Estradiol inhibits ongoing autoimmune neuroinflammation and NFkappaB-dependent CCL2 expression in reactive astrocytes.

Astroglial reactivity associated with increased production of NFkappaB-dependent proinflammatory molecules is an important component of the pathophysiology of chronic neurological disorders such as multiple sclerosis (MS). The use of estrogens as potential anti-inflammatory and neuroprotective drugs is a matter of debate. Using mouse experimental allergic encephalomyelitis (EAE) as a model of chronic neuroinflammation, we report that implants reproducing pregnancy levels of 17beta-estradiol (E2) alleviate ongoing disease and decrease astrocytic production of CCL2, a proinflammatory chemokine that drives the local recruitment of inflammatory myeloid cells. Immunohistochemistry and confocal imaging reveal that, in spinal cord white matter EAE lesions, reactive astrocytes express estrogen receptor (ER)alpha (and to a lesser extent ERbeta) with a preferential nuclear localization, whereas other cells including infiltrated leukocytes express ERs only in their membranes or cytosol. In cultured rodent astrocytes, E2 or an ERalpha agonist, but not an ERbeta agonist, inhibits TNFalpha-induced CCL2 expression at nanomolar concentrations, and the ER antagonist ICI 182,170 blocks this effect. We show that this anti-inflammatory action is not associated with inhibition of NFkappaB nuclear translocation but rather involves direct repression of NFkappaB-dependent transcription. Chromatin immunoprecipitation assays further indicate that estrogen suppresses TNFalpha-induced NFkappaB recruitment to the CCL2 enhancer. These data uncover reactive astrocytes as an important target for nuclear ERalpha inhibitory action on chemokine expression and suggest that targeting astrocytic nuclear NFkappaB activation with estrogen receptor alpha modulators may improve therapies of chronic neurodegenerative disorders involving astroglial neuroinflammation.

Tumor-like enlargement of the optic chiasm in an infant with Alexander disease.

We report a patient with infantile Alexander disease (AXD) due to the recurrent p.Arg79Cys GFAP mutation. In addition to typical AXD abnormalities, magnetic resonance imaging demonstrated a tumor-like lesion of the optic chiasm suggestive of a glioma. A transient papilloedema appeared during the follow-up and the lesion partially regressed despite a worsening of white matter involvement. Rare radiological and pathological tumor-like lesions have already been reported in AXD patients. This patient confirms that enlargement of the optic chiasm is a rare feature of AXD, possibly linked to abnormal astrocytic proliferation.

Autoimmunity against myelin oligodendrocyte glycoprotein is dispensable for the initiation although essential for the progression of chronic encephalomyelitis in common marmosets.

To elucidate the pathogenetic significance of myelin/oligodendrocyte glycoprotein (MOG)-specific autoreactivity in a genetically and immunologically heterogeneous nonhuman primate model of multiple sclerosis, we analyzed experimental autoimmune encephalomyelitis (EAE) in the outbred common marmoset (Callithrix jacchus). One sibling each of 5 bone marrow chimeric marmoset twins was immunized with myelin derived from wild-type (WT) C57BL/6 mice (WT myelin); the other sibling was immunized with myelin from MOG-deficient C57BL/6 mice (MOG -/- myelin). One twin pair developed acute EAE simultaneously; the 4 remaining twin siblings immunized with WT myelin developed chronic progressive EAE, whereas siblings of these 4 monkeys remained free of clinical disease signs. Many EAE-related abnormalities were identified in the CNS of both groups by magnetic resonance imaging and histologic analysis, but mean percentages of spinal cord demyelination were lower in monkeys immunized with MOG -/- myelin (8.2%) than in WT myelin-immunized animals (40.5%). There was a strong correlation between the development of overt clinical EAE and seropositivity for anti-MOG antibodies, but blood and lymph node T-cell proliferative responses showed no relationship to disease. These results indicate that the initiation of CNS inflammation and demyelination can take place in the absence of detectable autoimmunity against MOG, but the clinical progression and histopathologic severity depends on the presence of antibodies against MOG in this multiple sclerosis model.

Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease.

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.

T cell repertoire diversity is required for relapses in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis.

