Immunocytochemical studies of quaking mice support a role for the myelin-associated glycoprotein in forming and maintaining the periaxonal space and periaxonal cytoplasmic collar of myelinating Schwann cells

The myelin-associated glycoprotein (MAG) is an integral membrane glycoprotein that is located in the periaxonal membrane of myelin-forming Schwann cells. On the basis of this localization, it has been hypothesized that MAG plays a structural role in (a) forming and maintaining contact between myelinating Schwann cells and the axon (the 12-14-nm periaxonal space) and (b) maintaining the Schwann cell periaxonal cytoplasmic collar of myelinated fibers. To test this hypothesis, we have determined the immunocytochemical localization of MAG in the L4 ventral roots from 11-mo-old quaking mice. These roots display various stages in the association of remyelinating Schwann cells with axons, and abnormalities including loss of the Schwann cell periaxonal cytoplasmic collar and dilation of the periaxonal space of myelinated fibers. Therefore, this mutant provides distinct opportunities to observe the relationships between MAG and (a) the formation of the periaxonal space during remyelination and (b) the maintenance of the periaxonal space and Schwann cell periaxonal cytoplasmic collar in myelinated fibers. During association of remyelinating Schwann cells and axons, MAG was detected in Schwann cell adaxonal membranes that apposed the axolemma by 12-14 nm. Schwann cell plasma membranes separated from the axolemma by distances greater than 12-14 nm did not react with MAG antiserum. MAG was present in adaxonal Schwann cell membranes that apposed the axolemma by 12-14 nm but only partially surrounded the axon and, therefore, may be actively involved in the ensheathment of axons by remyelinating Schwann cells. To test the dual role of MAG in maintaining the periaxonal space and Schwann cell periaxonal cytoplasmic collar of myelinated fibers, we determined the immunocytochemical localization of MAG in myelinated quaking fibers that displayed pathological alterations of these structures. Where Schwann cell periaxonal membranes were not stained by MAG antiserum, the cytoplasmic side of the periaxonal membrane was "fused" with the cytoplasmic side of the inner compact myelin lamella and formed a major dense line. This loss of MAG and the Schwann cell periaxonal cytoplasmic collar usually resulted in enlargement of the 12-14-nm periaxonal space and ruffling of the apposing axolemma. In myelinated fibers, there was a strict correlation between the presence of MAG in the Schwann cell periaxonal membrane and (a) maintenance of the 12-14-nm periaxonal space, and (b) presence of the Schwann cell periaxonal cytoplasmic collar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Presence of the myelin-associated glycoprotein correlates with alterations in the periodicity of peripheral myelin

The myelin-associated glycoprotein (MAG) is an integral membrane protein (congruent to 100,000 mol wt) which is a minor component of purified peripheral nervus system (PNS) myelin. In the present study, MAG was localized immunocytochemically in 1-micrometer thick Epon sections of 7-d and adult rat peripheral nerves, and its localization was compared to that of the major structural protein (Po) of PNS myelin. To determine more precisely the localization of MAG, immunostained areas in 1 micrometer sections were traced on electron micrographs of identical areas from adjacently cut thin sections.l MAG was localized in periaxonal membranes. Schmidt-Lantermann incisures, paranodal membranes, and the outer mesaxon of PNS myelin sheaths. Compact regions of PNS myelin did not react with MAG antiserum. The results demonstrate MAG's presence in "'semi-compact" Schwann cell or myelin membranes that have a gap of 12-14 nm between extracellular leaflets and a spacing of 5 nm or more between cytoplasmic leaflets. In compact regions of the myelin sheath which do not contain MAG, the cytoplasmic leaflets are "fused" and form the major dense line, whereas the extracellular leaflets are separated by a 2.0 nm gap appearing as paired minor dense lines. Thus, it is proposed that MAG plays a role in maintaining the periaxonal space, Schmidt-Lantermann incisures, paranodal myelin loops, and outer mesaxon by preventing "complete" compaction of Schwann cell and myelin membranes. The presence of MAG in these locations also suggests that MAG may serve a function in regulating myelination in the PNS.     

Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve

The cellular and subcellular localization of the neural cell adhesion molecules L1, N-CAM, and myelin-associated glycoprotein (MAG), their shared carbohydrate epitope L2/HNK-1, and the myelin basic protein (MBP) were studied by pre- and post-embedding immunoelectron microscopic labeling procedures in developing mouse sciatic nerve. L1 and N-CAM showed a similar staining pattern. Both were localized on small, non-myelinated, fasciculating axons and axons ensheathed by non- myelinating Schwann cells. Schwann cells were also positive for L1 and N-CAM in their non-myelinating state and at the onset of myelination, when the Schwann cell processes had turned approximately 1.5 loops. Thereafter, neither axon nor Schwann cell could be detected to express the L1 antigen, whereas N-CAM was found in the periaxonal area and, more weakly, in compact myelin of myelinated fibers. Compact myelin, Schmidt-Lanterman incisures, paranodal loops, and finger-like processes of Schwann cells at nodes of Ranvier were L1-negative. At the nodes of Ranvier, the axolemma was also always L1- and N-CAM-negative. The L2/HNK-1 carbohydrate epitope coincided in its cellular and subcellular localization most closely to that observed for L1. MAG appeared on Schwann cells at the time L1 expression ceased. MAG was then expressed at sites of axon-myelinating Schwann cell apposition and non-compacted loops of developing myelin. When compaction of myelin occurred, MAG remained present only at the axon-Schwann cell interface; Schmidt- Lanterman incisures, inner and outer mesaxons, and paranodal loops, but not at finger-like processes of Schwann cells at nodes of Ranvier or compacted myelin. All three adhesion molecules and the L2/HNK-1 epitope could be detected in a non-uniform staining pattern in basement membrane of Schwann cells and collagen fibrils of the endoneurium. MBP was detectable in compacted myelin, but not in Schmidt-Lanterman incisures, inner and outer mesaxon, paranodal loops, and finger-like processes at nodes of Ranvier, nor in the periaxonal regions of myelinated fibers, thus showing a complementary distribution to MAG. These studies show that axon-Schwann cell interactions are characterized by the sequential appearance of cell adhesion molecules and MBP apparently coordinated in time and space. From this sequence it may be deduced that L1 and N-CAM are involved in fasciculation, initial axon-Schwann cell interaction, and onset of myelination, with MAG to follow and MBP to appear only in compacted myelin. In contrast to L1, N- CAM may be further involved in the maintenance of compact myelin and axon-myelin apposition of larger diameter axons.     

A Hypothesis About the Relationship of Myelin-Associated Glycoprotein’s Function in Myelinated Axons to its Capacity to Inhibit Neurite Outgrowth

The myelin-associated glycoprotein (MAG) is selectively localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths suggesting that it functions in glia–axon interactions in the PNS and CNS, and this is supported by much experimental evidence. In addition, MAG is now well known as one of several white matter inhibitors of neurite outgrowth in vitro and axonal regeneration in vivo, and this latter area of research has provided a substantial amount of information about neuronal receptors or receptor complexes for MAG. This article makes the hypothesis that the capacity of MAG to inhibit outgrowth of immature developing or regenerating neurites is an aberration of its normal physiological function to promote the maturation, maintenance, and survival of myelinated axons. The overview summarizes the literature on the function of MAG in PNS and CNS myelin sheaths and its role as an inhibitor of neurite outgrowth to put this hypothesis into perspective. Additional research is needed to determine if receptors and signaling systems similar to those responsible for MAG inhibition of neurite outgrowth also promote the maturation, maintenance, and survival of myelinated axons as hypothesized here, or if substantially different MAG-mediated signaling mechanisms are operative at the glia–axon junction. Special issue article in honor of Dr. George DeVries.     

Myelin-Associated Glycoprotein: Electron Microscopic Immunocytochemical Localization in Compact Developing and Adult Central Nervous System Myelin

Light microscopic immunocytochemical studies have shown that myelin-associated glycoprotein (MAG) is localized in myelin of the developing CNS; but in the adult, MAG appears to be restricted to periaxonal regions of myelinated fibers. To extend these observations, we embedded optic nerves of 15-day-old rats, adult rats, and an adult human in epon after aldehyde and osmium tetroxide fixation. After 5% H2O2 pretreatment, thin sections were immunostained with 1:250-1:5,000 rabbit antiserum to rat CNS MAG according to the avidin-biotin-peroxidase complex (ABC) method. Dense deposits of reaction product covered compact myelin in both developing and adult optic nerves. When we used 1:500, 1:1,000, and 1:2,000 anti-MAG, less intense immunostaining of myelin was found. We also obtained the same localization in compact myelin with the peroxidase-antiperoxidase (PAP) method. With 1:250 anti-MAG, dense deposits of reaction product were not observed on axolemmal membranes or on oligodendroglial membranes located periaxonally and paranodally. In thin sections of adult human optic nerve, anti-MAG also stained compact myelin intensely. When thin sections of rat and human optic nerves were treated with preimmune or absorbed serum, no immunostaining was observed. Immunoblot tests showed that our MAG antisera did not react with any non-MAG myelin proteins. In contrast with earlier light microscopic data, this study shows that MAG localization does not change during CNS development; both developing and adult compact myelin sheaths contain MAG. As many biochemical studies also show that MAG is present in compact myelin, we suggest that this 100,000 dalton glycoprotein now be called myelin glycoprotein (MGP) instead of MAG.     

