Endothelial Wnt/ß-catenin signaling reduces immune cell infiltration in multiple sclerosis.

Disruption of the blood-brain barrier (BBB) is a defining and early feature of multiple sclerosis (MS) that directly damages the central nervous system (CNS), promotes immune cell infiltration, and influences clinical outcomes. There is an urgent need for new therapies to protect and restore BBB function, either by strengthening endothelial tight junctions or suppressing endothelial vesicular transcytosis. Although wingless integrated MMTV (Wnt)/ß-catenin signaling plays an essential role in BBB formation and maintenance in healthy CNS, its role in BBB repair in neurologic diseases such as MS remains unclear. Using a Wnt/ß-catenin reporter mouse and several downstream targets, we demonstrate that the Wnt/ß-catenin pathway is up-regulated in CNS endothelial cells in both human MS and the mouse model experimental autoimmune encephalomyelitis (EAE). Increased Wnt/ß-catenin activity in CNS blood vessels during EAE progression correlates with up-regulation of neuronal Wnt3 expression, as well as breakdown of endothelial cell junctions. Genetic inhibition of the Wnt/ß-catenin pathway in CNS endothelium before disease onset exacerbates the clinical presentation of EAE, CD4+ T-cell infiltration into the CNS, and demyelination by increasing expression of vascular cell adhesion molecule-1 and the transcytosis protein Caveolin-1 and promoting endothelial transcytosis. However, Wnt signaling attenuation does not affect the progressive degradation of tight junction proteins or paracellular BBB leakage. These results suggest that reactivation of Wnt/ß-catenin signaling in CNS vessels during EAE/MS partially restores functional BBB integrity and limits immune cell infiltration into the CNS.

The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination.

Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination.

Multiple sclerosis: Serum-derived exosomes express myelin proteins.

BACKGROUND: Exosomes are small extracellular vesicles that provide cell-to-cell communication and are involved in immunoregulation. OBJECTIVE: To investigate serum exosomes for the presence of myelin proteins outside the central nervous system (CNS) and their role in multiple sclerosis (MS). METHODS: Serum, cerebrospinal fluid (CSF), and peripheral blood mononuclear cell (PBMC) samples were collected from 45 patients with relapsing-remitting MS (RRMS), 30 patients with secondary progressive MS (SPMS), and 45 healthy controls. Exosomes were isolated using a polymer formulation method, and their size, concentration, and CNS myelin protein contents were measured by a nanoparticle tracking analysis, enzyme-linked immunosorbent assays, and Western blot. RESULTS: We found that exosomes expressed three major myelin proteins, myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG). Exosomal content of MOG strongly correlated with disease activity and was highest in RRMS patients in relapse and in SPMS patients. Serum-derived exosomes induced proliferation of MOG-T cell receptor transgenic T cells confirming that serum exosomes maintained MOG immunogenicity. CONCLUSION: Exosomes isolated outside CNS tissue expressed myelin proteins, and the presence of MOG correlated strongly with disease activity. We conclude that exosomes might enhance and/or perpetuate anti-myelin immune reactions in MS and may provide novel markers of disease activity.

Myelin phagocytosis by astrocytes after myelin damage promotes lesion pathology.

