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OBJECTIVES To describe the neuropathological features of clinical syndromes associated with tomacula or focal myelin swellings in sural nerve biospies and to discuss possible common aetiopathological pathways leading to their formation in this group of neuropathies. METHODS Fifty two patients with sural nerve biopsies reported to show tomacula or focal myelin swellings were reviewed, light and electron microscopy were performed, and tomacula were analysed on teased fibre studies. Molecular genetic studies were performed on those patients who were available for genetic testing. RESULTS Thirty seven patients were diagnosed with hereditary neuropathy with liability to pressure palsies (HNPP), four with hereditary motor and sensory neuropathy type I (HMSN I) or Charcot-Marie-Tooth disease type 1 (CMT1), four with HMSN with myelin outfolding (CMT4B), three with IgM paraproteinemic neuropathy, three with chronic inflammatory demyelinating polyneuropathy (CIDP), and one with HMSN III (CMT3). CONCLUSIONS Most of these syndromes were shown to be related to genetic or immunological defects of myelin components such as peripheral myelin protein 22 (PMP22), myelin protein zero (P0), or myelin associated glycoprotein (MAG). These proteins share the HNK-1 epitope which has been implicated in cell adhesion processes. Impaired myelin maintenance may therefore contribute to the formation of tomacula and subsequent demyelination.

The adhesive cell surface molecule P0is the most abundant glycoprotein in peripheral nerve myelin and fulfills pivotal functions during myelin formation and maintenance. Mutations in the corresponding gene cause hereditary demyelinating neuropathies. In mice heterozygously deficient in P0(P0+/−mice), an established animal model for a subtype of hereditary neuropathies, T-lymphocytes are present in the demyelinating nerves. To monitor the possible involvement of the immune system in myelin pathology, we cross-bred P0+/−mice with null mutants for the recombination activating gene 1 (RAG-1) or with mice deficient in the T-cell receptor α-subunit. We found that in P0+/−mice myelin degeneration and impairment of nerve conduction properties is less severe when the immune system is deficient. Moreover, isolated T-lymphocytes from P0+/−mice show enhanced reactivity to myelin components of the peripheral nerve, such as P0, P2, and myelin basic protein. We hypothesize that autoreactive immune cells can significantly foster the demyelinating phenotype of mice with a primarily genetically based peripheral neuropathy.

Mice lacking the major Schwann cell myelin component P0 show a severe dysmyelination with pathological features reminiscent of the Déjérine-Sottas syndrome in humans. Previous morphological and electrophysiological studies on these mice did not only demonstrate a compromised myelination and myelin maintenance, but were suggestive of an impairment of axons as well. Here, we studied the axonal pathology in P0-deficient mice by quantitative electron microscopy. In addition, we investigated epidermal receptor end organs by immunocytochemistry and muscle pathology by histochemistry.In proximal sections of facial and femoral nerves, axon calibers were significantly reduced, whereas the number of myelin-competent axons was not diminished in 5- and 17-month-old P0-deficient mice. However, in distal branches of the femoral and sciatic nerve (digital nerves innervating the skin of the first toe) the numbers of myelin-competent axons were reduced by 70% in 6-month-old P0-deficient mice. Immunolabeling of foot pads revealed a corresponding loss of Merkel cells by 75%, suggesting that survival of these cells is dependent on the presence or maintenance of their innervating myelinated axons. In addition, quadriceps and gastrocnemius muscles showed pathological features indicative of denervation and axonal sprouting. These findings demonstrate that loss of an important myelin component can initiate degenerative mechanisms not only in the Schwann cell but also in the distal portions of myelinated axons, leading to the degeneration of specialized receptor end organs and impairment of muscle innervation.

By screening an olfactory bulb cDNA library using dopamine receptor probes, we isolated the cDNA coding for the rat counterpart of an orphan receptor known as Edg-2, homologous to G protein-coupled receptors. In situ hybridization analysis showed that Edg-2 mRNA expression is restricted to myelinated structures, e.g. corpus callosum or peripheral nerves. A weaker expression in various peripheral organs was also detected in newborns. A 3.8-kb transcript was found at high levels in highly myelinated brain structures and sciatic nerve, and, at lower levels, in poorly myelinated peripheral organs, consistent with its occurrence in Schwann cells in the peripheral nervous system. One hundred percent of Edg-2 mRNA-containing cells in the brain also expressed mRNA encoding myelin-basic-protein, a marker of oligodendrocytes. This restricted olygodendrocytes localization was confirmed by the absence of cellular colocalization of Edg-2 and glial fibrillary acidic protein, an astrocytic marker. During prenatal development, Edg-2 mRNA expression was high in the cortical neuroepithelium and meningeal layer at E16, extended later to other neuroepithelia, and disappeared shortly after birth. During brain postnatal development, Edg-2 mRNA expression in myelinated structures followed a caudo-rostral gradient, similar to that of myelination. Thus, Edg-2 is the first G protein-coupled receptor found to be selectively expressed in myelin-forming cells in the nervous system and its temporal expression pattern is consistent with a dual role (i) in neurogenesis, during embryonic development, and (ii) in myelination and myelin maintenance, during postnatal life.

Several glia-associated cell surface molecules have been implicated in myelin formation in the central (CNS) and peripheral nervous system (PNS). Recent studies in mice deficient for such molecules have been instrumental in understanding the role of these molecules during the formation of the spiraling loops around the axon, compaction of the spiraling loops, determination of the thickness of the myelin sheath, and myelin maintenance. In the PNS, the major peripheral myelin protein P0 and the peripheral myelin protein (PMP) 22 are involved in spiral formation as reflected by retarded myelin formation in mice deficient for the respective molecules. An involvement of the myelin-associated glycoprotein (MAG) in this process is detectable only in mice deficient in both P0 and MAG, suggesting that P0 can replace MAG during the formation of the spiraling loops. Myelin compaction is mediated by both P0 and the intracellular myelin component myelin basic protein (MBP). The determination of the correct myelin thickness is mediated by P0, MBP, and PMP22, with P0 and MBP fostering and PMP22 attenuating myelin growth. For the maintenance of the association of the Schwann cell and myelin with its ensheathed axon, the myelin components P0, PMP22, MAG, and Connexin 32 are crucial. In the CNS, recognition of oligodendrocytes and axons and the formation of the spiraling loops is mediated by MAG. MAG is additionally responsible for the maintenance of myelin. Myelin compaction is mediated by MBP and by PLP, which fulfills some analogous functions in the CNS as P0 in the PNS. These studies reveal that myelin-related cell surface molecules can play distinct but also partially overlapping roles during the formation and maintenance of myelin. GLIA 19:298–310, 1997. © 1997 Wiley-Liss, Inc.

Myelination by oligodendrocytes is a highly specialized process that relies on intimate interactions between the axon and the oligodendrocytes. Astrocytes have an important part in facilitating myelination in the CNS, however, comparatively less is known about how they affect myelination. This review therefore summarizes the literature and explores lingering questions surrounding differences between white matter and gray matter astrocytes, how astrocytes support myelination, how their dysfunction in pathological states contributes to myelin pathologies and how astrocytes may facilitate remyelination. We discuss how astrocytes in the white matter are specialized to promote myelination and myelin maintenance by clearance of extracellular ions and neurotransmitters and by secretion of pro-myelinating factors. Additionally, astrocyte–oligodendrocyte coupling via gap junctions is crucial for both myelin formation and maintenance, due to K + buffering and possibly metabolic support for oligodendrocytes via the panglial syncytium. Dysfunctional astrocytes aberrantly affect oligodendrocytes, as exemplified by a number of leukodystrophies in which astrocytic pathology is known as the direct cause of myelin pathology. Conversely, in primary demyelinating diseases, such as multiple sclerosis, astrocytes may facilitate remyelination. We suggest that specific manipulation of astrocytes could help prevent myelin pathologies and successfully restore myelin sheaths after demyelination.

