Harnessing the Lysosome Machinery to Counter Metal Toxicity

镉是一种有毒金属，对人类健康有重大危害。迫切需要发展 针对损伤的细胞和分子机制的治疗策略。我们的长期目标是 识别和表征镉的内源解毒机制，可以适应 治疗效果。使用强大的线虫模型系统，我们发现接触高饮食 锌通过激活主要转录调节因子TFEB刺激溶酶体的生物发生。 优雅女装)。镉与锌相似，接触镉会引起与锌相似的转录反应。 太过分了。我们提出了TFEB在镉的解毒中起重要作用的创新假说 通过增强溶酶体的生物发生，导致金属封存增加，并通过增强线粒体 质量。在这里，我们建议通过使用大范围的两个特定目标来严格地检验这一假设 遗传和细胞生物学技术。我们将利用线虫和临床相关的小鼠模型 并探索海藻糖作为一种激活TFEB的潜在治疗方法。目标1：描述 线虫和线虫镉暴露过程中HLH-30/TFEB信号轴的功能和调节 老鼠。我们将分析Cd的积累和溶酶体在Cd抗性中的作用。 优雅女装。使用功能增益和功能丧失方法的遗传分析将严格确定 HLH-30在镉中毒中的作用。镉暴露对HLH-30的调节将在 线虫。为了确定TFEB在小鼠镉暴露过程中的功能，我们将从基因上产生 靶向肝细胞或肾小管上皮细胞过度表达或缺乏TFEB的小鼠。肝或肾 镉暴露后的损伤将被测量。我们将确定镉暴露如何监管TFEB 小鼠肝细胞和近端肾小管细胞中的信号转导。我们将决定是否对 海藻糖是一种天然存在的非还原糖，可激活TFEB，将有效预防和 治疗镉所致小鼠肝肾毒性。海藻糖酶的遗传消融 将进行分析，以确定增加海藻糖生物利用度对镉诱导的毒性的效果。 目的2：通过分析线粒体和溶酶体来确定镉毒性的细胞机制 改建。我们将确定镉和HLH-30/TFEB活性如何影响线虫的线粒体， 老鼠和哺乳动物细胞。测量将包括线粒体ROS的生成、质量、超微结构、 和功能。这些研究将严格检验线粒体损伤在镉中起作用的模型。 毒性作用可被HLH-30/TFEB改善。我们将确定镉和HLH-30活性如何影响 利用最新的超分辨技术在线虫和哺乳动物细胞中进行溶酶体结构重构 显微镜。这些实验的成功完成将产生很大的影响，因为它阐明了 TFEB对镉的抗性和海藻糖作为一种缓解镉毒性的方法的可行性。

Cadmium is a toxic metal and a significant human health hazard. There is an urgent need to develop therapeutic strategies that target the cellular and molecular mechanisms of injury. Our long-term goal is to identify and characterize endogenous mechanisms of cadmium detoxification that can be adapted for therapeutic benefit. Using the powerful C. elegans model system, we discovered that exposure to high dietary zinc stimulates lysosome biogenesis by activating the master transcriptional regulator, TFEB (HLH-30 in C. elegans). Cadmium is similar to zinc, and cadmium exposure causes a transcriptional response similar to zinc excess. We propose the innovative hypothesis that TFEB plays an important role in cadmium detoxification by enhancing lysosome biogenesis, leading to increased metal sequestration, and by enhancing mitochondrial quality. Here we propose to rigorously test this hypothesis by conducting two Specific Aims using a wide range of genetic and cell biology techniques. We will exploit C. elegans as well as the clinically relevant mouse model and explore the use of trehalose as a potential therapeutic that activates TFEB. Aim 1: Characterize the function and regulation of the HLH-30/TFEB signaling axis during cadmium exposure in C. elegans and mice. We will analyze accumulation of cadmium and the role of lysosomes in cadmium resistance in C. elegans. Genetic analysis using gain-of-function and loss-of-function approaches will rigorously determine the function of hlh-30 during cadmium toxicity. The regulation of hlh-30 by cadmium exposure will be determined in C. elegans. To establish the function of TFEB during cadmium exposure in mice, we will generate genetically targeted mice that over-express or lack TFEB specifically in hepatocytes or in renal tubular cells. Liver or renal injury will be measured after cadmium exposure. We will determine how cadmium exposure regulates TFEB signaling in murine hepatocytes and proximal renal tubular cells. We will determine if administration of trehalose, a naturally occurring non-reducing sugar which activates TFEB, will be effective in preventing and treating cadmium-induced hepatotoxicity and nephrotoxicity in mice. Genetic ablation of the enzyme trehalase will be analyzed to determine the efficacy of increased trehalose bioavailability on cadmium-induced toxicity. Aim 2: Determine cellular mechanisms of cadmium toxicity by analyzing mitochondria and lysosome remodeling. We will determine how cadmium and hlh-30/TFEB activity effect mitochondria in C. elegans, mice, and mammalian cells. Measurements will include mitochondrial ROS generation, mass, ultrastructure, and function. These studies will rigorously test the model that mitochondrial damage plays a role in cadmium toxicity and can be ameliorated by HLH-30/ TFEB. We will determine how cadmium and hlh-30 activity effect lysosomal structural remodeling in C. elegans and mammalian cells using state-of-the-art super resolution microscopy. Successful completion of these experiments will have a high impact by elucidating the role of TFEB in cadmium resistance and the viability of trehalose as an approach to ameliorate cadmium toxicity.