Characterizing Cell and Zebrafish Models of Mitochondrial Complex V Deficiency

1.摘要。 线粒体复合物V（CV）亚单位基因突变导致多种严重的代谢疾病， 儿童健康和发育，中风，神经病，共济失调，视力丧失和心肌病。然而，在这方面， 关于心血管疾病的潜在机制和潜在治疗方法仍有许多需要了解的地方。我们 假设（a）CV亚单位突变引起其结构和功能的各种变化， 导致各种离散的生化缺陷，和（B）CV疾病严重程度直接受以下因素影响： mTORC1活性和细胞营养状态。这项工作的具体目标是[目标1]确定精确的 生物化学过程被CV缺乏破坏;[目的2]表征细胞营养素的影响， 通过AMPK/mTOR通路对CV调节的营养感应信号传导;[目的3]评价器官- 在斑马鱼脊椎动物模型动物中CV缺乏和靶向信号治疗的水平效应， CV的广泛进化保守性。方法将包括体外细胞评估线粒体 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），以及人类整合营养传感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、遗传性CV疾病和药理学CV抑制的细胞 （寡霉素）。细胞分析将响应于细胞营养物（葡萄糖， 亮氨酸）和mTORC 1活性（雷帕霉素，普罗布考）。我们还将生成和表征药理学 CV疾病的遗传（寡霉素）和遗传（吗啉代，CRISPR/Cas9）斑马鱼模型动物，其中 评估CV疾病的器官水平后遗症以及细胞治疗的潜在治疗效果 营养素和mTORC 1活性调节剂对CV功能的影响。这些研究将为 未来将开发临床诊断检测方法，以确认CV突变的致病性并评估潜力 治疗反应性，并告知疾病的器官特异性效应和新的潜在治疗方法。 CV疾病的脊椎动物模型动物。

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.