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Multiple sclerosis (MS) is a chronic central nervous system (CNS) disorder characterized by demyelination, neuronal damage, and oligodendrocyte depletion. Reliable biomarkers are essential for early diagnosis and disease management. Emerging research highlights the role of mitochondrial dysfunction and oxidative stress in CNS disorders, including MS, in which mitochondria are central to the degenerative process. Adenosine monophosphate-activated protein kinase (AMPK) regulates the mitochondrial energy balance and initiates responses in neurodegenerative conditions. This systematic review, following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, aimed to comprehensively assess the literature on AMPK pathways, mitochondrial dysfunction, and in vivo studies using MS animal models. The search strategy involved the use of AMPK syntaxes, MS syntaxes, and animal model syntaxes. The PubMed, Scopus, Web of Science, and Google Scholar databases were systematically searched on August 26, 2023 without publication year restrictions. The review identified and analyzed relevant papers to provide a comprehensive overview of the current state of related research. Eight studies utilizing various interventions and methodological approaches were included. Risk of bias assessment revealed some areas of low risk but lacked explicit reporting in others. These studies collectively revealed a complex relationship between AMPK, mitochondrial dysfunction, and MS pathogenesis, with both cuprizone and experimental autoimmune encephalomyelitis models demonstrating associations between AMPK and mitochondrial disorders, including oxidative stress and impaired expression of mitochondrial genes. These studies illuminate the multifaceted role of AMPK in MS animal models, involving energy metabolism, inflammatory processes, oxidative stress, and gene regulation leading to mitochondrial dysfunction. However, unanswered questions about its mechanisms and clinical applications underscore the need for further research to fully harness its potential in addressing MS-related mitochondrial dysfunction., (© 2024 Wiley Periodicals LLC.)

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis 1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY + p300 + memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY + p300 + astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Caspase-8 functions at the crossroad of programmed cell death and inflammation. Here, using genetic approaches and the experimental autoimmune encephalomyelitis model of inflammatory demyelination, we identified a negative regulatory pathway for caspase-8 in infiltrated macrophages whereby it functions to restrain interleukin (IL)-1β-driven autoimmune inflammation. Caspase-8 is partially activated in macrophages/microglia in active lesions of multiple sclerosis. Selective ablation of Casp8 in myeloid cells, but not microglia, exacerbated autoimmune demyelination. Heightened IL-1β production by caspase-8-deficient macrophages underlies exacerbated activation of encephalitogenic T cells and production of GM-CSF and interferon-γ. Mechanistically, IL-1β overproduction by primed caspase-8-deficient macrophages was mediated by RIPK1/RIPK3 through the engagement of NLRP3 inflammasome and was independent of cell death. When instructed by autoreactive CD4 T cells in the presence of antigen, caspase-8-deficient macrophages, but not their wild-type counterparts, released significant amount of IL-1β that in turn acted through IL-1R to amplify T cell activation. Moreover, the worsened experimental autoimmune encephalomyelitis progression in myeloid Casp8 mutant mice was completely reversed when Ripk3 was simultaneously deleted. Together, these data reveal a functional link between T cell-driven autoimmunity and inflammatory IL-1β that is negatively regulated by caspase-8, and suggest that dysregulation of the pathway may contribute to inflammatory autoimmune diseases, such as multiple sclerosis.

N-acylethanolamines (NAEs) are endogenous bioactive lipids reported to exert anti-inflammatory and neuroprotective effects mediated by cannabinoid receptors and peroxisome proliferator-activated receptors (PPARs), among others. Therefore, interfering with NAE signaling could be a promising strategy to decrease inflammation in neurological disorders such as multiple sclerosis (MS). Fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase (NAAA) are key modulators of NAE levels. This study aims to investigate and compare the effect of NAAA inhibition, FAAH inhibition, and dual inhibition of both enzymes in a mouse model of MS, namely the experimental autoimmune encephalomyelitis (EAE). Our data show that NAAA inhibition strongly decreased the hallmarks of the pathology. Interestingly, FAAH inhibition was less efficient in decreasing inflammatory hallmarks despite the increased NAE levels. Moreover, the inhibition of both NAAA and FAAH, using a dual-inhibitor or the co-administration of NAAA and FAAH inhibitors, did not show an added value compared to NAAA inhibition. Furthermore, our data suggest an important role of decreased activation of astrocytes and microglia in the effects of NAAA inhibition on EAE, while NAAA inhibition did not affect T cell recall. This work highlights the beneficial effects of NAAA inhibition in the context of central nervous system inflammation and suggests that the simultaneous inhibition of NAAA and FAAH has no additional beneficial effect in EAE., (© 2021. The American Society for Experimental NeuroTherapeutics, Inc.)

Multiple Sclerosis (MS) is a debilitating central nervous system disorder associated with inflammatory T cells. Activation and expansion of inflammatory T cells is thought to be behind MS relapses and influence disease severity. Protein arginine N-methyltransferase 5 (PRMT5) is a T cell activation-induced enzyme that symmetrically dimethylates proteins and promotes T cell proliferation. However, the mechanism behind PRMT5-mediated control of T cell proliferation and whether PRMT5 contributes to diseases severity is unclear. Here, we evaluated the role of PRMT5 on cyclin/cdk pairs and cell cycle progression, as well as PRMT5's link to disease severity in an animal model of relapsing-remitting MS. Treatment of T helper 1 (mTh1) cells with the selective PRMT5 inhibitor, HLCL65, arrested activation-induced T cell proliferation at the G1 stage of the cell cycle, suggesting PRMT5 promotes cell cycle progression in CD4 + T cells. The Cyclin E1/Cdk2 pair promoting G1/S progression was also decreased after PRMT5 inhibition, as was the phosphorylation of retinoblastoma. In the SJL mouse relapsing-remitting model of MS, the highest PRMT5 expression in central nervous system-infiltrating cells corresponded to peak and relapse timepoints. PRMT5 expression also positively correlated with increasing CD4 Th cell composition, disease severity and Cyclin E1 expression. These data indicate that PRMT5 promotes G1/S cell cycle progression and suggest that this effect influences disease severity and/or progression in the animal model of MS. Modulating PRMT5 levels may be useful for controlling T cell expansion in T cell-mediated diseases including MS., Competing Interests: MG-d-A has a PRMT5 inhibitor patent pending and is a PRMT5 inhibitor inventor on a licensing deal with Prelude Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amici, Osman and Guerau-de-Arellano.)

Perturbed expression of microRNAs (miRs) has been reported in different diseases including autoimmune and chronic inflammatory disorders. In this study, we investigated the expression of miR-25-3p and its targets in the central nervous system (CNS) tissue from mice with experimental autoimmune encephalomyelitis (EAE). We also analyzed the expression of miR-25 and its targets in activated macrophages and splenocytes. EAE was induced in 12-week old female C57BL/6 mice; using myelin oligodendrocyte glycoprotein 35-55/complete Freund's adjuvant (MOG35-55/CFA) protocol. The expression of miR-25-3p and its targets, as well as the expression of inflammatory cytokines, were analyzed. We next established primary macrophage cultures as well as splenocyte cultures and evaluated the levels of miR-25-3p and its target genes in these cells following activation with lipopolysaccharide (LPS) and anti-CD3/anti-CD28 antibodies, respectively. MiR-25-3p expression showed a strong positive correlation with the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 pro-inflammatory cytokines. The expression of phosphatase and tensin homolog (Pten) and Krüppel-like factor 4 (Klf4) was significantly reduced at the peak of the disease. Interestingly, Pten and Klf4 expression showed a significant negative correlation with miR-25-3p. Analysis of miR-25-3p expression in LPS-treated primary macrophages revealed significant upregulation in cells treated with 100ng/ml of LPS. This was associated with suppressed levels of miR-25-3p targets in these cells. However, anti-CD3/anti-CD28-stimulated splenocytes failed to show any alterations in miR-25-3p expression compared with vehicle-treated cells. Our results indicate that miR-25-3p expression is likely induced by inflammatory mediators during autoimmune neuroinflammation. This upregulation is associated with decreased levels of Pten and Klf4, genes with known roles in cell cycle regulation and inflammation.