Comparison of TCRalphabeta repertoires of myelin oligodendrocyte glycoprotein (MOG)-specific T lymphocytes in C57BL/6 and TdT-deficient littermates (TdT(-/-)) generated during experimental autoimmune encephalomyelitis (EAE) highlights a link between a diversified TCRalphabeta repertoire and EAE relapses. At the onset of the disease, the EAE-severity is identical in TdT(+/-) and TdT(-/-) mice and the neuropathologic public MOG-specific T cell repertoires express closely similar public Valpha-Jalpha and Vbeta-Jbeta rearrangements in both strains. However, whereas TdT(+/+) and TdT(+/-) mice undergo successive EAE relapses, TdT(-/-) mice recover definitively and the lack of relapses does not stem from dominant regulatory mechanisms. During the first relapse of the disease in TdT(+/-) mice, new public Valpha-Jalpha and Vbeta-Jbeta rearrangements emerge that are distinct from those detected at the onset of the disease. Most of these rearrangements contain N additions and are found in CNS-infiltrating T lymphocytes. Furthermore, CD4(+) T splenocytes bearing these rearrangements proliferate to the immunodominant epitope of MOG and not to other immunodominant epitopes of proteolipid protein and myelin basic protein autoantigens, excluding epitope spreading to these myelin proteins. Thus, in addition to epitope spreading, a novel mechanism involving TCRalphabeta repertoire diversification contributes to autoimmune progression.

Complex alternative splicing of the myelin oligodendrocyte glycoprotein gene is unique to human and non-human primates.

Myelin/oligodendrocyte glycoprotein (MOG) is a minor integral membrane protein specific to CNS myelin, encoded by a gene located in the major histocompatibility complex. MOG is an highly encephalitogenic autoantigen and a target for autoaggressive immune responses in CNS inflammatory demyelinating diseases. We performed transcriptomic analyses for a gene expressed only in mammalian CNS, myelin oligodendrocyte glycoprotein (MOG). Complex splicing patterns were exclusively found in primates and not in mice, unlike patterns found for other myelin protein genes. In addition to those shared with rodents, these multiple MOG isoforms likely support functions unique to the primate order, in particular maintenance of myelin structure, intracellular signaling, and modulation of CNS autoimmunity via exposure of specific MOG determinants. Developmentally, in human brain the splice variants of MOG appear at a late stage compared to the major isoform, coincidental with myelination and myelin maturation, unlike other myelin proteins. These findings are discussed within the framework of a biological basis for phenotype diversity in recent mammalian evolution and for the notoriously variable clinical expression of diseases such as multiple sclerosis.

Upregulation of alpha-synuclein in neurons and glia in inflammatory demyelinating disease.

A growing body of evidence suggests that axonal loss and neurodegeneration are responsible for the permanent neurological deficit that typically develops in the course of MS. To investigate the neurodegenerative component of MS pathogenesis, we examined the expression of alpha-synuclein, a protein whose accumulation is common to many neurodegenerative disorders, under conditions of immune-mediated inflammatory demyelination. alpha-Synuclein expression was examined in the spinal cord of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in rats using immunofluorescence and in situ hybridization and in postmortem tissues from cases of secondary progressive MS using immunohistochemistry. alpha-Synuclein upregulation was detected in neurons and glia in and close by lesions and in normal appearing spinal cord EAE tissue at the protein and mRNA levels. alpha-Synuclein positive neurons and glia appeared early, and their number was maximal during EAE exacerbations, but some expression was maintained throughout the course of EAE. In addition, increased alpha-synuclein expression was detected in neurons and glia in and close to MS lesions. Although the increased expression of alpha-synuclein was detected as a granular cytoplasmic labeling rather than inclusion bodies, this result does suggest that neuronal cell death in immune-mediated demyelinating disease may share some common features with other neurodegenerative conditions.

Persistence of autoreactive myelin oligodendrocyte glycoprotein (MOG)-specific T cell repertoires in MOG-expressing mice.

Experimental autoimmune encephalomyelitis, an experimental murine model for multiple sclerosis, is induced by stimulation of myelin-specific T lymphocytes. Myelin oligodendrocyte glycoprotein (MOG), a minor component of myelin proteins, is a potent autoantigen which contributes extensively to the anti-myelin response. In the present work, immunoscope analyses and sequencing of the oligoclonal expansions revealed anti-MOG Valpha and Vbeta public repertoires in lymphocytes infiltrating the CNS of wild-type (WT) mice. Moreover, a subset of CNS-infiltrating CD4+ T lymphocytes bearing the public Vbeta8.2 segment have an inflammatory phenotype strongly suggesting that it is encephalitogenic. We then observed that, in lymph node cells of MOG-deficient and WT animals, the Valpha and Vbeta public repertoires expressed by MOG-specific T cells are identical in both strains of mice and correspond to those found in the CNS of WT animals. These findings indicate that the MOG immunodominant determinant is unable to induce tolerance by deletion, and public anti-MOG T cell repertoires are selected for, regardless of the presence of MOG in the thymus and peripheral organs.

