Minority Predoctoral Fellowship Program

描述(申请人提供)：肠道Na/H交换器NHx-2调节线虫的细胞pH和营养吸收，功能丧失会导致脂肪储存减少和寿命延长。为了弄清这些表型在机制上是pH改变的结果还是热量限制的结果，我们进行了全球RNAi筛选，寻找改变肠道细胞pH的其他靶点。在45个克隆中，几乎四分之一的克隆确定了线粒体的功能；特别是ETC复合体I被很好地代表了，线粒体中涉及的蛋白质也是如此！核糖体的功能和调节线粒体的形态。进一步的测试表明，有几个克隆至少在一定程度上是以细胞非自主方式起作用的。在这里，我们建议首先使用一种新的细胞特异性RNAi系统来识别哪些细胞中发生了非自主功能。然后，我们将检测相关细胞中RNAi后体内线粒体的氧化还原电位、pH、形态和能量产生，以及靶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定位每个克隆，以直接测试细胞自主性。这些实验的结果将提供关于线粒体如何影响肠道细胞pH的机械性见解，以及这种耦合如何影响脂肪代谢和衰老。 在过去的十年里，肥胖已经成为一个紧迫的健康问题，特别是在年轻人中，也是患糖尿病和心脏病的主要风险因素之一。我们的研究旨在确定满足细胞能量需求的线粒体如何影响保存完好的遗传模式生物体中的pH归属。由于细胞pH值与营养吸收、脂肪积累和寿命有关，这种功能耦合可能代表着一种独特的细胞交流方式和抗肥胖治疗的新靶点。

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.