Compact Myelin Research Articles

Interactions between oligodendrocyte precursors control the onset of CNS myelination

The formation of CNS myelin is dependent on the differentiation of oligodendrocyte precursor cells (OPCs) and oligodendrocyte maturation. How the initiation of myelination is regulated is unclear, but it is likely to depend on the development of competence by oligodendrocytes and receptivity by target axons. Here we identify an additional level of control of oligodendrocyte maturation mediated by interactions between the different cellular components of the oligodendrocyte lineage. During development oligodendrocyte precursors mature through a series of stages defined by labeling with monoclonal antibodies A2B5 and O4. Newly differentiated oligodendrocytes begin to express galactocerebroside recognized by O1 antibodies and subsequently mature to myelin basic protein (MBP)-positive cells prior to formation of compact myelin. Using an in vitro brain slice culture system that supports robust myelination, the consequences of ablating cells at different stages of the oligodendrocyte lineage on myelination have been assayed. Elimination of all OPC lineage cells through A2B5+, O4+, and O1+ complement-mediated cell lysis resulted in a delay in development of MBP cells and myelination. Selective elimination of early OPCs (A2B5+) also unexpectedly resulted in delayed MBP expression compared to controls suggesting that early OPCs contribute to the timing of myelination onset. By contrast, elimination of differentiated (O1+) immature oligodendrocytes permanently inhibited the appearance of MBP+ cells suggesting that oligodendrocytes are critical to facilitate the maturation of OPCs. These data illuminate that the presence of intra-lineage feed-forward and feedback cues are important for timely myelination by oligodendrocytes.

Preserving and Restoring Optic Nerve Function

Preserving and Restoring Optic Nerve Function

Visualization of cytoplasmic diffusion within living myelin sheaths of CNS white matter axons using microinjection of the fluorescent dye Lucifer Yellow

The compactness of myelin allows for efficient insulation defining rapid propagation of action potentials, but also raises questions about how cytoplasmic access to its membranes is achieved, which is critical for physiological activity. Understanding the organization of cytoplasmic (‘water’) spaces of myelin is also important for diffusion MRI studies of CNS white matter. Using longitudinal slices of mature rat spinal cord, we monitored the diffusion of the water-soluble fluorescent dye Lucifer Yellow injected into individual oligodendrocytes or internodal myelin. We show that living myelin sheaths on CNS axons are fenestrated by a network of diffusionally interconnected cytoplasmic ‘pockets’ (1.9 ± 0.2 pockets per 10 μm sheath length, n = 58) that included Schmidt–Lanterman clefts (SLCs) and numerous smaller compartments. 3-D reconstructions of these cytoplasmic networks show that the outer cytoplasmic layer of CNS myelin is cylindrically ‘encuffing’, which differs from EM studies using fixed tissue. SLCs were found in different ‘open states’ and remained stable within a 1–2 hour observation period. Unlike the peripheral nervous system, where similarly small (< 500 Da) molecules diffuse along the whole myelin segment within a few minutes, in mature CNS this takes more than one hour. The slower cytoplasmic diffusion in CNS myelin possibly contributes to its known vulnerability to injury and limited capacity for repair. Our findings point to an elaborate cytoplasmic access to compact CNS myelin. These results could be of relevance to MRI studies of CNS white matter and to CNS repair/regeneration strategies.

Morphological Progression of Myelin Abnormalities in IgM-Monoclonal Gammopathy of Undetermined Significance Anti-Myelin-Associated Glycoprotein Neuropathy

