Association of a gliotoxic activity with active multiple sclerosis in US patients.

We recently found that cerebrospinal fluid (CSF) from multiple sclerosis (MS) patients contains a gliotoxic activity which induces programmed cell death of astrocytes and oligodendrocytes and could be the main contributing factor to the massive glial cell death seen in MS active lesions. A previous clinical study aimed at evaluating the gliotoxicity of CSF from a cohort of MS patients from France indicated that MS patients with the active form of the disease do indeed present significant CSF gliotoxicity. To extend this observation, the effect of 141 CSFs from United States patients with different neurological diseases (including 71 MS) was tested on immortalized astrocytes. A cell death assay showed that a gliotoxic activity is significantly present in the CSF from MS patients with the active forms. Thus, this gliotoxic activity may represent a critical pathogenic factor in the neuropathology of active MS by playing a role both in demyelinisation and alteration of the blood-brain barrier.     

Epidermal Growth Factor in the CNS: A Beguiling Journey from Integrated Cell Biology to Multiple Sclerosis. An Extensive Translational Overview

This article reviews the wealth of papers dealing with the different effects of epidermal growth factor (EGF) on oligodendrocytes, astrocytes, neurons, and neural stem cells (NSCs). EGF induces the in vitro and in vivo proliferation of NSCs, their migration, and their differentiation towards the neuroglial cell line. It interacts with extracellular matrix components. NSCs are distributed in different CNS areas, serve as a reservoir of multipotent cells, and may be increased during CNS demyelinating diseases. EGF has pleiotropic differentiative and proliferative effects on the main CNS cell types, particularly oligodendrocytes and their precursors, and astrocytes. EGF mediates the in vivo myelinotrophic effect of cobalamin on the CNS, and modulates the synthesis and levels of CNS normal prions (PrP^Cs), both of which are indispensable for myelinogenesis and myelin maintenance. EGF levels are significantly lower in the cerebrospinal fluid and spinal cord of patients with multiple sclerosis (MS), which probably explains remyelination failure, also because of the EGF marginal role in immunology. When repeatedly administered, EGF protects mouse spinal cord from demyelination in various experimental models of autoimmune encephalomyelitis. It would be worth further investigating the role of EGF in the pathogenesis of MS because of its multifarious effects.

     

A subgenomic segment of Theiler's murine encephalomyelitis virus RNA causes demyelination

The DA strain of Theiler's murine encephalomyelitis virus (TMEV) causes a persistent central nervous system (CNS) infection of mice with a restricted virus gene expression and induces an inflammatory demyelinating disease that is thought to be immune mediated and a model of multiple sclerosis (MS). The relative contribution of virus vis-à-vis the immune system in the pathogenesis of DA-induced white matter disease remains unclear, as is also true in MS. To clarify the pathogenesis of DA-induced demyelination, we used Cre/loxP technology to generate a transgenic mouse that has tamoxifen (Tm)-inducible expression of a subgenomic segment of DA RNA in oligodendrocytes and Schwann cells. Tm-treated young transgenic mice developed progressive weakness leading to death, with abnormalities of oligodendrocytes and Schwann cells and demyelination, but without inflammation, demonstrating that DA virus can play a direct pathogenic role in demyelination. Tm treatment of mice at a later age resulted in milder disease, with evidence of peripheral nerve remyelination and focal fur depigmentation; surviving weak mice had persistent expression of the recombined transgene in the CNS, suggesting that the DA subgenomic segment can cause cellular dysfunction but not death, possibly similar to the situation seen during DA virus persistence. These studies demonstrate that DA RNA or a DA protein(s) is toxic to myelin-synthesizing cells. This Cre/loxP transgenic system allows for spatially and temporally controlled expression of the viral transgene and is valuable for clarifying nonimmune (and immune) mechanisms of demyelination induced by TMEV as well as other viruses.     

Demyelinating and nondemyelinating strains of mouse hepatitis virus differ in their neural cell tropism

Some strains of mouse hepatitis virus (MHV) can induce chronic inflammatory demyelination in mice that mimics certain pathological features of multiple sclerosis. We have examined neural cell tropism of demyelinating and nondemyelinating strains of MHV in order to determine whether central nervous system (CNS) cell tropism plays a role in demyelination. Previous studies demonstrated that recombinant MHV strains, isogenic other than for the spike gene, differ in the extent of neurovirulence and the ability to induce demyelination. Here we demonstrate that these strains also differ in their abilities to infect a particular cell type(s) in the brain. Furthermore, there is a correlation between the differential localization of viral antigen in spinal cord gray matter and that in white matter during acute infection and the ability to induce demyelination later on. Viral antigen from demyelinating strains is detected initially in both gray and white matter, with subsequent localization to white matter of the spinal cord, whereas viral antigen localization of nondemyelinating strains is restricted mainly to gray matter. This observation suggests that the localization of viral antigen to white matter during the acute stage of infection is essential for the induction of chronic demyelination. Overall, these observations suggest that isogenic demyelinating and nondemyelinating strains of MHV, differing in the spike protein expressed, infect neurons and glial cells in different proportions and that differential tropism to a particular CNS cell type may play a significant role in mediating the onset and mechanisms of demyelination.     

