A novel mouse model of TDP-43 Proteinopathy in FTLD-ALS: Elucidating the role of TDP-43 acetylation in neurodegeneration and proteostasis impairment

项目摘要 额颞叶变性（FTLD）和肌萎缩侧索硬化症（ALS）是两种进行性 神经退行性疾病对与RNA/DNA结合蛋白TAR DNA相关的疾病谱的影响- 43 kDa结合蛋白（TDP-43）。许多患者表现出痴呆的中间表型， 运动神经元疾病（这里称为FTLD-ALS）。绝大多数纯FTLD和ALS病例是散发的 （sFTLD，sALS），没有家族史或已知的基因突变。超过50%的FTLD和90%的ALS 例表现出受影响的神经元的特征性病理：过度磷酸化，泛素化的包涵体， TDP-43 TDP-43病理学通常在其他神经退行性疾病中观察到，包括阿尔茨海默病和阿尔茨海默病。 帕金森氏病，表明一种共同的致病机制，连接TDP-43功能障碍和 神经变性TDP-43聚集体通常通过自噬降解，但在FTLD-ALS中，这种机制 失败，导致疾病进展。事实上，一些家族性FTLD-ALS病例是由以下突变引起的： 自噬相关蛋白TDP-43聚集及其神经毒性的机制仍然存在 我们对此知之甚少，尤其是在散发性疾病中。大多数动物模型依赖于疾病的过度表达- 相关的遗传变异;然而，这些可能局限于散发性疾病的普遍性。我们实验室 鉴定了在关键赖氨酸残基（Ac-K145）处乙酰化的TDP-43作为TDP-43病理学的驱动因素。Ac-K145 TDP- 43在sALS脊髓的病理包涵体中检测到。为了更好地模拟散发性疾病， 我们使用CRISPR/Cas9技术在内源性小鼠中插入K145 Q乙酰化模拟突变， Tardbp基因座（TDP-43 K145 Q），以产生散发性FTLD-ALS中TDP-43蛋白质病的新模型。TDP- 43 K145 Q小鼠表现出标志性病理学，如年龄依赖性认知障碍和 不溶性TDP-43在皮质和脊髓中。该项目旨在确定乙酰化模拟TDP的作用， 43例神经变性和自噬损伤。目标1将检验衰老加剧 TDP-43 K145 Q小鼠的神经退行性表型，使用认知和运动功能的行为测定， 皮质组织的神经病理学评估和运动单位功能的电诊断研究。目标2将 检验TDP-43 K145 Q sFTLD-ALS小鼠的原代皮层神经元中自噬通量受损的假设， 使用体外衰老范例以及自噬和生物化学和活体的药理学操作， 细胞成像技术。该项目的长期目标是更好地了解TDP背后的机制- 43-相关的神经变性，并揭示治疗干预的机会。这项工作将为我提供 在转化和基础科学研究方法的全面培训，我将补充我的 研究与指导临床活动照顾神经退行性疾病患者。顶级研究 和临床的机会，可在圣查佩尔山，旁边我的导师，博士托德科恩，和专家 合作者，将有助于启动我的职业生涯作为一个领先的医生，科学家在神经退行性疾病生物学。

Project Summary Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are two progressive neurodegenerative disorders on a spectrum of disease related to the RNA/DNA binding protein TAR DNA- binding Protein of 43 kDa (TDP-43). Many patients demonstrate an intermediate phenotype of dementia with motor neuron disease (here called FTLD-ALS). The vast majority of pure FTLD and ALS cases are sporadic (sFTLD, sALS), with no family history or known genetic mutation. More than 50% of all FTLD and 90% of all ALS cases manifest a characteristic pathology in affected neurons: hyperphosphorylated, ubiquitinated inclusions of TDP-43. TDP-43 pathology is often observed in other neurodegenerative disorders, including Alzheimer’s and Parkinson’s Diseases, suggesting a common pathogenic mechanism linking TDP-43 dysfunction and neurodegeneration. TDP-43 aggregates are normally degraded by autophagy, but in FTLD-ALS this machinery fails, contributing to disease progression. In fact, some familial FTLD-ALS cases are caused by mutations in autophagy-related proteins. The mechanisms behind TDP-43 aggregation and the neurotoxicity it imparts remain poorly understood, particularly in sporadic disease. Most animal models rely on overexpression of disease- associated genetic variants; however, these may be limited in generalizability to sporadic disease. Our lab identified TDP-43 acetylated at a key lysine residue (Ac-K145) as a driver of TDP-43 pathology. Ac-K145 TDP- 43 is detected in the pathologic inclusions in sALS spinal cord. With the goal of better modeling sporadic illness, we used CRISPR/Cas9 technology to insert a K145Q acetylation-mimic mutation in the endogenous mouse Tardbp locus (TDP-43K145Q) to generate a novel model of TDP-43 proteinopathy in sporadic FTLD-ALS. TDP- 43K145Q mice show hallmark pathologies, such as age-dependent cognitive impairment and accumulation of insoluble TDP-43 in the cortex and spinal cord. This project aims to determine the role of acetylation-mimic TDP- 43 in neurodegeneration and autophagy impairment. Aim 1 will test the hypothesis that aging exacerbates the neurodegenerative phenotype in TDP-43K145Q mice, using behavioral assays of cognitive and motor function, neuropathologic assessment of cortical tissue, and electrodiagnostic studies of motor unit function. Aim 2 will test the hypothesis that autophagic flux is impaired in primary cortical neurons of TDP-43K145Q sFTLD-ALS mice, using an in vitro aging paradigm alongside pharmacologic manipulation of autophagy and biochemical and live- cell imaging techniques. The long-term goal of this project is to better understand the mechanisms behind TDP- 43-related neurodegeneration and reveal opportunities for therapeutic intervention. This work will provide me with comprehensive training in both translational and basic science research methods, and I will complement my research with mentored clinical activities caring for neurodegenerative disease patients. The top-tier research and clinical opportunities available at UNC Chapel Hill, alongside my mentor, Dr. Todd Cohen, and expert collaborators, will help launch my career as a leading physician-scientist in neurodegenerative disease biology.