Autophagy and Drug-Induced Liver Injury

项目总结/摘要 对乙酰氨基酚（APAP）肝毒性是急性肝功能衰竭的最常见原因， 美国的然而，目前还没有针对APAP诱导的肝损伤的有效治疗策略。 特别是对于晚期患者。众所周知，APAP过量后，N-乙酰基-p- 苯醌亚胺（NAPQI），APAP的反应性代谢物，与细胞和线粒体蛋白结合， 在细胞谷胱甘肽耗尽后形成APAP-蛋白加合物（APAP-AD）， 线粒体功能障碍、氧化应激和随后的坏死。在上一个融资周期，我们 证明了自噬的激活，溶酶体降解途径的细胞适应性反应， 通过清除APAP-AD和受损的线粒体来保护APAP诱导的肝损伤。肝脏是一种非常 有活力的器官，有能力在受伤后修复和再生。我们还证明， 线粒体生物合成可以改善肝再生和从APAP诱导的肝损伤中恢复。 重要的是，我们的初步数据表明，转录因子EB（TFEB），一个主调节因子， 控制溶酶体的自噬和线粒体的再生的生物发生， 在APAP诱导的肝损伤过程中。因此，此次竞争性R 01更新的主要目标是 了解APAP损害肝脏中TFEB信号传导的分子机制。我们的中央 假设TFEB活化将导致溶酶体和线粒体的生物合成增加 抑制APAP诱导的肝损伤的进展并促进肝再生。 提出了两个具体的目标：1）确定APAP损害TFEB介导的 肝细胞中溶酶体和线粒体的生物发生;和2）确定TFEB 通过增加溶酶体的生物合成促进APAP诱导的肝损伤的恢复， 线粒体拟议的研究是创新的概念，转录程序， 在APAP诱导的肝损伤中，自噬-溶酶体途径和线粒体生物发生都受损。 我们将利用新的遗传动物模型，如肝脏特异性TFEB KO小鼠和腺相关病毒- 介导的TFEB和PGC-1α过表达的方法，专门研究TFEB和PGC-1α的作用。 PGC-1α在自噬清除损伤线粒体和促进新线粒体生物合成中的作用 逆转APAP诱导的肝损伤。此外，我们还将利用新开发的新分子工具， 准确监测和定量小鼠线粒体自噬和线粒体生物合成的分区变化 APAP后的肝脏我们提出的研究结果将导致对TFEB的深入了解- 介导的细胞适应性反应促进受损线粒体的自噬降解， 线粒体生物合成在APAP诱导的肝损伤逆转中的作用最终，这些知识 确定治疗APAP诱导的肝损伤和急性肝衰竭的新治疗靶点的潜力。

PROJECT SUMMARY / ABSTRACT Acetaminophen (APAP) hepatotoxicity is the most frequent cause of acute liver failure of any etiology in the United States. However, no effective therapeutic strategies for APAP-induced liver injury are currently available, especially for late presenting patients. It is well known that after APAP overdose, N-acetyl-p- benzoquinone imine (NAPQI), the reactive metabolite of APAP, binds to cellular and mitochondrial proteins to form APAP-protein adducts (APAP-AD) following the depletion of cellular glutathione, which triggers mitochondrial dysfunction, oxidant stress and subsequent necrosis. In our previous funding cycle, we demonstrated that activation of autophagy, a cellular adaptive response of lysosomal degradation pathway, protects against APAP-induced liver injury by removing APAP-AD and damaged mitochondria. Liver is a very dynamic organ that has the capacity to repair and regenerate after injury. We also demonstrated that increased mitochondrial biogenesis can improve liver regeneration and recovery from APAP-induced liver injury. Importantly, our preliminary data showed that the transcription factor EB (TFEB), a master regulator that governs both the biogenesis of lysosomes for autophagy and mitochondria for regeneration, was impaired during the course of APAP-induced liver injury. Therefore, the major goal of this competitive R01 renewal is to understand the molecular mechanisms by which APAP impairs TFEB signaling in the liver. Our central hypothesize is that activation of TFEB will lead to increased biogenesis of both lysosomes and mitochondria that inhibits the progression of APAP-induced liver injury and promotes the liver regeneration. Two specific aims are proposed: 1) determine the mechanisms by which APAP impairs TFEB-mediated biogenesis of lysosomes and mitochondria in hepatocytes; and 2) determine the mechanism(s) by which TFEB promotes the recovery from APAP-induced liver injury by increased biogenesis of lysosomes and mitochondria. The proposed research is innovative in the concept that a transcription program that governs both the autophagy-lysosomal pathway and mitochondrial biogenesis is impaired in APAP-induced liver injury. We will utilize novel genetic animal models such as liver-specific TFEB KO mice, and adeno-associated virus- mediated overexpression of TFEB and PGC-1α approaches to specifically investigate the role of TFEB and PGC-1α in autophagic removal of damaged mitochondrial and enhancing new mitochondria biogenesis in reversal of APAP-induced liver injury. Moreover, we will also utilize the newly developed new molecular tools to accurately monitor and quantify the zonated changes of mitophagy and mitochondrial biogenesis in mouse livers after APAP. Results from our proposed study will lead to the in-depth understanding of the TFEB- mediated cellular adaptive response in promoting autophagic degradation of damaged mitochondria and mitochondrial biogenesis in the reversal of APAP-induced liver injury. Ultimately, such knowledge has the potential of identifying novel therapeutic targets for treating APAP-induced liver injury and acute liver failure.