A novel microglial subset plays a key role in myelinogenesis in developing brain.

Microglia are resident macrophages of the central nervous system that contribute to homeostasis and neuroinflammation. Although known to play an important role in brain development, their exact function has not been fully described. Here, we show that in contrast to healthy adult and inflammation-activated cells, neonatal microglia show a unique myelinogenic and neurogenic phenotype. A CD11c+ microglial subset that predominates in primary myelinating areas of the developing brain expresses genes for neuronal and glial survival, migration, and differentiation. These cells are the major source of insulin-like growth factor 1, and its selective depletion from CD11c+ microglia leads to impairment of primary myelination. CD11c-targeted toxin regimens induced a selective transcriptional response in neonates, distinct from adult microglia. CD11c+ microglia are also found in clusters of repopulating microglia after experimental ablation and in neuroinflammation in adult mice, but despite some similarities, they do not recapitulate neonatal microglial characteristics. We therefore identify a unique phenotype of neonatal microglia that deliver signals necessary for myelination and neurogenesis.

Dimethyl fumarate decreases neurofilament light chain in CSF and blood of treatment naïve relapsing MS patients.

OBJECTIVES: In a prospective phase IV trial of the first-line oral treatment dimethyl fumarate (DMF), we examined dynamics of neurofilament light (NFL) chain in serum, plasma and cerebrospinal fluid (CSF) samples collected over 12 months from relapsing-remitting multiple sclerosis (RRMS) patients. NFL changes were related to disease activity. METHODS: We examined NFL levels by single-molecule array in 88 CSF, 348 plasma and 131 sera from treatment-naïve RRMS patients (n=52), healthy controls (n=23) and a placebo group matched by age, sex and NFL (n=52). Plasma/sera were collected at baseline, and 1, 3, 6 and 12 months after DMF. CSF samples were collected at baseline and 12 months after DMF. RESULTS: NFL concentration in CSF, plasma and serum correlated highly (p<0.0001 for all), but plasma levels were only 76.9% of paired serum concentration. After 12 months of DMF treatment, NFL concentration decreased by 73%, 69% and 55% in the CSF, serum and plasma (p<0.0001, respectively). Significant reduction in blood was observed after 6 and 12 months treatment compared with baseline (p<0.01 and p<0.0001, respectively) and to placebo (p<0.0001). Patients with NFL above the 807.5 pg/mL cut-off in CSF had 5.0-times relative risk of disease activity (p<0.001). CONCLUSIONS: This study provides Class II evidence that first-line DMF reduces NFL in both blood and CSF after 6 months and normalises CSF levels in 73% of patients. High NFL concentration in CSF after a year reflected disease activity. NFL levels were higher in serum than in plasma, which should be considered when NFL is used as a biomarker.

TNF deficiency causes alterations in the spatial organization of neurogenic zones and alters the number of microglia and neurons in the cerebral cortex.

BACKGROUND: Although tumor necrosis factor (TNF) inhibitors are used to treat chronic inflammatory diseases, there is little information about how long-term inhibition of TNF affects the homeostatic functions that TNF maintains in the intact CNS. MATERIALS AND METHODS: To assess whether developmental TNF deficiency causes alterations in the naïve CNS, we estimated the number of proliferating cells, microglia, and neurons in the developing neocortex of E13.5, P7 and adult TNF knock out (TNF-/-) mice and wildtype (WT) littermates. We also measured changes in gene and protein expression and monoamine levels in adult WT and TNF-/- mice. To evaluate long-term effects of TNF inhibitors, we treated healthy adult C57BL/6 mice with either saline, the selective soluble TNF inhibitor XPro1595, or the nonselective TNF inhibitor etanercept. We estimated changes in cell number and protein expression after two months of treatment. We assessed the effects of TNF deficiency on cognition by testing adult WT and TNF-/- mice and mice treated with saline, XPro1595, or etanercept with specific behavioral tasks. RESULTS: TNF deficiency decreased the number of proliferating cells and microglia and increased the number of neurons. At the same time, TNF deficiency decreased the expression of WNT signaling-related proteins, specifically Collagen Triple Helix Repeat Containing 1 (CTHRC1) and Frizzled receptor 6 (FZD6). In contrast to XPro1595, long-term inhibition of TNF with etanercept in adult C57BL/6 mice decreased the number of BrdU+ cells in the granule cell layer of the dentate gyrus. Etanercept, but not XPro1595, also impaired spatial learning and memory in the Barnes maze memory test. CONCLUSION: TNF deficiency impacts the organization of neurogenic zones and alters the cell composition in brain. Long-term inhibition of TNF with the nonselective TNF inhibitor etanercept, but not the soluble TNF inhibitor XPro1595, decreases neurogenesis in the adult mouse hippocampus and impairs learning and memory after two months of treatment.