Cellular therapy with mesenchymal stem cells (MSCs) is a huge challenge for scientists, as little translational relevance has been achieved. However, many studies using MSCs have proved their suppressive and regenerative capacity. Thus, there is still a need for a better understanding of MSCs biology and the establishment of newer protocols, or to test unexplored tissue sources. Here, we demonstrate that murine endometrial-derived MSCs (meMSCs) suppress Experimental Autoimmune Encephalomyelitis (EAE). MSC-treated animals had milder disease, with a significant reduction in Th1 and Th17 lymphocytes in the lymph nodes and in the central nervous system (CNS). This was associated with increased Il27 and Cyp1a1 expression, and presence of IL-10-secreting T CD4+ cells. At EAE peak, animals had reduced CNS infiltrating cells, histopathology and demyelination. qPCR analysis evidenced the down-regulation of several pro-inflammatory genes and up-regulation of indoleamine-2,3-dioxygenase (IDO). Consistently, co-culturing of WT and IDO-/- meMSCs with T CD4+ cells evidenced the necessity of IDO on the suppression of encephalitogenic lymphocytes, and IDO-/- meMSCs were not able to suppress EAE. In addition, WT meMSCs stimulated with IL-17A and IFN-γ increased IDO expression and secretion of kynurenines in vitro, indicating a negative feedback loop. Pathogenic cytokines were increased when CD4+ T cells from AhR-/- mice were co-cultured with WT meMSC. In summary, our research evidences the suppressive activity of the unexplored meMSCs population, and shows the mechanism depends on IDO-kynurenines-Aryl hydrocarbon receptor (AhR) axis. To our knowledge this is the first report evidencing that the therapeutic potential of meMSCs relying on IDO expression., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Macrophages are highly plastic, key regulators of inflammation. Deregulation of macrophage activation can lead to excessive inflammation as seen in inflammatory disorders like atherosclerosis, obesity, multiple sclerosis and sepsis. Targeting intracellular metabolism is considered as an approach to reshape deranged macrophage activation and to dampen the progression of inflammatory disorders. ATP citrate lyase (Acly) is a key metabolic enzyme and an important regulator of macrophage activation. Using a macrophage-specific Acly-deficient mouse model, we investigated the role of Acly in macrophages during acute and chronic inflammatory disorders. First, we performed RNA sequencing to demonstrate that Acly-deficient macrophages showed hyperinflammatory gene signatures in response to acute LPS stimulation in vitro . Next, we assessed endotoxin-induced peritonitis in myeloid-specific Acly-deficient mice and show that, apart from increased splenic Il6 expression, systemic and local inflammation were not affected by Acly deficiency. Also during obesity, both chronic low-grade inflammation and whole-body metabolic homeostasis remained largely unaltered in mice with Acly-deficient myeloid cells. Lastly, we show that macrophage-specific Acly deletion did not affect the severity of experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis. These results indicate that, despite increasing inflammatory responses in vitro , macrophage Acly deficiency does not worsen acute and chronic inflammatory responses in vivo. Collectively, our results indicate that caution is warranted in prospective long-term treatments of inflammatory disorders with macrophage-specific Acly inhibitors. Together with our earlier observation that myeloid Acly deletion stabilizes atherosclerotic lesions, our findings highlight that therapeutic targeting of macrophage Acly can be beneficial in some, but not all, inflammatory disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Verberk, van der Zande, Baardman, de Goede, Harber, Keuning, Lambooij, Otto, Zawistowska-Deniziak, de Vries, de Winther, Guigas and Van den Bossche.)

The exact mechanisms of multiple sclerosis (MS) development are still unknown, but the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice is associated with the violation of bone marrow hematopoietic stem cells (HSCs) differentiation profiles associated with the production of harmful for human's autoantibodies hydrolyzing myelin basic protein, myelin oligodendrocyte glycoprotein (MOG 35-55 ), and DNA. It was shown that IgGs from the sera of healthy humans and autoimmune patients oxidize many different compounds due to their H 2 O 2 -dependent peroxidase and oxidoreductase activity in the absence of H 2 O 2 . Here we first analyzed the change in the relative redox activities of IgGs antibodies from the blood of C57BL/6 mice over time at different stages of the EAE development. It was shown that the peroxidase activity of mice IgGs in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is on average 6.9-fold higher than the oxidoreductase activity. The peroxidase activity of IgGs increased during the spontaneous development of EAE during 40 days, 1.4-fold. After EAE development acceleration due to mice immunization with MOG 35-55 (5.3-fold), complexes of bovine DNA with methylated bovine serum albumin (DNA-metBSA; 3.5-fold), or with histones (2.6-fold), the activity was increased much faster. The increase in peroxidase activity after mice immunization with MOG 35-55 and DNA-metBSA up to 40 days of experiments was relatively gradual, while for DNA-histones complex was observed its sharp increase at the acute phase of EAE (14-20 days). All data show that IgGs' redox activities can play an important role in the protection of mice from toxic compounds and oxidative stress.

Proteoglycans (PGs) are one of the main components in the extracellular matrix of the central nervous system. Chondroitin sulfate (CS) is a glycosaminoglycan (GAG), which is composed of major PGs. Similar to keratin sulfate (KS), another GAG, CS inhibits axon regeneration. However, the influence of these GAGs on the pathogenicity of neuroimmunological diseases is unclear. Here, we induced experimental autoimmune encephalomyelitis (EAE) in mice lacking CS N-acetylgalactosaminyltransferase-1 (CSGalNAcT1-KO), an important enzyme for CS synthesis. In our study, CSGalNAcT1-KO mice showed milder EAE symptoms than those in wild-type (WT) mice. The recall response of antigen-specific lymphocytes showed that CSGalNAcT1-KO-derived lymphocytes had a milder cell proliferation response than that in WT-derived lymphocytes. These results suggest that CS contributes toward the induction phase of EAE. We previously performed EAE experiments in GlcNAc-6-O-sulfotransferase KO (GlcNAc6ST-KO) and C6ST1-KO mice, which had reduced KS and reduced CS-C, respectively. EAE in CSGalNAcT1-KO mice was more similar to that in GlcNAc6ST-KO mice than in C6ST1-KO mice. In conclusion, the distinct GAG sugar chains are associated with severe or mild phenotypes of EAE and are therefore potential new therapeutic targets for neuroimmunological diseases, including multiple sclerosis., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The experimental autoimmune encephalomyelitis (EAE) is a model that mimics multiple sclerosis in rodents. Evidence has suggested that the activation of indoleamine-2,3-dioxygenase (IDO), the rate-limiting enzyme in the kynurenine pathway (KP), plays a crucial role in inflammation-related diseases. The present study aimed to investigate the involvement of the inflammatory process and KP components in a model of EAE in mice. To identify the role of KP in EAE pathogenesis, mice received IDO inhibitor (INCB024360) at a dose of 200 mg/kg (per oral) for 25 days. We demonstrated that IDO inhibitor mitigated the clinical signs of EAE, in parallel with the reduction of cytokine levels (brain, spinal cord, spleen and lymph node) and ionized calcium-binding adaptor protein-1 (Iba-1) gene expression in the central nervous system of EAE mice. Besides, IDO inhibitor causes a significant decrease in the levels of tryptophan, kynurenine and neurotoxic metabolites of KP, such as 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) in the prefrontal cortex, hippocampus, spinal cord, spleen and lymph node of EAE mice. The mRNA expression and enzyme activity of IDO and kynurenine 3-monooxygenase (KMO) were also reduced by IDO inhibitor. These findings indicate that the inflammatory process concomitant with the activation of IDO/KP is involved in the pathogenic mechanisms of EAE. The modulation of KP is a promising target for novel pharmacological treatment of MS.

