Versican promotes T helper 17 cytotoxic inflammation and impedes oligodendrocyte precursor cell remyelination.

Remyelination failure in multiple sclerosis (MS) contributes to progression of disability. The deficient repair results from neuroinflammation and deposition of inhibitors including chondroitin sulfate proteoglycans (CSPGs). Which CSPG member is repair-inhibitory or alters local inflammation to exacerbate injury is unknown. Here, we correlate high versican-V1 expression in MS lesions with deficient premyelinating oligodendrocytes, and highlight its selective upregulation amongst CSPG members in experimental autoimmune encephalomyelitis (EAE) lesions modeling MS. In culture, purified versican-V1 inhibits oligodendrocyte precursor cells (OPCs) and promotes T helper 17 (Th17) polarization. Versican-V1-exposed Th17 cells are particularly toxic to OPCs. In NG2CreER:MAPTmGFP mice illuminating newly formed GFP+ oligodendrocytes/myelin, difluorosamine (peracetylated,4,4-difluoro-N-acetylglucosamine) treatment from peak EAE reduces lesional versican-V1 and Th17 frequency, while enhancing GFP+ profiles. We suggest that lesion-elevated versican-V1 directly impedes OPCs while it indirectly inhibits remyelination through elevating local Th17 cytotoxic neuroinflammation. We propose CSPG-lowering drugs as potential dual pronged repair and immunomodulatory therapeutics for MS.

Oxidized phosphatidylcholines found in multiple sclerosis lesions mediate neurodegeneration and are neutralized by microglia.

Neurodegeneration occurring in multiple sclerosis (MS) contributes to the progression of disability. It is therefore important to identify and neutralize the mechanisms that promote neurodegeneration in MS. Here, we report that oxidized phosphatidylcholines (OxPCs) found in MS lesions, previously identified as end-product markers of oxidative stress, are potent drivers of neurodegeneration. Cultured neurons and oligodendrocytes were killed by OxPCs, and this was ameliorated by microglia. After OxPC injection, mouse spinal cords developed focal demyelinating lesions with prominent axonal loss. The depletion of microglia that accumulated in OxPC lesions exacerbated neurodegeneration. Single-cell RNA sequencing of lesioned spinal cords identified unique subsets of TREM2high mouse microglia responding to OxPC deposition. TREM2 was detected in human MS lesions, and TREM2-/- mice exhibited worsened OxPC lesions. These results identify OxPCs as potent neurotoxins and suggest that enhancing microglia-mediated OxPC clearance via TREM2 could help prevent neurodegeneration in MS.

Neuregulin-1 beta 1 is implicated in pathogenesis of multiple sclerosis.

Multiple sclerosis is characterized by immune mediated neurodegeneration that results in progressive, life-long neurological and cognitive impairments. Yet, the endogenous mechanisms underlying multiple sclerosis pathophysiology are not fully understood. Here, we provide compelling evidence that associates dysregulation of neuregulin-1 beta 1 (Nrg-1ß1) with multiple sclerosis pathogenesis and progression. In the experimental autoimmune encephalomyelitis model of multiple sclerosis, we demonstrate that Nrg-1ß1 levels are abated within spinal cord lesions and peripherally in the plasma and spleen during presymptomatic, onset and progressive course of the disease. We demonstrate that plasma levels of Nrg-1ß1 are also significantly reduced in individuals with early multiple sclerosis and is positively associated with progression to relapsing-remitting multiple sclerosis. The functional impact of Nrg-1ß1 downregulation preceded disease onset and progression, and its systemic restoration was sufficient to delay experimental autoimmune encephalomyelitis symptoms and alleviate disease burden. Intriguingly, Nrg-1ß1 therapy exhibited a desirable and extended therapeutic time window of efficacy when administered prophylactically, symptomatically, acutely or chronically. Using in vivo and in vitro assessments, we identified that Nrg-1ß1 treatment mediates its beneficial effects in EAE by providing a more balanced immune response. Mechanistically, Nrg-1ß1 moderated monocyte infiltration at the blood-CNS interface by attenuating chondroitin sulphate proteoglycans and MMP9. Moreover, Nrg-1ß1 fostered a regulatory and reparative phenotype in macrophages, T helper type 1 (Th1) cells and microglia in the spinal cord lesions of EAE mice. Taken together, our new findings in multiple sclerosis and experimental autoimmune encephalomyelitis have uncovered a novel regulatory role for Nrg-1ß1 early in the disease course and suggest its potential as a specific therapeutic target to ameliorate disease progression and severity.

Autofluorescence spectroscopy as a proxy for chronic white matter pathology.

