Fibulin-2 is an extracellular matrix inhibitor of oligodendrocytes relevant to multiple sclerosis.

Impairment of oligodendrocytes and myelin contributes to neurological disorders including multiple sclerosis (MS), stroke, and Alzheimer's disease. Regeneration of myelin (remyelination) decreases the vulnerability of demyelinated axons, but this repair process commonly fails with disease progression. A contributor to inefficient remyelination is the altered extracellular matrix (ECM) in lesions, which remains to be better defined. We have identified fibulin-2 (FBLN2) as a highly upregulated ECM component in lesions of MS and stroke and in proteome databases of Alzheimer's disease and traumatic brain injury. Focusing on MS, the inhibitory role of FBLN2 was suggested in the experimental autoimmune encephalomyelitis (EAE) model, in which genetic FBLN2 deficiency improved behavioral recovery by promoting the maturation of oligodendrocytes and enhancing remyelination. Mechanistically, when oligodendrocyte progenitors were cultured in differentiation medium, FBLN2 impeded their maturation into oligodendrocytes by engaging the Notch pathway, leading to cell death. Adeno-associated virus deletion of FBLN2 in astrocytes improved oligodendrocyte numbers and functional recovery in EAE and generated new myelin profiles after lysolecithin-induced demyelination. Collectively, our findings implicate FBLN2 as a hitherto unrecognized injury-elevated ECM, and a therapeutic target, that impairs oligodendrocyte maturation and myelin repair.

Enhanced liver X receptor signalling reduces brain injury and promotes tissue regeneration following experimental intracerebral haemorrhage: roles of microglia/macrophages.

BACKGROUND: Inflammation-exacerbated secondary brain injury and limited tissue regeneration are barriers to favourable prognosis after intracerebral haemorrhage (ICH). As a regulator of inflammation and lipid metabolism, Liver X receptor (LXR) has the potential to alter microglia/macrophage (M/M) phenotype, and assist tissue repair by promoting cholesterol efflux and recycling from phagocytes. To support potential clinical translation, the benefits of enhanced LXR signalling are examined in experimental ICH. METHODS: Collagenase-induced ICH mice were treated with the LXR agonist GW3965 or vehicle. Behavioural tests were conducted at multiple time points. Lesion and haematoma volume, and other brain parameters were assessed using multimodal MRI with T2-weighted, diffusion tensor imaging and dynamic contrast-enhanced MRI sequences. The fixed brain cryosections were stained and confocal microscopy was applied to detect LXR downstream genes, M/M phenotype, lipid/cholesterol-laden phagocytes, oligodendrocyte lineage cells and neural stem cells. Western blot and real-time qPCR were also used. CX3CR1CreER: Rosa26iDTR mice were employed for M/M-depletion experiments. RESULTS: GW3965 treatment reduced lesion volume and white matter injury, and promoted haematoma clearance. Treated mice upregulated LXR downstream genes including ABCA1 and Apolipoprotein E, and had reduced density of M/M that apparently shifted from proinflammatory interleukin-1ß+ to Arginase1+CD206+ regulatory phenotype. Fewer cholesterol crystal or myelin debris-laden phagocytes were observed in GW3965 mice. LXR activation increased the number of Olig2+PDGFRα+ precursors and Olig2+CC1+ mature oligodendrocytes in perihaematomal regions, and elevated SOX2+ or nestin+ neural stem cells in lesion and subventricular zone. MRI results supported better lesion recovery by GW3965, and this was corroborated by return to pre-ICH values of functional rotarod activity. The therapeutic effects of GW3965 were abrogated by M/M depletion in CX3CR1CreER: Rosa26iDTR mice. CONCLUSIONS: LXR agonism using GW3965 reduced brain injury, promoted beneficial properties of M/M and facilitated tissue repair correspondent with enhanced cholesterol recycling.

Inflammation in an Animal Model of Multiple Sclerosis Leads to MicroRNA-25-3p Dysregulation Associated with Inhibition of Pten and Klf4.

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.

The benefits of neuroinflammation for the repair of the injured central nervous system.

Inflammation of the nervous system (neuroinflammation) is now recognized as a hallmark of virtually all neurological disorders. In neuroinflammatory conditions such as multiple sclerosis, there is prominent infiltration and a long-lasting representation of various leukocyte subsets in the central nervous system (CNS) parenchyma. Even in classic neurodegenerative disorders, where such immense inflammatory infiltrates are absent, there is still evidence of activated CNS-intrinsic microglia. The consequences of excessive and uncontrolled neuroinflammation are injury and death to neural elements, which manifest as a heterogeneous set of neurological symptoms. However, it is now readily acknowledged, due to instructive studies from the peripheral nervous system and a large body of CNS literature, that aspects of the neuroinflammatory response can be beneficial for CNS outcomes. The recognized benefits of inflammation to the CNS include the preservation of CNS constituents (neuroprotection), the proliferation and maturation of various neural precursor populations, axonal regeneration, and the reformation of myelin on denuded axons. Herein, we highlight the benefits of neuroinflammation in fostering CNS recovery after neural injury using examples from multiple sclerosis, traumatic spinal cord injury, stroke, and Alzheimer's disease. We focus on CNS regenerative responses, such as neurogenesis, axonal regeneration, and remyelination, and discuss the mechanisms by which neuroinflammation is pro-regenerative for the CNS. Finally, we highlight treatment strategies that harness the benefits of neuroinflammation for CNS regenerative responses.