Carbonic Anhydrase Immunostaining in Astrocytes in the Rat Cerebral Cortex

Carbonic anhydrase is known to occur in the choroid plexus, oligodendrocytes, and myelin, and to be virtually absent from neurons, in the mammalian CNS; however, there is significant controversy whether it is also present in astrocytes. When brain sections from adult rats were stained for simultaneous immunofluorescence of carbonic anhydrase and the astrocyte marker glutamine synthetase, both antigens were detected in the same glial cells in the cortical gray matter, whereas the oligodendrocytes and myelinated fibers in and adjacent to the white matter showed immunofluorescence only for carbonic anhydrase. Some glial cells in the gray matter also showed double immunofluorescence for carbonic anhydrase and glial fibrillary acidic protein. These results indicate that there is carbonic anhydrase in some astrocytes in the mammalian CNS.     

The immune system preferentially clears Theiler's virus from the gray matter of the central nervous system.

Infection of susceptible strains of mice with Daniel's (DA) strains of Theiler's murine encephalomyelitis virus (DAV) results in virus persistence in the central nervous system (CNS) white matter and chronic demyelination similar to that observed in multiple sclerosis. We investigated whether persistence is due to the immune system more efficiently clearing DAV from gray than from white matter of the CNS. Severe combined immunodeficient (SCID) and immunocompetent C.B-17 mice were infected with DAV to determine the kinetics, temporal distribution, and tropism of the virus in CNS. In early disease (6 h to 7 days postinfection), DAV replicated with similar kinetics in the brains and spinal cords of SCID and immunocompetent mice and in gray and white matter. DAV RNA was localized within 48 h in CNS cells of all phenotypes, including neurons, oligodendrocytes, astrocytes, and macrophages/microglia. In late disease (13 to 17 days postinfection), SCID mice became moribund and permitted higher DAV replication in both gray and white matter. In contrast, immunocompetent mice cleared virus from the gray matter but showed replication in the white matter of their brains and spinal cords. Reconstitution of SCID mice with nonimmune splenocytes or anti-DAV antibodies after establishment of infection demonstrated that both cellular and humoral immune responses decreased virus from the gray matter; however, the cellular responses were more effective. SCID mice reconstituted with splenocytes depleted of CD4+ or CD8+ T lymphocytes cleared virus from the gray matter but allowed replication in the white matter. These studies demonstrate that both neurons and glia are infected early following DAV infection but that virus persistence in the white matter is due to preferential clearance of virus from the gray matter by the immune system.     

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.     

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white-matter regions of rat brain, and in Vibratome sections as well as paraffin sections.     

NG2 cells generate both oligodendrocytes and gray matter astrocytes

NG2 glia constitute a fourth major glial cell type in the mammalian central nervous system (CNS) that is distinct from other cell types. Although circumstantial evidence suggests that some NG2 glia differentiate into oligodendrocytes, their in vivo fate has not been directly examined. We have used the bacterial artificial chromosome (BAC) modification technique to generate transgenic mice that express DsRed or Cre specifically in NG2-expressing (NG2+) cells. In NG2DsRedBAC transgenic mice, DsRed was expressed specifically in NG2+ cells throughout the postnatal CNS. When the differentiation potential of NG2+ cells in vitro was examined using DsRed+NG2+ cells purified from perinatal transgenic brains, the majority of the cells either remained as NG2+ cells or differentiated into oligodendrocytes. In addition, DsRed+NG2+ cells also differentiated into astrocytes. The in vivo fate of NG2 glia was examined in mice that were double transgenic for NG2creBAC and the Cre reporter Z/EG. In the double transgenic mice, the Cre reporter EGFP was detected in myelinating oligodendrocytes and in a subpopulation of protoplasmic astrocytes in the gray matter of ventrolateral forebrain but not in fibrous astrocytes of white matter. These observations suggest that NG2+ cells are precursors of oligodendrocytes and some protoplasmic astrocytes in gray matter.     