BACKGROUND: The balance of tissue injury and repair ultimately determines outcomes of chronic neurological disorders, such as progressive multiple sclerosis (MS). However, the extent of pathology can be difficult to detect, particularly when it is insidious and/or offset by tissue regeneration. OBJECTIVES: The objective of this research is to evaluate whether tissue autofluorescence-typically a source of contamination-provides a surrogate marker of white matter injury. METHODS: Tissue autofluorescence in autopsied specimens both experimental and clinical was characterized by spectral confocal microscopy and correlated to severity and chronicity as determined by standard histopathology. RESULTS: Months after cuprizone (CPZ)-induced demyelination, despite robust remyelination, autofluorescent deposits progressively accumulated in regions of prior pathology. Autofluorescent deposits (likely reflecting myelin debris remnants) were conspicuously localized to white matter, proportional to lesion severity, and displayed differential fluorescence over time. Strikingly, similar features were apparent also in autopsied MS tissue. CONCLUSION: Autofluorescence spectroscopy illuminates prior and ongoing white matter injury. The accumulation of autofluorescence in proportion to the extent of progressive atrophy, despite robust remyelination in the CPZ brain, provides important proof-of-concept of a phenomenon (insidious ongoing damage masked by mechanisms of tissue repair) that we hypothesize is highly relevant to the progressive phase of MS.

Correction to: Niacin-mediated rejuvenation of macrophage/microglia enhances remyelination of the aging central nervous system.

The article Niacin­mediated rejuvenation of macrophage/microglia enhances remyelination of the aging central nervous system, written by Khalil S. Rawji, Adam M.H. Young, Tanay Ghosh, Nathan J. Michaels, Reza Mirzaei, Janson Kappen, Kathleen L. Kolehmainen, Nima Alaeiilkhchi, Brian Lozinski, Manoj K. Mishra, Annie Pu, Weiwen Tang, Salma Zein, Deepak K. Kaushik, Michael B. Keough, Jason R. Plemel, Fiona Calvert, Andrew J. Knights, Daniel J. Gaffney, Wolfram Tetzlaff, Robin J. M. Franklin and V. Wee Yong, was originally published electronically on the publisher's internet.

Niacin-mediated rejuvenation of macrophage/microglia enhances remyelination of the aging central nervous system.

Remyelination following CNS demyelination restores rapid signal propagation and protects axons; however, its efficiency declines with increasing age. Both intrinsic changes in the oligodendrocyte progenitor cell population and extrinsic factors in the lesion microenvironment of older subjects contribute to this decline. Microglia and monocyte-derived macrophages are critical for successful remyelination, releasing growth factors and clearing inhibitory myelin debris. Several studies have implicated delayed recruitment of macrophages/microglia into lesions as a key contributor to the decline in remyelination observed in older subjects. Here we show that the decreased expression of the scavenger receptor CD36 of aging mouse microglia and human microglia in culture underlies their reduced phagocytic activity. Overexpression of CD36 in cultured microglia rescues the deficit in phagocytosis of myelin debris. By screening for clinically approved agents that stimulate macrophages/microglia, we have found that niacin (vitamin B3) upregulates CD36 expression and enhances myelin phagocytosis by microglia in culture. This increase in myelin phagocytosis is mediated through the niacin receptor (hydroxycarboxylic acid receptor 2). Genetic fate mapping and multiphoton live imaging show that systemic treatment of 9-12-month-old demyelinated mice with therapeutically relevant doses of niacin promotes myelin debris clearance in lesions by both peripherally derived macrophages and microglia. This is accompanied by enhancement of oligodendrocyte progenitor cell numbers and by improved remyelination in the treated mice. Niacin represents a safe and translationally amenable regenerative therapy for chronic demyelinating diseases such as multiple sclerosis.

Systematic screening of generic drugs for progressive multiple sclerosis identifies clomipramine as a promising therapeutic.

The treatment of progressive multiple sclerosis (MS) is unsatisfactory. One reason is that the drivers of disease, which include iron-mediated neurotoxicity, lymphocyte activity, and oxidative stress, are not simultaneously targeted. Here we present a systematic screen to identify generic, orally available medications that target features of progressive MS. Of 249 medications that cross the blood-brain barrier, 35 prevent iron-mediated neurotoxicity in culture. Of these, several antipsychotics and antidepressants strongly reduce T-cell proliferation and oxidative stress. We focus on the antidepressant clomipramine and found that it additionally inhibits B-lymphocyte activity. In mice with experimental autoimmune encephalomyelitis, a model of MS, clomipramine ameliorates clinical signs of acute and chronic phases. Histologically, clomipramine reduces inflammation and microglial activation, and preserves axonal integrity. In summary, we present a systematic approach to identify generic medications for progressive multiple sclerosis with the potential to advance rapidly into clinical trials, and we highlight clomipramine for further development.

