Differentially expressed hippocampal miRNAs with age and cognitive decline

描述（由申请人提供）：随着年龄的增长，认知能力下降会降低老年人的独立性、生活质量和生产力。虽然比阿尔茨海默氏症和帕金森氏症等神经退行性疾病更常见，但认知能力下降的病因仍不清楚。这项研究的中心假设是，随着年龄的增长和认知能力的下降，miRNA的差异表达通过调节蛋白质表达对突触可塑性和认知能力产生负面影响。我们以前的研究已经确定了海马mRNA和蛋白质表达的变化与老化和认知能力下降，使用Fischer 344 x Brown Norway（F1）大鼠，通过Morris水迷宫测试进行认知分层。有趣的是，在成年（12个月）和老年（26个月）认知完整，老年认知受损大鼠的比较中，我们已经确定了一些与神经元可塑性相关的蛋白质表达的变化，这些变化在转录水平上不受调节。这表明蛋白质表达调控的替代机制，包括miRNA的作用。在使用相同模型和行为测试的初步研究中，我们已经鉴定了随年龄调节的miRNA以及在认知完整和受损的老年动物之间特异性调节的miRNA。这些miRNAs的预测目标包括许多我们已经确定为下调但转录水平不变的蛋白质。这表明miRNA作为海马蛋白表达随认知能力下降和衰老而变化的调节机制之一的作用。 为了扩展我们的初步结果，在第一个具体目标中，将在成年和老年认知分层Fischer 344 x Brown Norway大鼠的CA 1、CA 2/3和DG海马亚区中确定所有已知大鼠miRNA的海马表达。这将提供海马miRNA表达与衰老和认知障碍的第一个全基因组检查。差异表达的miRNA将通过qPCR确认，潜在靶标将通过计算机模拟分析确定。在目标预测之后，所有的miRNA表达和目标数据将与我们现有的关于衰老和认知衰退的mRNA和蛋白质表达数据集相结合，以确定特定miRNA可能的调控点。 第二个具体目标是确定由差异表达的miRNA调控的特定蛋白质组。在神经元细胞培养物中，随着认知下降或衰老调节的特异性miRNA将过度/不足表达，以评估蛋白质表达的特异性和整体变化。直接/间接调节的区分将通过使用调节基因的3' UTR的报告基因测定来确定。不仅知道随着衰老和认知能力下降而调节的miRNA，而且知道它们已被确认的调节靶点，这将有助于理解改变miRNA表达对可塑性和认知的潜在影响。

DESCRIPTION (provided by applicant): Cognitive decline with aging reduces independence, quality of life, and productivity in older individuals. While much more common than neurodegenerative diseases, such as Alzheimer's and Parkinson's, the etiology of cognitive decline remains unknown. The central hypothesis of this proposed study is that differential expression of miRNAs with age and cognitive decline negatively impacts synaptic plasticity and cognition through the regulation of protein expression. Our previous studies have identified hippocampal mRNA and protein expression changes with aging and cognitive decline utilizing Fischer 344 x Brown Norway (F1) rats cognitively stratified by Morris Water Maze testing. Interestingly, in comparisons of Adult (12 month) and Aged (26 Month) Cognitively Intact, and Aged Cognitively Impaired rats we have identified a number of changes in protein expression related to neuronal plasticity that are not regulated at the transcript level. This suggests a role for alternate mechanisms of protein expression regulation, including miRNAs. In pilot studies using the same model and behavioral testing we have identified miRNAs regulated with age and miRNAs specifically regulated between cognitively intact and impaired aged animals. The predicted targets of these miRNAs included many of those proteins that we have identified as down regulated but whose transcript levels are unchanged. This suggests a role for miRNAs as one of the regulatory mechanisms underlying the hippocampal protein expression changes with cognitive decline and aging. To expand on our preliminary results, in the first specific aim, hippocampal expression of all known rat miRNAs will be determined in CA1, CA2/3 and DG hippocampal sub regions of Adult and Aged cognitively stratified Fischer 344 x Brown Norway rats. This will provide the first genome-wide examination of hippocampal miRNA expression with aging and cognitive impairment. Differentially expressed miRNAs will be confirmed by qPCR and potential targets will be determined by in silico analysis. Following target prediction, all miRNA expression and target data will be integrated with our existing mRNA and protein expression datasets on aging and cognitive decline to identify likely points of regulation by specific miRNAs. The second specific aim will determine specific sets of proteins regulated by differentially expressed miRNAs. Specific miRNAs regulated with cognitive decline or aging will be over/under-expressed in neuron cell culture to assess both specific and global changes in protein expression. Differentiation of direct/indirect regulation will be determined through reporter assays using the 3' UTR of regulated genes. Knowing not only the miRNAs regulated with aging and cognitive decline but their confirmed regulatory targets will allow for an understanding of the potential outcome of altered miRNA expression on plasticity and cognition.