Astrocytic dysfunctions have been recently identified in four leukosdystrophies without peripheral nervous system myelin involvement. Alexander disease, the first primary genetic astrocytic disorder identified, is due to dominant GFAP mutations. The presence of Rosenthal fibers throughout the CNS is the pathological hallmark of this disease. Neurological degradation, megalencephaly, and typical MRI pattern are characteristic of infantile sporadic patients. Nevertheless, clinical and MRI expression is large, including late onset forms which can be familial. Spongiform or cystic white matter CNS degeneration is present in the other three recessive disorders. The visualization of a white matter cystic breakdown on MRI has led to the identification of CACH/VWM and MLC diseases. CACH/VWM is due to mutations in one of the five subunits of EIF2B which compromise the astrocytic lineage. The clinical spectrum is large, from antenatal to adult forms, and several extraneurological organs can be affected. Mutations in MLC1, which is mainly expressed in astrocyte endfeet, produce megalencephaly, whereas the mild clinical course contrasts with severe MRI features. An increased concentration of NAA in the urine is sufficient to diagnose Canavan disease, which is due to mutations of the ASPA gene. These disorders highlight the role of astrocytes in myelination or myelin maintenance.

Myelination is an essential component of normal development in the brain. Thus, the factors which influence the onset of myelination need to be identified and monitored during development to insure adequate myelination. These factors may also play a role in remyelination attempts which occur as a result of demyelinating diseases. One factor known to be involved in myelination is iron. In this study, the cellular deposition of iron and the intracellular iron storage protein ferritin are examined in the brains of 1-month old piglets. Ferritin consists of 2 subunits (H and L chains) which occur in different ratios in different organs. The subunits are functionally distinct so their pattern of expression at the cellular level reveals information about the iron requirement of the cell and utilization versus storage. The H subunit of ferritin is expressed in abundance in oligodendrocytes within white-matter tracts whereas the L subunit in this .region is found only in endothelial cells of blood vessels. H-ferritin-positive cells occur in clearly defined patches which are scattered throughout the white-matter tracts. The cellular distribution of iron is identical to that of H ferritin. H-ferritin-positive cells are identified as oligodendrocytes on the basis of immunofluorescent colocalization with CNPase. Also, some of the H-ferritin-positive cells are positive for myelin basic protein. However, the distribution of CNPase-positive cells -is more even in the white matter than the patches of H-ferritin/iron-positive cells. The relationship between H-ferritin- and CNPase-positive cells indicates that the former are oligodendrocytes, but also reveals a subset of oligodendrocytes in the white matter. The results of this study provide insight into how the intracellular iron is managed in oligodendrocytes. The factors which initiate iron uptake and ferritin expression in a select population of oligodendrocytes, and the relationship of this select population to myelinogenesis and myelin maintenance, have yet to be identified.

Studying the function and malfunction of genes and proteins associated with inherited forms of peripheral neuropathies has provided multiple clues to our understanding of myelinated nerves in health and disease. Here, we have generated a mouse model for the peripheral neuropathy Charcot–Marie–Tooth disease type 4H by constitutively disrupting the mouse orthologue of the suspected culprit gene FGD4 that encodes the small RhoGTPase Cdc42-guanine nucleotide exchange factor Frabin. Lack of Frabin/Fgd4 causes dysmyelination in mice in early peripheral nerve development, followed by profound myelin abnormalities and demyelination at later stages. At the age of 60 weeks, this was accompanied by electrophysiological deficits. By crossing mice carrying alleles of Frabin/ Fgd4 flanked by loxP sequences with animals expressing Cre recombinase in a cell type-specific manner, we show that Schwann cell-autonomous Frabin/Fgd4 function is essential for proper myelination without detectable primary contributions from neurons. Deletion of Frabin/Fgd4 in Schwann cells of fully myelinated nerve fibres revealed that this protein is not only required for correct nerve development but also for accurate myelin maintenance. Moreover, we established that correct activation of Cdc42 is dependent on Frabin/Fgd4 function in healthy peripheral nerves. Genetic disruption of Cdc42 in Schwann cells of adult myelinated nerves resulted in myelin alterations similar to those observed in Frabin/Fgd4-deficient mice, indicating that Cdc42 and the Frabin/Fgd4–Cdc42 axis are critical for myelin homeostasis. In line with known regulatory roles of Cdc42, we found that Frabin/Fgd4 regulates Schwann cell endocytosis, a process that is increasingly recognized as a relevant mechanism in peripheral nerve pathophysiology. Taken together, our results indicate that regulation of Cdc42 by Frabin/Fgd4 in Schwann cells is critical for the structure and function of the peripheral nervous system. In particular, this regulatory link is continuously required in adult fully myelinated nerve fibres. Thus, mechanisms regulated by Frabin/Fgd4–Cdc42 are promising targets that can help to identify additional regulators of myelin development and homeostasis, which may crucially contribute also to malfunctions in different types of peripheral neuropathies. * Abbreviations : Dlg1 : disc large homologue 1 GEF : guanine nucleotide exchange factor PTEN : phosphatase and tensin homologue

Very long-chain fatty acids (VLCFAs) are fatty acids (FAs) with a chain-length of ≥22 carbons. Mammals have a variety of VLCFAs differing in chain-length and the number of double bonds. Each VLCFA exhibits certain functions, for example in skin barrier formation, liver homeostasis, myelin maintenance, spermatogenesis, retinal function and anti-inflammation. These functions are elicited not by free VLCFAs themselves, but through their influences as components of membrane lipids (sphingolipids and glycerophospholipids) or precursors of inflammation-resolving lipid mediators. VLCFAs are synthesized by endoplasmic reticulum membrane-embedded enzymes through a four-step cycle. The most important enzymes determining the tissue distribution of VLCFAs are FA elongases, which catalyze the first, rate-limiting step of the FA elongation cycle. Mammals have seven elongases (ELOVL1-7), each exhibiting a characteristic substrate specificity. Several inherited disorders are caused by mutations in genes involved in VLCFA synthesis or degradation. In this review, I describe the molecular mechanism of FA elongation and the responsible enzymes in mammals and yeast, as well as VLCFA-related disorders in human.

The pathogenesis of cobalamin (Cbl)-deficient (Cbl-D) neuropathy and the role of normal prions (PrPcs) in myelin maintenance are both subjects of debate. We have demonstrated that Cbl deficiency damages myelin by increasing tumor necrosis factor (TNF)-α, and decreasing epidermal growth factor (EGF) levels in the rat central nervous system (CNS). It is known that TNF-α and EGF regulate PrPc expression in vitro, and that myelin vacuolation, reactive astrocytosis and microglial activation are common to rat Cbl-D neuropathy and some prion diseases. We have shown that Cbl deficiency leads to high levels of PrPcs [particularly the octapeptide repeat (OR) domains] in the rat CNS thereby damaging the spinal cord (SC) myelin, and that chronic intra-cerebroventricular treatment with anti-OR antibodies normalizes SC myelin morphology. We have also found that PrPc levels are increased in the SC of Cbl-D rats by the time the myelin lesions appear, and that this increase is mediated by excess myelinotoxic TNF-α and prevented by EGF treatment, which has proved to be as effective as Cbl in preventing Cbl deficiency-induced lesions. Cbl stimulates PrPc mRNA-related synthesis in Cbl-D SC and duodenum, two rat tissues that are severely affected by Cbl deficiency. New PrPc synthesis is a common effect of various myelinotrophic agents, two of which (EGF and anti-TNF-α antibodies) also stimulate PrPc mRNA-related synthesis in the SC of Cbl-D rats.

