Modulation of neuronal atrophy in Huntington's disease

项目概要/摘要 亨廷顿病 (HD) 是一种由 CAG 三联体引起的常染色体显性神经退行性疾病 亨廷顿蛋白 (HTT) 基因外显子 1 中编码谷氨酰胺的扩展突变。突变 HTT (mHTT) 蛋白 破坏许多分子和细胞过程。 Ras 相关的 Rho GTPases 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。 p190RhoGAP 和 Rap 依赖性 RhoGAP 介导的长期 RhoA 失活 (ARAP3) 对于神经突生长至关重要。在我们使用体外 HD 纹状体细胞系和 HD 的初步研究中 我们在转基因小鼠中发现了在人类中发现的 ARAP3-RhoA 通路的显着异常 高清死后大脑。 HD 患者纹状体裂解物中的 Rho-GTPase 活性增加了 24 倍，而 ARAP3（带有 RhoGAP 结构域、锚蛋白重复序列和 PH 结构域 3 的 ArfGAP）是 RhoA 的负调节因子 显着下调。纹状体细胞中 ARAP3 的过度表达恢复了神经元的大小和功能 受到组成型活性突变体 RhoA 的影响。 AAV-shRNA ARAP3 的传递显着加剧 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-肌动蛋白应力纤维 形成和细胞骨架破坏，进而导致 HD 中的神经元萎缩和功能障碍。至 研究受损的 ARAP3-RhoA 通路是否是神经元的细胞和分子基础 由于萎缩是可逆的，我们提出了三个具体目标： 目标 1：研究 ARAP3 和 死后大脑、转基因动物模型和 HD 细胞系模型中的 RhoA 水平。我们会 确定纹状体中 ARAP3、RhoA 和细胞骨架结构的时空变化 蛋白质印迹、qPCR 和共聚焦显微镜与 3-D 重建图像分析相结合。目标 2： 确定 ARAP3 和 RhoA 通路之间的关系，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 基于 FRET 的 RhoA 生物传感器和活细胞成像，以确定 ARAP3 功能的丧失或增强如何影响 HD 纹状体细胞中的 RhoA 活性。目标 3：检查 ARAP3 和 RhoA 对神经元的体内作用 HD 小鼠模型中的萎缩和功能、运动活动和存活。我们将进行横截面 研究确定 ARAP3 功能丧失和 RhoA 功能增强对运动症状和 HD 小鼠的存活率。我们将分析纹状体中 F-肌动蛋白应力纤维的形成和 DARPP32 活性 小鼠的神经元。我们将进一步测量神经病理学变化，例如神经元大小/数量和 mHTT 聚合。我们的研究将确定神经元萎缩的新分子和细胞机制 探讨HD的发病机制并为HD的治疗提供方法。

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.