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，包括阿尔茨海默氏病、帕金森氏病、朊病毒和亨廷顿氏病，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，以及这与病理学的关系。这些问题将使用酵母，神经母细胞瘤细胞和原代皮质神经元表达FUS和TDP-43作为细胞模型的肌萎缩侧索硬化症和额颞叶痴呆。从酵母到人类神经元的细胞通路（如蛋白质重折叠、泛素蛋白酶体系统、分泌、囊泡运输和自噬）在细胞中的保守性使酵母能够以其强大的实验工具箱、短的世代时间和良好表征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。许多聚集并与神经退行性疾病相关的人类蛋白质在酵母中表达时也聚集并有毒。在酵母中通过遗传筛选鉴定的这种毒性的修饰剂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鉴定增强或抑制TDP-43或FUS毒性的新型遗传修饰剂。这些修饰物的同源物将在神经母细胞瘤细胞和原代神经元中测试类似的作用，并在患者中筛选突变。目的：本研究还通过以下方式探索了已知修饰剂的作用机制：确定Hsp 40伴侣蛋白Sis 1的哪些结构域是Sis 1过表达拯救细胞免于FUS或TDP-43毒性所必需的;检验过表达ATXN 2（一种应激颗粒polyQ扩增蛋白）通过增加TDP- 43对Sis 1的滴定而增强TDP-43毒性的假设，从而减少Sis 1介导的泛素化蛋白向蛋白酶体的递送;测试Hsp 104伴侣蛋白的过表达是否通过伴侣蛋白不平衡从其末端修剪聚集体而降低毒性;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。这一目标有可能确定新的人类疾病易感基因和治疗靶点。目的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TDP-43构象变体，并测试其对原代皮层神经元的致病作用。这一目标建立在我们的新发现基础上，即TDP-43可以作为朊病毒在酵母中繁殖。鉴定TDP-43的构象变体很重要，因为不同的变体可能影响疾病 不同的症状和症状。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来寻找具有增强TDP-43和FUS从头聚集能力的哺乳动物蛋白。目的III还考察了应激对TDP-43和FUS从头聚集的影响。了解串扰和应激如何影响TDP-43和FUS聚集可能导致抑制聚集的疗法。

 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.