Recent progress on the molecular organization ... - Wiley Online Library

Journal of the Peripheral Nervous System 7:1–12 (2002)

BUNGE MEMORIAL LECTURE

Recent progress on the molecular organization of myelinated axons Steven S. Scherer and Edgardo J. Arroyo Department of Neurology, Room 460 Stemmler Hall, 36th Street and Hamilton Walk, The University of Pennsylvania Medical Center, Philadelphia

Abstract The structure of myelinated axons was well described 100 years ago by Ramón y Cajal, and now their molecular organization is being revealed. The basal lamina of myelinating Schwann cells contains laminin-2, and their abaxonal/outer membrane contains two laminin-2 receptors, 64 integrin and dystroglycan. Dystroglycan binds utrophin, a short dystrophin isoform (Dp116), and dystroglycan-related protein 2 (DRP2), all of which are part of a macromolecular complex. Utrophin is linked to the actin cytoskeleton, and DRP2 binds to periaxin, a PDZ domain protein associated with the cell membrane. Non-compact myelin—found at incisures and paranodes—contains adherens junctions, tight junctions, and gap junctions. Nodal microvilli contain F-actin, ERM proteins, and cell adhesion molecules that may govern the clustering of voltage-gated Na channels in the nodal axolemma. Nav1.6 is the predominant voltage-gated Na channel in mature nerves, and is linked to the spectrin cytoskeleton by ankyrinG. The paranodal glial loops contain neurofascin 155, which likely interacts with heterodimers composed of contactin and Caspr/paranodin to form septate-like junctions. The juxtaparanodal axonal membrane contains the potassium channels Kv1.1 and Kv1.2, their associated 2 subunit, as well as Caspr2. Kv1.1, Kv1.2, and Caspr2 all have PDZ binding sites and likely interact with the same PDZ binding protein. Like Caspr, Caspr2 has a band 4.1 binding domain, and both Caspr and Caspr2 probably bind to the band 4.1B isoform that is specifically found associated with the paranodal and juxtaparanodal axolemma. When the paranode is disrupted by mutations (in cgt-, contactin-, and Caspr-null mice), the localization of these paranodal and juxtaparanodal proteins is altered: Kv1.1, Kv1.2, and Caspr2 are juxtaposed to the nodal axolemma, and this reorganization is associated with altered conduction of myelinated fibers. Understanding how axon-Schwann interactions create the molecular architecture of myelinated axons is fundamental and almost certainly involved in the pathogenesis of peripheral neuropathies. Key words: junctions, Schwann cells, cell adhesion molecules, neuropathy, channels

Introduction

encode components of the myelin sheath. Moreover, the maintenance of the myelinating phenotype appears to depend on a continuous relationship with an axon, as axotomy results in the down-regulation of myelin-related genes and the dedifferentiation of previously myelinating Schwann cells (Scherer and Salzer, 1996). Myelinating Schwann cells, in turn, organize the axonal membrane. Voltage-gated Na channels accumulate at the ends of developing myelin sheaths, and as two adjacent internodes elongate, these clusters fuse to form a node of Ranvier (Vabnick and Shrager, 1998). The Shaker-type K channels, Kv1.1 and Kv1.2, are subse-

Myelinating Schwann cells differentiate from immature Schwann cells in response to as yet undetermined axonal signals (Mirsky and Jessen, 1999). The acquisition of a myelinating phenotype is accompanied by altered expression of numerous genes, many of which Address correspondence to: Steven S. Scherer, M.D., Ph.D., Department of Neurology, Room 460 Stemmler Hall, 36th Street and Hamilton Walk, The University of Pennsylvania Medical Center, Philadelphia, PA 19104, USA. Tel: 215-573-3198; Fax: 215-573-4454; E-mail: [email protected] © 2002 Peripheral Nerve Society, Inc.

