An Animal Model for Chronic Meningeal Inflammation and Inflammatory Demyelination of the Cerebral Cortex.

Modeling chronic cortical demyelination allows the study of long-lasting pathological changes observed in multiple sclerosis such as failure of remyelination, chronically disturbed functions of oligodendrocytes, neurons and astrocytes, brain atrophy and cognitive impairments. We aimed at generating an animal model for studying the consequences of chronic cortical demyelination and meningeal inflammation. To induce long-lasting cortical demyelination and chronic meningeal inflammation, we immunized female Lewis rats against myelin oligodendrocyte glycoprotein (MOG) and injected lentiviruses for continuing overexpression of the cytokines TNFα and IFNγ in the cortical brain parenchyma. Immunization with MOG and overexpression of TNFα and IFNγ led to widespread subpial demyelination and meningeal inflammation that were stable for at least 10 weeks. We demonstrate here that immunization with MOG is necessary for acute as well as chronic cortical demyelination. In addition, long-lasting overexpression of TNFα and IFNγ in the brain parenchyma is sufficient to induce chronic meningeal inflammation. Our model simulates key features of chronic cortical demyelination and inflammation, reminiscent of human multiple sclerosis pathology. This will allow molecular, cellular and functional investigations for a better understanding of the adaptation mechanisms of the cerebral cortex in multiple sclerosis.

Molecular signature of slowly expanding lesions in progressive multiple sclerosis.

Multiple sclerosis is an immune-mediated chronic inflammatory disease of the CNS that leads to demyelinated lesions in the grey and white matter. Inflammatory, active demyelinating white matter lesions predominate in the relapsing-remitting disease stages, whereas in the progressive stage the so-called slowly expanding lesion is characteristic. These lesions show an accumulation of macrophages/microglia at their borders, mediating the ongoing myelin breakdown and axonal degeneration. The exact pathogenetic mechanisms of lesion progression in chronic multiple sclerosis are still not clear. In the present study, we performed a detailed immunological and molecular profiling of slowly expanding lesions (n = 21) from 13 patients aged between 30 to 74 years (five females and eight males), focusing on macrophage/microglia differentiation. By applying the microglia-specific marker TMEM119, we demonstrate that cells accumulating at the lesion edge almost exclusively belonged to the microglia lineage. Macrophages/microglia can be subdivided into the M1 type, which are associated with inflammatory and degenerative processes, and M2 type, with protective properties, whereby also intermediate polarization phenotypes can be observed. By using a panel of markers characterizing M1- or M2-type macrophages/microglia, we observed a preferential accumulation of M1-type differentiated cells at the lesion edge, indicating a crucial role of these cells in lesion progression. Additionally, unbiased RNA microarray analyses of macrodissected lesion edges from slowly expanding and chronic inactive lesions as well as normal-appearing white matter were performed. In slowly expanding lesions, we identified a total of 165 genes that were upregulated and 35 genes that were downregulated. The upregulated genes included macrophage/microglia-associated genes involved in immune defence and inflammatory processes. Among the upregulated genes were ALOX15B, MME and TNFRSF25. We confirmed increased expression of ALOX15B by quantitative PCR, and of all three genes on the protein level by immunohistochemistry. In conclusion, the present study characterized in detail slowly expanding lesions in progressive multiple sclerosis and demonstrated a preferential accumulation of resident microglia with M1 differentiation at the lesion edge. Microarray analysis showed an increased expression of genes related to immune function, metabolic processes as well as transcription/translation. Thus, these genes may serve as future therapeutic targets to impede lesion progression.

Cocapture of cognate and bystander antigens can activate autoreactive B cells.

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are associated with autoimmune central nervous system diseases like acute disseminated encephalomyelitis (ADEM). For ADEM, it is speculated that a preceding infection is the trigger of the autoimmune response, but the mechanism connecting the infection to the production of MOG antibodies remains a mystery. We reasoned that the ability of B cells to capture cognate antigen from cell membranes, along with small quantities of coexpressed "bystander" antigens, might enable B-cell escape from tolerance. We tested this hypothesis using influenza hemagglutinin as a model viral antigen and transgenic, MOG-specific B cells. Using flow cytometry and live and fixed cell microscopy, we show that MOG-specific B cells take up large amounts of MOG from cell membranes. Uptake of the antigen from the membrane leads to a strong activation of the capturing B cell. When influenza hemagglutinin is also present in the membrane of the target cell, it can be cocaptured with MOG by MOG-specific B cells via the B-cell receptor. Hemagglutinin and MOG are both presented to T cells, which in turn are activated and proliferate. As a consequence, MOG-specific B cells get help from hemagglutinin-specific T cells to produce anti-MOG antibodies. In vivo, the transfer of MOG-specific B cells into recipient mice after the cocapture of MOG and hemagglutinin leads to the production of class-switched anti-MOG antibodies, dependent on the presence of hemagglutinin-specific T cells. This mechanism offers a link between infection and autoimmunity.

