Defining the mechanisms of dipeptide repeat protein toxicity in C9orf72 ALS/FTD

定义 C9orf72 ALS/FTD 中二肽重复蛋白毒性的机制

• 批准号: 8984742
• 负责人: Aaron D. Gitler
• 金额: $ 44.3万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8984742
• 关键词: Affect; Antithymoglobulin; Area; Arginine; Autopsy; Binding; Biological; Brain; C9ORF72; Carrier Proteins; Cell physiology; Cells; Clinic; Collaborations; Development; Dipeptides; Disease; Enhancers; Family; Fibroblasts; Foundations; Future; Gene Deletion; Genes; Genetic; Genetic Screening; Glutaminase; Goals; Homologous Gene; Human; Impairment; Individual; Introns; Investigation; Karyopherins; Laboratories; Length; Mammalian Cell; Mediating; Modeling; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nuclear; Nuclear Import; Nuclear Pore Complex; Pathogenesis; Pathology; Patients; Polymers; Proline; Proteins; RNA; Research; Research Proposals; Role; Sampling; System; Testing; Therapeutic Intervention; Toxic effect; Translations; Validation; Yeast Model System; Yeasts; base; coping; genome-wide; genome-wide analysis; insight; interest; member; mouse model; mutation carrier; novel; novel therapeutic intervention; novel therapeutics; nucleocytoplasmic transport; programs; protein aggregation; public health relevance; research study; synergism; therapeutic target; yeast genetics

 DESCRIPTION (provided by applicant): The recent discovery of a mutation in the C9orf72 gene as the most common genetic cause of ALS and FTD (c9FTD/ALS) has opened up many new and exciting areas of investigation in the quest to understand ALS and FTD disease mechanisms and to develop effective disease-modifying strategies. The C9orf72 gene contains a polymorphic hexanucleotide repeat, GGGGCC, located in an intron. The repeat tract length in unaffected individuals, although variable, is typically between five and ten repeats and almost always fewer than 23 repeats. In c9FTD/ALS cases, the hexanucleotide repeat tract is expanded to hundreds or even thousands of repeats. Given the major contribution of this mutation to neurodegenerative disease, there is intense interest in defining the mechanism(s) by which GGGGCC repeat expansions in the C9orf72 gene cause ALS and FTD. An exciting new hypothesis has emerged to explain how the GGGGCC repeat expansions in C9orf72 could cause disease: Repeat-Associated Non-ATG (RAN) translation, which generates polymers of dipeptides derived from the sense and antisense C9orf72 RNA. These dipeptide repeat proteins are aggregation-prone and accumulate in the brain of affected C9orf72 mutation carriers. We have used a yeast model to explore the mechanisms by which C9orf72-derived dipeptide proteins cause cellular toxicity. In Preliminary Studies, we have performed two unbiased genome-wide screens and discovered potent modifiers of toxicity for one out of the five possible dipeptide products, proline-arginine (PR). Among the strongest modifiers are several karyopherin proteins, which mediate nuclear import of proteins, including FUS/TLS. This Co-PI research proposal employs complementary types of research that will allow for intellectual synergism between the Gitler laboratory at Stanford and the Petrucelli laboratory at the Mayo Clinic. We will use a combination of yeast genetics and cell biological experiments and validation in mammalian cells, mice, primary neurons, and human patient samples with the goal to test novel hypotheses about the mechanism by which C9orf72 dipeptide proteins cause neurodegeneration. In Aim 1, we will perform genetic screens in yeast to identify modifiers of C9orf72 dipeptide repeat protein toxicity. In Aim 2, we will validate findings from yeast in primar neurons and in human neurons generated by direct re-programming of patient cells. In Aim 3, we will perform mechanistic experiments to test the novel hypothesis that C9orf72-derived dipeptide repeat proteins interfere with karyopherin-mediated nuclear impairments and that this underlies the pathogenesis of c9FTD/ALS. Taken together, these findings will reveal key aspects of C9orf72 dipeptide repeat proteotoxicity central to ALS and FTD, and lay the foundation for novel therapeutic insights.

 描述（由申请人提供）：最近发现C9 orf 72基因突变是ALS和FTD（c9 FTD/ALS）最常见的遗传原因，这为了解ALS和FTD疾病机制以及开发有效的疾病修饰策略开辟了许多新的令人兴奋的研究领域。C9 orf 72基因含有位于内含子中的多态性六核苷酸重复序列GGGGCC。未受影响个体的重复序列长度虽然可变，但通常在5至10个重复序列之间，并且几乎总是少于23个重复序列。在c9 FTD/ALS病例中，六核苷酸重复序列扩展到数百甚至数千个重复序列。鉴于这种突变对神经退行性疾病的主要贡献，人们对确定C9 orf 72基因中GGGGCC重复扩增导致ALS和FTD的机制非常感兴趣。 一个令人兴奋的新假设已经出现，以解释C9 orf 72中GGGGCC重复扩增如何导致疾病：重复相关非ATG（RAN）翻译，它产生来自正义和反义C9 orf 72 RNA的二肽聚合物。这些二肽重复蛋白易于聚集，并在受影响的C9 orf 72突变携带者的大脑中积累。我们已经使用酵母模型来探索C9 orf 72衍生的二肽蛋白引起细胞毒性的机制。在初步研究中，我们进行了两次无偏的全基因组筛选，发现了五种可能的二肽产物之一脯氨酸-精氨酸（PR）的有效毒性调节剂。其中最强的修饰剂是几种karyopherin蛋白，其介导蛋白质的核输入，包括FUS/TLS。 该合作PI研究提案采用互补类型的研究，这将允许斯坦福大学的Gitler实验室和马约诊所的Petrucelli实验室之间的智力协同作用。我们将使用酵母遗传学和细胞生物学实验的组合，并在哺乳动物细胞，小鼠，原代神经元和人类患者样本中进行验证，目的是测试关于C9 orf 72二肽蛋白引起神经变性的机制的新假设。在目标1中，我们将在酵母中进行遗传筛选以鉴定C9 orf 72二肽重复蛋白毒性的修饰剂。在目标2中，我们将验证酵母在原代神经元和通过直接重编程患者细胞产生的人类神经元中的发现。在目的3中，我们将进行机制实验，以检验C9 orf 72衍生的二肽重复蛋白干扰核转运蛋白介导的核损伤的新假设，这是c9 FTD/ALS发病机制的基础。总之，这些发现将揭示C9 orf 72二肽重复蛋白毒性的关键方面，并为新的治疗见解奠定基础。