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quently excluded from the nodal axolemma and sequestered beneath the myelin sheath by the developing paranode (Vabnick et al., 1999). The analysis of mutations that affect the integrity of paranode, cgt-, contactin, and Casprnull mice, highlights the importance of this structure for the proper function of myelinated fibers (Bhat et al., 2001; Boyle et al., 2001; Poliak et al., 2001; Popko, 2000). In this review, we consider some of the recent findings relating to structure and function of myelinated axons in the PNS. We have focused on three emerging topics: the laminin-2 receptors of myelinating Schwann cells, the differences between compact and non-compact myelin, and the structure and function of the nodal region. We have compared some of these findings to those of Ramón y Cajal, and have emphasized how the molecular organization of myelinated fibers is related to various peripheral neuropathies.

was shown to be composed of a basal lamina and associated collagen fibers (Thomas and Olsson, 1993), and subsequently to contain laminin-2 (also known as merosin) and other associated proteins (Bunge, 1993). A series of investigations, largely in the laboratory of Richard and Mary Bunge (Bunge, 1993), indicate that axon-Schwann cells interactions are required for Schwann cells to assemble their extracellular matrix, and that extracellular matrix may be required for myelination (c.f., Podratz et al., 1998). Laminins are trimers of , , and subunits, each of which belongs to a gene family; the composition of laminin-2 is 211. Laminin-2 is found in the basal laminae of skeletal muscle and Schwann cells, and is required for their normal development, as shown by the phenotype of dystrophic mice. These mice lack the 2 chain because they are homozygous for lama2 mutations (Sunada et al., 1994; Xu et al., 1994), and have a mild dysmyelinating neuropathy in addition to a myopathy, that are both related to defective basal laminae. In humans, LamA2 mutations are the commonest cause of congenital myopathies. Because some of these patients also have mildly slowed conduction velocities (Shorer et al., 1995), laminin-2 is likely to be essential for the normal myelination in humans, too. Myelinating Schwann cells have two receptors for laminin-2, 64 integrin (Einheber et al., 1993; Feltri et al., 1994) and dystroglycan (Matsumura et al., 1997; Yamada et al., 1996). As shown in Fig. 1, 64 integrin is ex-

Myelinating Schwann cells are polarized According to Ramón y Cajal (1928), “The medullated nerve fibre, then, consists of an external solid membrane—sheath of Schwann—of a tubular cell—Schwann cell—of a fatty sheath, and of an axon.” He understood that the sheath was not part of the Schwann cell itself, and that it continued across nodes of Ranvier. With the advent of electron microscopy, the sheath of Schwann

Figure 1. Myelinating Schwann cells are polarized. The left panel is a confocal image of a transverse section of rat sciatic nerve, double-labeled with a rabbit antiserum against 4 integrin (FITC) and a mouse monoclonal antibody against MAG (TRITC). 4 integrin is localized around the entire circumference of the outer/abaxonal membrane, and MAG is localized on the inner/adaxonal membrane. Compact myelin is not stained and thus appears black. The circumferential organization of a myelinated axon is shown schematically on the right panel.

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pressed around the entire circumference of the abaxonal/ outer membrane of myelinating Schwann cells. Mice lacking either the 6 or the 4 integrin subunit die shortly after birth because of a profound skin defect, but myelination in co-cultures established from 4-null mice appears normal (Frei et al., 1999). In epithelia such as the skin, 64 heterodimers are found in hemi-desmosomes, which adhere epithelial cells to their basal lamina via intermediate filaments. Schwann cells, however, do not have hemi-desmosomes and 64 integrin may be linked to the actin cytoskeleton and not to intermediate filaments. Dystroglycan is composed of and subunits; these are products of the same gene. In skeletal muscle, dystroglycan binds to dystrophin, which, in turn, binds to the actin cytoskeleton; dystroglycan also forms a complex with , , , and sacroglycans and sarcospan (Ozawa et al., 1998; Straub and Campbell, 1997). Recently, Sherman and colleagues (Sherman et al., 2001) showed that dystroglycan-related protein 2 (DRP2) and periaxin form a complex with the dystroglycan receptor in myelinating Schwann cells (Fig. 2). As shown in Fig. 3A, DRP2 is found in large blocks separated by longitudinal and transverse bands that can be immunostained for caveolin-1, as previously shown by Mikol et al. (1999). These anatomical features were well known to Ramón y Cajal (1928) (Fig. 3B). Thus, in contrast to the continuous localization of 64 integrin, DRP2 is specifically localized to the Schwann cell cytoplasm that directly apposes the myelin sheath (Sherman et al., 2001). As depicted in Fig. 2, in myelinating Schwann cells DRP2/periaxin complexes are linked to a complex containing a short dystropin isoform (Dp116), utrophin, , , and sarcoglycan, but not or sarcoglycan or sar-

