Minority Predoctoral Fellowship Program

描述（由申请人提供）：肠道Na+/H+交换剂NHX-2调节线虫的细胞pH和营养吸收，功能丧失导致脂肪储存减少和寿命延长。为了了解这些表型是否在机制上是pH改变或热量限制的结果，对改变肠细胞pH值的其他靶点进行了全球RNAi筛选。在45个克隆中，近四分之一的克隆在线粒体中发现了功能；特别是ETC复合体I，线粒体中涉及的蛋白质也很有代表性！核糖体的功能及其对线粒体形态的调节。进一步的测试表明，几个克隆至少在部分细胞非自主行为。我们在此建议首先使用一种新的细胞特异性RNAi系统来确定在哪些细胞中发生非自主功能。然后，我们将在相关细胞中检测RNAi后线粒体氧化还原电位、pH值、形态和体内能量产生，以及目标蠕虫的脂肪摄取和寿命。最后，我们将在培养的线虫胚胎细胞中针对每个克隆直接测试细胞自主性。这些实验的结果将提供线粒体如何影响肠道细胞pH的机制见解，以及这种耦合如何影响脂肪代谢和衰老。

DESCRIPTION (provided by applicant): The intestinal Na+/H+ exchanger NHX-2 regulates both cellular pH and nutrient uptake in the nematode C. elegans, and loss-of-function leads to decreased fat stores and increased lifespan. In order to ask whether these phenotypes are mechanistically a result of altered pH or of caloric restriction, a global RNAi screen for other targets that alter intestinal cellular pH was performed. Almost a quarter of the 45 clones identified function in the mitochondria; in particular, ETC complex I was well-represented, as were proteins involved in mitochondria! ribosome function and in regulating mitochondrial morphology. Further testing suggested that several clones act at least in part cell non-autonomously. We propose here first to identify in which cells the non-autonomous functions occur using a novel system for cell specific RNAi. Then we will examine mitochondrial redox potential, pH, morphology, and energy production in vivo following RNAi in the relevant cells, as well as fat uptake and longevity in the targeted worms. Finally, we will target each clone in cultured nematode embryonic cells to directly test cell autonomy. The results of these experiments will provide mechanistic insights into how mitochondria influence intestinal cellular pH, as well as how this coupling influences fat metabolism and aging. Obesity has become a pressing health concern, particularly in younger people, over the last decade, and is one of the leading risk factors for developing diabetes and heart disease. Our research is aimed at defining how mitochondria, which meets the energy demands of the cell, influences pH homestasis in a well- conserved genetic model organism. Since cellular pH is linked to nutrient uptake, fat accumulation, and longevity, this functional coupling may represent a unique means of cellular communication and a novel target for anti-obesity therapeutics.