Evaluating Soluble EMMPRIN as a Marker of Disease Activity in Multiple Sclerosis: Studies of Serum and Cerebrospinal Fluid.

Extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is an inducer of matrix metalloproteinases and has roles in leukocyte activation and migration. We reported previously that in MS and its animal model, experimental autoimmune encephalomyelitis, cell surface-associated EMMPRIN was significantly elevated in leukocytes around inflammatory perivascular cuffs in the CNS. In this study we report that activated T-cells can secrete soluble form of EMMPRIN (sEMMPRIN) upon activation. As sEMMPRIN is also present in biological fluids, we determined whether sEMMPRIN is altered in the CSF and sera of MS subjects. Sera from individuals without neurological conditions served as controls, while CSFs collected from subjects undergoing discectomy, and without evidence of CNS pathology, were used as a comparator group. We found that serum levels of sEMMPRIN from clinically stable MS patients or other inflammatory conditions did not differ from control subjects. Paired serum and CSF samples demonstrated poor correlation of sEMMPRIN. Interestingly, sEMMPRIN levels were approximately 60% higher in CSFs compared to sera. sEMMPRIN CSF levels were significantly higher in secondary progressive compared to primary progressive subjects. Thus we conclude that measurement of sEMMPRIN in serum is not informative for disease activity in MS. The differential expression of sEMMPRIN in the CSF of primary and secondary progressive MS invites hypotheses of the still undefined roles of EMMPRIN in the CNS.

Impact of Minocycline on Extracellular Matrix Metalloproteinase Inducer, a Factor Implicated in Multiple Sclerosis Immunopathogenesis.

Extracellular matrix metalloproteinase inducer (EMMPRIN, CD147) is a transmembrane glycoprotein that is upregulated on leukocytes in active lesions in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Administration of anti-EMMPRIN Abs reduces the severity of EAE. Minocycline is a tetracycline antibiotic with immune-modulatory properties that decreases the severity of EAE; it was recently found to attenuate the conversion from a first demyelinating event to clinically definite MS in a phase III trial. We investigated whether and how minocycline affects the expression of EMMPRIN on T cells in culture and in mice afflicted with EAE. EMMPRIN expression in cultures of mouse splenocytes or human PBMCs was elevated upon polyclonal T cell activation, and this was reduced by minocycline correspondent with decreased P-Akt levels. An established MS medication, IFN-ß, also diminished EMMPRIN levels on human cells whereas this was not readily observed for fingolimod or monomethylfumarate. In EAE-afflicted mice, minocycline treatment significantly reduced EMMPRIN levels on splenic lymphocytes at the presymptomatic (day 7) phase, and prevented the development of disease. Day 7 spleen transcripts from minocycline-treated EAE mice had a significantly lower MMP-9/TIMP-1 ratio, and significantly lower MCT-1 and CD98 levels, factors associated with EMMPRIN function. Day 16 (peak clinical severity) CNS samples from EAE mice had prominent representation of inflammatory perivascular cuffs, inflammatory molecules and EMMPRIN, and these were abrogated by minocycline. Overall, minocycline attenuated the activation-induced elevation of EMMPRIN on T cells in culture and in EAE mice, correspondent with reduced immune function and EAE CNS pathology.

Interleukin-1ß orchestrates underlying inflammatory responses in microglia via Krüppel-like factor 4.

Microglia are the resident macrophages of the CNS, which secrete several pro- and anti-inflammatory cyto-chemokines including interleukin-1ß (IL-1ß), in response to pathogenic stimuli. Once secreted, IL-1ß binds to IL-1 receptor present on microglia and initiates the production of inflammatory cytokines in microglia. However, the detailed information regarding the molecular mechanisms of IL-1ß triggered inflammatory pathways in microglia is lacking. Our studies focused on the role of Krüppel-like factor 4 (Klf4) in mediating the regulation of pro-inflammatory gene expression upon IL-1ß stimulation in microglia. Our studies show that stimulation of microglia with IL-1ß robustly induces Klf4 via PI3K/Akt pathway which positively regulates the production of endogenous IL-1ß as well as other pro-inflammatory markers, cyclooxygenase-2, monocyte chemoattractant protein-1 and interleukin-6 (IL-6). In addition, we report that Klf4 negatively regulates the expression of inducible nitric oxide synthase, thereby playing a key role in regulating the immunomodulatory activities of microglia.

