Novel MicroRNAs in the Aging Mouse Brain Figure 4. The insulin signaling pathway is predicted to be targeted by many aging-regulated novel miRNA candidates. Multiple novel miRNA candidates are predicted to target each of the genes implicated in the pathway, and each novel miRNA candidate is predicted to target multiple genes in the pathway. In red: upregulated (.2.0-fold) novel miRNAs; in black: downregulated (.2.0-fold) novel miRNAs. doi:10.1371/journal.pone.0040028.g004 as the insulin signaling pathway. The insulin signaling pathway plays a central, conserved role in lifespan determination, with many genes affecting lifespan when mutated [31]. In mice, animals that are mutant for the insulin/insulin-like growth factor 1 receptor (IGF-1R) exhibit long life and stress resistance [32]. Additionally, increases in brain IGF-1R and compromised insulin receptor signaling have been associated with neurodegenerative diseases such as Alzheimer’s disease [33]. The novel miRNAs we report here may contribute to both normal and pathological aging through their potential functions in regulating the insulin signaling pathway. AGEMAP mRNA expression data did not show large changes in expression of putative novel miRNA-targeted insulin signaling pathway genes with age. However, 11 of the 43 insulin signaling pathway genes predicted to be targeted by downregulated novel miRNAs were not included in the AGEMAP analysis. One of these genes was Insr. Increased brain INSR expression with age has been shown by Muller et al. [29], and this is in agreement with the age-related reduction of novel miRNAs predicted to target Insr expression. We also looked at a list of genes showing increased expression with age in the neocortex generated by Lee et al. comparing mice at 5 and 30 months of age [6] and found that one gene, Akt2, was also predicted to be targeted by downregulated novel miRNAs we report here (Table S8, red cell). That miRNAs exhibit dynamic expression changes with age while mRNAs do not could be suggestive of a role for miRNAs in buffering molecular changes during aging. Moreover, miRNAs function by targeting multiple genes and working in cooperation with other miRNAs; and we do not fully understand how much individual miRNAs contribute to each mRNA’s expression. Further research is needed to elucidate the roles of miRNAs, individually and collectively, in aging. Many of the miRNAs that we detect in our study have been identified by others as brain-expressed miRNAs, thus validating our method for its ability to detect brain-expressed miRNAs. For example, miR-124 was found by Lagos-Quintana and colleagues as dominating the population of brain-expressed miRNAs in young mice [14]. While not the predominating miRNA, miR-124 is strongly represented in our set of aging brain-expressed miRNAs. Other brain-specific miRNAs [14] included in our dataset include miR-101, -127, -128, and -132. Among the most abundantly expressed miRNAs in our dataset were the let-7 family miRNAs. let-7 family members have been noted for their high representation in the mouse as well as primate brains [14,15]. Further, these miRNAs generally demonstrated increased expression with age in our dataset (Table S1 and S2). This trend is in agreement with the results of a recent study which used size-coded ligation-mediated PCR and found increased let-7 family miRNA expression levels in the aged mouse brain [34]. It is interesting to note that increased let-7b expression has been reported in aged mouse neural stem cells and maybe contributing to decreased stem cell function by targeting Hmga2 expression [35]. While let-7b expression was not significantly altered during brain aging in our dataset, it is possible that other let-7 family members, like let-7f which is increased more than 2-fold with age and shares identical seed sequences with let-7b, could play regulatory roles in brain aging. Some of the aging brain-associated miRNAs have been implicated in neurodegenerative diseases. For example, miR-101 and miR-433, both significantly downregulated in our dataset, have been linked to spinocerebellar ataxia type 1 (SCA1) and Parkinson’s disease, respectively [36,37]. miR-101 targets ataxin 1, responsible for SCA1, at both the mRNA and protein levels; miR- 433 targets fibroblast growth factor 20 which induces a-synuclein PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40028