To characterize the morphological progression of neuropathy associated with immunoglobulin M-monoclonal gammopathy of undetermined significance with anti-myelin-associated glycoprotein antibody, we assessed histopathologic features of sural nerve specimens from 15 patients, emphasizing widely spaced myelin (WSM), demyelination, and tomaculous changes. The frequency of WSM correlated with that of demyelination and tomaculous appearance in teased-fiber preparations. In longitudinal sections at nodes of Ranvier and paranodal regions, the spaces between terminal myelin loops, particularly those adjacent to the node of Ranvier, were widened, indicating an early change before demyelination, and there was concomitant swelling of terminal myelin loops. Some conspicuously swollen terminal myelin loops were detached from the paranodal axolemma, thereby widening the nodes of Ranvier. Tomacula coexisted frequently with redundant myelin loops and WSM, particularly in the outermost layer of myelin sheaths, suggesting that loosening of the outer layers contributes to their formation. By immunofluorescence microscopy, immunoglobulin M and myelin-associated glycoprotein were colocalized in paranodal regions and Schmidt-Lanterman incisures. Confocal analysis revealed colocalization of immunoglobulin M and complement product C3d corresponding to the area of WSM. Thus, morphological changes in terminal myelin loops, formation of WSM at paranodes, and subsequent dissociation from paranodal axolemma (which may be associated with activation of the complement pathway) likely contribute to demyelination in this condition. Loosening of compact myelin seems to contribute to tomacula formation.

Oligodendrocyte Myelin Glycoprotein Does Not Influence Node of Ranvier Structure or Assembly

Oligodendrocyte myelin glycoprotein (OMgp) is expressed by both neurons and oligodendrocytes in the CNS. It has been implicated in growth cone collapse and neurite outgrowth inhibition by signaling through the Nogo receptor and paired Ig-like receptor B (PirB). OMgp was also reported to be an extracellular matrix (ECM) protein surrounding CNS nodes of Ranvier and proposed to function as (1) an inhibitor of nodal collateral sprouting and (2) an important contributor to proper nodal and paranodal architecture. However, we show here that the anti-OMgp antiserum used in previous studies to define the functions of OMgp at nodes is not specific. Among all reported nodal ECM components, the antiserum exhibited strong cross-reactivity against versican V2 isoform, a chondroitin sulfate proteoglycan. Furthermore, the OMgp antiserum labeled OMgp-null nodes, but not nodes from versican V2-deficient mice, and preadsorption of the OMgp antiserum with recombinant versican V2 blocked nodal labeling. Analysis of CNS nodes in OMgp-null mice failed to reveal any nodal or paranodal defects, or increased nodal collateral sprouting, indicating that OMgp does not participate in CNS node of Ranvier assembly or maintenance. We successfully identified a highly specific anti-OMgp antibody and observed OMgp staining in white matter only after initiation of myelination. OMgp immunoreactivity decorated the surface of mature myelinated axons, but was excluded from compact myelin and nodes. Together, our results strongly argue against the nodal localization of OMgp and its proposed functions at nodes, and reveal OMgp's authentic localization relative to nodes and myelin.

Sp1 phosphorylation is involved in myelin basic protein gene transcription

Myelin basic protein (MBP), which helps form compact myelin sheets, is a major protein expressed during oligodendrocyte (OL) differentiation. Myelin basic protein expression is regulated mainly at the transcriptional level. Previous studies showed that the transcription factor Sp1 can activate the MBP promoter. Data from the laboratory also indicate that Sp1 is expressed highly in both growing and differentiated cells. Because this is true, we wanted to understand how Sp1 activity is regulated such that it increases MBP gene transcription only in differentiating cells. Phosphorylation is one major way to regulate transcription factor activity. Our results show that there is more Sp1 binding to the MBP promoter in differentiating OLs. Sp1 is also more phosphorylated in differentiating OLs than in precursor cells. Using inhibitors of different pathways, we found that the protein kinase C (PKC) modulator phorbol 12-myristate 13-acetate (PMA) can increase Sp1 phosphorylation when the cells are treated for 1 hr and can decrease Sp1 phosphorylation with long treatment (12 hr). The increased phosphorylation of Sp1 induced by PMA in short treatments could be abolished by the extracellular signal-regulated kinases (ERK) pathway inhibitor PD98059. This indicates that PKC phosphorylates Sp1 through the ERK pathway. Mutation of Sp1 threonines 453 and 739, which are phosphorylated by ERK, decreased MBP transcriptional activity. Furthermore, we found that PKC regulates Sp1 phosphorylation only in differentiating OLs. In conclusion, our results indicate that, in OLs, Sp1 phosphorylation can be regulated by PKC-ERK pathways. This phosphorylation is important for MBP transcription and oligodendrocyte differentiation.

