fig. 3-1B) and PMP22 (Fig. 3-1C) are colocalized at

interendothelial cell junctions. Immunolabeling of parallel nerve sections reveal that

occludin is barely detectable at these cell-cell contacts in the P10 nerve (Fig. 3-1D).

Claudin-5, a tight junction protein predominantly expressed in endothelia (Morita et al.,

1999b), exhibits a primarily diffuse, likely intracellular pattern (Fig. 3-1E). With

maturation of the nerve (at P20 and P70), both ZO-1 and PMP22 remain at endothelial

junctions (Fig. 3-1G, L, and 3-1H, M, respectively), while occludin-like

immunoreactivity gradually increases between P20 (Fig. 3-11) and P70 (Fig. 3-1N).

During the same developmental period, the subcellular distribution of claudin-5 becomes

more distinct at cell-cell junctions (Fig. 3-1J, 0). Secondary antibody alone (Fig. 3-1C')

and antigenic PMP22 peptide-adsorbed antibody (Fig. 3-1M') controls fail to label

endothelial cell contacts. Thus, the temporal expression of PMP22 at interendothelial

junctions of the developing rat nerve parallels that of ZO-1 and claudin-5, rather than of

occludin.








PMP22 in the Developing and Adult Rat Brain Microvasculature

Since PMP22 is present in endothelia and epithelia of various tissues (Notterpek

et al., 2001) and mRNA expression is reported in the CNS (Baechner et al., 1995;

Parmantier et al., 1995; 1997, Wulf and Suter, 1999), we investigated whether PMP22


A P1 B



o/o16
8- C 1B


0.
S1.4-
18P22 1.8kb X 12"
C4 10.

developing rat BBB. (A) PMP22 expression in various rat tissues and cell
0.6-



18Sand 18S ribosomal RNA (loading control). (1.8kb) Densitometric analysis of the



1 2 3 4

Figure 3-2. Expression of PMP22 mRNA is elevated in tissues and cells of the
developing rat BBB. (A) PMP22 expression in various rat tissues and cell
lines was investigated by Northern blot analysis. Total RNA from P1 rat
cortex (lane 1), BMV-enriched fraction (lane 2), choroid plexus tissue (lane 3)
and cultured choroid epithelia (lane 4) (10 stg/lane) were probed for PMP22
and 18S ribosomal RNA (loading control). (B) Densitometric analysis of the
blot was performed after correction for RNA loading. The level of PMP22
mRNA in the P1 cortex was arbitrarily set to a value of 1, allowing for
comparison of the samples.

is expressed in endothelial and/or epithelial cells of the brain (Fig. 3-2). Northern blot

analysis of rat brain cortex, isolated cortical microvessels and choroid plexuses, and

primary cultures of choroid epithelia was performed to compare the levels of PMP22









mRNA in the rat brain microvasculature (BMV) and choroid epithelia to that of total

cortex (Fig. 3-2A). Densitometric analysis of the blot, after correction for loading, is

shown in Figure 2B. Cultured rat Schwann cell RNA was used as a positive control for

the PMP22 hybridization (data not shown). In the P cortex, without meninges or choroid

plexuses, the level of PMP22 mRNA is very low (Fig. 3-2, lane 1). Compared to total

cortical RNA, isolated rat BMV (lane 2) and choroid plexus tissue (lane 3) from early

postnatal rats have an approximately 8-fold enrichment in PMP22 message (Fig. 3-2B).

The expression of PMP22 observed for choroid plexuses was confirmed in cultured

choroid epithelia (lane 4), which contains PMP22 message levels nearly 17-fold higher

than total cortex. Low levels of PMP22 mRNA are detected in the total cortex, BMV and

choroid plexus from the P70 rat brain (data not shown). These results demonstrate that

during early postnatal development, PMP22 message levels are elevated in BMV and

choroid plexus epithelia, as compared to total cortex.

The localization of PMP22 at intercellular junctions of the BMV was investigated

by double immunolabeling pressed preparations of E15, P and P70 rat brain cortices

(Fig. 3-3). As the Nissl stained samples reveal (Fig. 3-3A, F, K), this method of tissue

preparation preserves vessel continuity and optimizes the visualization of cell-cell

contacts (Utsumi et al., 2000). At the earliest age examined (El 5), ZO-1 (Fig. 3-3B) and

PMP22 (Fig. 3-3C) are already present at interendothelial contacts and persist throughout

development without obvious changes in levels or distribution (Fig. 3-3G and L; 3-3H

and M). On parallel sections, the localizations of the tight junction proteins occludin (Fig.

3-3D, I, N) and claudin-5 (Fig. 3-3E, J, 0) were also examined. In the E15 BMV,

occludin-like immunostaining is barely detectable (Fig. 3-3D), with a gradual increase in













E15


Nissi

A ?


ZO-1


PMP22


KI


P70


occludin


Figure 3-3. Endothelial cell contacts of the developing and adult rat BMV are
immunoreactive for PMP22. Rat brain cortices (1 mm3) were pressed, stained
with Nissl (A, F, and K), or coimmunostained for ZO-1 (B, G and L) and
PMP22 (C, H and M). Occludin (D, I and N) and claudin-5 (E, J and 0)
immunoreactivities were examined in parallel. In the E15 (A-E), P (F-J) and
P70 (K-0) rat cortices, PMP22 colocalizes with ZO-1 at endothelial cell
contacts of microvessels. During early development (E15 and P1), junctional
occludin-like immunoreactivity is less pronounced (D and I), as compared to
PMP22 (C and H). Endothelial claudin-5 expression is observed for all ages
studied (E, J and 0). No primary antibody control (C'), preimmune rabbit
serum (H') and peptide-adsorbed anti-PMP22 antibodies (M') fail to label the
cell contacts. Scale bar = 20tm (0).
signal between PI and P70 (Fig. 3-31, N). In comparison, similar to PMP22 and ZO-1,

claudin-5-like immunoreactivity (Fig. 3-3E, J, 0) is present throughout development. The

subcellular localization of claudin-5, in addition to being found at junctions, includes









significant amounts of diffuse staining, likely representing intracellular protein. At all

ages studied, the anti-rabbit secondary antibodies (Fig. 3-3C'), the preimmune rabbit

serum (Fig. 3-3H') and antigenic PMP22 peptide-adsorbed antibodies (Fig. 3-3M') fail to

label cell-cell contacts of the rat BMV.

To improve the subcellular resolution of PMP22-like immunoreactivity in brain

endothelia, microvessels isolated from P4 mouse cortices were dissociated and cultured

on fibronectin-coated glass coverslips. Semi-confluent BEC monolayers were double

immunostained for PMP22 and ZO-1 (Fig. 3-4). Whole anti-PMP22 rabbit serum (Fig.

3-4A') not only detects cell junctions (arrow), but also labels BEC nuclei (asterisk). In

order to establish the specificity of the PMP22 immunoreactivity pattern, antigenic

PMP22 peptide-purified antibodies were prepared and used to immunolabel the BEC

cultures (Fig. 3-4A, D). In agreement with the in vivo studies (Fig. 3-3), PMP22-like

immunoreactivity, detected with the peptide-purified antibody (Fig. 3-4A) colocalizes

with ZO-1 (Fig. 3-4B, C, arrows) at endothelial cell junctions. Low levels of PMP22 are

also seen in the cytoplasm of the cells, possibly reflecting the ER-Golgi fraction of the

protein. Significantly, the nuclear immunoreactivity of the whole PMP22 antiserum is

absent with the antigenic peptide-purified antibody. To corroborate the colocalization of

PMP22 with ZO-1 at cell contacts, cultured BECs were treated with 4 mM EGTA for 4

hours to induce the internalization and disassembly of endothelial junctions (Gumbiner

and Simons, 1986; Notterpek et al., 2001) (Fig. 3-4D-F). After calcium depletion,

intracellular vesicular structures are coimmunoreactive for PMP22 (Fig. 3-4D) and ZO-1

(Fig. 3-4E), likely representing internalized junctional complexes. The merged image









reveals PMP22- and ZO-1-containing vesicles (Fig. 3-4F, arrows) and perinuclear, ER-

Golgi PMP22-like immunoreactivity (Fig. 3-4F, arrowhead). These subcellular studies in


PMP22 ZO-1 merged




















Figure 3-4. In mouse BECs, PMP22 is a constituent of intercellular junctions. Affinity
purified anti-PMP22 antibodies were used to detect PMP22 (A) and ZO-1 (B)
in primary cultures of mouse BECs. Colocalization of PMP22 and ZO-1 at
intercellular junctions (arrows) is seen as yellow in the merged image (C).
Whole anti-PMP22 rabbit serum (A') labels intercellular junctions (arrow) and
cell nuclei (asterisk). Perturbation of endothelial junctions with EGTA causes
the internalization of PMP22 (D) and ZO-1 (E), resulting in
coimmunoreactive vesicular structures (yellow in F, arrows). Perinuclear
PMP22 immunoreactive regions that do not colocalize with ZO-1 likely
represent the ER-Golgi protein fraction (F, arrowhead). Hoechst dye was used
to label nuclei (blue in C and F). Scale bar = 20[tm (B).

cultured BECs further support the notion that PMP22 is a constituent of intercellular

junctions in the rodent BMV.

