Mitochondrial dynamics in acute lung injury

说明（由申请人提供）：意义。急性肺损伤（ALI）是导致死亡和发病的主要原因。我们的目标是确定可能导致治愈的机制。我们将重点关注肺泡线粒体机制，这是知之甚少。在ALI中，线粒体功能障碍可能是肺泡功能障碍的基础，导致肺损伤。功能障碍可能是由于线粒体Ca2+缓冲的丧失，其中Ca2+通过线粒体Ca2+单转运体（MCU）从细胞质扩散到线粒体基质，防止细胞质Ca2+ (cytCa2+)的促炎增加，从而保护免受损伤。更好地了解这些机制可能会导致MCU恢复线粒体Ca2+缓冲，作为LPS-ALI的治疗方法。建议。我们提出了线粒体Ca2+ (mitCa2+)决定表面活性剂分泌的新假设，因为线粒体Ca2+摄取增加了ATP和H2O2的产生。在LPS-ALI中，持续的cytCa2+增加导致线粒体Ca2+缓冲失败，导致Ca2+依赖性磷酸酶钙调磷酸酶的激活，钙调磷酸酶反过来去磷酸化并激活线粒体裂变蛋白DRP1。由此产生的线粒体分裂导致MCU的废除。钙调磷酸酶也促进肌动蛋白解聚。这些作用共同诱导肺泡功能障碍，表现为表面活性剂分泌的减少。具体的目标。具体目的是确定MCU在肺泡表面活性物质分泌中的作用（目的1），并确定MCU的丧失在多大程度上加剧了lps诱导的ALI肺损伤（目的2）。我们将建立MCU作为一个特定的Ca2+通道的作用，以及它在表面活性剂分泌中的作用。我们将评估LPS-ALI中线粒体Ca2+缓冲丧失的分子机制，我们将考虑基于骨髓间充质间质细胞（BMSC）的治疗方法，旨在通过线粒体受损的肺泡恢复Ca2+缓冲，作为肺修复的一种策略。的方法。我们将通过对离体灌注小鼠肺的活共聚焦和双光子显微镜以及对离体肺泡2型（AT2）细胞的研究来实现这些目标。我们的检测将包括：(1)完整肺泡和分离AT2细胞中的cytCa2+和mitCa2+；(2)单细胞成像通过检测Ca2+、ATP、H2O2和F-actin栅栏来调节表面活性剂的分泌；(3) MCU和Ca2+缓冲内毒素ALI通过肺和AT2细胞特异性突变体和siRNA的表达；(4)在骨髓间充质干细胞中表达MCU突变体，以验证MCU恢复恢复线粒体Ca2+缓冲的假设；(5)通过线粒体转移介导MCU过表达作为LPS-ALI的保护手段。初步的数据。我们发现(1)MCU调节cytCa2+和mitCa2+以及表面活性剂的分泌，(2)内毒素ALI通过激活DRP1下调MCU来阻断线粒体Ca2+缓冲，(3)在内毒素ALI中，肺泡转染突变MCU会恶化小鼠的存活，而填充长度MCU的表达会增加小鼠的存活。这些和其他关于我们测量策略的初步数据支持项目的可行性。的影响。该项目将首次系统地了解微处理器作为肺泡功能决定因素的作用。MCU的丧失在多大程度上加剧了ALI， MCU的恢复在多大程度上促进了肺修复，这将是第一次被理解。对ALI的发病机制将有新的认识。

DESCRIPTION (provided by applicant): Significance. Acute lung injury (ALI) is major cause of mortality and morbidity. Our goal is to define mechanisms that might lead to a cure. We will focus on alveolar mitochondrial mechanisms, which are poorly understood. In ALI, mitochondrial dysfunction might underlie alveolar dysfunction, leading to lung injury. The dysfunction might follow loss of mitochondrial Ca2+ buffering in which Ca2+ diffuses from cytosol to mitochondrial matrix across the mitochondrial Ca2+ uniporter (MCU), preventing proinflammatory increase of the cytosolic Ca2+ (cytCa2+), hence protecting against injury. Better understanding of these mechanisms might lead to restoration of mitochondrial Ca2+ buffering by MCU as a therapy for LPS-ALI. Proposal. We propose the novel hypothesis that mitochondrial Ca2+ (mitCa2+) determine surfactant secretion, because the mitochondrial Ca2+ uptake increases ATP and H2O2 production. In LPS-ALI, sustained cytCa2+ increase causes failure of mitochondrial Ca2+ buffering leading to the activation of the Ca2+-dependent phosphatase, calcineurin, which in turn, dephosphorylates and activates the mitochondrial fission protein, DRP1. The resulting mitochondrial fission leads to abrogation of MCU. Calcineurin also promotes actin depolymerization. Together these effects induce alveolar dysfunction as reflected in loss of surfactant secretion. Specific Aims. The specific aims are to determine the role of the MCU in alveolar surfactant secretion (Aim 1) and to determine the extent to which loss of MCU exacerbates lung injury in LPS-induced ALI (Aim 2). We will establish the role of the MCU as a specific Ca2+ channel, as well as its role in surfactant secretion. We will evaluate molecular mechanisms underlying the loss of mitochondrial Ca2+ buffering in LPS-ALI and we will consider bone-marrow-derived mesenchymal stromal cell (BMSC)-based therapeutic approaches aimed at reinstating Ca2+ buffering by mitochondria damaged alveoli as a strategy for lung repair. Approach. We will achieve these aims by live confocal and two-photon microscopy of isolated perfused mouse lungs and by studies in isolated alveolar type 2 (AT2) cells. Our determinations will include: (1) cytCa2+ and mitCa2+ in intact alveoli and in isolated AT2 cells; (2) regulation of surfactant secretion by single cell imaging through determinations of Ca2+, ATP, H2O2 and the F-actin fence; (3) MCU and Ca2+ buffering in endotoxin ALI through lung and AT2 cell expressions of specific mutants and siRNA; (4) Expression of MCU mutants in BMSCs to test the hypothesis that MCU restitution re-instates mitochondrial Ca2+ buffering; (5) MCU overexpression by mitochondrial transfer as a means to protect against LPS-ALI. Preliminary data. We show (1) MCU regulates cytCa2+ and mitCa2+ and surfactant secretion, (2) endotoxin ALI blocks mitochondrial Ca2+ buffering by downregulating MCU through activation of DRP1, and that (3) while alveolar transfection of a mutant MCU worsens mouse survival in endotoxin ALI, expression of a fill length MCU increases mouse survival. These and other preliminary data on our measurement strategies support feasibility of the project. Impact. This project will provide the first systematic understanding of the role of the MCU as a determinant of alveolar function. The extent to which loss of the MCU exacerbates ALI, and the extent to which MCU reinstatement promotes lung repair will be understood for the first time. Outstandingly new understanding of the pathogenesis of ALI will be achieved.