A proteome map of axoglial specializations isolated and purified from human central nervous system

Compact myelin, the paranode, and the juxtaparanode are discrete domains that are formed on myelinated axons. In humans, neurological disorders associated with loss of myelin, including Multiple Sclerosis, often also result in disassembly of the node of Ranvier. Despite the importance of these domains in the proper functioning of the CNS, their molecular composition and assembly mechanism remains largely unknown. We therefore performed a large-scale proteomics MudPIT screen for the identification of proteins in human myelin and axogliasomal fractions. We identified over 1,000 proteins in these fractions. Since even minor perturbations in neuron-glial interactions can uncouple the glial support of axons, the proteome map presented here can be used as a reference library for "myelin health" and disease states, including white matter disorders such as leukodystrophies and multiple sclerosis.

CNS-Resident Glial Progenitor/Stem Cells Produce Schwann Cells as well as Oligodendrocytes during Repair of CNS Demyelination

After central nervous system (CNS) demyelination-such as occurs during multiple sclerosis-there is often spontaneous regeneration of myelin sheaths, mainly by oligodendrocytes but also by Schwann cells. The origins of the remyelinating cells have not previously been established. We have used Cre-lox fate mapping in transgenic mice to show that PDGFRA/NG2-expressing glia, a distributed population of stem/progenitor cells in the adult CNS, produce the remyelinating oligodendrocytes and almost all of the Schwann cells in chemically induced demyelinated lesions. In contrast, the great majority of reactive astrocytes in the vicinity of the lesions are derived from preexisting FGFR3-expressing cells, likely to be astrocytes. These data resolve a long-running debate about the origins of the main players in CNS remyelination and reveal a surprising capacity of CNS precursors to generate Schwann cells, which normally develop from the embryonic neural crest and are restricted to the peripheral nervous system.

Pals1 Is a Major Regulator of the Epithelial-Like Polarization and the Extension of the Myelin Sheath in Peripheral Nerves

Diameter, organization, and length of the myelin sheath are important determinants of the nerve conduction velocity, but the basic molecular mechanisms that control these parameters are only partially understood. Cell polarization is an essential feature of differentiated cells, and relies on a set of evolutionarily conserved cell polarity proteins. We investigated the molecular nature of myelin sheath polarization in connection with the functional role of the cell polarity protein pals1 (Protein Associated with Lin Seven 1) during peripheral nerve myelin sheath extension. We found that, in regard to epithelial polarity, the Schwann cell outer abaxonal domain represents a basolateral-like domain, while the inner adaxonal domain and Schmidt-Lanterman incisures form an apical-like domain. Silencing of pals1 in myelinating Schwann cells in vivo resulted in a severe reduction of myelin sheath thickness and length. Except for some infoldings, the structure of compact myelin was not fundamentally affected, but cells produced less myelin turns. In addition, pals1 is required for the normal polarized localization of the vesicular markers sec8 and syntaxin4, and for the distribution of E-cadherin and myelin proteins PMP22 and MAG at the plasma membrane. Our data show that the polarity protein pals1 plays an essential role in the radial and longitudinal extension of the myelin sheath, likely involving a functional role in membrane protein trafficking. We conclude that regulation of epithelial-like polarization is a critical determinant of myelin sheath structure and function.

Interleukin-6 upregulates the expression of PMP22 in cultured rat Schwann cells via a JAK2-dependent pathway

The interleukin-6 (IL-6) family of cytokines is thought to be involved in the development and regeneration of peripheral nerves; however, their roles in myelination remain unclear. In this study, we examined the effects of IL-6 on the expression of genes for compact myelin proteins using Schwann cell cultures prepared by multiple explantation of adult rat sciatic nerves. In semi-quantitative reverse transcription-polymerase chain reaction analysis, stimulation of Schwann cells with IL-6 significantly increased the mRNA level of peripheral myelin protein 22 (PMP22), but not those of myelin protein zero and myelin basic protein. The increase in PMP22 mRNA was markedly suppressed by AG490, a Janus kinase 2 (JAK2) inhibitor, but not significantly by PD098059, a mitogen-activated protein kinase inhibitor. Immunocytochemical staining revealed that IL-6 enhanced immunoreactivities for the phosphorylated forms of both JAK2 and signal transducer and activator of transcription 3 (STAT3), as well as that for PMP22. These results indicate that IL-6 can enhance PMP22 production in Schwann cells via a JAK2-dependent pathway by probably activating STAT3 and thus may contribute to myelination.