Astrocytes are key players in the pathology of multiple sclerosis and can assume beneficial and detrimental roles during lesion development. The triggers and timing of the different astroglial responses in acute lesions remain unclear. Astrocytes in acute multiple sclerosis lesions have been shown previously to contain myelin debris, although its significance has not been examined. We hypothesized that myelin phagocytosis by astrocytes is an early event during lesion formation and leads to astroglial immune responses. We examined multiple sclerosis lesions and other central nervous system pathologies with prominent myelin injury, namely, progressive multifocal leukoencephalopathy, metachromatic leukodystrophy and subacute infarct. In all conditions, we found that myelin debris was present in most astrocytes at sites of acute myelin breakdown, indicating that astroglial myelin phagocytosis is an early and prominent feature. Functionally, myelin debris was taken up by astrocytes through receptor-mediated endocytosis and resulted in astroglial NF-κB activation and secretion of chemokines. These in vitro results in rats were validated in human disease where myelin-positive hypertrophic astrocytes showed increased nuclear localization of NF-κB and elevated chemokine expression compared to myelin-negative, reactive astrocytes. Thus, our data suggest that myelin uptake is an early response of astrocytes in diseases with prominent myelin injury that results in recruitment of immune cells. This first line response of astrocytes to myelin injury may exert beneficial or detrimental effects on the lesion pathology, depending on the inflammatory context. Modulating this response might be of therapeutic relevance in multiple sclerosis and other demyelinating conditions.

Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation, and less remyelination following cuprizone exposure.

The TAM (Tyro3, Axl, and MerTK) family of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and ProS1, are important for innate immune responses and central nervous system (CNS) homeostasis. While only Gas6 directly activates Axl, ProS1 activation of Tyro3/MerTK can indirectly activate Axl through receptor heterodimerization. Therefore, we generated Gas6-/- Axl-/- double knockout (DKO) mice to specifically examine the contribution of this signaling axis while retaining ProS1 signaling through Tyro3 and MerTK. We found that naïve young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3-weeks recovery from a 6-week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Iba1+ microglia/macrophages between the groups of mice. However, at 6-weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6-Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6-Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS1/Tyro3/MerTK signaling.

Multiple sclerosis: Presence of serum antibodies to lipids and predominance of cholesterol recognition.

A lot of available data on lipid immunology in multiple sclerosis (MS) have been derived from studies using synthetic lipids, therefore the role of lipids in the immunopathogenesis of MS remains poorly defined. The present study on the lipid response in MS was performed on native lipids from autopsied brain tissue. For this, lipid fractions (n = 9) were prepared from MS (n = 3) and control (n = 2) white matter according to the Folch procedure and were characterized depending on their solubility in chloroform/methanol. TLC showed that, in brain from MS cases, neutral lipids were rich in cholesterol and cholesterol esters while lipids from control brains displayed a predominance of phospholipids. MS serum IgG and IgM were found to bind to MS brain lipid fractions with a higher efficacy (p < 0.05) than the control serum. F(ab)2 fractionation revealed that MS serum IgG binding depended on a specific antibody-type of recognition. Pre-adsorption of serum with cholesterol, galactocerebrosides, sulfitides, and phosphatidylinositol prior to ELISA with MS brain lipids, showed that cholesterol diminished IgG and IgM binding up to 70%. Experiments with synthetic lipids confirmed the predominance of cholesterol binding by MS serum. Our results demonstrate that IgG and IgM fractions from MS serum specifically and predominantly recognize native cholesterol and cholesterol esters isolated from the brain tissue of patients with MS. © 2017 Wiley Periodicals, Inc.

Astrocytic TYMP and VEGFA drive blood-brain barrier opening in inflammatory central nervous system lesions.

In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood-brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood-brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood-brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood-brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood-brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP as an astrocyte-derived permeability factor, and suggest TYMP and VEGFA together promote blood-brain barrier breakdown.

Brain glycolipids suppress T helper cells and inhibit autoimmune demyelination.

The CNS is considered an immune privileged site because its repertoire of highly immunogenic molecules remains unseen by the immune system under normal conditions. However, the mechanism underlying the inhibition of immune reactions within the CNS environment is not known, particularly in regions containing myelin, which contains several potent proteins and lipids that are invariably recognized as foreign by immune system cells. Sulfatides constitute a major component of myelin glycolipids and are known to be capable of raising an immune response. In this study, the effect of sulfatides on mouse T cell function and differentiation was analyzed in vitro and in vivo. We found profound inhibition of sulfatide-dependent T cell proliferation which was particularly pronounced in naive T helper (Th) cells. The inhibitory effect of sulfatides on T cell function was CD1d-independent and was not related to apoptosis or necrosis but did involve the induction of anergy as confirmed by the upregulation of early growth response 2 transcription factor. A glycolipid 3-sulfate group was essential for the T cell suppression, and the T cell inhibition was galectin-4-dependent. Sulfatide stimulation in vitro led to prominent suppression of Th17 differentiation, and this was related to a decrease in susceptibility to disease in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. Thus, we have defined a novel mechanism of negative regulation of T cell function by endogenous brain-derived glycolipids, a family of molecules traditionally deemphasized in favor of myelin proteins in studies of CNS autoimmunity.