In this study, we have investigated the contribution of oligodendrocytic connexin47 (Cx47) and astrocytic Cx30 to panglial gap junctional networks as well as myelin maintenance and function by deletion of both connexin coding DNAs in mice. Biocytin injections revealed complete disruption of oligodendrocyte-to-astrocyte coupling in the white matter of 10- to 15-d-old Cx30/Cx47 double-deficient mice, while oligodendrocyte-to-oligodendrocyte coupling was maintained. There were no quantitative differences regarding cellular networks in acute brain slices obtained from Cx30/Cx47 double-null mice and control littermates, probably caused by the upregulation of oligodendrocytic Cx32 in Cx30/Cx47 double-deficient mice. We observed early onset myelin pathology, and ∼40% of Cx30/Cx47 double-deficient animals died within 42 to 90 d after birth, accompanied by severe motor impairments. Histological and ultrastructural analyses revealed severe vacuolization and myelination defects in all white matter tracts of the CNS. Furthermore, Cx30/Cx47 double-deficient mice exhibited a decreased number of oligodendrocytes, severe astrogliosis, and microglial activation in white matter tracts. Although less affected concerning motor impairment, surviving double-knock-out (KO) mice showed behavioral alterations in the open field and in the rotarod task. Vacuole formation and thinner myelin sheaths were evident also with adult surviving double-KO mice. Since interastrocytic coupling due to Cx43 expression and interoligodendrocytic coupling because of Cx32 expression are still maintained, Cx30/Cx47 double-deficient mice demonstrate the functional role of both connexins for interastrocytic, interoligodendrocytic, and panglial coupling, and show that both connexins are required for maintenance of myelin.

Background: Chronic or binge ethanol exposures during development can cause fetal alcohol spectrum disorder (FASD) which consists of an array of neurobehavioral deficits, together with structural, molecular, biochemical, and neurotransmitter abnormalities in the brain. Previous studies showed that perinatal neurodevelopmental defects in FASD are associated with inhibition of brain insulin and insulin-like growth factor (IGF) signaling. However, it is not known whether sustained abnormalities in adolescent brain structure and function are mediated by the same phenomena. Aims:Using an early postnatal (3rd trimester equivalent) binge ethanol exposure model, we assessed neurobehavioral function, structure, and the integrity of insulin/IGF signaling in young adolescent cerebella. Methods:Long Evans male rats were treated with 50 μl of saline (vehicle) or 2 mg/kg of ethanol by i.p. injection on postnatal days (P) 2, 4, 6, and 8. On P19-20, rats were subjected to rotarod testing of motor function, and on P30, they were sacrificed to harvest cerebella for histological, molecular, and biochemical studies. Results: Binge ethanol exposures impaired motor function, caused sustained cerebellar hypocellularity, and reduced neuronal and oligodendrocyte gene expression. These effects were associated with significant deficits in insulin and IGF signaling, including impaired receptor binding, reduced Akt, and increased GSK-3β activation. Conclusions: FASD-associated neurobehavioral, structural, and functional abnormalities in young adolescent brains may be mediated by sustained inhibition of insulin/IGF-1 signaling needed for cell survival, neuronal plasticity, and myelin maintenance.

Oligodendrocytes generate large amounts of myelin by extension of their cell membranes. Though lipid is the major component of myelin, detailed lipid metabolism in the maintenance of myelin is not understood. We reported previously that miR-32 might be involved in myelin maintenance (Shin et al., 2009). Here we demonstrate a novel role for miR-32 in oligodendrocyte function and development through the regulation of SLC45A3 (solute carrier family 45, member 3) and other downstream targets such as CLDN-11. miR-32 is highly expressed in the myelin-enriched regions of the brain and mature oligodendrocytes, and it promotes myelin protein expression. We found that miR-32 directly regulates the expression of SLC45A3 by binding to the complementary sequence on the 3'UTR of cldn11 and slc45a3. As a myelin-enriched putative sugar transporter, SLC45A3 enhances intracellular glucose levels and the synthesis of long-chain fatty acids. Therefore, overexpression of SLC45A3 triggers neutral lipid accumulation. Interestingly, both overexpression and suppression of SLC45A3 reduces myelin protein expression in mature oligodendrocytes and alters oligodendrocyte morphology, indicating that tight regulation of SLC45A3 expression is necessary for the proper maintenance of myelin proteins and structure. Taken together, our data suggest that miR-32 and its downstream target SLC45A3 play important roles in myelin maintenance by modulating glucose and lipid metabolism and myelin protein expression in oligodendrocytes.

Backgrounds: Charcot–Marie–Tooth neuropathy has been reported to be associated with renal diseases, mostly focal segmental glomerulosclerosis (FSGS). However, the common mechanisms underlying the neuropathy and FSGS remain unknown. Mutations in INF2 were recently identified in patients with autosomal dominant FSGS. INF2 encodes a formin protein that interacts with the Rho-GTPase CDC42 and myelin and lymphocyte protein (MAL) that are implicated in essential steps of myelination and myelin maintenance. We therefore hypothesized that INF2 may be responsible for cases of Charcot–Marie–Tooth neuropathy associated with FSGS., Methods: We performed direct genotyping of INF2 in 16 index patients with Charcot–Marie–Tooth neuropathy and FSGS who did not have a mutation in PMP22 or MPZ, encoding peripheral myelin protein 22 and myelin protein zero, respectively. Histologic and functional studies were also conducted., Methods: We identified nine new heterozygous mutations in 12 of the 16 index patients (75%), all located in exons 2 and 3, encoding the diaphanous-inhibitory domain of INF2. Patients presented with an intermediate form of Charcot–Marie–Tooth neuropathy as well as a glomerulopathy with FSGS on kidney biopsy. Immunohistochemical analysis revealed strong INF2 expression in Schwann-cell cytoplasm and podocytes. Moreover, we demonstrated that INF2 colocalizes and interacts with MAL in Schwann cells. The INF2 mutants perturbed the INF2–MAL–CDC42 pathway, resulting in cytoskeleton disorganization, enhanced INF2 binding to CDC42 and mislocalization of INF2, MAL, and CDC42., Conclusions: INF2 mutations appear to cause many cases of FSGS-associated Charcot–Marie–Tooth neuropathy, showing that INF2 is involved in a disease affecting both the kidney glomerulus and the peripheral nervous system. These findings provide new insights into the pathophysiological mechanisms linking formin proteins to podocyte and Schwann-cell function., Funded by the Agence Nationale de la Recherche and others.Supported by grants from the Association pour l'Utilisation du Rein Artificiel (to Dr. Antignac), Association Française contre les Myopathies (ANR-08-GENOPAT-017-01, to Dr. Antignac), Agence Nationale de la Recherche (PodoNet project number ANR-07-E-RARE-011-01 in the ERA-Net Consortium [JTC2007], to Dr. Antignac, and ANR-06-MRAR-024-01, to Dr. Leguern), Fondation pour la Recherche Médicale (project number DMP 2010-11-20-386, to Dr. Antignac, and doctoral funding, to Dr. Boyer), Association des Malades du Syndrome Néphrotique (to Dr. Mollet), Fonds de la Recherche en Santé du Québec (Fellowship Training Award to Dr. Benoit), and Ministerio de Ciencia e Innovación (BFU2009-07886 and CONSOLIDER COAT CSD2009-00016, to Dr. Alonso).