Figure 3. Cytoplasmic domains in myelinating Schwann cells. Panel A is a confocal image of three teased fibers from a rat sciatic nerve, double-labeled with a rabbit antiserum against DRP2 (gift of Dr. Diane Sherman; FITC) and a mouse monoclonal antibody against caveolin-1 (TRITC). A node (apposed arrowheads) and two nuclei (n) are indicated. Panel B is a drawing made by Ramón y Cajal (1928) of teased fibers from an adult cat, stained with reduced silver (used with permission of Oxford University Press). He subdivided the Schwann cell cytoplasm into a “perinuclear mass”, “longitudinal stripes”, and “transverse trabeculae”; as shown in panel A, the Schwann cell membrane in these regions contains caveolin-1 but not DRP2. Scale bar: 10 m.

cospan (Imumura et al., 2000; Sherman et al., 2001). Whereas utrophin is probably directly linked to the actin cytoskeleton, Dp116 and DRP2 lack an actin-binding domain. The importance of this complex in myelinating Schwann cells remains to be fully elucidated, but it is clear that dystrophin as well as , , , and sarcoglycan are essential for skeletal muscle cells (Ozawa et al., 1998; Straub and Campbell, 1997). Dp116 may be essential for proper myelination, as a splice site mutation in the human dystrophin gene that abolishes dystrophin expression in peripheral nerve has been reported to cause a demyelinating neuropathy (Comi et al., 1995). Periaxin is clearly essential, as periaxin-null mice develop a demyelinating neuropathy (Gillespie et al., 2000), and recessive PRX mutations in humans cause inherited demyelinating neuropathy (Boerkoel et al., 2000; Guilbot et al., 2001). Dystroglycan also binds to an isoform of agrin that is found in the basal laminae of Schwann cells (Yamada et al., 1996). Finally, dystroglycan has another important connection to peripheral neuropathy, as it appears to be a receptor for Mycobacterium leprae, an intracellular pathogen of Schwann cells (Rambukkana, 2001).

Figure 2. The dystroglycan receptor complex of myelinating Schwann cells. The dystroglycan receptor is linked to three members of the dystrophin family in myelinating Schwann cells, utrophin, DRP2, and Dp116. Of these, only utrophin has an actin-binding domain, whereas DPR2 binds periaxin, which is shown binding to itself via its PDZ domains. Three sacroglycans, , , and , associate with dystroglycan. The figure was modified from one kindly provided by Drs. Diane Sherman and Peter Brophy.

The PNS myelin sheath The organization of a myelinated fiber is shown in Fig. 4A. It depicts two internodes; one has been unrolled to reveal its trapezoidal shape. The figure shows that myelin sheath itself can be divided into two domains, compact 3

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and non-compact myelin, each containing a non-overlapping set of proteins (Fig. 4B). Compact myelin forms the bulk of the myelin sheath, and Fig. 5 depicts its molecular organization. Interaction of P0 tetramers in cis and trans (Shapiro et al., 1996) are essential for myelin compaction, as the extracellular space (between the intraperiod lines) is widened in Mpz-null myelin sheaths (Giese et al., 1992). Even mice that are heterozygous for a null Mpz mutation have focal areas of widened myelin (Samsam et al., 2002); this is also a hallmark of some human MPZ mutations (Gabreels-Festen et al., 1996). Hence, both MPZ/Mpz alleles may be required for proper myelination; the loss of one allele causes haplotype insufficiency.

