Mitochondrial Porin in Bioenergetic Defects in Huntingtons Disease

描述(申请人提供)：亨廷顿病(HD)是一种遗传性神经退行性疾病，与CAG三联体的异常扩张有关，该三联体编码亨廷顿蛋白的多谷氨酰胺结构域，亨廷顿蛋白是一种在各种组织中表达的350 kDa蛋白质。Htt基因突变与导致HD神经功能异常的神经元丢失之间的机制联系尚未确定，但线粒体功能障碍已成为HD发病机制中的一个原因。尽管进行了广泛的研究，但HD患者线粒体功能障碍的机制仍不清楚。本研究的总体目标是阐明线粒体孔蛋白，也称为电压依赖性阴离子通道(VDAC)，在突变亨廷顿蛋白(MHTT)诱导的线粒体功能障碍和mHTT表达神经元中的异常线粒体碎裂中的作用。在拟议的研究中，我们将检验一种新的假说，即mHTT与VDAC结合并抑制代谢物通过OMM的运输，导致线粒体功能障碍、钙处理缺陷、线粒体氧化应激和线粒体分裂增加。我们将解决以下问题：(1)mHTT是否通过与通道结合而降低VDAC的转运活性？(2)mHTT对VDAC的抑制是否与呼吸抑制、去极化和线粒体超氧阴离子O2-的积累有关？(3)mHTT是否通过抑制VDAC而导致线粒体对钙离子损伤的敏感性增加和钙摄取能力降低？(4)在表达mHTT的培养神经元中，VDAC抑制是否导致线粒体氧化应激和线粒体分裂增加？为了回答这些问题，我们将使用VDAC重组的巨型蛋白脂质体，结合电生理膜片钳技术和谷胱甘肽-S-转移酶-聚Q融合蛋白。我们将使用从野生型小鼠和转基因和敲入HD小鼠模型中分离的突触和非突触纯化的脑线粒体，并结合现代药理学、生化和生物能量学方法。为了分析线粒体动力学，我们将使用活细胞、激光旋转圆盘共聚焦显微镜，然后使用复杂的图像处理和定量3D图像渲染，应用于培养的纹状体和皮质神经元，这些神经元来自野生型和HD小鼠，线粒体通过线粒体靶向荧光蛋白可视化。在本研究计划结束时，我们将确定VDAC抑制在mHTT暴露的线粒体功能障碍、钙处理缺陷、线粒体氧化应激和线粒体分裂增强中的作用。因此，我们的研究将为HD患者线粒体功能障碍的分子机制提供新的、至关重要的知识，并为以后的HD研究搭建一个平台。这将为创造旨在改善线粒体功能和HD神经元存活的治疗方法奠定坚实的基础。最重要的是，这将极大地帮助发展新的治疗策略，以减轻HD患者的神经功能障碍，显著减少HD患者的痛苦，改善他们的生活质量，并减轻家庭和整个社会的情感和经济负担。

DESCRIPTION (provided by applicant): Huntington's Disease (HD) is an inherited, neurodegenerative disorder associated with the abnormal expansion of CAG triplet that encodes a polyglutamine domain in huntingtin, a 350 kDa protein expressed in various tissues. A mechanistic link between Htt gene mutation and neuronal loss leading to neurological abnormalities in HD has not yet been determined, but mitochondrial dysfunction has emerged as a causal factor involved in HD pathogenesis. Despite extensive studies, the mechanisms of mitochondrial dysfunction in HD remain unclear. The overall objectives of the proposed study are to clarify the role of mitochondrial porin, also known as voltage-dependent anion channel (VDAC), in mutant huntingtin (mHtt)-induced mitochondrial dysfunction and abnormal mitochondrial fragmentation in mHtt-expressing neurons. In the proposed study, we will test a novel hypothesis that mHtt binds to VDAC and inhibits metabolite transport across the OMM, leading to mitochondrial dysfunction, Ca2+ handling defects, mitochondrial oxidative stress, and augmented mitochondrial fission. We will address the following questions: (1) Does mHtt diminish VDAC transport activity by binding to the channel? (2) Is VDAC inhibition accountable for respiratory suppression, depolarization, and accumulation of superoxide anion O2¿ - in mitochondria exposed to mHtt? (3) Does mHtt result in increased susceptibility to mitochondrial Ca2+-induced injury and decreased Ca2+ uptake capacity by inhibiting VDAC? (4) Does VDAC inhibition lead to mitochondrial oxidative stress and augmented mitochondrial fission in cultured neurons expressing mHtt? To answer these questions we will use VDAC-reconstituted giant proteoliposomes in conjunction with electrophysiological patch-clamp technique and glutathione-S-transferase (GST)-polyQ fusion proteins. We will use synaptic and non-synaptic purified brain mitochondria isolated from wild-type mice and transgenic and knock-in HD mouse models in combination with modern pharmacological, biochemical, and bioenergetic methodologies. To analyze mitochondrial dynamics, we will use live-cell, laser spinning-disk confocal microscopy followed by sophisticated image processing and quantitative 3D image rendering applied to cultured striatal and cortical neurons derived from wild-type and HD mice with mitochondria visualized by mitochondrially targeted fluorescent proteins. At the conclusion of this research program, we will establish the role of VDAC inhibition in mitochondrial dysfunction, Ca2+ handling defects, mitochondrial oxidative stress, and augmented fission in mitochondria exposed to mHtt. Thus, our study will provide novel, vital knowledge about molecular mechanisms of mitochondrial dysfunction in HD and build a platform for future HD research. This will lay a solid foundation for creating treatments aimed at improving mitochondrial functioning and neuronal survival in HD. Most importantly, this will immensely help in the development of new therapeutic strategies to alleviate neurological deficits in HD and significantly diminish suffering of HD patients, improve quality of their life, and lessen the emotional and financial burden on the family and the whole society.