Mechanisms of Neurodegeneration in KIF5A ALS/FTD

KIF5A ALS/FTD 神经退行性变的机制

• 批准号: 10740732
• 负责人: Jonathan Robert Brent
• 金额: $ 20.82万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740732
• 关键词: ALS patients; Adaptor Signaling Protein; Affect; Age; Amyotrophic Lateral Sclerosis; Applications Grants; Axon; Axonal Transport; Behavior; Behavioral; Binding; Biochemical; Biology; Brain; C-terminal; CRISPR/Cas technology; Cells; Cessation of life; Complex; Cytoplasmic Granules; Cytoskeleton; Data; Defect; Development; Development Plans; Disease; Distal; Doctor of Medicine; Doctor of Philosophy; Exclusion; Exons; Fluorescence Microscopy; Foundations; Frontotemporal Dementia; Functional disorder; Funding; Future; Genetic; Genetic Engineering; Genetically Engineered Mouse; Gliosis; Goals; Health; Homeostasis; Human; Hyperactivity; Image; Imaging Techniques; In Vitro; Induced pluripotent stem cell derived neurons; Kinesin; Knowledge; Label; Language; Longevity; Lysosomes; Maintenance; Manuscripts; Mentors; Mentorship; Microtubules; Mitochondria; Morphology; Motor; Motor Neuron Disease; Motor Neurons; Mus; Mutation; Nerve Degeneration; Neurites; Neurodegenerative Disorders; Neuromuscular Junction; Neurons; Organelles; Pathogenesis; Pathogenicity; Pathology; Patients; Pattern; Physicians; Post-Translational Protein Processing; Preparation; Production; Proteins; Proteomics; Publications; RNA; RNA Interference; RNA Splicing; Records; Regulation; Research; Resources; Role; Scientist; Site; Slide; Solid; Spinal Cord; Splice-Site Mutation; Structure; Synapses; Techniques; Testing; Therapeutic; Time; Tissues; Training; Transmission Electron Microscopy; Universities; Western Blotting; Work; age related; aged; career development; causal variant; cell motility; cognitive function; cohort; confocal imaging; frontotemporal lobar dementia amyotrophic lateral sclerosis; gain of function; gain of function mutation; genetic regulatory protein; human disease; in vivo; induced pluripotent stem cell; insight; live cell imaging; mouse model; mutant; neurofilament; neuron loss; novel; programs; protein TDP-43; protein aggregation; rational design; skills; small molecule; tool; trafficking; ultra high resolution

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the dysfunction and death of motor neurons. Some ALS patients develop widespread neuronal damage leading to Frontotemporal Dementia (FTD), which is characterized by progressive behavioral change and language decline. Significant overlap exists between ALS and FTD regarding the genetic causes, suggesting that they may share common pathogenic mechanisms. Recently, mutations in motor protein, KIF5A, were identified in patients with ALS, ALS/FTD, and FTD alone. These concentrate in the splicing regulatory sites flanking exon 27 leading to its exclusion and abnormal KIF5A C-terminal domain structure. Our preliminary findings suggest that KIF5A ALS/FTD mutations result in the production of a constitutively active protein that causes disease through toxic gain of function. This proposal will test the hypothesis that KIF5A ALS/FTD is caused by gain of function of KIF5A activity in axonal transport and cytoskeletal regulation. To ensure that our work will be highly disease relevant we will utilize human iPSC derived motor neurons and mouse models harboring KIF5A ALS/FTD mutation. In Aim 1: we will define the effects of KIF5A ALS mutations on the assembly and motility of motor/cargo complexes. Specifically, we will utilize live-cell imaging of cargo motility, assessment of cargo distribution by IHC and transmission electron microscopy (TEM), candidate-based biochemical assessment of interactions with cargo adaptors, and unbiased assessment of interactions with potentially novel cargo adaptors and regulatory proteins using proximity labelling. In Aim 2: we will characterize the effects of KIF5A ALS/FTD mutation on cytoskeletal structure using IHC and TEM, live-imaging of MT dynamics and sliding, and analysis of MT post-translational modifications. In Aim 3: we will develop a mouse model of KIF5A ALS/FTD and perform analysis of longevity, behavior, and pathology. The mouse model will be an invaluable resource to validate findings from our in vitro studies and to advance knowledge regarding key features of neurodegeneration in KIF5A ALS/FTD. My ultimate goal is to become a successful physician-scientist with an independent research program investigating the mechanisms of neurodegeneration in motor neuron disease with a specific focus on axonal transport and cytoskeletal dynamics. Northwestern University has unique strengths in the study of ALS, FTD, and cytoskeletal biology and an outstanding commitment to the development of physician-scientists. The mentorship team includes renowned scientists with strong records in mentorship including Han-Xiang Deng M.D., Ph.D. (Primary Mentor) and Robert Kalb M.D (co-mentor). The career development plan focuses on broadening the awardee’s portfolio of research publications and presentations, which will be necessary for successful competition for RO1 funding. Career development activities will bolster grantsmanship and manuscript preparation skills and provide extensive didactic training in advanced imaging techniques, proteomics, cytoskeletal biology and ALS/FTD degenerative mechanisms.

