权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

项目总结