Duplication or deletion of PMP22 is the most common cause of inherited demyelinating neuropathy (Murakami et al., 1996; Suter and Snipes, 1995), causing Charcot-Marie-Tooth disease type 1A (CMT1A; affected individuals have 3 copies of PMP22) and hereditary neuropathy with liability to pressure palsies (HNPP; affected individuals have 1 copy of PMP22), respectively. Although the function PMP22 is unknown, these results provide strong evidence that the amount of PMP22 in compact myelin is critical, which has been directly confirmed (Vallat et al., 1996). Because perturbations in the stoichiometry of either P0 or PMP22 appears to alter the stability of the myelin sheath, compact myelin has been Figure 4. The organization of a myelinated axon. Panel A depicts an “unrolled” myelinating Schwann cell, revealing the regions that form compact and non-compact myelin. Tight junctions are depicted as two continuous (green) lines; these form a circumferential belt and are also found in incisures. Gap junctions are depicted as orange ovals; these are found between the rows of tight junctions, and are more numerous in the inner aspects of incisures and paranodes. Adherens junctions are depicted as purple ovals; these are more numerous in the outer aspects of incisures and paranodes. The nodal, paranodal, and juxtaparanodal regions of the axonal membrane are colored blue, red, and green, respectively. The figure was modified from Arroyo and Scherer (2000), with permission of SpringerVerlag. Panel B depicts the proteins of compact and non-compact myelin. Compact myelin contains P0, PMP22, and MBP; noncompact myelin contains E-cadherin, MAG, DM20, Cx32, and an unknown claudin.

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dri et al., 1982). The claudin(s) forming these tight junctions remains to be determined. The gap junctions in myelinating Schwann cells contain connexin32 (Cx32) (Bergoffen et al., 1993; Chandross et al., 1996; Scherer et al., 1995). A role for gap junctions in the myelin sheath was not established until it was discovered that mutations in the gene encoding Cx32, GJB1, cause X-linked CMT (CMTX) (Bergoffen et al., 1993). Dye transfer studies in living myelinated fibers provide functional evidence that gap junctions mediate a radial pathway of diffusion across incisures (Balice-Gordon et al., 1998). A radial pathway would be advantageous as it provides a much shorter pathway (up to 1000-fold), owing to the geometry of the myelin sheath. Disruption of this radial pathway may be the reason that GJB1 mutations cause CMTX. However, the pathway and the rate of 5,6carboxyfluorescein diffusion in Gjb1/cx32-null mice did not appear to be different than in wild type mice (BaliceGordon et al., 1998), implying that another connexin(s) forms functional gap junctions in PNS myelin sheaths. The existence of a radial pathway provides further evidence that the resistance of myelin is not as high as commonly conceived (Funch and Faber, 1984). The lateral borders of the Schwann cell cytoplasm have microvilli (Fig. 6). Microvilli contain F-actin (Trapp et al., 1989), and as shown in Figs. 6 and 7, ezrin, radixin, and moesin (Hayashi et al., 1999; Melendez-Vasquez et al., 2001; Scherer et al., 2001), the defining members of the ERM family of proteins. ERM proteins bind to actin filaments mainly via their C-termini, and can associate with a number of different integral membrane proteins via their N-termini. ERM proteins also form head-to-tail oligomers with themselves and with merlin, the product of the NF2 locus. It remains to be determined whether ERM proteins are associated with integral membrane proteins in Schwann cell microvilli, and whether their phosphorylation regulates their function in this setting (Hayashi et al., 1999). As shown in Fig. 8A, ERM proteins are also colocalized with F-actin in incisures and in the inner mesaxon (Scherer et al., 2001; Trapp et al., 1989). Strands of ERM/F-actin staining within the non-compact myelin appear to form a spiral pattern (Scherer et al., 2001), owing to the spiral nature of the myelin sheath itself, strikingly reminiscent of what was once known as the “apparatus of Rezzonico” (Fig. 8B).

