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

项目摘要/摘要 蛋白质稳态，或蛋白质稳态，对神经细胞和分子过程以及 蛋白质平衡机制中的紊乱与神经退行性蛋白聚集体有关。这个项目 将研究含伴侣激素的TCP-1(CCT)复合体在调节细胞骨架调节中的作用 在动态平衡和蛋白病理性疾病中的作用以及这些作用如何机械地影响树突状 开发和维护。这两个具体目标将：(1)考察有条件现金转移如何促进 直接和间接调节微管的复杂树枝和(2)解析 CCT如何减少体内神经致病蛋白聚集体的积累及其与基因的相互作用 用突变体紫杉素和亨廷丁保存乔木形态。CCT是一种胞质多亚单位伴侣蛋白， 新折叠蛋白、胞浆中的错误折叠蛋白和突变的聚集性蛋白常见 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。 我们首次证明了果蝇IV类多树突触中单个CCT亚单位突变体 神经元导致了树突分支的严重减少。我已经进行了进一步的实验，表明 CCT突变会导致树突状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 微管是通过直接或间接的方式存在的，目前尚不清楚。此外，初步数据显示， SkpA-F-box-cullin E3泛素连接酶组分cullin1与CCT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 细胞内稳态中树突状细胞骨架调控的相关分子途径。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。我还完成了初步研究，发现减少了 由于突变的亨廷顿蛋白和Aaxin蛋白的表达，导致树突状分支。已知CCT可与 这些突变蛋白在体外可以减轻聚集，但这种关系在体内还没有得到检验。 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化技术，如蛋白质印迹和免疫共沉淀，我将 研究CCT对体内突变蛋白聚集体的改善作用，并研究CCT如何 在动态平衡中支持树突形成的关系在以下背景下被维持或错乱 疾病。除了研究计划的目标外，我的培训目标还包括高级技术培训 将成像/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 活动/网络。我已经组建了一个由科学和技术顾问组成的专家团队， 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。

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.