Epithelial Junctions of Choroid Plexus are PMP22 Immunoreactive

Epithelial cells of the choroid plexus are crucial to the establishment and

maintenance of the blood-CSF barrier (Segal, 2000). In the adult mouse and rat brain,









occludin PMP22 ZO-1
AL


E15





P70


in vitro



Figure 3-5. In the choroid plexus, PMP22 is a junctional constituent of epithelia.
Cryosections from E15 (A-C) and P70 (D-F) rat brains were fixed and
coimmunolabeled for occludin (A and D) and PMP22 (B and E). Parallel
sections were immunostained for ZO-1 (C and F). In the E15 rat brain,
PMP22 (B) colocalizes with occludin (A) at the ZO-1 immunoreactive (C)
junctions of the budding choroid plexus (A-C, arrows). However, occludin is
absent from the adjacent PMP22 and ZO-1 immunoreactive ventricular
surface (A-C, arrowheads). In the P70 choroid plexus, occludin, PMP22, and
ZO-1 remain at the choroid epithelial junctions (D-F, respectively, arrows).
Preimmune serum (B') and peptide-adsorbed anti-PMP22 antibody (E')
controls fail to label cell-cell contacts. Confluent primary cultures of choroid
epithelia were immunostained with affinity purified anti-PMP22 (G) and
anti-ZO-1 (H) antibodies. Colocalization at intercellular junctions was
detected (G and H, arrows). Whole anti-PMP22 rabbit serum (G') labels
intercellular junctions (arrow) and cell nuclei (asterisk). Nuclei were stained
with Hoechst dye (blue in A, C, D, F, and H). Scale bar = 50[tm (F).
occludin, ZO-1 and various claudins are present at interepithelial junctions of the choroid

(Lippoldt et al., 2000; Wolburg et al., 2001). As PMP22 is detected at epithelial cell

contacts of the rat colon (Notterpek et al., 2001), and PMP22 message is elevated in

choroid epithelia (Fig. 3-2), we also investigated the expression and localization of









PMP22 in choroid plexus tissue (Fig. 3-5). Cryosections of E15 (Fig. 3-5A-C) and P70

(Fig. 3-5D-F) rat brains were double immunolabeled with monoclonal anti-occludin (Fig.

3-5A and D) and polyclonal anti-PMP22 (Fig. 3-5B and E) antibodies. In the E15 rat

brain, occludin (Fig. 3-5A, arrows) and PMP22 (Fig. 3-5B, arrows), as well as ZO-1 (Fig.

3-5C, arrows), are readily detected at cell junctions of the budding choroid plexus. In

contrast, occludin is absent from the anti-PMP22 and anti-ZO-1 immunoreactive

neuroepithelial layer of the periventricular region (Fig. 3-5A, B, C, arrowheads) (see

below). In the P70 rat brain, occludin (Fig. 3-5D), as well as PMP22 (Fig. 3-5E), remain

at interepithelial cell contacts of the mature choroid plexus (Fig. 3-5D, E, arrows).

Immunolabeling of parallel sections reveals that the expression of ZO-1 (Fig. 3-5F,

arrow) persists at these junctions. Secondary antibody alone (data not shown),

preimmune serum (Fig. 3-5B') and antigenic PMP22 peptide-adsorbed antiserum (Fig.

3-5E') controls do not label cell contacts.

In parallel with the in vivo studies, we investigated the subcellular localization of

PMP22 in purified cultures of choroid plexus epithelia (Fig. 3-5G and H). Confluent

monolayers were coimmunolabeled with peptide-purified polyclonal anti-PMP22 (Fig.

3-5G) and monoclonal anti-ZO-1 (Fig. 3-5H) antibodies. As observed in the mouse BECs

(Fig. 3-4A'), the anti-PMP22 whole rabbit serum labels the cell junctions, as well as the

nuclei of choroid epithelia (Fig. 3-5G', arrow and asterisk, respectively). However, the

nuclear staining is largely abolished by using the antigenic peptide-purified PMP22

antibody (Fig. 3-5G). Similar to the rat brain tissue (Fig. 3-5A-F), PMP22 and ZO-1 are

present at cell-cell contacts of cultured mouse choroid epithelia (Fig. 3-5G and H,









arrows). Together, these results identify PMP22 as a constituent of intercellular junctions

in the developing and adult rodent choroid epithelia.

PMP22 is Detected at Neuroepithelial Junctions

Neuroepithelial cell junctions lose expression of occludin during neural tube

invagination (Aaku-Saraste et al., 1996), but continue to express ZO-1 and P-catenin


p D







E F


Figure 3-6. Neuroepithelial cell junctions of the embryonic rat brain are immunoreactive
for PMP22. Cryosections of E15 rat brain were fixed and coimmunostained
for ZO-1 (A) and PMP22 (B), occludin (C) and PMP22 (D), or 3-catenin (E)
and PMP22 (F). At the apical surface of the ventricular zone, ZO-1 (A) and
P-catenin (E), but not occludin (C) colocalize with PMP22 (B, D, and F).
Nuclei were stained with Hoechst dye (blue in A, C and E). V: Ventricular
space. Scale bar = 17[tm (A).

(Chenn et al., 1998; Manabe et al., 2002). In agreement, in the E15 rat brain, we did not

detect occludin at the ventricular zone, while PMP22-like immunoreactivity is readily









visible (Fig. 3-5A, B, arrowheads). To further investigate the distribution of PMP22 at

the neuroepithelial cell layer, E15 rat brain cryosections were processed for double

immunolabeling with ZO-1 (Fig. 3-6A), occludin (Fig. 3-6C), or P-catenin (Fig. 3-6E)

and PMP22 (Fig. 3-6B, D, F) antibodies. As the higher magnification view of this region

reveals, PMP22 is colocalized with ZO-1 (Fig. 3-6A) and P-catenin (Fig. 3-6E) at the

apical surface of the neuroepithelial cell layer. In agreement with previous investigations

in the chicken (Aaku-Saraste et al., 1996), occludin (Fig. 3-6C) is not detected at these

cell junctions. In comparison to ZO-1 and PMP22, the P-catenin-like immunoreactivity,

although concentrated at the apical surface of the ventricle, extends along the lateral

contacts of the neuroepithelial cell layer (Fig. 3-6E). The observed distribution patterns

for ZO-1 and P-catenin are in agreement with previous studies in the developing

mammalian brain (Chenn et al., 1998; Manabe et al., 2002). These data illustrate that, at

the subventricular region, PMP22 is present at unique neuroepithelial cell-cell contacts,

which lack classical tight junctions (Mollgard et al., 1987; Aaku-Saraste et al., 1996).

Discussion

This study identifies PMP22 as a constituent of intercellular contacts of the BNB

and BBB. The colocalization with known junctional proteins in the developing and adult

rat sciatic nerve, BMV and choroid epithelia, suggests a role for PMP22 in the

establishment of the BNB and BBB. Additionally, the presence of PMP22 at occludin-

negative, specialized adherens-like junctions of the embryonic rat neuroepithelia may

indicate a ubiquitous role at intercellular contacts.

PMP22 is a broadly distributed membrane protein with documented expression in

a variety of developing and mature tissues, including epithelial cells of the lung and









intestine (Taylor et al., 1995; Baechner et al., 1995; Wulf et al., 1999). The detection of

PMP22 at the paraventricular region of the El 5 rat brain is consistent with prior reports

of neuroepithelial mRNA expression (Baechner et al., 1995; Parmantier et al., 1997).

Additionally, we observed both PMP22 message and protein in endothelial cells of the

developing BMV and choroid epithelia. Whereas PMP22 remains localized to

intercellular junctions of the adult rat BBB, message levels are reduced, as compared to

early postnatal ages. This discrepancy between mRNA levels and detection of protein in

the adult rodent brain could result from a low turnover rate of PMP22 at stable cell

contacts and/or an increased half-life for the mRNA. The elevated expression of PMP22

in the developing rodent brain, prior to the maturation of the BBB, might reflect de novo

junction formation or remodeling. Endothelial cell-cell junctions undergo a similar

structural remodeling following shear stress (Ogunrinade, 2002). Supporting a role for

PMP22 in the assembly of intercellular junctions, PMP22 mRNA is significantly

upregulated after prolonged (24-48 hours) laminar shear stress in human umbilical vein

and cardiac microvascular endothelial cells (Bongrazio et al., 2000).

The molecular composition of tight junctions at the BBB is rapidly being

elucidated (Wolburg and Lippoldt, 2002). For all ages and tissues examined, we

consistently detected PMP22 together with ZO-1, a broadly distributed structural

constituent of cell junctions. Previous studies show ZO-1 in the developing mouse BMV

as early as E15, but not at E9 (Nico et al., 1999). It remains to be determined whether

prior to E15 in the rat brain or P10 in the nerve, PMP22 and ZO-1 are targeted to

junctions simultaneously or sequentially. Nonetheless, at both locations their junctional

localization precedes that of occludin. Our findings are in agreement with earlier reports









describing junctional occludin in the rat BMV at P1 (Utsumi et al., 2000) and elevated

occludin immunoreactivity at P70, as compared to P8 (Hirase et al., 1997). Similar to the

BBB, the expression of occludin at endothelial cell junctions of the BNB lags behind

PMP22 and ZO-1. It has been reported that the rat peripheral nerve vasculature becomes

more restrictive to Evans Blue albumin and horseradish peroxidase, between P13-18 in

the rat (Smith et al., 2001), coinciding with our observation of increased occludin

expression. Occludin is thought to regulate tight junction physiology; however, the

precise function of occludin at intercellular junctions has not been definitively established

(Saitou et al., 2000).

The expression pattern of PMP22 was also compared to claudin-5; a tight

junction-associated integral membrane protein present in embryonic mouse BMV (Nitta

et al., 2003) and cultured BECs (Chen et al., 1998). In all of the studied nerve and brain

samples, both PMP22 and claudin-5 are present at endothelial junctions; however, a

notable fraction of claudin-5 is intracellular at the younger ages. Thus, in the rodent CNS

and PNS, we identified PMP22 as an early constituent of intercellular contacts, prior to

structural maturation of the BNB and BBB. The temporospatial expression pattern of

PMP22 is similar to ZO-1 and claudin-5, but not occludin.

