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.

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.

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.

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.