Figure 5. The localization of PNS myelin proteins in compact myelin. The left panel is an electron micrograph of compact myelin, which consists of alternating layers known as the intraperiod line (which is actually a double line) and the major dense line. The right panel is a schematic depiction of how apposed cell membranes create the intraperiod and major dense lines. The disposition of P0 tetramers, PMP22 dimers, and MBP monomers, as well as the glycolipids galactocerebroside and sulfatide are shown. The approximate thickness of the lipid bilayers, as well as the intracellular and extracellular spaces is indicated (Vonasek et al., 2001).

likened to a liquid crystal composed of highly ordered proteins and lipids. How other point mutations of MPZ and PMP22 cause inherited demyelinating neuropathies remains to be determined, as these mutant proteins have toxic effects that are not seen in null alleles. The main finding to date in this regard is that most mutant PMP22 proteins are retained in the endoplasmic reticulum, and do not reach compact myelin (D’Urso et al., 1998; Naef et al., 1997; Naef and Suter, 1999; Notterpek et al., 1997; Tobler et al., 1999). Protein-protein interactions may also play a role, as P0 forms tetramers and PMP22 forms dimers; PMP22 may also interact with P0 (D’Urso et al., 1999; Shapiro et al., 1996; Tobler et al., 1999). Non-compact myelin is found in paranodes (the lateral borders of the myelin sheath) and in Schmidt-Lanterman incisures. As depicted in Fig. 4, non-compact myelin contains junctional specializations between the layers of the myelin sheath, so-called “reflexive” or “autotypic” junctions (Arroyo and Scherer, 2000). Some of these reflexive junctions are also found in inner and outer mesaxons. Adherens junctions are most numerous in the outer mesaxon as well as in outermost layers of the paranodes and incisures; these contain E-cadherin, -catenin, and -catenin, and are likely linked to the actin cytoskeleton (Fannon et al., 1995; Hall and Williams, 1970). Tight junction strands enclosing gap junction-like plaques have been found by freeze fracture electron microscopy (San-

Specializations at nodes In spite of the differences between myelinating Schwann cells and oligodendrocytes and their myelin sheaths, the organization of the axon itself is quite similar in the PNS and CNS (Fig. 6A). Voltage-gated Na channels are highly concentrated in the nodal axolemma (Ellisman and Levinson, 1982; Haimovich et al., 1984); these belong to a multi-gene family, but the 5

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Figure 7. ERM proteins in Schwann cell microvilli. Panels A-D show a confocal reconstruction of a teased fiber from a rat sciatic nerve (fixed for 60 minutes in 4% paraformaldehyde), labeled with a pan-ERM antiserum (A), a mouse monoclonal antibody against Na channels (B), and a rat monoclonal antibody against a phosphorylated epitope of neurofilament heavy (NF-H, C); the merged image is shown in panel D. The insets in panels A and B show the superimposed NF-H staining. At the node (double arrowheads), note that the ERM antibody stains a larger diameter disk than does the Na channel antibody. Panel E shows a single 0.5 m thick optical section from a section through a node of Ranvier; taken from an unfixed ventral root, double-labeled with a rabbit antiserum against ezrin (TRITC) and a mouse monoclonal antibody against Na channels (FITC). Note that the ring of ERM staining is larger than the ring of Na channel staining. Scale bar: A-D, 10 m; E, 1 m. From Scherer et al. (2001), with permission of Wiley-Liss.

Figure 6. Nodal specializations in PNS myelin sheaths. Panel A is a schematic depiction of the node, paranode, and juxtaparanode. Panel B is a schematic drawing of possible cis and trans interactions between the molecular components of nodes (modified from Arroyo and Scherer (2000), used with permission of Springer-Verlag).

