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可能改善中风预后的治疗化合物。 年长的雌性。关注这一群体至关重要，因为研究表明，更年期后，女性 有更大的风险和更严重的缺血性中风。赞助商实验室之前的研究表明， 生殖衰老(无循环)(RS)雌性大鼠有更大的梗塞和更严重的感觉运动障碍 而不是年轻的雌性。此外，对RS女性进行雌激素替代会加剧中风损害。因此， 这种动物模型复制了人类人群中与中风相关的后果的关键特征， 产生了一个具有翻译潜力的模型，在这个模型中可以测试神经治疗化合物。 赞助商实验室先前的研究表明，生殖性衰老(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。这包括减少的容量 谷氨酸清除，营养因子分泌减少，炎性细胞因子释放增加， 与年轻的女性相比。此外，这些老化的星形胶质细胞还表现出组蛋白甲基化减少。 H3K4染色质区域，导致包括microRNA mir-17-92在内的几个基因转录减少 集群。最近，microRNAs作为治疗各种疾病的潜在疗法而广受欢迎， 包括缺血性中风。 这项提议的初步数据显示，mir-17-92星系团的成员miR-20a-3p 在中年女性星形胶质细胞中表达下调。此外，静脉注射miR-20a-3p模拟四个 大脑中动脉阻塞(MCAO)后数小时，脑梗塞体积缩小，感觉运动减弱 中年女性的损害，表明miR-20a-3p可能与中风有机械联系 结果。这提出了一种耐人寻味的可能性，在年轻女性中，内源性星形胶质细胞来源的miR- 20A-3P可能被“分配”到其他细胞，以改善中风预后。这一假设将在 以下目标：目标1将确定miR-20a-3p是否改变星形胶质细胞胞外成分 囊泡(EVS)和miR-20a-3p修饰的EVS将提供神经保护。星形胶质细胞已被证明 卒中后通过EVS释放神经保护因子、细胞器和RNA翻译机制。在硅胶中 分析预测miR-20a-3p可能调控炎症介质和线粒体基因。因此，miR- 20A-3P可能会极大地改变电动汽车的组成。在AIM 2中，复制缺陷型腺病毒 构建物将仅用于增加星形胶质细胞中miR-20a-3p的水平，并确定这是否 概述了静脉注射miR-20a-3p的神经保护作用。使用临床相关的中风模型，一组 行为分析和现代基因转移技术，这项提议将批判性地检查 修饰老化的星形胶质细胞将促进神经保护。

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.