Analysis of autophagy and mitochondrial homeostasis in a human iPS model of NCL

NCL 人类 iPS 模型中自噬和线粒体稳态的分析

• 批准号: 8509955
• 负责人: John Francois Staropoli
• 金额: $ 15.87万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8509955
• 关键词: Address; Adolescent; Affect; Alzheimer' s Disease; Autophagocytosis; Basic Science; Bioenergetics; Biological Assay; Biology; Blindness; Cell Culture Techniques; Cell Line; Cell model; Cells; Cessation of life; Child; Childhood; Clinic; Clinical; Clinical Pathology; Collaborations; Complement; Complex; Core Facility; Databases; Defect; Disease; Electron Microscopy; Enzymes; Event; Family member; Fibroblasts; Foundations; Functional disorder; Funding; General Hospitals; Genes; Genetic; Genetic Research; Genus Hippocampus; Homeostasis; Human; Human Genetics; Huntington Disease; Image; Impaired cognition; Impairment; Inborn Genetic Diseases; Inherited; Institutional Review Boards; Joints; Knock-in Mouse; Label; Life; Lysosomal Storage Diseases; Massachusetts; Measurement; Mentors; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Proton-Translocating ATPases; Modeling; Molecular; Morphology; Motor; Movement Disorders; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neuronal Ceroid-Lipofuscinosis; Neurons; Oxidative Phosphorylation; Oxidative Stress; Parkinson Disease; Pathologic; Pathology; Pathway interactions; Patients; Physicians; Polymerase; Process; Proteins; Quality Control; Reporter; Research; Resources; Retinal Degeneration; Role; Scientist; Seizures; Solid; Spielmeyer-Vogt Disease; Staging; System; Technology; Therapeutic; Therapeutic Intervention; Training; Training Activity; Transcriptional Regulation; Transmission Electron Microscopy; Vacuole; Variant; Work; biobank; career; career development; cell type; cellular imaging; effective intervention; extracellular; induced pluripotent stem cell; insight; interest; mitochondrial autophagy; molecular pathology; nervous system disorder; neuron loss; next generation sequencing; novel; precursor cell; premature; proband; programs; progressive neurodegeneration; public health relevance; repository; respiratory; stem; therapeutic target; tissue culture; tool

DESCRIPTION (provided by applicant): As a physician-scientist with training in clinical and molecular pathology, I plan to pursue my long-standing interest in inherited forms of neurodegeneration and develop an independent, R01-funded academic career at the Massachusetts General Hospital (MGH). My graduate background on the relationship between protein processing defects and neurodegeneration provides a solid foundation for my proposed work on the role of autophagy in the neuronal ceroid lipofuscinoses (NCLs; also known as Batten Disease). The MGH-CHGR (Center for Human Genetic Research) Joint Program in NCL Disorders will serve as the focal point of my career development and training activities. The Joint Program includes (1) the basic research labs of my primary mentor Dr. Susan Cotman and my co-mentor Dr. Marcy MacDonald, (2) a clinical component (MGH Batten Disease Center of Excellence) directed by my co-mentor Dr. Katherine Sims, (3) a reference lab for NCLs and other rare neurologic disorders, and (4) a tissue culture core facility that includes an IRB-approved bio repository of fibroblast and lymphoblastic cell lines from NCL probands and family members. The NCLs comprise a group of at least 12 distinct, currently untreatable lysosomal storage diseases with clinical features that include progressive motor and cognitive decline, retinal degeneration and visual loss in most cases, seizures, movement disorder, and eventual premature death. I propose to build on insights gained from a genetically precise knock-in murine model of NCL that replicates the most common mutation among the NCL disorders. Cerebellar cell lines derived from this model have demonstrated that end-stage pathology is preceded by impairments in autophagy as well as by altered mitochondrial morphology, decreased basal ATP levels, increased sensitivity to oxidative stress, and altered transcriptional regulation of mitochondrial oxidative phosphorylation under basal conditions. In this application, I propose to use novel, human iPS-derived neuronal culture models of NCL to address the hypothesis that impaired autophagic turnover of mitochondria (mitophagy), and thus impaired mitochondrial quality control, is a critical early component of pathophysiology in NCL patients. Specifically, I will investigate autophagy and mitochondrial biology in this neuronal system by electron microscopy, dynamic imaging of mitophagy, and measurement of bioenergetics parameters, including basal and maximal respiratory capacity. In addition, I will use an extensive database that I developed to identify atypical and molecularly undefined NCL patients with early clinic pathologic evidence of mitochondrial disease. I will use next- generation sequencing technology to identify potential causal variants in a core subset of these patients, an approach that will likely generate new hypotheses about the genetic interaction between NCL and mitochondrial biology and suggest novel targets for therapeutic intervention.

描述（由申请人提供）：作为一名接受过临床和分子病理学培训的医生-科学家，我计划在马萨诸塞州总医院（MGH）继续我对遗传性神经变性形式的长期兴趣，并发展独立的R 01资助的学术生涯。我在蛋白质加工缺陷和神经变性之间的关系的研究生背景提供了一个坚实的基础，我提出的工作对神经元蜡样脂褐质沉积症（NCL的作用自噬;也被称为巴顿病）。MGH-CHGR（人类遗传研究中心）NCL疾病联合项目将作为我职业发展和培训活动的重点。联合计划包括（1）我的主要导师Susan Cotman博士和我的共同导师Marcy MacDonald博士的基础研究实验室，（2）临床部分（MGH Batten疾病卓越中心）由我的共同导师凯瑟琳西姆斯博士指导，（3）NCL和其他罕见神经系统疾病的参考实验室，和（4）组织培养核心设施，包括IRB批准的来自NCL先证者和家族成员的成纤维细胞和淋巴母细胞系的生物储存库。 NCL包括一组至少12种不同的目前无法治疗的溶酶体贮积病，其临床特征包括进行性运动和认知下降、视网膜变性和视力丧失（大多数情况下）、癫痫发作、运动障碍和最终过早死亡。我建议建立从遗传精确敲入小鼠模型的NCL复制最常见的突变之间的NCL疾病的见解。从该模型衍生的小脑细胞系已经证明，终末期病理之前是自噬损伤以及线粒体形态改变，基础ATP水平降低，对氧化应激的敏感性增加，以及基础条件下线粒体氧化磷酸化的转录调节改变。 在本申请中， 我建议使用新的，人类iPS衍生的神经元培养模型的NCL来解决这一假设，即受损的线粒体自噬营业额（线粒体自噬），从而受损的线粒体质量控制，是一个关键的早期组成部分的病理生理学在NCL患者。具体来说，我将研究自噬和线粒体生物学在这个神经元系统的电子显微镜，动态成像的线粒体自噬，和测量的生物能量学参数，包括基础和最大呼吸能力。此外，我将使用一个广泛的数据库，我开发，以确定非典型和分子未定义的NCL患者与线粒体疾病的早期临床病理证据。我将使用下一代测序技术来识别这些患者的核心子集中的潜在因果变异，这种方法可能会产生关于NCL和线粒体生物学之间遗传相互作用的新假设，并提出治疗干预的新靶点。