Deciphering RNA based mechanisms of neurodegeneration

描述（由申请人提供）：ALS是一种成人发作的进行性神经系统疾病，其特征在于皮质和脊髓中运动神经元的选择性变性和死亡。近年来，几种RNA结合蛋白与运动神经元疾病有关，包括senataxin，angiogenin，TDP-43和FUS。这些发现导致了目前神经元变性机制模型的范式转变，并表明ALS的重要组成部分可能是由于RNA代谢失调。目前，TDP-43已成为迄今为止已知的大多数ALS病例的共同特征，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、特定蛋白质伴侣和RNA靶点的哪些方面产生了失调。该探索性建议旨在通过关注TDP-43与候选RNA结合蛋白库之间的物理和功能连接来测试ALS中RNA失调的假设。 我们将在我们实验室开发的基于TDP-43的ALS果蝇模型中进行所提出的研究，该模型与人类病理学具有显著的相似性。在合作者的帮助下，我们的研究将扩展到从ALS患者获得的人类细胞和组织样本。从果蝇和人类细胞获得的初步数据显示，TDP-43与FMRP形成复合物，FMRP是一种RNA结合蛋白，在局部翻译中具有既定作用，并与脆性X综合征有关。我们还发现TDP-43与PABP共定位于运动神经元的应激颗粒中。与野生型TDP-43相反，A315 T突变体与人类患者的ALS相关，与候选RNA结合蛋白（包括FMRP和PABP）表现出差异共定位和遗传相互作用。我们假设ALS部分源于RNA失调，并建议通过分子，电生理学，遗传学和实时成像方法的组合来测试这一点。在目的1中，我们将建立TDP-43变体（野生型和突变型）和几个神经元RNA颗粒成分的共定位，活的贩运研究，生化纯化和细胞分级在正常条件下或诱导的细胞应激之间的关系。在目标2中，我们将使用遗传相互作用的方法结合一系列表型测定，以建立TDP-43与神经元RNA颗粒组分（包括FMRP和已建立的应激颗粒和P体标记物）相关的生理意义。 拟议的实验将提供对RNA调控的哪些方面（例如，应激颗粒组装、组成、翻译）被致病突变干扰，并将鉴定在体内调节TDP-43神经毒性的特异性RNA结合蛋白。鉴于我们在基于RNA的神经元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，可以测试将RNA失调与神经退行性变联系起来的这一新颖而令人兴奋的假设，并确定ALS的新治疗靶点。

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.