Selective in vivo removal of pathogenic anti-MAG autoantibodies, an antigen-specific treatment option for anti-MAG neuropathy.

Anti-MAG (myelin-associated glycoprotein) neuropathy is a disabling autoimmune peripheral neuropathy caused by monoclonal IgM autoantibodies that recognize the carbohydrate epitope HNK-1 (human natural killer-1). This glycoepitope is highly expressed on adhesion molecules, such as MAG, present in myelinated nerve fibers. Because the pathogenicity and demyelinating properties of anti-MAG autoantibodies are well established, current treatments are aimed at reducing autoantibody levels. However, current therapies are primarily immunosuppressive and lack selectivity and efficacy. We therefore hypothesized that a significant improvement in the disease condition could be achieved by selectively neutralizing the pathogenic anti-MAG antibodies with carbohydrate-based ligands mimicking the natural HNK-1 glycoepitope 1. In an inhibition assay, a mimetic (2, mimHNK-1) of the natural HNK-1 epitope blocked the interaction of MAG with pathogenic IgM antibodies from patient sera but with only micromolar affinity. Therefore, considering the multivalent nature of the MAG-IgM interaction, polylysine polymers of different sizes were substituted with mimetic 2. With the most promising polylysine glycopolymer PL84(mimHNK-1)45 the inhibitory effect on patient sera could be improved by a factor of up to 230,000 per epitope, consequently leading to a low-nanomolar inhibitory potency. Because clinical studies indicate a correlation between the reduction of anti-MAG IgM levels and clinical improvement, an immunological surrogate mouse model for anti-MAG neuropathy producing high levels of anti-MAG IgM was developed. The observed efficient removal of these antibodies with the glycopolymer PL84(mimHNK-1)45 represents an important step toward an antigen-specific therapy for anti-MAG neuropathy.

The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.

Fibroblast growth factor signalling in multiple sclerosis: inhibition of myelination and induction of pro-inflammatory environment by FGF9.

Remyelination failure plays an important role in the pathophysiology of multiple sclerosis, but the underlying cellular and molecular mechanisms remain poorly understood. We now report actively demyelinating lesions in patients with multiple sclerosis are associated with increased glial expression of fibroblast growth factor 9 (FGF9), which we demonstrate inhibits myelination and remyelination in vitro. This inhibitory activity is associated with the appearance of multi-branched 'pre-myelinating' MBP+ / PLP+ oligodendrocytes that interact with axons but fail to assemble myelin sheaths; an oligodendrocyte phenotype described previously in chronically demyelinated multiple sclerosis lesions. This inhibitory activity is not due to a direct effect of FGF9 on cells of the oligodendrocyte lineage but is mediated by factors secreted by astrocytes. Transcriptional profiling and functional validation studies demonstrate that these include effects dependent on increased expression of tissue inhibitor of metalloproteinase-sensitive proteases, enzymes more commonly associated with extracellular matrix remodelling. Further, we found that FGF9 induces expression of Ccl2 and Ccl7, two pro-inflammatory chemokines that contribute to recruitment of microglia and macrophages into multiple sclerosis lesions. These data indicate glial expression of FGF9 can initiate a complex astrocyte-dependent response that contributes to two distinct pathogenic pathways involved in the development of multiple sclerosis lesions. Namely, induction of a pro-inflammatory environment and failure of remyelination; a combination of effects predicted to exacerbate axonal injury and loss in patients.

Myelin-associated glycoprotein gene mutation causes Pelizaeus-Merzbacher disease-like disorder.

Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by mutations or rearrangements in PLP1. It presents in infancy with nystagmus, jerky head movements, hypotonia and developmental delay evolving into spastic tetraplegia with optic atrophy and variable movement disorders. A clinically similar phenotype caused by recessive mutations in GJC2 is known as Pelizaeus-Merzbacher-like disease. Both genes encode proteins associated with myelin. We describe three siblings of a consanguineous family manifesting the typical infantile-onset Pelizaeus-Merzbacher disease-like phenotype slowly evolving into a form of complicated hereditary spastic paraplegia with mental retardation, dysarthria, optic atrophy and peripheral neuropathy in adulthood. Magnetic resonance imaging and spectroscopy were consistent with a demyelinating leukodystrophy. Using genetic linkage and exome sequencing, we identified a homozygous missense c.399C>G; p.S133R mutation in MAG. This gene, previously associated with hereditary spastic paraplegia, encodes myelin-associated glycoprotein, which is involved in myelin maintenance and glia-axon interaction. This mutation is predicted to destabilize the protein and affect its tertiary structure. Examination of the sural nerve biopsy sample obtained in childhood in the oldest sibling revealed complete absence of myelin-associated glycoprotein accompanied by ill-formed onion-bulb structures and a relatively thin myelin sheath of the affected axons. Immunofluorescence, cell surface labelling, biochemical analysis and mass spectrometry-based proteomics studies in a variety of cell types demonstrated a devastating effect of the mutation on post-translational processing, steady state expression and subcellular localization of myelin-associated glycoprotein. In contrast to the wild-type protein, the p.S133R mutant was retained in the endoplasmic reticulum and was subjected to endoplasmic reticulum-associated protein degradation by the proteasome. Our findings identify involvement of myelin-associated glycoprotein in this family with a disorder affecting the central and peripheral nervous system, and suggest that loss of the protein function is responsible for the unique clinical phenotype.

Interleukin-22 is increased in multiple sclerosis patients and targets astrocytes.

BACKGROUND: Increasing evidences link T helper 17 (Th17) cells with multiple sclerosis (MS). In this context, interleukin-22 (IL-22), a Th17-linked cytokine, has been implicated in blood brain barrier breakdown and lymphocyte infiltration. Furthermore, polymorphism between MS patients and controls has been recently described in the gene coding for IL-22 binding protein (IL-22BP). Here, we aimed to better characterize IL-22 in the context of MS. METHODS: IL-22 and IL-22BP expressions were assessed by ELISA and qPCR in the following compartments of MS patients and control subjects: (1) the serum, (2) the cerebrospinal fluid, and (3) immune cells of peripheral blood. Identification of the IL-22 receptor subunit, IL-22R1, was performed by immunohistochemistry and immunofluorescence in human brain tissues and human primary astrocytes. The role of IL-22 on human primary astrocytes was evaluated using 7-AAD and annexin V, markers of cell viability and apoptosis, respectively. RESULTS: In a cohort of 141 MS patients and healthy control (HC) subjects, we found that serum levels of IL-22 were significantly higher in relapsing MS patients than in HC but also remitting and progressive MS patients. Monocytes and monocyte-derived dendritic cells contained an enhanced expression of mRNA coding for IL-22BP as compared to HC. Using immunohistochemistry and confocal microscopy, we found that IL-22 and its receptor were detected on astrocytes of brain tissues from both control subjects and MS patients, although in the latter, the expression was higher around blood vessels and in MS plaques. Cytometry-based functional assays revealed that addition of IL-22 improved the survival of human primary astrocytes. Furthermore, tumor necrosis factor α-treated astrocytes had a better long-term survival capacity upon IL-22 co-treatment. This protective effect of IL-22 seemed to be conferred, at least partially, by a decreased apoptosis. CONCLUSIONS: We show that (1) there is a dysregulation in the expression of IL-22 and its antagonist, IL-22BP, in MS patients, (2) IL-22 targets specifically astrocytes in the human brain, and (3) this cytokine confers an increased survival of the latter cells.

DARC shuttles inflammatory chemokines across the blood-brain barrier during autoimmune central nervous system inflammation.

The Duffy antigen/receptor for chemokines, DARC, belongs to the family of atypical heptahelical chemokine receptors that do not couple to G proteins and therefore fail to transmit conventional intracellular signals. Here we show that during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, the expression of DARC is upregulated at the blood-brain barrier. These findings are corroborated by the presence of a significantly increased number of subcortical white matter microvessels staining positive for DARC in human multiple sclerosis brains as compared to control tissue. Using an in vitro blood-brain barrier model we demonstrated that endothelial DARC mediates the abluminal to luminal transport of inflammatory chemokines across the blood-brain barrier. An involvement of DARC in experimental autoimmune encephalomyelitis pathogenesis was confirmed by the observed ameliorated experimental autoimmune encephalomyelitis in Darc(-/-) C57BL/6 and SJL mice, as compared to wild-type control littermates. Experimental autoimmune encephalomyelitis studies in bone marrow chimeric Darc(-/-) and wild-type mice revealed that increased plasma levels of inflammatory chemokines in experimental autoimmune encephalomyelitis depended on the presence of erythrocyte DARC. However, fully developed experimental autoimmune encephalomyelitis required the expression of endothelial DARC. Taken together, our data show a role for erythrocyte DARC as a chemokine reservoir and that endothelial DARC contributes to the pathogenesis of experimental autoimmune encephalomyelitis by shuttling chemokines across the blood-brain barrier.

