Pharmacologic treatment of mitochondrial complex I dysfunction in C. elegans

描述(由申请人提供)：线粒体呼吸链(RC)中复合体I的功能障碍是一组令人惊讶地频繁出现的多系统疾病的基础，这些疾病折磨着所有年龄和种族。相对不能客观地评估调节疾病表现差异很大的细胞机制，在很大程度上阻碍了对受影响患者的有效治疗的评估和实施。我们假设，对线粒体RC功能障碍的细胞后果的药理学调节将为RC功能障碍的常见亚型提供有效的治疗，而不考虑个别的致病原因。这项建议的总体目标是阐明药物调节RC功能障碍的代谢后果的机制，利用线虫动物模型提供的固有研究优势。这项建议的具体目的是：SA1：确定药物调节线虫PPAR/SIRT1途径是否会减弱甚至逆转复杂I功能障碍的代谢后果；SA2：在翻译的线虫复杂I功能障碍动物模型中表征“线粒体鸡尾酒”成分的体内后果。在SA1和SA2的RC复合体I突变体中，将分别研究直接调节PPAR/SIRT1相关信号通路的5种药物以及四大类(维生素、抗氧化剂、复合体I翻译后激活剂和中间代谢调节剂)中常见的15种“线粒体鸡尾酒”成分。将进行生物信息学整合，以确定每种药物对5种体内表型的总体疗效，这两个目标都是：(A)寿命；(B)通过表达谱芯片分析代谢途径；(C)使用稳定同位素通过中间代谢途径进行通量；(D)通过荧光显微镜测定线粒体膜电位；以及(E)通过荧光显微镜测定相对线粒体氧化负荷。通过体内功能分析，对含有RNA干扰诱导的单个PPAR/SIRT1途径基因敲除的复杂I突变体，将探索可识别的表型反应的机制。通过这种方式，目前缺乏疗效或危害的科学或临床证据的对人类RC疾病有假定益处的药物可以在线虫中以低成本和高通量的方式进行客观和非侵入性的筛选。这项转译研究可能会提出特定的治疗靶点和潜在有效的药理制剂，以减轻人类线粒体疾病的全球后遗症。 与公共卫生相关：线粒体复合体I功能障碍发生在一个频率惊人、多种多样且基本上无法治愈的遗传性疾病中，这些疾病困扰着所有年龄和种族。秀丽隐杆线虫提供了一个强大的遗传模型，在该模型中评估复杂的i功能障碍和候选治疗的全球影响。这项转化研究可能会展示有效的药物疗法及其特定的机制，以潜在地减轻人类线粒体疾病的次要后果。

DESCRIPTION (provided by applicant): Dysfunction of complex I in the mitochondrial respiratory chain (RC) underlies an astonishingly frequent group of multi-systemic disorders that afflict all ages and ethnicities. A relative inability to objectively assess cellular mechanisms that mediate widely variable disease manifestations has largely prohibited evaluation and implementation of effective therapies in affected patients. We hypothesize that pharmacologic modulation of the cellular consequences of mitochondrial RC dysfunction will offer effective therapies for common subgroups of RC dysfunction, irrespective of individual pathogenic cause. The overall goal of this proposal is to elucidate mechanisms by which pharmacologic agents modulate the metabolic consequences of RC dysfunction, capitalizing on the inherent investigative advantages that the C. elegans animal model provides. The Specific Aims of this proposal are to: SA1: Determine whether pharmacologic modulation of the PPAR/SIRT1 pathway in C. elegans will attenuate, or even reverse, the metabolic consequences of complex I dysfunction; and SA2: Characterize in vivo consequences of "mitochondrial cocktail" components in a translational C. elegans animal model of complex I dysfunction. Five pharmacologic agents that directly modulate PPAR/SIRT1-related signaling pathways as well as fifteen common "mitochondrial cocktail" components in four major classes (vitamins, antioxidants, complex I post- translational activator, and intermediary metabolic modifiers) will be studied in RC complex I mutants in SA1 and SA2, respectively. Bioinformatics integration will be performed to discern the overall efficacy of each pharmacologic agent on 5 in vivo phenotypes for both aims: (a) lifespan; (b) metabolic pathway profiling by expression microarray analysis; (c) flux through intermediary metabolic pathways using stable isotopes; (d) mitochondrial membrane potential by fluorescence microscopy; and (e) relative mitochondrial oxidant burden by fluorescence microscopy. Mechanisms underlying discernible phenotypic responses will be explored by in vivo functional analyses in complex I mutants harboring RNA interference-induced knockdown for individual PPAR/SIRT1 pathway genes. In this manner, drugs having postulated benefit for human RC disease that currently lack scientific or clinical evidence of either efficacy or harm can be objectively and non- invasively screened in a low-cost and high-throughput fashion in C. elegans. This translational research may suggest specific therapeutic targets and potentially effective pharmacologic agents to mitigate the global sequelae of human mitochondrial disease. PUBLIC HEALTH RELEVANCE: Mitochondrial complex I dysfunction occurs in an astonishingly frequent, varied, and largely untreatable group of genetic disorders afflicting all ages and ethnicities. Caenorhabditis elegans offers a robust genetic model in which to assess the global impact of complex I dysfunction and therapeutic candidates. This translational research may demonstrate effective pharmacologic therapies, and their specific mechanisms, to potentially mitigate the secondary consequences of human mitochondrial disease.