Mfn2介导的线粒体内质网结构偶联在高铜诱导鸭肾组织自噬中的作用

Mitofusin 2 (Mfn2) is a key protein that regulates the machinery of mitochondria-endoplasmic reticulum physical coupling. The latest studies have found that mitochondria-endoplasmic reticulum physical coupling plays an important role in the regulation of mitochondrial morphology, cellular calcium signaling, and autophagy. However, it is not reported that the role of Mfn2-mediated mitochondria-endoplasmic reticulum physical coupling in renal autophagy induced by high level of copper in duck. Our previous studies have shown that high level of copper could induce oxidative damage, mitochondrial dysfunction, and increase autophagic level in hepatocytes. In this study, we will establish the models of isolated renal tubular epithelial cell culture and animal model of high level of copper, respectively. And then techniques of gene knockdown and overexpression, transcriptome, proteomics, laser confocal microscopy and flow cytometry, etc will be used. The changes of mitochondria associated endoplasmic reticulum membrane (MAM), mitochondrial function and morphology, endoplasmic reticulum stress and morphology, autophagosomes, and the mRNA and protein expression of MAM-related and autophagy-related signaling molecules will be detected. This study will elucidate the mechanism of Mfn2-mediated mitochondria-endoplasmic reticulum physical coupling in renal autophagy induced by high level of copper in duck. The results of this study will lay a foundation for further development of copper toxicity molecular pathology.

线粒体融合蛋白2（Mfn2）是调控线粒体内质网结构偶联机制的关键蛋白。最新研究发现，线粒体内质网结构偶联在线粒体形态调控、细胞钙信号传导、自噬等机制中扮演着重要角色。但是Mfn2介导的线粒体内质网结构偶联在高铜诱导鸭肾组织自噬中的作用尚不清楚。前期研究表明高铜可以诱导细胞发生氧化损伤、线粒体功能障碍、自噬水平升高。本课题在建立离体肾小管上皮细胞培养模型和在体高铜动物模型的基础上，应用基因敲低与过表达、转录组、蛋白组、激光共聚焦显微和流式细胞等技术，检测线粒体相关内质网膜（MAM）形态学变化、线粒体功能和形态学变化、内质网应激和形态学变化、自噬体以及MAM和自噬相关信号分子mRNA和蛋白的表达，阐明Mfn2介导线粒体内质网偶联在高铜诱导鸭肾组织自噬中的作用机制，为进一步开展铜毒性分子病理学奠定基础。

线粒体融合蛋白2（Mfn2）是调控线粒体内质网结构偶联机制的关键蛋白。最新研究发现，线粒体内质网结构偶联在线粒体形态调控、细胞钙信号传导、自噬等机制中扮演着重要角色。但是Mfn2介导的线粒体内质网结构偶联在高铜诱导鸭肾组织自噬中的作用尚不清楚。本项目利用建立的离体肾小管上皮细胞培养模型和在体高铜动物模型研究Mfn2介导线粒体内质网偶联在高铜诱导鸭肾组织自噬中的作用机制。项目研究发现，高铜可诱导鸭肾组织和肾小管上皮细胞线粒体相关内质网膜（MAM）间距增加、MAM结构数量较少、自噬体数量增加，进而导致MAM功能障碍和自噬水平升高。进一步研究发现，Mfn2在该过程中发挥重要作用。我们构建了Mfn2过表达细胞模型，发现Mfn2过表达可使高铜诱导的MAM功能障碍和自噬水平上升更为严重。将提取出的MAM进行转录组和蛋白组测序，发现高铜调控MAM主要通过钙信号通路、脂肪酸代谢通路和内质网中蛋白质加工过程通路，所富集到的差异蛋白很可能是高铜引起的鸭肾损伤的靶向蛋白质。通过以上研究阐明了Mfn2介导的线粒体内质网结构偶联在高铜诱导鸭肾组织自噬中的作用及分子机理，为铜中毒的发病机制研究和防治具有重要价值。