An essential role of MAG in mediating axon-myelin attachment in Charcot-Marie-Tooth 1A disease.

Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary demyelinating peripheral neuropathy caused by the duplication of the PMP22 gene. Demyelination precedes the occurrence of clinical symptoms that correlate with axonal degeneration. It was postulated that a disturbed axon-glia interface contributes to altered myelination consequently leading to axonal degeneration. In this study, we examined the expression of MAG and Necl4, two critical adhesion molecules that are present at the axon-glia interface, in sural nerve biopsies of CMT1A patients and in peripheral nerves of mice overexpressing human PMP22, an animal model for CMT1A. We show an increase in the expression of MAG and a strong decrease of Necl4 in biopsies of CMT1A patients as well as in CMT1A mice. Expression analysis revealed that MAG is strongly upregulated during peripheral nerve maturation, whereas Necl4 expression remains very low. Ablating MAG in CMT1A mice results in separation of axons from their myelin sheath. Our data show that MAG is important for axon-glia contact in a model for CMT1A, and suggest that its increased expression in CMT1A disease has a compensatory role in the pathology of the disease. Thus, we demonstrate that MAG together with other adhesion molecules such as Necl4 is important in sustaining axonal integrity.

Coronin 1-mediated naive T cell survival is essential for the development of autoimmune encephalomyelitis.

Autoimmune encephalomyelitis is a disease of the CNS that can develop when an initial peripheral inflammatory stimulus is followed by infiltration and reactivation of T lymphocytes in the CNS. We report a crucial role for coronin 1, which is essential for maintenance of the naive T cell pool, for the development of murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. In the absence of coronin 1, immunization with myelin oligoglycoprotein (MOG(35-55)) peptide largely failed to induce EAE symptoms, despite normal mobilization of leukocyte subsets in the blood, as well as effector cytokine expression comparable with wild-type T cells on polyclonal stimulation. Susceptibility of coronin 1-deficient mice to EAE induction was restored by transfer of wild-type CD4(+) T cells, suggesting that the observed resistance of coronin 1-deficient mice to EAE development is T cell intrinsic. Importantly, although coronin 1-deficient regulatory T cells (Tregs) showed a suppressor activity comparable with wild-type Tregs, Treg depletion failed to restore EAE development in coronin 1-deficient animals. These results suggest a hitherto unrecognized role of naive T cells in the development of autoimmune encephalomyelitis and reveal coronin 1 as a crucial modulator of EAE induction.

VIP17/MAL expression modulates epithelial cyst formation and ciliogenesis.

The polarized organization of epithelial cells is required for vectorial solute transport and may be altered in renal cystic diseases. Vesicle integral protein of 17 kDa (VIP17/MAL) is involved in apical vesicle transport. VIP17/MAL overexpression in vivo results in renal cystogenesis of unknown etiology. Renal cystogenesis can occur as a consequence of defects of the primary cilium. To explore the role of VIP17/MAL in renal cystogenesis and ciliogenesis, we examined the polarization and ciliary morphology of wild-type and VIP17/MAL overexpressing Madin-Darby canine kidney renal epithelial cells grown in two-dimensional (2D) and three-dimensional (3D) cyst culture. VIP17/MAL is apically localized when expressed in cells maintained in 2D and 3D culture. VIP17/MAL overexpressing cells produce more multilumen cysts compared with controls. While the distributions of basolateral markers are not affected, VIP17/MAL expression results in aberrant sorting of the apical marker gp135 to the primary cilium. VIP17/MAL overexpression is also associated with shortened or absent cilia. Immunofluorescence analysis performed on kidney sections from VIP17/MAL transgenic mice also demonstrates fewer and shortened cilia within dilated lumens (P < 0.01). These studies demonstrate that VIP17/MAL overexpression results in abnormal cilium and cyst development, in vitro and in vivo, suggesting that VIP17/MAL overexpressing mice may develop cysts secondary to a ciliary defect.