LIF receptor signaling limits immune-mediated demyelination by enhancing oligodendrocyte survival

Multiple sclerosis (MS) is a disabling inflammatory demyelinating disease of the central nervous system (CNS) that primarily affects young adults. Available therapies can inhibit the inflammatory component of MS but do not suppress progressive clinical disability. An alternative approach would be to inhibit mechanisms that drive the neuropathology of MS, which often includes the death of oligodendrocytes, the cells responsible for myelinating the CNS. Identification of molecular mechanisms that mediate the stress response of oligodendrocytes to optimize their survival would serve this need. This study shows that the neurotrophic cytokine leukemia inhibitory factor (LIF) directly prevents oligodendrocyte death in animal models of MS. We also demonstrate that this therapeutic effect complements endogenous LIF receptor signaling, which already serves to limit oligodendrocyte loss during immune attack. Our results provide a novel approach for the treatment of MS.     

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.     

In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

A demyelinating disease induced in C57B1/6N mice by intracranial injection of a coronavirus (murine hepatitis virus strain A59) is followed by functional recovery and efficient CNS myelin repair. To study the biological properties of the cells involved in this repair process, glial cells were isolated and cultured from spinal cords of these young adult mice during demyelination and remyelination. Using three-color immunofluorescence combined with [3H]thymidine autoradiography, we have analyzed the antigenic phenotype and mitotic potential of individual glial cells. We identified oligodendrocytes with an antibody to galactocerebroside, astrocytes with an antibody to glial fibrillary acidic protein, and oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells with the O4 antibody. Cultures from demyelinated tissue differed in several ways from those of age-matched controls: first, the total number of O-2A lineage cells was strikingly increased; second, the O-2A population consisted of a higher proportion of O4-positive astrocytes and cells of mixed oligodendrocyte-astrocyte phenotype; and third, all the cell types within the O-2A lineage showed enhanced proliferation. This proliferation was not further enhanced by adding PDGF, basic fibroblast growth factor (bFGF), or insulin-like growth factor I (IGF-I) to the defined medium. However, bFGF and IGF-I seemed to influence the fate of O-2A lineage cells in cultures of demyelinated tissue. Basic FGF decreased the percentage of cells expressing galactocerebroside. In contrast, IGF-I increased the relative proportion of oligodendrocytes. Thus, O-2A lineage cells from adult mice display greater phenotypic plasticity and enhanced mitotic potential in response to an episode of demyelination. These properties may be linked to the efficient remyelination achieved in this demyelinating disease.     

Role of natural killer cells as immune effectors in encephalitis and demyelination induced by Theiler's virus

Infection of susceptible mice (SJL) with Theiler's murine encephalitis virus (TMEV) causes a biphasic disease characterized by gray matter inflammation followed by late chronic demyelination. The role of NK cells was studied in this model by using susceptible (SJL) or resistant (C57BL/10) mice. CNS TMEV titer were higher in SJL compared with C57BL/10 mice, correlating with a 50% lower NK cell activity in the SJL than in the C57BL/10 mice. When resistant (C57BL/10) mice were depleted of NK cells using either mAb NK1.1 or polyclonal anti-asialo-GM1, TMEV induced the development of diffuse encephalitis and meningitis early in the postinfection period (days 6 to 11). However, the second phase of TMEV-induced CNS disease (demyelination) was observed only in resistant C57BL/10 mice treated with anti-asialo-GM1. Experiments with beige/beige mice of C57BL/10 background showed a mild degree of gray matter inflammation but no demyelination. In conclusion, NK cells are critical effectors in protecting against TMEV-induced gray matter disease, whereas a different population of either NK1.1- NK cells, or other activated lymphocytes may be critical in resistance/susceptibility to demyelination.     

IFN-γ Signaling to Astrocytes Protects from Autoimmune Mediated Neurological Disability

Demyelination and axonal degeneration are determinants of progressive neurological disability in patients with multiple sclerosis (MS). Cells resident within the central nervous system (CNS) are active participants in development, progression and subsequent control of autoimmune disease; however, their individual contributions are not well understood. Astrocytes, the most abundant CNS cell type, are highly sensitive to environmental cues and are implicated in both detrimental and protective outcomes during autoimmune demyelination. Experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice expressing signaling defective dominant-negative interferon gamma (IFN-γ) receptors on astrocytes to determine the influence of inflammation on astrocyte activity. Inhibition of IFN-γ signaling to astrocytes did not influence disease incidence, onset, initial progression of symptoms, blood brain barrier (BBB) integrity or the composition of the acute CNS inflammatory response. Nevertheless, increased demyelination at peak acute disease in the absence of IFN-γ signaling to astrocytes correlated with sustained clinical symptoms. Following peak disease, diminished clinical remission, increased mortality and sustained astrocyte activation within the gray matter demonstrate a critical role of IFN-γ signaling to astrocytes in neuroprotection. Diminished disease remission was associated with escalating demyelination, axonal degeneration and sustained inflammation. The CNS infiltrating leukocyte composition was not altered; however, decreased IL-10 and IL-27 correlated with sustained disease. These data indicate that astrocytes play a critical role in limiting CNS autoimmune disease dependent upon a neuroprotective signaling pathway mediated by engagement of IFN-γ receptors.     

