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.     

Mechanisms of immunopathology in murine models of central nervous system demyelinating disease

Many disorders of the CNS, such as multiple sclerosis (MS), are characterized by the loss of the myelin sheath surrounding nerve axons. MS is associated with infiltration of inflammatory cells into the brain and spinal cord, which may be the primary cause of demyelination or which may be induced secondary to axonal damage. Both the innate and adaptive arms of the immune system have been reported to play important roles in myelin destruction. Numerous murine demyelinating models, both virus-induced and/or autoimmune, are available, which reflect the clinical and pathological variability seen in human disease. This review will discuss the immunopathologic mechanisms involved in these demyelinating disease models.     

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.     

Oxydative phosphorylation in sciatic nerve myelin and its impairment in a model of dysmyelinating peripheral neuropathy

Myelin sheath is the proteolipid membrane wrapping the axons of CNS and PNS. We have shown data suggesting that CNS myelin conducts oxidative phosphorylation (OXPHOS), challenging its role in limiting the axonal energy expenditure. Here, we focused on PNS myelin. Samples were: (i) isolated myelin vesicles (IMV) from sciatic nerves, (ii) mitochondria from primary Schwann cell cultures, and (iii) sciatic nerve sections, from wild type or Charcot‐Marie‐Tooth type 1A (CMT1A) rats. The latter used as a model of dys‐demyelination. O₂ consumption and activity of OXPHOS proteins from wild type (Wt) or CMT1A sciatic nerves showed some differences. In particular, O₂ consumption by IMV from Wt and CMT1A 1‐month‐old rats was comparable, while it was severely impaired in IMV from adult affected animals. Mitochondria extracted from CMT1A Schwann cell did not show any dysfunction. Transmission electron microscopy studies demonstrated an increased mitochondrial density in dys‐demyelinated axons, as to compensate for the loss of respiration by myelin. Confocal immunohistochemistry showed the expression of OXPHOS proteins in the myelin sheath, both in Wt and dys‐demyelinated nerves. These revealed an abnormal morphology. Taken together these results support the idea that also PNS myelin conducts OXPHOS to sustain axonal function.     

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.     

Axonal pathology in myelin disorders

Myelination provides extrinsic trophic signals that influence normal maturation and long-term survival of axons. The extent of axonal involvement in diseases affecting myelin or myelin forming cells has traditionally been underestimated. There are, however, many examples of axon damage as a consequence of dysmyelinating or demyelinating disorders. More than a century ago, Charcot described the pathology of multiple sclerosis (MS) in terms of demyelination and relative sparing of axons. Recent reports demonstrate a strong correlation between inflammatory demyelination in MS lesions and axonal transection, indicating axonal loss at disease onset. Disruption of axons is also observed in experimental allergic encephalomyelitis and in Theiler's murine encephalomyelitis virus disease, two animal models of inflammatory demyelinating CNS disease. A number of dysmyelinating mouse mutants with axonal pathology have provided insights regarding cellular and molecular mechanisms of axon degeneration. For example, the myelin-associated glycoprotein and proteolipid protein have been shown to be essential for mediating myelin-derived trophic signals to axons. Patients with the inherited peripheral neuropathy Charcot-Marie Tooth disease type 1 develop symptomatic progressive axonal loss due to abnormal Schwann cell expression of peripheral myelin protein 22. The data summarized in this review indicate that axonal damage is an integral part of myelin disease, and that loss of axons contributes to the irreversible functional impairment observed in affected individuals. Early neuroprotection should be considered as an additional therapeutic option for these patients.     

Glial grafting for demyelinating disease

Remyelination of demyelinated central nervous system (CNS) axons is considered as a potential treatment for multiple sclerosis, and it has been achieved in experimental models of demyelination by transplantation of pro-myelinating cells. However, the experiments undertaken have not addressed the need for tissue-type matching in order to achieve graft-mediated remyelination since they were performed in conditions in which the chance for graft rejection was minimized. This article focuses on the factors determining survival of allogeneic oligodendrocyte lineage cells and their contribution to the remyelination of demyelinating CNS lesions. The immune status of the CNS as well as the suitability of different models of demyelination for graft rejection studies are discussed, and ways of enhancing allogeneic oligodendrocyte-mediated remyelination are presented. Finally, the effects of glial graft rejection on host remyelination are described, highlighting the potential benefits of the acute CNS inflammatory response for myelin repair.     