Malat1 long noncoding RNA regulates inflammation and leukocyte differentiation in experimental autoimmune encephalomyelitis.

In this study, we investigated the contributions of the MALAT1 long noncoding RNA to autoimmune neuroinflammation in central nervous system tissues from patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE). Expression of MALAT1 was decreased in the spinal cords of EAE mice as well as in stimulated splenocytes and primary macrophages. MALAT1 downregulation by specific siRNAs enhanced the polarization of macrophages towards the M1 phenotype. Interestingly, siRNA-mediated MALAT1 downregulation shifted the pattern of T-cell differentiation towards a Th1/Th17 cell profile and decreased differentiation towards a Tregs phenotype. Proliferation of T-cells was also increased following MALAT1 downregulation. These data point to a potential anti-inflammatory effect for MALAT1 in the context of autoimmune neuroinflammation.

MicroRNA-150 targets PU.1 and regulates macrophage differentiation and function in experimental autoimmune encephalomyelitis.

PU.1 is a transcription factor which is expressed in myeloid cells. Herein, we investigated the expression of PU.1 and its potentially targeting miRNAs in the central nervous system (CNS) of mice with experimental autoimmune encephalitis (EAE) and in cultured primary macrophages. PU.1 levels where highly induced in EAE spinal cords and in activated macrophages; this was associated with a significant reduction in miR-150-5p levels at chronic phase of disease and in activated cells. Luciferase assays confirmed the PU.1-miR-150-5p interaction. Overexpression of miR-150-5p in macrophages decreased the expression of proinflammatory cytokines and shifted the polarization of macrophages away from the M1-like phenotype.

miR-181 interacts with signaling adaptor molecule DENN/MADD and enhances TNF-induced cell death.

MicroRNAs are small noncoding RNAs, which regulate the expression of protein coding transcripts through mRNA degradation or translational inhibition. Numerous reports have highlighted the role of miRNAs in regulating cell death pathways including the expression of genes involved in the induction of apoptosis. Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine which can send pro-death signals through its receptor TNFR1. Diverse adaptor molecules including DENN/MADD adaptor protein have been shown to modulate TNF-α pro-death signaling via recruitment of MAP kinases to TNFR1 and activation of pro-survival NFκB signaling. Herein, we investigated the role of microRNA-181 (miR-181) in regulating DENN/MADD expression levels and its subsequent effects on TNF-α-induced cell death. Using bioinformatics analyses followed by luciferase reporter assays we showed that miR-181 interacts with the 3' UTR of DENN/MADD transcripts. miR-181 overexpression also led to decreased endogenous DENN/MADD mRNA levels in L929 murine fibroblasts. Flow cytometric analysis of miR-181 transfected cells showed this miRNA accentuates mitochondrial membrane potential loss caused by TNF-α. These findings were associated with enhanced apoptosis of L929 cells following TNF-α treatment. Overall, these data point to the potential role of miR-181 in regulating TNF-α pro-death signaling, which could be of importance from pathogenesis and therapeutic perspectives in inflammatory disorders associated with tissue degeneration and cell death.

MicroRNA-142 regulates inflammation and T cell differentiation in an animal model of multiple sclerosis.

BACKGROUND: MicroRNAs have emerged as an important class of modulators of gene expression. These molecules influence protein synthesis through translational repression or degradation of mRNA transcripts. Herein, we investigated the potential role of miR-142a isoforms, miR-142a-3p and miR-142a-5p, in the context of autoimmune neuroinflammation. METHODS: The expression levels of two mature isoforms of miR-142 were measured in the brains of patients with multiple sclerosis (MS) and the CNS tissues from mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Expression analyses were also performed in mitogen and antigen-stimulated splenocytes, as well as macrophages and astrocytes using real-time RT-PCR. The role of the mature miRNAs was then investigated in T cell differentiation by transfection of CD4+ T cells, followed by flow cytometric analysis of intracellular cytokines. Luciferase assays using vectors containing the 3'UTR of predicted targets were performed to confirm the interaction of miRNA sequences with transcripts. Expression of targets were then analyzed in activated splenocytes and MS/EAE tissues. RESULTS: Expression of miR-142-5p was significantly increased in the frontal white matter from MS patients compared with white matter from non-MS controls. Likewise, expression levels of miR-142a-5p and miR-142a-3p showed significant upregulation in the spinal cords of EAE mice at days 15 and 25 post disease induction. Splenocytes stimulated with myelin oligodendrocyte glycoprotein (MOG) peptide or anti-CD3/anti-CD28 antibodies showed upregulation of miR-142a-5p and miR-142a-3p isoforms, whereas stimulated bone marrow-derived macrophages and primary astrocytes did not show any significant changes in miRNA expression levels. miR-142a-5p overexpression in activated lymphocytes shifted the pattern of T cell differentiation towards Th1 cells. Luciferase assays revealed SOCS1 and TGFBR1 as direct targets of miR-142a-5p and miR-142a-3p, respectively, and overexpression of miRNA mimic sequences suppressed the expression of these target transcripts in lymphocytes. SOCS1 levels were also diminished in MS white matter and EAE spinal cords. CONCLUSIONS: Our findings suggest that increased expression of miR-142 isoforms might be involved in the pathogenesis of autoimmune neuroinflammation by influencing T cell differentiation, and this effect could be mediated by interaction of miR-142 isoforms with SOCS1 and TGFBR-1 transcripts.