Macrophages in CNS Remyelination: Friend or Foe?

Hematogenous macrophages and resident brain microglia are agents of demyelination in multiple sclerosis (MS) and paradoxically may also participate in remyelination. In vitro studies have shown that macrophage enrichment of aggregate brain cultures promotes myelination per se and enhances the capacity to remyelinate following a demyelinating episode. It has been hypothesized that remyelination in MS is implemented by surviving dedifferentiated oligodendrocytes or by newly recruited progenitors that migrate, proliferate and synthesize myelin in response to signalling molecules in the local environment. We postulate that macrophage-derived cytokines or growth factors may directly or indirectly promote oligodendroglial proliferation and differentiation, contributing to myelin repair in inflammatory demyelinating disease.     

Multiple sclerosis - remyelination failure as a cause of disease progression

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system (CNS) that affects worldwide about 2.5 million people. The morphological correlates of the disease are multiple lesions in brain and spinal cord which are characterized by demyelination, inflammation, gliosis and axonal damage. The underlying cause for the permanent neurological deficits in MS patients is axonal loss. Demyelinated axons are prone to damage due to the lack of trophic support by myelin sheaths and oligodendrocytes, as well as the increased vulnerability to immune mediated attacks. Remyelination occurs, but especially in chronic lesions is frequently limited to a small rim at the lesion border. Current treatment strategies are based on anti-inflammatory or immunomodulatory drugs and have the potential to reduce the numbers of newly evolving lesions, although as yet no treatment strategy exists to influence or prevent the progressive disease phase. Therefore, the development of neuroprotective treatment options, such as the promotion of endogenous remyelination is an attractive strategy. A prerequisite for the development of such new treatments is the understanding of the mechanisms leading to remyelination and the reasons for insufficient endogenous repair in chronic MS. This review will therefore provide an overview of the current concepts regarding remyelination in the rodent and human CNS. We will also summarize a selected number of inhibitory pathways and non-disease related factors which may contribute to remyelination failure in chronic MS.     

Gas6 increases myelination by oligodendrocytes and its deficiency delays recovery following cuprizone-induced demyelination

Multiple sclerosis (MS) is a complex demyelinating disease of the central nervous system. Current research has shown that at least in some cases, the primary insult in MS could be directed at the oligodendrocyte, and that the earliest immune responses are primarily via innate immune cells. We have identified a family of receptor protein tyrosine kinases, known as the TAM receptors (Tyro3, Axl and Mertk), as potentially important in regulating both the oligodendrocyte and immune responses. We have previously shown that Gas6, a ligand for the TAM receptors, can affect the severity of demyelination in mice, with a loss of signalling via Gas6 leading to decreased oligodendrocyte survival and increased microglial activation during cuprizone-induced demyelination. We hypothesised TAM receptor signalling would also influence the extent of recovery in mice following demyelination. A significant effect of the absence of Gas6 was detected upon remyelination, with a lower level of myelination after 4 weeks of recovery in comparison with wild-type mice. The delay in remyelination was accompanied by a reduction in oligodendrocyte numbers. To understand the molecular mechanisms that drive the observed effects, we also examined the effect of exogenous Gas6 in in vitro myelination assays. We found that Gas6 significantly increased myelination in a dose-dependent manner, suggesting that TAM receptor signalling could be directly involved in myelination by oligodendrocytes. The reduced rate of remyelination in the absence of Gas6 could thus result from a lack of Gas6 at a critical time during myelin production after injury. These findings establish Gas6 as an important regulator of both CNS demyelination and remyelination.     

Galanin transgenic mice with elevated circulating galanin levels alleviate demyelination in a cuprizone-induced MS mouse model

Multiple Sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS) with a presumed autoimmune etiology. Approved treatments for MS are immunoregulatory and are able to reduce the inflammatory components of the disease. However, these treatments do not suppress progressive clinical disability. Approaches that directly protect myelin-producing oligodendrocytes and enhance remyelination are likely to improve long-term outcomes and reduce the rate of axonal damage. Galanin (GAL) is a bioactive neuropeptide that is widely distributed throughout the nervous system and has diverse neuromodulatory effects. In this study, using the cuprizone (CPZ) demyelination model of MS, we demonstrate that GAL has pronounced neuroprotective effects with respect to demyelination and remyelination. Using our GAL transgenic mouse (GAL-Tg), we identified a novel attenuation of OLs against CPZ induced demyelination, which was exerted independently of progenitor cells. Alleviation of myelin breakdown in the GAL-Tg mice was observed to be significant. Furthermore, we observed changes in the expression of the GAL receptor GalR1 during the demyelination and remyelination processes. Our data strongly indicate that GAL has the capacity to influence the outcome of primary insults that directly target OLs, as opposed to cases where immune activation is the primary pathogenic event. Taken together, these results suggest that GAL is a promising next-generation target for the treatment of MS.     