HINT1 peptide/Hsp70 complex induces NK-cell-dependent immunoregulation in a model of autoimmune demyelination.

Heat shock proteins (Hsps) interact with the immune system and have been shown to contribute to immunoregulation. As efficient chaperones, Hsps bind many peptides and these complexes have many yet-to-be-clarified functions. We have shown that Hsp70 is complexed within the mouse CNS with peptide CLAFHDISPQAPTHFLVIPK derived from histidine triad nucleotide-binding protein-1 (HINT138₋57/Hsp70). Only this complex, in contrast to other peptides complexed with Hsp70, was able to prevent experimental autoimmune encephalomyelitis (EAE) by induction of immunoregulatory mechanisms dependent on NK cells. Pretreatment of proteolipid protein peptide 139₋151(PLP139₋151) sensitized SJL/J mice with HINT138₋57/Hsp70 prevented the development of EAE, suppressed PLP139₋151-induced T-cell proliferation, and blocked secretion of IL-17. HINT138₋57 /Hsp70 stimulation of NK cells depended on synergistic activation of two NK-cell receptors, CD94 and NKG2D. NK cells with depleted CD94 or with blocked NKG2D did not inhibit PLP139₋151-induced spleen cell (SC) proliferation. The HINT138₋57/Hsp70 complex enhanced surface expression of the NKG2D ligand-H60. Downstream signaling of CD94 and NKG2D converged at the adaptor proteins DAP10 and DAP12, and in response to HINT138₋57 /Hsp70 stimulation, expression of DAP10 and DAP12 was significantly increased in NK cells. Thus, we have shown that the HINT138₋57 /Hsp70 complex affects NK-cell function by enhancing NK-cell-dependent immunoregulation in the EAE model of autoimmune demyelination.

Accelerated repair of demyelinated CNS lesions in the absence of non-muscle myosin IIB.

The oligodendrocyte (OL), the myelinating cell of the central nervous system, undergoes dramatic changes in the organization of its cytoskeleton as it differentiates from a precursor (oligodendrocyte precursor cells) to a myelin-forming cell. These changes include an increase in its branching cell processes, a phenomenon necessary for OL to myelinate multiple axon segments. We have previously shown that levels and activity of non-muscle myosin II (NMII), a regulator of cytoskeletal contractility, decrease as a function of differentiation and that inhibition of NMII increases branching and myelination of OL in coculture with neurons. We have also found that mixed glial cell cultures derived from NMIIB knockout mice display an increase in mature myelin basic protein-expressing OL compared with wild-type cultures. We have now extended our studies to investigate the role of NMIIB ablation on myelin repair following focal demyelination by lysolecithin. To this end, we generated an oligodendrocyte-specific inducible knockout model using a Plp-driven promoter in combination with a temporally activated CRE-ER fusion protein. Our data indicate that conditional ablation of NMII in adult mouse brain, expedites lesion resolution and remyelination by Plp+ oligodendrocyte-lineage cells when compared with that observed in control brains. Taken together, these data validate the function of NMII as that of a negative regulator of OL myelination in vivo and provide a novel target for promoting myelin repair in conditions such as multiple sclerosis.

Proteomic analysis of active multiple sclerosis lesions reveals therapeutic targets.

