RNA dysregulation in neurodegeneration

 描述（由申请人提供）：近年来，越来越清楚的是，RNA失调是肌萎缩侧索硬化症（ALS）和相关神经退行性疾病的病理生理学的关键因素。事实上，已经在病理性聚集体中鉴定了几种RNA结合蛋白，并且还显示其具有导致ALS的突变。其中，与绝大多数ALS病例相关的TDP-43涉及RNA代谢的几个方面，包括剪接、运输、在应激颗粒（SG）中的储存和翻译。这些发现表明TDP-43、RNA SGs、翻译和疾病之间存在密切联系。尽管最近的研究已经提供了关于TDP-43和ALS中RNA应激颗粒之间关系的见解，但我们目前对TDP-43在翻译中的作用、其mRNA翻译靶点的身份及其对疾病的贡献的了解仍然很少。这项研究的长期目标是确定TDP-43与神经系统中翻译之间的机制联系，并确定RNA失调对神经退行性疾病病理生理学的贡献。我们最近证明，TDP-43调节futsch/MAP1B在果蝇中的定位和翻译，并且在果蝇运动神经元中发现的缺陷与ALS脊髓中发现的缺陷非常相似。使用基于TDP-43的ALS的果蝇模型，我们还发现FMRP（一种完善的翻译调节剂）通过减少TDP-43聚集和恢复特异性mRNA靶标的翻译而具有神经保护性。此外，我们已经确定了几个新的候选mRNA翻译靶点，包括hsc70 - 4 mRNA，一种控制突触囊泡（SV）运输的分子伴侣，以及其他参与突触功能的候选人。值得注意的是，通过过表达恢复运动神经元中的Hsc70 - 4水平挽救了由ALS相关突变体TDP-43引起的突触囊泡内吞缺陷。基于这些发现，我们假设TDP-43作为运动神经元中的翻译调节剂，并且蛋白质合成的失调有助于ALS的病理生理学。我们的假设将在三个具体目标中得到严格检验。首先，我们将确定TDP-43和翻译机制之间的物理和功能相互作用。其次，我们将使用标记核糖体亲和纯化（TRAP）来鉴定TDP-43在运动神经元中的mRNA翻译靶点，然后使用核糖体足迹来确定TDP-43影响翻译的哪个阶段。第三，我们将建立由TDP-43引起的突触缺陷，并将确定通过在运动神经元中过表达恢复候选靶标是否挽救TDP-43依赖性毒性。我们在果蝇模型中的发现将在ALS脊髓中得到验证。这项研究有望为TDP-43的翻译功能提供新的见解，以确定生理学上重要的和疾病相关的蛋白质和mRNA翻译靶点，从而可以确定急需的分子靶点和途径，对ALS和相关神经退行性疾病具有治疗潜力。

 DESCRIPTION (provided by applicant): In recent years, it has become increasingly clear that RNA dysregulation is a critical contributor to the pathophysiology of amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases. Indeed, several RNA binding proteins have been identified in pathologic aggregates and have also been shown to harbor mutations causative of ALS. Among these, TDP-43, which is linked to the vast majority of ALS cases, has been implicated in several aspects of RNA metabolism including splicing, transport, storage in stress granules (SGs) and translation. These findings suggest an intimate link between TDP-43, RNA SGs, translation and disease. Although recent studies have provided insights into the relationship between TDP-43 and RNA stress granules in ALS, our current knowledge of TDP-43's role in translation, the identity of its mRNA translation targets and their contribution to disease remain poorly understood. The long-term goal of this research is to determine the mechanistic connections between TDP-43 and translation in the nervous system, and to establish the contribution of RNA dysregulation to the pathophysiology of neurodegenerative diseases. We have recently demonstrated that TDP-43 regulates the localization and translation of futsch/MAP1B in Drosophila and defects identified in fly motor neurons are remarkably similar to those found in ALS spinal cords. Using a Drosophila model of ALS based on TDP-43 we also found that FMRP, a well- established translational regulator is neuroprotective by reducing TDP-43 aggregation and restoring the translation of specific mRNA targets. Furthermore, we have identified several new candidate mRNA translation targets including hsc70-4 mRNA, a molecular chaperone that controls synaptic vesicle (SV) trafficking as well as additional candidates implicated in synaptic function. Notably, restoring Hsc70-4 levels in motor neurons by overexpression rescues synaptic vesicle endocytosis defects caused by ALS associated mutant TDP-43. Based on these findings we hypothesize that TDP-43 acts as a translational regulator in motor neurons and that dysregulation of protein synthesis contributes to the pathophysiology of ALS. Our hypothesis will be critically tested in three specific aims. First, we will determine physical and functional interactions between TDP-43 and the translation machinery. Second, we will identify mRNA translation targets of TDP-43 in motor neurons using tagged ribosome affinity purification (TRAP), then will use ribosome footprinting to determine what stage of translation is impacted by TDP-43. Third, we will establish the synaptic defects caused by TDP- 43 and will determine whether restoring candidate targets by overexpression in motor neurons rescues TDP-43 dependent toxicity. Our findings in the Drosophila model will be validated in ALS spinal cords. This research is expected to provide novel insights into TDP-43's function in translation, to identify physiologically significant and disease relevant protein partnrs and mRNA translation targets, which in turn may pinpoint much needed molecular targets and pathways with therapeutic potential for ALS and related neurodegenerative diseases.