Antibodies to native myelin oligodendrocyte glycoprotein are serologic markers of early inflammation in multiple sclerosis.

Myelin oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed in CNS oligodendrocytes and outermost myelin lamellae. Anti-MOG Abs cause myelin destruction (demyelination) in animal models of multiple sclerosis (MS); however, such pathogenic Abs have not yet been characterized in humans. Here, a method that specifically detects IgG binding to human MOG in its native, membrane-embedded conformation on MOG-transfected mammalian cells was used to evaluate the significance of these auto Abs. Compared with healthy controls, native MOG-specific IgGs were most frequently found in serum of clinically isolated syndromes (P < 0.001) and relapsing-remitting MS (P < 0.01), only marginally in secondary progressive MS (P < 0.05), and not at all in primary progressive MS. We demonstrate that epitopes exposed in this cell-based assay are different from those exposed on the refolded, extracellular domain of human recombinant MOG tested by solid-phase ELISA. In marmoset monkeys induced to develop MS-like CNS inflammatory demyelination, IgG reactivity against the native membrane-bound MOG is always detected before clinical onset of disease (P < 0.0001), unlike that against other myelin constituents. We conclude that (i) epitopes displayed on native, glycosylated MOG expressed in vivo are early targets for pathogenic Abs; (ii) these Abs, which are not detected in solid-phase assays, might be the ones to play a pathogenic role in early MS with predominant inflammatory activity; and (iii) the cell-based assay provides a practical serologic marker for early detection of CNS autoimmune demyelination including its preclinical stage at least in the primate MS model.

Axon loss is responsible for chronic neurological deficit following inflammatory demyelination in the rat.

Axonal loss is now considered a consistent feature of MS pathology and evidence suggests that its accumulation may be the pathological correlate for the development of irreversible disability. In this study, we investigated the features of axonal loss in myelin autoimmunity and tested the hypothesis that loss of axons determines permanent neurological impairment in a model of inflammatory demyelination that closely mimics the pathology and course of MS. EAE was induced in DA rats by injection of recombinant mouse MOG with IFA. Animals that developed progressive EAE were killed at several time points after disease onset and animals that followed a chronic relapsing-remitting course of EAE were killed at approximately 4 months, exhibiting varying degrees of residual disability. Toluidine blue staining of semithin sections and immunohistochemistry for OX-42 were used to quantify demyelination, remyelination, inflammation and axonal loss in the spinal cord of MOG-EAE rats. In progressive EAE, the degree of axon loss, demyelination and inflammation all correlated significantly with clinical severity scores and a causative role for macrophages in the pathogenesis of axonal injury is suggested. However, in the chronic stage of relapsing-remitting EAE, in rats having suffered a variable number of relapses, only axonal loss correlated significantly with clinical severity scores. In addition, both axonal loss and clinical severity scores correlated with the number of relapses. These findings imply that secondary, or 'bystander', axonal loss is the main determinant of irreversible neurological disability in MOG-EAE and make the model a useful tool for the investigation of mechanisms of axonal loss and the evaluation of the benefits of neuroprotective therapies under conditions of antibody-mediated inflammatory demyelination.

Native myelin oligodendrocyte glycoprotein promotes severe chronic neurological disease and demyelination in Biozzi ABH mice.

Myelin oligodendrocyte glycoprotein (MOG) is a powerful encephalitogen for experimental autoimmune demyelination. However, the use of MOG peptides or recombinant proteins representing part of the protein fails to fully address the possible pathogenic role of the full-length myelin-derived protein expressing post-translational modifications. Immunization of mice with central nervous system tissues from wild-type (WT) and MOG-deficient (MOG(-/-)) mice demonstrates that MOG in myelin is necessary for the development of chronic demyelinating experimental autoimmune encephalomyelitis (EAE) in mice. While immunization with WT spinal cord homogenate (SCH) resulted in a progressive EAE phenotype, MOG(-/-) SCH induced a mild self-limiting acute disease. Following acute EAE with MOG(-/-) SCH, mice developed T cell responses to recombinant mouse MOG (rmMOG), indicating that MOG released from myelin is antigenic; however, the lack of chronic disease indicates that such responses were not pathogenic. Chronic demyelinating EAE was observed when MOG(-/-) SCH was reconstituted with a dose of rmMOG comparable to MOG in myelin (2.5% of total white matter-derived protein). These data reveal that while immunization with the full-length post-translational modified form of MOG in myelin promotes the development of a more chronic autoimmune demyelinating neurological disease, MOG (and/or other myelin proteins) released from myelin during ongoing disease do not induce destructive autoimmunity.