Dicer ablation in oligodendrocytes provokes neuronal impairment in mice

MicroRNAs (miRNAs) regulate gene expression and have many roles in the brain, but a role in oligodendrocyte (OL) function has not been demonstrated. A Dicer floxed conditional allele was crossed with the proteolipid protein promoter-driven inducible Cre allele to generate inducible, OL-specific Dicer-floxed mice. OL-specific Dicer mutants show demyelination, oxidative damage, inflammatory astrocytosis and microgliosis in the brain, and eventually neuronal degeneration and shorter lifespan. miR-219 and its target ELOVL7 (elongation of very long chain fatty acids protein 7) were identified as the main molecular components that are involved in the development of the phenotype in these mice. Overexpressing ELOVL7 results in lipid accumulation, which is suppressed by miR-219 co-overexpression. In Dicer mutant brain, excess lipids accumulate in myelin-rich brain regions, and the peroxisomal beta-oxidation activity is dramatically reduced. Postnatal Dicer ablation in mature OLs results in inflammatory neuronal degeneration through increased demyelination, lipid accumulation, and peroxisomal and oxidative damage, and therefore indicates that miRNAs play an essential role in the maintenance of lipids and redox homeostasis in mature OLs that are necessary for supporting axonal integrity as well as the formation of compact myelin.

Dysmyelinated axons in shiverer mice are highly vulnerable to α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated toxicity

Glutamate excitotoxicity plays a role in white matter injury in many neurological diseases. Oligodendrocytes in particular are highly vulnerable to excitotoxicity, mediated through activation of AMPA/kainate receptors. Myelin may also be injured independently via NMDA ( N-methyl- d-aspartic acid) receptors located on peripheral oligodendroglial processes. Central axons are susceptible to glutamate receptor activation in vivo, but it is unclear whether this is mediated directly by activation of receptors expressed on axons, or indirectly through glutamate toxicity of myelin or neighboring glial cells. We examined axonal vulnerability in mice deficient in myelin basic protein (shiverer), also expressing yellow fluorescent protein (YFP) in a subset of axons. YFP fluorescence, EM, and mouse behavior were assessed 24 h after microstereotactical injections of S-AMPA or NMDA into lumbar dorsal columns. S-AMPA injection led to impaired rotarod performance and widespread axonal degeneration and was more pronounced in shiverer mice than controls. In contrast, NMDA injection did not cause axonal injury or behavioral changes in either group. These results indicate that spinal cord axons in vivo are vulnerable to toxicity mediated by AMPA but not NMDA receptors. The presence of compact myelin is not required for excitotoxic axon damage, and its absence may increase vulnerability. Further understanding of AMPA receptor-mediated axonal toxicity may provide new targets for neuroprotective therapy in WM diseases.

Glutamate receptor‐mediated ischemic injury of premyelinated central axons

Ischemic injury of axons is a feature of periventricular leukomalacia, a pathological correlate of cerebral palsy. Recent evidence suggests that axons are damaged before they receive the first layer of compact myelin. Here we examine the cellular mechanisms underlying ischemic-type injury of premyelinated central axons. Two-thirds of axons in the postnatal day 10 (P10) rat optic nerve are small premyelinated axons (<0.4microm in diameter), and one-third have undergone radial expansion in preparation for glial contact and the onset of myelination. Compound action potential recording and quantitative electron microscopy were used to examine the effect of modeled ischemia (oxygen-glucose deprivation) upon these two axon populations. Glutamate receptor (GluR) expression was investigated using polymerase chain reaction (PCR) and immunostaining approaches at the confocal light and ultrastructural levels. Oxygen-glucose deprivation produced action potential failure and focal breakdown of the axolemma of small premyelinated axons at sites of contact with oligodendrocyte processes, which were also disrupted. The resulting axon loss was Ca(2+)-dependent, Na(+)- and Cl(-)-independent, and required activation of N-methyl-D-aspartic acid (NMDA) and non-NMDA GluRs. NMDA receptor expression was localized to oligodendrocyte processes at sites of contact with premyelinated axons, in addition to expression within compact myelin. No periaxonal NMDA receptor expression was observed on oligodendrocyte processes ensheathing large premyelinated axons and no protective effect of GluR block was observed in these axons. NMDA receptor-mediated injury to oligodendrocyte processes navigating along small premyelinated axons precedes damage to the underlying axon, a phenomena that is lost following radial expansion and subsequent oligodendrocyte ensheathment.