Remyelination in the CNS: from biology to therapy

Remyelination involves reinvesting demyelinated axons with new myelin sheaths. In stark contrast to the situation that follows loss of neurons or axonal damage, remyelination in the CNS can be a highly effective regenerative process. It is mediated by a population of precursor cells called oligodendrocyte precursor cells (OPCs), which are widely distributed throughout the adult CNS. However, despite its efficiency in experimental models and in some clinical diseases, remyelination is often inadequate in demyelinating diseases such as multiple sclerosis (MS), the most common demyelinating disease and a cause of neurological disability in young adults. The failure of remyelination has profound consequences for the health of axons, the progressive and irreversible loss of which accounts for the progressive nature of these diseases. The mechanisms of remyelination therefore provide critical clues for regeneration biologists that help them to determine why remyelination fails in MS and in other demyelinating diseases and how it might be enhanced therapeutically.     

Exogenous Schwann Cells Migrate,Remyelinate and Promote Clinical Recovery in Experimental Auto-Immune Encephalomyelitis

Schwann cell (SC) transplantation is currently being discussed as a strategy that may promote functional recovery in patients with multiple sclerosis (MS) and other inflammatory demyelinating diseases of the central nervous system (CNS). However this assumes they will not only survive but also remyelinate demyelinated axons in the chronically inflamed CNS. To address this question we investigated the fate of transplanted SCs in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in the Dark Agouti rat; an animal model that reproduces the complex inflammatory demyelinating immunopathology of MS. We now report that SCs expressing green fluorescent protein (GFP-SCs) allografted after disease onset not only survive but also migrate to remyelinate lesions in the inflamed CNS. GFP-SCs were detected more frequently in the parenchyma after direct injection into the spinal cord, than via intra-thecal delivery into the cerebrospinal fluid. In both cases the transplanted cells intermingled with astrocytes in demyelinated lesions, aligned with axons and by twenty one days post transplantation had formed Pzero protein immunoreactive internodes. Strikingly, GFP-SCs transplantation was associated with marked decrease in clinical disease severity in terms of mortality; all GFP-SCs transplanted animals survived whilst 80% of controls died within 40 days of disease.     

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.     

IL-17A Promotes Granulocyte Infiltration,Myelin Loss,Microglia Activation,and Behavioral Deficits During Cuprizone-Induced Demyelination

Recent evidence suggests a pivotal role of the proinflammatory cytokine interleukin - 17A (IL-17) in demyelinating autoimmune diseases of the central nervous system (CNS) such as multiple sclerosis (MS). Nevertheless, it remains unclear if this cytokine exerts direct effects on CNS resident cells during MS or modulates the function of infiltrating immune cells towards a more detrimental phenotype. Here, we investigated the effects of locally produced IL-17 during experimental demyelination of the CNS using the cuprizone (CPZ) model in mice with (GF/IL17) or without transgenic production of IL-17 by astrocytes in the CNS. During early demyelination, GF/IL17 mice demonstrated enhanced activity and decreased anxiety-related behavior in the elevated plus maze suggesting a more severe disease course. Furthermore, in GF/IL17 mice, toxic demyelination was accelerated and synthesis of myelin proteins was reduced. Early demyelination was accompanied by an increased ratio of infiltrating granulocytes in GF/ILl17 mice. The presence of IL-17 during CPZ treatment increased the accumulation of activated microglia and sustained microglial proliferation during myelin loss. Taken together, our results argue for a detrimental role of IL-17 during demyelinating diseases.     

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.     

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.     

Mediators of oligodendrocyte differentiation during remyelination

Myelin, a dielectric sheath that wraps large axons in the central and peripheral nervous systems, is essential for proper conductance of axon potentials. In multiple sclerosis (MS), autoimmune-mediated damage to myelin within the central nervous system (CNS) leads to progressive disability primarily due to limited endogenous repair of demyelination with associated axonal pathology. While treatments are available to limit demyelination, no treatments are available to promote myelin repair. Studies examining the molecular mechanisms that promote remyelination are therefore essential for identifying therapeutic targets to promote myelin repair and thereby limit disability in MS. Here, we present our current understanding of the critical extracellular and intracellular pathways that regulate the remyelinating capabilities of oligodendrocyte precursor cells (OPCs) within the adult CNS.     