N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these Naaa CD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in Naaa CD11b+ than in control mice. Naaa CD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The proinflammatory cytokine interleukin 1 (IL-1) is crucially involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Herein, we studied the role of IL-1 signaling in blood-brain barrier (BBB) endothelial cells (ECs), astrocytes and microglia for EAE development, using mice with the conditional deletion of its signaling receptor IL-1R1. We found that IL-1 signaling in microglia and astrocytes is redundant for the development of EAE, whereas the IL-1R1 deletion in BBB-ECs markedly ameliorated disease severity. IL-1 signaling in BBB-ECs upregulated the expression of the adhesion molecules Vcam-1, Icam-1 and the chemokine receptor Darc, all of which have been previously shown to promote CNS-specific inflammation. In contrast, IL-1R1 signaling suppressed the expression of the stress-responsive heme catabolizing enzyme heme oxygenase-1 (HO-1) in BBB-ECs, promoting disease progression via a mechanism associated with deregulated expression of the IL-1-responsive genes Vcam1, Icam1 and Ackr1 (Darc). Mechanistically, our data emphasize a functional crosstalk of BBB-EC IL-1 signaling and HO-1, controlling the transcription of downstream proinflammatory genes promoting the pathogenesis of autoimmune neuroinflammation.

Anti-CD20-mediated B-cell depletion effectively reduces acute multiple sclerosis (MS) flares. Recent data shows that antibody-mediated extinction of B cells as a lasting immune suppression, harbors the risk of developing humoral deficiencies over time. Accordingly, more selective, durable and reversible B-cell-directed MS therapies are needed. We here tested inhibition of Bruton's tyrosine kinase (BTK), an enzyme centrally involved in B-cell receptor signaling, as the most promising approach in this direction. Using mouse models of MS, we determined that evobrutinib, the first BTK inhibiting molecule being developed, dose-dependently inhibited antigen-triggered activation and maturation of B cells as well as their release of pro-inflammatory cytokines. Most importantly, evobrutinib treatment functionally impaired the capacity of B cells to act as antigen-presenting cells for the development of encephalitogenic T cells, resulting in a significantly reduced disease severity in mice. In contrast to anti-CD20, BTK inhibition silenced this key property of B cells in MS without impairing their frequency or functional integrity. In conjunction with a recent phase II trial reporting that evobrutinib is safe and effective in MS, our mechanistic data highlight therapeutic BTK inhibition as a landmark towards selectively interfering with MS-driving B-cell properties.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15, THOP1) is a metallopeptidase ubiquitously distributed in mammalian tissues. Beyond its previously well characterized role in major histocompatibility class I (MHC-I) antigen presentation, the recent characterization of the THOP1 C57BL6/N null mice (THOP1 -/- ) phenotype suggests new key functions for THOP1 in hyperlipidic diet-induced obesity, insulin resistance and non-alcoholic liver steatosis. Distinctive levels of specific intracellular peptides (InPeps), genes and microRNAs were observed when comparing wild type C57BL6/N to THOP1 -/- fed either standard or hyperlipidic diets. A possible novel mechanism of action was suggested for InPeps processed by THOP1, which could be modulating protein-protein interactions and microRNA processing, thus affecting the phenotype. Together, research into the biochemical and biomedical significance of THOP1 suggests that degradation by the proteasome is a step in the processing of various proteins, not merely for ending their existence. This allows many functional peptides to be generated by proteasomal degradation in order to, for example, control mRNA translation and the formation of protein complexes., Competing Interests: The authors declare no conflict of interest.

Cluster of differentiation 226 (CD226) molecules play a crucial role in the activation of effector CD4 + T cells during the immune response process, but a cell-intrinsic function of CD226 in CD4 + T subsets is not clear. In this study, we showed that Cd226 -/- mice were resistant to myelin oligodendrocyte glycoprotein peptide 35-55 (MOG 35-55 )-induced experimental autoimmune encephalomyelitis (EAE) with highly expressed IL-10 + CD4 + T cells and downregulated IL-17A + CD4 + T cells when compared with wild-type (WT) mice. Th17 cell infiltration into the central nervous system (CNS) was largely decreased in the absence of CD226 during EAE. CD226 deficiency facilitated the proliferation of regulatory T cells (Tregs), with increased numbers of Tregs observed in EAE mice, and supported the elevated induced regulatory T cell (iTregs) proliferation in vitro . The Akt and Erk signaling pathways were shown to be involved in Cd226 -/- Treg proliferation and function in vivo and in vitro . These findings collectively indicate that CD226 is a key molecule regulating the Treg-mediated suppression of autoimmune responses by inhibiting Treg proliferation. Thus, the results of this study identify additional mechanisms by which CD226 regulates Treg functions in EAE and supports the potential therapeutic effects of anti-CD226 molecules on autoimmune diseases., (Copyright © 2020 Wang, Yi, Fang, Jin, Ma, Shen, Li, Liang, Xiong, Li, Zeng, Jiang, Jin and Chen.)

Multiple sclerosis (MS) is the most common autoimmune disease of the CNS. The etiology of MS is still unclear but it is widely recognized that both genetic and environmental factors contribute to its pathogenesis. Immune signaling and responses are critically regulated by ubiquitination, a posttranslational modification that is promoted by ubiquitinating enzymes and inhibited by deubiquitinating enzymes (DUBs). Genome-wide association studies (GWASs) identified that polymorphisms in or in the vicinity of two human DUB genes TNFAIP3 and USP18 were associated with MS susceptibility. Studies with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, have provided biological rationale for the correlation between these DUBs and MS. Additional studies have shown that other DUBs are also involved in EAE by controlling distinct cell populations. Therefore, DUBs are emerging as crucial regulators of MS/EAE and might become potential therapeutic targets for the clinical treatment of MS.