MicroRNAs (miRNAs or miRs) are a class of endogenous small non-coding RNAs that consist of about 22 nucleotides and play critical roles in various biological processes, including cell proliferation, differentiation, apoptosis, and tumorigenesis. In recent years, some specific miRNA, such as miR-219, miR-138, miR-9, miR-23, and miR-19b were found to participate in the regulation of oligodendrocyte (OL) differentiation and myelin maintenance, as well as in the pathogenesis of demyelination-related diseases (e.g., multiple sclerosis, ischemic stroke, and leukodystrophy). These miRNAs control their target mRNA or regulate the protein levels of some signaling pathways, and participate in OL differentiation and the pathogenesis of demyelination-related diseases. During pathologic processes, the expression levels of specific miRNAs are dynamically altered. Therefore, miRNAs act as diagnostic and prognostic indicators of defects in OL differentiation and demyelination-related diseases, and they can provide potential targets for therapeutic drug development.

The pathogenesis of cobalamin (Cbl)-deficient (Cbl-D) neuropathy is not clear, nor is the role of prions (PrP(C)) in myelin maintenance. However, as it is known that Cbl deficiency damages myelin by increasing tumor necrosis factor (TNF)-α and decreasing epidermal growth factor (EGF) levels in rat spinal cord (SC), and that TNF-α and EGF regulate PrP(C) expression in vitro, we investigated whether Cbl deficiency modifies SC PrP(C) and PrP(C)-mRNA levels in Cbl-D rats. PrP(C) levels had increased by the time myelin lesions appeared. This increase was mediated by excess myelinotoxic TNF-α and prevented by EGF, which proved to be as effective as Cbl in preventing Cbl deficiency-induced lesions. There were no significant changes in hepatic PrP(C) levels of Cbl-D rats. Anti-octapeptide repeat (OR) region antibodies normalized SC myelin morphology. Cbl deficiency greatly reduced SC PrP(C)-mRNA levels, which were subsequently increased by Cbl and EGF. Cbl deficiency-induced excess OR is myelin-damaging, but new PrP(C) synthesis is a common effect of different myelinotrophic agents.

Too Much of a Good Thing In peripheral nerves, the insulating myelin sheath speeds up electrical conductivity by allowing impulses to skip down the axon from node to node. Axons signal using neuregulin to get the Schwann cells to begin their wrap-around insulation project. But when is enough myelin too much? Cotter et al. (p. 1415 , published online 6 May) have now found the signal that stops further rounds of myelin insulation. In developing mice, the proteins Dlg1 (mammalian disc large 1) and PTEN (phosphatase and tensin homolog) were involved in calling a halt to insulation during early development. The balance between not enough and too much myelin insulation is controlled by opposing signals, which together optimize both the myelination and the velocity of nerve conduction.

This paper summarizes neuroendocrine effects on myelination and their possible relevance for the pathogenesis of multiple sclerosis (MS). Steroid hormones known as neurosteroids are synthesized in the human central nervous system (CNS) and exert local effects on glial and neuronal tissue. Progesterone derivatives seem to act as promyelinating factors in the slow but continuous process of myelin maintenance in the adult human brain. Diminished production of these myelin-promoting factors may lead to the formation of structurally altered and less stable myelin, resulting in the observed pathology of the normal-appearing white matter (NAWM) in MS. Dysmyelination, characterized by an altered myelin protein composition, reduced myelin content and increased vulnerability of the myelin sheath, precedes the formation of inflammatory lesions and the clinical onset of disease. Defects in the myelin sheath first occur in mechanically strained areas of the brain, where myelin turnover is physiologically increased. The continuous exposure of myelin proteins, normally sheltered from immunosurveillance, will lead to microglia activation and phagocytosis of myelin. Phagocytic cells from the brain and myelin material may drain to cervical lymph nodes with subsequent priming of T-cells. Finally, heterogenous focal auto-inflammatory reactions contribute to the clinical symptoms of the disease. Neurosteroids influence the biochemical composition of myelin proteins and promote myelin renewal. These promyelinating neurosteroidal functions seem to be impaired in the MS brain. Contrary to the view of auto-inflammatory demyelination being a causative factor in MS pathogenesis, it is argued here that widespread dysmyelination in the adult human brain precedes and induces a focal immune response to various myelin compounds.

Membrane-membrane interactions between axons and Schwann cells are required for initial myelin formation in the peripheral nervous system. However, recent studies of double myelination in sympathetic nerve have indicated that myelin sheaths continue to exist after complete loss of axonal contact (Kidd, G. J., and J. W. Heath. 1988. J. Neurocytol. 17:245-261). This suggests that myelin maintenance may be regulated either by diffusible axonal factors or by nonaxonal mechanisms. To test these hypotheses, axons involved in double myelination in the rat superior cervical ganglion were destroyed by chronic guanethidine treatment. Guanethidine-induced sympathectomy resulted in a Wallerian-like pattern of myelin degeneration within 10 d. In doubly myelinated configurations the axon, inner myelin sheath (which lies in contact with the axon), and approximately 75% of outer myelin sheaths broke down by this time. Degenerating outer sheaths were not found at later periods. It is probably that outer sheaths that degenerated were only partially displaced from the axon at the commencement of guanethidine treatment. In contrast, analysis of serial sections showed that completely displaced outer internodes remained ultrastructurally intact. These internodes survived degeneration of the axon and inner sheath, and during the later time points (2-6 wk) they enclosed only connective tissue elements and reorganized Schwann cells/processes. Axonal regeneration was not observed within surviving outer internodes. We therefore conclude that myelin maintenance in the superior cervical ganglion is not dependent on direct axonal contact or diffusible axonal factors. In addition, physical association of Schwann cells with the degenerating axon may be an important factor in precipitating myelin breakdown during Wallerian degeneration.

Brain white matter myelin abnormalities and cell membrane fatty acid abnormalities have been implicated in schizophrenia and other psychiatric disorders. We investigated in young adults with a psychotic disorder (n=12) whether (poly)unsaturated fatty acid concentrations in erythrocyte membranes are related to an MRI measure of brain white matter, which depends on the degree of myelination. A significant correlation was found between total (poly)unsaturated fatty acid concentration and fractional anisotropy of a fronto-temporal white matter tract (r=0.503, P=0.048). Unsaturated fatty acids may be necessary for the myelinating activity of oligodendrocytes or for myelin maintenance. These results warrant further investigation.

