Mitochondrial Porin in Bioenergetic Defects in Huntingtons Disease

亨廷顿病生物能缺陷中的线粒体孔蛋白

• 批准号: 8416946
• 负责人: Nickolay Brustovetsky
• 金额: $ 32.68万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8416946
• 关键词: Address; Affect; American; Applications Grants; Binding; Biochemical; Bioenergetics; Biological Assay; Brain; Brain Injuries; Cells; Chimeric Proteins; Co-Immunoprecipitations; Confocal Microscopy; Corpus striatum structure; Cytosol; Data; Defect; Development; Emotional; Face; Family; Foundations; Future; Gene Mutation; Genetic Risk; Glutathione S-Transferase; Goals; Huntington Disease; Individual; Inherited; Injury; Inner mitochondrial membrane; Knock-in Mouse; Knowledge; Laboratories; Lasers; Lead; Life; Link; Literature; Membrane Potentials; Methodology; Mitochondria; Molecular; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurologic; Neurons; Outer Mitochondrial Membrane; Oxidative Stress; Patch-Clamp Techniques; Pathogenesis; Pathway interactions; Patients; Play; Predisposition; Proteins; Research; Respiration; Role; Scheme; Societies; Solid; Superoxides; Surface Plasmon Resonance; Synapses; Testing; Therapeutic; Three-Dimensional Image; Tissues; Transgenic Organisms; Triplet Multiple Birth; United States; Voltage-Dependent Anion Channel; Wild Type Mouse; base; design; fighting; human Huntingtin protein; image processing; improved; mitochondrial dysfunction; mitochondrial membrane; mouse model; mutant; neuron loss; neuronal survival; neurotoxicity; novel; novel therapeutics; patch clamp; polyglutamine; porin; programs; proteoliposomes; reconstitution; respiratory; therapeutic target; uptake

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.

描述（由申请人提供）：亨廷顿病（HD）是一种遗传性神经退行性疾病，与编码亨廷顿蛋白（一种在多种组织中表达的 350 kDa 蛋白）中的多谷氨酰胺结构域的 CAG 三联体异常扩增相关。 Htt 基因突变与导致 HD 神经系统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 发病机制中的一个致病因素。尽管进行了大量研究，HD 线粒体功能障碍的机制仍不清楚。该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表达 mHtt 的神经元中异常线粒体碎片中的作用。在拟议的研究中，我们将测试一个新的假设，即 mHtt 与 VDAC 结合并抑制代谢物跨 OMM 的运输，导致线粒体功能障碍、Ca2+ 处理缺陷、线粒体氧化应激和线粒体裂变增强。我们将解决以下问题：(1) mHtt 是否会通过绑定到通道来减少 VDAC 传输活动？ (2) VDAC 抑制是否会导致暴露于 mHtt 的线粒体中的呼吸抑制、去极化和超氧阴离子 O2¿ - 的积累？ (3) mHtt 是否会通过抑制 VDAC 来增加对线粒体 Ca2+ 诱导损伤的敏感性并降低 Ca2+ 摄取能力？ (4) VDAC 抑制是否会导致表达 mHtt 的培养神经元中的线粒体氧化应激和线粒体裂变增强？为了回答这些问题，我们将使用 VDAC 重建的巨型蛋白脂质体，结合电生理膜片钳技术和谷胱甘肽-S-转移酶 (GST)-polyQ 融合蛋白。我们将使用从野生型小鼠以及转基因和敲入 HD 小鼠模型中分离的突触和非突触纯化脑线粒体，并结合现代药理学、生化和生物能量方法。为了分析线粒体动力学，我们将使用活细胞激光转盘共聚焦显微镜，然后对来自野生型和 HD 小鼠的培养纹状体和皮质神经元进行复杂的图像处理和定量 3D 图像渲染，其中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 抑制在线粒体功能障碍、Ca2+ 处理缺陷、线粒体氧化应激和暴露于 mHtt 的线粒体中增强裂变中的作用。因此，我们的研究将为HD线粒体功能障碍的分子机制提供新颖、重要的知识，并为未来的HD研究搭建平台。这将为创造旨在改善 HD 线粒体功能和神经元存活的治疗方法奠定坚实的基础。最重要的是，这将极大地有助于开发新的治疗策略，以减轻HD的神经功能缺损，并显着减少HD患者的痛苦，提高他们的生活质量，并减轻家庭和整个社会的情感和经济负担。