l-tryptophan (Trp), an essential amino acid for mammals, is the precursor of a wide array of immunomodulatory metabolites produced by the kynurenine and serotonin pathways. The kynurenine pathway is a paramount source of several immunoregulatory metabolites, including l-kynurenine (Kyn), the main product of indoleamine 2,3-dioxygenase 1 (IDO1) that catalyzes the rate-limiting step of the pathway. In the serotonin pathway, the metabolite N -acetylserotonin (NAS) has been shown to possess antioxidant, antiinflammatory, and neuroprotective properties in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). However, little is known about the exact mode of action of the serotonin metabolite and the possible interplay between the 2 Trp metabolic pathways. Prompted by the discovery that NAS neuroprotective effects in EAE are abrogated in mice lacking IDO1 expression, we investigated the NAS mode of action in neuroinflammation. We found that NAS directly binds IDO1 and acts as a positive allosteric modulator (PAM) of the IDO1 enzyme in vitro and in vivo. As a result, increased Kyn will activate the ligand-activated transcription factor aryl hydrocarbon receptor and, consequently, antiinflammatory and immunoregulatory effects. Because NAS also increased IDO1 activity in peripheral blood mononuclear cells of a significant proportion of MS patients, our data may set the basis for the development of IDO1 PAMs as first-in-class drugs in autoimmune/neuroinflammatory diseases., Competing Interests: The authors declare no competing interest.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) exhibits neuropathological and immunological dysfunctions similar to those found in multiple sclerosis (MS) and has been used as an animal model of MS. Inflammatory infiltrates and oxidative stress have been linked to the development of both diseases. Ethanolamine plasmalogen derivates have been shown to be powerful antioxidants and immunomodulators. Therefore, the objective of this study was to analyse inflammatory infiltrates, the state of the oxidative defences and the possible protective effects of calcium, magnesium and phosphate ethanolamine (EAP) in the CR-EAE rat hippocampus. To this aim, we evaluated, by immunohistochemistry, T cell infiltrates, Iba-1 + (a marker of activated microglia) immunoreactivity and TUNEL (+) cells. We also measured the protein levels and activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione reductase (GR). In addition, reduced (GSH) and oxidized (GSSG) glutathione levels, lipid peroxidation and cholesterol as well as desmosterol content were determined. We found an increase in T cell infiltrates and Iba1 + immunoreactivity, lipid peroxidation, SOD, GP and GR activities as well as enhanced cholesterol levels and a decrease in CAT activity, GSH and desmosterol levels in the first and second attack in the CR-EAE rat hippocampus. Pretreatment of CR-EAE rats with EAP led to a delay in the onset of the clinical signs of the disease as well as a decrease in inflammatory infiltrates and alterations of the antioxidant defences in the hippocampus. Altogether, the present results suggest a protective role of EAP in the CR-EAE rat hippocampus.

Oligodendrocytes (OLs) are the myelinating glia of the central nervous system. Injury to OLs causes myelin loss. In demyelinating diseases, such as multiple sclerosis, the remyelination is hindered principally due to a failure of the oligodendrocyte precursor cells (OPCs) to differentiate into mature OLs. To identify inducers of OPC to OL differentiation, a high-throughput screening based on myelin basic protein expression using neural progenitor cells-derived OPCs has been performed and, PD0325901-an MEK (MAPK kinase) inhibitor-is found to significantly enhance OPC to OL differentiation in a dose- and time-dependent manner. Other MEK inhibitors also display similar effect, indicating blockade of MAPK-ERK signaling is sufficient to induce OPC differentiation into OLs. PD0325901 facilitates the formation of myelin sheaths in OPC-neuron co-culture in vitro. And in experimental autoimmune encephalomyelitis model and cuprizone-induced demyelination model, PD0325901 displays significant therapeutic effect by promoting myelin regeneration. Our results suggest that targeting the MAPK-ERK pathway might be an intriguing way to develop new therapies for demyelinating diseases., (© 2019 The Authors. Glia published by Wiley Periodicals, Inc.)

Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4+ T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4+ T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4+ T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.

Inducible nitric oxide synthase (iNOS) plays a critical role in the regulation of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Previous studies have shown that iNOS plays pathogenic as well as regulatory roles in MS and EAE. However, how does iNOS alters the pathophysiology of the central nervous system (CNS) in neuronal autoimmunity is not clearly understood. In the present work, we show that treatment of mice with L-NAME, an iNOS inhibitor, during the antigen-priming phase primarily alters brain pathology, while in the subsequent effector phase of the immune response, the spinal cord is involved. Inhibition of iNOS during the priming phase of the immune response promotes the infiltration of pathogenic CD11b + F4/80 - Gr-1 + cells, but there is low recruitment of regulatory CD11b + F4/80 + cells in the brain. Inhibition of iNOS during the effector phase shows similar pathogenic alterations in the spinal cord, instead of in the brain. Treatment of wild-type mice with L-NAME or mice having genetic deficiency of iNOS show lower MHC-II expression on the dendritic cells, but not on macrophages. Our data suggest that iNOS has a critical regulatory role during antigen-priming as well as in the effector phase of EAE, and inhibition iNOS at different stages of the immune response can differentially alter either the brain or spinal cord pathology. Understanding the cellular and molecular mechanisms through which iNOS functions could help to design a better strategies for the clinical management of neuroinflammation and neuronal autoimmunity.

Glutathione peroxidase 4 (GPx4) is the only enzyme capable of reducing toxic lipid hydroperoxides in biological membranes to the corresponding alcohols using glutathione as the electron donor. GPx4 is the major inhibitor of ferroptosis, a non-apoptotic and iron-dependent programmed cell death pathway, which has been shown to occur in various neurological disorders with severe oxidative stress. In this study, we investigate whether GPx4 expression is altered in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). The results clearly show that mRNA expression for all three GPx4 isoforms (cytoplasmic, mitochondrial and nuclear) decline in multiple sclerosis gray matter and in the spinal cord of MOG 35-55 peptide-induced EAE. The amount of GPx4 protein is also reduced in EAE, albeit not in all cells. Neuronal GPx4 immunostaining, mostly cytoplasmic, is lower in EAE spinal cords than in control spinal cords, while oligodendrocyte GPx4 immunostaining, mainly nuclear, is unaltered. Neither control nor EAE astrocytes and microglia cells show GPx4 labeling. In addition to GPx4, two other negative modulators of ferroptosis (γ-glutamylcysteine ligase and cysteine/glutamate antiporter), which are critical to maintain physiological levels of glutathione, are diminished in EAE. The decrease in the ability to eliminate hydroperoxides was also evidenced by the accumulation of lipid peroxidation products and the reduction in the proportion of the docosahexaenoic acid in non-myelin lipids. These findings, along with presence of abnormal neuronal mitochondria morphology, which includes an irregular matrix, disrupted outer membrane and reduced/absent cristae, are consistent with the occurrence of ferroptotic damage in inflammatory demyelinating disorders., (© 2018 International Society for Neurochemistry.)

Phospholipases A2 (PLA 2 ) are a diverse group of enzymes that cleave the fatty acids of membrane phospholipids. They play critical roles in pathogenesis of neurodegenerative diseases such as multiple sclerosis by enhancing oxidative stress and initiating inflammation. The levels of PLA 2 activity in MS patients compared to controls and role of inhibiting PLA 2 activity on severity scores in different experimental models are not comprehensively assessed in the light of varying evidence from published studies. The objective of this systematic review is to determine the association between PLA 2 activity and multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). We performed a systematic review of six studies that assessed PLA 2 activity in MS patients compared to controls and nine studies that assessed PLA 2 activity in EAE. sPLA 2 nor Lp-PLA 2 activity were not increased in MS compared to controls in five of those six studies. A difference in sPLA 2 activity was only found in a study that measured the enzyme activity in urine. However, inhibiting cPLA 2 or sPLA 2 led to lower clinical severity or no signs of EAE in mice, and a lower incidence of EAE lesions compared to animals without cPLA 2 inhibition. These findings indicate that PLA 2 appears to play a role in the pathogenesis of EAE., (Copyright © 2018. Published by Elsevier B.V.)