Macrophage migration inhibitory factor (MIF) modulates trophic signaling through interaction with serine protease HTRA1.

Macrophage migration inhibitory factor (MIF), a small conserved protein, is abundant in the immune- and central nervous system (CNS). MIF has several receptors and binding partners that can modulate its action on a cellular level. It is upregulated in neurodegenerative diseases and cancer although its function is far from clear. Here, we report the finding of a new binding partner to MIF, the serine protease HTRA1. This enzyme cleaves several growth factors, extracellular matrix molecules and is implicated in some of the same diseases as MIF. We show that the function of the binding between MIF and HTRA1 is to inhibit the proteolytic activity of HTRA1, modulating the availability of molecules that can change cell growth and differentiation. MIF is therefore the first endogenous inhibitor ever found for HTRA1. It was found that both molecules were present in astrocytes and that the functional binding has the ability to modulate astrocytic activities important in development and disease of the CNS.

Systemically administered anti-TNF therapy ameliorates functional outcomes after focal cerebral ischemia.

BACKGROUND: The innate immune system contributes to the outcome after stroke, where neuroinflammation and post-stroke systemic immune depression are central features. Tumor necrosis factor (TNF), which exists in both a transmembrane (tm) and soluble (sol) form, is known to sustain complex inflammatory responses associated with stroke. We tested the effect of systemically blocking only solTNF versus blocking both tmTNF and solTNF on infarct volume, functional outcome and inflammation in focal cerebral ischemia. METHODS: We used XPro1595 (a dominant-negative inhibitor of solTNF) and etanercept (which blocks both solTNF and tmTNF) to test the effect of systemic administration on infarct volume, functional recovery and inflammation after focal cerebral ischemia in mice. Functional recovery was evaluated after one, three and five days, and infarct volumes at six hours, 24 hours and five days after ischemia. Brain inflammation, liver acute phase response (APR), spleen and blood leukocyte profiles, along with plasma microvesicle analysis, were evaluated. RESULTS: We found that both XPro1595 and etanercept significantly improved functional outcomes, altered microglial responses, and modified APR, spleen T cell and microvesicle numbers, but without affecting infarct volumes. CONCLUSIONS: Our data suggest that XPro1595 and etanercept improve functional outcome after focal cerebral ischemia by altering the peripheral immune response, changing blood and spleen cell populations and decreasing granulocyte infiltration into the brain. Blocking solTNF, using XPro1595, was just as efficient as blocking both solTNF and tmTNF using etanercept. Our findings may have implications for future treatments with anti-TNF drugs in TNF-dependent diseases.

Absence of miRNA-146a Differentially Alters Microglia Function and Proteome.

Background: MiR-146a is an important regulator of innate inflammatory responses and is also implicated in cell death and survival. Methods: By sorting CNS resident cells, microglia were the main cellular source of miR-146a. Therefore, we investigated microglia function and phenotype in miR-146a knock-out (KO) mice, analyzed the proteome of KO and wild-type (WT) microglia by LC-MS/MS, and examined miR-146a expression in different brain lesions of patients with multiple sclerosis (MS). Results: When stimulated with LPS or myelin in vitro, microglia from KO mice expressed higher levels of IL-1ß, TNF, IL-6, IL-10, CCL3, and CCL2 compared to WT. Stimulation increased migration and phagocytosis of WT but not KO microglia. CD11c+ microglia were induced by cuprizone (CPZ) in the WT mice but less in the KO. The proteome of ex vivo microglia was not different in miR-146a KO compared to WT mice, but CPZ treatment induced differential and reduced protein responses in the KO: GOT1, COX5b, CRYL1, and cystatin-C were specifically changed in KO microglia. We explored discriminative features of microglia proteomes: sparse Partial Least Squares-Discriminant Analysis showed the best discrimination when control and CPZ-treated conditions were compared. Cluster of ten proteins separated WT and miR-146a KO microglia after CPZ: among them were sensomes allowing to perceive the environment, Atp1a3 that belongs to the signature of CD11c+ microglia, and proteins related to inflammatory responses (S100A9, Ppm1g). Finally, we examined the expression of miR-146a and its validated target genes in different brain lesions of MS patients. MiR-146 was upregulated in all lesion types, and the highest expression was in active lesions. Nineteen of 88 validated target genes were significantly changed in active lesions, while none were changed in NAWM. Conclusion: Our data indicated that microglia is the major source of miR-146a in the CNS. The absence of miR-146a differentially affected microglia function and proteome, and miR-146a may play an important role in gene regulation of active MS lesions.

