SUMMARY/ABSTRACT MicroRNAs are a growing family of conserved molecules involved in normal biological processes including cell growth and differentiation and pathological conditions such as cancer. MiR-124 is expressed specifically in the CNS but not other tissues and plays an important role in the regulation of the genes involved in differentiation of neurons. We found that miR-124 was expressed only in normal CNS-resident macrophages, but not in inflammatory macrophages or normal macrophages isolated from blood, spleen, bone marrow, peritoneal cavity or liver. We also found that overexpression of miR-124 deactivated inflammatory macrophages and converted them into microglia-like cells. Further analysis demonstrated that miR-124 inhibited macrophage activation by targeting CEBP, a transcription factor responsible for the differentiation of myeloid linage cells. We applied these new basic findings related to miR-124 to EAE which serves as a model of MS. We found that microRNA 124 (miR-124) is expressed in microglia cells but not in peripheral macrophages isolated from mice with EAE. Furthermore, we found that i.v. injection of liposomes containing miR-124 markedly suppressed clinical EAE and inhibited the infiltration of encephalitogenic T cells and inflammatory macrophages into the CNS. Based on these findings we hypothesize that brain-specific miR-124 mediates phenotype switching of inflammatory macrophages into a non-inflammatory phenotype, which results in suppression of autoimmune inflammation. Our data suggest that miR-124 provides a new avenue to understand basic mechanisms of microglia/monocyte biology and provides a novel therapeutic approach for diseases such as MS. We will address these specific aims in our proposal AIM 1: How does miR-124 affect the phenotype and function of macrophages? We will investigate how transfection of M with miR-124 in vitro affects 1) polarization of macrophages into M1 or M2 types and secretion of pro- and anti-inflammatory cytokines; 2) the ability of M to stimulate or inhibit proliferation and cytokine production by autoimmune T cells. AIM 2 : What is the role of miR-124 in promoting quiescence of microglia cells in the CNS microenvironment? We found that co-culture of macrophages with astroglial and neuronal cell lines deactivated macrophages and induced expression of miR-124 in these cells. We will utilize this co-culture system to investigate factors that result in upregulation in the expression of miR-124 in microglia/macrophages in CNS. AIM 3: What are the mechanisms of EAE suppression by peripheral administration of miR-124? We investigated which cell types are affected by i.v. injection of miR-124 and found that miR-124 directly affects macrophages but not DCs, or T and B cells. We will investigate whether suppression of EAE is related to induction of regulatory subsets of myeloid cells or T cells in vivo and will investigate the treatment of relapsing and progressive EAE mouse models with miR-124. 1