Biochemical demonstration of the myelin-associated glycoprotein in the peripheral nervous system

Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [3H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, W1, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [3H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [14C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and W1 fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole LIS-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.     

Biochemical Demonstration of the Myelin-Associated Glycoprotein in the Peripheral Nervous System

Abstract: Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [³H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, WI, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [³H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [¹⁴C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and Wl fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole US-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.     

Myelin-Associated Glycoprotein and Other Proteins in Trembler Mice

The myelin-associated glycoprotein (MAG) and other myelin proteins were quantitated in homogenates of whole sciatic nerve from adult and 20-day-old Trember mice. In the nerves of adult mice, the concentration of MAG was increased from 1.1 ng/micrograms of total protein in the controls to 1.4 ng/micrograms protein in the Tremblers. By contrast, the concentrations of P0 glycoprotein and myelin basic proteins were reduced to 27% and 20% of control levels, respectively. Immunoblots demonstrated that P2 was also greatly reduced in the Trembler nerves. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was 65% of the control level. Immunoblot analysis showed that MAG had a higher than normal apparent Mr in the sciatic nerves of the Trembler mice, but its apparent Mr was normal in the brains of these mutants. In 20-day-old Tremblers, the P0 and myelin basic protein were reduced slightly less to about 40% of the level in the nerves of age-matched controls. CNP and MAG levels were not significantly different from those in controls, and MAG exhibited a shift toward higher apparent Mr similar to that in the adults. The maintenance of high MAG levels despite the severe deficit of myelin, as reflected by the decrease of the major myelin proteins, is consistent with the immunocytochemical localization of MAG in periaxonal Schwann cell membranes, Schmidt-Lantermann incisures, lateral loops, and the outer mesaxon and its absence from compact myelin. The abnormal form of MAG in the peripheral nervous system (PNS) of the Trembler mice may contribute to the pathology in this mutant.     

Myelin-associated glycoprotein (MAG): past, present and beyond

The myelin-associated glycoprotein (MAG) is a type I transmembrane glycoprotein localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths where it functions in glia-axon interactions. It contains five immunoglobulin (Ig)-like domains and is in the sialic acid-binding subgroup of the Ig superfamily. It appears to function both as a ligand for an axonal receptor that is needed for the maintenance of myelinated axons and as a receptor for an axonal signal that promotes the differentiation, maintenance and survival of oligodendrocytes. Its function in the maintenance of myelinated axons may be related to its role as one of the white matter inhibitors of neurite outgrowth acting through a receptor complex involving the Nogo receptor and/or gangliosides containing 2,3-linked sialic acid. MAG is expressed as two developmentally regulated isoforms with different cytoplasmic domains that may activate different signal transduction pathways in myelin-forming cells. MAG contains a carbohydrate epitope shared with other glycoconjugates that is a target antigen in autoimmune peripheral neuropathy associated with IgM gammopathy and has been implicated in a dying back oligodendrogliopathy in multiple sclerosis.     

Schwann cell myelination requires timely and precise targeting of P(0) protein

This report investigated mechanisms responsible for failed Schwann cell myelination in mice that overexpress P(0) (P(0)(tg)), the major structural protein of PNS myelin. Quantitative ultrastructural immunocytochemistry established that P(0) protein was mistargeted to abaxonal, periaxonal, and mesaxon membranes in P(0)(tg) Schwann cells with arrested myelination. The extracellular leaflets of P(0)-containing mesaxon membranes were closely apposed with periodicities of compact myelin. The myelin-associated glycoprotein was appropriately sorted in the Golgi apparatus and targeted to periaxonal membranes. In adult mice, occasional Schwann cells myelinated axons possibly with the aid of endocytic removal of mistargeted P(0). These results indicate that P(0) gene multiplication causes P(0) mistargeting to mesaxon membranes, and through obligate P(0) homophilic adhesion, renders these dynamic membranes inert and halts myelination.     

Microtubule-associated protein 1B: a neuronal binding partner for myelin-associated glycoprotein.