Experimental Demyelination and Axonal Loss Are Reduced in MicroRNA-146a Deficient Mice.

Background: The cuprizone (CPZ) model of multiple sclerosis (MS) was used to identify microRNAs (miRNAs) related to in vivo de- and remyelination. We further investigated the role of miR-146a in miR-146a-deficient (KO) mice: this miRNA is differentially expressed in MS lesions and promotes differentiation of oligodendrocyte precursor cells (OPCs) during remyelination, but its role has not been examined during demyelination. Methods: MicroRNAs were examined by Agilent Mouse miRNA Microarray in the corpus callosum during CPZ-induced demyelination and remyelination. Demyelination, axonal loss, changes in number of oligodendrocytes, OPCs, and macrophages/microglia was compared by histology/immunohistochemistry between KO and WT mice. Differential expression of target genes and proteins of miR-146a was analyzed in the transcriptome (4 × 44K Agilent Whole Mouse Genome Microarray) and proteome (liquid chromatography tandem mass spectrometry) of CPZ-induced de- and remyelination in WT mice. Levels of proinflammatory molecules in the corpus callosum were compared in WT versus KO mice by Meso Scale Discovery multiplex protein analysis. Results: miR-146a was increasingly upregulated during CPZ-induced de- and remyelination. The absence of miR-146a in KO mice protected against demyelination, axonal loss, body weight loss, and atrophy of thymus and spleen. The number of CNP+ oligodendrocytes was increased during demyelination in the miR-146a KO mice, while there was a trend of increased number of NG2+ OPCs in the WT mice. miR-146a target genes, SNAP25 and SMAD4, were downregulated in the proteome of demyelinating corpus callosum in WT mice. Higher levels of SNAP25 were measured by ELISA in the corpus callosum of miR-146a KO mice, but there was no difference between KO and WT mice during demyelination. Multiplex protein analysis of the corpus callosum lysate revealed upregulated TNF-RI, TNF-RII, and CCL2 in the WT mice in contrast to KO mice. The number of Mac3+ and Iba1+ macrophages/microglia was reduced in the demyelinating corpus callosum of the KO mice. Conclusion: During demyelination, absence of miR-146a reduced inflammatory responses, demyelination, axonal loss, the number of infiltrating macrophages, and increased the number of myelinating oligodendrocytes. The number of OPCs was slightly higher in the WT mice during remyelination, indicating a complex role of miR-146a during in vivo de- and remyelination.

Orthologous proteins of experimental de- and remyelination are differentially regulated in the CSF proteome of multiple sclerosis subtypes.

OBJECTIVE: Here, we applied a multi-omics approach (i) to examine molecular pathways related to de- and remyelination in multiple sclerosis (MS) lesions; and (ii) to translate these findings to the CSF proteome in order to identify molecules that are differentially expressed among MS subtypes. METHODS: To relate differentially expressed genes in MS lesions to de- and remyelination, we compared transcriptome of MS lesions to transcriptome of cuprizone (CPZ)-induced de- and remyelination. Protein products of the overlapping orthologous genes were measured within the CSF by quantitative proteomics, parallel reaction monitoring (PRM). Differentially regulated proteins were correlated with molecular markers of inflammation by using MesoScale multiplex immunoassay. Expression kinetics of differentially regulated orthologous genes and proteins were examined in the CPZ model. RESULTS: In the demyelinated and remyelinated corpus callosum, we detected 1239 differentially expressed genes; 91 orthologues were also differentially expressed in MS lesions. Pathway analysis of these orthologues suggested that the TYROBP (DAP12)-TREM2 pathway, TNF-receptor 1, CYBA and the proteasome subunit PSMB9 were related to de- and remyelination. We designed 129 peptides representing 51 orthologous proteins, measured them by PRM in 97 individual CSF, and compared their levels between relapsing (n = 40) and progressive MS (n = 57). Four proteins were differentially regulated among relapsing and progressive MS: tyrosine protein kinase receptor UFO (UFO), TIMP-1, apolipoprotein C-II (APOC2), and beta-2-microglobulin (B2M). The orthologous genes/proteins in the mouse brain peaked during acute remyelination. UFO, TIMP-1 and B2M levels correlated inversely with inflammation in the CSF (IL-6, MCP-1/CCL2, TARC/CCL17). APOC2 showed positive correlation with IL-2, IL-16 and eotaxin-3/CCL26. CONCLUSIONS: Pathology-based multi-omics identified four CSF markers that were differentially expressed in MS subtypes. Upregulated TIMP-1, UFO and B2M orthologues in relapsing MS were associated with reduced inflammation and reflected reparatory processes, in contrast to the upregulated orthologue APOC2 in progressive MS that reflected changes in lipid metabolism associated with increased inflammation.