Myelin oligodendrocyte glycoprotein is expressed in the peripheral nervous system of rodents and primates.

The myelin oligodendrocyte glycoprotein (MOG) is a minor CNS myelin-specific protein that is an important candidate autoantigen in multiple sclerosis. We now report that MOG mRNA transcripts are present in the peripheral nervous system of rodents and primates at levels approximately ten-fold lower than in brain as demonstrated by real time PCR. A major source of this signal are Schwann cells which are also shown to express MOG protein within their cytoplasm in vitro by immunohistochemistry. Expression of MOG by Schwann cells associated with tissue innervation may account for the widespread distribution of low levels of MOG mRNA transcripts, and potentially may provide a source of antigen that can influence the composition and function of the MOG-specific immune repertoire.

Evidence for cytokine dysregulation in multiple sclerosis: peripheral blood mononuclear cell production of pro-inflammatory and anti-inflammatory cytokines during relapse and remission.

We investigated circulating anti-inflammatory and pro-inflammatory cytokines, and their ex vivo PBMC production in the absence or presence of the neuroantigens myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) and T cell mitogen (PHA) in MS patients in relapse and remission, patients with other neurological disorders (OND) and normal healthy controls. MS patients in relapse exhibited significantly increased PBMC production of TNF-alpha spontaneously compared with MS remission and healthy controls and with MBP compared with MS remission. Patients in relapse had significantly increased spontaneous, PHA- and MBP-induced PBMC IL-1beta production compared with remission MS, and was increased compared (PHA only) with OND and healthy controls. In relapse there was also significantly increased PBMC IFN-gamma production (PHA only) compared with remission and a significantly lower production of biologically active TGF-beta1 (PHA only) compared with remission MS and OND. In contrast, MS patients in remission produced significantly less spontaneous and MBP-induced TNF-alpha, spontaneous, PHA- and MBP-induced IL-1beta and PHA-induced IFN-gamma together with increased production of biologically active TGF-beta1. MOG non-specifically increased PBMC TNF-alpha and IL-1beta production in all groups. Pro-inflammatory cytokines in corresponding plasma samples were undetectable whilst the concentration of biologically active TGF-beta1 was the reverse of ex vivo PBMC findings. The increase in biologically active TGF-beta1 production ex vivo in OND patients, despite active disease, compared with the low level in the MS relapse may indicate a regulatory defect in MS. We conclude that the balance between biologically active TGF-beta1 and the pro-inflammatory TNF-alpha, IL-1beta and IFN-gamma is dysregulated during MS relapse-remission and that normal counter-regulatory mechanisms during the relapse phase are defective.

Myelin/oligodendrocyte glycoprotein-deficient (MOG-deficient) mice reveal lack of immune tolerance to MOG in wild-type mice.

We studied the immunological basis for the very potent encephalitogenicity of myelin/oligodendrocyte glycoprotein (MOG), a minor component of myelin in the CNS that is widely used to induce experimental autoimmune encephalomyelitis (EAE). For this purpose, we generated a mutant mouse lacking a functional mog gene. This MOG-deficient mouse presents no clinical or histological abnormalities, permitting us to directly assess the role of MOG as a target autoantigen in EAE. In contrast to WT mice, which developed severe EAE following immunization with whole myelin, MOG-deficient mice had a mild phenotype, demonstrating that the anti-MOG response is a major pathogenic component of the autoimmune response directed against myelin. Moreover, while MOG transcripts are expressed in lymphoid organs in minute amounts, both MOG-deficient and WT mice show similar T and B cell responses against the extracellular domain of MOG, including the immunodominant MOG 35-55 T cell epitope. Furthermore, no differences in the fine specificity of the T cell responses to overlapping peptides covering the complete mouse MOG sequence were observed between MOG+/+ and MOG-/- mice. In addition, upon adoptive transfer, MOG-specific T cells from WT mice and those from MOG-deficient mice are equally pathogenic. This total lack of immune tolerance to MOG in WT C57BL/6 mice may be responsible for the high pathogenicity of the anti-MOG immune response as well as the high susceptibility of most animal strains to MOG-induced EAE.

