Uncovering the Roles of Chaperonin CCT in Neural Health and Disease.

揭示伴侣蛋白 CCT 在神经健康和疾病中的作用。

• 批准号: 10607652
• 负责人: Erin Lottes
• 金额: $ 4.13万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-07 至 2025-02-06
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607652
• 关键词: 4D Imaging; Actins; Afferent Neurons; Atrophic; Attenuated; Biochemical; Biological; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Complex; Coupled; Cullin Proteins; Cytoskeleton; Cytosol; Data; Dendrites; Development; Disease; Drosophila genus; Drosophila melanogaster; Educational process of instructing; Ensure; Environment; Exhibits; F Box Domain; Genetic; Goals; Growth; Health; Homeostasis; Human; Huntington Disease; Huntington gene; Image; Imaging Techniques; In Vitro; Individual; Institution; Investigation; Knowledge; Larva; Lead; Link; Machado-Joseph Disease; Maintenance; Mediating; Mentors; Microscopy; Microtubules; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Monitor; Morphology; Movement; Mutation; Nerve Degeneration; Nervous System; Nervous System Physiology; Neurodegenerative Disorders; Neurons; Neuropathy; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Pharmacology Study; Phenotype; Pilot Projects; Process; Protein Biochemistry; Proteins; Publishing; RNA Interference; Regulation; Reporter Genes; Research; Research Infrastructure; Resolution; Ribosomal Protein S6 Kinase; Role; Shapes; Signal Pathway; Signal Transduction; Spinocerebellar Ataxias; Stable Populations; System; Techniques; Testing; Time; Training; Transgenic Organisms; Tubulin; Visualization; Western Blotting; Work; career development; chaperonin; experimental study; in vivo; knock-down; live cell imaging; misfolded protein; mutant; neural; neurogenetics; novel; overexpression; polypeptide; postmitotic; preservation; protein aggregation; protein function; proteostasis; reconstruction; response; superresolution microscopy; ubiquitin-protein ligase; ultra high resolution

Project Summary/Abstract Protein homeostasis, or proteostasis, is critical to neuronal cellular and molecular processes and derangements in proteostasis machinery have been linked to neurodegenerative protein aggregates. This project will investigate roles of the Chaperonin-Containing TCP-1 (CCT) complex in regulating cytoskeletal modulation in both homeostatic and proteinopathic disease conditions and how those roles mechanistically impact dendrite development and maintenance. The two specific aims will (1) examine how CCT facilitates the formation of complex dendritic arbors through secondary regulation of microtubules both directly and indirect and (2) parse how CCT attenuates the accumulation of neuropathogenic protein aggregates in vivo and genetically interacts with mutant Ataxin and Huntingtin to preserve arbor morphology. CCT is a cytosolic multi-subunit chaperone that folds de novo proteins, misfolded proteins in the cytosol, and mutant aggregate-prone proteins commonly associated with neurodegenerative diseases such as Huntington’s Disease and Spinocerebellar Ataxia (SCA). We were first to demonstrate that individual CCT subunit mutants in Drosophila Class IV multidendritic sensory neurons caused severe reductions in dendritic branching. I have carried out further experiments showing that CCT mutants results in underlying changes to the dendritic cytoskeleton, especially disrupting microtubules. While it is well-established that CCT folds tubulin, whether and how CCT is influencing the assembly of microtubules through direct or indirect means is unknown. Furthermore, preliminary data reveals a putative relationship between Cullin1, a component of the SkpA-F-box-Cullin E3 ubiquitin ligase, and CCT in the regulation of microtubules. A common target of Cullin1 and CCT is TORC1, and thus, I propose to investigate the associated molecular pathway in the regulation of the dendritic cytoskeleton in cellular homeostasis. For these analyses, I will leverage our expertise in neurogenetics, phenotypic analyses, time-lapse 4D imaging, neuromorphometrics and drug pharmacology studies. I have also completed pilot studies that reveal reductions in dendritic branching due to expression of mutant Huntingtin and Ataxin proteins. CCT is known to interact with these mutant proteins in vitro and mitigate aggregation, but the relationship has not been examined in vivo in neurons. Using advanced imaging techniques including time-lapse imaging, expansion and super-resolution microscopy as well as traditional biochemical techniques like Western blot and co-immunoprecipitation, I will investigate the ameliorative effects of CCT on mutant protein aggregates in vivo and examine how the relationships that support dendritic formation in homeostasis are maintained or deranged in the context of disease. Beyond the goals of the research plan, my training goals include new technical training in advanced imaging/microscopy and protein biochemistry coupled to mentoring/teaching activities and career development activities/networking. I have assembled an expert team of scientific and technical advisors which coupled to institutional environment and research infrastructure will support and advance my overall training goals.

项目概要/摘要 蛋白质稳态或蛋白质稳态对于神经元细胞和分子过程至关重要 蛋白质稳态机制的紊乱与神经退行性蛋白质聚集有关。 将研究含伴侣蛋白的 TCP-1 (CCT) 复合物在调节细胞骨架调节中的作用 在稳态和蛋白质疾病条件下以及这些作用如何机械地影响树突 开发和维护的两个具体目标将（1）研究 CCT 如何促进形成。 通过直接和间接微管的二级调节来形成复杂的树突状乔木，以及（2）解析 CCT 如何减弱体内神经病原蛋白聚集体的积累以及遗传相互作用 与突变体 Ataxin 和 Huntingtin 一起保持乔木形态 CCT 是一种胞质多亚基伴侣。 从头折叠蛋白质、细胞质中错误折叠的蛋白质以及常见的易聚集突变蛋白质 与亨廷顿病和脊髓小脑性共济失调（SCA）等神经退行性疾病有关。 我们首先证明了果蝇 IV 类多树突感觉中的单个 CCT 亚基突变体 神经元导致树突分支严重减少，我进行了进一步的实验，表明这一点。 CCT 突变体导致树突细胞骨架发生根本性变化，尤其是破坏微管。 虽然 CCT 折叠微管蛋白已是公认的事实，但 CCT 是否以及如何影响微管蛋白的组装 此外，初步数据揭示了一个假设。 Cullin1（SkpA-F-box-Cullin E3 泛素连接酶的一个组成部分）与 CCT 之间的关系 Cullin1 和 CCT 的共同靶点是 TORC1，因此我建议进行研究。 细胞稳态中树突状细胞骨架调节的相关分子途径。 在这些分析中，我将利用我们在神经遗传学、表型分析、延时 4D 成像方面的专业知识， 我还完成了神经形态计量学和药物药理学研究，揭示了减少的情况。 已知突变亨廷顿蛋白和 Ataxin 蛋白的表达会导致树突分支。 这些突变蛋白在体外可以减轻聚集，但这种关系尚未在体内得到检验 使用先进的成像技术，包括延时成像、扩展和超分辨率。 显微镜以及传统的生化技术，如蛋白质印迹和免疫共沉淀，我会 研究 CCT 对体内突变蛋白聚集体的改善作用，并研究如何 在稳态中支持树突形成的关系在以下情况下得以维持或紊乱 除了研究计划的目标之外，我的培训目标还包括高级新技术培训。 成像/显微镜和蛋白质生物化学与指导/教学活动和职业发展相结合 我组建了一个由科学和技术顾问组成的专家团队，并与他们合作。 制度环境和研究基础设施将支持和推进我的总体培训目标。