Exploring the toxicity of aggregates associated with protein-misfolding diseases

探索与蛋白质错误折叠疾病相关的聚集体的毒性

基本信息

批准号：
9324268
负责人：
SUSAN W LIEBMAN
金额：
$ 37.16万
依托单位：
UNIVERSITY OF NEVADA RENO
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-08-01 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9324268
关键词：
Address Affect Alzheimer&apos s Disease Amyloid beta-Protein Amyotrophic Lateral Sclerosis Autophagocytosis Autopsy Biological Models Cell Line Cell model Cells Creutzfeldt-Jakob Syndrome Cytoplasmic Granules Detection Disease Disease Progression Fiber Frontotemporal Dementia Gene Targeting Generations Genetic Genetic Screening Genome Goals Grant Heritability Human Huntington Disease Huntington gene In Vitro Lead Limb-Girdle Muscular Dystrophies Mammalian Cell Mediating Methods Modeling Molecular Chaperones Molecular Conformation Mutation Nerve Degeneration Neurodegenerative Disorders Neurons Parkinson Disease Pathogenicity Pathology Pathway interactions Patients PrP Prions Property Protein Structure Initiative Proteins Resistance Risk Factors Spinocerebellar Ataxias Stress Susceptibility Gene Symptoms System Testing Time Tissues Titrations Toxic effect Toxicity Tests Translating Ubiquitin Variant Work Yeast Model System Yeasts alpha synuclein brain tissue cDNA Library design disorder risk experimental study high throughput screening human disease insight multicatalytic endopeptidase complex mutation screening neuroblastoma cell novel overexpression personalized medicine polyglutamine prevent protein TDP-43 protein aggregate protein misfolding public health relevance sarkosyl superoxide dismutase 1 tau Proteins therapeutic target trafficking yeast prion yeast protein

项目摘要

DESCRIPTION (provided by applicant): Many neurodegenerative conditions including Alzheimer's, Parkinson's, prion and Huntington's diseases are associated with specific protein aggregates that form as a result of protein misfolding. To prevent or treat these conditions we must understand what causes the proteins to aggregate, and how this is associated with pathology. These questions will be addressed using yeast, neuroblastoma cells and primary cortical neurons expressing FUS and TDP-43 as cellular models of Amyotrophic lateral sclerosis and Fronto-temporal dementia. The conservation in cells from yeast to human neurons of cellular pathways such as protein refolding, the ubiquitin proteasome system, secretion, vesicular trafficking, and autophagy, has allowed yeast with its powerful experimental toolbox, short generation time, and well-characterized genome, to address aspects of neurodegenerative disease that involve these fundamental systems. This has provided new insight into protein misfolding neurodegenerative disease. Many human proteins that aggregate and are associated with neurodegenerative disease, also aggregate and are toxic when expressed in yeast. Modifiers of this toxicity identified by genetic screens in yeast have been shown, remarkably, to be new or previously known human disease risk factors. In aim I, newly designed high-throughput screens in yeast will identify novel genetic modifiers that enhance or suppress toxicity of TDP-43 or FUS. Homologs of these modifiers will then be tested for similar effects in neuroblastoma cells and primary neurons and screened for mutations in patients. Aim I also explores the mechanisms of action of known modifiers by: determining which domains of the Hsp40 chaperone Sis1 are required in order for Sis1 overexpression to rescue cells from FUS or TDP-43 toxicity; testing the hypothesis that over- expression of ATXN2, a stress granule polyQ expansion protein, enhances TDP-43 toxicity by increasing TDP- 43's titration of Sis1, thereby reducing Sis1 mediated delivery of ubiquitinated proteins to the proteasome; testing if overexpression of the Hsp104 chaperone, reduces toxicity by trimming aggregates from their ends via chaperone imbalance; determining if TDP-43 or FUS aggregates are toxic because they co-aggregate with essential proteins thereby inactivating them. This aim has the potential to identify new human disease susceptibility genes and therapeutic targets. The goal of aim II is to establish and characterize different heritable TDP-43 con- formational variants in yeast and neuroblastoma cells and to test their pathogenic effects on primary cortical neurons. This aim builds on our new discovery that TDP-43 can propagate as a prion in yeast. Identifying con- formational variants of TDP-43 is important because different variants are likely to affect disease progression and symptoms differently. As cross-talk between heterologous proteins can enhance de novo aggregation, aim III uses a candidate and screen approach to find mammalian proteins with the ability to enhance de novo aggregation of TDP-43 and FUS. Also aim III examines the effects of stress on de novo aggregation of TDP-43 and FUS. Under- standing how cross-talk and stress influence TDP-43 and FUS aggregation may lead to therapies that inhibit aggregation.

描述（由适用提供）：许多神经退行性疾病，包括阿尔茨海默氏症，帕金森氏症，prion和亨廷顿疾病，与由于蛋白质错误折叠而形成的特定蛋白质聚集体有关。为了防止或治疗这些疾病，我们必须了解导致蛋白质汇总的是什么，以及与病理学的相关性。这些问题将使用酵母，神经母细胞瘤细胞和原发性皮质神经元和表达FUS和TDP-43的原发性皮质神经元作为肌萎缩性侧面硬化症和额叶痴呆的细胞模型。细胞从酵母到人类神经元的细胞中的保存，例如蛋白质重塑，泛素蛋白质体系统，分泌，囊泡运输和自噬等细胞途径，通过其强大的实验工具箱，短生成时间和良好的基因组，以解决这些疾病疾病的各个方面，这些基因组允许酵母。这为蛋白质错误折叠神经退行性疾病提供了新的见解。许多人类蛋白质构成并与神经退行性疾病有关，在酵母中表达时也有毒性。通过酵母中遗传筛选确定的这种毒性的修饰符已显着，是新的或以前已知的人类疾病危险因素。在AIM I中，新设计的酵母中新设计的高通量屏幕将确定新型的遗传修饰符，以增强或抑制TDP-43或FUS的毒性。然后，将对这些修饰剂的同源物在神经母细胞瘤细胞和原发性神经元中的类似作用进行测试，并筛选患者的突变。 AIM I还通过以下方式探索已知修饰符的作用机理：确定哪些HSP40伴侣SIS1的域是需要SIS1过表达的，以从FUS或TDP-43毒性中拯救细胞；测试了以下假设：ATXN2的过表达（应力颗粒PolyQ膨胀蛋白）通过增加TDP-43的SIS1滴定来增强TDP-43的毒性，从而减少了SIS1介导的泛素化蛋白的递送到蛋白质组；测试如果HSP104伴侣的过表达，通过通过伴侣不平衡从末端修剪骨料来降低毒性；确定TDP-43或FUS聚集体是否有毒，因为它们与必需蛋白共聚集，从而使它们失活。该目标有可能识别新的人类疾病敏感性基因和治疗靶标。 AIM II的目标是在酵母和神经母细胞瘤细胞中建立和表征不同的可遗传性TDP-43结构变体，并测试其对原发性皮质神经元的致病作用。这个目标是基于我们的新发现，即TDP-43可以作为酵母中的素数传播。识别TDP-43的构造变体很重要，因为不同的变体可能会影响疾病进展和符号不同。由于异源蛋白之间的串扰可以增强从头聚集，因此AIM III使用候选和筛选方法找到具有增强TDP-43和FUS NOVE聚集能力的哺乳动物蛋白。还要瞄准III，以检查压力对TDP-43和FUS的从头聚集的影响。透视和压力如何影响TDP-43和FUS聚集可能导致抑制聚集的疗法。