Although homologous to the claudin family (Notterpek et al., 2001; Takeda et al.,

2001) and present at apical intercellular junctions (Notterpek et al., 2001), functional

studies to date suggest a distinct role for PMP22 at these locations. In C6 glioma cells or

L fibroblasts, unlike the claudins (Furuse et al., 1998b), PMP22 overexpression does not

induce the formation of tight junction-like strands (Takeda et al., 2001; Notterpek et al.,

2001). The detection of PMP22 in immature rat nerve and BMV suggests involvement









early in the formation of cell-cell contacts. The presence of PMP22 at the apical surface

of the neuroepithelial cell layer of the embryonic rat brain, which lack classical tight

junctions (Mollgard et al., 1987; Aaku-Saraste et al., 1996), support a role for the protein

either at adherens junctions or at the unique apical 'strap-junctions' (Mollgard et al.,

1987). Together, these findings imply that, unlike the claudins, PMP22 may not directly

take part in the formation of paracellular resistance at the BNB and BBB; but instead may

participate in the establishment and/or maintenance of cell polarity and cell-cell adhesion.

In agreement with this notion, VAB-9, a recently identified PMP22/epithelial membrane

protein (EMP)/claudin family member, is an adherens junction protein crucial for the

proper development of C. elegans (Simske et al., 2003).

If PMP22 is indeed a crucial constituent of epithelial and endothelial cell

junctions, then one might expect to see widespread pathology when the protein is

misexpressed. Nonetheless, the well-documented phenotype of PMP22 mutations,

deletion or duplication is the demyelination of peripheral nerves (Gabreels-Festen and

Wetering, 1999). Similar to the occludin-null mice (Saitou et al., 2000), PMP22-deficient

mice are viable with no overt morphological CNS pathology (Adlkofer et al., 1995),

suggestive of some functional redundancy for this protein at intercellular junctions of the

BBB. However, mice homozygous for PMP22 mutations often display seemingly non-

PNS related pathologies such as seizure and reduced growth rate (Henry et al., 1983;

Suter et al., 1992a; Notterpek et al., 1997, Isaacs et al., 2000). A less understood

phenotype associated with PMP22 misexpression is CNS demyelination in a subset of

hereditary neuropathy with liability to pressure palsies (HNPP) patients (Amato et al.,

1996; Schneider et al., 2000; Dackovic et al., 2001). Since PMP22 is not expressed in









oligodendrocytes (Baechner et al., 1995: Parmantier et al., 1995; 1997), it is unlikely that

the protein has a direct role in CNS myelin. Based on our finding of PMP22 at

intercellular junctions of BMV and choroid epithelium, it is possible that the CNS

pathology reported in some HNPP patients is the result of a compromised BBB.

How might the localization of PMP22 at the BNB relate to the demyelinating phenotype

of PMP22 neuropathies? Myelinated Schwann cells of the PNS share several

characteristics with endothelia and epithelia, including polarization, distinct membrane

domains (Bunge and Bunge, 1983) and close membrane apposition to create discrete

compartments to restrict ion flow, all likely established by intermembranous junctions

(Poliak et al., 2002; Scherer and Arroyo, 2002). It is conceivable that the myelin

pathology observed in PMP22-associated neuropathies (Gabreels-Festen and Wetering,

1999), in part, is a result of disrupted PMP22 function at junction-like structures within

peripheral myelin or at the BNB. Perturbation of the BNB is a feasible hypothesis for the

etiology of at least some of the nerve pathology observed in patients with PMP22-

associated neuropathies. Elevated levels of endoneurial macrophages are found in the

PMP22-mutant TrJ mice (Misko et al., 2002) and macrophage-associated demyelination

is detected in a Charcot-Marie-Tooth disease type IA patient with a PMP22 duplication

(Vital et al., 2003). However, similar observations in PO and connexin-32 mutant mice

suggest macrophage infiltration may be common to several hereditary peripheral

neuropathies (Carenini et al., 2001; Kobsar et al., 2002).

The results described here support the notion that PMP22 is a constituent of

epithelial and endothelial intercellular junctions. Furthermore, our findings suggest a role

for PMP22 early in the establishment and/or maintenance of cell-cell contacts. Future






56


investigations will attempt to establish the function of PMP22 at these locations, which

may also help to clarify the role of the protein in peripheral nerves.














CHAPTER 4
MODULATION OF EPITHELIAL MORPHOLOGY, MONOLAYER
PERMEABILITY AND CELL MIGRATION BY GAS3/PMP22

Introduction

The tetraspan glycoprotein peripheral myelin protein 22 (PMP22), also known as

gi ,i\ ihi arrest-specific gene-3 (gas-3), has proposed roles in peripheral nerve myelin

formation, cell-cell interactions and cell proliferation (Suter and Snipes, 1995). Although

the highest expression levels are found in myelin-forming Schwann cells, PMP22 mRNA

can be detected in a multitude of developing and mature non-neural tissues; including

epithelia of the intestine (Taylor et al., 1995; Baechner et al., 1995; Wulf et al., 1999) and

the choroid plexus (Roux et al., 2004). The specific role of PMP22 in Schwann cell

biology remains undefined; although, it is known that altered expression is associated

with heritable demyelinating peripheral neuropathies (reviewed by Naef and Suter, 1998).

Similarly, the function of the protein at these non-neural locations remains undetermined.

To date, in vitro studies have identified a role for PMP22 in the regulation of cell

proliferation and morphology. In Schwann cells, elevated expression delays the transition

from GO/G1 to the S phase of the cell cycle (Zoidl et al., 1995), and can lead to apoptosis

in some instances (Fabretti et al., 1995, Zoidl et al., 1997). Conversely, reduced PMP22

mRNA levels are associated with enhanced DNA synthesis and entry into the S+G2/M

phases (Zoidl et al. 1995). In NIH3T3 fibroblasts, PMP22 overexpression regulates cell

spreading, an effect that is dependent on the Rho-GTPase pathway (Brancolini et al.,

1999). Recent studies have detected exogenous PMP22 in ADP-ribosylation factor 6









(Arf-6) positive plasma membrane-endosomal recycling vacuoles prior to apoptosis or

changes in cell shape (Chies et al., 2003). This pathway is known to be involved in

modulating the actin cytoskeleton, cell polarity, adhesion and migration (Donaldson,

2003). Together, these findings support the notion that PMP22 has a significant role in

basic cellular processes, extending beyond an involvement in Schwann cell myelination.

We previously described PMP22 as a constituent of apical intercellular junctions

in epithelial and endothelial cells (Notterpek et al., 2001; Roux et al., 2004). While

PMP22 shares significant amino acid homology with members of the claudin

superfamily, overexpression of the protein in L-fibroblasts (Notterpek et al., 2001) or C6

glioma cells (Takeda et al., 2001) did not induce the formation of tight junction strands.

Nonetheless, PMP22 might function in the establishment and maintenance of

ion-selective paracellular barriers. Transmembrane proteins of the apical junctional

complex such as the claudins, occludin and the junctional adhesion molecules (JAMs)

(reviewed by Gonzalez-Mariscal et al., 2003) all participate in the regulation of junctional

permeabililty. In addition, based on the findings of Brancolini and colleagues (Brancolini

et al., 1999; Brancolini et al., 2000; Chies et al., 2003), PMP22 might be involved in the

regulation of epithelial proliferation and/or cell migration, dynamic processes that

involve changes in cell adhesion and morphology. In support of this possibility, PMP22

contains the carbohydrate L2/HNK1 adhesion/recognition epitope in the 1st extracellular

loop (Snipes et al., 1993; Schachner et al., 1995).

In this report, we examined the role of PMP22 in several facets of epithelial cell

biology, including proliferation, cell shape and migration. An elevated level of PMP22

alters the migration of epithelia and reduces the formation of lamellipodial protrusions.









The transepithelial electrical resistance (TER) and paracellular flux are also affected in

these cultures. Application of PMP22 peptides has a similar effect on the TER and the

permeability of MDCK monolayers. Together, these results indicate that PMP22 plays a

role in modulating growth, morphology, migration and paracellular permeability in

epithelial cells.

Materials and Methods

Cell Culture

MDCK type I (high resistance) and type II (low resistance) cells were grown in

Eagle's minimum essential medium supplemented with 5% FCS or Dulbecco's modified

Eagle's medium with 10% FCS, respectively. Cells were cultured on 6.5 or 12 mm Costar

Transwell filters (0.4 |tm pores) (Coming Incorporated) or tissue culture dishes, and

maintained at 37C and 5% CO2. For transepithelial electrical resistance (TER) or

paracellular flux studies (see below), MDCK II monolayers were grown on filters (3x105

cells/cm2) in low calcium media (see below) for 48 h to ensure confluency prior to

calcium addition. TER measurements were recorded every 24 h after calcium addition

until 6 days post-plating at which time TER levels had reached a steady-state. After

plating MDCK I cells on filters (3x105 cells/cm2), the medium was replaced every 24 h

until the 6th day when TER levels had reached a steady-state. For the calcium-switch

assay (Gumbiner and Simons, 1986), cells were treated with EDTA (4 mM) containing

media for 4 h (Notterpek et al., 2001), or for 18 h in calcium- and magnesium-free media

with 5% Chelex (Sigma) treated FCS (Suzuki et al., 2001).









Establishment of Stable Transgene Expressing Cells

The human PMP22 (kind gift of Dr. Clare Huxley, Imperial College School of

Science, Technology and Medicine) and human occludin (Van Itallie and Anderson,

1997) ORFs were inserted into the pLNCX-II retroviral vector (Clontech). Transgene

expression is regulated by the cytomegalovirus (CMV) promoter. Following transient

transfection with LipofectamineTM and PLUSTM reagent (Invitrogen), MDCK cells were

treated with 1.1 mg/ml Geneticin (G418 sulfate) (Gibco) for four weeks to establish a

population of stably expressing cells. Subclones of the stably expressing MDCK II cells

were monitored for transgene expression by immunoblotting as described below. Three

subclones were established for each construct. Where indicated, 2.5 mM sodium butyrate

was added for 20 h to induce the transgene expression under the CMV promoter (Gorman

et al., 1983).

