Mitochondrial calcification in juvenile dermatomyositis

项目摘要 线粒体是参与新陈代谢、炎症和细胞死亡的细胞内细胞器。线粒体 也在调节细胞内钙水平，清除细胞内钙和无机钙方面发挥着重要作用 磷酸盐，将其以晶体形式(羟基磷灰石)困在线粒体中，将细胞从 细胞内钙超载。在成人病例中观察到线粒体内钙化。 皮肌炎(DM)以及皮肤钙质沉着症的实验模型，这是一种虚弱的表现 幼年型糖尿病(JDM)。然而，线粒体内的潜在机制和后果 钙化还没有被研究过。虽然线粒体主要存在于细胞内，但也可以挤出 在ROS介导的损伤时，参与诱导炎症。这项申请的前提是 JDM患者线粒体钙化促进线粒体排泄、钙结晶堆积 (钙质沉着症)和炎症。为了研究这一假设，我们有两个明确的目标。第一个目标是看起来 探讨了在体外导致线粒体钙化和挤出的机制。我们假设 线粒体ROS的产生将支持线粒体的钙化和挤出，有助于 炎症和钙质沉着。在实验中，原代人类骨骼肌细胞将在钙中孵化 富含应激剂的介质，包括缺氧、TLR激动剂或JDM血清和线粒体 采用流式细胞术、定量聚合酶链式反应、免疫荧光显微镜和代谢组学等方法进行功能评价。质量 基于光谱的磷酸蛋白质组学将被用来确定线粒体涉及哪些途径 钙化。包括线粒体ROS在内的潜在靶点将通过使用小分子来阻断，例如 MitoTEMPO。第二个目标将研究挤出的线粒体被清除的机制。我们 假设钙化的线粒体被吞噬，但不被降解，留在胞浆中 隔室触发TLR9和炎症体激活。简而言之，线粒体将与 原代人单核细胞和中性粒细胞的摄取、细胞内定位、信号转导 途径(磷蛋白质组学)和细胞因子诱导。参与线粒体诱导的关键途径- 介导的炎症，例如DNA传感器TLR9和cGAS，以及炎症体的激活，将是 使用化学抑制剂进行靶向治疗。JDM儿童和健康人的细胞支持能力 线粒体的清除量将在体外进行比较。最后，将分析JDM免疫细胞是否存在 用流式细胞术和共聚焦显微镜观察体内含羟基磷灰石的线粒体。我们相信 我们的建议具有很高的创新性和重要意义，因为i)我们将定义涉及以下内容的基本机制 线粒体钙化；以及ii)我们将首次探索挤出的线粒体是否以及如何起作用 与JDM的炎症和钙质沉着有关。这些发现也可能适用于其他情况，即 钙化过程突出，包括动脉粥样硬化。

Project Summary Mitochondria are intracellular organelles involved in metabolism, inflammation and cell death. Mitochondria also play an important role in regulating intracellular calcium levels, scavenging cytosolic calcium and inorganic phosphate, trapping it in its crystalline form (hydroxyapatite) within the mitochondria to rescue the cell from cytosolic Ca2+ overload. Intramitochondrial calcification has been observed in case reports of adult dermatomyositis (DM) as well as in experimental models of cutaneous calcinosis, a debilitating manifestation of juvenile DM (JDM). However, the underlying mechanisms and consequences of intramitochondrial calcification have not been investigated. Though mainly found intracellularly, mitochondria can be extruded upon ROS-mediated damage, partaking in induction of inflammation. The premise of this application is that JDM patients have mitochondrial calcification promoting mitochondrial extrusion, calcium crystal accumulation (calcinosis), and inflammation. To investigate this hypothesis we have two specific aims. The first aim will look into mechanisms contributing to mitochondrial calcification and extrusion in vitro. We hypothesize that mitochondrial ROS generation will support mitochondrial calcification and extrusion, contributing to inflammation and calcinosis. Experimentally, primary human skeletal muscle cells will be incubated in calcium rich medium in presence of stress agents, including hypoxia, TLR agonists, or JDM sera, and mitochondrial function assessed using flow cytometry, qPCR, immunofluorescence microscopy and metabolomics. Mass spectrometry-based phosphoproteomics will be used to establish which pathways are involved in mitochondrial calcification. Potential targets, including mitochondrial ROS, will be blocked by using small molecules, e.g. mitoTEMPO. The second aim will investigate mechanisms by which extruded mitochondria are cleared. We hypothesize that calcified mitochondria are phagocytosed, but not degraded, remaining in the cytosolic compartment triggering TLR9 and inflammasome activation. In brief, mitochondria will be incubated with primary human monocytes and neutrophils and assessed for uptake, intracellular localization, signaling pathways (phosphoproteomics) and cytokine induction. Key pathways involved in induction of mitochondrial- mediated inflammation, e.g. DNA sensors TLR9 and cGAS, as well as inflammasome activation, will be targeted using chemical inhibitors. The capacity of cells from JDM children and healthy individuals to support clearance of mitochondria will be compared in vitro. Finally, JDM immune cells will be analyzed for presence of hydroxyapatite-containing mitochondria in vivo using flow cytometry and confocal microscopy. We believe that our proposal is highly innovative and significant as i) we will define underlying mechanisms involved in mitochondrial calcification; and ii) we will for the first time explore if, and how, extruded mitochondria contribute to inflammation and calcinosis in JDM. These findings may also be applicable to other conditions in which the calcification process is prominent, including atherosclerosis.