Axonal contact plays a critical role in initiating myelin formation by Schwann cells. However, recent studies of “double myelination” have indicated that myelin maintenance continues in Schwann cells completely displaced from physical contact with the axon. This raises the possibility either that diffusible trophic factors are produced by the axon, or that the axon is not required for myelin maintenance by these displaced Schwann cells. To test these hypotheses, the axons involved in double myelination in the mouse superior cervical ganglion (SCG) were transected surgically by a transganglionic lesion. The inferior pole of the SCG was resected to limit axonal regeneration. This method produced a typical Wallerian pattern of degeneration in the superior pole, without compromising the blood supply or introducing nonspecific trauma. EM analysis at 1 and 5 d postoperatively showed that initially the axon degenerated, followed by breakdown of the inner myelin sheath. In those configurations where the outer Schwann cell was only partly displaced from the axon, the outer myelin sheath degenerated simultaneously. However, in completely displaced internodes the outer sheath survived degeneration of the axon and inner sheath. Outer internodes remained intact for at least 5 weeks after transection (the longest time point in this study), at which time they enclosed reorganized processes of the inner Schwann cells, their basal lamina, and numerous collagen fibrils. Axonal regeneration within surviving outer internodes was rare and was characterized by the development of typical Remak ensheathment by the inner Schwann cells. We conclude that in the mouse SCG, myelin maintenance does not depend on the continued presence of the axon. These data suggest further that myelin breakdown in Wallerian degeneration may be initiated by mechanisms other than absence of a viable axon.

Proteolipid protein (PLP), the major protein of central nervous system myelin, contains approximately 2 mol of covalently bound fatty acids. In this study, the in vivo turnover rate of the acyl chains bound to PLP was determined in 40-day-old rats after a single intracranial injection of [3H]palmitic acid. The apparent half-life of total fatty acids bound to PLP was approximately 7 days. After correction for acyl chain interconversion, the half-life of palmitate bound to PLP was only 3 days. This turnover rate is much more rapid than that of the protein moiety calculated under the same experimental conditions (t1/2 = 1 month). Additional evidence for the dynamic metabolism of acyl groups was provided by experiments in brain tissue slices which showed that acylation of PLP occurs in adult animals as well as during active myelination. Acylation of endogenous PLP in purified myelin and its subfractions was also studied during rat brain development using either [3H]palmitoyl-CoA or [3H]palmitic acid plus ATP and CoA. Labeling of endogenous PLP with [3H]palmitoyl-CoA was observed as early as 10 days postnatal and continued at the same rate throughout development. When [3H]palmitic acid was used as precursor in the presence of both ATP and CoA, esterification of myelin PLP occurred rapidly in adult animals, indicating that both nonacylated PLP and acyl-CoA ligase are present in myelin. Finally, pulse-chase experiments in a cell-free system showed that PLP-bound fatty acids turn over with a half-life shorter than 10 min. These observations are consistent with the concept that acylation of myelin PLP is a dynamic process involved mainly in myelin maintenance and function.

Within the nervous system, expression of the intriguing giant protein AHNAK had been reported so far only for blood-brain barrier forming vascular endothelium. In a screen for genes upregulated after spinal cord injury, we recently identified ahnak as being highly expressed by non-neuronal cells invading the lesion, delimiting the interior surface of cystic cavities in front of barrier-forming astrocytes. Here, we show for the first time that AHNAK is constitutively expressed in peripheral nervous system, notably by myelinating Schwann cells (SCs), in which we investigated its function. During sciatic nerve development, AHNAK is redistributed from adaxonal toward abaxonal SC compartments in contact with basement membrane. AHNAK labeling on myelinated fibers from adult nerve delineates the so-called "Cajal bands," constituting the residual peripheral SC cytoplasm. Its distribution pattern is complementary to that of periaxin, known to be involved in the myelination process. In vitro, nonconfluent cultured primary SCs seeded on laminin express high levels of AHNAK concentrated in their processes, whereas at confluence, AHNAK is downregulated together with laminin receptor dystroglycan. AHNAK silencing by siRNA interference affects SC morphology and laminin-substrate attachment, as well as expression and distribution of dystroglycan. Thus, our results clearly show the implication of AHNAK in SC adhesion to laminin, probably via targeting of the dystroglycan-associated receptor complex. These findings are of high interest regarding the importance of SC-basal lamina interactions for myelination and myelin maintenance, and open up new perspectives for investigations of the molecular mechanisms underlying demyelinating neuropathies.

SUMMARYDuplication of the gene encoding lamin B1 (LMNB1) with increased mRNA and protein levels has been shown to cause severe myelin loss in the brains of adult-onset autosomal dominant leukodystrophy patients. Similar to many neurodegenerative disorders, patients with adult-onset autosomal dominant leukodystrophy are phenotypically normal until adulthood and the defect is specific to the central nervous system despite the ubiquitous expression pattern of lamin B1. We set out to dissect the molecular mechanisms underlying this demyelinating phenotype. Increased lamin B1 expression results in disturbances of inner nuclear membrane proteins, chromatin organization and nuclear pore transport in vitro. It also leads to premature arrest of oligodendrocyte differentiation, which might be caused by reduced transcription of myelin genes and by mislocalization of myelin proteins. We identified the microRNA miR-23 as a negative regulator of lamin B1 that can ameliorate the consequences of excessive lamin B1 at the cellular level. Our results indicate that regulation of lamin B1 is important for myelin maintenance and that miR-23 contributes to this process, at least in part, by downregulating lamin B1, therefore establishing novel functions of lamin B1 and miR-23 in the regulation of oligodendroglia development and myelin formation in vitro.

Copper deficiency myelopathy (CDM) is an increasingly recognised mimic of subacute combined degeneration (SCD) of the cord due to cobalamin (vitamin B(12)) deficiency. It has been suggested that copper deficiency induces myelopathy through dysfunction of cytochrome oxidase, which is known to be copper-dependent. However, cytochrome oxidase is not cobalamin-dependent, so this hypothesis fails to explain the phenotypic similarity between CDM and SCD. We propose that the first step in a final common pathway of CDM and SCD is dysfunction of the methylation cycle. This cycle includes both copper and cobalamin-dependent enzymes and catalyses the net transfer of a methyl group from methyltetrahydrofolate to a variety of macromolecules, including myelin proteins. Dysfunction of the cycle might therefore cause failure of myelin maintenance and ultimately myelopathy. One step of the methylation cycle is catalysed by methionine synthase, which is known to be cobalamin-dependent. Nitrous oxide specifically inhibits this enzyme by inactivating methylcobalamin, causing SCD in animals and humans. Both animal and human data suggest that methionine synthase also requires copper, implying that the enzyme may be involved in the pathogenesis of CDM. Another enzyme involved in the methylation cycle, S-adenosylhomocysteine hydrolase, may be regulated by copper. Although this enzyme is not cobalamin-dependent, its potential impairment in copper deficiency may contribute to the overall dysfunction of the methylation cycle. In cases of congenital deficiencies of methylation cycle enzymes, spinal and cerebral demyelination was observed, providing further support for a critical role of the methylation cycle in myelination. Biochemical dysfunction of the methylation cycle has been reported in HIV myelopathy, which has pathological parallels with SCD. This raises the possibility that other demyelinating myelopathies might involve an impairment of the methylation cycle. Our hypothesis could be tested by measuring CSF concentrations of methylation cycle intermediates in cases of CDM, as these reflect spinal cord tissue levels. If it were confirmed, the hypothesis would not only provide a plausible explanation for the phenotypic similarity between CDM and SCD, but might also open up further therapeutic options such as methionine and betaine supplementation.

