Synthetic Reconstruction of Human Chaperone Networks in Yeast Models of Neurodegeneration

神经退行性酵母模型中人类伴侣网络的综合重建

基本信息

批准号：
10591799
负责人：
Edward Matthew Barbieri
金额：
$ 11.53万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-15 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10591799
关键词：
Advisory Committees Age Aging Alzheimer&apos s disease related dementia Biochemistry Biological Assay Brain Diseases CRISPR-mediated transcriptional activation Cell model Cells Cellular Assay Collaborations Complement Complex Cytoplasm Engineering Enzymes Eukaryota Flow Cytometry Frontotemporal Dementia Funding Future Genes Genetic Genetic Techniques Goals Heat shock proteins Human In Vitro Individual Learning Skill Libraries Maps Measures Mentors Messenger RNA Modeling Molecular Chaperones Molecular Genetics Mutation Nerve Degeneration Neurodegenerative Disorders Neurons Organism Pathologic Pathology Pennsylvania Phase Phenotype Population Positioning Attribute Postdoctoral Fellow Preparation Prevention Process Proteins Proteome RNA Splicing Reporter Genes Research Specificity Stress System TDP-43 aggregation Testing Toxic effect Training Translating Triplet Multiple Birth United States National Institutes of Health Universities Work Yeast Model System Yeasts combat combinatorial experimental study genetic approach genetic technology genetic variant heat-shock proteins 110 in vitro Assay insight misfolded protein mutant neuronal survival novel strategies overexpression post-doctoral training programs protein TDP-43 protein expression protein misfolding protein purification proteostasis reconstruction screening skill acquisition skills targeted treatment tau Proteins tau aggregation therapeutic target tool

项目摘要

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.

项目概要在衰老的神经元中，关键的错误折叠蛋白质积累成聚集体是许多神经元的标志。神经退行性疾病。例如，TDP43 的病理形式错误定位到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积累痴呆症（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，尚不清楚分子伴侣网络如何无法对抗 ADRD 蛋白的病理形式，例如 TDP43、FUS 和 TAU。研究伴侣网络的一个主要挑战是组合复杂性。这典型的 Hsp70 网络由 54 个 Hsp40、12 个 Hsp70 和 16 个 Hsp110 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。 Hsp40-Hsp70-Hsp110 蛋白的独特组合假设赋予复杂人类蛋白质组中不同错误折叠蛋白质的特异性。这假设已被广泛接受，但由于技术限制，它从未被直接检验过。这个NIH K99/R00 提案概述了通过构建第一个详尽的伴侣地图来直接检验这一假设的计划网络针对 ADRD 相关蛋白 TDP43、FUS 和 TAU。为了实现这一目标，目标 1 将利用 Edward Barbieri 博士开发的一种新基因技术可表达和研究所有 12,155 种可能的基因 ADRD 酵母模型中人类 Hsp40-Hsp70-Hsp110 网络的组合。确定的伴侣由于其在酵母中具有针对 TDP43 的活性，将在目标 2 中使用人体细胞和体外测定进一步研究。 Barbieri 博士将通过人体细胞模型研究 TDP43 活性伴侣对细胞质 TDP43 的影响聚集并评估分子伴侣是否恢复 HEK-293T 中 mRNA 剪接中的天然 TDP43 功能细胞和神经元。利用体外生物化学，他将测量伴侣活性以预防和逆转 TDP43 聚合。在目标 3 的 R00 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 Hsp40 对来扩展在酵母中研究的伴侣网络小热休克蛋白。最后，Barbieri 博士将结合他在 K99 阶段学到的技能，开发一个屏幕直接在 ADRD 人类细胞模型中组合过表达所有 194 个人类伴侣进行研究天然背景下的蛋白质稳态网络。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 博士将执行 Shorter 指导下的 K99 阶段宾夕法尼亚大学实验室，世界一流的生物化学实验室，在伴侣研究方面拥有专业知识和ADRD。这是 Barbieri 博士获得生物化学新技能的理想培训环境。此外， Barbieri 博士组建了一个咨询委员会，提供 ADRD 方面的专业知识和人类的正式培训。细胞测定。新技能将补充他目前在分子遗传学方面的专业知识以及他概述的培训该计划将为 Barbieri 博士实现其独立职位的目标提供必要的准备。