An animal model for late onset chronic demyelination disease caused by failed terminal differentiation of oligodendrocytes

Various animal models are available for studying human multiple sclerosis (MS). Most of them model the initial phase of MS,including the immune-triggered attack of the myelin membrane and/or oligodendrocytes and, occasionally, demonstrate there mission and relapsing phases. However, few mimic the late chronic demyelinating phase. Overexpression of the proteolipid protein gene (Plp) causes a unique demyelinating disorder in mice in which normal-appearing myelin forms early in life and chronic demyelination occurs later. We found that remyelination is severely affected in this late demyelinating phase, but is not caused by deprivation of oligodendrocyte progenitors expressing PDGF receptor alpha (PDGFRa) and Olig2, which are present at an even higher number in the demyelinated white matter of the mutants than in wild-type controls. Furthermore, mature oligodendrocytes containing PLP were observed, but failed to remyelinate. The ability of oligodendrocytes from older transgenic animals to produce a myelin membrane-like structure was not impaired when cultured in vitro, which indicates that the lack of remyelination is not simply caused by changes in the intrinsic properties of the oligodendrocytes. Glial activation also occurred much earlier than active demyelination in mutant mice. Thus, in addition to intrinsic mechanisms, extrinsic mechanisms might also have an important role in defects of remyelination. These features are also observed in patients at a late stage of MS, leading to chronic demyelinating lesions. Thus, this mouse model partly mimics the late stage of MS and can be used to study the cause of inhibition of remyelination.     

Amyloid precursor protein at node of Ranvier modulates nodal formation

Amyloid precursor protein (APP), commonly associated with Alzheimer disease, is upregulated and distributes evenly along the injured axons, and therefore, also known as a marker of demyelinating axonal injury and axonal degeneration. However, the physiological distribution and function of APP along myelinated axons was unknown. We report that APP aggregates at nodes of Ranvier (NOR) in the myelinated central nervous system (CNS) axons but not in the peripheral nervous system (PNS). At CNS NORs, APP expression co-localizes with tenascin-R and is flanked by juxtaparanodal potassium channel expression demonstrating that APP localized to NOR. In APP-knockout (KO) mice, nodal length is significantly increased, while sodium channels are still clustered at NORs. Moreover, APP KO and APP-overexpressing transgenic (APP TG) mice exhibited a decreased and an increased thickness of myelin in spinal cords, respectively, although the changes are limited in comparison to their littermate WT mice. The thickness of myelin in APP KO sciatic nerve also increased in comparison to that in WT mice. Our observations indicate that APP acts as a novel component at CNS NORs, modulating nodal formation and has minor effects in promoting myelination.     

Synergistic activity of interleukin-17 and tumor necrosis factor-α enhances oxidative stress-mediated oligodendrocyte apoptosis

Th1 cytokine-induced loss of oligodendrocytes (OLs) is associated with axonal loss in CNS demyelinating diseases such as multiple sclerosis (MS)that contributes to neurological disabilities in affected individuals. Recent studies indicated that, in addition to Th1-phenotype cytokines including tumor necrosis factor (TNF)-α, Th17 phenotype cytokine, interleukin (IL)-17 also involved in the development of MS. In this study, we investigated the direct effect of IL-17 on the survival of OLs in the presence of TNF-α and individually in vitro settings. Our findings suggest that IL-17 alone, however, was not able to affect the survival of OLs, but it exacerbates the TNF-α-induced OL apoptosis as compared with individual TNF-α treatment. This effect of cytokines was ascribed to an inhibition of cell-survival mechanisms, co-localization of Bid/Bax proteins in the mitochondrial membrane and caspase 8 activation mediated release of apoptosis inducing factor from mitochondria in treated OLs. In addition, cytokine treatment disturbed the mitochondrial membrane potential in OLs with corresponding increase in the generation of reactive oxygen species, which were attenuated by N-acetyl cysteine treatment. In addition, combining of these cytokines induced cell-cycle arrest at G1/S phases in OL-like cells and inhibited the maturation of OL progenitor cells that was attenuated by peroxisome proliferator-activated receptor-γ/-β agonists. Collectively, these data provide initial evidence that IL-17 exacerbates TNF-α-induced OL loss and inhibits the differentiation of OL progenitor cells suggesting that antioxidant- or peroxisome proliferator-activated receptor agonist-based therapies have potential to limit CNS demyelination in MS or other related demyelinating disorders.     

