Thyroid hormone alleviates demyelination induced by cuprizone through its role in remyelination during the remission period

Multiple sclerosis (MS) is a disease induced by demyelination in the central nervous system, and the remission period of MS is crucial for remyelination. In addition, abnormal levels of thyroid hormone (TH) have been identified in MS. However, in the clinic, insufficient attention has been paid to the role of TH in the remission period. Indeed, TH not only functions in the development of the brain but also affects myelination. Therefore, it is necessary to observe the effect of TH on remyelination during this period. A model of demyelination induced by cuprizone (CPZ) was used to observe the function of TH in remyelination during the remission period of MS. Through weighing and behavioral tests, we found that TH improved the physical symptoms of mice impaired by CPZ. Supplementation of TH led to the repair of myelin as detected by immunohistochemistry and western blot. In addition, a sufficient TH supply resulted in an increase in myelinated axons without affecting myelin thickness and g ratio in the corpus callosum, as detected by electron microscopy. Double immunostaining with myelin basic protein and neurofilament 200 (NF200) showed that the CPZ-induced impairment of axons was alleviated by TH. Conversely, insufficient TH induced by 6-propyl-2-thiouracil resulted in the enlargement of mitochondria. Furthermore, we found that an adequate supply of TH promoted the proliferation and differentiation of oligodendrocyte lineage cells by immunofluorescence, which was beneficial to remyelination. Further, we found that TH reduced the number of astrocytes without affecting microglia. Conclusively, it was shown that TH alleviated demyelination induced by CPZ by promoting the development of oligodendrocyte lineage cells and remyelination. The critical time for remyelination is the remission period of MS. TH plays a significant role in alleviating demyelination during the remission period in the clinical treatment of MS.     

The remyelination Philosopher's Stone: stem and progenitor cell therapies for multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease that leads to oligodendrocyte loss and subsequent demyelination of the adult central nervous system (CNS). The pathology is characterized by transient phases of recovery during which remyelination can occur as a result of resident oligodendroglial precursor and stem/progenitor cell activation. However, myelin repair efficiency remains low urging the development of new therapeutical approaches that promote remyelination activities. Current MS treatments target primarily the immune system in order to reduce the relapse rate and the formation of inflammatory lesions, whereas no therapies exist in order to regenerate damaged myelin sheaths. During the last few years, several transplantation studies have been conducted with adult neural stem/progenitor cells and glial precursor cells to evaluate their potential to generate mature oligodendrocytes that can remyelinate axons. In parallel, modulation of the endogenous progenitor niche by neural and mesenchymal stem cell transplantation with the aim of promoting CNS progenitor differentiation and myelination has been studied. Here, we summarize these findings and discuss the properties and consequences of the various molecular and cell-mediated remyelination approaches. Moreover, we address age-associated intrinsic cellular changes that might influence the regenerative outcome. We also evaluate the extent to which these experimental treatments might increase the regeneration capacity of the demyelinated human CNS and hence be turned into future therapies.     

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.     

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.     

Connexins-Mediated Glia Networking Impacts Myelination and Remyelination in the Central Nervous System

In the central nervous system (CNS), the glial gap junctions are established among astrocytes (ASTs), oligodendrocytes (OLs), and/or between ASTs and OLs due to the expression of membrane proteins called connexins (Cxs). Together, the glial cells form a network of communicating cells that is important for the homeostasis of brain function for its involvement in the intercellular calcium wave propagation, exchange of metabolic substrates, cell proliferation, migration, and differentiation. Alternatively, Cxs are also involved in hemichannel function and thus participate in gliotransmission. In recent years, pathologic changes of oligodendroglia or demyelination found in transgenic mice with different subsets of Cxs or pharmacological insults suggest that glial Cxs may participate in the regulation of the myelination or remyelination processes. However, little is known about the underlying mechanisms. In this review, we will mainly focus on the functions of Cx-mediated gap junction channels, as well as hemichannels, in brain glial cells and discuss the way by which they impact myelination and remyelination. These aspects will be considered at the light of recent genetic and non-genetic studies related to demyelination and remyelination.     

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.     

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.     

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.     

Castration attenuates myelin repair following lysolecithin induced demyelination in rat optic chiasm: an evaluation using visual evoked potential, marker genes expression and myelin staining

Multiple sclerosis (MS) is a demyelinating disease that affects the central nervous system. MS is the most common neurological disorder in young adults with a greater incidence among females. Male gonadal hormones have a protective effect on neural system development and myelin maturation. In this study, we investigate the effect of castration on lysolecithin-induced demyelination and remyelination processes using visual evoked potentials, in addition to measuring the expressions of Olig2, MBP, Nogo-A and GFAP mRNAs as oligodendrocyte or astrocyte markers; and histological assessments by myelin-specific staining. We observed more expanded demyelination with delayed repair process in castrated rats. Expression levels of the aforementioned marker genes confirmed histological and electrophysiological observations. Our results showed a pivotal role for endogenous male hormones in the context of demyelinating insults. It may also account for the different prognosis of MS between male and female genders and provide new insights for therapeutic treatments.     