Nav1.6 channel appears to be the main one expressed at nodes (Caldwell et al., 2000). The gene encoding this channel is mutated in mice with motor endplate disease (med), a recessively inherited disease that cause respiratory paralysis (Burgess et al., 1995). Nav1.6 is probably not the only voltage-gated Na channel at nodes, because mixed nerve conduction velocity is only minimally slowed in med mice (Duchen and Stefani, 1971; Rieger et al., 1984); indeed, Nav1.2, Nav1.8, and Nav1.9 have also been reported in nodes (Boiko et al., 2001; Fjell et al., 2000; Kaplan et al., 2001). Two splice variants of ankyrinG, 480 and 270 kDa, anchor voltage-gated Na channels at nodes (Kordeli et al., 1990; Kordeli et al., 1995). These isoforms are distinguished by their membrane-binding domain composed of ANK repeats, a spectrin-binding domain, and a serine/ threonine-rich domain (Zhang and Bennett, 1996). As depicted in Fig. 6B, ankyrinG also interacts with the cytoplasmic domains of neurofascin 186 kDa (NF186) and Nr-CAM (Davis and Bennett, 1994; Davis et al., 1996;

Davis et al., 1993; Srinivasan et al., 1988), and with spectrin (Koenig and Repasky, 1985; Trapp et al., 1989). A splice variant of spectrin IV (IV 4) is specifically localized to nodes (Berghs et al., 2000), a number of different spontaneous mutations in the murine spectrin 4 gene cause quivering, in which altered ion channel distribution has been noted (Parkinson et al., 2001). In keeping with the idea that ankyrinG links voltage-gated Na channels, NF186, and Nr-CAM to the spectrin cytoskeleton, inactivation of the ankyrinG gene in the cerebellum reduces the amount of voltage-gated Na channels and neurofascin in the initial segments of granule cells and Purkinje cells, respectively (Zhou et al., 1998a). Although these workers were unable to visualize a reduced number of Na channels in Purkinje cell initial segments, the diminished ability of these cells to initiate axon potentials is consistent with this idea. Since initial segments and nodes share many molecular characteris6

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(Ichimura and Ellisman, 1991; Sandri et al., 1982; Thomas et al., 1993). In freeze-fracture, the paranodal loops of the myelin sheath contain rows of large particles that are in register with a double row of smaller particles on the axolemma; these particles are thought to connect the terminal loops to the axon. These particles correspond to the so-called “terminal bands” seen by transmission electron microscopy, and have been more recently termed septate-like junctions, as they resemble invertebrate septate junctions (Einheber et al., 1997). Septate junctions may function similarly to vertebrate tight junctions, forming intercellular junctions that prevent the diffusion of small molecules and ions. Septate-like junctions, however, do not prevent the diffusion of lanthanum or even microperoxidase (molecular mass 5 kDa) into the periaxonal space (Feder, 1971; Hirano et al., 1969; MacKenzie et al., 1984); hence are not “tight” in the conventional sense. The molecular organization of the paranode is depicted in Fig. 6B. Heterodimers of contactin and Caspr are localized to the paranodal axolemma in myelinated fibers of the PNS and CNS (Einheber et al., 1997; Menegoz et al., 1997; Rios et al., 2000). These contactin/Caspr heterodimers co-localize with an isoform of neurofascin, NF155 (Tait et al., 2000). All of these molecules are likely to be components of septate-like junctions, as in both contactin- and Caspr-null mice, the absence of either contactin or Caspr results in the loss of septate-like junctions (Bhat et al., 2001; Boyle et al., 2001). A neurofascin-null mouse has not yet been reported, but the targeted disruption of UDP-galactose ceramide galactosyltransferase gene (cgt) have provided a glimpse of the likely phenotype in myelinating Schwann cells (Bosio et al., 1996; Coetzee et al., 1996; 1998). CGT is necessary for the synthesis of galactocerebroside and sulfatide, which are glycolipids found in the myelin sheath (Fig. 5). Besides lacking these glycolipids, the paranodes in cgt-null mice lack septate-like junctions (Bosio et al., 1998; Dupree et al., 1998).

