Functional and pathological interactions of TDP-43

项目摘要 蛋白质的细胞内含物是绝大多数神经退行性疾病的主要标志。在 常见的肌萎缩侧索硬化症（ALS）、阿尔茨海默病相关痴呆（额颞 痴呆;边缘优势年龄相关性TDP-43脑病（LATE））以及某些形式的 阿尔茨海默病，43 kDa的人类TAR DNA结合蛋白（TDP-43）形成神经元内 集料.在母基金中，我们详细研究了TDP-43的结构生物学，以获得新的见解 疾病的机制和潜在的新疗法。重要的是，在一个基因组中有几十个错义突变， TDP-43的聚集倾向结构域已在ALS的家族性和散发性病例中发现， 额颞叶痴呆在父母奖，我们专注于两个热点，TDP-43聚集性疾病的地区 突变。在这份补充提案中，我们将利用实验室中可用的工具，重点研究第三种新的 发现了与ALS相关的突变和致病突变的热点，但目前尚未提出 生物物理/生化机制进行了探讨。新提出的实验符合以下范围： 父母奖和TDP-43突变改变分子结构的总体假设， 相互作用，导致TDP-43功能障碍。作为理论基础，11种与ALS相关的突变， 在第378-390位残基中发现了额颞叶痴呆，包括家族性ALS中的四个残基，这表明了因果关系。 此外，我们以前的数据表明，该区域在使接触稳定功能方面具有潜在作用 交互.我们将检验残基378-390参与TDP的生物学自身相互作用的假设。 43，被这个热点中的突变破坏，并且热点中的翻译后修饰 调节这种重要的相互作用。在补充目的1中，我们假设该区域形成疏水性的 接触介导的动态相互作用，这些相互作用被疾病相关的 该区域的突变导致聚集。使用我们建立的核磁共振光谱技术配对 通过体外相分离，我们将确定该位点接触的结构细节，并评估 疾病突变的机制作用。在目标2中，我们将测试已知的TDP-43翻译后蛋白质的影响。 在该区域中的修饰对相互作用和对TDP-43聚集的影响，以及 突变对翻译后修饰的影响，反之亦然（即串扰）。这些研究 为预防阿尔茨海默病中TDP-43的毒性破坏的策略设计提供未来的输入 相关的痴呆症和ALS。这些研究也是URM博士论文计划的重要组成部分 研究生。这里提出了一个全面的培训和职业发展计划， 学生的职业目标是在分子理解方面做出持续和有影响力的贡献， 神经退行性疾病的治疗。

Project Summary Cellular inclusions of proteins are primary hallmarks of a great majority of neurodegenerative diseases. In common forms of amyotrophic lateral sclerosis (ALS), Alzheimer’s disease related dementias (frontotemporal dementia; limbic-predominant age-related TDP-43 encephalopathy (LATE)) as well as some forms of Alzheimer’s disease, the essential human TAR DNA binding protein of 43 kDa (TDP-43) forms intraneuronal aggregates. In the parent grant, we examine in detail the structural biology of TDP-43 to yield new insights into the mechanism of disease and potential new therapies. Importantly, dozens of missense mutations in an aggregation-prone domain of TDP-43 have been found in familial and sporadic cases of ALS and frontotemporal dementia. In the parent award, we focus on two hotspots, regions of TDP-43 clustering disease mutations. In this supplement proposal, we will use the tools available in the laboratory to focus on a third new hotspot where mutations associated with and causative for ALS are found but currently no proposed biophysical / biochemical mechanism has been probed. The new proposed experiments fit within the scope of the parent award and the overall hypothesis that mutations in TDP-43 alter the molecular structure and interactions, leading to TDP-43 dysfunction. As rationale, 11 mutations associated with ALS and frontotemporal dementia are found in residues 378-390, including four in familial ALS, suggesting causation. Furthermore, our previous data indicate a potential role for this region in making contacts stabilizing functional interactions. We will test the hypothesis that residues 378-390 participate in biological self-interactions of TDP- 43 that are disrupted by mutations in this hot spot and that post translational modifications in the hot spot modulate this important interaction. In supplement Aim 1, we hypothesize that this region forms hydrophobic contacts mediating dynamic interactions, and that these interactions are disrupted by disease-associated mutations in this region leading to aggregation. Using our established NMR spectroscopy techniques paired with in vitro phase separation, we will determine the structural details of contacts at this site and evaluate the mechanistic role of disease mutations. In Aim 2, we will test the impact of known TDP-43 post-translational modifications in this region on the interactions and the resulting impact on TDP-43 aggregation, as well as the impact of mutations on the post-translational modification and vice versa (i.e. cross-talk). These studies provide future input to the design of strategies to prevent toxic disruption of TDP-43 in Alzheimer’s disease related dementias and ALS. These studies also form an important part of the PhD thesis plan for a URM graduate student. A comprehensive training and career development plan is presented here to prepare the student for his career goal to make sustained and impactful contributions in the molecular understanding and treatment of neurodegenerative diseases.