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

• 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
• 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

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。 线粒体复合体V(CV)亚单位基因突变可导致多种严重的代谢性疾病 儿童健康和发育，有中风、神经病变、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的治疗方法，仍有许多有待了解。我们 假设(A)CV亚单位突变引起其结构和功能的各种变化，这可能 导致各种离散的生化缺陷，以及(B)心血管疾病的严重程度直接受 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 [目标2]表征细胞营养和营养物质的影响 通过AMPK/mTOR途径调节CV的营养传感信号；和[目标3]评估器官- 斑马鱼脊椎动物模型中CV缺乏和靶向信号治疗的水平效应 CV的广泛进化守恒。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)，以及整合的营养感知信号网络中中央节点的活动 成纤维细胞，使用来自健康个体的细胞，基于遗传的心血管疾病，以及药物的心血管抑制 (寡霉素)。细胞分析将响应细胞营养的调节(葡萄糖， 亮氨酸)和mTORC1活性(雷帕霉素、普罗布考)。我们还将生成和表征药理学 (寡霉素)和遗传性(Morolino，CRISPR/Cas9)斑马鱼CV病模型动物 评价心血管疾病的器官水平后遗症以及细胞因子的潜在治疗效果 营养素和mTORC1活性调节剂对心血管功能的影响。这些研究将为以下方面奠定基础 这为今后开发临床诊断方法以确认CV突变的致病性和评估其潜力奠定了基础 治疗反应性，并在一项新的疾病和潜在治疗方法中告知特定器官的影响 心血管疾病的脊椎动物模型动物。