Primary Antibodies

Monoclonal mouse anti-occludin, anti-ZO-1 (Zymed Laboratories, Inc.),

anti-p-catenin (BD Transduction Labs), anti-E-cadherin clone rrl (Developmental Studies

Hybridoma Bank), anti-a-tubulin, anti-actin (Sigma), anti-GP-135 (kind gift from Dr.

George Ojakian, SUNY Downstate Medical Center), rat anti-E-cadherin (Zymed), and

rabbit polyclonal anti-occludin (Zymed), anti-PMP22 and affinity purified anti-PMP22

antibodies (Notterpek et al., 2001; Roux et al., 2004) were used.

Immunofluorescent Labeling

MDCK cells plated on glass coverslips or 12 mm Transwells were grown to

confluency and fixed with either 3% PFA followed by a 1 min incubation in 100% -20C

acetone (for PMP22 detection) or 1% PFA followed by permeabilization with 0.2%









Triton X-100 (TX-100) (junctions staining). For z-plane imaging of confluent

monolayers, filters were fixed with 4% PFA followed by 5 min 100% methanol at -20C

and then frozen in liquid nitrogen-cooled N-methylbutane prior to cryosectioning filters

along the z-plane. Samples were blocked in 10% normal goat serum in PBS. After

incubation with primary antibodies, Alexa FITC-conjugated anti-mouse IgG and Texas

red-conjugated anti-rabbit IgG (Molecular Probes) antibodies were added. Nuclei were

stained with Hoechst dye (10 [tg/ml) (Molecular Probes). Actin filaments were visualized

with FITC-conjugated phalloidin (Molecular Probes). Coverslips were mounted by using

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

attached to a Nikon Eclipse 800 microscope and formatted for printing by using Adobe

PHOTOSHOP 5.0. Images were measured using Spot Advanced 3.5.

BrdU Incorporation

The DNA synthesis rate of subconfluent MDCK II cells plated on glass coverslips

was analyzed using a BrdU labeling and detection kit (Roche) optimized for

immunofluorescence of adherent cells following the manufacturer's recommended

protocol. The percentage of BrdU positive cells was determined by counting in 4 random

fields (0.8 mm2)and comparing to the total number of cells visualized by Hoechst

staining. More than 500 cells were counted for each condition. The percentage was

calculated from the ratio between BrdU positive and total cells.

Epithelial Cyst Formation

To generate MDCK II cysts, we followed an established protocol (Pollack et al.,

1998). Briefly, MDCK II cells (5X104 cells/ml) were suspended in 2 mg/ml rat tail

collagen type I (Sigma) on 6.5 mm Transwells (0.4 [tm). After the collagen gel solidified









at 370C, medium was added to the apical and basal chambers and was replaced every 48

h. Eight days after plating, the collagen gels were removed from the filters and fixed in

4% PFA prior to freezing in liquid-nitrogen cooled N-methylbutane. Cryosections (7 [tm)

were fixed on glass slides in 100% methanol for 5 min at -200C prior to blocking and

immunolabeling as above.

Cell Surface Biotinylation and Western Blotting

For Western blotting, monolayers of confluent MDCK cells were lysed

(Notterpek et al., 2001) and where indicated, treated with N-glycosidase (PNGase F)

(Pareek et al., 1997). Samples were separated by SDS-PAGE and transferred to

nitrocellulose membrane (Bio-Rad) prior to immunoblotting. Bound HRP-conjugated

anti-rabbit or anti-mouse secondary antibodies (Sigma) were detected using ECL reagents

(Perkin Elmer).

For the detection of PMP22 at the cell surface, confluent monolayers of stably

expressing MDCK II cells (6 cm dish) were biotinylated with biocytin hydrazide (Lisanti

et al., 1989; Prince et al., 1993). Monolayers were rinsed with PBS containing 10 mM

CaCl2 and 1 mM MgCl2 (PBS-CM) and incubated with 10 mM NaIO4 in PBS-CM for 30

min at 40C in the dark while rocking. After rinsing with PBS-CM, cell monolayers were

kept in the dark for 1 h at 230C with 2 mM biocytin hydrazide (Pierce). Following

extensive rinsing in PBS-CM, cells were lysed for affinity precipitation in 3.2 ml 4C

NP-40 buffer (25 mM Hepes/NaOH pH 7.4, 150 mM NaCl, 4 mM EDTA, 25 mM, NaF,

1% NP-40, 1 mM Na3VO4, IX Complete protease inhibitor (Roche Diagnostics))

(modified from Sakakibara et al., 1997), scraped from the culture dish and gently rocked

for 30 min at 40C. Cell lysates were centrifuged 10,000 g for 30 min at 40C and the









supernatants reserved. The pellet was solubilized in 320 [tl SDS buffer (25 mM Hepes,

pH 7.4, 4 mM EDTA, 25 mM NaF, 1% SDS, 1 mM Na3VO4) by sonication on ice. To the

solubilized pellet, 9 volumes of NP-40 buffer was added, passed through a 27-gauge

needle 10 times on ice, and incubated for 30 min at 40C. The lysates were centrifuged

10,000 g for 30 min at 40C and the supernatants reserved. ImmunoPure immobilized

streptavadin beads (Pierce) suspended in lysis buffer, were added to the cell lysate and

gently rocked for 2 h at 40C. Streptavadin beads were washed 4 times with NP-40 buffer

and boiled in 30 [tl SDS gel sample buffer (Notterpek et al., 1997). After brief

centrifugation, the 30 [tl of sample buffer was removed and treated with PNGase F, as

above. Samples were processed for immunoblotting with anti-PMP22 antibodies, as

above.

Measurement of Junctional Permeability

TER was measured in 370C culture media using an EVOM Epithelial

voltohmmeter with an STX-2 electrode (World Precision Instruments, Inc.). The TER

values were calculated by subtracting the background TER of blank filters and

normalized by the area of the monolayer. Steady state TER measurements (N=9 wells per

construct) were detected 6 days after cell plating under the described culture conditions.

To measure nonionic paracellular flux, FITC-dextran of 3 and 40 kD (Molecular Probes)

was dissolved in P-buffer (Balda et al. 1996) at a concentration of 10 mg/ml. Apical and

basolateral compartments of cells cultured on Transwell filters (N=3-4 wells per

construct) were rinsed with P-buffer and allowed to equilibrate for 10 min. The 3 and 40

kD FITC-dextran stock solutions (25 and 50 [tg/[tl, respectively) was added to the apical

chamber and the cells were incubated at 370C for 30 min. By sampling the basal media,









the amount of FITC-dextran diffusion from the apical to the basal chamber was measured

in a VersaFluor fluorometer (Bio-Rad). A standard curve was used to convert relative

fluorescent units to the concentration of dextran in solution.

Peptide Perturbation

HPLC-purified peptides were purchased from United Biochemical Research, Inc.

Peptides corresponding to a portion of the 1st (aa 45-63)

(NH2-SALGAVQHCYSSSVSEWLQ-COOH) (PMP22-1st) and the entire 2nd loop (aa

117-132) (NH2-YTVRHEWHVNTDYSY-COOH) (PMP22-2nd) of murine PMP22 were

chosen. A scrambled peptide using the same amino acids as the 2nd loop of PMP22

(NH2-HDEYVSNTHWYRSYTV-COOH) (scrambled-2nd) served as a control. A 44 aa

peptide corresponding to the 2nd extracellular loop of the chicken occludin (Occ-2nd) was

used as a positive control (Wong and Gumbiner, 1997). Peptides were dissolved in

DMSO (10 mM) and added to calcium-containing media at the indicated concentrations.

Monolayers (N=3-4 wells per condition) were fed every 24 h with fresh

peptide-containing medium.

Wound Migration Studies

Wound assays using MDCK cells have previously been reported (Fenteany et al.

2000; Sabo et al., 2001). Highly confluent MDCK II monolayers on either glass

coverslips or tissue culture plastic wells were wounded with a 200 [l pipette tip (Sabo et

al., 2001). Long scratches and short wounds were made prior to rinsing the monlayer in

fresh media to remove detached cells. At various time points after wounding, wound

areas were imaged with a Nikon DS camera attached to a Nikon Eclipse TS100 inverted

microscope. In order to ensure that identical areas were imaged between time points,









multiple positioning-marks were made at the center of the denuded surface with a small

needle. Relative wound areas (N=3 per construct) were measured with the NIH image

analysis program. Alternatively, the mean distance migrated along the wound edge (N=6,

measurements in 2 separate fields) was determined using Adobe PHOTOSHOP 5.0. The

Rho-kinase inhibitor Y-27632 (10 tpM) (Calbiochem) was applied to MDCK monolayers

2 h after wounding and monolayers were fixed for staining after an additional 3 h

(Omelchenko et al., 2003). Scatter factor (SF) was obtained by culturing highly confluent

NIH3T3 fibroblasts in DMEM with 1% FCS for 72 hr, followed by 0.45 |tm filtration.

Prior to use, SF was tested confirming its ability to induce the dispersion of small

colonies of neo-MDCK cells (Stoker et al., 1987).

Statistical Analysis

Where indicated, means and standard deviations (SD) were calculated and

statistical significance was determined by unpaired 2-tail t test using GraphPad Prism 4.0.

