A specific microRNA (Mir-107) is a potential therapeutic target in Alzheimer's di

描述（由申请人提供）：摘要微小RNA（miRNAs）是最近发现的在神经生物学中起基础作用的小调节RNA。初步结果表明，一种特定的miRNA，miR-107的表达减少，可能有助于阿尔茨海默病（AD）的发病机制，通过代谢途径。这些是关于可能导致AD的特定途径中的miRNAs的第一个发表的数据。我们将测试一种特定的策略来靶向这一途径用于AD治疗。假设#1：MiR-107表达在AD中非常早期降低，这增加了BACE 1表达，因此增加了AD患者脑中神经毒性Abeta肽的量。假设#2：苯扎贝特治疗通过增加miR-107水平降低AD型病理学。具体目标#1：充分表征miRNA对BACE 1的调控。子目标a.一种新的技术将直接测试BACE 1 mRNA是否是miRNA靶点。这种生物化学方法涉及使用我们的单克隆抗Argonaute抗体的免疫共沉淀。子目标B。将进行组织培养研究以准确评估BACE 1 mRNA 3 'UTR的哪些部分构成miRNA靶标。子目标c.“敲入”和“敲低”技术将用于改变人培养细胞中特异性的miR-107水平，以确定miR-107表达变化对BACE 1蛋白、C99多肽和A2肽水平的影响。实验最初将在H4和SH-SY 5 Y细胞上进行。具体目标#2：表征miR-107对葡萄糖代谢的影响，并将miR-107和其他miRNA的表达与AD病理学原位关联。将进行组织培养研究，以评估改变代谢的药物治疗如何影响miR-107表达，并评估miR-107的细胞变化如何影响特定代谢中间体的水平。子目标B。人脑原位杂交将用于了解miR-107表达如何与AD和非AD痴呆的病理标志相关。具体目标#3：评价苯扎贝特在体外和体内增加miR-107水平和减少BACE 1蛋白和A2肽形成的作用。苯扎贝特是一种口服给药，耐受性良好的药物。子目标a.在培养细胞中的初步结果表明，苯扎贝特引起miR-107表达增加，也诱导BACE 1蛋白的下调。苯扎贝特作用的具体机制将使用操纵miR-107水平的实验来表征。子目标B。将向小鼠- APPNLh/NLh x PS1 P264 L/P264 L人源化APP敲入突变体-施用苯扎贝特，所述小鼠是AD型淀粉样蛋白生成的优良模型（1），以证明苯扎贝特在体内调节miR-107、BACE 1、A2水平和AD型神经病理学中的功效。公共卫生相关性：MicroRNA是最近发现的在人类大脑中发挥基本功能的分子。这项研究首次证明了一种特定的microRNA可能在阿尔茨海默病中发挥重要作用。提出了一项利用这一新发现的研究计划，以开发和评估一种新的治疗阿尔茨海默病风险患者的方法。

DESCRIPTION (provided by applicant): Abstract MicroRNAs (miRNAs) are recently-discovered small regulatory RNAs that play fundamental roles in neurobiology. Preliminary results indicate that decreased expression of a particular miRNA, miR-107, may contribute to Alzheimer's disease (AD) pathogenesis through a metabolic pathway. These are the first published data pertaining to miRNAs in a specific pathway that may contribute to AD. We will test a specific strategy to target this pathway for AD therapy. Hypothesis #1: MiR-107 expression is decreased very early in AD, which increases BACE1 expression, and hence increases the amount of neurotoxic Abeta peptides in AD patients' brains. Hypothesis #2: Bezafibrate treatment decreases AD-type pathology by increasing levels of miR-107. Specific Aim #1: Characterize fully the regulation of BACE1 by miRNAs. Sub-Aim a. A novel technique will test directly whether BACE1 mRNA is a miRNA target. This biochemical approach involves co- immunoprecipitation using our monoclonal anti-Argonaute antibody. Sub-Aim b. Tissue culture studies will be performed to evaluate exactly which parts of the BACE1 mRNA 3'UTR constitute miRNA targets. Sub-Aim c. `Knock-in' and `knock-down' techniques will be used to alter miR-107 levels specifically in human cultured cells, to determine the effects of miR-107 expression changes on the levels of BACE1 protein, C99 polypeptide, and A2 peptide. Experiments will be performed initially on H4 and SH-SY5Y cells. Specific Aim #2: Characterize the impact of miR-107 on glucose metabolism and correlate the expression of miR-107 and other miRNAs with AD pathology in situ Sub-Aim a. Tissue culture studies will be performed to assess how pharmacological treatments that alter metabolism affect miR-107 expression, and to evaluate how cellular changes in miR-107 influence the levels of specific metabolic intermediaries. Sub-Aim b. Human brain in situ hybridization will be used to understand how miR-107 expression relates to pathological hallmarks of AD and non-AD dementia. Specific Aim #3: Evaluate bezafibrate for increasing miR-107 levels and decreasing BACE1 protein and A2 peptide(s) formation in vitro and in vivo. Bezafibrate is an orally-administered, well-tolerated medication. Sub-Aim a. Preliminary results in cultured cells demonstrated that bezafibrate causes increased miR-107 expression and also induced down-regulation of BACE1 protein. The specific mechanism of bezafibrate action will be characterized using experiments in which the levels of miR-107 are manipulated. Sub-Aim b. Bezafibrate will be administered to mice - APPNLh/NLh x PS1P264L/P264L humanized APP knock-in mutants - that are an excellent model of AD-type amyloidogenesis (1), to demonstrate in vivo the efficacy of bezafibrate in modulating miR-107, BACE1, A2 levels, and AD-type neuropathology. PUBLIC HEALTH RELEVANCE: MicroRNAs are recently-discovered molecules that serve fundamental functions in the human brain. This research demonstrates for the first time that a particular microRNA may play an important role in Alzheimer's disease. A research program is proposed which exploits this new discovery, in order to develop and evaluate a novel therapy for patients at risk for Alzheimer's disease.