Background/aims: Multiple sclerosis (MS) is one of the most common autoimmune disorders of the central nervous system (CNS) and the leading cause of neurological disability among young adults in the Western world. We have previously shown that the acid sphingomyelinase plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis., Methods: We induced adoptively transferred EAE in wildtype and acid sphingomyelinase-deficient mice. In addition, we immunized mice with MOG aa35-55 to induce active EAE and treated the mice with amitriptyline, a functional inhibitor of the acid sphingomyelinase. We investigated symptoms of EAE, blood-brain barrier integrity and neuroinflammation., Results: In the model of adoptively transferred EAE we demonstrate that expression of acid sphingomyelinase in the recipients rather than on transferred encephalitogenic T cells contributes to the clinical development of EAE symptoms. To test if pharmacological targeting of acid sphingomyelinase can be explored for the development of novel therapies for MS, we inhibited acid sphingomyelinase with amitriptyline in mice in which EAE was induced by active immunization. We demonstrate that pharmacological inhibition of acid sphingomyelinase using amitriptyline protects against the development of EAE and markedly attenuates the characteristic detrimental neuroinflammatory response., Conclusion: The studies identify the acid sphingomyelinase as a novel therapeutic target for treating MS patients., Competing Interests: The authors declare to have no conflict of interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

NLRP3 inflammasome plays a critical spatiotemporal role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). This study reports a mechanistic insight into noncanonical NLRP3 inflammasome activation in microglia for the effector stage of EAE. Microglia-specific deficiency of ASC (apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment [CARD] domain) attenuated T cell expansion and neutrophil recruitment during EAE pathogenesis. Mechanistically, TLR stimulation led to IRAKM-caspase-8-ASC complex formation, resulting in the activation of caspase-8 and IL-1β release in microglia. Noncanonical inflammasome-derived IL-1β produced by microglia in the CNS helped to expand the microglia population in an autocrine manner and amplified the production of inflammatory cytokines/chemokines. Furthermore, active caspase-8 was markedly increased in the microglia in the brain tissue from patients with multiple sclerosis. Taken together, our study suggests that microglia-derived IL-1β via noncanonical caspase-8-dependent inflammasome is necessary for microglia to exert their pathogenic role during CNS inflammation.

Microsomal prostaglandin E synthetase-1 (mPGES-1) is an inducible terminal enzyme for the production of prostaglandin E₂ (PGE₂). In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, mPGES-1 is induced in vascular endothelial cells (VECs) around inflammatory foci and facilitates inflammation, demyelination, and paralysis. Therefore, we investigated the role of CD31-positive VECs in mPGES-1-mediated EAE aggravation using immunohistochemical analysis and imaging of wild-type (wt) and mPGES - 1 -deficient ( mPGES-1 -/- ) mice. We demonstrated that EAE induction facilitated vascularity in inflammatory lesions in the spinal cord, and this was significantly higher in wt mice than in mPGES-1 -/- mice. In addition, endothelial interleukin-1β (IL-1β) production was significantly higher in wt mice than in mPGES-1 -/- mice. Moreover, endothelial PGE₂ receptors (E-prostanoid (EP) receptors EP1⁻4) were expressed after EAE induction, and IL-1β was induced in EP receptor-positive VECs. Furthermore, IL-1 receptor 1 expression on VECs was increased upon EAE induction. Thus, increased vascularity is one mechanism involved in EAE aggravation induced by mPGES-1. Furthermore, mPGES-1 facilitated the autocrine function of VECs upon EP receptor induction and IL-1β production, modulating mPGES-1 induction in EAE.

Phosphodiesterase-5 inhibitors (PDE5Is) have been shown to modulate cell death/cell survival in different in vivo and in vitro models of disease by activating many signaling pathways. This review aimed at elucidating how PDE5Is can inhibit apoptosis. In this study, we describe many signaling pathways involved with the mechanism of action of PDE5Is that ultimately inhibit apoptosis and thus promote cell survival.

Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase (PDH) is the key bifurcation enzyme, which in its active dephosphorylated form advances the oxidative phosphorylation from glycolytic pathway. The transcriptional factor, inducible cAMP early repressor/cAMP response element modulator (ICER/CREM), has been shown to be induced in Th17 cells and to be overexpressed in CD4 + T cells from the patients with systemic lupus erythematosus (SLE). We found that glycolysis and lactate production in in vitro Th17-polarized T cells was reduced and that the expression of pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), an enzyme that converts the inactive PDH to its active form, and PDH enzyme activity were increased in Th17 cells from ICER/CREM-deficient animals. ICER was found to bind to the Pdp2 promoter and suppress its expression. Furthermore, forced expression of PDP2 in CD4 + cells reduced the in vitro Th17 differentiation, whereas shRNA-based suppression of PDP2 expression increased in vitro Th17 differentiation and augmented experimental autoimmune encephalomyelitis. At the translational level, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4 + T cells from lupus-prone MRL/ lpr mice and patients with SLE suppressed Th17 differentiation. These data demonstrate the direct control of energy production during Th17 differentiation in health and disease by the transcription factor ICER/CREM at the PDH metabolism bifurcation level., Competing Interests: The authors declare no conflict of interest.

Angiogenesis, a prominent feature of pathology, is known to be guided by factors secreted by living cells around a lesion. Although many cells are disrupted in a response to injury, the relevance of degenerating cells in pathological angiogenesis is unclear. Here, we show that the release of lactate dehydrogenase A (LDHA) from degenerating neurons drives central nervous system (CNS) angiogenesis. Silencing neuronal LDHA expression suppressed angiogenesis around experimental autoimmune encephalomyelitis (EAE)- and controlled cortical impact-induced lesions. Extracellular LDHA-mediated angiogenesis was dependent on surface vimentin expression and vascular endothelial growth factor receptor (VEGFR) phosphorylation in vascular endothelial cells. Silencing vimentin expression in vascular endothelial cells prevented angiogenesis around EAE lesions and improved survival in a mouse model of glioblastoma. These results elucidate novel mechanisms that may mediate pathologic angiogenesis and identify a potential molecular target for the treatment of CNS diseases involving angiogenesis., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Oxidative stress and mitochondrial dysfunction are important determinants of neurodegeneration in secondary progressive multiple sclerosis (SPMS). We previously showed that febuxostat, a xanthine oxidase inhibitor, ameliorated both relapsing-remitting and secondary progressive experimental autoimmune encephalomyelitis (EAE) by preventing neurodegeneration in mice. In this study, we investigated how febuxostat protects neuron in secondary progressive EAE. A DNA microarray analysis revealed that febuxostat treatment increased the CNS expression of several mitochondria-related genes in EAE mice, most notably including GOT2, which encodes glutamate oxaloacetate transaminase 2 (GOT2). GOT2 is a mitochondrial enzyme that oxidizes glutamate to produce α-ketoglutarate for the Krebs cycle, eventually leading to the production of adenosine triphosphate (ATP). Whereas GOT2 expression was decreased in the spinal cord during the chronic progressive phase of EAE, febuxostat-treated EAE mice showed increased GOT2 expression. Moreover, febuxostat treatment of Neuro2a cells in vitro ameliorated ATP exhaustion induced by rotenone application. The ability of febuxostat to preserve ATP production in the presence of rotenone was significantly reduced by GOT2 siRNA. GOT2-mediated ATP synthesis may be a pivotal mechanism underlying the protective effect of febuxostat against neurodegeneration in EAE. Accordingly, febuxostat may also have clinical utility as a disease-modifying drug in SPMS.