Understanding the neuropathology of multiple sclerosis (MS) is essential for improved therapies. Therefore, identification of targets specific to pathological types of MS may have therapeutic benefits. Here we identify, by laser-capture microdissection and proteomics, proteins unique to three major types of MS lesions: acute plaque, chronic active plaque and chronic plaque. Comparative proteomic profiles identified tissue factor and protein C inhibitor within chronic active plaque samples, suggesting dysregulation of molecules associated with coagulation. In vivo administration of hirudin or recombinant activated protein C reduced disease severity in experimental autoimmune encephalomyelitis and suppressed Th1 and Th17 cytokines in astrocytes and immune cells. Administration of mutant forms of recombinant activated protein C showed that both its anticoagulant and its signalling functions were essential for optimal amelioration of experimental autoimmune encephalomyelitis. A proteomic approach illuminated potential therapeutic targets selective for specific pathological stages of MS and implicated participation of the coagulation cascade.

The astrocyte in multiple sclerosis revisited.

Among the constituent cell types of the multiple sclerosis (MS) plaque, the astrocyte has been the least considered as a player in the pathogenesis of the lesion. Traditionally, it has been assigned a secondary scarring role with little or no role in lesion formation or repair. However, the recent upsurge of interest in the demyelinating condition neuromyelitis optica (NMO) has resulted in NMO being identified as the first disease of myelin in which primary damage to astrocytes, resulting from a humoral immune response that forms against the water channel aquaporin-4, has been documented. This finding in NMO prompted us to re-examine data and material from cases of MS displaying active lesions. Our reappraisal revealed unambiguous early damage to perivascular astrocyte end-feet and to hypertrophic astrocytes in the adjacent parenchyma, but whether this was a primary event was difficult to evaluate due to concomitant edema and inflammation in these acute lesions. The astrocyte damage was long-lasting since resolving lesions displaying remyelination also showed defects in the integrity of the astrocytic covering around blood vessels. Analysis of our findings and of the astrocytic literature supports multiple roles for the astrocyte in the evolution of changes encountered in MS depending upon lesion stage and lesion topography. At variance with the somewhat inhibitory role of the astrocyte is the abundant and growing evidence for this cell to actively participate in both lesion development and repair. We propose that the unequivocal selective early involvement of the astrocyte in MS lesions may have therapeutic relevance

Proapoptotic and antiapoptotic actions of Stat1 versus Stat3 underlie neuroprotective and immunoregulatory functions of IL-11.

Current therapies for multiple sclerosis target inflammation but do not directly address oligodendrocyte protection or myelin repair. The gp130 family cytokines ciliary neurotrophic factor, leukemia inhibitory factor, and IL-11 have been identified as oligodendrocyte growth factors, and IL-11 is also strongly immunoregulatory, but their underlying mechanisms of action are incompletely characterized. In this study, we demonstrate that these effects of IL-11 are mediated via differential regulation of apoptosis in oligodendrocytes versus Ag-presenting dendritic cells (DCs), and are dependent on lineage-specific activity of the transcription factors Stat1 versus Stat3. Focal demyelinating lesions induced in cerebral cortices of IL-11Rα(-/-) mice using stereotactic microinjection of lysolecithin were larger than in controls, and remyelination was delayed. In IL-11Rα(-/-) mice, lesions displayed extensive oligodendrocyte loss and axonal transection, and increased infiltration by inflammatory cells including CD11c(+) DCs, CD3(+) lymphocytes, and CD11b(+) phagocytes. In oligodendrocyte progenitor cell (OPC) cultures, IL-11 restricted caspase 9 activation and apoptosis, and it increased myelination in OPC-neuron cocultures. Importantly, siRNA inhibition of Stat1 enhanced the antiapoptotic effects of IL-11 on OPCs, but IL-11 induced apoptosis in the presence of Stat3 silencing. In contrast, IL-11 augmented caspase activation and apoptosis in cultures of CD11c(+) DCs, but not in CD11b(+) or CD3(+) cells. Inhibition of Stat3 exacerbated the proapoptotic effects of IL-11 on DCs, whereas they were ablated in Stat1(-/-) cultures. Collectively, these findings reveal novel mechanisms underlying the actions of a neuroprotective and immunoregulatory member of the gp130 cytokine family, suggesting avenues to enhance oligodendrocyte viability and restrict CNS inflammation in multiple sclerosis.