Results

PMP22 Overexpression Alters Epithelial Cell Proliferation and Morphology

To investigate the role of PMP22 in epithelial cells, human PMP22 (hPMP22)

was overexpressed in the pLNCX-2 vector under the control of the CMV promoter in

MDCK II cells (PMP22-MDCK). These low resistance kidney-derived cells are

frequently used for studies of polarized epithelia (Stevenson et al., 1988). In total cell

lysates (T), using an antibody optimized to detect the human protein, PMP22 is faintly

observed at ~25kD (Fig. 4-1A, arrowhead). Upon PNGase F treatment, the protein is

detected more prominently at 18kD (Fig. 4-1A, arrow). Enhanced immunoreactivity of

PMP22 after endoglycosidase treatment has been previously observed (Fabretti et al.,









1995). When the cell lysate is fractionated into detergent soluble (S) and detergent

insoluble fractions (I), the overexpressed human PMP22 protein is highly enriched in the

detergent-insoluble pool. A similar detergent solubility profiles has been observed


A PMP22 neo B C
PN"F T + -S+- +- + neo PMP22 10a
25- P S I I a .s
13- -< -1 8-m
0,


neo PMP22

D E F_


i
C


200 E


1100


neo PMP22


Figure 4-1. Altered epithelial cell proliferation and morphology by PMP22. (A) Stable,
neo and human PMP22 (hPMP22) expressing MDCK II cells were lysed in
3% SDS or 0.5% TX-100 buffer, and total lysates (T), TX-100 soluble (S) and
insoluble (I) fractions (30 [tg/lane) were analyzed with (+) and without (-)
PNGase F digestion. The glycosylated hPMP22 protein is detected at -26 kD
(arrowhead), and following PNGase F treatment becomes readily visible,
migrating at -18 kD (arrow). Compared to total lysates, human PMP22 is
enriched in the detergent insoluble pellet. (B) Plasma membrane targeting of
hPMP22 was determined by cell surface biotinylation, followed by PNGase F
treatment and immunoblotting with anti-hPMP22 antibodies. The majority of
biotinylated hPMP22 is detected in the 1% NP-40 insoluble (I) fraction
(arrow). Molecular mass, in kDs. (C) As measured by BrdU incorporation in
subconfluent cultures, compared to neo cells, hPMP22 expression reduces
DNA synthesis by 32.99.5%, (*, P<0.004). (D) In confluent PMP22-MDCK
monolayers, the cell density is 51.78.4% of the neo cultures (*, P<0.004). (E)
The reduced cell density in confluent PMP22 monolayers is in agreement with
an increase in nuclear area. Hoechst staining of representative cultures is
shown (Bar, 15 [tm). Quantification of nuclei reveals -1.5-fold increase in
nuclear dimension of PMP22-MDCK cells as compared to neo controls (*, P
<0.0001). (F) The apical area of the PMP22-MDCK cells, outlined by ZO-1
immunostaining, is significantly larger than in neo cells (*, P<0.0004). Error
bars in C, D, E and F, show means SD. P-values were determined by t test.


PMP22









previously for both the endogenous canine and exogenous myc-tagged rat PMP22 in

MDCK cells (Notterpek et al., 2001). Vector-only cells (neo) are not immunoreactive

with the anti-human PMP22 antibody (Fig. 4-1A).

We next examined the targeting of human PMP22 to the cell surface, utilizing

biotinylation and subsequent precipitation (Ryan et al., 2002) (Fig. 4-1B). Endoglysidase

treatment of the precipitated protein revealed hPMP22 at the plasma membrane. Since the

majority of hPMP22 is found in the detergent insoluble fraction (arrow), the cell-surface

protein is likely accumulated at apical intercellular junctions and/or, as reported in

Schwann cells, possibly in lipid rafts (Erne et al., 2002; Hasse et al., 2002).

Since PMP22 is known to modulate cell cycle progression (Schneider et al., 1988;

Manfioletti et al., 1990; Zoidl et al., 1995; 1997; Karlsson et al., 1999), we next examined

how hPMP22 might affect epithelial proliferation. Similar to previous reports, in

subconfluent cultures, elevated levels of hPMP22 resulted in a 33% reduction of BrdU

incorporation, as compared to neo cells (Fig. 4-1C). At confluency, 51.7+8.4% fewer

cells are in the PMP22-MDCK cultures, compared to controls (Fig. 4-1D). The lower cell

density of PMP22 monolayers is readily visible by Hoechst imaging of nuclei from

confluent filter-grown cultures (Fig. 4-1E). On images taken at the same magnification,

the PMP22 cell nuclei appear larger and, when quantified, reveal an approximately

1.5-fold increase in area (graph, Fig. 4-1E). As predicted from a confluent monolayer

with reduced cell density, the apical surface area of the PMP22 cells, as determined by

ZO-1 immunostaining (see below), is -1.9-fold larger than neo controls (Fig. 4-1F).

These morphological characteristics of PMP22-MDCK cultures suggest the monolayer

consists of fewer cells with a flattened morphology.








PMP22 Does Not Alter Epithelial Polarity or the Localization of Junctional Proteins

To further evaluate the altered epithelial morphology induced by hPMP22

overexpression, confluent filter-grown monolayers were double immunostained with


A B
neo neo PMP22


PMP22


C neo PMP22 D s

udcludin



0-catenIn --act-


Figure 4-2. Protein polarity and functional constituents in PMP22-MDCK. (A)
Confluent, filter-grown neo- and PMP22-MDCK monolayers were
immunostained and examined by from the z-plane by cryosectioning. In neo
and PMP22-expressing monolayers, the protein polarity of GP-135 (green)
and E-cadherin (red) are apical and basolateral, respectively. The flattened
morphology of the PMP22 cells is apparent (Bar, 10 pm). (B) In a 3-D
collagen matrix polarization model, both cultures form multicellular cysts
with apical GP-135 (green) oriented towards the center. Nuclei are visualized
by Hoechst dye (blue) (Bar, 15 [tm). (C) Compared to neo cultures, an
increased level of PMP22 immunoreactivity is detected at intercellular
junctions of PMP22-MDCK cells, when the images are taken at a constant
exposure time. Parallel monolayers were also immunostained for a
representative tight and adherens junction constituent, ZO-1 and 8-catenin,
respectively. Both junction proteins appear similarly localized in neo and
PMP22 cells. Bar, 20 |tm. (D) Immunoblotting of 0.5% TX-100 soluble (S)
and insoluble (I) fractions (20 [tg/lane) reveals comparable levels and
detergent solubilities for occludin, 0-catenin, as well as actin, between
confluent neo- and PMP22-MDCK monolayers. Occludin appears slightly
elevated in the detergent insoluble fraction of the PMP22-MDCK sample.









anti-E-cadherin and anti-GP135 antibodies following z-plane cryosectioning. As

expected, in neo-MDCK cultures, GP135 is apical (Ojakian and Schwimmer, 1988) and

E-cadherin labels the lateral borders (Fig. 4-2A). While the PMP22-MDCK cells are

flattened, the protein polarity is similar to that of the neo controls. We also examined

protein polarity in a three-dimensional (3-D) model of epithelial cysts (Ojakia and

Schwimmer, 1994). In this model, MDCK cells form a polarized multicellular structure

in which the apical surface faces a lumen. After sectioning and immunostaining for

GP135, similar to neo cells, PMP22 cells formed cysts with normal polarity (Fig. 4-2B).

As PMP22 is a protein constituent of apical intercellular junctions (Notterpek et

al., 2001; Roux et al., 2004), we next investigated how PMP22 overexpression affects the

localization and detergent solubility properties of tight and adherens-junction molecules

(Fig. 4-2 C and D). As compared to neo cells, an elevated level of PMP22-like

immunoreactivity is associated with the cell-cell contacts of PMP22-MDCK monolayers

(Fig. 4-2C). The expression and localization of tight junction-associated occludin, as

well as the adherens junction-associated P-catenin, appear similar between the control

neo and PMP22-MDCK samples (Fig. 4-2C). Similarly, the localization of claudin-1,

ZO-1 and E-cadherin was unaltered by PMP22 (data not shown). As described above

(Fig. 4-1F), the larger apical dimensions of the PMP22 cells are apparent by the

junctional immunostaining.

To further examine the levels and detergent-solubility characteristics of

junction-associated proteins, parallel samples were processed for immunoblotting with

the indicated antibodies (Fig. 4-2D). The three examined proteins, occludin, P-catenin

and actin, show similar expression levels and detergent-solubility properties between the








neo and PMP22-MDCK cells, with a slight increase for occludin in the PMP22 sample.

Thus, the overexpression of PMP22 does not drastically alter epithelial protein polarity,

nor the levels or localization of representative tight and adherens junction molecules.

Paracellular Permeability is Altered by PMP22 Expression

Overexpression of the tight junction-associated occludin in MDCK cells revealed

a role for this protein in regulating paracellular resistance (Balda et al., 1996; 2000;


A B
175- 70 *
E In W &PMP22 r: so-
g 125 I 50

7 S- 30"


20
neo PMP22 Occ
tiUme (days)
C Dr

200- J 30"

E 150" 20-
a 100" Z 15'

0 S"
neo PMP22 Occ 0 MW
I neo PMP22 Occ
Figure 4-3. Altered paracellular permeability of epithelial monolayers by hPMP22
expression. (A) TER recordings beginning two days after the addition of
calcium, from newly-confluent MDCK monolayers are shown. Three different
clones of neo, PMP22 and Occ cells (N=3 filters per clone) were used for the
quantification. Compared to neo cultures, the TER is elevated in PMP22 and
Occ cells. (B) At steady-state, six days after plating, PMP22- and Occ-MDCK
monolayers have increased TER, compared to neo cells (*, P<0.0001). (C)
Treatment with sodium butyrate (20 h), further enhances the differences in
TER values between control and PMP22, as well as Occ monolayers (*,
P<0.0001). (D) The paracellular flux of 3kD nonionic FITC-dextran by
PMP22- and Occ-MDCK monolayers is significantly elevated (*, P<0.0001),
as compared to neo cells. Error bars show means + SD. P-values were
determined by t-test.









