Study of the ER-mitochondria interface as a new target in diabetic retinopathy

描述（由申请人提供）：内质网（ER）和线粒体是调节细胞代谢的主要隔室。令人信服的证据表明，这两个细胞器密切相互作用和协调，以维持细胞的代谢完整性。因此，紊乱的ER稳态（或ER应激）和线粒体功能障碍通常在病理状况如神经退行性疾病和糖尿病中共存。在糖尿病视网膜病变（DR）中，高血糖足以诱导ER应激和氧化应激，两者都有助于视网膜炎症、血管渗漏、细胞凋亡和最终的新血管形成和神经元变性。有趣的是，除了功能性相互作用之外，ER和线粒体通过ER的高度特化的亚结构域（称为线粒体相关ER膜（MAM））物理和生化互连。尽管MAM的确切作用机制仍不清楚，但新出现的证据表明MAM是脂质和蛋白质代谢以及钙信号传导的关键位点。几项研究 显示MAM功能和/或结构缺陷负面影响线粒体ATP产生，增加ROS产生，加剧ER应激并导致细胞凋亡。然而，MAM在健康和患病视网膜和视网膜细胞中的作用尚未研究。这项初步研究的总体目标是确定MAM在糖尿病视网膜细胞代谢中的作用。在目标1中，我们将使用创新的方法来表征MAM在暴露于糖尿病损伤的视网膜细胞和糖尿病动物视网膜中的结构和生化变化。我们将确定MAM改变对DR中氧化应激、ER应激、炎症、血管损伤和视网膜细胞死亡的功能后果。在目的2中，我们将使用专门精制的最先进的蛋白质组学技术来分析正常和糖尿病视网膜中MAM的蛋白质谱。MAM蛋白的这种全面和公正的测定将证实MAM在调节视网膜代谢中的作用，并探索MAM在与DR发病机制相关的信号通路中的功能意义。我们预计，这一新颖而令人兴奋的项目将为未来关于Escheria-ER调节的机制研究提供必要的数据，这也可能填补我们对糖尿病诱导的视网膜细胞代谢缺陷的理解中的空白，并确定DR的新治疗靶点。

DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) and mitochondria are the primary compartments that regulate cellular metabolism. Compelling evidence suggests that these two organelles closely interact and coordinate to maintain the cell's metabolic integrity. Accordingly, disturbed ER homeostasis (or ER stress) and mitochondrial dysfunction often co-exist in pathological conditions such as neurodegenerative diseases and diabetes. In diabetic retinopathy (DR), hyperglycemia is sufficient to induce ER stress and oxidative stress, both contributing to retinal inflammation, vascular leakage, apoptosis, and ultimate neovascularization and neuronal degeneration. Intriguingly, additional to functional interactions, the ER and mitochondria are physically and biochemically interconnect through a highly specialized subdomain of the ER named the mitochondria- associated ER membrane (MAM). Although the exact mechanism of action of MAM remains obscure, emerging evidence suggests that MAM is a critical site for lipid and protein metabolism and calcium signaling. Several studies show that defective MAM function and/or structure negatively affects mitochondrial ATP production, increases ROS generation, exacerbates ER stress and leads to apoptosis. However, the role of MAM in healthy and diseased retina and retinal cells has not been studied. The overall goal of this pilot study is to establish a role of MAM in retinal cell metabolism in diabetes. In Aim 1, we will use innovative approaches to characterize the structural and biochemical changes in MAM in retinal cells exposed to diabetic insults and in the retina from diabetic animals. We will determine the functional consequence of MAM alterations on oxidative stress, ER stress, inflammation, vascular injury, and retinal cell death in DR. In Aim 2, we will use a specifically refined state-of-the-art proteomic technology to analyze the protein profile of MAM from normal and diabetic retinas. This comprehensive and unbiased assay of MAM proteins will confirm the role of MAM in regulation of retinal metabolism and explore the functional implication of MAM in signaling pathways related to DR pathogenesis. We anticipate that this novel and exciting project will generate essential data for future mechanistic study on mitochondria-ER regulation, which may also fill in a gap in our understanding of diabetes- induced metabolic defects in retinal cells and identify new therapeutic targets for DR.