Transcriptional profiling of microdissected areas of active multiple sclerosis lesions reveals activation of heat shock protein genes.

Heat shock proteins (HSPs) are stress-responsive proteins that serve as important molecules contributing to cellular "protein triage." We and others have reported an increase of selected HSPs in multiple sclerosis (MS) lesions. However, the exact expression pattern of HSP family genes in MS is not known. The aim of our research was to assess global transcriptional changes of all gene members of the HSP families within MS lesions and associated normal-appearing white matter (NAWM). To this end, we used laser capture microdissection (LCM) to isolate defined regions of chronic-active MS lesions (n = 5), one of the most common types of MS lesions. To identify changes in HSP genes in relation to different areas of the plaque, we used genome-wide microarray analysis. We detected a significant change in the transcriptional profile of the demyelinated region compared with NAWM. In particular, overall expression of different HSP genes was upregulated in different areas of chronic-active lesion. These changes were linked to an upregulation of heat shock factor 4 (HSF4). This is the first global analysis of transcriptional changes in HSPs in the central nervous system during MS. The results support a relationship between HSP activation and lesion activity.

Blocking angiotensin-converting enzyme induces potent regulatory T cells and modulates TH1- and TH17-mediated autoimmunity.

The renin-angiotensin-aldosterone system (RAAS) is a major regulator of blood pressure. The octapeptide angiotensin II (AII) is proteolytically processed from the decapeptide AI by angiotensin-converting enzyme (ACE), and then acts via angiotensin type 1 and type 2 receptors (AT1R and AT2R). Inhibitors of ACE and antagonists of the AT1R are used in the treatment of hypertension, myocardial infarction, and stroke. We now show that the RAAS also plays a major role in autoimmunity, exemplified by multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Using proteomics, we observed that RAAS is up-regulated in brain lesions of MS. AT1R was induced in myelin-specific CD4+ T cells and monocytes during autoimmune neuroinflammation. Blocking AII production with ACE inhibitors or inhibiting AII signaling with AT1R blockers suppressed autoreactive TH1 and TH17 cells and promoted antigen-specific CD4+FoxP3+ regulatory T cells (Treg cells) with inhibition of the canonical NF-kappaB1 transcription factor complex and activation of the alternative NF-kappaB2 pathway. Treatment with ACE inhibitors induces abundant CD4+FoxP3+ T cells with sufficient potency to reverse paralytic EAE. Modulation of the RAAS with inexpensive, safe pharmaceuticals used by millions worldwide is an attractive therapeutic strategy for application to human autoimmune diseases.

Notch1 signaling plays a role in regulating precursor differentiation during CNS remyelination.

In the developing CNS, Notch1 and its ligand, Jagged1, regulate oligodendrocyte differentiation and myelin formation, but their role in repair of demyelinating lesions in diseases such as multiple sclerosis remains unresolved. To address this question, we generated a mouse model in which we targeted Notch1 inactivation to oligodendrocyte progenitor cells (OPCs) using Olig1Cre and a floxed Notch1 allele, Notch1(12f). During CNS development, OPC differentiation was potentiated in Olig1Cre:Notch1(12f/12f) mice. Importantly, in adults, remyelination of demyelinating lesions was also accelerated, at the expense of proliferation within the progenitor population. Experiments in vitro confirmed that Notch1 signaling was permissive for OPC expansion but inhibited differentiation and myelin formation. These studies also revealed that astrocytes exposed to TGF-beta1 restricted OPC maturation via Jagged1-Notch1 signaling. These data suggest that Notch1 signaling is one of the mechanisms regulating OPC differentiation during CNS remyelination. Thus, Notch1 may represent a potential therapeutical avenue for lesion repair in demyelinating disease.

Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation.

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-beta 1 (TGF-beta 1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-beta 1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.

Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis.

Microarray analysis of multiple sclerosis (MS) lesions obtained at autopsy revealed increased transcripts of genes encoding inflammatory cytokines, particularly interleukin-6 and -17, interferon-gamma and associated downstream pathways. Comparison of two poles of MS pathology--acute lesions with inflammation versus 'silent' lesions without inflammation--revealed differentially transcribed genes. Some products of these genes were chosen as targets for therapy of experimental autoimmune encephalomyelitis (EAE) in mice. Granulocyte colony-stimulating factor is upregulated in acute, but not in chronic, MS lesions, and the effect on ameliorating EAE is more pronounced in the acute phase, in contrast to knocking out the immunoglobulin Fc receptor common gamma chain where the effect is greatest on chronic disease. These results in EAE corroborate the microarray studies on MS lesions. Large-scale analysis of transcripts in MS lesions elucidates new aspects of pathology and opens possibilities for therapy.

Multiple Sclerosis: circRNA Profile Defined Reveals Links to B-Cell Function.

BACKGROUND AND OBJECTIVES: To investigate the total circular RNA (circRNA) profile in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy controls (HCs). METHODS: Hybridization microarray was used to define the circRNA profile in peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with RRMS (10 in relapse and 10 in remission) and 10 HCs. We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-PCR with an independent cohort of 47 patients with RRMS (19 in relapse and 28 in remission) and 27 HCs. RESULTS: Microarray analysis revealed 914 transcripts to be differentially expressed between patients with RRMS in relapse and HCs (p < 0.05). We validated 3 circRNAs from 5 showing highest levels of differential expression in the RRMS relapse vs HC group: hsa_circRNA_101348, hsa_circRNA_102611, and hsa_circRNA_104361. Their expression was significantly increased during relapse in RRMS (p = 0.0002, FC = 2.9; p = 0.01, FC = 1.6; and p = 0.001, FC = 1.5, respectively) and in patients showing gadolinium enhancement on brain MRI (hsa_circRNA_101348, p = 0.0039, FC = 2.4; hsa_circRNA_104361, p = 0.029, FC = 1.7). Bioinformatic analysis revealed 15 microRNAs interacting with these circRNAs in a complementary manner and led to the discovery and validation of 3 protein-coding RNAs upregulated in patients with RRMS during relapse. Two of these, AK2 and IKZF3, have previously been implicated in B-cell function. DISCUSSION: circRNAs display a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNA in the known disturbed B-cell activity in RRMS and thus represent a novel biomarker for monitoring relapse activity.

The endocannabinoid anandamide protects neurons during CNS inflammation by induction of MKP-1 in microglial cells.

Endocannabinoids are released after brain injury and believed to attenuate neuronal damage by binding to CB(1) receptors and protecting against excitotoxicity. Such excitotoxic brain lesions initially result in primary destruction of brain parenchyma, which attracts macrophages and microglia. These inflammatory cells release toxic cytokines and free radicals, resulting in secondary neuronal damage. In this study, we show that the endocannabinoid system is highly activated during CNS inflammation and that the endocannabinoid anandamide (AEA) protects neurons from inflammatory damage by CB(1/2) receptor-mediated rapid induction of mitogen-activated protein kinase phosphatase-1 (MKP-1) in microglial cells associated with histone H3 phoshorylation of the mkp-1 gene sequence. As a result, AEA-induced rapid MKP-1 expression switches off MAPK signal transduction in microglial cells activated by stimulation of pattern recognition receptors. The release of AEA in injured CNS tissue might therefore represent a new mechanism of neuro-immune communication during CNS injury, which controls and limits immune response after primary CNS damage.