MiR-15/107 microRNAs are important genetic regulators in Alzheimer disease

描述（由申请人提供）：miR-15/107 miRNA组是同源microrna (miRNA)，具有重叠的基因调控作用，在阿尔茨海默病（AD）大脑中下调。我们的基本假设是miR- 15/107基因的增强（上调）将有助于减少BACE1和tau激酶（PKA和cdk5调节蛋白）的脑细胞表达。我们有强有力的初步证据支持这一假设。本研究计划的总体目标是获得开发未来基于mirna的脑疗法所需的信息，并增加我们对mirna在AD中的作用的了解。治疗策略必须包括刺激内源性miRNA的产生，或者采用外源引入RNA或病毒载体的“基因治疗”方法。在开发与miR-15/107基因相关的脑疗法之前，主要的挑战是了解改变人脑中miR-15/107表达的上游刺激，以及确定由这些mirna调节的下游基因，包括评估操纵miR-15/107相关mirna的“脱靶效应”的潜力。因此，我们提出在人脑和大鼠原代脑源性培养细胞中进行新的实验。本研究项目的具体目的：具体目的#1：确定调节miR-15/107基因表达的特定ad相关信号通路。目的1A：根据我们的初步结果，在培养的大鼠原代脑细胞中进行基于假设的研究，发现葡萄糖水平和离散神经炎症刺激特异性地改变miR-15/107基因表达，这可能为基于mirna的治疗策略提供建议。目标1B：在人脑中进行全面的miR-15/107表达谱分析，以了解与AD风险相关的参数如何与miR-15/107基因表达改变相关，因为miRNA神经化学研究需要锚定在人脑数据中。特异性目标#2：确定原代培养大鼠脑细胞中miR-15/107基因调控的确切“下游”mRNA靶点。初步数据表明，miR-15/107基因可能是人脑代谢变化和tau磷酸化增加之间的关键机制联系。使用我们实验室开发和优化的方法，我们将对原代培养的大鼠脑细胞中的miR-15/107 miRNA靶点进行全面分析。特定目标#3：在原代培养的脑细胞中使用RNA序列修饰来操纵和最小化miRNAs的“脱靶”效应。培养的神经元将用含有miR-15/107基因相关改变序列的慢病毒载体进行转导。我们已经产生了表达慢病毒的miRNA，并转导了原代培养的神经元，以帮助优化miRNA为基础的脑治疗的潜在影响。总体目标是生产具有最佳“靶向”和最小“脱靶”影响的试剂，以最大限度地发挥基于小RNA的治疗方法的潜在价值。

DESCRIPTION (provided by applicant): The miR-15/107 miRNA group are paralogous microRNAs (miRNAs), with overlapping gene regulatory impact, that are downregulated in Alzheimer's disease (AD) brains. Our underlying hypothesis is that miR- 15/107 gene augmentation (up-regulation) will help to reduce brain cellular expression of BACE1 and tau kinases (PKA and CDK5-regulating proteins). We have strong preliminary evidence in support of this hypothesis. The overall goal of the present research proposal is to obtain information necessary to develop future miRNA-based brain therapies and to increase our knowledge about miRNAs' role in AD. Therapeutic strategies must involve either stimulating endogenous miRNA production, or, alternatively, a "gene therapy" approach with exogenously introduced RNA or viral vectors. The key challenges, before developing brain therapies related to miR-15/107 genes, are 1> to understand upstream stimuli that alter miR-15/107 expression in human brains, and 2> define downstream genes regulated by these miRNAs, including an assessment of the potential for manipulating the "off-target effects" of miR-15/107-related miRNAs. We thus propose novel experiments in human brain and in primary rat brain-derived cultured cells. Specific Aims for this research project: Specific Aim #1: Define the specific AD-relevant signaling pathways that modulate miR-15/107 gene expression. Aim 1A: Perform hypothesis-based studies in cultured rat primary brain cells anchored in our preliminary results finding that glucose levels and discrete neuroinflammatory stimuli specifically alter miR-15/107 gene expression which may suggest strategies for miRNA-based therapies. Aim 1B: Perform comprehensive miR-15/107 expression profiling in human brain to understand how parameters linked with AD risk correlate with altered miR-15/107 gene expression because miRNA neurochemical studies need to be anchored in human brain data. Specific Aim #2: Define the exact "downstream" mRNA targets regulated by miR-15/107 genes in primary cultured rat brain cells. Preliminary data indicate that miR-15/107 genes may be a key mechanistic link between metabolic changes and increased tau phosphorylation in human brains. Using methods that were developed and optimized in our laboratory, we will perform comprehensive analyses of miR-15/107 miRNA targets in primary cultured rat brain cells. Specific Aim #3: Manipulate and minimize "off-target" effects of miRNAs using RNA sequence modifications in primary cultured brain cells. Cultured neurons will be transduced with lentivirus vectors harboring altered sequences related to miR-15/107 genes. We have already generated miRNA expressing lentivirus and transduced primary cultured neurons to help optimize the potential impact of miRNA-based therapies in the brain. The overall goal is to produce reagents with optimal "on-target" and minimal "off-target" impact, in order to maximize the potential value of small RNA- based therapeutics.