Myelin-associated glycoprotein (MAG) is expressed in periaxonal membranes of myelinating glia where it is believed to function in glia-axon interactions by binding to a component of the axolemma. Experiments involving Western blot overlay and coimmunoprecipitation demonstrated that MAG binds to a phosphorylated neuronal isoform of microtubule-associated protein 1B (MAP1B) expressed in dorsal root ganglion neurons (DRGNs) and axolemma-enriched fractions from myelinated axons of brain, but not to the isoform of MAP1B expressed by glial cells. The expression of some MAP1B as a neuronal plasma membrane glycoprotein (Tanner, S.L., R. Franzen, H. Jaffe, and R.H. Quarles. 2000. J. Neurochem. 75:553-562.), further documented here by its immunostaining without cell permeabilization, is consistent with it being a binding partner for MAG on the axonal surface. Binding sites for a MAG-Fc chimera on DRGNs colocalized with MAP1B on neuronal varicosities, and MAG and MAP1B also colocalized in the periaxonal region of myelinated axons. In addition, expression of the phosphorylated isoform of MAP1B was increased significantly when DRGNs were cocultured with MAG-transfected COS cells. The interaction of MAG with MAP1B is relevant to the known role of MAG in affecting the cytoskeletal structure and stability of myelinated axons.     

Schwann cells infected with a recombinant retrovirus expressing myelin-associated glycoprotein antisense RNA do not form myelin.

To elucidate the role of myelin-associated glycoprotein (MAG) in the axon-Schwann cell interaction leading to myelination, neonatal rodent Schwann cells were infected in vitro with a recombinant retrovirus expressing MAG antisense RNA or MAG sense RNA. Stably infected Schwann cells and uninfected cells were then cocultured with purified sensory neurons under conditions permitting extensive myelination in vitro. A proportion of the Schwann cells infected with the MAG antisense virus did not myelinate axons and expressed lower levels of MAG than control myelinating Schwann cells, as measured by immunofluorescence. Electron microscopy revealed that the affected cells failed to segregate large axons and initiate a myelin spiral despite having formed a basal lamina, which normally triggers Schwann cell differentiation. Cells infected with the MAG sense virus formed normal compact myelin. These observations strongly suggest that MAG is the critical Schwann cell component induced by neuronal interaction that initiates peripheral myelination.     

Myelin-Associated Glycoprotein and Related Glycoconjugates in Developing Cat Peripheral Nerve: A Correlative Biochemical and Morphometric Study

The expression and accumulation of the myelin-associated glycoprotein (MAG) and other glycoconjugates have been studied during myelination in the developing cat peripheral nervous system. The glycoconjugates studied have in common a similar carbohydrate determinant which is bound by many antibodies, including the mouse monoclonal antibody HNK-1, and human IgM paraproteins from patients with neuropathy. In addition to MAG, the reactive glycoconjugates include a 60-kilodalton (kD) glycoprotein and a group of 20-26 kD glycoproteins, as well as a group of recently identified acidic glycolipids, the major one of which is sulfate-3-glucuronyl paragloboside (SGPG). The accumulation of these glycoproteins and glycolipids is compared with the established myelin proteins P0, P1, and P2 and with morphometric indices of myelin volume and axonal perimeter. The study demonstrates that MAG appears and accumulates very early during myelination, being present at 15% of the maximum level prior to the appearance of P0, and at 80% of the maximum level when P0 is at 30% of its maximum level. In the adult, the level of MAG falls to 60% maximum. The 60 kD and 20-26 kD glycoproteins accumulate at the same time as or later than P0, suggesting that they are either compact myelin proteins or in membranes closely associated with compact myelin. SGPG accumulates with P0 early in myelination, but falls to 60% of maximum in the adult. By comparing biochemical and morphometric data, we demonstrate that P0 and other compact myelin proteins accumulate synchronously with the increase in myelin area. MAG accumulation, however, is closely related to changes in axonal perimeter, consistent with a predominant localization of MAG to the periaxonal membranes in the peripheral nervous system.     

Distribution of the myelin-associated glycoprotein and P0 protein during myelin compaction in quaking mouse peripheral nerve

Ultrastructural studies have shown that during early stages of Schwann cell myelination mesaxon membranes are converted to compact myelin lamellae. The distinct changes that occur in the spacing of these Schwann cell membranes are likely to be mediated by the redistribution of (a) the myelin-associated glycoprotein, a major structural protein of mesaxon membranes; and (b) P0 protein, the major structural protein of compact myelin. To test this hypothesis, the immunocytochemical distribution of these two proteins was determined in serial 1-micron-thick Epon sections of ventral roots from quaking mice and compared to the ultrastructure of identical areas in an adjacent thin section. Ventral roots of this hypomyelinating mouse mutant were studied because many fibers have a deficit in converting mesaxon membranes to compact myelin. The results indicated that conversion of mesaxon membranes to compact myelin involves the insertion of P0 protein into and the removal of the myelin-associated glycoprotein from mesaxon membranes. The failure of some quaking mouse Schwann cells to form compact myelin appears to result from an inability to remove the myelin-associated glycoprotein from their mesaxon membranes.     

