Role of Astrocytic miR-20a-3p as a Potential Therapeutic for Ischemic Stroke

项目摘要 该项目的目标是测试一种有潜力改善中风预后的治疗化合物， 年长的女性关注这一人口统计数据至关重要，因为研究表明，绝经后， 患缺血性中风的风险和严重程度更高。申办者实验室的先前研究表明， 生殖性衰老（非周期性）（RS）雌性大鼠的梗死面积更大，感觉运动障碍更严重 比年轻的女性。此外，雌激素替代RS女性加重中风损害。因此 该动物模型复制了在人群中观察到的中风相关后果的关键特征， 产生具有翻译潜能的模型，其中可以测试神经治疗化合物。 申办方实验室的先前研究表明，生殖衰老（RS）雌性大鼠的星形胶质细胞 表现出与年龄相关的变化，导致中风后恢复较差。这包括减少的能力， 谷氨酸清除，营养因子分泌减少，炎性细胞因子释放增加， 与年轻女性相比。此外，这些老化的星形胶质细胞还显示出减少的组蛋白甲基化， H3 K4染色质区域，导致包括microRNA mir-17-92在内的几个基因的转录减少 集群微RNA最近作为多种病症的潜在治疗剂而受到欢迎， 包括缺血性中风 该提议的初步数据显示，miR-20 a-3 p，mir-17-92簇的成员，显著地 在中年女性的星形胶质细胞中下调。此外，静脉注射miR-20 a-3 p模拟了四种 大脑中动脉闭塞（MCAo）后24小时，梗死体积减少，感觉运动减弱， 在中年女性中，miR-20 a-3 p可能与中风有机械联系。 结果。这提出了一个有趣的可能性，即在年轻女性中，内源性星形胶质细胞来源的miR- 20 a-3 p可以“分布”到其他细胞以改善中风结果。这一假设将在 目的1：确定miR-20 a-3 p是否改变星形胶质细胞外基质的组成， 研究表明，miR-20 a-3 p修饰的EV将产生神经保护作用。已经证明星形胶质细胞 中风后通过EV释放神经保护因子、细胞器和RNA翻译机器。计算机模拟 分析预测miR-20 a-3 p可能调节炎症介质和线粒体基因。因此，miR- 20 a-3 p可能会彻底改变电动汽车的组成。在aim 2中，复制缺陷型腺病毒 构建体将用于仅在星形胶质细胞中增加miR-20 a-3 p水平，并确定这是否 概括了iv miR-20 a-3 p的神经保护作用。使用临床相关的中风模型， 行为分析和现代基因转移技术，这项建议将严格审查是否 老化星形胶质细胞的修饰将促进神经保护。

Project Summary The goal of this project is to test a therapeutic compound that has the potential to improve stroke outcomes in older females. It is critical to focus on this demographic because studies show that after menopause, women have greater risk and severity of ischemic stroke. Previous studies from the sponsor's lab have shown that reproductively senescent (acyclic) (RS) female rats have larger infarct and worse sensorimotor impairments than younger females. Moreover, estrogen replacement to RS females exacerbates stroke impairment. Thus this animal model replicates key features of stroke-related consequences seen in the human population, resulting in a model with translational potential, in which neurotherapeutic compounds can be tested. Prior research from the sponsor's lab has shown that astrocytes from reproductively senescent (RS) female rats exhibit age-related changes that contribute to poorer recovery after stroke. This includes a reduced capacity for glutamate clearance, decreased secretion of trophic factors, and increased release of inflammatory cytokines, compared to younger females. Furthermore, these aging astrocytes also display reduced histone methylation of the H3K4 chromatin region, resulting in less transcription of several genes including the microRNA mir-17-92 cluster. MicroRNAs have recently gained popularity as potential therapeutics for a variety of conditions, including ischemic stroke. Preliminary data for this proposal shows that miR-20a-3p, a member of the mir-17-92 cluster is dramatically down regulated in astrocytes of middle-aged females. Furthermore, iv injection of miR-20a-3p mimics four hours after middle cerebral artery occlusion (MCAo) decreased infarct volume and attenuated sensorimotor impairment in middle-aged females, suggesting that the miR-20a-3p could be mechanistically linked to stroke outcomes. This raises the intriguing possibility that in younger females, endogenous astrocyte-derived miR- 20a-3p may be `distributed' to other cells to improve stroke outcomes. This hypothesis will be tested in the following aims: Aim 1 will determine whether miR-20a-3p alters the composition of astrocytice extracellular vesicles (EVs) and that miR-20a-3p modified EVs will confer neuroprotection. Astrocytes have been shown to release neuroprotective factors, organelles and RNA translational machinery through EVs after stroke. In silico analysis predicts that miR-20a-3p may regulate inflammatory mediators and mitochondrial genes. Thus, miR- 20a-3p could drastically change the composition of the EVs. In aim 2, replication-deficient adenovirus constructs will be used to increase miR-20a-3p levels only in astrocytes and determine whether this recapitulates the neuroprotective effect of iv miR-20a-3p. Using clinically relevant stroke models, a battery of behavioral assays, and modern gene transfer techniques, this proposal will critically examine whether modification of the aging astrocyte will promote neuroprotection.