Oligodendrocyte precursor cells modify the neural cell adhesion molecule (NCAM) by the attachment of polysialic acid (PSA). Upon further differentiation into mature myelinating oligodendrocytes, however, oligodendrocyte precursor cells down-regulate PSA synthesis. In order to address the question of whether this down-regulation is a necessary prerequisite for the myelination process, transgenic mice expressing the polysialyltransferase ST8SiaIV under the control of the proteolipid protein promoter were generated. In these mice, postnatal down-regulation of PSA in oligodendrocytes was abolished. Most NCAM-120, the characteristic NCAM isoform in oligodendrocytes, carried PSA in the transgenic mice at all stages of postnatal development. Polysialylated NCAM-120 partially co-localized with myelin basic protein and was present in purified myelin. The permanent expression of PSA-NCAM in oligodendrocytes led to a reduced myelin content in the forebrains of transgenic mice during the period of active myelination and in the adult animal. In situ hybridizations indicated a significant decrease in the number of mature oligodendrocytes in the forebrain. Thus, down-regulation of PSA during oligodendrocyte differentiation is a prerequisite for efficient myelination by mature oligodendrocytes. Furthermore, myelin of transgenic mice exhibited structural abnormalities like redundant myelin and axonal degeneration, indicating that the down-regulation of PSA is also necessary for myelin maintenance.

Transferrin, the iron carrier protein, has been shown to be involved in oligodendroglial cell differentiation in the central nervous system but little is known about its role in the peripheral nervous system. In the present work, we have studied the presence of transferrin and of its mRNA in rat sciatic nerves and in Schwann cells isolated at embryonic and adult ages as well as during the regeneration process that follows nerve crush. We have also studied the correlation between the expression of the mRNAs of transferrin and the expression of mature myelin markers in the PNS. We show that transferrin is present in whole sciatic nerves at late stages of embryonic life as well as at postnatal day 4 and in adult rats. We demonstrate for the first time, that in normal conditions, the transferrin mRNA is expressed in Schwann cells isolated from sciatic nerves between embryonic days 14 and 18, being absent at later stages of development and in adult animals. In adult rats, 3 days after sciatic nerve crushing, the mRNA of transferrin is expressed in the injured nerve, but 7 days after injury its expression disappears. Transferrin protein in the sciatic nerve closely follows the expression of its mRNA indicating that under these circumstances, it appears to be locally synthesized. Transferrin in the PNS could have a dual role. During late embryonic ages it could be locally synthesized by differentiating Schwann cells, acting as a pro-differentiating factor. A similar situation would occur during the regeneration that follows Wallerian degeneration. In the adult animals on the other hand, Schwann cells could pick up transferrin from the circulation or/and from the axons, sub serving possible trophic actions closely related to myelin maintenance.

Publisher Summary Hereditary spastic paraparesis (HSP) represents a group of conditions in which the prominent feature is a progressive spastic paraparesis. The most useful way of classifying HSP is genetically to one of the current HSP gene loci. There are currently 28 spastic paraplegia (SPG) loci. Advances in recent years identifying the genes at 11 of these loci have suggested that disruption in any of the following—axonal transport, cytoskeleton regulation, mitochondrial function, myelin maintenance and assembly, and neuronal migration—may cause axonal damage in HSP. The major neuropathological feature of pure HSP is a length dependent axonopathy within the central nervous system (CNS). HSP can also be classified clinically according to the mode of inheritance, age of onset, or clinical phenotype. HSP is inherited most often as an autosomal dominant trait, with autosomal recessive and X-linked inheritance occurring rarely and very rarely, respectively. Identifying the mode of inheritance is complicated by an increasing recognition of reduced penetrance, most often described in spastin-related HSP. Two broad HSP phenotypes—pure HSP and complicated HSP—have been used to clinically classify HSP.

Onset of myelination in Schwann cells is governed by several transcription factors, including Krox20/Egr2, and mutations affectingKrox20result in various human hereditary peripheral neuropathies, including congenital hypomyelinating neuropathy (CHN) and Charcot-Marie-Tooth disease (CMT). Similar molecular information is not available on the process of myelin maintenance. We have generated conditionalKrox20mutations in the mouse that allowed us to develop models for CHN and CMT. In the latter case, specific inactivation ofKrox20in adult Schwann cells results in severe demyelination, involving rapid Schwann cell dedifferentiation and increased proliferation, followed by an attempt to remyelinate and a block at the promyelinating stage. These data establish that Krox20 is not only required for the onset of myelination but that it is also crucial for the maintenance of the myelinating state. Furthermore, myelin maintenance appears as a very dynamic process in which Krox20 may constitute a molecular switch between Schwann cell myelination and demyelination programs.

Over the last 15 years, a number of mutations in a variety of genes have been identified that lead to inherited motor and sensory neuropathies (HMSN), also called Charcot-Marie-Tooth disease (CMT). In this review we will focus on the molecular and cellular mechanisms that cause the Schwann cell pathologies observed in dysmyelinating and demyelinating forms of CMT. In most instances, the underlying gene defects alter primarily myelinating Schwann cells followed by secondary axonal degeneration. The first set of proteins affected by disease-causing mutations includes the myelin components PMP22, P0/MPZ, Cx32/GJB1, and periaxin. A second group contains the regulators of myelin gene transcription EGR2/Krox20 and SOX10. A third group is composed of intracellular Schwann cells proteins that are likely to be involved in the synthesis, transport and degradation of myelin components. These include the myotubularin-related lipid phosphatase MTMR2 and its regulatory binding partner MTMR13/SBF2, SIMPLE, and potentially also dynamin 2. Mutations affecting the mitochondrial fission factor GDAP1 may indicate an important contribution of mitochondria in myelination or myelin maintenance, whereas the functions of other identified genes, including NDRG1, KIAA1985, and the tyrosyl-tRNA synthase YARS, are not yet clear. Mutations in GDAP1, YARS, and the pleckstrin homology domain of dynamin 2 lead to an intermediate form of CMT that is characterized by moderately reduced nerve conduction velocity consistent with minor myelin deficits. Whether these phenotypes originate in Schwann cells or in neurons, or whether both cell types are directly affected, remains a challenging question. However, based on the advances in systematic gene identification in CMT and the analyses of the function and dysfunction of the affected proteins, crucially interconnected pathways in Schwann cells in health and disease have started to emerge. These networks include the control of myelin formation and stability, membrane trafficking, intracellular protein sorting and quality control, and may extend to mitochondrial dynamics and basic protein biosynthesis.

Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by genetic deficiency of galactocerebrosidase (GALC) activity. Failure in catalyzing the degradation of its major substrate, galactocerebroside, in oligodendrocytes (OLs) and Schwann cells leads to death of these myelinating cells, progressive demyelination, and early demise of GLD patients. Transplantation of bone marrow cells and umbilical cord blood have been attempted as a means of enzyme replacement and have shown limited success. It remains unknown whether or how these therapies support survival of GALC-deficient OLs and myelin maintenance. We report that, upon transplantation, GALC-deficient OLs from the twitcher mouse, a model of GLD, achieved widespread myelination in the brain and spinal cord of the myelin-deficient shiverer mouse, which was preserved for the life of the host. GALC immunohistochemistry showed direct evidence for GALC transfer from the shiverer environment to the engrafted mutant OLs in vivo . These findings suggest that the mutant OLs can internalize exogenous GALC and maintain stable myelin, demonstrating that exogenous enzyme replacement will be a key strategy in the therapy of GLD.

