Nitric oxide and mitochondrial biogenesis in sepsis

描述（由申请人提供）：本修订后的竞争性续期申请旨在了解细菌性败血症中一氧化氮（NO）的产生如何导致线粒体损伤并调节线粒体质量控制，从而防止器官衰竭。在之前的资助周期中，我们发现NF-kB依赖性NO合成酶（iNOS/NOS2）的toll样受体（TLR）激活通过主要的共激活因子PGC-11和单磷酸腺苷（AMP）激活的蛋白激酶（AMPK）参与肝脏线粒体生物发生的调节。我们在脓毒症小鼠中的初步数据表明：NOS2诱导导致AMPK激活，AMPK有助于激活线粒体生物发生，但并不总是在ATP耗尽后，NOS2缺乏减少AMPK上调和线粒体生物发生，并增加TLR激活后的凋亡和炎症。因此，AMPK似乎激活线粒体生物发生，同时对抗败血症中的细胞凋亡和炎症。相反，过多的NO与线粒体损伤和NO信号特异性的丧失独立相关，这是由NO物种（NOx）对蛋白质和核酸的化学攻击引起的。我们假设NOS2部分通过AMPK起作用，在ATP产生失败之前对线粒体生物发生的转录程序进行适当调节，以维持线粒体质量控制并防止脓毒症中的细胞死亡。要验证这一假设，需要定义NO依赖的转录控制机制、AMPK的作用以及相关模型系统中线粒体NO的化学生物学。我们的方法将侧重于肝脏作为前哨器官和一个遗传因子- NOS2-作为线粒体周转和细胞存活的定量影响。目的1：通过定量NOS2基因滴定研究，确定NOS2在严重脓毒症期间对肝脏线粒体生物发生和细胞存活的作用。1一个。使用NOS2滴定法确定nox介导的mtDNA与蛋白质损伤、呼吸能力、高能代谢物水平和败血症诱导的肝细胞死亡之间的关系。1 b。通过CREB和/或NRF-1诱导，以及调控NRF-21 （GABPA）和/或PGC-11表达，评估NOS2表达对脓毒症线粒体生物发生的调节作用。目的2：了解脓毒症中肝脏AMPK激活与NOS2、线粒体生物发生转录程序和细胞凋亡预防的关系。2 a。确定no依赖性AMPK激活是否通过CREB促进脓毒症中的线粒体生物发生和/或通过抑制促凋亡蛋白Bad和BNIP3的磷酸化来阻止细胞凋亡。2 b。确定AMPK在脓毒症中的药理激活是否可以独立于NOS2促进线粒体生物发生和/或抑制细胞凋亡。这些研究将在NOS2调控线粒体生物发生的生理机制以及AMPK在多大程度上调控线粒体生物发生的背景下，为脓毒症中nox诱导的线粒体损伤提供新的见解。这意味着，NO对AMPK的调节将在MODS中发挥关键的挽救作用，这一知识将允许合理的新药理学方法来支持线粒体功能，同时最大限度地减少NOx的附带损害。

DESCRIPTION (provided by applicant): This revised competitive renewal application seeks to understand how nitric oxide (NO) production in bacterial sepsis causes both mitochondrial damage and regulates mitochondrial quality control, which protects against organ failure. In the previous grant cycle, we discovered that toll-like receptor (TLR) activation of NF-kB- dependent NO synthase (iNOS/NOS2) is involved in the regulation of hepatic mitochondrial biogenesis through the major co-activator, PGC-11 and the adenosine monophosphate (AMP)-activated protein kinase (AMPK). Our preliminary data in mice with sepsis indicate that: NOS2 induction leads to AMPK activation, AMPK helps to activate mitochondrial biogenesis, but not always after ATP depletion, and NOS2 deficiency diminishes AMPK up-regulation and mitochondrial biogenesis and increases apoptosis and inflammation in response to TLR activation. Thus, AMPK appears to activate mitochondrial biogenesis while opposing apoptosis and inflammation in sepsis. In contrast, too much NO is independently associated with mitochondrial damage and loss of NO signal specificity by chemical attack of NO species (NOx) on proteins and nucleic acids. We hypothesize that NOS2, acting in part through AMPK, is required for apposite regulation of the transcriptional program of mitochondrial biogenesis before the failure of ATP production in order to maintain mitochondrial quality control and prevent cell death in sepsis. A test of this hypothesis requires definitions of NO-dependent transcriptional control mechanisms, the role of AMPK, and the chemical biology of mitochondrial NO in relevant model systems. Our approach will focus on the liver as a sentinel organ and on one genetic factor- NOS2- as a quantitative influence on mitochondrial turnover and cell survival. We plan to test these Specific Aims: Aim 1: To determine the role of NOS2 on hepatic mitochondrial biogenesis and cell survival during severe sepsis through quantitative NOS2 gene titration studies. 1A. Define the relationships between NOx-mediated mtDNA and protein damage, respiratory capacity, high-energy metabolite levels, and sepsis-induced hepatic cell death using NOS2 titration. 1B. Assess the importance of NOS2 expression on the regulation of mitochondrial biogenesis in sepsis through CREB and/or NRF-1 induction and regulation of NRF-21 (GABPA) and/or PGC-11 expression. Aim 2: To understand hepatic AMPK activation in sepsis in relation to NOS2, the transcriptional program of mitochondrial biogenesis, and prevention of apoptosis. 2A. Determine if NO-dependent AMPK activation promotes mitochondrial biogenesis in sepsis through CREB and/or prevents apoptosis by inhibitory phosphorylation of pro-apoptotic proteins, Bad and BNIP3. 2B. Determine if pharmacological activation of AMPK in sepsis can promote mitochondrial biogenesis and/or inhibit apoptosis independently of NOS2. These studies will provide new insights into NOx-induced mitochondrial damage in sepsis in the context of physiological mechanisms by which NOS2 regulates mitochondrial biogenesis and the extent to which it is orchestrated by AMPK. By implication, NO regulation of AMPK would play a critical salvage role in MODS, and this knowledge would allow rational new pharmacological approaches to support mitochondrial function while minimizing collateral damage by NOx.