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In contrast to peripheral macrophages, microglia in the central nervous system (CNS) exhibit a specific deactivated phenotype; however, it is not clear how this phenotype is maintained. Two alternative hypotheses were postulated recently: (a) microglia differ from peripheral macrophages being derived from the yolk sac (YS), whereas peripheral macrophages originate from bone marrow (BM); (b) microglia acquire a specific phenotype under the influence of the CNS microenvironment. We have previously shown that microglia express miR-124, which was also induced in BM-derived macrophages co-cultured with a neurons. We here investigated the possibility of horizontal transfer of the neuron-specific microRNAs miR-124 and miR-9 from primary neurons to microglia/macrophages. We found that after incubation with neuronal conditioned media (NCM), macrophages downregulated activation markers MHC class II and CD45. Neither cultured adult microglia nor YS- and BM-derived macrophages demonstrated intrinsic levels of miR-124 expression. However, after incubation with NCM, miR-124 was induced in both YS- and BM-derived macrophages. Biochemical analysis demonstrated that the NCM contained miR-124 and miR-9 in complex with small proteins, large high-density lipoproteins (HDLs), and exosomes. MiR-124 and miR-9 were promptly released from neurons, and this process was inhibited by tetrodotoxin, indicating an important role of neuronal electric activity in secretion of these microRNAs. Incubation of macrophages with exogenous miR-124 resulted in efficient translocation of miR-124 into the cytoplasm. This study demonstrates an important role of neuronal miRNAs in communication of neurons with microglia, which favors the hypothesis that microglia acquire a specific phenotype under the influence of the CNS microenvironment., (© 2018 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.)

MicroRNA-132 is markedly downregulated in Alzheimer's disease (AD) and related tauopathies, and its levels are closely associated with tau pathology in AD. Whether and how miR-132 contributes to pathology in these neurodegenerative diseases remains unclear. Here, we show that miR-132 and its paralogue miR-212 directly regulate the expression of neuronal nitric oxide synthase (NOS1) through the primate-specific binding site. Inhibition of miR-132 in primary human neurons and neural cells leads to increased NOS1 levels and triggers excessive production of nitric oxide, followed by aberrant S-nitrosylation (SNO) of specific proteins associated with neurodegeneration and tau pathology, such as cyclin-dependent kinase 5, dynamin-related protein 1, and glyceraldehyde-3-phosphate dehydrogenase. This, in turn, increases tau phosphorylation at disease associated Ser396, Ser404, and Ser202 sites, and impairs neural viability. Our findings indicate that downregulation of miR-132/212 disturbs the balance of S-nitrosylation and induces tau phosphorylation in a NOS1-dependent way, and thereby may contribute to the pathogenesis of AD and other tauopathies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Monocytic cells exhibit a high level of heterogeneity and have two distinct modes of their activation: 1) classical M1 path associated with inflammation and tissue damage, and 2) alternative M2 path. Although it has been demonstrated that M2 macrophages play an important role in the regulation of the allergic immune responses, tissue maintenance and repair, little is known about the mechanisms that determine the M2 phenotype. We have previously shown that miR-124 is expressed in microglia that exhibit the M2 phenotype and overexpression of miR-124 in macrophages resulted in downregulation of a number of M1 markers (MHC class II, CD86) and up-regulation of several M2 markers (Fizz1, Arg1). We further investigated whether the polarization of macrophages towards the M2 phenotype induced miR-124 expression. We found that exposure of cells to IL-4 and IL-13 resulted in the upregulation of miR-124 in macrophages. We also demonstrated that IL-4 induced expression of three miR-124 precursor transcripts with predominant expression of pri-miR-124.3, suggesting regulation of miR-124 expression by IL-4 on a transcriptional level. Expression of miR-124 in microglia did not depend on IL-4 and/or IL-13, whereas expression of miR-124 in lung resident macrophages was IL-4 and IL-13-dependent and was upregulated by systemic administration of IL-4 or during allergic inflammation. Upregulation of several M2 markers (CD206, Ym1) and downregulation of the M1 markers (CD86, iNOS, TNF) in M2-polarized macrophages was abrogated by a miR-124 inhibitor, suggesting that this microRNA contributed to the M2 phenotype development and maintenance. Finally we showed that human CD14(+)CD16(+) intermediate monocytes, which are found in increased numbers in patients with allergies and bronchial asthma, expressed high levels of miR-124 and exhibited other properties of M2-like cells. Thus, our study suggests that miR-124 serves as a regulator of the M2 polarization in various subsets of monocytic cells both in vitro and in vivo.

