Innovating Yeast and Human Genetics Approaches to Define Mechanisms of Neurodegenerative Disease

这是一项建议，将我们三个由NINDS资助的R01项目合并为一个更大的R35奖项，使我们有时间和灵活性来探索与人类神经退行性疾病相关的重要生物学问题。在过去的八年里，我的实验室使用酵母和人类遗传学的组合来定义ALS、FTD和帕金森病的新机制。这些实验导致了发现ataxin 2中长多谷氨酰胺膨胀是ALS的主要遗传风险因素，发现了RNA套索脱支酶作为TDP-43蛋白病的有效治疗靶点，这是解释C9orf72突变如何导致神经退化和看似不同的帕金森氏病基因之间意外连接的新细胞途径。在初步研究中，我们发现小鼠体内ataxin 2的遗传减少大大延长了TDP-43转基因小鼠的存活时间(寿命增加了80%)。我们建议进行研究，以探索ataxin 2如何预防TDP-43蛋白病变，在其他小鼠模型(如C9orf72和FUS/TLS)中测试ataxin 2，并在人类细胞模型中寻找针对ataxin 2的反义寡核苷酸。虽然我们之前的工作源于酵母模型和遗传修饰物筛选，但现在我们提出了一项雄心勃勃的进步-使用CRISPR/Cas9基因激活和失活文库在人类细胞中进行全基因组修饰物筛选。我们已经用C9orf72模型进行了三次试点筛选，并确定了几种有效的修饰物，我们将在初级神经元和小鼠模型中进行验证。我计划将我的实验室带入这个新的方向，用TDP-43、FUS、α-突触核蛋白、ataxin 2和进一步的C9orf72模型(例如，RNA和dprs)在人类细胞中进行CRISPR筛查。我们也对RAN(重复相关的非ATG)翻译过程感兴趣，它已经成为几种核苷酸重复疾病(包括c9ALS/FTD)的一个重要方面。我们建议通过实验来发现RAN翻译的分子机制，以设计特定的抑制剂，我们已经确定了至少两个似乎是RAN翻译所必需的基因。我们共同提出了一个雄心勃勃的研究计划，旨在定义人类神经退行性疾病的新机制，然后加紧工作，将这些机制转化为新的疗法，以帮助治疗这些毁灭性的疾病。

This is a proposal to combine our three NINDS funded R01 projects into one larger R35 award, enabling us the time and flexibility to explore important biological questions relevant to human neurodegenerative diseases. Over the past eight years, my laboratory has used a combination of yeast and human genetics to define novel mechanisms of ALS, FTD, and Parkinson's disease. These experiments have led to the discovery of ataxin 2 intermediate-length polyglutamine expansions as a major genetic risk factor for ALS, the discovery of RNA lariat debranching enzyme as a powerful therapeutic target for TDP-43 proteinopathies, a new cellular pathway to explain how C9orf72 mutations could cause neurodegeneration and unexpected connections between seemingly distinct Parkinson's disease genes. In preliminary studies, we have found that genetic reduction of ataxin 2 in mouse profoundly extends survival of TDP-43 transgenic mice (>80% increase in lifespan). We propose studies to explore how ataxin 2 protects against TDP-43 proteinopathy, test ataxin 2 in other mouse models (e.g., C9orf72 and FUS/TLS), and to pursue antisense oligonucleotides targeting ataxin 2 in human cell models. While our previous work has stemmed from yeast models and genetic modifier screens, we now propose an ambitious advance – performing genomewide modifier screens in human cells using CRISPR/Cas9 gene activation and inactivation libraries. We have already performed three pilot screens with C9orf72 models and have identified several potent modifiers, which we will validate in primary neurons and mouse models. I plan to take my lab into this new direction by performing CRISPR screens in human cells with TDP-43, FUS, alpha-synuclein, ataxin 2, and further C9orf72 models (e.g., RNA vs. DPRs). We are also interested in the process of RAN (repeat-associated non-ATG) translation, which has emerged as a powerful facet of several nucleotide-repeat diseases (including c9ALS/FTD). We propose experiments to discover the molecular mechanisms of RAN translation in order to design specific inhibitors and we have already identified at least two genes that seem to be required for RAN translation. Together, we present an ambitious research program aimed at defining novel mechanisms of human neurodegenerative diseases and then intensely working to translate those mechanisms to novel therapies to help treat these devastating conditions.