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.

项目总结/摘要 蛋白质稳态或蛋白质稳态对神经元细胞和分子过程至关重要， 蛋白质稳定机制的紊乱与神经变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 在稳态和蛋白质病变疾病中的作用，以及这些作用如何机械地影响树突 开发和维护。这两个具体目标将（1）研究有条件现金援助如何促进 通过直接和间接的微管的次级调节复杂的树突状乔木和（2）解析 CCT如何减弱体内神经致病性蛋白聚集体的积累和遗传相互作用 用突变的共济失调蛋白和亨廷顿蛋白来保持乔木形态。CCT是一种胞质多亚基伴侣， 新折叠蛋白质、胞质中的错误折叠蛋白质和突变的聚集倾向蛋白质通常 与神经退行性疾病如亨廷顿病和脊髓小脑共济失调（SCA）相关。 我们首次证明了果蝇IV类多树突感觉神经元中的单个CCT亚基突变体 神经元引起严重减少树突分支。我进行了进一步的实验， CCT突变体导致树突细胞骨架的潜在变化，特别是破坏微管。 虽然CCT折叠微管蛋白是公认的，但CCT是否以及如何影响微管蛋白的组装， 微管通过直接或间接的手段是未知的。此外，初步数据显示， Cullin 1，SkpA-F-box-Cullin E3泛素连接酶的一个组分，和CCT之间的关系， 微管的调节。Cullin 1和CCT的共同目标是TORC 1，因此，我建议研究 树突状细胞骨架在细胞内稳态调节中的相关分子通路。为 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， 神经形态计量学和药物药理学研究。我还完成了试点研究， 在树突状分支由于突变亨廷顿蛋白和共济失调蛋白的表达。已知CCT与 这些突变蛋白在体外和减轻聚集，但这种关系还没有在体内检查， 神经元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物化学技术，如蛋白质印迹和免疫共沉淀，我将 研究CCT对体内突变蛋白质聚集体的改善作用，并检查CCT如何影响突变蛋白质聚集体。 支持树突形成的关系在体内平衡中维持或紊乱， 疾病除了研究计划的目标之外，我的培训目标还包括先进的新技术培训。 成像/显微镜和蛋白质生物化学结合辅导/教学活动和职业发展 活动/网络。我召集了一个科学和技术顾问专家小组， 制度环境和研究基础设施将支持和促进我的总体培训目标。