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.

项目摘要在衰老的神经元中，关键错误折叠蛋白的积累成骨料是许多人的标志神经退行性疾病。例如，TDP43的病理形式被错误地定位于细胞质并积聚在额叶痴呆（FTD）和其他与阿尔茨海默氏病有关的聚集体中痴呆症（ADRD）。高度复杂的酶网络称为分子伴侣，打击了这些过程。在ADRD中，未知伴侣网络如何反对ADRD蛋白的病理形式 TDP43，FUS和TAU。研究伴侣网络的主要挑战是组合复杂性。这规范HSP70网络由54 Hsp40、12 HSP70和16 HSP110基因变体组成，创建景观为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在复杂的人蛋白质组中对不同错误折叠蛋白的特异性具有特异性。这假设被广泛接受，但由于技术局限性从未直接进行测试。此NIH K99/R00 提案概述了通过构建伴侣的第一张详尽地图直接检验该假设的计划反对ADRD相关蛋白TDP43，FUS和TAU的网络。为了实现这一目标，AIM 1将利用 Edward Barbieri博士开发的一种新的遗传技术来表达和研究所有可能的12,155 ADRD酵母模型中人类HSP40-HSP70-HSP110网络的组合。确定的伴侣因为在酵母中具有针对TDP43的活性将在AIM 2中进一步研究人类细胞和体外测定。借助人类细胞模型，Barbieri博士将研究TDP43活性伴侣对细胞质TDP43的影响聚合并评估伴侣是否还原两个HEK-293T中mRNA剪接中的本机TDP43功能细胞和神经元。使用体外生物化学，他将测量伴侣活动以预防和逆转 TDP43聚合。在AIM 3的R00阶段，Barbieri博士将应用伴侣网络屏幕进行研究 Tau聚集，他将通过包括HSP40对和小型HSP。最后，Barbieri博士将结合他在K99阶段所学的技能，以开发一个屏幕直接在ADRD的人类细胞模型中，与所有194个人类伴侣过表达的组合以研究本地上下文中的Proteostasis网络。该提案中概述的实验将共同确定关键伴侣作为ADRD的治疗靶标。 Barbieri博士将在较短的宾夕法尼亚大学的实验室，世界一流的生物化学实验室，具有专业知识和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，以提供人类的ADRD和正式培训的专业知识细胞测定。新技能将补充他目前在分子遗传学方面的专业知识，并概述了培训计划将为Barbieri博士提供必要的准备，以实现其独立职位的目标。