Fumarate decreases edema volume and improves functional outcome after experimental stroke.

BACKGROUND: Oxidative stress and inflammation exacerbate tissue damage in the brain after ischemic stroke. Dimethyl-fumarate (DMF) and its metabolite monomethyl-fumarate (MMF) are known to stimulate anti-oxidant pathways and modulate inflammatory responses. Considering these dual effects of fumarates, we examined the effect of MMF treatment after ischemic stroke in mice. METHODS: Permanent middle cerebral artery occlusion (pMCAO) was performed using adult, male C57BL/6 mice. Thirty minutes after pMCAO, 20mg/kg MMF was administered intravenously. Outcomes were evaluated 6, 24 and 48h after pMCAO. First, we examined whether a bolus of MMF was capable of changing expression of kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor (Nrf)2 in the infarcted brain. Next, we studied the effect of MMF on functional recovery. To explore mechanisms potentially influencing functional changes, we examined infarct volumes, edema formation, the expression of heat shock protein (Hsp)72, hydroxycarboxylic acid receptor 2 (Hcar2), and inducible nitric oxide synthase (iNOS) in the infarcted brain using real-time PCR and Western blotting. Concentrations of a panel of pro- and anti-inflammatory cytokines (IFNγ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, TNF) were examined in both the infarcted brain tissue and plasma samples 6, 24 and 48h after pMCAO using multiplex electrochemoluminiscence analysis. RESULTS: Administration of MMF increased the protein level of Nrf2 6h after pMCAO, and improved functional outcome at 24 and 48h after pMCAO. MMF treatment did not influence infarct size, however reduced edema volume at both 24 and 48h after pMCAO. MMF treatment resulted in increased Hsp72 expression in the brain 6h after pMCAO. Hcar2 mRNA levels increased significantly 24h after pMCAO, but were not different between saline- and MMF-treated mice. MMF treatment also increased the level of the anti-inflammatory cytokine IL-10 in the brain and plasma 6h after pMCAO, and additionally reduced the level of the pro-inflammatory cytokine IL-12p70 in the brain at 24 and 48h after pMCAO. CONCLUSIONS: A single intravenous bolus of MMF improved sensory-motor function after ischemic stroke, reduced edema formation, and increased the levels of the neuroprotective protein Hsp72 in the brain. The early increase in IL-10 and reduction in IL-12p70 in the brain combined with changes in systemic cytokine levels may also contribute to the functional recovery after pMCAO.

BID Mediates Oxygen-Glucose Deprivation-Induced Neuronal Injury in Organotypic Hippocampal Slice Cultures and Modulates Tissue Inflammation in a Transient Focal Cerebral Ischemia Model without Changing Lesion Volume.

The BH3 interacting-domain death agonist (BID) is a pro-apoptotic protein involved in death receptor-induced and mitochondria-mediated apoptosis. Recently, it has also been suggested that BID is involved in the regulation of inflammatory responses in the central nervous system. We found that BID deficiency protected organotypic hippocampal slice cultures in vitro from neuronal injury induced by oxygen-glucose deprivation. In vivo, BID-knockout (KO) mice and wild type (WT) mice were subjected to 60 min of transient middle cerebral artery occlusion (tMCAO) to induce focal cerebral ischemia, and allowed to recover for 24 h. Infarct volumes and functional outcome were assessed and the inflammatory response was evaluated using immunofluorescence, Western blotting, quantitative PCR (qPCR) and Mesoscale multiplex analysis. We observed no difference in the infarct volume or neurological outcome between BID-KO and WT mice. The inflammatory response was reduced by BID deficiency as indicated by a change in microglial/leukocyte response. In conclusion, our data suggest that BID deficiency is neuroprotective in an in vitro model and modulates the inflammatory response to focal cerebral ischemia in vivo. However, this is not translated into a robust neuroprotection in vivo.