Synthetic Reconstruction of Human Chaperone Networks in Yeast Models of Neurodegeneration

项目摘要 在老化的神经元中，关键错误折叠蛋白质聚集成聚集体是许多神经元的标志。 神经退行性疾病例如，病理形式的TDP 43错误定位于细胞质 并在额颞叶痴呆（FTD）和其他阿尔茨海默病相关疾病中聚集 痴呆症（ADRD）。被称为分子伴侣的高度复杂的酶网络对抗这些过程。 在ADRD中，尚不清楚伴侣蛋白网络如何对ADRD蛋白的病理形式失效，例如 TDP 43、FUS和TAU。研究伴侣网络的一个主要挑战是组合的复杂性。的 典型的Hsp 70网络由54个Hsp 40、12个Hsp 70和16个Hsp 110基因变体组成，形成了一个景观 12,155种可能的蛋白质表达组合。Hsp 40-Hsp 70-Hsp 110蛋白的独特组合 被假设为赋予对复杂的人类蛋白质组中不同错误折叠蛋白质的特异性。这 这一假设被广泛接受，但由于技术限制，从未得到直接检验。此NIH K99/R 00 一项提案概述了一项计划，通过构建伴侣的第一个详尽地图来直接测试这一假设 针对ADRD相关蛋白TDP 43、FUS和TAU的网络。为了实现这一目标，aim 1将利用 爱德华·巴比里博士开发的一种新的遗传技术， 人Hsp 40-Hsp 70-Hsp 110网络在ADRD酵母模型中的组合。监护人确认了 因为在酵母中具有抗TDP 43的活性，将在目的2中使用人细胞和体外测定进一步研究。 在人类细胞模型中，Barbieri博士将研究TDP 43活性分子伴侣对细胞质TDP 43的影响。 在HEK-293 T细胞和HEK-293 T细胞中， 细胞和神经元。利用体外生物化学，他将测量分子伴侣的活性，以预防和逆转 TDP 43聚集。在aim 3的R 00阶段，Barbieri博士将应用伴侣网络筛选进行研究 TAU聚集，他将通过包括Hsp 40对来扩展酵母中研究的伴侣蛋白网络， 小HSP最后，Barbieri博士将联合收割机结合他在K99阶段学到的技能， 在ADRD的人细胞模型中直接组合过表达所有194种人分子伴侣，以研究 蛋白质稳态网络。总之，本提案中概述的实验将确定关键的 分子伴侣作为ADRD的治疗靶点。Barbieri博士将在短期指导下进行K99阶段 实验室在宾夕法尼亚大学，一个世界一流的生物化学实验室，在伴侣蛋白的研究专业知识 和ADRD。这是Barbieri博士获得生物化学新技能的理想培训环境。此外，委员会认为， 博士Barbieri组建了一个咨询委员会，为ADRD提供专业知识，并对人类进行正式培训。 细胞测定。新的技能将补充他目前在分子遗传学方面的专业知识， 该计划将为Barbieri博士实现独立职位的目标提供必要的准备。

Project Summary In aging neurons, the accumulation of key misfolded proteins into aggregates is a hallmark of many neurodegenerative diseases. For example, pathological forms of TDP43 become mislocalized to the cytoplasm and accumulate in aggregates in frontotemporal dementia (FTD) and other Alzheimer Disease-related Dementias (ADRD). Highly intricate networks of enzymes called molecular chaperones combat these processes. In ADRD, it is unknown how the chaperone networks fail against pathological forms of ADRD proteins such as TDP43, FUS, and TAU. A major challenge to studying chaperone networks is the combinatorial complexity. The canonical Hsp70 network consists of 54 Hsp40, 12 Hsp70, and 16 Hsp110 gene variants, creating a landscape of 12,155 possible protein expression combinations. Unique combinations of the Hsp40-Hsp70-Hsp110 proteins are hypothesized to confer specificity for different misfolded proteins in the complex human proteome. This hypothesis is widely accepted but it has never been directly tested due to technical limitations. This NIH K99/R00 proposal outlines a plan to directly test this hypothesis by building the first exhaustive map of a chaperone network against the ADRD-associated proteins TDP43, FUS, and TAU. To achieve this goal, aim 1 will leverage a new genetic technique developed by Dr. Edward Barbieri to express and study all 12,155 possible combinations of the human Hsp40-Hsp70-Hsp110 network in yeast models of ADRD. The chaperones identified as having activity against TDP43 in yeast will be further studied in aim 2 using human cells and in vitro assays. With human cell models, Dr. Barbieri will study the effect of the TDP43-active chaperones on cytoplasmic TDP43 aggregation and assess if the chaperones restore native TDP43 function in mRNA splicing in both HEK-293T cells and neurons. Using in vitro biochemistry, he will measure chaperone activity for prevention and reversal of TDP43 aggregation. During the R00 phase in aim 3 Dr. Barbieri will apply the chaperone network screen to study the TAU aggregation and he will expand the chaperone networks studied in yeast by including Hsp40 pairs and small HSPs. Lastly, Dr. Barbieri will combine the skills he learns during the K99 phase to develop a screen for combinatorially overexpressing all 194 human chaperones directly in human cell models of ADRD to study proteostasis networks in the native context. Together, the experiments outlined in this proposal will identify key chaperones as therapeutic targets for ADRD. Dr. Barbieri will perform the K99 phase mentored in the Shorter lab at the University of Pennsylvania, a world class biochemistry lab with expertise in the study of chaperones and ADRD. This is an ideal training setting for Dr. Barbieri to acquire new skills in biochemistry. Furthermore, Dr. Barbieri assembled an advisory committee to provide expertise in ADRD and formal training in the human cell assays. The new skills will complement his current expertise in molecular genetics, and his outlined training plan will provide the necessary preparation for Dr. Barbieri to progress to his goal of an independent position.