An endogenous lectin "CSL" interacts with glycoprotein components in peripheral nervous system myelin

An endogenous mannose binding lectin isolated from the rat cerebellum, CSL, was localized using immunocytochemical techniques in adult and in developing rat sciatic nerve. The lectin is present in Schwann cell cytoplasm and in compact myelin. It is present very early in Schwann cells and persists throughout postnatal sciatic nerve development. Endogenous ligands for the lectin were detected using iodinated-CSL binding to proteins blotted after polyacrylamide gel electrophoresis. Probably PO and MAG glycoproteins are specifically bound by CSL in contrast with numerous other Concanavalin A binding glycoproteins. A 31 kDa glycoprotein identified in purified preparations of axons of young rats also reacts with CSL. Based on the present developmental biochemical and immunochemical studies, an hypothetical scheme is proposed for the molecular basis of axon-Schwann cell interactions and of stabilization of compact myelin.     

Degradation products of myelin-oligodendrocyte-associated proteins in a light CNS subcellular fraction

The presence of degradation products of the myelin/oligodendrocyte glycoprotein (MOG) and a new myelin/oligodendrocyte associated protein, FD1, defined by a monoclonal antibody was established in a subfraction (the floating fraction, or FF) of adult rabbit CNS. The histochemical distribution of FD1 was determined by indirect immunofluorescense using conventional and confocal microscopy. FD1 was found to be present in oligodendrocytes, and at the outer rim of CNS myelin sheaths. Strong antibody reactivity was noted at nodes of Ranvier, as well as in regions with a high nodal density. No staining of compact myelin was seen. In the PNS, inner and outer cytoplasmic compartments of the Schwann cells as well as their cell bodies were stained, with no staining of compact myelin. The FF has previously been shown to be highly enriched in Marchi-positive bodies. These structures are situated paranodally in the CNS of myelinated nerve fibers, and their presence has been interpreted as reflections of myelin breakdown and turnover occurring in association with myelin sheath segments situated close to nodes at Ranvier in adult, normal vertebrate CNS. The present findings extend previous observations of partially degraded myelin-associated proteins in the FF, and give further results indicating that Marchi-positive bodies are aspects of intermediate stages in myelin catabolism.     

Axonal regulation of Schwann cell integrin expression suggests a role for alpha 6 beta 4 in myelination

Ensheathment and myelination of axons by Schwann cells in the peripheral nervous system requires contact with a basal lamina. The molecular mechanism(s) by which the basal lamina promotes myelination is not known but is likely to reflect the activity of integrins expressed by Schwann cells. To initiate studies on the role of integrins during myelination, we characterized the expression of two integrin subunits, beta 1 and beta 4, in an in vitro myelination system and compared their expression to that of the glial adhesion molecule, the myelin-associated glycoprotein (MAG). In the absence of neurons, Schwann cells express significant levels of beta 1 but virtually no beta 4 or MAG. When Schwann cells are cocultured with dorsal root ganglia neurons under conditions promoting myelination, expression of beta 4 and MAG increased dramatically in myelinating cells, whereas beta 1 levels remained essentially unchanged. (In general agreement with these findings, during peripheral nerve development in vivo, beta 4 levels also increase during the period of myelination in sharp contrast to beta 1 levels which show a striking decrease.) In cocultures of neurons and Schwann cells, beta 4 and MAG appear to colocalize in nascent myelin sheaths but have distinct distributions in mature sheaths, with beta 4 concentrated in the outer plasma membrane of the Schwann cell and MAG localized to the inner (periaxonal) membrane. Surprisingly, beta 4 is also present at high levels with MAG in Schmidt-Lanterman incisures. Immunoprecipitation studies demonstrated that primary Schwann cells express beta 1 in association with the alpha 1 and alpha 6 subunits, while myelinating Schwann cells express alpha 6 beta 4 and possibly alpha 1 beta 1. beta 4 is also downregulated during Wallerian degeneration in vitro, indicating that its expression requires continuous Schwann cell contact with the axon. These results indicate that axonal contact induces the expression of beta 4 during Schwann cell myelination and suggest that alpha 6 beta 4 is an important mediator of the interactions of myelinating Schwann cells with the basal lamina.