Modulation of neuronal atrophy in Huntington's disease

项目总结/摘要 亨廷顿病（Huntington's disease，HD）是一种由CAG三联体引起的常染色体显性遗传性神经退行性疾病 亨廷顿蛋白（HTT）基因外显子1中编码谷氨酰胺的扩增突变。突变HTT（mHTT）蛋白 破坏了许多分子和细胞过程。Ras相关的Rho GTP酶是分子开关 调节许多过程，包括细胞增殖、分化、迁移、转录和 肌动蛋白动力学p190 RhoGAP和Rap-dependent RhoGAP介导的RhoA长期失活 （ARAP 3）是神经突生长所必需的。在我们的初步研究中，使用体外HD纹状体细胞系和HD 在转基因小鼠中，我们发现了我们在人类中发现的ARAP 3-RhoA通路的显著异常， 高清死后大脑。在HD患者的纹状体裂解物中，Rho-GT3活性增加了24倍， ARAP 3（ArfGAP with RhoGAP Domain，ankyrin repeat and PH Domain 3）是RhoA的负调控因子， 大幅下调。纹状体细胞中ARAP 3的过表达恢复了神经元的大小和功能， 受到了一种活性突变体RhoA的影响AAV-shRNA ARAP 3的递送显著加剧了 YAC 128小鼠纹状体中的神经元萎缩。基于这些发现，我们提出了一个新的假设 ARAP 3功能和RhoA活性的改变可能导致可逆的F-肌动蛋白应力纤维， 形成和细胞骨架破坏，这又导致HD中的神经元萎缩和功能障碍。到 研究受损的ARAP 3-RhoA通路是否是神经元损伤的细胞和分子基础。 我们提出了三个具体目标：目标1：研究ARAP 3的改变， RhoA水平在死后的大脑，转基因动物模型，和细胞系模型的HD。我们将 确定ARAP 3，RhoA和纹状体细胞骨架结构的时空变化， 蛋白质印迹、qPCR和共聚焦显微镜结合3-D重建图像分析。目标2： 确定ARAP 3和RhoA通路之间的关系，并确定分子和细胞 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）的RhoA生物传感器和活细胞成像，以确定ARAP 3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：研究ARAP 3和RhoA对神经元凋亡的影响。 HD小鼠模型中的萎缩和功能、运动活动和存活率。我们将进行横向的 确定运动症状中ARAP 3功能丧失和RhoA功能获得的研究， HD小鼠的存活率。我们将分析纹状体中F-actin应力纤维的形成和DARPP 32的活性， 小鼠的神经元。我们将进一步测量神经病理学变化，如神经元大小/数量和mHTT 聚合来我们的研究将确定神经元萎缩的新的分子和细胞机制， HD的发病机制，并提供HD的治疗方法。

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.