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

 描述(申请人提供)：最近发现的C9orf72基因突变是ALS和FTD最常见的遗传原因(C9FTD/ALS)，在寻求了解ALS和FTD的疾病机制和开发有效的疾病改进策略方面，开辟了许多新的和令人兴奋的研究领域。C9orf72基因包含一个位于内含子的多态六核苷酸重复序列GGGGCC。在未患病的个体中，重复道长度虽然可变，但通常在5到10个重复之间，几乎总是少于23个重复。在c9FTD/ALS病例中，六核苷酸重复序列扩大到数百甚至数千个重复。鉴于这种突变对神经退行性疾病的主要贡献，人们对定义C9orf72基因中GGGGGCC重复扩张导致ALS和FTD的机制非常感兴趣(S)。一个令人兴奋的新假说已经出现，可以解释C9orf72中GGGGCC重复扩张如何导致疾病：重复相关的非ATG(RAN)翻译，它产生来自正义和反义C9orf72 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C9orf72突变携带者的大脑中积累。我们使用酵母模型来探索C9orf72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倚的全基因组筛查，发现了五种可能的二肽产品中的一种具有毒性的有效修饰剂，即脯氨酸-精氨酸(PR)。其中最强的修饰物是几种核粘附素蛋白，它们介导蛋白质的核输入，包括FUS/TLS。这项联合PI研究计划采用了互补类型的研究，这将允许斯坦福大学的吉特勒实验室和梅奥诊所的佩特鲁切利实验室之间的智力协同。我们将结合酵母遗传学和细胞生物学实验，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C9orf72二肽蛋白导致神经退化机制的新假说。在目标1中，我们将在酵母中进行遗传筛选，以确定C9orf72二肽重复蛋白毒性的修饰因子。在目标2中，我们将验证酵母在初级神经元和通过直接对患者细胞重新编程而产生的人类神经元中的发现。在目标3中，我们将进行机械实验来验证这一新的假设，即C9orf72衍生的二肽重复蛋白干扰核粘附素介导的核损伤，这是C9FTD/ALS发病机制的基础。综上所述，这些发现将揭示C9orf72二肽重复蛋白毒性的关键方面，对ALS和FTD至关重要，并为新的治疗见解奠定基础。

 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.