Phloroglucinol derivative compound 21 attenuates cuprizone-induced multiple sclerosis mice through promoting remyelination and inhibiting neuroinflammation

Multiple sclerosis(MS) is a chronic autoimmune demyelinating disease in the central nervous system. The myelin loss is mainly caused by dysfunction of oligodendrocytes and inflammatory responses of microglia and astrocytes further aggravate the demyelination. Current therapies for MS focus on suppressing the overactivated immune response but cannot halt the disease progress, so effective drugs are urgently needed. Compound 21 is a phloroglucinol derivative that has been proved to have an outstanding anti-inflammatory effect. The purpose of the present study is to investigate whether this novel compound is effective in MS. The cuprizone-induced model was used in this study to mimic the pathological progress of MS. The results showed that Compound 21 significantly improved the neurological dysfunction and motor coordination impairment. Luxol Fast Blue staining and myelin basic protein immunostaining demonstrated that Compound 21 remarkably promoted remyelination. In addition,Compound 21 significantly promoted oligodendrocytes differentiation. Furthermore, we found that Compound 21 decreased microglia and astrocytes activities and the subsequent neuroinflammatory response, indicating that the anti-inflammatory effect of Compound 21 was also involved in its neuro-protection. All the data prove that Compound 21 exerts protective effect on MS through promoting remyelination and suppressing neuroinflammation, indicating that Compound 21 might be a potential drug candidate for MS treatment.     

Minocycline reduces remyelination by suppressing ciliary neurotrophic factor expression after cuprizone‐induced demyelination

Remyelination is disrupted in demyelinating diseases such as multiple sclerosis, but the underlying pathogenetic mechanisms are unclear. In this study, we employed the murine cuprizone model of demyelination, in which remyelination occurs after removal of the toxin from the diet, to examine the cellular and molecular changes during demyelination and remyelination. Microglia accumulated in the corpus callosum during weeks 2–4 of the cuprizone diet, and these cells remained activated 2 weeks after the change to the normal diet. To examine the role of microglia in remyelination, mice were treated with minocycline to inactivate these cells after cuprizone‐induced demyelination. Minocycline treatment reduced the number of CC1‐positive oligodendrocytes, as well as levels of myelin basic protein (MBP) and CNPase in the remyelination phase. The expression of CNTF mRNA in the corpus callosum increased after 4 weeks on the cuprizone diet and remained high 2 weeks after the change to the normal diet. Minocycline suppressed CNTF expression during the remyelination phase on the normal diet. Primary culture experiments showed that CNTF was produced by microglia in addition to astrocytes. In vitro, CNTF directly affected the differentiation of oligodendrocytic cells. These findings suggest that minocycline reduces remyelination by suppressing CNTF expression by microglia after cuprizone‐induced demyelination.     

Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination

Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal role for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis.     

Promotion of Remyelination by Sulfasalazine in a Transgenic Zebrafish Model of Demyelination

Most of the axons in the vertebrate nervous system are surrounded by a lipid-rich membrane called myelin, which promotes rapid conduction of nerve impulses and protects the axon from being damaged. Multiple sclerosis (MS) is a chronic demyelinating disease of the CNS characterized by infiltration of immune cells and progressive damage to myelin and axons. One potential way to treat MS is to enhance the endogenous remyelination process, but at present there are no available treatments to promote remyelination in patients with demyelinating diseases.Sulfasalazine is an anti-inflammatory and immune-modulating drug that is used in rheumatology and inflammatory bowel disease. Its anti-inflammatory and immunomodulatory properties prompted us to test the ability of sulfasalazine to promote remyelination. In this study, we found that sulfasalazine promotes remyelination in the CNS of a transgenic zebrafish model of NTR/MTZ-induced demyelination. We also found that sulfasalazine treatment reduced the number of macrophages/microglia in the CNS of demyelinated zebrafish larvae, suggesting that the acceleration of remyelination is mediated by the immunomodulatory function of sulfasalazine. Our data suggest that temporal modulation of the immune response by sulfasalazine can be used to overcome MS by enhancing myelin repair and remyelination in the CNS.     