The presence of immunoglobulin (Ig)M antibody against myelin associated glycoprotein (MAG) has been associated with autoimmune demyelinating, sensorimotor neuropathies. Approximately 50% of patients with IgM paraproteinemia and associated peripheral neuropathy possess antibodies against MAG. These autoantibodies are thought to interfere with the process of myelination, myelin maintenance, or axon‐Schwann cell interaction. The detection of these autoantibodies is useful to the clinician and is suggestive of active demyelination in a peripheral neuropathy. Our objective in this study was to compare the results obtained using three different methods (dual enzyme immunoassay [EIA], immunofluorescent antibody [IFA] and Western blot [WB]) for detecting IgM antibody against MAG in patients suspected of having autoimmune demyelinating neuropathies. Since the dual EIA utilized two different antigens, results from this assay were separated into two groups: MAG and sulfate‐3‐glucuronyl paragloboside (SGPG). When compared to WB (gold standard), percent agreement, sensitivity, and specificity for EIA and IFA are as follows: MAG EIA (68.3, 100.0, and 60.6); SGPG EIA (95.1, 100.0, and 93.9); and myelin IFA (97.6, 100.0, and 97.0). The authors conclude that the SGPG EIA and myelin IFA compared well with the standard WB method when detecting IgM antibody against MAG (100 kD). Many sera demonstrated reactivity on the MAG EIA that were negative by WB (100 kD glycoprotein). The authors recommend screening for MAG IgM in suspected patient sera by SGPG EIA or myelin IFA and utilizing these same methods to titer sera confirmed positive by WB. J. Clin. Lab. Anal. 18:247–250, 2004. © 2004 Wiley‐Liss, Inc.

NDRG1 is an intracellular protein that is induced under a number of stress and pathological conditions, and it is thought to be associated with cell growth and differentiation. Recently, human NDRG1 was identified as a gene responsible for hereditary motor and sensory neuropathy-Lom (classified as Charcot-Marie-Tooth disease type 4D), which is characterized by early-onset peripheral neuropathy, leading to severe disability in adulthood. In this study, we generated mice lacking Ndrg1 to analyze its function and elucidate the pathogenesis of Charcot-Marie-Tooth disease type 4D. Histological analysis showed that the sciatic nerve of Ndrg1-deficient mice degenerated with demyelination at about 5 weeks of age. However, myelination of Schwann cells in the sciatic nerve was normal for 2 weeks after birth. Ndrg1-deficient mice showed muscle weakness, especially in the hind limbs, but complicated motor skills were retained. In wild-type mice, NDRG1 was abundantly expressed in the cytoplasm of Schwann cells rather than the myelin sheath. These results indicate that NDRG1 deficiency leads to Schwann cell dysfunction, suggesting that NDRG1 is essential for maintenance of the myelin sheaths in peripheral nerves. These mice will be used for future analyses of the mechanisms of myelin maintenance.

The estrogen receptor (ER) is a member of a superfamily of ligand-regulated transcription factors that were thought to localize primarily to the nucleus; however, a membrane-associated ER that can initiate rapid non-genomic cell-signaling events has been identified recently in various cells. The presence of the ER in myelin has not been reported although the nuclear form has been detected in oligodendrocytes. We have shown that an ER with similarities to ERbeta is present in isolated central nervous system (CNS) myelin, the myelin sheath in spinal cord and brain sections, and the oligodendrocyte plasma membrane using two-dimensional (2D) PAGE, mass spectrometry, peptide mass fingerprinting, Western blotting of 1D and 2D gels, and confocal microscopy. Caveolin-1 was also shown to be present in isolated CNS myelin and oligodendrocyte plasma membranes, where it was partially colocalized with ER. After Triton X-100 extraction of myelin, the ER was present in an insoluble low-density glycosphingolipid-enriched fraction and even more in a higher density fraction also containing caveolin and cytoskeletal elements, suggesting that the membrane form of ER may be associated with caveolin or the radial component of myelin. The discovery of the ER in the oligodendrocyte plasma membrane and within the myelin sheath indicates a potential role for estrogen in myelin maintenance or functions.

Publisher Summary This chapter reviews the structure, regulation, and biological function of peripheral myelin protein 22 (PMP22) in myelination, myelin maintenance, and disease. The PMP22 is a minor component of the myelin sheath of peripheral nerves. It belongs to a family of membrane proteins that is characterized by four hydrophobic domains and conserved amino acid motifs. PMP22 plays a crucial functional role in peripheral nerves based on the observation that genetic alterations in the PMP22 gene lead to various forms of myelination deficiencies in humans and rodents. PMP22 is widely expressed in neural and nonneural tissues during embryonic development and in the adult. PMP22 appears to be a valuable marker for PNS progenitors in rat embryos, but not for early mouse-neural-crest derivatives. The genomic structure of the human PMP22 gene has been determined. The chapter further discusses the regulation of PMP22 and its role in hereditary motor and sensory neuropathies in human and rodents.

The myelin-associated glycoprotein, a minor component of myelin in the central and peripheral nervous system, has been implicated in the formation and maintenance of myelin. Although the analysis of MAG null mutants confirms this view, the phenotype of this mutant is surprisingly subtle. In the CNS of MAG-deficient mice, initiation of myelination, formation of morphologically intact myelin sheaths and to a minor extent, integrity of myelin is affected. In the PNS, in comparison, only maintenance of myelin is impaired. Recently, the large isoform of MAG has been identified as the functionally important isoform in the CNS, whereas the small MAG isoform is sufficient to maintain the integrity of myelinated fibers in the PNS. Remarkably, none of the different defects in the MAG mutant is consistently associated with each myelinated fiber. These observations suggest that other molecules performing similar functions as MAG might compensate, at least partially, for the absence of MAG in the null mutant.

A Western blot analysis showed connexin32 (Cx32) to be present in the myelin membrane. Cx32 mRNA rapidly decreased in the distal segments of crushed sciatic nerves and thereafter returned to normal, as did P0 mRNA. Both Cx32 and P0 mRNAs were detectable in cultured Schwann cells and were enhanced by the addition of forskolin. The developmental profile showed that Cx32 mRNA had thus markedly increased by the time that myelination had become most active, and thereafter only slowly increased unlike P0. Cx32 mRNA seems to parallel myelin size, which thus suggests that Cx32 supports myelin maintenance as a gap junction protein that facilitates the intramyelin exchange of small molecules.

The sciatic nerve of the mouse mutant with Niemann-Pick type C disease (NPC mouse) was investigated using light and electron microscopy, and teased-fiber preparations. As early as postnatal day 20, when clinical symptoms were not yet apparent, focal paranodal swellings with an accumulation of small myelin figures in the Schwann cell cytoplasm were noted. These paranodal changes were more pronounced in the distal segment and became progressively conspicuous with increasing age. The morphometric analysis revealed a hypomyelination of large myelinated fibers in the NPC nerves at 70 days, whereas an essentially similar histogram pattern was noted in both control and NPC nerves at 20 days, suggesting progressively defective utilization of cholesterol in the NPC nerves with age. Intraxonal accumulation of dense bodies was noted in older mice, but no segmental demyelination or Wallerian type of axonal degeneration was observed at any age. The changes noted in the paranodal regions in the NPC mouse closely resemble those found in rats treated with an inhibitor of cholesterol biosynthesis, as well as those seen in remodeling fibers during an early stage of peripheral nerve development. Thus, the morphological changes seen in the sciatic nerve of the NPC mouse may be an expression of perturbation in myelin maintenance as a result of defective cholesterol metabolism.