Theophylline Induces Remyelination and Functional Recovery in a Mouse Model of Peripheral Neuropathy.

Charcot-Marie-Tooth disease (CMT) is a large group of inherited peripheral neuropathies that are primarily due to demyelination and/or axonal degeneration. CMT type 1A (CMT1A), which is caused by the duplication of the peripheral myelin protein 22 (PMP22) gene, is a demyelinating and the most frequent CMT subtype. Hypermyelination, demyelination, and secondary loss of large-caliber axons are hallmarks of CMT1A, and there is currently no cure and no efficient treatment to alleviate the symptoms of the disease. We previously showed that histone deacetylases 1 and 2 (HDAC1/2) are critical for Schwann cell developmental myelination and remyelination after a sciatic nerve crush lesion. We also demonstrated that a short-term treatment with Theophylline, which is a potent activator of HDAC2, enhances remyelination and functional recovery after a sciatic nerve crush lesion in mice. In the present study, we tested whether Theophylline treatment could also lead to (re)myelination in a PMP22-overexpressing mouse line (C22) modeling CMT1A. Indeed, we show here that a short-term treatment with Theophylline in C22 mice increases the percentage of myelinated large-caliber axons and the expression of the major peripheral myelin protein P0 and induces functional recovery. This pilot study suggests that Theophylline treatment could be beneficial to promote myelination and thereby prevent axonal degeneration and enhance functional recovery in CMT1A patients.

Global deprivation of brain-derived neurotrophic factor in the CNS reveals an area-specific requirement for dendritic growth.

Although brain-derived neurotrophic factor (BDNF) is linked with an increasing number of conditions causing brain dysfunction, its role in the postnatal CNS has remained difficult to assess. This is because the bdnf-null mutation causes the death of the animals before BDNF levels have reached adult levels. In addition, the anterograde axonal transport of BDNF complicates the interpretation of area-specific gene deletion. The present study describes the generation of a new conditional mouse mutant essentially lacking BDNF throughout the CNS. It shows that BDNF is not essential for prolonged postnatal survival, but that the behavior of such mutant animals is markedly altered. It also reveals that BDNF is not a major survival factor for most CNS neurons and for myelination of their axons. However, it is required for the postnatal growth of the striatum, and single-cell analyses revealed a marked decreased in dendritic complexity and spine density. In contrast, BDNF is dispensable for the growth of the hippocampus and only minimal changes were observed in the dendrites of CA1 pyramidal neurons in mutant animals. Spine density remained unchanged, whereas the proportion of the mushroom-type spine was moderately decreased. In line with these in vivo observations, we found that BDNF markedly promotes the growth of cultured striatal neurons and of their dendrites, but not of those of hippocampal neurons, suggesting that the differential responsiveness to BDNF is part of a neuron-intrinsic program.

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

EEF1A1 deacetylation enables transcriptional activation of remyelination.

Remyelination of the peripheral and central nervous systems (PNS and CNS, respectively) is a prerequisite for functional recovery after lesion. However, this process is not always optimal and becomes inefficient in the course of multiple sclerosis. Here we show that, when acetylated, eukaryotic elongation factor 1A1 (eEF1A1) negatively regulates PNS and CNS remyelination. Acetylated eEF1A1 (Ac-eEF1A1) translocates into the nucleus of myelinating cells where it binds to Sox10, a key transcription factor for PNS and CNS myelination and remyelination, to drag Sox10 out of the nucleus. We show that the lysine acetyltransferase Tip60 acetylates eEF1A1, whereas the histone deacetylase HDAC2 deacetylates eEF1A1. Promoting eEF1A1 deacetylation maintains the activation of Sox10 target genes and increases PNS and CNS remyelination efficiency. Taken together, these data identify a major mechanism of Sox10 regulation, which appears promising for future translational studies on PNS and CNS remyelination.

Increased HLA-DR expression and cortical demyelination in MS links with HLA-DR15.