Promoting myelin repair and return of function in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS. Conduction block in demyelinated axons underlies early neurological symptoms, but axonal transection and neuronal loss are believed to be responsible for more permanent chronic deficits. Several therapies are approved for treatment of relapsing-remitting MS, all of which are immunoregulatory and clinically proven to reduce the rate of lesion formation and exacerbation. However, existing approaches are only partially effective in preventing the onset of disability in MS patients, and novel treatments to protect myelin-producing oligodendrocytes and enhance myelin repair may improve long-term outcomes. Studies in vivo in genetically modified mice have assisted in the characterization of mechanisms underlying the generation of neuropathology in MS patients, and have identified potential avenues for oligodendrocyte protection and myelin repair. However, no treatments are yet approved that target these areas directly, and in addition, the relationship between demyelination and axonal transection in the lesions of the disease remains unclear. Here, we review translational research targeting oligodendrocyte protection and myelin repair in models of autoimmune demyelination, and their potential relevance as therapies in MS.     

Glia Disease and Repair—Remyelination

The inability of the mammalian central nervous system (CNS) to undergo spontaneous regeneration has long been regarded as a central tenet of neurobiology. However, although this is largely true of the neuronal elements of the adult mammalian CNS, save for discrete populations of granular neurons, the same is not true of its glial elements. In particular, the loss of oligodendrocytes, which results in demyelination, triggers a spontaneous and often highly efficient regenerative response, remyelination, in which new oligodendrocytes are generated and myelin sheaths are restored to denuded axons. Yet, remyelination in humans is not without limitation, and a variety of demyelinating conditions are associated with sustained and disabling myelin loss. In this review, we will review the biology of remyelination, including the cells and signals involved; describe when remyelination occurs and when and why it fails and the consequences of its failure; and discuss approaches for therapeutically enhancing remyelination in demyelinating diseases of both children and adults, both by stimulating endogenous oligodendrocyte progenitor cells and by transplanting these cells into demyelinated brain.     

Vitamins E and D3 Attenuate Demyelination and Potentiate Remyelination Processes of Hippocampal Formation of Rats Following Local Injection of Ethidium Bromide

Cognitive deficits have been observed in patients with multiple sclerosis (MS) due to hippocampal insults. Antioxidant vitamins D and E are suggested for patients suffering from neurodegenerative diseases like MS, while their mechanisms of action are not well understood. Here, we have tried to study the effects of these vitamins on demyelination, cell death, and remyelination of rat hippocampus following local ethidium bromide (EB) injection. Animals received 100 mg/kg vitamin E or 5 μg/kg of vitamin D3 for 2, 7, or 28 days. The extent of demyelination, myelin staining intensity, and expression of myelin basic protein and caspase-3 were investigated using histological and immunoblotting verification. Administration of EB alone caused demyelination, cell death, and afterward an endogenous repair. Vitamins E and D3 reduced the EB-induced damage and increased the endogenous remyelination of hippocampus. Although the anti-apoptotic effect of these vitamins and protection against demyelination were predictable based on their antioxidant effect, our results indicated the positive effect of vitamins E and D3 on process of remyelination by endogenous progenitor cells and supported their possible therapeutic effects in the context of demyelinating diseases like MS.     

Oligodendrocytes assist in the maintenance of sodium channel clusters independent of the myelin sheath

To ensure rapid and efficient impulse conduction, myelinated axons establish and maintain specific protein domains. For instance, sodium (Na+) channels accumulate in the node of Ranvier; potassium (K+) channels aggregate in the juxtaparanode and neurexin/caspr/paranodin clusters in the paranode. Our understanding of the mechanisms that control the initial clustering of these proteins is limited and less is known about domain maintenance. Correlative data indicate that myelin formation and/or mature myelin-forming cells mediate formation of all three domains. Here, we test whether myelin is required for maintaining Na+ channel domains in the nodal gap by employing two demyelinating murine models: (1) cuprizone ingestion, which induces complete demyelination through oligodendrocyte toxicity; and (2) ceramide galactosyltransferase deficient mice, which undergo spontaneous adult-onset demyelination without oligodendrocyte death. Our data indicate that the myelin sheath is essential for long-term maintenance of sodium channel domains; however, oligodendrocytes, independent of myelin, provide a partial protective influence on the maintenance of nodal Na+ channel clusters. Thus, we propose that multiple mechanisms regulate the maintenance of nodal protein organization. Finally, we present evidence that following the loss of Na+ channel clusters the chronological progression of expression and reclustering of Na+ channel isoforms during the course of CNS remyelination recapitulates development.     

