Deciphering RNA based mechanisms of neurodegeneration

描述(申请人提供)：肌萎缩侧索硬化症是一种成人起病的进行性神经系统疾病，其特征是皮质和脊髓中运动神经元的选择性变性和死亡。近年来，一些与运动神经元病相关的RNA结合蛋白被发现，包括感觉神经毒素、血管生成素、TDP-43和FUS。这些发现导致了当前神经元退化机制模型的范式转变，并表明ALS的一个重要组成部分可能是由于RNA代谢的失调。目前，TDP-43已成为迄今已知的大多数ALS病例的共同分母，但其导致神经元变性的机制仍知之甚少。这项研究的长期目标是破译TDP-43在神经系统中利用的基于RNA的机制，并确定与ALS相关的TDP-43突变对RNA代谢、特定蛋白质伙伴和RNA靶标的哪些方面的失调。这一探索性建议旨在通过关注TDP-43与一系列候选RNA结合蛋白之间的物理和功能联系来检验ALS中RNA失调的假说。我们将在基于我们实验室开发的TDP-43的ALS果蝇模型上进行拟议的研究，该模型与人类病理学具有显著的相似之处。在合作者的帮助下，我们的研究将扩展到从ALS患者身上获得的人类细胞和组织样本。从果蝇和人类细胞中获得的初步数据表明，TDP-43与FMRP形成复合体，FMRP是一种RNA结合蛋白，在局部翻译中具有既定的作用，并与脆性X综合征有关。我们还发现TDP-43与PABP共存于运动神经元的应激颗粒中。与野生型TDP-43不同，已与人类患者ALS连锁的A315T突变体与候选RNA结合蛋白包括FMRP和PABP表现出不同的共定位和遗传相互作用。我们假设肌萎缩侧索硬化症部分源于RNA失调，并建议通过分子、电生理学、遗传和活体成像方法的组合来验证这一点。在目标1中，我们将通过共定位、活转运研究、生化纯化和正常条件下或诱导细胞应激下的细胞分级来建立TDP-43突变体(野生型和突变型)与几种神经元RNA颗粒成分的关系。在目标2中，我们将使用遗传交互作用方法结合一系列表型分析来建立TDP-43‘S与神经元核糖核酸颗粒成分的生理意义，包括FMRP和已建立的应激颗粒和P体标记物。拟议的实验将深入了解核糖核酸调控的哪些方面(例如，应激颗粒组装、组成、翻译)受到致病突变的干扰，并将识别在体内调节TDP-43‘S神经毒性的特定核糖核酸结合蛋白。鉴于我们在基于RNA的神经机制和果蝇遗传学方面的广泛专业知识，以及来自专家合作者团队的支持，我们处于独特的地位，可以测试这一将RNA失调与神经退化联系起来的新的和令人兴奋的假说，并确定ALS的新治疗靶点。 与公共卫生相关：肌萎缩侧索硬化症运动神经元变性的分子机制在很大程度上仍不清楚。这项提议的重点是测试将肌萎缩侧索硬化症与RNA失调联系起来的新假说。我们将在基于RNA结合蛋白TDP-43的ALS果蝇模型中使用分子、遗传和功能方法的组合。这些实验将识别与TDP-43物理上相关的特定RNA结合蛋白，调节其在体内的神经毒性，并有可能为ALS提供新的治疗靶点。

DESCRIPTION (provided by applicant): ALS is an adult onset, progressive neurological disorder characterized by selective degeneration and death of motor neurons in the cortex and the spinal cord. In recent years, several RNA binding proteins have been linked to motor neuron disease, including senataxin, angiogenin, TDP-43 and FUS. These findings led to a paradigm shift in the current models for neuronal degeneration mechanisms and suggest that a significant component of ALS may be due to dysregulation of RNA metabolism. At present, TDP-43 has emerged as a common denominator for the majority of ALS cases known to date, however the mechanisms by which it causes neuronal degeneration remain poorly understood. The long-term goals of this research are to decipher the RNA-based mechanisms utilized by TDP-43 in the nervous system and to identify what aspects of RNA metabolism, specific protein partners and RNA targets are dysregulated by TDP-43 mutations linked to ALS. This exploratory proposal aims to test the hypothesis of RNA dysregulation in ALS by focusing on the physical and functional connections between TDP-43 and a repertoire of candidate RNA binding proteins. We will perform the proposed studies in a Drosophila model of ALS based on TDP-43 that we developed in our laboratory and which bears remarkable similarities to the human pathology. With help from collaborators our studies will be extended to human cells and tissue samples obtained from ALS patients. Preliminary data obtained from Drosophila and human cells show that TDP-43 forms a complex with FMRP, an RNA binding proteins with an established role in local translation and implicated in Fragile X syndrome. We also found that TDP-43 colocalizes with PABP in stress granules in motor neurons. In contrast to wild-type TDP-43, the A315T mutant, which has been linked to ALS in human patients, exhibits differential colocalization and genetic interactions with candidate RNA binding proteins including FMRP and PABP. We hypothesize that ALS stems in part from RNA dysregulation and propose to test this through a combination of molecular, electrophysiology, genetic and live imaging approaches. In Aim 1 we will establish the relationship between TDP-43 variants (wild-type and mutant) and several neuronal RNA granule components using colocalization, live trafficking studies, biochemical purifications and cellular fractionations under normal conditions or induced cellular stress. In Aim 2 we will use genetic interaction approaches in conjunction with a battery of phenotypic assays to establish the physiological significance of TDP-43's association with neuronal RNA granule components, including FMRP and established stress granule and P body markers. The proposed experiments will provide insights into what aspects of RNA regulation (e.g., stress granules assembly, composition, translation) are perturbed by disease causing mutations and will identify specific RNA binding proteins that modulate TDP-43's neurotoxicity in vivo. Given our extensive expertise in RNA based neuronal mechanisms and Drosophila genetics as well as the support from a team of expert collaborators we are uniquely positioned to test this novel and exciting hypothesis linking RNA dysregulation to neurodegeneration and to identify novel therapeutic targets for ALS. PUBLIC HEALTH RELEVANCE: The molecular mechanisms underlying motor neuron degeneration in ALS remain largely elusive. This proposal is focused on testing new hypotheses linking ALS to RNA dysregulation. We will employ a combination of molecular, genetic and functional approaches in a Drosophila model of ALS based on the RNA binding protein TDP-43. These experiments will identify specific RNA binding proteins that physically associate with TDP-43, modulate its neurotoxicity in vivo and have the potential to provide novel therapeutic targets for ALS.