MicroRNAs in the brain: it's regulatory role in neuroinflammation.

MicroRNAs (miRNAs) are single-stranded noncoding regions of approximately 21 nucleotides that regulate protein synthesis by targeting mRNAs for translational repression or degradation at the post-transcriptional level. These classes of RNAs are highly conserved across species and are known to regulate several protein-coding genes in humans. Therefore, their dysregulation is synonymous with inflammation, autoimmunity, neurodegeneration, viral infections, heart diseases, and cancer, among other conditions. Recent years have witnessed considerable amount of research interest in studies on miRNA-mediated modulation of gene function during neuroinflammation. This review is a meticulous compilation of information on biogenesis of miRNAs and their role in neuroinflammatory diseases. Further, their potential as markers of inflammatory diseases or novel therapeutic agents against neuroinflammation has also been discussed in detail.

Changes in cytosolic Ca2+ levels correspond to fluctuations of lactate levels in crosstalk of astrocyte-neuron cell lines.

Neurons and astrocytes differentially express isoenzymes of lactate dehydrogenase (LDH). The metabolic consequences for the variations in mRNA expression of LDH isoenzyme subtypes in neurons and astrocytes control cerebral vasoregulation. Moreover, cellular signalling consequences for functional neurovascular control may also be dependent on LDH isoenzyme subtype profiles. Initial computer simulations revealed glutamate-induced calcium waves in connected astrocytes, and showed concomitant changes in the expression of nitric oxide synthase (NOS) and lactic acid metabolism. To validate these findings, the nature and extent of glutamate-dependent signalling crosstalk in murine cell lines were investigated through correlated lactate levels and calcium upregulation. Neuro2A and C8D1A cells were separately treated with timed supernatant extracts from each other and their LDH1 and LDH5 isoenzyme responses were recorded. Western blot analysis showed LDH1/LDH5 isoenzyme ratio in the astrocytes to be positively correlated with Neuro2A-derived lactate levels estimated by the amplitude of 1.33-ppm spectral peak in 1H-NMR, and LDH1/LDH5 isoenzyme ratio in neurons is negatively correlated with CSD1A-derived lactate levels. Significant modulations of the calcium-responsive protein pCamKII levels were also observed in both cell lines, particularly correlations between pCamKII and lactate in C8D1A cells, thus explaining the calcium dependence of the lactate response. Together, these observations indicate that lactate is a key indicator of the metabolic state of these cell types, and may be a determinant of release of vasoregulatory factors.

Krüppel-like factor 4, a novel transcription factor regulates microglial activation and subsequent neuroinflammation.

BACKGROUND: Activation of microglia, the resident macrophages of the central nervous system (CNS), is the hallmark of neuroinflammation in neurodegenerative diseases and other pathological conditions associated with CNS infection. The activation of microglia is often associated with bystander neuronal death. Nuclear factor-κB (NF-κB) is one of the important transcription factors known to be associated with microglial activation which upregulates the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2) and other pro-inflammatory cytokines. Recent studies have focused on the role of Krüppel-like factor 4 (Klf4), one of the zinc-finger transcription factors, in mediating inflammation. However, these studies were limited to peripheral system and its role in CNS is not understood. Our studies focused on the possible role of Klf4 in mediating CNS inflammation. METHODS: For in vitro studies, mouse microglial BV-2 cell lines were treated with 500 ng/ml Salmonella enterica lipopolysacchride (LPS). Brain tissues were isolated from BALB/c mice administered with 5 mg/kg body weight of LPS. Expressions of Klf4, Cox-2, iNOS and pNF-κB were evaluated using western blotting, quantitative real time PCR, and reverse transcriptase polymerase chain reactions (RT-PCRs). Klf4 knockdown was carried out using SiRNA specific for Klf4 mRNA and luciferase assays and electromobility shift assay (EMSA) were performed to study the interaction of Klf4 to iNOS promoter elements in vitro. Co-immunoprecipitation of Klf4 and pNF-κB was done in order to study a possible interaction between the two transcription factors. RESULTS: LPS stimulation increased Klf4 expression in microglial cells in a time- and dose-dependent manner. Knockdown of Klf4 resulted in decreased levels of the pro-inflammatory cytokines TNF-α, MCP-1 and IL-6, along with a significant decrease in iNOS and Cox-2 expression. NO production also decreased as a result of Klf4 knockdown. We found that Klf4 can potentially interact with pNF-κB and is important for iNOS and Cox-2 promoter activity in vitro. CONCLUSIONS: These studies demonstrate the role of Klf4 in microglia in mediating neuroinflammation in response to the bacterial endotoxin LPS.