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)在健康衰老和阿尔茨海默病(AD)期间在人脑中发挥重要作用。MiRNAs是调节基因表达的小RNA。大多数miRNAs通过与“靶标”mRNAs杂交来发挥作用。我们和其他人观察到了阿尔茨海默病脑组织中miRNA表达模式的变化。在miRNAs减少的各种模型中，神经退化会迅速发生。MiRNA介导的神经保护和神经变性的机制尚不清楚，部分原因是miRNAs的大多数信使核糖核酸靶标仍然未知。不幸的是，还没有合适的技术来实验表明哪些mRNAs是miRNA靶标。我们开发了一种高通量分析方法来识别人脑组织中的miRNA靶标。在这里，我们建议使用这一新的检测方法来更好地了解AD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取脑组织，其特征包括临床评估濒临死亡、短时间的死亡时间和最先进的神经病理学。临床队列包括非痴呆对照组、轻度认知障碍对照组、阿尔茨海默病和非阿尔茨海默病对照组的脑组织。2.优化了一种准确、特异、直接鉴定miRNA靶标的生化方法。共免疫沉淀微量RNA分析程序(CoMAP)已经在细胞培养中进行了优化，并初步在脑组织中进行了检测。CoMAP将对特定目标1中描述的脑组织进行CoMAP。CoMAP数据的分析将结合来自相同标本的mRNA微阵列、miRNA微阵列、临床数据和病理数据。这些分析将集中于发现与AD治疗和诊断相关的miRNA靶点，并阐明辅助miRNA神经保护功能的信使核糖核酸靶点。原始和分析的数据集，以及CoMAP分析本身，将免费与其他研究人员共享。这些特定的目标旨在回答以下基本问题：7在健康的脑老化中，哪些mRNAs是miRNA靶标？7在阿尔茨海默病和其他大脑状态改变中，哪些mRNAs是miRNA靶标？7mRNAs靶标的变化与miRNAs表达的变化如何相关？ 公共卫生相关性：该项目的目标是使用一种新的技术来更好地了解阿尔茨海默病的原因，以及为什么有些人在衰老过程中没有阿尔茨海默病。我们将使用一种名为共免疫沉淀微RNA分析程序(CoMAP)的方法，在人类大脑中表征一种新发现的高影响水平的基因调控，称为microRNAs。我们认为，我们可以产生直接或间接为阿尔茨海默病患者的诊断和治疗做出贡献的信息。

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.