McCarthy et al., 1996; 2000). Since PMP22 is similarly localized to intercellular

junctions of MDCK cells (Notterpek et al., 2001), we investigated whether the TER of

the hPMP22-expressing epithelial monolayers is altered compared to neo controls. Two

days after the addition of calcium, PMP22- and occludin-expressing cells have a high

TER level indicative of newly-confluent monolayers, after which the TER decreases to

steady-state by day four. (Fig. 4-3A). Occludin expressing monolayers (Occ-MDCK),

used as a positive control, have TERs slightly more elevated than the hPMP22 cells. By

four days after calcium addition, the monolayers reach a steady-state level of confluency

and the TER of the hPMP22 and occludin expressing cells is -1.6-fold higher than the

neo control (Fig. 4-3B). To further induce transgene expression, confluent monolayers

were treated with sodium butyrate for 20 h (Gorman et al., 1983). As indicated in Fig.

4-3C, both the PMP22- and Occ-MDCK monolayers exhibit a 3.2- and 4.1-fold increase

in TER, respectively, as compared to butyrate-treated neo cells. A similar phenomenon

has been observed previously in epithelial monolayers overexpressing occludin (Balda et

al., 1996; 2000; McCarthy et al., 1996; 2000).

As overexpression of junctional proteins has been shown to alter paracellular flow

of nonionic molecules (Balda et al., 1996; 2000; McCarthy et al., 1996; 2000), we

performed a dextran flux assay on confluent PMP22-MDCK monolayers. As compared to

neo controls, the flux of a 3kD nonionic FITC-labeled dextran is elevated -17-fold in

both the PMP22- and Occ-MDCK monolayers (Fig. 4-3D). These results indicate that

elevated expression of PMP22 results in altered permeability of MDCK monolayers.









Epithelial Monolayer Permeability is Perturbed by PMP22 Peptides

An alternative approach to elucidate the role of junctional proteins is to apply

peptides that correspond to extracellular domains of endogenous proteins (Wong, 1997;


B 110"
100-
a 80-
70o
60-
*j 50"
S40-
-- 30
I-- 20
lo


*-I


0- < r *
0
n' p


C
7500-

E
6 5000-
E

I-U
01


0 30 55
Washout time (hrs)


Figure 4-4. Perturbation of epithelial monolayer TER by PMP22 peptides. (A) Peptides
corresponding to a portion of the 1st (1) and the entire 2n (2) extracellular
loops of murine PMP22, a scrambled 2nd loop and the 2nd extracellular loop of
chicken occludin were applied to confluent MDCK monolayers after a
calcium-switch. (B) Twenty hours after the addition of the peptides, the TER
of PMP22-2nd peptide treated monolayers (32 [LM) remains low compared to
naive, DMSO, PMP22-1st or scrambled-2nd treated cells (*, P<0.0001). TER
reformation by monolayers exposed to Occ-2nd peptide (16 [LM) is also
significantly perturbed (**, P<0.0006). (C) A dosage curve for PMP22-2nd
peptide reveals an effective concentration range for TER disruption between 8
to 32 aM. Error bars show means + SD. P-values were determined by t test.
(D) Following a 20h PMP22-2nd peptide treatment, and subsequent washout,
the monolayers regain a TER similar to untreated samples.


E
S5000

S2500
I-


I


IIII


0 10 20 30
PMP22-2nd [pM]









Wong and Gumbiner, 1997; Lacaz-Vieira et al., 1999; Chung et al., 2001; Vietor et al.,

2001; Tavelin et al., 2003; Lee et al., 2004). As the canine PMP22 has not been cloned,

we designed peptides representing portions of the 1st and 2nd extracellular domains of

the mouse protein (Fig. 4-4A). While PMP22 has homology to some members of the

claudin family, the proteins are less similar in the extracellular domains (Align software,

data not shown). Confluent MDCK I monolayers were treated with the indicated

peptides, following a calcium-switch. Twenty hours after the re-addition of calcium with

the corresponding peptides (324M PMP22-1st, -2nd, PMP22-scrambled-2nd, or 16tM

Occ-2nd), the TER of the monolayers was recorded (Fig. 4-4B). As expected based on the

literature (Wong, 1997; Wong and Gumbiner, 1997; Vietor et al., 2001), the Occ-2nd loop

peptide inhibited TER recovery by 50.48.7%. Similarly, the PMP22-2nd loop peptide

diminished TER recovery by 91.30.4%. Application of vehicle (DMSO), PMP22-1st or

scrambled-2nd peptides had no significant effect on the TER compared to naive cells (Fig.

4-4B). A concentration curve for PMP22-2nd peptide identified an effective range of TER

disruption between 8 and 32 [tM (Fig. 4-4C). The washout of PMP22-2nd peptide from

MDCK monolayers results in the restoration of the TER to levels similar to controls (Fig.

4-4D). Therefore, the disruptive effect of the PMP22-2nd loop peptide on the monolayer

TER is reversible.

Next, we examined the paracellular flux and the morphology of peptide-treated

monolayers (Fig. 4-5). The flux of the 3 kD, but not the 40 kD, FITC-dextran is

significantly elevated in both the PMP22-2nd- and Occ-2nd-loop treated monolayers,

indicating a size selective disturbance of paracellular permeability (Fig. 4-5A). The

observed 3-fold increase in the flow of the 3kD dextran in Occ-2nd treated cultures is in











A
2
U
N








B


1=13kD
40OkD





l-e ni'


Figure 4-5. An increased paracellular flux of epithelial monolayers by PMP22 peptides.
(A) MDCK monolayers were treated with the indicated peptides and the
paracellular flux of 3 kD, and 40 kD FITC-labeled nonionic dextrans were
determined. In PMP22-2"d (*, P<0.0001) and Occ-2nd treated monolayers (**,
P<0.002) the paracellular flow of the 3kD, but not the 40kD, dextran is
significantly elevated. Error bars show means + SD. P-values were determined
by t test. (B) In PMP22-2nd and scrambled-2nd peptide-treated monolayers (32
[M), the localization of ZO-1 and E-cadherin remain comparable. Bar, 20 |tm.

agreement with previous reports (Wong and Gumbiner, 1997). In order to determine if

the PMP22-2nd peptide treatment alters the distribution of junctional constituents, the

localization of ZO-1 and E-cadherin was examined in parallel peptide-treated monolayers

(Fig. 4-5B). Both ZO-1 and E-cadherin appear unaltered following treatment with

PMP22-2nd or scrambled-2nd peptides. These findings indicate that the PMP22-2nd


S rI

949 #r 4f9









peptide reduces monolayer permeability without radically altering major constituents of

apical intercellular junctions.

PMP22 Expression Slows the Migration of Epithelial Monolayers

Injured epithelial monolayers down-regulate ZO-1 and occludin mRNAs (Cao et

al., 2002), suggestive of junctional remodeling. After observing that the expression of


A

neo



PMP22


Figure 4-6. Wound healing is altered by PMP22 in epithelial monolayers. (A) Confluent
neo- and PMP22-MDCK monolayers were wounded with a pipette tip and the
migration of the cells into the wound area (borders outlined in black) was
evaluated at 2 and 24h. Neo-MDCK monolayers nearly close the wound by 24
h, while PMP22-MDCK cells are unable to similarly reduce the wound area.
Bar, 800 tm. (B) Quantification of wounding experiments reveal that
compared to naive and neo cells, PMP22-MDCK cells are significantly less
competent to migrate (*, P<0.0003). Error bars show means + SD. P-values
determined by t test. (C) Hoechst staining of parallel samples shows dispersed
nuclei along the wound edge in the neo cultures. In comparison, the nuclei of
the PMP22-MDCK cells appear densely packed and uniform throughout the
image. Bar, 150 tm.









hPMP22 alters the morphology and permeability of epithelial monolayers, we

hypothesized that the protein might modulate the dynamic processes involved in

epithelial migration. To test this idea, hPMP22-overexpressing epithelial cells were

observed in a two-dimensional wound-migration assay (Fig. 4-6A) (Fenteany et al.,

2000). By phase microscopy 24 hours after wounding, the neo cultures nearly close the

denuded area (Fig. 4-6A, top panels). In comparison, the rate of monolayer closure is

visibly reduced in PMP22-MCDK cells (Fig. 4-6A, bottom panels). Indeed, within a 24h

period, PMP22 monolayers exhibit a 60.3+6% reduction in wound closure as compared

to neo cells (Fig. 4-6 B). At higher magnification of Hoechst stained samples, the neo

monolayers display a typical wave of migrating cells at the wound edge that appear

flattened and spread out (Sheffers et al., 2003; Matsubayashi et al., 2004) (Fig. 4-6C). In

contrast, the nuclei of PMP22-expressing cells are more compact at the wound edge and

are more uniformly spaced throughout the monolayer.

In response to monolayer wounding, migrating MDCK cells maintain cell-cell

contacts, form an actin purse-string along the wound edge and pull multiple cell rows

forward in by Rac-dependent lamellipodial crawling (Fenteany et al., 2000).

Lamellipodial protrusion by leader cells, but not the formation of an actin purse-string, is

required by MDCK monolayers to close a wound (Fenteany et al., 2000). Therefore,

utilizing immunolabeling we examined these two structures in PMP22-MDCK cells (Fig.

