Autophagy and Drug-Induced Liver Injury

项目摘要/摘要 扑热息痛(APAP)肝毒性是引起急性肝功能衰竭的最常见原因。 美国。然而，目前尚无有效的治疗APAP所致肝损伤的策略。 可供选择，尤其适用于出现较晚的患者。众所周知，APAP过量后，N-乙酰-p- 苯醌亚胺(NAPQI)是APAP的反应性代谢产物，它与细胞和线粒体蛋白结合，从而 在细胞谷胱甘肽耗尽后形成APAP-蛋白质加合物(APAP-AD)，从而触发 线粒体功能障碍、氧化应激和随后的坏死。在我们之前的融资周期中，我们 证明了自噬的激活，溶酶体降解途径的一种细胞适应性反应， 通过清除APAP-AD和损伤的线粒体来保护APAP诱导的肝损伤。肝脏是一种非常 损伤后具有修复和再生能力的动态器官。我们还证明，增加了 线粒体生物发生可促进肝再生和APAP所致肝损伤的恢复。 重要的是，我们的初步数据显示，转录因子EB(TFEB)，一个主要的调节因子， 控制自噬的溶酶体的生物发生和再生的线粒体的生物发生都受到了损害 在APAP诱导的肝损伤过程中。因此，本次竞争性R01续展的主要目标是 了解APAP损害肝脏TFEB信号的分子机制。我们的中央 假设TFEB的激活将导致溶酶体和线粒体的生物发生增加 抑制APAP所致肝损伤的进展，促进肝再生。 提出了两个具体的目标：1)确定APAP损害TFEB介导的机制 肝细胞中溶酶体和线粒体的生物发生；2)确定TFEB的作用机制(S) 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。这项拟议的研究是创新的，因为一个转录程序管理 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到损害。 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TFEB和PGC-1α过表达的方法特异性研究TFEB和PGC-1的作用 PGC-1α在自噬清除受损线粒体和促进新线粒体生物发生中的作用 逆转APAP所致肝损伤。此外，我们还将利用新开发的新分子工具来 准确监测和量化小鼠有丝分裂和线粒体生物发生的带状变化 APAP后的肝脏。我们建议的研究结果将有助于深入了解TFEB- 介导的细胞适应性反应促进受损线粒体的自噬降解 线粒体生物发生在APAP诱导的肝损伤逆转中的作用归根结底，这种知识具有 寻找治疗APAP所致肝损伤和急性肝功能衰竭的新治疗靶点的可能性。

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.