Harnessing the Lysosome Machinery to Counter Metal Toxicity

镉是一种有毒金属，对人体健康有重大危害。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 确定和表征镉解毒的内源性机制， 治疗益处。使用强大的C。elegans模型系统，我们发现暴露于高饮食 锌通过激活主转录调节因子TFEB（在C. elegans）。镉与锌相似，镉暴露引起的转录反应与锌相似 过度我们提出了TFEB在镉解毒过程中发挥重要作用的创新假说 通过增强溶酶体生物发生，导致增加的金属螯合，以及通过增强线粒体 质量.在这里，我们建议严格测试这一假设进行两个具体的目标，使用广泛的 基因和细胞生物学技术。我们将使用C。以及临床相关的小鼠模型 并探索海藻糖作为激活TFEB的潜在治疗剂的用途。目标1：描述 HLH-30/TFEB信号轴在镉暴露过程中的功能和调节线虫和 小鼠本研究将分析镉的积累和溶酶体在镉抗性中的作用。 优雅使用功能获得和功能丧失方法的遗传分析将严格确定 HL-30在镉中毒中的作用镉暴露对hlh-30的调节将在 C.优雅为了确定TFEB在小鼠镉暴露过程中的功能，我们将从基因上产生TFEB。 在肝细胞或肾小管细胞中特异性过表达或缺乏TFEB的靶向小鼠。肝或肾 将在镉暴露后测量损伤。我们将确定镉暴露如何调节TFEB 在鼠肝细胞和近端肾小管细胞中的信号传导。我们将决定， 海藻糖是一种天然存在的非还原糖，可激活TFEB， 治疗镉诱导的小鼠肝毒性和肾毒性。海藻糖酶的基因消除 将进行分析，以确定增加海藻糖生物利用度对镉诱导毒性的功效。 目的2：通过对线粒体和溶酶体的分析，确定镉毒性的细胞机制 重塑我们将确定镉和hLH-30/TFEB活性如何影响C.优雅， 小鼠和哺乳动物细胞。测量将包括线粒体ROS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发挥作用的模型 HLH-30/ TFEB可改善其毒性。我们将确定镉和hlh-30活性如何影响 C.利用最先进的超分辨率技术 显微镜成功完成这些实验将通过阐明以下作用产生很大影响： TFEB在镉抗性中的作用以及海藻糖作为改善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.