Myelin is a multispiraled extension of glial membrane that surrounds axons. How glia extend a surface many-fold larger than their body is poorly understood. Schwann cells are peripheral glia and insert radial cytoplasmic extensions into bundles of axons to sort, ensheath, and myelinate them. Laminins and β1 integrins are required for axonal sorting, but the downstream signals are largely unknown. We show that Schwann cells devoid of β1 integrin migrate to and elongate on axons but cannot extend radial lamellae of cytoplasm, similar to cells with low Rac1 activation. Accordingly, active Rac1 is decreased in β1 integrin–null nerves, inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells of transgenic mice delays axonal sorting and impairs myelination. Finally, expressing active Rac1 in β1 integrin–null nerves improves sorting. Thus, increased activation of Rac1 by β1 integrins allows Schwann cells to switch from migration/elongation to the extension of radial membranes required for axonal sorting and myelination.

Nodes of Ranvier are specialized axonal domains, at which voltage-gated sodium channels cluster. How axons cluster molecules in discrete domains is mostly unknown. Both axons and glia probably provide constraining mechanisms that contribute to domain formation. Proper sodium channel clustering in peripheral nerves depends on contact from Schwann cell microvilli, where at least one molecule, gliomedin, binds the sodium channel complex and induces its clustering. Furthermore, mice lacking Schwann cell dystroglycan have aberrant microvilli and poorly clustered sodium channels. Dystroglycan could interact at the basal lamina or at the axon-glial surface. Because dystroglycan is a laminin receptor, and laminin 2 mutations [merosin-deficient congenital muscular dystrophy (MDC1A)] cause reduced nerve conduction velocity, we asked whether laminins are involved. Here, we show that the composition of both laminins and the dystroglycan complex at nodes differs from that of internodes. Mice defective in laminin 2 have poorly formed microvilli and abnormal sodium clusters. These abnormalities are similar, albeit less severe, than those of mice lacking dystroglycan. However, mice lacking all Schwann cell laminins show severe nodal abnormalities, suggesting that other laminins compensate for the lack of laminin 2. Thus, although laminins are located at a distance from the axoglial junction, they are required for proper clustering of sodium channels. Laminins, through their specific nodal receptors and cytoskeletal linkages, may participate in the formation of mechanisms that constrain clusters at nodes. Finally, abnormal sodium channel clusters are present in a patient with MDC1A, providing a molecular basis for the reduced nerve conduction velocity in this disorder.

Schwann cells integrate signals deriving from the axon and the basal lamina to myelinate peripheral nerves. Integrin α6β4 is a laminin receptor synthesized by Schwann cells and displayed apposed to the basal lamina. α6β4 integrin expression in Schwann cells is induced by axons at the onset of myelination, and rise in adulthood. The β4 chain has a uniquely long cytoplasmic domain that interacts with intermediate filaments such as dystonin, important in peripheral myelination. Furthermore, α6β4 integrin binds peripheral myelin protein 22, whose alteration causes the most common demyelinating hereditary neuropathy. All these data suggest a role for α6β4 integrin in peripheral nerve myelination. Here we show that ablating α6β4 integrin specifically in Schwann cells of transgenic mice does not affect peripheral nerve development, myelin formation, maturation or regeneration. However, consistent with maximal expression in adult nerves, α6β4 integrin-null myelin is more prone to abnormal folding with aging. When the laminin receptor dystroglycan is also ablated, major folding abnormalities occur, associated with acute demyelination in some peripheral nervous system districts. These data indicate that, similar to its role in skin, α6β4 integrin confers stability to myelin in peripheral nerves.

SUMMARYRadial sorting allows the segregation of axons by a single Schwann cell (SC) and is a prerequisite for myelination during peripheral nerve development. Radial sorting is impaired in models of human diseases, congenital muscular dystrophy (MDC) 1A, MDC1D and Fukuyama, owing to loss-of-function mutations in the genes coding for laminin 2, Large or fukutin glycosyltransferases, respectively. It is not clear which receptor(s) are activated by laminin 211, or glycosylated by Large and fukutin during sorting. Candidates are 1 integrins, because their absence phenocopies laminin and glycosyltransferase deficiency, but the topography of the phenotypes is different and 1 integrins are not substrates for Large and fukutin. By contrast, deletion of the Large and fukutin substrate dystroglycan does not result in radial sorting defects. Here, we show that absence of dystroglycan in a specific genetic background causes sorting defects with topography identical to that of laminin 211 mutants, and recapitulating the MDC1A, MDC1D and Fukuyama phenotypes. By epistasis studies in mice lacking one or both receptors in SCs, we show that only absence of 1 integrins impairs proliferation and survival, and arrests radial sorting at early stages, that 1 integrins and dystroglycan activate different pathways, and that the absence of both molecules is synergistic. Thus, the function of dystroglycan and 1 integrins is not redundant, but is sequential. These data identify dystroglycan as a functional laminin 211 receptor during axonal sorting and the key substrate relevant to the pathogenesis of glycosyltransferase congenital muscular dystrophies.

Myelinating glial cells exhibit a spectacular cytoarchitecture, because they polarize on multiple axes and domains. How this occurs is largely unknown. The dystroglycan-dystrophin complex is required for the function of myelin-forming Schwann cells. Similar to other tissues, the dystroglycan complex in Schwann cells localizes with different dystrophin-family members in specific domains, thus promoting polarization. We show here that cleavage of dystroglycan by matrix-metalloproteinases 2 and 9, an event that is considered pathological in most tissues, is finely and dynamically regulated in normal nerves and modulates dystroglycan complex composition and the size of Schwann cell compartments. In contrast, in nerves of Dy2j/2j mice, a model of laminin 211 deficiency, metalloproteinases 2 and 9 are increased causing excessive dystroglycan cleavage and abnormal compartments. Pharmacological inhibition of cleavage rescues the cytoplasmic defects of Dy2j/2j Schwann cells. Thus, regulated cleavage may be a general mechanism to regulate protein complex composition in physiological conditions, while unregulated processing is pathogenic and a target for treatment in disease.