Exploring the toxicity of aggregates associated with protein-misfolding diseases

 描述(由申请人提供)：许多神经退行性疾病，包括阿尔茨海默氏症、帕金森氏症、普里昂病和亨廷顿病，都与特定的蛋白质聚集体有关，这些聚集体是蛋白质错误折叠的结果。为了预防或治疗这些疾病，我们必须了解是什么导致蛋白质聚集，以及这与病理有何关联。这些问题将使用酵母、神经母细胞瘤细胞和表达FUS和TDP-43的初级皮质神经元作为肌萎缩侧索硬化症和额颞痴呆的细胞模型来解决。从酵母到人类神经元的细胞通路的保守性，如蛋白质折叠、泛素蛋白酶体系统、分泌、囊泡运输和自噬，使酵母能够以其强大的实验工具箱、较短的世代时间和特征良好的基因组来解决涉及这些基本系统的神经退行性疾病的各个方面。这为蛋白质错误折叠神经退行性疾病提供了新的见解。许多与神经退行性疾病相关的人类蛋白质聚集在一起，在酵母中表达时也会聚集并具有毒性。值得注意的是，通过酵母中的遗传筛选确定的这种毒性的修饰物已经被证明是新的或以前已知的人类疾病风险因素。在目标I中，新设计的酵母高通量筛选将识别增强或抑制TDP-43或FUS毒性的新型遗传修饰物。然后将测试这些修饰物的同源物在神经母细胞瘤细胞和原代神经元中的类似作用，并筛选患者的突变。目的我还探索了已知修饰剂的作用机制，方法包括：确定HSP40伴侣蛋白SIS1的哪些结构域是过表达所必需的，以便将细胞从FUS或TDP-43毒性中拯救出来；测试应激颗粒多聚Q扩展蛋白ATXN 2的过表达，通过增加SIS1的TDP-43‘S滴定来增强TDP-43的毒性，从而减少SIS1介导的泛素化蛋白向蛋白酶体的输送；测试Hsp104伴侣蛋白的过表达是否通过通过伴侣蛋白失衡从其末端剪裁聚集体来降低毒性；确定TDP-43或FUS聚集体是否有毒，因为它们与必要的蛋白质共聚集，从而使它们失活。这一目标有可能识别新的人类疾病易感基因和治疗靶点。AIM II的目标是在酵母和神经母细胞瘤细胞中建立和鉴定不同的可遗传的TDP-43构象变异，并测试它们对初级皮质神经元的致病作用。这一目标建立在我们的新发现基础上，即TDP-43可以作为普恩在酵母中繁殖。识别TDP-43的构象变体很重要，因为不同的变体可能会影响疾病 进展和症状不同。由于异源蛋白之间的串扰可以增强从头聚集，Aim III使用候选和筛选的方法来寻找能够增强TDP-43和FUS从头聚集的哺乳动物蛋白质。Aim III还检测了压力对TDP-43和FUS从头聚集的影响。了解串扰和压力如何影响TDP-43和FUS聚集可能导致抑制聚集的治疗。

 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.