Adult Ceramide Synthase 2 (CERS2)-deficient Mice Exhibit Myelin Sheath Defects, Cerebellar Degeneration, and Hepatocarcinomas

(Dihydro)ceramide synthase 2 (cers2, formerly called lass2) is the most abundantly expressed member of the ceramide synthase gene family, which includes six isoforms in mice. CERS2 activity has been reported to be specific toward very long fatty acid residues (C22-C24). In order to study the biological role of CERS2, we have inactivated its coding region in transgenic mice using gene-trapped embryonic stem cells that express lacZ reporter DNA under control of the cers2 promoter. The resulting mice lack ceramide synthase activity toward C24:1 in the brain as well as the liver and show only very low activity toward C18:0-C22:0 in liver and reduced activity toward C22:0 residues in the brain. In addition, these mice exhibit strongly reduced levels of ceramide species with very long fatty acid residues (>or=C22) in the liver, kidney, and brain. From early adulthood on, myelin stainability is progressively lost, biochemically accompanied by about 50% loss of compacted myelin and 80% loss of myelin basic protein. Starting around 9 months, both the medullary tree and the internal granular layer of the cerebellum show significant signs of degeneration associated with the formation of microcysts. Predominantly in the peripheral nervous system, we observed vesiculation and multifocal detachment of the inner myelin lamellae in about 20% of the axons. Beyond 7 months, the CERS2-deficient mice developed hepatocarcinomas with local destruction of tissue architecture and discrete gaps in renal parenchyma. Our results indicate that CERS2 activity supports different biological functions: maintenance of myelin, stabilization of the cerebellar as well as renal histological architecture, and protection against hepatocarcinomas.

Unusual Charcot–Marie–Tooth phenotype due to a mutation within the intracellular domain of myelin protein zero

Myelin protein zero (MPZ/P0) constitutes a major component of compact peripheral myelin. We report a family with a missense mutation, c.700G>T p.Asp234Tyr (deviant nomenclature: c.670G>T, p.Asp224Tyr), within the intracellular domain of myelin protein zero, who has distal sensorimotor symptoms, cramps, restless legs syndrome, neuropathic pain, and carpal tunnel syndrome. The index patient responded to intravenous immunoglobulin and immunosuppression, so there may be a possible secondary autoimmune process, probably triggered by altered antigen presentation due to mutated MPZ protein.

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP-CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein-protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP-CaM interaction, including a 3D model of the complex between full-length proteins.

SH3TC2/KIAA1985 protein is required for proper myelination and the integrity of the node of Ranvier in the peripheral nervous system

Charcot-Marie-Tooth disease type 4C (CMT4C) is an early-onset, autosomal recessive form of demyelinating neuropathy. The clinical manifestations include progressive scoliosis, delayed age of walking, muscular atrophy, distal weakness, and reduced nerve conduction velocity. The gene mutated in CMT4C disease, SH3TC2/KIAA1985, was recently identified; however, the function of the protein it encodes remains unknown. We have generated knockout mice where the first exon of the Sh3tc2 gene is replaced with an enhanced GFP cassette. The Sh3tc2(DeltaEx1/DeltaEx1) knockout animals develop progressive peripheral neuropathy manifested by decreased motor and sensory nerve conduction velocity and hypomyelination. We show that Sh3tc2 is specifically expressed in Schwann cells and localizes to the plasma membrane and to the perinuclear endocytic recycling compartment, concordant with its possible function in myelination and/or in regions of axoglial interactions. Concomitantly, transcriptional profiling performed on the endoneurial compartment of peripheral nerves isolated from control and Sh3tc2(DeltaEx1/DeltaEx1) animals uncovered changes in transcripts encoding genes involved in myelination and cell adhesion. Finally, detailed analyses of the structures composed of compact and noncompact myelin in the peripheral nerve of Sh3tc2(DeltaEx1/DeltaEx1) animals revealed abnormal organization of the node of Ranvier, a phenotype that we confirmed in CMT4C patient nerve biopsies. The generated Sh3tc2 knockout mice thus present a reliable model of CMT4C neuropathy that was instrumental in establishing a role for Sh3tc2 in myelination and in the integrity of the node of Ranvier, a morphological phenotype that can be used as an additional CMT4C diagnostic marker.