Nutritional factors and aging in demyelinating diseases

Demyelination is a pathological process characterized by the loss of myelin around axons. In the central nervous system, oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Remyelination is a regenerative process by which myelin sheaths are restored to demyelinated axons, resolving functional deficits. This process is often deficient in demyelinating diseases such as multiple sclerosis (MS), and the reasons for the failure of repair mechanisms remain unclear. The characterization of these mechanisms and the factors involved in the proliferation, recruitment, and differentiation of oligodendroglial progenitor cells is key in designing strategies to improve remyelination in demyelinating disorders. First, a very dynamic combination of different molecules such as growth factors, cytokines, chemokines, and different signaling pathways is tightly regulated during the remyelination process. Second, factors unrelated to this pathology, i.e., age and genetic background, may impact disease progression either positively or negatively, and in particular, age-related remyelination failure has been proven to involve oligodendroglial cells aging and their intrinsic capacities among other factors. Third, nutrients may either help or hinder disease progression. Experimental evidence supports the anti-inflammatory role of omega-6 and omega-3 polyunsaturated fatty acids through the competitive inhibition of arachidonic acid, whose metabolites participate in inflammation, and the reduction in T cell proliferation. In turn, vitamin D intake and synthesis have been associated with lower MS incidence levels, while vitamin D–gene interactions might be involved in the pathogenesis of MS. Finally, dietary polyphenols have been reported to mitigate demyelination by modulating the immune response.     

Remyelination induced by a DNA aptamer in a mouse model of multiple sclerosis

Multiple sclerosis (MS) is a debilitating inflammatory disease of the central nervous system (CNS) characterized by local destruction of the insulating myelin surrounding neuronal axons. With more than 200 million MS patients worldwide, the absence of treatments that prevent progression or induce repair poses a major challenge. Anti-inflammatory therapies have met with limited success only in preventing relapses. Previous screening of human serum samples revealed natural IgM antibodies that bind oligodendrocytes and promote both cell signaling and remyelination of CNS lesions in an MS model involving chronic infection of susceptible mice by Theiler's encephalomyelitis virus and in the lysolecithin model of focal demyelination. This intriguing result raises the possibility that molecules with binding specificity for oligodendrocytes or myelin components may promote therapeutic remyelination in MS. Because of the size and complexity of IgM antibodies, it is of interest to identify smaller myelin-specific molecules with the ability to promote remyelination in vivo. Here we show that a 40-nucleotide single-stranded DNA aptamer selected for affinity to murine myelin shows this property. This aptamer binds multiple myelin components in vitro. Peritoneal injection of this aptamer results in distribution to CNS tissues and promotes remyelination of CNS lesions in mice infected by Theiler's virus. Interestingly, the selected DNA aptamer contains guanosine-rich sequences predicted to induce folding involving guanosine quartet structures. Relative to monoclonal antibodies, DNA aptamers are small, stable, and non-immunogenic, suggesting new possibilities for MS treatment.     

Geissoschizine Methyl Ether, an Alkaloid from the Uncaria Hook, Improves Remyelination After Cuprizone-Induced Demyelination in Medial Prefrontal Cortex of Adult Mice

Accumulating evidence indicates that the medial prefrontal cortex (mPFC) is a site of myelin and oligodendrocyte abnormalities that contribute to psychotic symptoms of schizophrenia. The development of therapeutic approaches to enhance remyelination, a regenerative process in which new myelin sheaths are formed on demyelinated axons, may be an attractive remedial strategy. Geissoschizine methyl ether (GM) in the Uncaria hook, a galenical constituent of the traditional Japanese medicine yokukansan (Yi-gan san), is one of the active components responsible for the psychotropic effects of yokukansan, though little is known about the mechanisms underlying the effects of either that medicine or GM itself. In the present study, we employed a cuprizone (CPZ)-induced demyelination model and examined the cellular changes in response to GM administration during the remyelination phase in the mPFC of adult mice. Using the mitotic marker 5-bromo-2′-deoxyuridine (BrdU), we demonstrated that CPZ treatment significantly increased the number of BrdU-positive NG2 cells, as well as microglia and mature oligodendrocytes in the mPFC. Newly formed oligodendrocytes were increased by GM administration after CPZ exposure. In addition, GM attenuated a decrease in myelin basic protein immunoreactivity caused by CPZ administration. Taken together, our findings suggest that GM administration ameliorated the myelin deficit by mature oligodendrocyte formation and remyelination in the mPFC of CPZ-fed mice. The present findings provide experimental evidence supporting the role for GM and its possible use as a remedy for schizophrenia symptoms by promoting the differentiation of progenitor cells to and myelination by oligodendrocytes.     