Potential role of CD4+ T cell-mediated apoptosis of activated astrocytes in Theiler's virus-induced demyelination.

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) into susceptible mouse strains results in a chronic, immune-mediated demyelinating disease similar to human multiple sclerosis. Here, we examined the role of astrocytes as an APC population in TMEV-induced demyelination and assessed the potential consequences of T cell activation following Ag presentation. IFN-gamma-pretreated astrocytes were able to process and present all the predominant T cell epitopes of TMEV to virus-specific T cell hybridomas, clones, as well as bulk T cells. Despite low levels of proliferation of T cells due to prostaglandins produced by astrocytes, such Ag presentation by activated astrocytes induced the production of IFN-gamma, a representative proinflammatory cytokine, in TMEV-specific Th cell clones derived from the CNS of virus-infected mice. Furthermore, these Th cell clones mediate lysis of the astrocytes in vitro in a Fas-dependent mechanism. TUNEL staining of CNS tissue demonstrates the presence of apoptotic GFAP+ cells in the white matter of TMEV-infected mice. These results strongly suggest that astrocytes could play an important role in the pathogenesis of TMEV-induced demyelination by activating T cells, subsequently leading to T cell-mediated apoptosis of astrocytes and thereby compromising the blood-brain barrier.     

Macrophage Infiltration, but Not Apoptosis, Is Correlated with Immune-Mediated Demyelination following Murine Infection with a Neurotropic Coronavirus

Mice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis that is in large part immune mediated. Potential mechanisms of immune activity were assessed using an adoptive transfer system. Mice deficient in recombinase-activating gene function (RAG1(-/-)), defective in B- and T-cell maturation, become persistently infected with MHV but do not develop demyelination. Adoptive transfer of splenocytes from mice immunized to MHV into RAG1(-/-) mice infected with an attenuated strain of the virus results in the rapid and progressive development of demyelination. Most striking, adoptive transfer resulted, within 5 to 6 days, in extensive recruitment of activated macrophages/microglia to sites of demyelination within the spinal cord. Clearance of virus antigen occurred preferentially from the gray matter of the spinal cord. Apoptotic cells were identified in both the gray and white matter of the central nervous system (CNS) from RAG1(-/-) mice before and after adoptive transfer, with a moderate increase in number, but not distribution, of apoptotic cells following the development of demyelination. These results suggest that apoptosis following MHV-JHM infection of the murine CNS is not sufficient to cause demyelination. These results, showing that macrophage recruitment and myelin destruction occur rapidly after immune reconstitution of RAG(-/-) mice, suggest that this will be a useful system for investigating MHV-induced demyelination.     

Induction of caspase-dependent apoptosis in cultured rat oligodendrocytes by murine coronavirus is mediated during cell entry and does not require virus replication

Murine coronavirus mouse hepatitis virus (MHV) causes demyelination of the central nervous system (CNS) in rats and mice. Apoptotic oligodendrocytes have been detected in the vicinity of the CNS demyelinating lesions in these animals. However, whether MHV can directly induce oligodendrocyte apoptosis has not been documented. Here, we established a rat oligodendrocyte culture that is morphologically and phenotypically indistinguishable from the primary rat oligodendrocytes. Using this culture, we showed that mature rat oligodendrocytes were permissive to MHV infection but did not support productive virus replication. Significantly, oligodendrocytes infected with both live and ultraviolet light-inactivated viruses underwent apoptosis to a similar extent, which was readily detectable at 24 h postinfection as revealed by apoptotic bodies and DNA fragmentation, indicating that MHV-induced apoptosis is mediated during the early stages of the virus life cycle and does not require virus replication. Prior treatment of cells with the lysosomotropic agents NH(4)Cl and chloroquine as well as the vacuolar proton pump-ATPase inhibitor bafilomycin A1, all of which block the acidification of the endosome, prevented oligodendrocytes from succumbing to apoptosis induced by MHV mutant OBLV60, which enters cells via endocytosis, indicating that fusion between the viral envelope and cell membranes triggers the apoptotic cascade. Treatment with the pan-caspase inhibitor Z-VAD-fmk blocked MHV-induced apoptosis, suggesting an involvement of the caspase-dependent pathway. Our results, thus, for the first time provide unequivocal evidence that infection of oligodendrocytes with MHV directly results in apoptosis. This finding provides an explanation for the destruction of oligodendrocytes and the damage of myelin sheath in MHV-infected CNS and suggests that oligodendrocyte apoptosis may be one of the underlying mechanisms for the pathogenesis of MHV-induced demyelinating diseases in animals.     

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.