Figure 8. Internodal localization of ERM proteins in myelinating Schwann cells. Panel A is a confocal reconstruction of two teased fibers from adult rat sciatic nerves immunolabeled with a pan ERM rabbit antiserum. The inner mesaxon is indicated (arrows). Scale bar: 10 m. From Scherer et al. (2001), with permission of Wiley-Liss. Panel B is a drawing made by Ramón y Cajal (1928) of teased fibers following “silver impregnation after fixation in formol-uranium . . . showing the apparatus of Rezzonico” (used with permission of Oxford University Press).

tics, the nodal membranes of Purkinje cells are probably similarly affected. Molecular interactions between Schwann cells and axons may define the locations of nodes. Bennett and colleagues (Bennett et al., 1997; Davis et al., 1996; Lambert et al., 1997) have proposed that NF186 and NrCAM have heterophilic interactions with other CAMs on the microvilli as depicted in Fig. 6B. This suggestion is in accord with the ultrastructural data showing tethering of the microvilli to the nodal axolemma (Ichimura and Ellisman, 1991; Raine, 1982). Neurofascin and NrCAM appear to be localized at presumptive nodes before ankyrinG and voltage-dependent Na channels (Lambert et al., 1997). The observation that ERM proteins are associated with clusters of voltage-gated Na channels in developing nerves also supports this idea (Melendez-Vasquez et al., 2001). Other molecular interactions that may serve to localize Na channels: the extracellular domain of the Na channel 2 subunit may interact in with tenascin-C and tenascin-R (Xiao et al., 1999), and extracellular matrix molecules that have been reported to be localized to the nodal region in the PNS and CNS (Bartsch et al., 1993; Ffrench-Constant et al., 1986; Rieger et al., 1986).

Specializations at juxtaparanodes By freeze-fracture electron microscopy, the axolemma in the region extending 10-15 m from the paranode contains clusters of 5-6 particles (Miller and Da Silva, 1977; Rosenbluth, 1976; Stolinski et al., 1981; Stolinski et al., 1985; Tao-Cheng and Rosenbluth, 1984). The distribution of these juxtaparanodal particles corresponds to the distribution of delayed rectifying K channels (Chiu and Ritchie, 1980), Kv1.1 and Kv1.2 and their associated 2 subunit (Gulbis et al., 1999; Mi et al., 1995; Rasband et al., 1998; Vabnick and Shrager, 1998; Wang et al., 1993; Zhou et al., 1998b). Kv1.1 and Kv1.2 subunits can freely mix in varying proportions to form tetramers, the functional channels (Hopkins et al., 1994), and the size

Specializations at paranodes At paranodes, the lateral edge of the myelin sheath spirals around the axon, forming the axoglial junctions 7

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of the particles (10 nm in diameter) compares well to the expected size of a tetramer (Kreusch et al., 1998). Although Kv1.1 and Kv1.2 channels appear to be concealed under the myelin sheath (Hildebrand et al., 1994; Kocsis et al., 1983), juxtaparanodal K channels are thought to have an important physiological function, dampening the excitability of myelinated fibers. The finding that Kv1.1-null mice have abnormal impulse generators near the neuromuscular junctions supports this idea (Smart et al., 1998; Zhou et al., 1998b). Similarly, mutations in the human Kv1.1 gene, KCNA1, cause a form of familial episodic ataxia that is associated with ectopic impulse generators somewhere within the peripheral nerve (Adelman et al., 1995; Browne et al., 1994; Brunt and Van Weerden, 1990; Van Dyke et al., 1975; Zerr et al., 1998). A homologue of Caspr, Caspr2 is localized to the juxtaparanodes of myelinated fibers in both the CNS and the PNS, colocalizing with Kv1.1/1.2/2 (Poliak et al., 1999). Caspr2 and Caspr have similar structures especially in the extracellular region, but only Caspr2 has an intracellular PDZ domain. Kv1.1 and Kv1.2, both of which also have PDZ domains, form a complex with Caspr2, probably mediated by a protein with multiple PDZ binding sites. Although transcellular connections between the juxtaparanodal axonal membrane and the myelin sheath have not been described, it is possible that Caspr2 has a binding partner (Peles and Salzer, 2000). Caspr and Caspr2 both have a cytoplasmic Band 4.1 protein binding site, and Band 4.1B protein is specifically localized to paranodes and juxtaparanodes (Ohara et al., 2000; Parra et al., 2000).