Current in vitro models to investigate the consequence of oligodendrocyte-specific loss-of-function mutations on myelination are primarily limited to co-culture experiments, which do not accurately recapitulate the complex in vivo environment. Here, we describe the development of an in vitro model of myelination and myelin maintenance in which oligodendrocyte precursor cells are transplanted into organotypic cerebellar slice cultures derived from dysmyelinated shiverer mice. Compared to neuron-oligodendrocyte co-cultures, organotypic slices more closely mimic the environment in vivo, while utilizing a genetic background that allows for straight-forward identification of myelin generated by transplanted cells. We show at the ultrastructural level that the myelin generated by wild-type transplanted oligodendrocytes is compact and terminates in cytoplasmic loops that form paranodal junctions with the axon. This myelination results in the appropriate sequestering of axonal proteins into specialized domains surrounding the nodes of Ranvier. We also demonstrate the applicability of this approach for xenograft transplantation of oligodendrocyte precursor cells derived from rat or human sources. This method provides a time-efficient and cost-effective adjunct to conditional knockout mouse lines or in vivo transplantation models to study oligodendrocyte-specific loss-of-function mutations. Furthermore, the approach can be readily used to assess the effect of pharmacological manipulations on myelin, providing a tool to better understand myelination and develop effective therapeutic strategies to treat myelin-related diseases.

Membrane depolarization and changes in ionic fluxes have been implicated in the signaling mechanisms between neurons and glial cells. We report here that K+-induced depolarization of cultured ovine oligodendrocytes (OLGs) decreases the phosphorylation of myelin basic protein (MBP) and 2′3′-cyclic nucleotide phosphohydrolase (CNPase). Membrane depolarization and decrease in phosphorylation of MBP and CNPase can also be elicited by inhibition of the inward rectifier with Ba2+ but not by inhibition of outward K+ channels with 4-aminopyridine or tetraethylammonium. These findings demonstrate that modulation of K+ currents can influence phosphorylation states of OLG proteins. Tumor necrosis factor-α (TNF-α), an immune peptide implicated in autoimmune demyelinating diseases, also inhibits the phosphorylation of these proteins. In contrast to elevated [K+]0, TNF-α does not decrease the stimulatory effect of protein kinase C activators or phosphatase inhibitors on MBP and CNPase phosphorylation, suggesting that depolarizing agents and TNF-α act via distinct mechanisms. We postulate that the presence of elevated extracellular K+ and/or cytokines under certain pathological conditions can perturb OLG function by altering the phosphorylation states of their proteins and perhaps affect myelin maintenance, contributing to demyelination. © 1994 Wiley-Liss, Inc.

The childhood leukodystrophies are characterized by neonatal or childhood deficiencies in myelin production or maintenance; these may be due to hereditary defects in genes for myelin maintenance, as in Pelizaeus-Merzbacher disease, or to enzymatic deficiencies resulting in substrate misaccumulation or misprocessing, as in the lysosomal storage disorders. Regardless of their respective etiologies, these disorders are essentially all manifested by a profound deterioration in neurological function with age. A congenital deficit in forebrain myelination is also noted in children with the periventricular leukomalacia of cerebral palsy, which yields a more static morbidity. In light of the wide range of disorders to which congenital hypomyelination or postnatal demyelination may contribute, and the relative homogeneity of oligodendrocytes and their progenitors, the leukodystrophies may be especially attractive targets for cell-based therapeutic strategies. As a result, glial progenitor cells, which can give rise to new myelinogenic oligodendrocytes, have become of great interest as potential vectors for the restoration of myelin to the dysmyelinated brain and spinal cord. In addition, by distributing throughout the neuraxis after perinatal graft, and giving rise to astrocytes as well as oligodendrocytes, glial progenitor cells may be of great utility in rectifying the dysmyelination-associated enzymatic deficiencies of the lysosomal storage disorders.

Formation and maintenance of myelin sheaths in the peripheral nervous system are regulated by unknown molecular interactions that are thought to depend upon physical contact between Schwann cells and axons. However, recent studies describing axons surrounded by two concentric myelin internodes in the superior cervical ganglion (SCG) of normal rodents have demonstrated that the outer myelin internodes are maintained without physical contact with the axon. To determine whether the centrally enclosed axon has a trophic effect in maintaining these remote outer internodes, we have produced axonal degeneration by surgical or chemical means. The results indicate that maintenance of myelin internodes totally displaced from axonal contact depends neither upon the presence of the axon nor on diffusible axonal factors. A further implication of these studies is that myelin breakdown during Wallerian degeneration is regulated by a positive signal which originates in degenerating nerves, rather than solely by loss of axonal trophic substances.

The molecular organization of surface galactose residues in glycoproteins of the intact myelin sheath was investigated using the enzymatic membrane probe, galactose oxidase. Rat spinal cords treated under physiological conditions with this nonpermanent probe were labeled specifically in galactose residues by reduction with tritiated sodium borohydride. The enzymatically modified proteins from isolated myelin were analyzed electrophoretically and their specific radioactivities determined. Results indicated tritium label associated with a surprising variety of high molecular weight proteins. The most extensively labeled peak corresponded to the major myelin glycoprotein as indicated by the coincidence of tritium label with that of [14C]fucose used as an internal marker for the glycoproteins. The radioactivity associated with this protein was 1.1 to 2.7 times higher after treatment with galactose oxidase when compared to reduction in the absence of the enzyme and 1.4 to 4.8 times higher when oxidized and reduced after prior treatment with neuraminidase. The results suggest a complex heterogeneity of minor glycoproteins associated with isolated myelin. It is concluded that from this complexity of glycoproteins, a major glycoprotein is at least partially localized on the external surface of either the intact myelin sheath or the closely associated oligodendroglial plasma membrane. Such a localization of this glycoprotein and the probable localization of the other glycoproteins enhances their potential role in specific interactions in the process of mpyelination or myelin maintenance.

Degeneration of the distal axons of long and large-diameter peripheral nerve fibers is perhaps the most common of all toxic peripheral nerve disorders. Degeneration commonly spreads centrally along affected nerve tracts in a dying-back fashion (1). Axonal disorders of this type are grouped under the term central-peripheral distal axonopathy to emphasize the contemporaneous onset of distal retrograde axonal degeneration in long nerve-fiber tracts in both the central nervous system (CNS) and the peripheral nervous system (PNS) (2). Axonopathy refers exclusively to the primary degeneration of axons, although associated changes may occur in corresponding neuronal perikarya. Schwann cells and oligodendrocytes enveloping affected axons in the PNS and CNS, respectively, commonly undergo secondary changes, induding mitosis and loss of myelin maintenance, while fibroblasts and astrocytes become involved later in the degenerative process. Distal axonopathy can result from a single or, more commonly, repeated exposure to a variety of chemically unrelated agents, or it can occur with apparent spontaneity in a number of abnormal nutritional or metabolic states (3). Although the biochemical mechanisms underlying axonal degeneration are unknown, some progress has been made in recent years in understanding the biochemical events that precede and accompany axon degeneration in certain toxic neuropathies, notably those associated with repeated exposure to acryla­ mide or aliphatic ,),-diketones. Multidisciplinary investigation of these neurop­ athies in laboratory animals and organotypic tissue cultures has revealed several previously unknown basic mechanisms that may be involved in the maintenance of the axon and the initiation and regulation of the degenerative