RNA Dominant Mechanisms in ALS

描述（由申请人提供）： RNA稳态和RNA结合蛋白功能的改变是神经退行性变发病机制的关键方面。对于由核苷酸重复序列引起的RNA显性疾病，RNA的影响是直接的，重复序列本身通过封存引发毒性 某些蛋白质。对于其他神经退行性病症，包括肌萎缩侧索硬化（ALS），RNA结合蛋白如TDP-43形成神经元聚集体，其充当疾病的病理标志。 最近，一种新的内含子GGGGCC核苷酸重复扩增（C9 ALS）被确定为ALS最常见的已知原因。C9 ALS与TDP-43病理学相关，但也表现出重复RNA积聚到核灶中。我们提出了一组研究，利用我们在RNA显性疾病的专业知识，询问如何GGGGCC重复electin疾病在C9 ALS。我们假设GGGGCC重复序列作为RNA是固有毒性的，并且它们通过螯合特定的RNA结合蛋白引起毒性。本研究的直接目标是生成和表征C9 ALS的果蝇模型，并在体外和体内鉴定与GGGGCC重复序列相互作用的蛋白质。研究计划/方法：为了更好地理解C9 ALS的发病机制，我们将首先在果蝇中产生GGGGCC重复模型，使我们能够跟踪表达组织内内含子的剪接和定位。我们将确定 如果GGGGCC RNA重复赋予神经变性并影响寿命和运动神经元功能。然后，我们将采取一种候选方法来确定GGGGCC重复毒性的遗传抑制因子。 在一组平行研究中，我们将使用体外和基于细胞培养的技术来凭经验鉴定与GGGGCC重复序列结合的蛋白质。然后，我们将验证这些候选人与果蝇和患者细胞中的RNA内含物共定位。影响/临床相关性：所有形式的ALS和FXTAS目前都无法治疗，并对退伍军人造成显著的发病率和死亡率。C9 ALS重复序列的发现提供了一个独特的机会，可以将RNA显性疾病的最新进展快速转化为这种新的疾病。该项目的长期目标是鉴定阻断GGGGCC重复RNA与其同源RNA结合蛋白之间相互作用的小分子。然后将评估这些化合物在动物模型中预防GGGGCC RNA毒性的能力，并最终用于治疗C9 ALS患者。总而言之，我们的方法是新颖的、可行的，并且对ALS和相关GGGGCC相关神经系统疾病患者具有很高的潜在影响。

DESCRIPTION (provided by applicant): Alterations in RNA homeostasis and RNA binding protein functions underlie critical aspects of pathogenesis in neurodegeneration. For RNA dominant disorders caused by nucleotide repeats, the effects of RNA are direct, with the repeats themselves eliciting toxicity via the sequestration of certain proteins. For other neurodegenerative conditions, including amyotrophic lateral sclerosis (ALS), RNA binding proteins such as TDP-43 form neuronal aggregates that serve as pathologic hallmarks of disease. Recently, a novel intronic GGGGCC nucleotide repeat expansion (C9ALS) was identified as the most common known cause of ALS. C9ALS is associated with TDP-43 pathology but also exhibits accumulation of repeat RNA into nuclear foci. We propose a set of studies that take advantage of our expertise in RNA dominant disorders to interrogate how the GGGGCC repeat elicits disease in C9ALS. We hypothesize that GGGGCC repeats are inherently toxic as RNA, and that they elicit toxicity by sequestering specific RNA binding proteins. The immediate goals of this study are to generate and characterize a drosophila model of C9ALS and to identify proteins which interact with the GGGGCC repeat in vitro and in vivo. Research Plan/Methods: To better understand C9ALS pathogenesis, we will first generate an GGGGCC repeat model in Drosophila that allows us to track both the splicing and localization of the intron within expressing tissues. We will determine if GGGGCC RNA repeats confer neurodegeneration and affect lifespan and motor neuron function. We will then take a candidate approach to identify genetic suppressors of GGGGCC repeat toxicity. In a parallel set of studies, we will use in vitro and cell culture based techniqes to empirically identify proteins that bind to the GGGGCC repeat. We will then validate that these candidates colocalize with RNA inclusions in Drosophila and patient cells. Impact/Clinical Relevance: All forms of ALS and FXTAS are currently untreatable and impart significant morbidity and mortality upon Veterans. The C9ALS repeat discovery provides a unique opportunity to rapidly translate recent advances in RNA dominant diseases to this new disorder. The long term goal of this project is to identify small molecules that block interactions between GGGGCC repeat RNA with its cognate RNA binding proteins. Such compounds will then be evaluated for their ability to prevent GGGGCC RNA toxicity in animal models and eventually utilized to treat patients with C9ALS. Taken together, our approach is novel, feasible, and of high potential impact to patients with ALS and related GGGGCC associated neurological disorders.