Sustained Axon–Glial Signaling Induces Schwann Cell Hyperproliferation, Remak Bundle Myelination, and Tumorigenesis

Type III neuregulins exposed on axon surfaces control myelination of the peripheral nervous system. It has been shown, for example, that threshold levels of type III beta1a neuregulin dictate not only the myelination fate of axons but also myelin thickness. Here we show that another neuregulin isoform, type III-beta3, plays a distinct role in myelination. Neuronal overexpression of this isoform in mice stimulates Schwann cell proliferation and dramatically enlarges peripheral nerves and ganglia-which come to resemble plexiform neurofibromas-but have no effect on myelin thickness. The nerves display other neurofibroma-like properties, such as abundant collagen fibrils and abundant dissociated Schwann cells that in some cases produce big tumors. Moreover, the organization of Remak bundles is dramatically altered; the small-caliber axons of each bundle are no longer segregated from one another within the cytoplasm of a nonmyelinating Schwann cell but instead are close packed and the whole bundle wrapped as a single unit, frequently by a compact myelin sheath. Because Schwann cell hyperproliferation and Remak bundle degeneration are early hallmarks of type I neurofibromatosis, we suggest that sustained activation of the neuregulin pathway in Remak bundles can contribute to neurofibroma development.

Structural Polymorphism and Multifunctionality of Myelin Basic Protein

Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly wrapped multilamellar structure around neurons enabling rapid and efficient signal propagation. The gene of oligodendrocyte lineage (golli) gives rise to a variety of developmentally regulated splice isoforms of myelin basic protein (MBP), denoted golli for early forms and classic for later ones. In mature myelin, the predominant splice isoform of classic MBP is 18.5 kDa; its central role is to maintain the structural integrity of the myelin sheath, by holding together the apposing cytoplasmic leaflets of the oligodendrocyte membrane in a tight, spiral, multilamellar arrangement. This protein's extreme physicochemical properties, net charge of +19 at neutral pH, low proportion of hydrophobic residues, alternating regions of predicted intrinsic disorder and order, induced folding upon association with membranes and other proteins, and diversification via combinatorial post-translational modifications, define not only its role as a molecular Velcro in compact myelin, but as a multifunctional hub that may also bind to a number of other proteins and small molecule ligands in myelinating oligodendrocytes. In particular, MBP may link the underlying cytoskeleton and proteins containing SH3 domains to the membrane, allowing it to transduce transmembrane signals to the cytosol. These associations are facilitated by MBP being an intrinsically disordered protein, creating a large effective protein surface, and by the formation of transient and/or induced ordered secondary structure elements for molecular recognition. These processes can be modulated by a molecular barcode of numerous post-translational modifications and interactions with proteins such as calmodulin. In the human demyelinating disease multiple sclerosis, an aberrant pattern of modifications may contribute to demyelination and confound inherent attempts at repair. The conformational dynamics of the various isoforms and modified variants of MBP and their interactions with other proteins potentially allow them to participate in events coupling extracellular signals to cytoskeletal organization during myelination or remyelination. Various biophysical and cell biological approaches are beginning to elucidate these properties of MBP and are leading to a new understanding of the role of this protein as a linker and/or hub in structural and signaling networks in oligodendrocytes and myelin.

P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca(2+)](i)) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca(2+)](i) is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca(2+) into CMT1A SC. Correction of the altered [Ca(2+)](i) in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca(2+)](i) and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.