4-7A). As expected, 24 h after monolayer wounding, neo cells (top row) have an actin-

belt along the wound edge, with periodic breaks (arrowhead on the right) representative

of migrating cells. Leader cell lamellipodia are observed in the neo monolayers by









A acin


I



B


CT
8

I
01
._'
2


neo PMP22


Figure 4-7. Lamellipodial protrusion in migrating epithelial monolayers is reduced by
PMP22. (A) The distribution of actin, a-tubulin and E-cadherin was examined
by fluorescence microscopy along the wound edge at 24 h post-wounding. In
neo cultures, an actin purse-string and lamellipodial protrusions (arrowheads)
are detected (top row). In wounded PMP22 samples, the actin purse-string is
uninterrupted and a-tubulin and E-cadherin appear concentrated along the
wound edge. Lamellipodial protrusions into the wound space are largely
absent in the PMP22-MDCK monolayers. Bar, 40 |tm. (B) A 3 h treatment of
wounded monolayers with Y-27632, a Rho kinase inhibitor, induces extensive
lamellipodial protrusions (arrows) along the wound edge of neo cells,
visualized by fluorescent labeling of actin. In PMP22-MDCK monolayers,
Y-27632 is unable to bring about a similar response, as cells with lamellipodia
are sparse (arrow). Bar, 50 |tm. (C) Quantification of epithelial migration after
wounding (5 h), in the absence and presence of scatter factor (SF). In normal
culture medium, neo cells migrate faster than PMP22-MDCK monolayers.
The addition of SF to the medium significantly increases the migration of neo
and PMP22-MDCK cells (*, P<0.0003; **, P<0.0001, respectively). Error
bars show means + SD. P-values were determined by t test.


a-tubulln E-cadhffln











tubulin and E-cadherin labeling (arrowheads). An actin purse-string is continuous along

the migrating edge of PMP22 monolayers (Fig. 4-7A, bottom panel); however, breaks or

perturbations in this actin-belt are largely absent. Additionally, the tubulin and E-

cadherin immunoreactivities appear concentrated along the leading edge in PMP22 cells

of the wound, and the monolayers have fewer cells extending lamellipodia into the

wound area.

Since lamellipodial protrusion is crucial for MDCK monolayer migration, we

examined if inhibition of Rho-kinase, known to induce lamellipodial expansion in

wounded epithelial monolayers (Omelchenko et al., 2003), could overcome the effects of

PMP22 overexpression (Fig. 4-7B). As expected, a three hour treatment of neo-MDCK

cells with a Rho kinase inhibitor (Y-27632) leads to increased lamellipodial-like cell

protrusion, visualized by actin-phalloidin fluorescent imaging (Fig. 4-7B, arrows). In

comparison, PMP22-MDCK cells appear resistant to the formation of lamellipodia, with

few cell protrusions apparent along the wound edge (arrow). Thus, the expression of

PMP22 results in the reduced migration of MDCK cells after wounding.

Fibroblast-derived scatter-factor (SF), has been shown to induce an epithelial to

mesenchymal transition (EMT) in MDCK cells (Stoker et al. 1987). Therefore, we

investigated whether SF is capable of overriding the inhibitory effect of PMP22

expression on MDCK monolayer migration. When cultured in SF, neo and PMP22 cells

migrate a similar distance five hours after a scratch wound (Fig. 4-7C, gray bars).

Compared to monolayers in normal medium (Fig. 4-7C, white bars), wound closure in the

presence of SF increases by 1.8- and 4.7- fold for the neo and PMP22-MDCK cells,









respectively. These results indicate that while PMP22-MDCK cells are refractory to the

effects of Rho kinase inhibition, they are competent to migrate after SF-induced EMT.

Discussion

The described results indicate that PMP22 plays a role in several aspects of

epithelial biology. The overexpression of PMP22 reduces the proliferation and final cell

density of epithelial monolayers, and induces flattened cell morphology. Monolayers of

such cultures have increased TER and paracellular flux of nonionic dextrans. In

agreement, a PMP22 peptide disrupts the reformation of paracellular resistance following

calcium-switch. The migration of epithelial monolayers is also reduced by PMP22

overexpression, possibly due to a deficiency in lamellipodial forming leader cells. These

results suggest that PMP22 takes part in a pathway by which apical cell junctions regulate

the proliferation and morphology of epithelial cells, and modulate paracellular

permeability and cell motility.

Cell junction-associated proteins have previously been shown to influence cell

proliferation. For example, in addition to regulating paracellular permeability (Balda and

Matter, 2000; Reichert et al., 2000), elevated levels of ZO-1 reduce proliferation and cell

density in MDCK cells (Balda et al., 2003). This effect is thought to result from

sequestration of the transcription factor ZONAB, a ZO-1 binding partner, from the cell

nucleus (Balda et al., 2003). Currently, it is unknown through which pathway the

transmembrane protein PMP22 elicits a similar response in the MDCK model. In

Schwann cells and fibroblasts, in addition to growth arrest, overexpression of PMP22 by

retroviral and transient transfection has been shown to induce apoptosis (Fabretti et al.,

1995; Zoidl et al., 1997), a process counteracted by exogenous Bcl-2 (Brancolini et al.,









1999). In our stably transduced cell populations, increased apoptosis as judged by

Hoechst staining was not observed, possibly due to a lower level of PMP22 expression.

It has been reported in several cell types that overexpression of PMP22 affects

cellular morphology (Brancolini et al., 1999; 2000; Chies et al., 2003). The altered cell

shape observed in PMP22-MDCK monolayers is likely the consequence of reduced cell

density at confluency, in which a flattened morphology is necessary for maintaining

functional cell-cell junctions. The elevated TER might be the result of this phenomenon,

as confluent monolayers with reduced cell density have less total tight junctional space

(Marcial et al., 1984). Since paracellular junctions are more permeable than the cell itself

(Stefani and Cereijido, 1983), an elevated TER would be expected. Yet, the reduced cell

density reported after ZO-1 overexpression did not significantly alter the TER of the

MDCK monolayers (Balda et al., 2000). Therefore, PMP22's effect on monolayer

resistance is not entirely based on altered morphology. In accordance, as shown here and

by others (Balda et al., 1996; 2000; McCarthy et al., 1996; 2000), elevated levels of

occludin increased the TER, but did not significantly alter the cell morphology or density

of confluent monolayers.

A role for PMP22 in modulating paracellular flow is supported by the increased

ionic and nonionic permeability following exposure to the PMP22-2nd loop peptide. The

reduced TER and increased flux of small dextrans may indicate that the 2nd loop peptide

disrupts homotypic interactions of PMP22. Indeed, PMP22 is known to form dimers and

larger oligomers in vivo and in vitro (Tobler et al., 1999; 2002). As the extracellular

domains of PMP22 share no significant homology with the claudins, a potential direct

effect on claudins is unlikely. However, the peptides may be perturbing the function of









other, as of yet undetermined, epithelial junction-associated proteins that are binding

partners for PMP22.

The localization of exogenous and endogenous PMP22 at apical intercellular

junctions (Notterpek et al., 2001; Roux et al., 2004), combined with the effects of

elevated PMP22 expression or PMP22 peptides on paracellular permeability, supports the

notion that PMP22 is a functional constituent of the apical junctional complex. Although

it has not been determined ultrastructurally whether PMP22 is at tight or adherens

junctions, the protein is capable of altering both the ionic and nonionic permeability of

epithelial monolayers. Similar effects have been attained in previous studies when the

expression of the tight junction protein occludin (Balda et al., 1996; 2000; McCarthy et

al., 1996; 2000) was modulated. As PMP22 is detected at intercellular contacts of rat

neuroepithelia (Roux et al., 2004), cells devoid of classical tight junctions (Mollgard et

al., 1987; Aaku-Saraste et al., 1996), the role of the protein might not be exclusive to

tight junctions. In this respect, PMP22 is similar to ZO-1, a junctional protein that in

some cell types exists at sites other than the tight junctional complex (Itoh et al., 1993).

Epithelial cells maintain physical contacts during wound closure, while they

extend Rac-GTPase-dependent lamellipodia (Fenteany et al., 2000). In normal epithelia,

lamellipodial protrusion is promoted by Y-27632, likely by disrupting the actin marginal

bundles along the wound edge (Omelchenko et al., 2003). PMP22 expression however,

even after Y-27632 treatment, prevents the lamellipodial formation. This suggests that an

elevated level of the protein interferes with the signaling for lamellipodial protrusion,

possibly by acting via the actin cytoskeleton directly or indirectly by modulating the Rac

GTPase pathway. In comparison, the same MDCK cells are capable of migrating similar









to controls following the application of SF. This result suggests that the inhibitory action

of PMP22 on wound-induced migration is likely dependent upon epithelial cell-cell

contacts, as SF induces a junction-disrupting EMT (Nusrat et al., 1994, Grisendi et al.,

1998).

Since PMP22 is predominantly expressed in myelinating Schwann cells, what

relevance do our studies in epithelia have to our understanding of the protein's function

in the PNS? The growth arrest properties of PMP22 appear to occur independent of cell

type, but have yet to be directly linked to PMP22-associated disease pathology. Cell

morphology is drastically altered during myelination, an event that involves extensive

membrane expansion; however, PMP22 is not required for myelin wrapping (Adlkofer et

al., 1995). Cell migration is also crucial to proper nerve development (reviewed in

Lobsiger et al., 2002). Nonetheless, our results show that when epithelia undergo EMT

and migrate as individual cells, the overexpression of PMP22 has no inhibitory effect.

Typically thought of as a component of compact myelin (Haney et al., 1996), PMP22 has

not yet been localized to tight junctions of PNS myelin. Based on the effects of PMP22

on epithelial paracellular permeability, the protein could have a similar role in PNS

myelin at claudin-1 and -5-positive autotypic tight junctions (Poliak et al., 2002).

Therefore, modulating PMP22 in an epithelial model may provide some clues as to the

function of the protein in PNS myelin.

In addition to identifying the participation of PMP22 in epithelial cell biology, we

established an in vitro model that is amenable to further experimentation. Utilizing this

system, the specific activity of various PMP22 domains can be dissected by examining

their effects on epithelial permeability and migration. It will be equally important to






83


identify binding partners of PMP22 at intercellular junctions in order to fully understand

how the protein signals such global changes in epithelial cell biology.














CHAPTER 5
CONCLUSIONS

Overview of Findings

At the time that these studies began, PMP22 was viewed primarily as a myelin

protein involved in peripheral nerve pathology. With little known about its function,

especially in myelinating Schwann cells, the majority of research has focused on

characterizing nerve pathology and the mechanism of disease with the goal to ameliorate

or prevent neuropathy. The research presented here has sought to examine fundamental

properties of non-neural PMP22 complementing other efforts by providing knowledge

about the function of PMP22. With its widespread and extensive expression pattern

throughout development and maturity, an understanding of the role for PMP22 in basic

cell biology is a justified endeavor that may lead to novel discoveries about undefined

cellular processes.

