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.

项目摘要/摘要 蛋白稳态或蛋白抑制作用对于神经元细胞和分子过程至关重要 蛋白质机械中的进化与神经退行性蛋白质聚集体有关。这个项目 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 在稳态和蛋白质恶性疾病状况以及这些角色如何机械影响树突状态下 开发和维护。这两个具体目标将（1）检查CCT如何促进形成 通过直接和间接的微管的二级调节和（2）解析，复杂的树突状乔木。 CCT如何减弱在体内和遗传相互作用的神经病性蛋白质聚集体的积累 用突变的共生蛋白和亨廷顿来保存乔木形态。 CCT是一种胞质的多含量链酮 从头蛋白质折叠，细胞质中的错误折叠蛋白和突变骨料蛋白通常是蛋白 与神经退行性疾病（如亨廷顿氏病和脊髓脑性共济失调（SCA））相关。 我们首先证明了果蝇IV类多发性感觉的单个CCT亚基突变体 神经元导致树突分支严重降低。我进行了进一步的实验，表明 CCT突变体导致树突状细胞骨架的基本变化，尤其是破坏微管的变化。 虽然CCT折叠微管蛋白的确定性，但CCT是否影响了CCT的组装 通过直接或间接均值的微管是未知的。此外，初步数据揭示了一个假定的 Cullin1（Skpa-f-box-cullin e3 ubiquitin连接酶的组件）与CCT中的CULLIN1之间的关系 微管的调节。 Cullin1和CCT的一个共同目标是TORC1，因此，我建议调查 在细胞稳态中树突状细胞骨架调节中的相关分子途径。为了 这些分析，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， 神经形态测量和药物药理学研究。我还完成了揭示减少的试点研究 在树突状分支中，由于突变体狩猎蛋白和共生蛋白蛋白的表达。 CCT已知与 这些突变蛋白在体外并减轻聚集，但在体内尚未在体内检查这种关系 神经元。使用高级成像技术，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，例如Western印迹和共免疫沉淀，我将 研究CCT对体内突变蛋白聚集体的改善作用，并检查如何如何 在体内平衡中支持树突形成的关系的关系是在 疾病。除了研究计划的目标之外，我的培训目标还包括高级的新技术培训 成像/显微镜和蛋白质生物化学与心理/教学活动和职业发展相连 活动/网络。我组建了一个科学和技术顾问的专家团队，该团队与 机构环境和研究基础设施将支持和推进我的整体培训目标。