Developing RNA therapeutics for TDP-43 proteinopathy in ALS/FTD

项目摘要 我的长期职业目标是作为一名学术研究人员解决神经退行性疾病。肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD），一种阿尔茨海默病相关性痴呆（ADRD），都是以神经元损失为特征的致命性神经退行性疾病。ALS的主要特征是由运动神经元损失引起的运动损伤，而FTD的主要症状包括由额叶和颞叶皮质神经元损失引起的个性、行为和语言变化。ALS和FTD存在于一个疾病谱中，其中一些患者同时具有这两种疾病的特征。几乎所有ALS患者和大约一半FTD患者共有的分子标志是RNA结合蛋白TDP-43的病理性聚集。最近已经确定TDP-43的溶解度通过其与RNA的结合而增加。我利用纯化TDP-43的体外聚集试验的初步数据表明，TDP-43中的错义突变可以改变RNA阻止TDP-43聚集的能力。基于这些发现，我假设ALS/FTD模型中TDP-43的异常聚集是由于TDP-43：RNA相互作用的改变。直接操纵TDP-43的RNA相互作用可以挽救疾病表型。本提案的目的是通过以下方式评估疾病中是否存在TDP-43的RNA相互作用差异：1）检查短RNA在体外防止疾病相关TDP-43变体聚集的能力; 2）确定ALS/FTD患者源性神经元与对照神经元中TDP-43结合的RNA是否存在改变。该提案还旨在3）确定结合TDP-43的短RNA的施用是否可以挽救ALS/FTD患者源性神经元中的疾病表型。拟议的实验将提供关于TDP-43和RNA在疾病中相互作用的严重缺乏的信息，以及治疗这些相互作用的策略。拟议的实验将大大有助于我的培训，使我能够获得新技术的专业知识，如电子显微镜，交联免疫沉淀（CLIP）和测序数据分析。我的培训环境将促进该提案的成功，结合肖特实验室的生化专业知识和宾夕法尼亚大学和神经科学研究生组内部的高度合作环境。这些研究将通过支持我发展成为一名为神经退行性疾病患者开发治疗方法的学术研究人员来促进我的科学和职业目标。

PROJECT SUMMARY My long-term career goal is to tackle neurodegeneration as an academic researcher. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), an Alzheimer's Disease Related Dementia (ADRD), are both fatal neurodegenerative disorders characterized by neuronal loss. ALS is primarily characterized by motor impairments stemming from loss of motor neurons, whereas the main symptoms of FTD include changes in personality, behavior, and language stemming from loss of cortical neurons in the frontal and temporal lobes. ALS and FTD exist on a disease spectrum, where some patients present with features of both diseases. A molecular hallmark shared by almost all ALS patients and approximately half of FTD patients is the pathological aggregation of the RNA-binding protein TDP-43. It has recently been established that the solubility of TDP-43 is increased by its binding to RNA. My preliminary data utilizing in vitro aggregation assays with purified TDP-43 indicate that missense mutations in TDP-43 can alter the ability of RNA to prevent aggregation of TDP-43. Based on these findings, I hypothesize that aberrant aggregation of TDP-43 in ALS/FTD models is due to alterations in TDP-43:RNA interactions. and that directly manipulating the RNA interactions of TDP-43 can rescue disease phenotypes. The goal of this proposal is to assess if differences in the RNA interactions of TDP-43 are present in disease by 1) examining the ability of short RNAs to prevent aggregation of disease-linked TDP-43 variants in vitro and 2) determining if there are alterations in the RNAs that TDP-43 binds to in ALS/FTD patient-derived neurons versus control neurons. The proposal also aims to 3) determine if administration of short RNAs that bind TDP-43 can rescue disease phenotypes in ALS/FTD patient-derived neurons. The proposed experiments will provide critically lacking information on the interactions between TDP-43 and RNA in disease as well as strategies to target these interactions therapeutically. The proposed experiments will substantially contribute to my training, allowing me to gain expertise in new techniques such as electron microscopy, crosslinking immunoprecipitation (CLIP), and sequencing data analysis. My training environment will foster success for the proposal, combining the biochemical expertise of the Shorter laboratory and the exceedingly collaborative environment at the University of Pennsylvania and within the Neuroscience Graduate Group. These studies will facilitate both my scientific and career goals by supporting my evolution into an academic researcher who develops therapeutics for patients with neurodegenerative disease.