Demyelination induces transport of ribosome-containing vesicles from glia to axons: evidence from animal models and MS patient brains

Glial cells were previously proven capable of trafficking polyribosomes to injured axons. However, the occurrence of such transfer in the general pathological context, such as demyelination-related diseases, needs further evidence. Since this may be a yet unidentified universal contributor to axonal survival, we study putative glia–axonal ribosome transport in response to demyelination in animal models and patients in both peripheral and central nervous system. In the PNS we investigate whether demyelination in a rodent model has the potential to induce ribosome transfer. We also probe the glia–axonal ribosome supply by implantation of transgenic Schwann cells engineered to produce fluorescent ribosomes in the same demyelination model. We furthermore examine the presence of axonal ribosomes in mouse experimental autoimmune encephalomyelitis (EAE), a well-established model for multiple sclerosis (MS), and in human MS autopsy brain material. We provide evidence for increased axonal ribosome content in a pharmacologically demyelinated sciatic nerve, and demonstrate that at least part of these ribosomes originate in the transgenic Schwann cells. In the CNS one of the hallmarks of MS is demyelination, which is associated with severe disruption of oligodendrocyte–axon interaction. Here, we provide evidence that axons from spinal cords of EAE mice, and in the MS human brain contain an elevated amount of axonal ribosomes compared to controls. Our data provide evidence that increased axonal ribosome content in pathological axons is at least partly due to glia-to-axon transfer of ribosomes, and that demyelination in the PNS and in the CNS is one of the triggers capable to initiate this process.     

Galectin-3 drives oligodendrocyte differentiation to control myelin integrity and function

Galectins control critical pathophysiological processes, including the progression and resolution of central nervous system (CNS) inflammation. In spite of considerable progress in dissecting their role within lymphoid organs, their functions within the inflamed CNS remain elusive. Here, we investigated the role of galectin-glycan interactions in the control of oligodendrocyte (OLG) differentiation, myelin integrity and function. Both galectin-1 and -3 were abundant in astrocytes and microglia. Although galectin-1 was abundant in immature but not in differentiated OLGs, galectin-3 was upregulated during OLG differentiation. Biochemical analysis revealed increased activity of metalloproteinases responsible for cleaving galectin-3 during OLG differentiation and modulating its biological activity. Exposure to galectin-3 promoted OLG differentiation in a dose- and carbohydrate-dependent fashion consistent with the 'glycosylation signature' of immature versus differentiated OLG. Accordingly, conditioned media from galectin-3-expressing, but not galectin-3-deficient (Lgals3(-/-)) microglia, successfully promoted OLG differentiation. Supporting these findings, morphometric analysis showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio in the corpus callosum and striatum of Lgals3(-/-) compared with wild-type (WT) mice. Moreover, the myelin structure was loosely wrapped around the axons and less smooth in Lgals3(-/-) mice versus WT mice. Behavior analysis revealed decreased anxiety in Lgals3(-/-) mice similar to that observed during early demyelination induced by cuprizone intoxication. Finally, commitment toward the oligodendroglial fate was favored in neurospheres isolated from WT but not Lgals3(-/-) mice. Hence, glial-derived galectin-3, but not galectin-1, promotes OLG differentiation, thus contributing to myelin integrity and function with critical implications in the recovery of inflammatory demyelinating disorders.     

Cdk2 loss accelerates precursor differentiation and remyelination in the adult central nervous system

The specific functions of intrinsic regulators of oligodendrocyte progenitor cell (OPC) division are poorly understood. Type 2 cyclin-dependent kinase (Cdk2) controls cell cycle progression of OPCs, but whether it acts during myelination and repair of demyelinating lesions remains unexplored. Here, we took advantage of a viable Cdk2(-/-) mutant mouse to investigate the function of this cell cycle regulator in OPC proliferation and differentiation in normal and pathological conditions. During central nervous system (CNS) development, Cdk2 loss does not affect OPC cell cycle, oligodendrocyte cell numbers, or myelination. However, in response to CNS demyelination, it clearly alters adult OPC renewal, cell cycle exit, and differentiation. Importantly, Cdk2 loss accelerates CNS remyelination of demyelinated axons. Thus, Cdk2 is dispensable for myelination but is important for adult OPC renewal, and could be one of the underlying mechanisms that drive adult progenitors to differentiate and thus regenerate myelin.