Impact of Airway Inflammation on Mitochondria

气道炎症对线粒体的影响

基本信息

批准号：
10385779
负责人：
Y. S. Prakash
金额：
$ 68.37万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-15 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10385779
关键词：
Acute Affect Airway Disease Asthma Awareness Biogenesis COVID-19 pandemic Chemicals Consumption Contractile Proteins Endoplasmic Reticulum Enzymes Experimental Designs Failure Generations Goals Human Individual Inflammation Inflammatory Inositol Mediating Messenger RNA Mitochondria Molecular Chaperones Muscle Mitochondria Oxidative Stress PINK1 gene Parkin Pathway interactions Persons Phosphorylation Plasmids Proteins RNA Splicing Reactive Oxygen Species Research Respiratory Disease Signal Transduction Site Small Interfering RNA Smooth Muscle Myocytes Stress Structure TNF gene Testing Transfection Ubiquitination airway inflammation cytokine density endoplasmic reticulum stress knock-down loss of function meetings mutant novel overexpression pandemic disease protein expression respiratory respiratory smooth muscle response

项目摘要

The impact of acute airway inflammation is mediated by pro-inflammatory cytokines (e.g., TNFα), and underlies a number of respiratory diseases. A fundamental question is why are some individuals more susceptible than others to the negative impact of airway inflammation. We will explore a novel homeostatic mechanism, which protects airway smooth muscle (hASM) cells from the negative impact of inflammation-induced reactive oxygen species (ROS) formation and protein unfolding (endoplasmic reticulum (ER) stress). We believe that a failure in this homeostatic mechanism leads to increased ROS formation thereby exacerbating oxidative and ER stress. Overall Hypothesis: TNFα-induced ROS formation and protein unfolding activates the pIRE1α/XBP1s ER stress pathway in hASM, which initiates a homeostatic response directed towards increasing mitochondrial biogenesis and mitochondrial volume density to reduce O2 consumption and ROS formation by individual mitochondrion, while still meeting the increase in ATP demand – sharing the energetic load across mitochondria. Furthermore, reduced Mfn2 disrupts mitochondrial tethering to the ER, thereby decreasing mitochondrial Ca2+ influx and maximum respiratory capacity of mitochondria. Aim 1: TNFα-induced activation of pIRE1α/XBP1s ER stress pathway increases mitochondrial volume density and reduces O2 consumption and ROS formation per mitochondrion. In hASM cells, the downstream impact of TNFα-induced activation of the pIRE1α/XBP1s ER stress pathway will be explored using transfection of a non-phosphorylatable IRE1α mutant plasmid (DP-IRE1α) or an unspliceable XBP1 (uXBP1) mRNA. In addition, we will examine the effects of siRNA knockdown of PGC1α and Mfn2 overexpression on TNFα-induced changes in mitochondrial biogenesis, mitochondrial volume density, O2 consumption and ROS formation. Aim 2: TNFα-induced reduction in Mfn2 disrupts mitochondrial tethering to ER, decreases mitochondrial Ca2+ influx and reduces maximum respiratory capacity of mitochondria. In hASM cells, we will examine the impact of DP-IRE1α or uXBP1 mRNA transfection and siRNA Mfn2 knockdown on TNFα-induced disruption of mitochondrial/ ER tethering, decreased mitochondrial Ca2+ influx and reduced maximum respiratory capacity of mitochondria. Aim 3: The impact of TNFα on activation of the pIRE1α/XBP1s ER stress pathway and downstream effects are mitigated by ROS scavenging and chemical chaperone treatment. In hASM cells, the mitigating effects of ROS scavenging and chemical chaperone treatment on TNFα-induced activation of the pIRE1α/XBP1s ER stress pathway will be examined.

急性气道炎症的影响是由促炎细胞因子（例如 TNFα）介导的，是许多呼吸系统疾病的基础，一个基本问题是为什么会出现某些呼吸系统疾病。比其他人更容易受到气道炎症负面影响的人。探索一种新的稳态机制，保护气道平滑肌（hASM）细胞免受炎症引起的活性氧（ROS）形成的负面影响和蛋白质展开（内质网（ER）应激）。我们认为这种稳态失败。机制导致 ROS 形成增加，从而加剧氧化和 ER 应激。总体假设：TNFα 诱导的 ROS 形成和蛋白质解折叠激活 hASM 中的 pIRE1α/XBP1s ER 应激通路，启动稳态反应增加线粒体生物发生和线粒体体积密度以减少 O2 单个线粒体的消耗和 ROS 形成，同时仍满足 ATP 需求——在线粒体之间分担能量负荷此外，Mfn2 减少。破坏线粒体与内质网的束缚，从而减少线粒体 Ca2+ 流入线粒体的最大呼吸能力。目标 1：TNFα 诱导的 pIRE1α/XBP1s 内质网应激途径激活增加线粒体体积密度并减少每个线粒体的 O2 消耗和 ROS 形成。在 hASM 细胞中，TNFα 诱导的 pIRE1α/XBP1s ER 激活的下游影响将通过转染不可磷酸化的 IRE1α 突变体来探索应激途径此外，我们将检查质粒 (DP-IRE1α) 或不可剪接的 XBP1 (uXBP1) mRNA。 siRNA 敲低 PGC1α 和 Mfn2 过表达对 TNFα 诱导的变化的影响线粒体生物合成、线粒体体积密度、O2 消耗和 ROS 形成。目标 2：TNFα 诱导的 Mfn2 减少破坏线粒体与 ER 的束缚，减少线粒体 Ca2+ 流入并降低线粒体的最大呼吸能力。在 hASM 细胞中，我们将检查 DP-IRE1α 或 uXBP1 mRNA 转染的影响和 siRNA Mfn2 敲低可减少 TNFα 诱导的线粒体/ ER 束缚破坏线粒体 Ca2+ 流入并降低线粒体最大呼吸能力。目标 3：TNFα 对 pIRE1α/XBP1s ER 应激通路激活的影响 ROS 清除和化学伴侣处理可减轻下游影响。在 hASM 细胞中，ROS 清除和化学伴侣处理的缓解作用将检查 TNFα 诱导的 pIRE1α/XBP1s ER 应激途径激活。