Multiple sclerosis (MS) is a T cell autoimmune, inflammatory, and demyelinating disease of the central nervous system (CNS). Currently available therapies have partially effective actions and numerous side reactions. Inosine, an endogenous purine nucleoside, has immunomodulatory, neuroprotective, and analgesic properties. Herein, we evaluated the effect of inosine on the development and progression of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. Inosine (1 or 10 mg/kg, i.p.) was administrated twice a day for 40 days. Immunological and inflammatory responses were evaluated by behavioral, histological, immunohistochemical, ELISA, RT-PCR, and Western blotting analysis. The administration of inosine exerted neuroprotective effects against EAE by diminishing clinical signs, including thermal and mechanical hyperalgesia, as well as weight loss typical of the disease. These beneficial effects of inosine seem to be associated with the blockade of inflammatory cell entry into the CNS, especially lymphocytes, thus delaying the demyelinating process and astrocytes activation. In particular, up-regulation of IL-17 levels in the secondary lymphoid tissues, a result of EAE, was prevented by inosine treatment in EAE mice. Additionally, inosine consistently prevented A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. Altogether, these results allow us to propose that this endogenous purine might be a putative novel and helpful tool for the prevention of autoimmune and neurodegenerative diseases, such as MS. Thus, inosine could have considerable implications for future therapies of MS, and this study may represent the starting point for further investigation into the role of inosine and adenosinergic receptors in neuroinflammation processes. Graphical Abstract Preventive treatment with inosine inhibits the development and progression of EAE in C57Bl/6 mice. Furthermore, neuroinflammation and demyelinating processes were blocked by inosine treatment. Additionally, inosine consistently inhibited IL-17 levels in peripheral lymphoid tissue, as well as IL-4 levels and A2AR up-regulation in the spinal cord, likely, through an ERK1-independent pathway. EAE: experimental autoimmune encephalomyelitis; MS: multiple sclerosis; A2AR: adenosine A2A receptor; IL-17: interleukin-17; IL-4: interleukin-4.

Background: Multiple sclerosis (MS), a complex disease associated with multifocal demyelination of the central nervous system and poorly understood etiology. It has been previously indicated that many factors, including oxidative stress and p38MAPK-SGK1 pathway, contribute to the pathogenesis of MS., Methods: This study, using an experimental autoimmune encephalomyelitis (EAE) model system, was aimed at investigating the molecular mechanisms determining interaction p38MAPK-SGK1 pathway and oxidative stress in MS pathogenesis. C57BL/6 mice was immunized with MOG35-55 peptide for EAE induction, which was followed by determination of the effect of treatment with classic p38 inhibitor SB203580 and antioxidant tempol on the development and progression of EAE., Results: Our experiments showed a dynamic change of immune inflammation, oxidative stress and p38MAPK-SGK1 pathway involvement in EAE demonstrating that p38MAPK-SGK1 pathway and oxidative stress contribute to the demyelination in central nerve system caused by Th17 inflammatory responses in a synergistic way. The administration of SB203580 and Tempol both markedly suppressed the progression of EAE. Furthermore, tempol showed a strong inhibiting effect to the p38MAPK-SGK1 pathway similar to SB203580 suggesting that oxidative stress exacerbates EAE via the activation of p38MAPK-SGK1 pathway., Conclusion: Cumulatively, our results show that oxidative stress p38MAPK-SGK1 signaling pathway may be a central player in EAE and even in MS.

The catabolism of tryptophan to immunosuppressive and neuroactive kynurenines is a key metabolic pathway regulating immune responses and neurotoxicity. The rate-limiting step is controlled by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO). IDO is expressed in antigen presenting cells during immune reactions, hepatic TDO regulates blood homeostasis of tryptophan and neuronal TDO influences neurogenesis. While the role of IDO has been described in multiple immunological settings, little is known about TDO's effects on the immune system. TDO-deficiency is neuroprotective in C. elegans and Drosophila by increasing tryptophan and specific kynurenines. Here we have determined the role of TDO in autoimmunity and neurodegeneration in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We created reporter-TDO mice for in vivo imaging to show that hepatic but not CNS TDO expression is activated during EAE. TDO deficiency did not influence myelin-specific T cells, leukocyte infiltration into the CNS, demyelination and disease activity. TDO-deficiency protected from neuronal loss in the spinal cord but not in the optic nerves. While this protection did not translate to an improved overt clinical outcome, our data suggest that spatially distinct neuroprotection is conserved in mammals and support TDO as a potential target for treatment of diseases associated with neurodegeneration.

Targeting Polymerase-1 (POL1) transcription machinery is a new strategy for suppression of multiple sclerosis (MS) disease activity that is based on suppression of ribosomal biogenesis and subsequent activation of apoptosis. We developed an oral POL1 inhibiting compound RAM-589.555, that suppress ribosomal biogenesis as an innovative therapeutic approach to ameliorate MS. RAM-589.555 shows high permeability, specificity to POL1 pathway, ability to induce apoptosis and to inhibit proliferation and viability of activated lymphocytes both in-vitro and in-vivo. Moreover, oral administration of RAM-589.555 blocks ribosomal RNA transcription and significantly suppresses and ameliorates experimental autoimmune encephalomyelitis (EAE)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Plasmacytoid dendritic cells (pDCs) have been shown to both mediate and prevent autoimmunity, and the regulation of their immunogenic versus tolerogenic functions remains incompletely understood. Here we demonstrate that, compared to other cells, pDCs are the major expressors of Indoleamine-2,3-dioxygenase (IDO) in steady-state lymph nodes (LNs). IDO expression by LN pDCs was closely dependent on MHCII-mediated, antigen-dependent, interactions with Treg. We further established that IDO production by pDCs was necessary to confer suppressive function to Tregs. During EAE development, IDO expression by pDCs was required for the generation of Tregs capable of dampening the priming of encephalitogenic T cell and disease severity. Thus, we describe a novel crosstalk between pDCs and Tregs: Tregs shape tolerogenic functions of pDCs prior to inflammation, such that pDCs in turn, promote Treg suppressive functions during autoimmunity., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aberrant canonical Wnt-β-catenin signaling has been reported in multiple sclerosis (MS), although the results are controversial. The present study aimed to examine the role of the Wnt-β-catenin pathway in experimental MS and also to test moringin (4-[α-L-rhamnopyranosyloxy]-benzyl isothiocyanate), resulting from exogenous myrosinase hydrolysis of the natural phytochemical glucomoringin 4(α-L-rhamnosyloxy)-benzyl glucosinolate as a modulator of neuroinflammation via the β-catenin-PPARγ axis. Experimental autoimmune encephalomyelitis (EAE), the most common model of MS, was induced in C57BL/6 mice by immunization with MOG 35-55 . Released moringin (10 mg/kg glucomoringin +5 μL myrosinase/mouse) was administered daily for 1 week before EAE induction and continued until mice were killed on day 28 after EAE induction. Our results clearly showed that the Wnt-β-catenin pathway was downregulated in the EAE model, whereas moringin pretreatment was able to avert this. Moringin pretreatment normalizes the aberrant Wnt-β-catenin pathway, resulting in GSK3β inhibition and β-catenin upregulation, which regulates T-cell activation (CD4 and FoxP3), suppresses the main inflammatory mediators (IL-1β, IL-6, and COX2), through activation of PPARγ. In addition, moringin attenuates apoptosis by reducing the expression of the Fas ligand and cleaved caspase 9, and in parallel increases antioxidant Nrf2 expression in EAE mice. Taken together, our results provide an interesting discovery in identifying moringin as a modulator of the Wnt-β-catenin signaling cascade and as a new potential therapeutic target for MS treatment., Competing Interests: The authors report no conflicts of interest in this work.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease in the murine central nervous system (CNS) and recapitulates the clinical and pathological features of human multiple sclerosis (MS). Glutamate carboxipeptidase II (GCPII), an enzyme expressed exclusively on astrocytes, is known to affect the disease progression of various neurological disorders by producing glutamate. Despite several findings indicating possible link between glutamate and MS/EAE, however, the involvement of astrocyte or GCPII on glutamate excitotoxicity has not received much attention in MS/EAE. When we examined GCPII expression during EAE progression in this study, we observed significantly elevated GCPII expression in peak stage of disease localized mainly in astrocytes. Intrigued by these results, we tried a potent GCPII inhibitor, 2-phosphonomethyl pentanedioic acid (2-PMPA), on EAE mice and noticed markedly attenuated EAE clinical signs along with significantly inhibited infiltration of inflammatory cells into CNS. Furthermore, 2-PMPA dampened the function of Th1 cell lineage and down-regulated mGluR1 expression in both periphery and CNS contributing to glutamate-mediated immune regulation. Our observations identify a sequence of events triggering EAE through GCPII overexpression, which may offer a novel therapeutic approach to the treatment of MS., (© 2016 Federation of European Biochemical Societies.)