Antibody-mediated CNS demyelination II. Focal spinal cord lesions induced by implantation of an IgM antisulfatide-secreting hybridoma

We showed previously that spinal cord implants of hybridoma cells (O1) that secrete an IgM antigalactocerebroside cause focal multiple-sclerosis-like plaques of demyelination followed by remyelination to form “shadow plaques” (Rosenbluth et al., 1999). The antibody in that case was directed against a glycolipid present in mature oligodendrocytes and myelin but not in precursor cells. We now report the effects of implanting a different hybridoma (O4) that secretes IgM antibodies directed against sulfatide, a constituent not only of mature myelin and oligodendrocytes but also of late precursor cells, in order to determine whether this hybridoma too would generate focal demyelination and would, in addition, block remyelination. Our results show that focal plaques of demyelination indeed appear after O4 implantation, and that remyelination does occur, but only in cases where the hybridoma cells have degenerated, probably through host rejection. The occurrence of remyelination suggests that oligodendrocyte precursor cells are capable of migrating in rapidly from adjacent areas or that early precursors, not yet expressing sulfatide, remain undamaged within the lesions. In cases where intact hybridoma cells persist at lesion sites, remyelination does not occur. Failure of remyelination in this model thus appears to result from the continuing presence of antimyelin antibodies rather than from depletion of oligodendrocyte precursors.     

Generation of demyelination models by targeted ablation of oligodendrocytes in the zebrafish CNS

Demyelination is the pathological process by which myelin sheaths are lost from around axons, and is usually caused by a direct insult targeted at the oligodendrocytes in the vertebrate central nervous system (CNS). A demyelinated CNS is usually remyelinated by a population of oligodendrocyte progenitor cells, which are widely distributed throughout the adult CNS. However, myelin disruption and remyelination failure affect the normal function of the nervous system, causing human diseases such as multiple sclerosis. In spite of numerous studies aimed at understanding the remyelination process, many questions still remain unanswered. Therefore, to study remyelination mechanisms in vivo, a demyelination animal model was generated using a transgenic zebrafish system in which oligodendrocytes are conditionally ablated in the larval and adult CNS. In this transgenic system, bacterial nitroreductase enzyme (NTR), which converts the prodrug metronidazole (Mtz) into a cytotoxic DNA cross-linking agent, is expressed in oligodendrocyte lineage cells under the control of the mbp and sox10 promoter. Exposure of transgenic zebrafish to Mtz-containing media resulted in rapid ablation of oligodendrocytes and CNS demyelination within 48 h, but removal of Mtz medium led to efficient remyelination of the demyelinated CNS within 7 days. In addition, the demyelination and remyelination processes could be easily observed in living transgenic zebrafish by detecting the fluorescent protein, mCherry, indicating that this transgenic system can be used as a valuable animal model to study the remyelination process in vivo, and to conduct high-throughput primary screens for new drugs that facilitate remyelination.     

Direct visualization of membrane architecture of myelinating cells in transgenic mice expressing membrane‐anchored EGFP

Myelinogenesis is a complex process that involves substantial and dynamic changes in plasma membrane architecture and myelin interaction with axons. Highly ramified processes of oligodendrocytes in the central nervous system (CNS) make axonal contact and then extrapolate to wrap around axons and form multilayer compact myelin sheathes. Currently, the mechanisms governing myelin sheath assembly and axon selection by myelinating cells are not fully understood. Here, we generated a transgenic mouse line expressing the membrane‐anchored green fluorescent protein (mEGFP) in myelinating cells, which allow live imaging of details of myelinogenesis and cellular behaviors in the nervous systems. mEGFP expression is driven by the promoter of 2'‐3'‐cyclic nucleotide 3'‐phosphodiesterase (CNP) that is expressed in the myelinating cell lineage. Robust mEGFP signals appear in the membrane processes of oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system (PNS), wherein mEGFP expression defines the inner layers of myelin sheaths and Schmidt‐Lanterman incisures in adult sciatic nerves. In addition, mEGFP expression can be used to track the extent of remyelination after demyelinating injury in a toxin‐induced demyelination animal model. Taken together, the membrane‐anchored mEGFP expression in the new transgenic line would facilitate direct visualization of dynamic myelin membrane formation and assembly during development and process remodeling during remyelination after various demyelinating injuries. genesis 52:341–349, 2014. © 2014 Wiley Periodicals, Inc.     

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.