Characterizing Cell and Zebrafish Models of Mitochondrial Complex V Deficiency

线粒体复合物 V 缺陷的细胞和斑马鱼模型的表征

基本信息

批准号：
10172889
负责人：
Rebecca Ganetzky
金额：
$ 16.21万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10172889
关键词：
ATP Hydrolysis ATP Synthesis Pathway Affect Animal Model Animals Ataxia Behavior Biochemical Biochemical Process Bioenergetics Blindness Brain CRISPR/Cas technology Caenorhabditis elegans Cardiomyopathies Cells Child Development Child Health Clinical Confocal Microscopy Defect Development Diabetes Mellitus Diagnosis Disease Electron Transport Embryo Energy-Generating Resources Ensure Exposure to Eye FRAP1 gene Failure Fibroblasts Fishes Foundations Free Radical Formation Functional disorder Future Gel Gene Mutation Genes Genetic Genetic Diseases Genome Genotype Glucose Goals Health Heart Homeostasis Human Impairment In Vitro Incubated Individual Inherited Inner mitochondrial membrane Laboratories Laboratory Research Larva Leucine Liver Membrane Potentials Metabolic Diseases Methodology Methods Mitochondria Mitochondrial DNA Mitochondrial Respiratory Chain Deficiencies Modeling Mutation Myocardium Neuropathy Nuclear Nutrient Nutritional Oligomycins Organ Oxidative Stress Pathogenicity Pathway interactions Patients Pharmaceutical Preparations Pharmacology Phenotype Physiological Physiology Probucol Process Production Pump Regulation Research Retinitis Pigmentosa Role Saccharomyces cerevisiae Severity of illness Signal Transduction Sirolimus Source Specificity Stroke Structure Swimming Testing Therapeutic Effect Tissues Transgenic Organisms Treatment Efficacy Whole Organism Work Zebrafish alpha ketoglutarate base body system clinical assay development clinical diagnostics clinical phenotype detection of nutrient diagnostic assay improved in vitro Model in vivo inhibitor/antagonist mitochondrial membrane mutant novel oligomycin sensitivity-conferring protein response targeted treatment treatment response variant of unknown significance

项目摘要

1. ABSTRACT. Mitochondrial complex V (CV) subunit gene mutations cause a variety of severe metabolic diseases that impair child health and development, with strokes, neuropathy, ataxia, vision loss, and cardiomyopathy. However, much remains to be learned about underlying mechanisms and potential therapies for CV diseases. We Hypothesize that (a) CV subunit mutations evoke assorted changes in its structure and function that may result in a variety of discrete biochemical defects, and (b) CV disease severity is directly influenced by mTORC1 activity and cellular nutrient status. Specific Aims of this work are to [Aim 1] Identify the precise biochemical processes disrupted by CV deficiency; [Aim 2] Characterize the impact of cellular nutrients and nutrient-sensing signaling through the AMPK/mTOR pathway on CV regulation; and [Aim 3] Evaluate organ- level effects of CV deficiency and targeted signaling therapies in a zebrafish vertebrate model animal, given extensive evolutionary conservation of CV. Methods will include in vitro cellular assessment of mitochondrial CV structure and function (blue native gel), mitochondrial physiology (mitochondrial membrane potential, oxidative stress), and activities of central nodes in the integrated nutrient-sensing signaling network in human fibroblasts, using cells from healthy individuals, genetic-based CV diseases, and pharmacologic CV inhibition (oligomycin). Cellular analyses will be performed in response to modulation of cellular nutrients (glucose, leucine) and mTORC1 activity (rapamycin, probucol). We will also generate and characterize pharmacologic (oligomycin) and genetic (morpholino, CRISPR/Cas9) zebrafish model animals of CV disease in which to evaluate the organ-level sequelae of CV diseases as well as the potential therapeutic effects of cellular nutrients and mTORC1 activity regulators on CV functions. These studies will establish the foundation on which to future develop clinical diagnostic assays to confirm CV mutation pathogenicity and evaluate potential treatment responsiveness, and inform organ-specific effects of disease and potential therapies in a novel vertebrate model animal of CV disease.

1. 摘要。线粒体复合物 V (CV) 亚基基因突变会导致多种严重代谢疾病，损害人体健康儿童健康和发育，包括中风、神经病、共济失调、视力丧失和心肌病。然而，关于心血管疾病的潜在机制和潜在疗法，还有很多东西有待了解。我们假设 (a) CV 亚基突变引起其结构和功能的各种变化，这些变化可能导致各种离散的生化缺陷，并且（b）CV疾病的严重程度直接受到以下因素的影响 mTORC1 活性和细胞营养状态。这项工作的具体目标是 [目标 1] 确定准确的 CV 缺乏会扰乱生化过程； [目标 2] 表征细胞营养素的影响和通过 AMPK/mTOR 通路调节 CV 的营养感应信号； [目标 3] 评估器官 CV 缺陷和靶向信号传导治疗对斑马鱼脊椎动物模型动物的水平影响，给定 CV 的广泛进化保守性。方法将包括线粒体的体外细胞评估 CV 结构和功能（蓝色天然凝胶）、线粒体生理学（线粒体膜电位、氧化应激）以及人体综合营养感应信号网络中的中心节点的活动成纤维细胞，使用来自健康个体的细胞、基于遗传的 CV 疾病和药物 CV 抑制（寡霉素）。细胞分析将根据细胞营养物质（葡萄糖、亮氨酸）和 mTORC1 活性（雷帕霉素、普罗布考）。我们还将产生并表征药理学 CV 疾病（寡霉素）和遗传（吗啉、CRISPR/Cas9）斑马鱼模型动物评估心血管疾病的器官水平后遗症以及细胞的潜在治疗效果营养素和 mTORC1 活性调节剂对 CV 功能的影响。这些研究将奠定基础未来将开发临床诊断方法来确认 CV 突变致病性并评估潜力治疗反应，并以新颖的方式告知疾病的器官特异性影响和潜在的疗法 CV病脊椎动物模型动物。