Alzheimer's disease (AD) is a multifactorial and fatal neurodegenerative disorder for which the mechanisms leading to profound neuronal loss are incompletely recognized. MicroRNAs (miRNAs) are recently discovered small regulatory RNA molecules that repress gene expression and are increasingly acknowledged as prime regulators involved in human brain pathologies. Here we identified two homologous miRNAs, miR-132 and miR-212, downregulated in temporal cortical areas and CA1 hippocampal neurons of human AD brains. Sequence-specific inhibition of miR-132 and miR-212 induces apoptosis in cultured primary neurons, whereas their overexpression is neuroprotective against oxidative stress. Using primary neurons and PC12 cells, we demonstrate that miR-132/212 controls cell survival by direct regulation of PTEN, FOXO3a and P300, which are all key elements of AKT signaling pathway. Silencing of these three target genes by RNAi abrogates apoptosis caused by the miR-132/212 inhibition. We further demonstrate that mRNA and protein levels of PTEN, FOXO3a, P300 and most of the direct pro-apoptotic transcriptional targets of FOXO3a are significantly elevated in human AD brains. These results indicate that the miR-132/miR-212/PTEN/FOXO3a signaling pathway contributes to AD neurodegeneration.

MicroRNAs (miRNAs) are a class of small (∼22 nucleotides) noncoding RNAs involved in the regulation of gene expression at the post-translational level. It is estimated that 30-90% of human genes are regulated by miRNAs, which makes these molecules of great importance for cell growth, activation, and differentiation. Microglia is CNS-resident cells of a myeloid lineage that play an important role in immune surveillance and are actively involved in many neurologic pathologies. Although the exact origin of microglia remains enigmatic, it is established that primitive macrophages from a yolk sac populate the brain and spinal cord in normal conditions throughout development. During various pathological events such as neuroinflammation, bone marrow derived myeloid cells also migrate into the CNS. Within the CNS, both primitive macrophages from the yolk sac and bone marrow derived myeloid cells acquire a specific phenotype upon interaction with other cell types within the CNS microenvironment. The factors that drive differentiation of progenitors into microglia and control the state of activation of microglia and bone marrow-derived myeloid cells within the CNS are not well understood. In this review we will summarize the role of miRNAs during activation and differentiation of myeloid cells. The role of miR-124 in the adaptation of microglia and macrophages to the CNS microenvironment will be further discussed. We will also summarize the role of miRNAs as modulators of activation of microglia and microphages. Finally, we will describe the role of miR-155 and miR-124 in the polarization of macrophages towards classically and alternatively activated phenotypes., (Copyright © 2012 Wiley Periodicals, Inc.)

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)(1). These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)(2). Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers(3). The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation(4) and pathologies such as inflammation(5). MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages(6) and microglia(7). In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

MicroRNAs are a family of regulatory molecules involved in many physiological processes, including differentiation and activation of cells of the immune system. We found that brain-specific miR-124 is expressed in microglia but not in peripheral monocytes or macrophages. When overexpressed in macrophages, miR-124 directly inhibited the transcription factor CCAAT/enhancer-binding protein-α (C/EBP-α) and its downstream target PU.1, resulting in transformation of these cells from an activated phenotype into a quiescent CD45(low), major histocompatibility complex (MHC) class II(low) phenotype resembling resting microglia. During experimental autoimmune encephalomyelitis (EAE), miR-124 was downregulated in activated microglia. Peripheral administration of miR-124 in EAE caused systemic deactivation of macrophages, reduced activation of myelin-specific T cells and marked suppression of disease. Conversely, knockdown of miR-124 in microglia and macrophages resulted in activation of these cells in vitro and in vivo. These findings identify miR-124 both as a key regulator of microglia quiescence in the central nervous system and as a previously unknown modulator of monocyte and macrophage activation.