Novel MicroRNAs Differentially Expressed during Aging in the Mouse Brain Sachi Inukai, Alexandre de Lencastre, Michael Turner¤, Frank Slack* Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States of America Abstract MicroRNAs (miRNAs) are endogenous small RNA molecules that regulate gene expression post-transcriptionally. Work in Caenorhabditis elegans has shown that specific miRNAs function in lifespan regulation and in a variety of age-associated pathways, but the roles of miRNAs in the aging of vertebrates are not well understood. We examined the expression of small RNAs in whole brains of young and old mice by deep sequencing and report here on the expression of 558 known miRNAs and identification of 41 novel miRNAs. Of these miRNAs, 75 known and 18 novel miRNAs exhibit greater than 2.0- fold expression changes. The majority of expressed miRNAs in our study decline in relative abundance in the aged brain, in agreement with trends observed in other miRNA studies in aging tissues and organisms. Target prediction analysis suggests that many of our novel aging-associated miRNAs target genes in the insulin signaling pathway, a central node of aging- associated genetic networks. These novel miRNAs may thereby regulate aging-related functions in the brain. Since many mouse miRNAs are conserved in humans, the aging-affected brain miRNAs we report here may represent novel regulatory genes that also function during aging in the human brain. Citation: Inukai S, de Lencastre A, Turner M, Slack F (2012) Novel MicroRNAs Differentially Expressed during Aging in the Mouse Brain. PLoS ONE 7(7): e40028. doi:10.1371/journal.pone.0040028 Editor: Michael Watson, The Roslin Institute, University of Edinburgh, United Kingdom Received December 14, 2011; Accepted June 1, 2012; Published July 23, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: A.d.L. was supported by a National Research Service Award Postdoctoral Fellowship from the National Institutes of Health (NIH; 1F32AG030851). This work was also supported by grants from the Ellison Medical Foundation and the NIH (AG033921). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: frank.slack@yale.edu ¤ Current address: United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, United States of America Introduction Aging is a complex process that manifests a variety of characteristic and evolutionarily conserved changes. In higher organisms, aging is often accompanied by cognitive decline and is also cited as the primary risk factor for many neurodegenerative disorders including Alzheimer’s disease, Parkinson’s disease, and trinucleotide repeat disorders [1–3]. While some physiological differences between young and aged brains have been described, such as the differential recruitment of specific brain regions and synaptic loss [4,5], the mechanisms involved in brain aging at the molecular level are not well understood. Gene expression profiling studies of the brain have been conducted in mice, rats, chimpanzees, and humans and have shown that specific genes and pathways are affected by aging [6– 10]. For example, studies in mice have reported that genes involved in inflammatory and stress responses, as well as many proteases involved in neuropeptide metabolism, show altered expression [6,9]. How the many affected pathways and processes are regulated and fit into the overall picture of brain aging remain to be elucidated. MicroRNAs (miRNAs) are endogenous small RNA molecules that control gene expression post-transcriptionally, primarily through binding to complementary target sequences in the 39 untranslated regions (UTRs) of mRNAs. Work in Caenorhabditis elegans has demonstrated that miRNAs function in aging and a variety of age-associated pathways and processes [11–13]. Brain- specific/2enriched miRNAs have been identified [14] and associated with brain development and neurodegeneration [15]. Furthermore, central nervous system-expressed miRNAs have been shown to demonstrate a wide variety of spatial and temporal expression patterns [16,17]. However, neither their specific functions nor the gene regulatory networks with which they are associated have been studied in detail, and we do not understand how miRNAs are affected in the aging brain. In this study, we used deep-sequencing technology to investigate the expression changes of miRNAs during aging in the mouse brain. Our results show that many miRNAs, including several novel miRNA candidates, are significantly differentially expressed during aging with a global downward trend of miRNA expression, in agreement with other studies of miRNA expression changes during aging [12,18]. Furthermore, predicted targets of these age- associated miRNAs have known roles in aging processes, such as in insulin signaling. Together, our study provides further support for the proposed roles for miRNAs in aging and age-related events in the murine brain. Results Mouse Brain Small RNA Cloning and Sequencing In order to understand potential contributions of miRNAs to aging processes in the mouse brain, we first examined their expression changes using deep sequencing with Solexa technology. cDNA libraries from small RNAs were prepared from whole brains of young and old mice (5 months and 24–25 months, PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40028