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.     

p53 Induction by Tumor Necrosis Factor-α and Involvement of p53 in Cell Death of Human Oligodendrocytes

Oligodendrocytes (OLs) and their myelin membranes are the primary targets in the autoimmune disease multiple sclerosis (MS). The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) has been implicated as a mediator of OL cell injury. TNF-alpha is detectable within MS lesions and induces apoptosis of mature human OLs in vitro. One possible mechanism by which TNF-alpha mediates cell death is through the activation of c-jun N-terminal kinase (JNK). We have previously shown that treatment of human OLs with TNF-alpha leads to activation of JNK. Here we provide evidence that p53, a regulator of the cell cycle and apoptosis, is a mediator of TNF-alpha-induced apoptosis of OLs. Although p53 was undetectable by western blot analysis in adult human OLs, its levels increased within 24 h after TNF-alpha treatment (100 ng/ml). The induced p53 was immunolocalized to the nucleus prior to the appearance of significant numbers of apoptotic cells. Overexpression of p53 by adenovirus-mediated gene transfer into human OLs in vitro resulted in marked apoptosis as revealed by in situ cleavage of DNA (TUNEL positive), decreased mitochondrial function, and release of lactate dehydrogenase into the culture medium. These in vitro studies demonstrate that increased p53 levels are associated with apoptosis of human OLs. The findings further implicate p53 as a target for the JNK pathway activated during TNF-alpha-mediated cell death of human adult OLs.     

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.     

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.     

Mesenchymal stem cell mediated effects on microglial phenotype in cuprizone-induced demyelination model

Microglial cells have an essential role in neurodegenerative disorders, such as multiple sclerosis. They are divided into two subgroups: M1 and M2 phenotypes. Mesenchymal stem cells (MSC), with neuroprotective and immunomodulating properties, could improve these diseases. We evaluate the immunomodulating effects of MSC on microglial phenotypes and the improvement of demyelination in a cuprizone (CPZ) model of multiple sclerosis (MS). For inducing the chronic demyelination model, C57BL6 mice were given a diet with 0.2% CPZ (w/w) for 12 weeks. In the MSC group, cells were transplanted into the right lateral ventricle of mice. The expression of targeted genes was assessed by real-time polymerase chain reaction. M1 and M2 microglial phenotypes were assessed by immunohistochemistry of inducible nitric oxide synthase (iNOS) and Arg-1, respectively. Remyelination was studied by luxal fast blue (LFB) staining and electron microscopy (EM). We found that MSC transplantation reduced the expression level of M1-specific messenger RNA (mRNA; iNOS and CD86) but increased the expression level of M2 specific genes (CD206, Arg-1, and CX3CR1) in comparison to the CPZ group. Moreover, cell therapy significantly decreased the M1 marker (iNOS⁺ cells), but M2 marker (Arg-1⁺ cells) significantly increased in comparison with the CPZ group. In addition, MSC treatment significantly increased the CX3CL1 expression level in comparison with the CPZ group and led to improvement in remyelination, which was confirmed by LFB and EM images. The results showed that MSC transplantation increases the M2 and decreases the M1 phenotype in MS. This change was accompanied by decrease in demyelination and axonal injury and indicated that MSCs have a positive effect on MS by modification of microglia cells.     

Leonurine suppresses neuroinflammation through promoting oligodendrocyte maturation

Focal inflammation and remyelination failure are major hallmarks of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we found that leonurine, a bioactive alkaloid, alleviated EAE disease severity along with reduced central nervous system inflammation and myelin damage. During the pathogenesis of EAE, leonurine dramatically suppressed the recruitment of encephalitogenic T cells into the central nervous system, whereas did not impair periphery immune responses and microglia activation. Mechanistically, leonurine protected mice against demyelination along with enhanced remyelination through promoting the maturation of oligodendrocytes in both EAE and cuprizone‐induced demyelination mouse models. Moreover, we identified that the expression of demethylase jumonji domain‐containing protein D3 was significantly enhanced upon treatment of leonurine, which suppressed the trimethylation of histone H3 lysine‐27 and enhanced oligodendrocyte maturation accordingly. Collectively, our study identified the therapeutic effect of leonurine on EAE model, which potentially represents a promising therapeutic strategy for multiple sclerosis, even other demyelination disorders.     

