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

项目总结 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变问题。肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTD)是一种阿尔茨海默病相关痴呆(ADRD)，都是以神经元丢失为特征的致死性神经退行性疾病。ALS的主要特征是运动神经元丢失导致的运动障碍，而FTD的主要症状包括由于额叶和颞叶皮质神经元丢失而导致的个性、行为和语言的改变。ALS和FTD存在于一个疾病谱上，其中一些患者同时具有这两种疾病的特征。几乎所有ALS患者和大约一半的FTD患者共有的一个分子特征是RNA结合蛋白TDP-43的病理聚集。最近发现，TDP-43通过与RNA结合而增加了其溶解度。我的初步数据利用纯化的TDP-43的体外聚集分析表明，TDP-43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。该建议的目标是评估TDP-43的RNA相互作用是否在疾病中存在差异，方法是：1)检测短RNA在体外防止疾病相关TDP-43变体聚集的能力；2)确定在ALS/FTD患者来源的神经元和对照神经元中，TDP-43结合的RNA是否有变化。该提案还旨在3)确定注射结合TDP-43的短RNA是否可以挽救ALS/FTD患者来源的神经元的疾病表型。拟议的实验将提供关于TDP-43和RNA在疾病中的相互作用以及针对这些相互作用的治疗策略的严重缺乏的信息。拟议中的实验将对我的培训有很大帮助，使我能够获得新技术方面的专业知识，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提案的成功，将较短实验室的生化专业知识与宾夕法尼亚大学和神经科学研究生小组内高度合作的环境相结合。这些研究将促进我的科学和职业目标，支持我演变为一名学术研究人员，为神经退行性疾病患者开发治疗方法。

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.