Type I Interferon (IFN-I) is critical for antiviral and antitumor defense. Additionally, IFN-I has been used for treating multiple sclerosis (MS), a chronic autoimmune disease of the central nervous system (CNS). Recently, we reported that 2'-5' oligoadenylate synthetase-like 1 (OASL1) negatively regulates IFN-I production upon viral infection and tumor challenge. Therefore, OASL1 deficient (Oasl1(-)(/)(-)) mice are resistant to viral infections and tumor challenge. In this study, we examined whether OASL1 plays a negative role in the development of autoimmune MS by using Oasl1(-)(/)(-) mice and a murine MS model, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). Oasl1(-)(/)(-) mice showed enhanced resistance to EAE development compared to wild-type (WT) mice. Additionally, EAE-induced Oasl1(-)(/)(-) mice showed fewer infiltrated immune cells such as T cells and macrophages in the CNS and less CNS inflammation, compared to WT mice. Collectively, these results indicate that OASL1 deficiency suppresses the development of MS-like autoimmunity and suggest that negative regulators of IFN-I could be good therapeutic targets for treating MS in humans., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The immunomodulatory drug FTY720 is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that requires activation by sphingosine kinase 2 (SK-2) to induce T cell homing to secondary lymphoid tissue. In this study, we have investigated the role of SK-2 in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. We show that SK-2 deficiency reduced clinical symptoms of EAE. Furthermore, in SK-2-deficient mice, the protective effect of FTY720 on EAE was abolished, while the non-prodrug FTY720-derivative ST-968 was still fully active. Protection was paralleled by reduced numbers of T-lymphocytes in blood and a reduced blood-brain-barrier leakage. This correlated with reduced mRNA expression of ICAM-1, VCAM-1, but enhanced expression of PECAM-1. A similar regulation of permeability and of PECAM-1 was seen in primary cultures of isolated mouse brain vascular endothelial cells and in a human immortalized cell line upon SK-2 knockdown. In summary, these data demonstrated that deletion of SK-2 exerts a protective effect on the pathogenesis of EAE in C57BL/6 mice and that SK-2 is essential for the protective effect of FTY720 but not of ST-968. Thus, ST-968 is a promising novel immunomodulatory compound that may be a valuable alternative to FTY720 under conditions where SK-2 activity is limited., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), peripherally developed myelin-reactive T lymphocytes stimulate myeloid cells (ie, microglia and infiltrated macrophages) to trigger an inflammatory reaction in the central nervous system, resulting in demyelination and neurodegeneration. IκB kinase β (IKKβ) is a kinase that modulates transcription of inflammatory genes. To investigate the pathogenic role of IKKβ in MS, we developed strains in which IKKβ was conditionally ablated in myeloid cells and established active or passive EAE in these animals. Deficiency of IKKβ in myeloid cells ameliorated EAE symptoms and suppressed neuroinflammation, as shown by decreased infiltration of T lymphocytes and macrophages and reduced inflammatory gene transcription in the spinal cord at the peak or end stage of EAE. Myeloid deficiency of IKKβ also reduced the transcription of Rorc or Il17 genes in T lymphocytes isolated from lymph nodes, spleen, and spinal cord of EAE mice. Moreover, cultured splenocytes isolated from myeloid IKKβ-deficient EAE mice released less IL-17, interferon-γ, and granulocyte-macrophage colony-stimulating factor after treatment with myelin peptide than splenocytes from IKKβ wild-type EAE mice. Thus, deficiency of myeloid IKKβ attenuates the severity of EAE by inhibiting both the neuroinflammatory activity and the activation of encephalitogenic T lymphocytes. These results suggest IKKβ may be a potential target for MS patients, especially when neuroinflammation is the primary problem., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Myelin oligodendrocyte glycoprotein peptide fragment 35-55 (MOG35-55) is a major autoantigen inducing experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis that is characterized by blood-brain barrier (BBB) disruption. Various experimental approaches have employed MOG35-55 in vivo; however, in vitro BBB models using MOG35-55 are rarely reported. We investigated MOG35-55 exposure effects with complete Freund's adjuvant (CFA) and pertussis toxin (PTX) on brain endothelial cells and elucidated the relationships among NADPH oxidase, MMP-9, ICAM-1, and VCAM-1. These 4 factors significantly increased in MOG35-55+CFA+PTX-exposed endothelial cells compared with the control cells. NADPH oxidase inhibition using apocynin reduced MMP-9 activity, ICAM-1, and VCAM-1. MMP-9 inhibitor I decreased expression of ICAM-1 and VCAM-1, and both anti-ICAM-1 and anti-VCAM-1 inhibited MMP-9 activity. Inhibitions of MMP-9, ICAM-1, and VCAM-1 did not change NADPH oxidase activity. Although inhibition of these 4 factors decreased BBB permeability in cells, inhibition of NADPH oxidase exhibited the highest decrease among these. NADPH oxidase directly influenced MMP-9, ICAM-1, and VCAM-1, but not vice versa. MMP-9 and the cell adhesion molecules reversibly affected each other. In conclusion, NADPH oxidase-derived superoxide elevated expression of MMP-9, ICAM-1, and VCAM-1, and these interactions can finally result in increases of BBB permeability in MOG35-55+CFA+PTX-exposed endothelial cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Altering the metabolism of immune cells is an attractive strategy to modify their activity during autoimmunity in MS. We investigated the effect of modulating fatty acid metabolism in an animal model of MS, EAE. Alpha-methylacyl-CoA racemase (AMACR) converts R-configuration branched fatty acids into the S-configuration, thereby preparing them for β-oxidation. We observed a significant, disease-dependent elevation of AMACR expression in monocytes and T cells from blood, draining lymph nodes and spleen of EAE mice during the preclinical phase. In vitro analysis revealed that the proliferation of T cells was inhibited in AMACR KO mice, but T-cell polarization was switched toward a pathogenic state involving the production of more IFN-γ and IL-17, but less IL-4. These opposing effects appeared to cancel out each other in vivo, because AMACR KO EAE mice showed a marginal increase in the severity of early clinical symptoms. AMACR was not regulated in the white blood cells of MS patients. Our data show that AMACR is regulated in immune cells during EAE, but it is not a suitable target for the treatment of MS due to its opposing effects., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Prostaglandins and leukotrienes, bioactive mediators generated by cyclooxygenases (COX) and 5-lipoxygenase (5-LO) from arachidonic acid, play an essential role in neuroinflammation. High levels of LTB4 and PGE2 and increased expression of COX and 5-LO, as well as high expression of PGE2 receptors were reported in multiple sclerosis (MS) patients and in experimental autoimmune encephalomyelitis (EAE). Prostaglandins and leukotrienes have an interdependent and compensatory role in EAE, which led to the concept of therapy using dual COX/5-LO inhibitors. The plant derived flavocoxid, a dual COX/5-LO inhibitor with anti-inflammatory and antioxidant properties, manufactured as a prescription pharmaconutrient, was reported to be neuroprotective in models of transient ischemic stroke and brain injury. The present study is the first report on prophylactic and therapeutic effects of flavocoxid in EAE. The beneficial effects correlate with reduced expression of proinflammatory cytokines and of COX2 and 5-LO in spinal cords and spleens of EAE mice. The protective mechanisms include: 1. reduction in expression of MHCII/costimulatory molecules and production of proinflammatory cytokines; 2. promotion of the M2 phenotype including IL-10 expression and release by macrophages and microglia; 3. inhibition of Th1 and Th17 differentiation through direct effects on T cells. The direct inhibitory effect on Th1/Th17 differentiation, and promoting the development of M2 macrophages and microglia, represent novel mechanisms for the flavocoxid anti-inflammatory activity. As a dual COX/5-LO inhibitor with antioxidant properties, flavocoxid might be useful as a potential therapeutic medical food agent in MS patients., (Copyright © 2015. Published by Elsevier Inc.)