By following clues such as homology to the claudin superfamily of tight junction

proteins (Chapter 2, Notterpek et al., 2001; Takeda et al., 2001) and expression in

epithelial (Baechner et al., 1995; Wulf et al., 1999) and endothelial cells (Bongrazio et

al., 2000), we correctly hypothesized that PMP22 is a constituent of cell-cell contacts in

epithelia and endothelia (Chapter 2, Notterpek et al., 2001). Subsequently, we

characterized the expression and localization of PMP22 in the developing and adult

blood-nerve and blood-brain barriers (BBB) (Chapter 3, Roux et al., 2004), The BBB is a

system extensively researched for its involvement in several CNS disorders (reviewed by









Neuwelt, 2004). As a result of those studies, PMP22 was detected early in development

at junctions of the brain vasculature and choroidal epithelia. Additionally, the protein was

found at neuroepithelial intercellular junctions, substantiating previous reports of PMP22

mRNA expression in the neuroepithelium (Baechner et al., 1995; Parmantier et al., 1995;

1997). These cell-cell junctions are thought to be requisite for neurogenesis (Chenn and

McConnell, 1995; Manabe et al., 2002). Finally, we began to investigate the function of

the protein in epithelia, where we found evidence for its involvement in regulating the

cell cycle and cell morphology. However, perhaps most importantly, these studies led to

the discovery of a novel role for PMP22 in the modulation of junction permeability and

cell migration, and identified an in vitro model in which to further investigate the

function of the protein.

Unresolved Issues

As with most scientific research, these studies have led to many as of yet

unanswered questions. With regards to specific subcellular localization, it is clear that

PMP22 does not display a pattern of immunolabeling similar to E-cadherin or 1-catenin,

typical adherens junction proteins detected at lateral cell contacts. Instead, the protein

colocalizes with ZO-1, occludin and claudin-1 at apical intercellular junctions, suggestive

of a tight junction protein. However, this issue is complicated by the detection of the

protein at apical neuroepithelial cell junctions, sites which are thought to be devoid of

classical tight junctions (Mollgard et al., 1987; Aaku-Saraste et al., 1996). These results

suggest that PMP22, like ZO-1 (Itoh et al., 1993), might be capable of existing at either

type of junction depending upon the cell type. This hypothesis may explain its expression

in cells, such as fibroblasts (Manfioletti et al., 1990), which lack tight junctions, but

contain ZO-1 (Itoh et al., 1993; Chapter 2, Notterpek et al., 2001). Nonetheless, we still









do not know at the ultrastructural level, whether PMP22 resides primarily at the tight or

adherens junctions of epithelia or endothelia. The most direct way to resolve this

uncertainty is to attempt ultrastructural immunolabeling, ideally on freeze-fractured

samples.

Another unresolved topic concerns non-PNS related consequences to peripheral

neuropathy-associated PMP22 misexpression. In humans, the only reported clinical

pathology seemingly unrelated to peripheral neuropathy is the CNS demyelination

reported in a small subset of patients deficient in PMP22 expression (Amato et al., 1996;

Schneider et al., 2000; Dackovic et al., 2001). This phenotype remains infrequently

reported in humans; however, a seizure-like behavior, suggestive of CNS pathology, is

also observed in homozygous PMP22-deficient and Tr-J mouse models. Additionally,

other organ systems, besides the PNS, have not been extensively examined for pathology

in mouse models for PMP22-misexpression. A likely candidate for such studies would be

the homozygous Tr-J mouse that is not viable beyond three weeks postnatal (Henry et al.,

1983). Of course, the possibility exists that there is a redundancy of function for PMP22

outside of the Schwann cell, with the family of epithelial membrane proteins being the

most likely candidates based on homology (Taylor et al., 1995; 1996; Lobsiger et al.,

1996). A similar redundancy likely occurs in mice deficient in claudin-14, a protein

expressed in several tissues, but with pathology detected in only cochlear hair cells (Ben-

Yoseph et al., 2003). If any redundancy can be identified in vitro, the establishment of

double knockout transgenic animals may allow for in vivo analysis.

Since PMP22 is localized to apical intercellular junctions, it was perhaps not

unexpected that junctional permeability would be affected by overexpression of the









protein (Chapter 4). Nonetheless, it still remains unknown how PMP22 induces this

effect. Based on its homology to the claudins, it is temping to assume that PMP22 itself

acts to modulate the barrier properties of the epithelial monolayer. However, it may be

more likely that PMP22 modulates, as of yet unknown, binding partners that themselves

function to maintain paracellular resistance. Possible candidates for this role would

certainly include the claudins or occludin. To date, attempts to co-immunoprecipitate

proteins with PMP22 have proven difficult, largely due to the protein's insolubility.

Future Studies

The studies described in Chapter 4 identified that an elevated PMP22 level

increases both the TER and paracellular flux and inhibits the proper migration of MDCK

monolayers; however, it remains unknown how a reduction in PMP22 expression will

affect these processes. Future studies may take advantage of an inducible antisense or

RNA-silencing technology to create a transient decrease in the level of the protein.

Hopefully, if a functional redundancy for PMP22 exists, it will not occur rapidly, but

instead require a lack of PMP22 expression during cellular development and

differentiation.

It is hoped that the studies described here will lead to a clearer understanding of

the mechanisms by which PMP22 modulates such seemingly diverse cellular processes as

proliferation, morphology, junctional permeability and epithelial migration. One

approach to this issue is to find binding partners for PMP22, either by hypothesis driven

testing or using a more comprehensive 'shotgun' technique, such as a yeast two hybrid

assay. Myelin protein zero (D'Urso et al., 1999) and P2X7 (Wilson et al., 2002), were

identified as proteins that interact with PMP22 by these approaches, respectively. Once









binding partners are discovered, it is possible that the modulation of their function, if

known, by PMP22 could account for the effects of altered PMP22 expression. Based on

its extensive expression pattern and ability to associate with two seemingly unrelated

proteins, it is conceivable that PMP22 may act as a transmembrane protein-chaperone,

possibly involved in the targeting or stoicheometry of other proteins within specific

membrane domains.

An alternative approach to investigate the function of PMP22 is to dissect the

signaling mechanisms by which the protein modulates cellular behaviors. For example, it

may be informative to evaluate the activation or inactivation of the major GTPase

pathways, Rho, Rac and Cdc42, following an increase or decrease in PMP22 expression.

A role for the RhoA-GTPase pathway in PMP22-induced altered cellular morphology has

already been identified in fibroblasts (Brancolini et al., 1999; Chies et al., 2003). In these

studies, it appears that active RhoA, either artificially induced or naturally occurring, is

able to counteract the effects of PMP22 overexpression on cell morphology. In addition

to cell morphology, the GTPase pathways are crucial to maintenance of paracellular

permeability (reviewed in Hopkins et al., 2000) and wound closure (Fenteany et al.,

2000) in epithelial cells. Overexpression of PMP22 produces a flattened morphology and

inhibits lamellipodial formation required for wound closure (Chapter 4), phenotypes

similar to that found in MDCK cells expressing a dominant negative Rac-GTPase

(Takaishi et al., 1997, Fenteany et al., 2000, respectively). These same GTPases are likely

involved in the migration and immense membrane expansion required for PNS myelin

formation (Melendez-Vasquez et al., 2004).









In parallel to investigating binding partners and signaling pathways involved in

the PMP22 modulation of epithelial biology, the MDCK cell model can be used to

identify protein domains or specific amino acids crucial to its normal functions. Based on

the peptide perturbation experiments in Chapter 4, the 2nd loop of PMP22 would be a

prime candidate for site directed mutagenesis, a technique to create proteins with altered

amino acids. The charged amino acids of both the 1st and 2nd loop should be analyzed as

these have been shown to mediate the specific ionic selectivity of several claudins

(Colegio et al., 2002; 2003; Van Itallie et al., 2003; Yu et al., 2003). As glycosylated

PMP22 is found at the cell surface in epithelia (Chapter 4), it may be insightful to

compare the effects of the wild type protein to a mutant lacking the proper glycosylation

motif. Previous studies in Schwann cells have shown that the non-glycosylated protein is

capable of proper targeting (Ryan et al., 2002); however, in Cos7 cells the protein is

unable to induce as significant a change in cell morphology (Brancolini et al., 2000).

Another unique domain of PMP22 is the short carboxyl tail of charged amino acids (Fig

1-1). Previous studies have shown that this domain does not act as an ER retrieval motif

(Brancolini et al., 2000), but no functional significance has yet to be ascribed to this

domain. It is unlikely that this carboxyl region would serve as a PDZ binding domain

(Gonzalez-Mariscal et al., 2003), a feature common to many junctional proteins, as it is

not similar to previously identified sequences and is rather short and likely very close to

the membrane. Perhaps with the exception of proliferation, a quantifiable functional

analysis of PMP22 in in vitro Schwann cells remains elusive. Thus, it may be tempting to

study neuropathy-associated PMP22 point mutants in epithelia since junctional

permeability and wound migration are quantifiable characteristics. Unfortunately, many









of the diseased-linked point mutant proteins appear to have defects in trafficking and are

unable to reach the cell surface. In such situations, it is unclear if the results are due to

disrupted function or targeting. Only after a non-neural clinical pathology can be

identified in PMP22 mutant mice, or humans, would it seem worthwhile to examine the

effects of the altered protein on epithelial biology.

In summary, the studies described in this dissertation have identified PMP22, for

the first time, as a constituent of apical intercellular junctions in epithelia and endothelia,

and have provided novel evidence of a functional role in epithelial biology. The findings

of this work will affect future efforts of scientists investigating the role for PMP22 in

hereditary peripheral neuropathies, as well as those seeking to understand basic epithelial

or endothelial cell biology.















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