Modulation of neuronal atrophy in Huntington's disease

项目摘要/摘要 亨廷顿病(HD)是一种常染色体显性遗传性神经退行性疾病，由CAG三胞胎引起 编码亨廷顿(Huntingtin，HTT)基因第一外显子谷氨酰胺的扩张性突变。突变型HTT(MHTT)蛋白 扰乱了许多分子和细胞过程。RAS相关的Rho GTP酶是分子开关 它调节一系列过程，包括细胞增殖、分化、迁移、转录和 肌动蛋白动力学。P190RhoGAP和Rap依赖的RhoGAP介导的RhoA长期失活 (ARAP3)是神经突起生长所必需的。在我们使用体外HD纹状体细胞系和HD进行的初步研究中 在转基因小鼠身上，我们发现了在人类中发现的ARAP3-RhoA途径的惊人异常 高清尸检大脑。HD患者纹状体裂解物中Rho-GTP酶活性增加24倍，而 RhoA的负调控因子ARAP3(ArfGAP具有RhoGAP结构域、Ankyrin Repeat和PH结构域3) 大幅下调了监管。纹状体细胞中ARAP3的过表达恢复了神经元的大小和功能 都受到一种结构性活性突变体RhoA的影响。AAV-shRNA ARAP3的交付显著恶化 YAC128小鼠纹状体神经元萎缩。基于这些发现，我们提出了一个新的假设 改变ARAP3功能和RhoA活性导致潜在可逆的F-肌动蛋白应激纤维 形成和细胞骨架的破坏，进而导致HD的神经元萎缩和功能障碍。至 研究ARAP3-RhoA通路受损是否是神经细胞和分子基础的基础 我们提出了三个具体目标：目标1：研究ARAP3和ARAP3的变化 RhoA在死后脑、转基因动物模型和HD细胞系模型中的水平。我们会 测定纹状体ARAP3、RhoA和细胞骨架结构的时空变化 免疫印迹、定量聚合酶链式反应、共聚焦显微镜结合三维重建图像分析。目标2：实现 确定ARAP3与RhoA途径的关系，并鉴定分子和细胞 HD中神经元萎缩的机制。我们将使用时间分辨荧光共振能量转移 (FRET)基于RhoA生物传感器和活细胞成像，以确定ARAP3功能的丧失或获得如何影响 HD纹状体细胞中的RhoA活性。目的3：检测ARAP3和RhoA对神经细胞的体内作用 HD小鼠模型中的萎缩和功能、运动活动和存活率。我们将执行横截面 ARAP3功能丧失和RhoA功能获得对运动症状和运动功能影响的研究 HD小鼠的存活率。我们将分析纹状体中F-肌动蛋白应激纤维的形成和DARPP32的活性 小鼠的神经元。我们将进一步测量神经病理变化，如神经元大小/数量和mHTT 聚合。我们的研究将确定新的分子和细胞机制的神经元萎缩。 为HD的发病机制研究提供了一条新的治疗途径。

Project Summary/Abstract Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disease caused by a CAG triplet expansion mutation coding for glutamine in exon 1 of the Huntingtin (HTT) gene. Mutant HTT (mHTT) protein disrupts a number of molecular and cellular processes. The Ras-related Rho GTPases are molecular switches that regulate a number of processes, including cell proliferation, differentiation, migration, transcription, and actin dynamics. Long-term RhoA inactivation mediated by p190RhoGAP and a Rap-dependent RhoGAP (ARAP3) is essential for neurite outgrowth. In our preliminary studies using in vitro HD striatal cell lines and HD transgenic mice, we discovered striking abnormalities of the ARAP3-RhoA pathway that we found in human HD postmortem brain. Rho-GTPase activity was increased 24-fold in the striatal lysates of HD patients while ARAP3 (ArfGAP with RhoGAP Domain, ankyrin repeat and PH Domain 3), a negative regulator of RhoA, was significantly down regulated. ARAP3 overexpression in striatal cells restored neuronal size and function that were affected by a constitutively active mutant RhoA. Delivery of AAV-shRNA ARAP3 significantly exacerbated neuronal atrophy in the striatum of YAC128 mice. Based on these findings we propose a novel hypothesis that altered ARAP3 function and RhoA activity cause potentially reversible F-actin stress fiber formation and cytoskeletal disruption which in turn lead to neuronal atrophy and dysfunction in HD. To investigate whether impaired ARAP3-RhoA pathway underlies the cellular and molecular basis of neuronal atrophy that is reversible, we propose three specific aims: Aim 1: To investigate alteration of ARAP3 and RhoA levels in postmortem brains, transgenic animal models, and cell line models of HD. We will determine the spatiotemporal change of ARAP3, RhoA, and cytoskeleton structures in the striatum by using Western blot, qPCR, and confocal microscopy combined with 3-D reconstruction image analysis. Aim 2: To determine the relationship between ARAP3 and the RhoA pathway, and identify molecular and cellular mechanisms of neuronal atrophy in HD. We will use time-resolved fluorescence resonance energy transfer (FRET)-based RhoA biosensor and live cell imaging to identify how loss or gain of ARAP3 function affects the RhoA activity in HD striatal cells. Aim 3: To examine the in vivo effects of ARAP3 and RhoA on neuronal atrophy and function, motor activity, and survival in HD mouse models. We will perform cross sectional studies to determine the loss of ARAP3 function and the gain of RhoA function on motor symptoms and survival rates in HD mice. We will analyze F-actin stress fiber formation and DARPP32 activity in the striatal neurons of mice. We will further measure neuropathological changes such as neuronal size/number and mHTT aggregation. Our studies will identify novel molecular and cellular mechanisms of neuronal atrophy in the pathogenesis of HD and provide a therapeutic approach for HD.