Intra- and Inter-individual Variability of microRNA Levels in Human Cerebrospinal Fluid: Critical Implications for Biomarker Discovery.

MicroRNAs are emerging as promising biomarkers for diagnosis of various diseases. Notably, cerebrospinal fluid (CSF) contains microRNAs that may serve as biomarkers for neurological diseases. However, there has been a lack of consistent findings among CSF microRNAs studies. Although such inconsistent results have been attributed to various technical issues, inherent biological variability has not been adequately considered as a confounding factor. To address this critical gap in our understanding of microRNA variability, we evaluated intra-individual variability of microRNAs by measuring their levels in the CSF from healthy individuals at two time points, 0 and 48 hours. Surprisingly, the levels of most microRNAs were stable between the two time points. This suggests that microRNAs in CSF may be a good resource for the identification of biomarkers. However, the levels of 12 microRNAs (miR-19a-3p, miR-19b-3p, miR-23a-3p, miR-25a-3p, miR-99a-5p, miR-101-3p, miR-125b-5p, miR-130a-3p, miR-194-5p, miR-195-5p, miR-223-3p, and miR-451a) were significantly altered during the 48 hours interval. Importantly, miRNAs with variable expression have been identified as biomarkers in previous studies. Our data strongly suggest that these microRNAs may not be reliable biomarkers given their intrinsic variability even within the same individual. Taken together, our results provide a critical baseline resource for future microRNA biomarker studies.

miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1).

BACKGROUND: Loss-of-function mutations in PINK1 and PARKIN are the most common causes of autosomal recessive Parkinson's disease (PD). PINK1 is a mitochondrial serine/threonine kinase that plays a critical role in mitophagy, a selective autophagic clearance of damaged mitochondria. Accumulating evidence suggests mitochondrial dysfunction is one of central mechanisms underlying PD pathogenesis. Therefore, identifying regulatory mechanisms of PINK1 expression may provide novel therapeutic opportunities for PD. Although post-translational stabilization of PINK1 upon mitochondrial damage has been extensively studied, little is known about the regulation mechanism of PINK1 at the transcriptional or translational levels. RESULTS: Here, we demonstrated that microRNA-27a (miR-27a) and miR-27b suppress PINK1 expression at the translational level through directly binding to the 3'-untranslated region (3'UTR) of its mRNA. Importantly, our data demonstrated that translation of PINK1 is critical for its accumulation upon mitochondrial damage. The accumulation of PINK1 upon mitochondrial damage was strongly regulated by expression levels of miR-27a and miR-27b. miR-27a and miR-27b prevent mitophagic influx by suppressing PINK1 expression, as evidenced by the decrease of ubiquitin phosphorylation, Parkin translocation, and LC3-II accumulation in damaged mitochondria. Consequently, miR-27a and miR-27b inhibit lysosomal degradation of the damaged mitochondria, as shown by the decrease of the delivery of damaged mitochondria to lysosome and the degradation of cytochrome c oxidase 2 (COX2), a mitochondrial marker. Furthermore, our data demonstrated that the expression of miR-27a and miR-27b is significantly induced under chronic mitophagic flux, suggesting a negative feedback regulation between PINK1-mediated mitophagy and miR-27a and miR-27b. CONCLUSIONS: We demonstrated that miR-27a and miR-27b regulate PINK1 expression and autophagic clearance of damaged mitochondria. Our data further support a novel negative regulatory mechanism of PINK1-mediated mitophagy by miR-27a and miR-27b. Therefore, our results considerably advance our understanding of PINK1 expression and mitophagy regulation and suggest that miR-27a and miR-27b may represent potential therapeutic targets for PD.

miR-186 is decreased in aged brain and suppresses BACE1 expression.

Accumulation of amyloid ß (Aß) in the brain is a key pathological hallmark of Alzheimer's disease (AD). Because aging is the most prominent risk factor for AD, understanding the molecular changes during aging is likely to provide critical insights into AD pathogenesis. However, studies on the role of miRNAs in aging and AD pathogenesis have only recently been initiated. Identifying miRNAs dysregulated by the aging process in the brain may lead to novel understanding of molecular mechanisms of AD pathogenesis. Here, we identified that miR-186 levels are gradually decreased in cortices of mouse brains during aging. In addition, we demonstrated that miR-186 suppresses ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1) expression by directly targeting the 3'UTR of Bace1 mRNA in neuronal cells. In contrast, inhibition of endogenous miR-186 significantly increased BACE1 levels in neuronal cells. Importantly, miR-186 over-expression significantly decreased Aß level by suppressing BACE1 expression in cells expressing human pathogenic mutant amyloid precursor protein. Taken together, our data demonstrate that miR-186 is a potent negative regulator of BACE1 in neuronal cells and it may be one of the molecular links between brain aging and the increased risk for AD during aging. We identified that miR-186 levels are gradually decreased in mouse cortices during aging. Furthermore, we demonstrated that miR-186 is a novel negative regulator of beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) expression in neuronal cells. Therefore, we proposed that reduction in miR-186 levels during aging may lead to the up-regulation of BACE1 in the brain, thereby increasing a risk for Alzheimer's disease in aged individuals. Read the Editorial Highlight for this article on page 308.