肌萎缩侧索硬化症(ALS)是一种以运动功能障碍和死亡为特征的致命疾病 神经元。一些ALS患者出现广泛的神经元损伤，导致额颞部痴呆(FTD)， 它的特点是逐渐的行为变化和语言的衰退。存在显著的重叠 ALS和FTD之间关于遗传原因的关系，表明它们可能具有共同的致病机理 机制。最近，在ALS、ALS/FTD患者中发现了运动蛋白KIF5A突变。 只有FTD。这些基因集中在外显子27两侧的剪接调控位点，导致其被排除和 KIF5A C-末端结构域结构异常。我们的初步发现表明KIF5A ALS/FTD突变 导致一种具有结构性活性的蛋白质的产生，该蛋白质通过有毒的功能获得而致病。这 提案将检验KIF5A ALS/FTD是由轴突中KIF5A活动的功能增强引起的假设 运输和细胞骨架调节。为了确保我们的工作与疾病高度相关，我们将利用人类 IPSC来源于运动神经元和携带KIF5A ALS/FTD突变的小鼠模型。在目标1中：我们将定义 KIF5A ALS突变对马达/货运复合体组装和运动的影响具体来说，我们将 利用活细胞成像的货物运动，评估货物分布的IHC和传输电子 显微镜(TEM)，基于候选人的与货物转接器相互作用的生化评估，且无偏见 使用邻近标记评估与潜在的新的货物适配器和调节蛋白的相互作用。 在目标2中，我们将利用IHC和IHC来表征KIF5A ALS/FTD突变对细胞骨架结构的影响 TM、MT动力学和滑动的实时成像，以及MT平移后修饰的分析。《目标3：我们》 将建立KIF5A ALS/FTD小鼠模型，并进行寿命、行为和病理分析。这个 小鼠模型将是验证我们体外研究结果并推动其发展的宝贵资源 关于KIF5A ALS/FTD中神经退行性变的关键特征的知识。我的最终目标是成为一名 成功的内科科学家，拥有独立的研究计划，研究 运动神经元病中的神经变性，特别关注轴突运输和细胞骨架动力学。 西北大学在ALS、FTD和细胞骨架生物学的研究方面具有独特的优势 对医学科学家的发展做出了杰出的承诺。导师团队包括著名的 具有良好导师记录的科学家包括邓汉祥医学博士、博士(主要导师)和罗伯特 卡尔布医学博士(共同导师)。职业发展计划的重点是扩大获奖者的研究组合 出版物和演示文稿，这将是成功竞争RO1资金所必需的。职业生涯 发展活动将加强赠款和手稿准备技能，并提供广泛的 先进成像技术、蛋白质组学、细胞骨架生物学和ALS/FTD退行性疾病的教学培训 机制。