Send Orders of Reprints at reprints@benthamscience.net MicroRNA 2013, 2, 000-000 1 2211-5366/13 $58.00+.00 © 2013 Bentham Science Publishers Prevention of Lymphocyte Neurotoxic Effects by microRNA Delivery A. Pulliero 1 , B. Marengo 2 , D. Fenoglio 3 , A. Parodi 3 , C. Cereda 4 , C. Domenicotti 2 , S. Orcesi 6 , J. Galli 5 , I. Olivieri 6 , G. Filaci 3 , U. Balottin 6,7 , E. Fazzi 5 and A. Izzotti* ,1 1 Department of Health Sciences, University of Genoa – Genoa Italy; 2 Department of Experimental Medicine, University of Genoa, Italy; 3 Centre of Excellence for Biomedical Research (CEBR), University of Genoa; Department of Internal Medicine (DiMI), University of Genoa, Genoa, Italy; 4 Laboratory of Experimental Neurobiology; 5 Unit of Child Neu- rology and Psychiatry, Ospedali Civili, Mother and Child Department, University of Brescia, Italy; 6 Child Neurology and Psychiatry Unit; 7 Department of Brain and Behavioral Sciences, Unit of Child Neurology and Psychiatry, Univer- sity of Pavia, Italy Abstract: Aicardi Goutieres Syndrome (AGS) is characterized by mutations occurring in genes encoding RNAses. AGS mutations silence intracellular RNases resulting in an intracellular overload of short RNAs arresting the physiological production of microRNA required for brain development. MiR-219 is typically down-regulated in Aicardi Goutieres Syn- drome (AGS). The goal of this study is to investigate miR-219 role in protecting astrocytes co-cultured with AGS-mutated lymphocytes. These lymphocytes display neurotoxic properties due to the presence of AGS-mutation and to their activa- tion by interpheron-alpha (IFN). Obtained results provide the evidence that astrocytes transfected with microRNA-219 are protected from the neurotoxic action of AGS lymphocytes activated by IFN-alpha. In particular, the miR-219 transfection increased astrocyte viability, growth, and differentiation while decreasing cell necrosis and apoptosis. Thus, microRNA- 219 transfection is a valuable strategy in order to confer resistance to astrocytes towards lymphocyte-induced neurotoxic- ity especially in the presence of IFN-alpha, whose levels are typically increased in the cerebrospinal fluid of AGS patients. Keywords: Aicardi Goutieres Syndrome, interferon-alpha, microRNA, neurotoxicity, miR-219 transfection, lymphocytes. INTRODUCTION MicroRNAs (miRNAs) are small, non-coding RNA molecules, with an average size of 22bp, that participate in the developmental control of gene expression at post- transcriptional level [1-4]. MicroRNAs activate the RNA in- hibitor silencing complex (RISC) and associated RNAses [5]. Mutations occuring in genes which encode RNAses cause Aicardi-Goutieres syndrome (AGS). These AGS muta- tions silence RNAse activity, thus inducing accumulation of endogenous RNAs, mainly consisting of short RNAs and microRNAs. Our previous results indicate that microRNA overload occurs in AGS patients [6]. This up-regulation in- hibits microRNA turnover, hindering the synthesis of the novel microRNAs required for the differentiation and myeli- nation of the brain during the initial period of postnatal life. These pathogenic mechanisms result in the cerebrospinal fluid alterations including lymphocytosis and elevated inter- feron-alpha levels (IFN-alpha) [6]. Recently miR-138, miR-338, and in particular miR-219, have all been demonstrated to be highly enriched in mature myelinating oligodendrocytes and to act as important regu- lators of oligodendrocyte differentiation, therefore providing new targets for myelin repair in demyelination-related dis- eases [7-9]. Indeed, miR-219 is necessary to support axonal integrity and the formation and maintenance of compact myelin [10]. The imbalance between myelin-directed *Address corresponding to this author at the Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132, Genoa, Italy; Tel: 0039-010-3538522; Fax: 0039-010-353-8504; E-mail: izzotti@unige.it extracellular proteases and their specific inhibitors typically occurs in young patients affected by AGS, a genetic disease associated with progressive severe leukodystrophy and mi- croencephalia observed during the first year of life [11]. In AGS patients, DICER inhibition, induced by IFN-alpha, is a mechanism that contributes to the occurrence of leukodys- trophy [8]. DICER1 failure inhibits the production of novel mature microRNA including miR-219 and miR-338, oli- godendrocyte-specific miRNAs identified in the spinal cord. In fact, over-expression of these miRNAs is per se sufficient to promote oligodendrocyte differentiation [8]. In addition, Dicer1 knockout mice, typically exhibit a disrupted normal central nervous system myelination [12]. This phenotype is due to the lack of expression of three microRNAs (miR-138, miR-219, and miR-338) that are required for oligodendro- cyte differentiation. Dicer is also involved in vascular devel- opment and regulates vascular remodeling by modulating vascular smooth muscle cells proliferation [13]. Interes- tingly, miR-219 is the most abundant miRNA in mature oligodendrocytes [14], is highly expressed in white matter of cerebellum and corpus callosum, but is reduced in brain oligodendrocyte of Dicer mutant mice [10]. MiR-219 and miR-338 target and inhibit a number of genes known to regulate neuronal differentiation, such the pro-neuronal differentiation factors NeuroD1, Isl1, and Otx2 [8]. In AGS patients, the inhibition of microRNA catabolism, related to the silencing mutations occurring in RNaseH genes, results in a negative feedback towards the whole mi- croRNA machinery [6]. This situation then brings about the blockage of microRNA turnover which, in turn, blocks the