Figure 9. Altered localization of paranodal and juxtaparanodal in cgt-null mice. Panel A is a photomicrograph of a ventral root from a cgt-null mouse, double-labeled with a rabbit antiserum against Caspr (TRITC) and a mouse monoclonal antibody against Kv1.2 (FITC). Scale bar: 10 m. Panel B is a schematic representation of the reorganization of the axonal membrane in cgt-null mice. In cgt-null mice, Kv1.2 is localized to paranodes, whereas Caspr is diffusely localized throughout the internode. Septate-like junctions/transverse bands are absent in both the CNS and the PNS, and axoglial junctions are disorganized in the CNS.

axonal membrane (Fig. 9). Contactin and Caspr are not restricted to the paranode, but are more diffusely localized in the internodal membrane, and NF155 is not restricted to the paranodal loops (Bhat et al., 2001; Boyle et al., 2001; Dupree et al., 1999; Poliak et al., 2001). Further, Kv1.1, Kv1.2, and Caspr2 are mislocalized to the paranodal axonal membrane. The apposition of Kv1.1 and Kv1.2 likely results in the inefficient axonal conduction of myelinated fibers, as conduction velocities are slowed in contactin-, Caspr, and cgt-null mice (Bosio et al., 1996; Coetzee et al., 1996). In keeping with this suggestion, K channel blockers have extraordinary effects in these mutant mice (Bhat et al., 2001; Boyle et al., 2001; Coetzee et al., 1996).

The internodal region The internodal axonal membrane lacks the conspicuous specializations of the nodal region. Nevertheless, in the PNS, intramembranous particles similar to those of the juxtaparanodal region are found apposing the internal mesaxon and incisures of the myelin sheath (Stolinski and Breathnach, 1982; Stolinski et al., 1981; Stolinski et al., 1985). In accord with these findings, the internodal membranes of PNS axons have a tripartite strand (consisting of a central strand of Caspr/contactin staining, flanked by strands of Kv1.1/1.2/2/Caspr2-immunoreactivity) that apposes the inner mesaxon and the innermost aspect of incisures (Arroyo et al., 1999; Rios et al., 2000). These results suggest that Caspr2 could be localized to the juxtaparanodal and internodal membrane by a trans-interaction with a protein expressed by the myelinating Schwann cell.

Conclusion In summary, the intricate localization of numerous axonal proteins is highly related to the structure of the overlying myelin sheath. This organization is disrupted by a number of mutations that affect various components of the myelinated axons, and functional consequences have been established. How the molecular architecture of myelinated fibers is disrupted in other

Disrupted septate-like junctions and disorganized axonal membranes The lack of septate-like junctions in contactin-, Caspr, and cgt-null mice leads to a profound reorganization of the 8

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inherited and acquired neuropathies may provide insight into their pathogenesis.

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Acknowledgements This paper is based on The Third Richard P. Bunge Memorial Lecture, given by S.S.S. to the Peripheral Nerve Society. Our work is supported by the NIH (NS37100, NS34528, and NS08075), the Juvenile Diabetes Foundation, and the Charcot-Marie-Tooth Association. We thank Drs. Rita Balice-Gordon, Linda Bone, Peter Brophy, Bill Chiu, Suzanne Deschênes, Laura Feltri, Kurt Fischbeck, and David Gutmann, Albee Messing, Dan Mikol, David Paul, Ori Peles, Brian Popko, Jim Salzer, Diane Sherman, Larry Wrabetz, and Lei Zhou for their contributions to various aspects of the work reported here.

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