OBJECTIVE: To investigate molecular changes in multiple sclerosis (MS) normal-appearing cortical gray matter (NAGM). METHODS: We performed a whole-genome gene expression microarray analysis of human brain autopsy tissues from 64 MS NAGM samples and 42 control gray matter samples. We further examined our cases by HLA genotyping and performed immunohistochemical and immunofluorescent analysis of all human brain tissues. RESULTS: HLA-DRB1 is the transcript with highest expression in MS NAGM with a bimodal distribution among the examined cases. Genotyping revealed that every case with the MS-associated HLA-DR15 haplotype also shows high HLA-DRB1 expression and also of the tightly linked HLA-DRB5 allele. Quantitative immunohistochemical analysis confirmed the higher expression of HLA-DRB1 in HLA-DRB1*15:01 cases at the protein level. Analysis of gray matter lesion size revealed a significant increase of cortical lesion size in cases with high HLA-DRB1 expression. CONCLUSIONS: Our data indicate that increased HLA-DRB1 and -DRB5 expression in the brain of patients with MS may be an important factor in how the HLA-DR15 haplotype contributes to MS pathomechanisms in the target organ.

Immunoglobulin M deposition in cutaneous nerves of anti-myelin-associated glycoprotein polyneuropathy patients correlates with axonal degeneration.

Anti-myelin-associated glycoprotein (MAG) neuropathy is an antibody-mediated polyneuropathy. We correlated clinical features, immunoglobulin (Ig) M blood levels, IgM deposition and axonal degeneration in skin biopsies of anti-MAG neuropathy patients. By confocal microscopy, IgM deposits were found exclusively within perineurium-enclosed nerves; they were not found on single, non-perineurium-ensheathed myelinated axons. There was a linear correlation between IgM accumulation in nerve fascicles with IgM blood levels but not with anti-MAG antibody titer or disease duration. Axons with specific IgM deposits had signs of axonal damage, including neurofilament disintegration. Nodal structures were intact even at sites where the axons showed pathologic changes. Ultrastructural analysis revealed degeneration of myelinating Schwann cells. Taken together, these findings suggest that in anti-MAG neuropathy patients, IgM deposits are entrapped within cutaneous perineurium-ensheathed nerve bundles where they accumulate in the endoneurial space. High local IgM levels in the endoneurium may be required for IgM deposition on myelin and subsequent axonal injury and degeneration. This study underlines the importance of early, effective anti-B-cell treatments for preventing progression of this neuropathy.

The raft-associated protein MAL is required for maintenance of proper axon--glia interactions in the central nervous system.

The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. We show that genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode--axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered. Initial formation of paranodal regions appeared normal, but abnormalities became detectable when MAL started to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.

Normal-appearing white matter in multiple sclerosis is in a subtle balance between inflammation and neuroprotection.

Multiple sclerosis is a chronic inflammatory disease of the CNS. Although progressive axonal injury and diffuse inflammatory damage has been shown in the chronic phase of the disease, little is known about the molecular mechanisms underlying these pathological processes. In order to identify these mechanisms, we have studied the gene expression profile in non-lesion containing tissue, the so-called normal-appearing white matter (NAWM). We performed differential gene expression analysis and quantitative RT-PCR on subcortical white matter from 11 multiple sclerosis and 8 control cases. Differentially expressed genes were further analysed in detail by in situ hybridization and immunofluorescence studies. We show that genes known to be involved in anti-inflammatory and protective mechanisms such as STAT6, JAK1, IL-4R, IL-10, Chromogranin C and Hif-1alpha are consistently upregulated in the multiple sclerosis NAWM. On the other hand, genes involved in pro-inflammatory mechanisms, such as STAT4, IL-1beta and MCSF, were also upregulated but less regularly. Immunofluorescence colocalization analysis revealed expression of STAT6, JAK1, IL-4R and IL-13R mainly in oligodendrocytes, whereas STAT4 expression was detected predominantly in microglia. In line with these data, in situ hybridization analysis showed an increased expression in multiple sclerosis NAWM of HIF-1alpha in oligodendrocytes and HLA-DRalpha in microglia cells. The consistency of the expression levels of STAT6, JAK1, JAK3 and IL-4R between the multiple sclerosis cases suggests an overall activation of the STAT6-signalling pathway in oligodendrocytes, whereas the expression of STAT4 and HLA-DRalpha indicates the activation of pro-inflammatory pathways in microglia. The upregulation of genes involved in anti-inflammatory mechanisms driven by oligodendrocytes may protect the CNS environment and thus limit lesion formation, whereas the activation of pro-inflammatory mechanisms in microglia may favour disease progression. Altogether, our data suggests an endogenous inflammatory reaction throughout the whole white matter of multiple sclerosis brain, in which oligodendrocytes actively participate. This reaction might further influence and to some extent facilitate lesion formation.