Novel assay identifies all microRNA targets in Alzheimer and normal aged brains

描述（由申请人提供）：我们假设microRNAs （miRNAs）在人类大脑健康衰老和阿尔茨海默病（AD）中发挥重要作用。mirna是调节基因表达的小rna。大多数mirna通过与“靶”mrna杂交而起作用。我们和其他人已经观察到阿尔茨海默病脑组织中miRNA表达模式的改变。在mirna减少的各种模型中，神经变性很快发生。mirna介导的神经保护和神经退行性变的机制尚不清楚，部分原因是大多数mirna的mRNA靶点仍然未知。不幸的是，目前还没有合适的技术来实验表明哪些mrna是miRNA的靶标。我们开发了一种高通量测定方法来鉴定人类脑组织中的miRNA靶点。在这里，我们建议使用这种新的分析来更好地了解阿尔茨海默病的神经化学。我们的研究计划有以下具体目标：1。从肯塔基大学阿尔茨海默病中心脑库获得脑组织，该脑组织具有完整的特征，包括接近死亡的临床评估、短的死后间隔和最先进的神经病理学。临床队列将包括非痴呆对照组、轻度认知障碍对照组、阿尔茨海默病对照组和非阿尔茨海默病痴呆对照组的脑组织。2. 优化一种新的生化分析准确，特异，直接的miRNA目标鉴定。这种共免疫沉淀MicroRNA测定程序（CoMAP）已在细胞培养和脑组织中进行了初步优化。CoMAP将在Specific Aim #1中描述的脑组织上进行。3. CoMAP数据的分析将包括来自相同标本的mRNA微阵列、miRNA微阵列、临床数据和病理数据的数据。这些分析将侧重于发现与阿尔茨海默病治疗和诊断相关的miRNA靶点，以及阐明miRNA具有神经保护功能的mRNA靶点。原始和分析数据集以及CoMAP分析本身将与其他研究人员免费共享。这些特定目标旨在回答以下基本问题：7在健康的大脑衰老中，miRNA的靶标是哪些mrna ？在阿尔茨海默病和其他改变的大脑状态中，miRNA靶向哪些mrna ？mRNA靶点的变化如何与mirna表达的变化相关？

DESCRIPTION (provided by applicant): We hypothesize that microRNAs (miRNAs) play important roles in the human brain during healthy aging and in Alzheimer's disease (AD). MiRNAs are small RNAs that regulate gene expression. Most miRNAs act through hybridization with "target" mRNAs. We and others have observed an altered pattern of miRNA expression in AD brain tissue. In various models where miRNAs are reduced, neurodegeneration ensues quickly. The mechanisms of miRNA-mediated neuroprotection and neurodegeneration are poorly understood, partly because most of the mRNA targets of miRNAs are still unknown. Unfortunately, there has been no suitable technique to indicate experimentally which mRNAs are miRNA targets. We developed a high-throughput assay to identify miRNA targets in human brain tissue. Here we propose to use this novel assay to better understand the neurochemistry of AD. Our research program has the following Specific Aims: 1. Obtain brain tissue from the University of Kentucky Alzheimer's Disease Center Brain Bank that is thoroughly characterized including clinical evaluations near death, short post-mortem intervals, and state-of-the-art neuropathology. Clinical cohorts will include brain tissue of non- demented controls, mild cognitive impairment controls, AD, and non-AD dementia controls. 2. Optimize a novel biochemical assay for accurate, specific, and direct miRNA target identification. This assay, the Co-immunoprecipitation MicroRNA Assay Procedure (CoMAP), has been optimized in cell culture and preliminarily in brain tissue. CoMAP will be performed on the brain tissue described in Specific Aim #1. 3. Analyses of CoMAP data will incorporate data from mRNA microarray, miRNA microarray, clinical data, and pathological data referent to the same specimens. These analyses will focus on discovering miRNA targets relevant to AD treatment and diagnosis, and on elucidating the mRNA targets that subserve the neuroprotective functions of miRNA. The raw and analyzed datasets, and the CoMAP assay itself, will be shared freely with other investigators. These Specific Aims are intended to answer the following fundamental questions: 7 What mRNAs are miRNA targets in healthy brain aging? 7 What mRNAs are miRNA targets in Alzheimer's disease and other altered brain states? 7 How do changes in mRNA targets correlate with changes in expression of miRNAs? PUBLIC HEALTH RELEVANCE: The objective of this project is to use a novel technique to better understand the causes of Alzheimer's disease and why some people remain Alzheimer's disease-free during aging. We will characterize in human brains a newly-discovered high impact level of gene regulation, which are called microRNAs, using a method called a Co-immunoprecipitation MicroRNA Assay Procedure (CoMAP). We think that we can produce information that directly or indirectly contributes to diagnostics and therapeutics for Alzheimer's disease patients.