Cognitive impairment is common in multiple sclerosis (MS). Unfortunately, the synaptic and molecular mechanisms underlying MS-associated cognitive dysfunction are largely unknown. We explored the presence and the underlying mechanism of cognitive and synaptic hippocampal dysfunction during the remission phase of experimental MS. Experiments were performed in a chronic-relapsing experimental autoimmune encephalomyelitis (EAE) model of MS, after the resolution of motor deficits. Immunohistochemistry and patch-clamp recordings were performed in the CA1 hippocampal area. The hole-board was utilized as cognitive/behavioural test. In the remission phase of experimental MS, hippocampal microglial cells showed signs of activation, CA1 hippocampal synapses presented an impaired long-term potentiation (LTP) and an alteration of spatial tests became evident. The activation of hippocampal microglia mediated synaptic and cognitive/behavioural alterations during EAE. Specifically, LTP blockade was found to be caused by the reactive oxygen species (ROS)-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We suggest that in the remission phase of experimental MS microglia remains activated, causing synaptic dysfunctions mediated by NADPH oxidase. Inhibition of microglial activation and NADPH oxidase may represent a promising strategy to prevent neuroplasticity impairment associated with active neuro-inflammation, with the aim to improve cognition and counteract MS disease progression.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model that reproduces cardinal signs of clinical, histopathological, and immunological features found in Multiple Sclerosis (MS). Mast cells are suggested to be involved in the main inflammatory phases occurring during EAE development, possibly by secreting several autacoids and proteases. Among the latter, the chymase mouse mast cell protease 4 (mMCP-4) can contribute to the inflammatory response by producing endothelin-1 (ET-1). The aim of this study was to determine the impact of mMCP-4 on acute inflammatory stages in EAE. C57BL/6 wild type (WT) or mMCP-4 knockout (KO) mice were immunized with MOG35-55 plus complete Freund's adjuvant followed by pertussis toxin. Immunized WT mice presented an initial acute phase characterized by progressive increases in clinical score, which were significantly reduced in mMCP-4 KO mice. In addition, higher levels of spinal myelin were found in mMCP-4 KO as compared with WT mice. Finally, whereas EAE triggered significant increases in brain levels of mMCP-4 mRNA and immunoreactive ET-1 in WT mice, the latter peptide was reduced to basal levels in mMCP-4 KO congeners. Together, the present study supports a role for mMCP-4 in the early inflammatory phases of the disease in a mouse model of MS.

In addition to myelin loss and oligodendrocyte injury, axonal damage is a major cause of irreversible neurological disability in multiple sclerosis (MS). A series of studies have demonstrated that Rho kinase (ROCK) is involved in synaptic plasticity of neurons. Here, we found that ROCK activity in MS serum was elevated compared with serum from healthy controls. In experimental autoimmune encephalomyelitis (EAE), ROCK activity was also increased in serum, spleen, brain and spinal cord. Neuron injury with scratch and TNF-α stimulation induced the up-regulation of ROCK activity. When serum of MS patients was co-cultured with mouse cortical neurons in vitro, MS serum caused neurite shortening and reduction of cell viability, while the addition of Fasudil partially restored synaptic morphology of neurons, revealing that MS sera inhibited neurite outgrowth and synapse formation. The expression of synaptophysin was decreased in MS serum-neurons, and elevated in the presence of Fasudil. In contrast, the expression of phosphorylated collapsin response mediator protein-2 (CRMP-2) was elevated in MS serum-neurons and decreased in the presence of Fasudil. However, the addition of anti-ROCK I/II mixed antibodies in MS serum partially declined ROCK activity, but did not improve neurite outgrowth of neurons, revealing that Fasudil should prevent synaptic damage possibly through inhibiting intracellular ROCK activation mediated with MS serum. Our results indicate that axonal loss in MS may be related to increased ROCK activity. Fasudil could promote synaptogenesis and thus may contribute to preventing irreversible neurological disability associated with MS.

Alpha/beta-hydrolase domain 6 (ABHD6) is a novel 2-arachidonoylglycerol (2-AG) hydrolytic enzyme, that can fine-tune the endocannabinoid signaling in the central nervous system. Recently we and others have demonstrated the protective effect of ABHD6 inhibition in the animal models of traumatic brain injury and epileptic seizures. In this study, we investigated the role of targeting ABHD6 in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Post-symptom treatment with an ABHD6 inhibitor WWL70 increased the brain levels of 2-AG and ameliorated the clinical signs of EAE, T cells infiltration, microglia activation and the expression of activated leukocyte cell adhesion molecules. The production of iNOS, COX-2, TNF-α and IL-1β and the phosphorylation of NF-κB were also significantly reduced by WWL70 treatment. The neuroprotective effect of WWL70 was demonstrated by increased survival of mature oligodendrocytes, reduced demyelination and axonal loss in WWL70 treated EAE mouse spinal cord. The therapeutic effect of WWL70 on EAE was absent by co-administration of CB2 receptor antagonist, but not CB1 receptor antagonist. Consistently, WWL70 did not afford any protection in CB2 receptor knockout mice after EAE induction. Given the increased expression of ABHD6 in microglia/macrophages, but not in T cells, we speculated that inhibition of ABHD6 might enhance 2-AG signaling particularly in microglia/macrophages to exert anti-inflammatory effects via activation of CB2 receptors. These results suggest that inhibition of ABHD6 might be used as an ideal strategy for the treatment of MS and other neurodegenerative diseases., (Published by Elsevier Ltd.)

The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b(+) cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an "oxidative stress memory" both in the periphery and CNS compartments, in chronic neuroinflammation.

Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that plays an important role in multiple sclerosis (MS). However, mechanisms by which CNTF expression could be increased in the brain are poorly understood. Recently we have discovered anti-inflammatory and immunomodulatory activities of sodium benzoate (NaB), a metabolite of cinnamon and a widely-used food additive. Here, we delineate that NaB is also capable of increasing the mRNA and protein expression of CNTF in primary mouse astrocytes and oligodendrocytes and primary human astrocytes. Accordingly, oral administration of NaB and cinnamon led to the upregulation of astroglial and oligodendroglial CNTF in vivo in mouse brain. Induction of experimental allergic encephalomyelitis, an animal model of MS, reduced the level of CNTF in the brain, which was restored by oral administration of cinnamon. While investigating underlying mechanisms, we observed that NaB induced the activation of protein kinase A (PKA) and H-89, an inhibitor of PKA, abrogated NaB-induced expression of CNTF. The activation of cAMP response element binding (CREB) protein by NaB, the recruitment of CREB and CREB-binding protein to the CNTF promoter by NaB and the abrogation of NaB-induced expression of CNTF in astrocytes by siRNA knockdown of CREB suggest that NaB increases the expression of CNTF via the activation of CREB. These results highlight a novel myelinogenic property of NaB and cinnamon, which may be of benefit for MS and other demyelinating disorders.