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)是两种进行性疾病 与RNA/DNA结合蛋白TAR DNA-DNA相关的一系列疾病的神经退行性疾病 43 kDa结合蛋白(TDP-43)。许多患者表现为中度痴呆症表型 运动神经元病(这里称为FTLD-ALS)。绝大多数单纯的FTLD和ALS病例是零星的 (sFTLD，sALS)，无家族史或已知基因突变。超过50%的FTLD和90%的ALS 2例受累神经元表现出典型的病理改变：过度磷酸化、泛素化的包涵体。 TDP-43。TDP-43病理常见于其他神经退行性疾病，包括阿尔茨海默氏症和 帕金森病，提示一种共同的致病机制，将TDP-43功能障碍与 神经退行性变。TDP-43聚集体通常通过自噬降解，但在FTLD-ALS中，这种机器 失败，导致疾病进展。事实上，一些家族性FTLD-ALS病例是由基因突变引起的 自噬相关蛋白。TDP-43聚集及其神经毒性背后的机制仍然存在 人们对此知之甚少，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，这些变异对散发性疾病的泛化能力可能有限。我们的实验室 确定关键赖氨酸残基(Ac-K145)上的TDP-43乙酰化是TDP-43病理的驱动因素。AC-K145 TDP- 43在SALS脊髓的病理包裹体中检测到。为了更好地模拟零星疾病， 我们使用CRISPR/Cas9技术在内源性小鼠中插入了K145Q乙酰化模拟突变 TardBP基因座(TDP-43K145Q)，以产生散发性FTLD-ALS中TDP-43蛋白病变的新模型。TDP- 43K145Q小鼠表现出显著的病理改变，如年龄依赖性认知障碍和脑积聚 皮质和脊髓中的不溶性TDP-43。该项目旨在确定乙酰化模拟TDP的作用- 43例为神经变性和自噬障碍。目标1将检验衰老会加剧 TDP-43K145Q小鼠的神经退行性表型，采用认知和运动功能的行为分析， 皮质组织的神经病理学评估，以及运动单位功能的电诊断研究。目标2将 验证TDP-43K145Q sFTLD-ALS小鼠原代皮质神经元自噬通量受损的假设， 使用体外衰老范例，结合自噬、生化和活体的药理操作-- 细胞成像技术。这个项目的长期目标是更好地了解TDP背后的机制- 43与神经变性相关，并揭示了治疗干预的机会。这份工作将为我提供 经过翻译和基础科学研究方法的全面培训，我将补充我的 指导神经退行性疾病患者的临床活动的研究。一流的研究 与我的导师、专家托德·科恩博士一起，在北卡罗来纳大学教堂山分校提供临床机会 合作者们，将帮助我开始作为神经退行性疾病生物学领域的领先内科科学家的职业生涯。

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.