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

描述(申请人提供)：内质网(ER)和线粒体是调节细胞新陈代谢的主要部分。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，内质网稳态紊乱(或内质网应激)和线粒体功能障碍通常在神经退行性疾病和糖尿病等病理条件下共存。在糖尿病视网膜病变(DR)中，高血糖足以诱导内质网应激和氧化应激，两者都会导致视网膜炎症、血管渗漏、细胞凋亡，最终导致新生血管和神经元变性。有趣的是，除了功能相互作用外，内质网和线粒体还通过内质网一个高度专业化的亚域相互连接，称为线粒体相关的内质网膜(MAM)。虽然MAM的确切作用机制尚不清楚，但新的证据表明，MAM是脂质和蛋白质代谢以及钙信号转导的关键部位。几项研究 研究表明，MAM功能和/或结构缺陷对线粒体ATP的产生产生负面影响，增加ROS的产生，加剧内质网应激并导致细胞凋亡。然而，MAM在健康和疾病的视网膜和视网膜细胞中的作用尚未被研究。这项先导性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暴露于糖尿病侮辱的视网膜细胞和糖尿病动物视网膜中MAM的结构和生化变化。我们将确定MAM改变对DR的氧化应激、内质网应激、炎症、血管损伤和视网膜细胞死亡的功能后果。在Aim 2中，我们将使用一种特别精炼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。这种对MAM蛋白的全面和公正的分析将证实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线粒体-ER调节的机械性研究提供必要的数据，这也可能填补我们对糖尿病引起的视网膜细胞代谢缺陷的理解空白，并为DR确定新的治疗靶点。

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.