Second generation S1P pathway modulators: Research strategies and clinical developments

Multiple Sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 2010, fingolimod/FTY720 (Gilenya, Novartis) was approved as the first oral treatment for relapsing forms of MS. Fingolimod causes down-modulation of S1P1 receptors on lymphocytes which prevents the invasion of autoaggressive T cells into the CNS. In astrocytes, down-modulation of S1P1 by the drug reduces astrogliosis, a hallmark of MS, thereby allowing restoration of productive astrocyte communication with other neural cells and the blood brain barrier. Animal data further suggest that the drug directly supports the recovery of nerve conduction and remyelination. In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by the drug. Fingolimod binds to 4 of the 5 known S1P receptor subtypes, and significant efforts were made over the past 5 years to develop next generation S1P receptor modulators and determine the minimal receptor selectivity needed for maximal therapeutic efficacy in MS patients. Other approaches considered were competitive antagonists of the S1P1 receptor, inhibitors of the S1P lyase to prevent S1P degradation, and anti-S1P antibodies. Below we discuss the current status of the field, and the functional properties of the most advanced compounds. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Cuprizone demyelination of the corpus callosum in mice correlates with altered social interaction and impaired bilateral sensorimotor coordination

For studies of remyelination in demyelinating diseases, the cuprizone model of CC (corpus callosum) demyelination has experimental advantages that include overall size, proximity to neural stem cells of the subventricular zone, and correlation with a lesion predilection site in multiple sclerosis. In addition, cuprizone treatment can be ended to allow more direct analysis of remyelination than with viral or autoimmune models. However, CC demyelination lacks a useful functional correlate in rodents for longitudinal analysis throughout the course of demyelination and remyelination. In the present study, we tested two distinct behavioural measurements in mice fed 0.2% cuprizone. Running on a ‘complex'' wheel with varied rung intervals requires integration between cerebral hemispheres for rapid bilateral sensorimotor coordination. Maximum running velocity on the ‘complex'' wheel decreased during acute (6 week) and chronic (12 week) cuprizone demyelination. Running velocity on the complex wheel distinguished treated (for 6 weeks) from non-treated mice, even after a 6-week recovery period for spontaneous remyelination. A second behavioural assessment was a resident–intruder test of social interaction. The frequency of interactive behaviours increased among resident mice after acute or chronic demyelination. Differences in both sensorimotor coordination and social interaction correlated with demonstrated CC demyelination. The wheel assay is applicable for longitudinal studies. The resident–intruder assay provides a complementary assessment of a distinct modality at a specific time point. These behavioural measurements are sufficiently robust for small cohorts as a non-invasive assessment of demyelination to facilitate analysis of subsequent remyelination. These measurements may also identify CC involvement in other mouse models of central nervous system injuries and disorders.     

Remyelination — An effective means of neuroprotection

Remyelination following central nervous system (CNS) demyelination restores rapid saltatory conduction of action potentials and contributes to the maintenance of axonal integrity. This robust regenerative phenomenon stands in contrast to the limited repair capacity that is characteristic of CNS neuronal injury. However, despite its efficiency in experimental models and some clinical diseases, remyelination failure becomes an increasingly pronounced feature of the pathology of chronic multiple sclerosis (MS) lesions. Chronic demyelination predisposes axons to atrophy, an irreversible event that is a major pathological correlate of progressive functional decline. This has created a compelling case for developing therapies that promote remyelination: evidence from experimental animal models suggests that hormones may have a beneficial role to play in this regard.     

Prox1 Inhibits Proliferation and Is Required for Differentiation of the Oligodendrocyte Cell Lineage in the Mouse

Central nervous system injury induces a regenerative response in ensheathing glial cells comprising cell proliferation, spontaneous axonal remyelination, and limited functional recovery, but the molecular mechanisms are not fully understood. In Drosophila, this involves the genes prospero and Notch controlling the balance between glial proliferation and differentiation, and manipulating their levels in glia can switch the response to injury from prevention to promotion of repair. In the mouse, Notch1 maintains NG2 oligodendrocyte progenitor cells (OPCs) in a progenitor state, but what factor may enable oligodendrocyte (OL) differentiation and functional remyelination is not understood. Here, we asked whether the mammalian homologue of prospero, Prox1, is involved. Our data show that Prox1 is distributed in NG2+ OPCs and in OLs in primary cultured cells, and in the mouse spinal cord in vivo. siRNA prox1 knockdown in primary OPCs increased cell proliferation, increased NG2+ OPC cell number and decreased CC1+ OL number. Prox1 conditional knockout in the OL cell lineage in mice increased NG2+ OPC cell number, and decreased CC1+ OL number. Lysolecithin-induced demyelination injury caused a reduction in CC1+ OLs in homozygous Prox1-/- conditional knockout mice compared to controls. Remarkably, Prox1-/- conditional knockout mice had smaller lesions than controls. Altogether, these data show that Prox1 is required to inhibit OPC proliferation and for OL differentiation, and could be a relevant component of the regenerative glial response. Therapeutic uses of glia and stem cells to promote regeneration and repair after central nervous system injury would benefit from manipulating Prox1.