Experimental autoimmune encephalomyelitis mobilizes neural progenitors from the subventricular zone to undergo oligodendrogenesis in adult mice.

The destiny of the mitotically active cells of the subventricular zone (SVZ) in adult rodents is to migrate to the olfactory bulb, where they contribute to the replacement of granular and periglomerular neurons. However, these adult neural progenitors also can be mobilized in periventricular white matter and triggered to differentiate into astrocytes and oligodendrocytes in response to lysolecithin-induced demyelination. To mimic the environmental conditions of multiple sclerosis, we assessed the proliferation, migration, and differentiation potential of adult SVZ progenitor cells in response to experimental autoimmune encephalomyelitis (EAE) in mice. Inflammation and demyelination were observed in all mouse brains after EAE induction. EAE induced cell proliferation throughout the brain and especially within the lesions. Proliferating cells were neural progenitors, astrocytes, and oligodendrocyte precursors. EAE enhanced the migration of SVZ-derived neural progenitors to the olfactory bulb and triggered their mobilization in the periventricular white matter. The mobilized cells gave rise to neurons, astrocytes, and oligodendrocytes in the olfactory bulb but essentially to astrocytes and oligodendrocytes in the lesioned white matter. Our data indicate that the adult mouse SVZ is a source of newly generated oligodendrocytes and thus may contribute, along with oligodendrocyte precursors, to the replacement of oligodendrocytes in inflammatory demyelinating diseases of the central nervous system such as multiple sclerosis.

Pathological and regulatory effects of anti-myelin antibodies in experimental allergic encephalomyelitis in mice.

Neurological deficit in experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS) is probably a consequence of synergy between T and B cell responses to CNS antigens. During the demyelinating phase of chronic relapsing EAE in ABH mice, anti-myelin oligodendrocyte glycoprotein (MOG) responses were increased compared to the inflammatory acute phase, but such levels did not correlate with the severity of clinical disease. The pathogenicity of antibodies (Ab) to MOG, myelin basic protein (MBP), proteolipid protein (PLP) and galactocerebroside (GalC) was investigated in vivo following injection at the onset of EAE. An IgG2a monoclonal Ab (mAb), clone Z12, directed to MOG augmented clinical disease and demyelination in ABH and C57BL/6 mice, but not MOG knock-out mice. No effect was observed with F(ab(2))' fragments of Z12 or with the anti-MOG IgG1 mAbs, clones Y10 or 8-18C5. Cobra venom factor partially reduced the augmenting effect of mAb Z12 suggesting a role for complement. The pathogenic effect of anti-myelin Abs was not restricted to MOG since an anti-GalC mAb exacerbated inflammation in the CNS while an MBP mAb (clone 22) reduced clinical disease. Taken together, these data provide further evidence that myelin-reactive Abs generated during autoimmune neurological disease may play an important role not only in the pathogenesis of disease but also the regulation of myelin-targeted autoimmune disease.

The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS.

Remyelination of primary demyelinated lesions is a common feature of experimental models of multiple sclerosis (MS) and is also suggested to be the normal response to demyelination during the early stages of MS itself. Many lines of evidence have shown that remyelination is preceded by the division of endogenous oligodendrocyte precursor cells (OPCs) in the lesion and its borders. It is suggested that this rapid response of OPCs to repopulate the lesion site and their subsequent differentiation into new oligodendrocytes is the key to the rapid remyelination. Antibodies to the NG2 chondroitin sulphate proteoglycan have proved exceedingly useful in following and quantitating the response of endogenous OPCs to demyelination. Here we review the literature on the response of NG2-expressing OPCs to demyelination and provide some new evidence on their response to the chronic inflammatory demyelinating environment seen in recombinant myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) in the DA rat. NG2-expressing OPCs responded to the inflammatory demyelination in this model by becoming reactive and increasing in number in a very focal manner. Evidence of NG2+ OPCs in lesioned areas beginning to express the oligodendrocyte marker CNP was also seen. The response of OPCs appeared to occur following successive relapses but did not always lead to remyelination, with areas of chronic demyelination observed in the spinal cord. The presence of OPCs in the adult human CNS is clearly of vital importance for repair in multiple sclerosis (MS). As in rat tissue, the antibody labels an evenly distributed cell population present in both white and grey matter, distinct from HLA-DR+ microglia. NG2+ cells are sparsely distributed in the centre of chronic MS lesions. These cells apparently survive demyelination and exhibit a multi-processed or bipolar morphology in the very hypocellular environment of the lesion.