PRO-APOPTOTIC GAPDH NUCLEAR ACCUMULATION AND RETINAL CELL DEATH

描述（由申请人提供）：糖尿病视网膜病变是世界范围内致盲的主要原因。目前没有治疗这种疾病的方法。因此，了解糖尿病视网膜病变发展的潜在机制将有助于促进有效治疗的发展。通过未知的高葡萄糖诱导机制发生的视网膜细胞死亡促成了该疾病。糖酵解酶、甘油醛-3-磷酸脱氢酶（GAPDH）（一种胞质酶）的核积累是早期促凋亡事件。以前，我们的实验室已经表明，GAPDH核积累发生在视网膜穆勒（神经胶质）细胞在体外和体内高血糖条件下。由于GAPDH缺乏核定位信号（NLS），GAPDH核积累的确切机制是不确定的。然而，最近的研究已经提出了一个NLS含有E3泛素连接酶七缺席同源物-1（siah-1）在脂多糖（LPS）诱导的促凋亡GAPDH在神经元和巨噬细胞核积累的作用。这些研究表明，IPS诱导GAPDH的翻译后修饰，如S-亚硝基化，从而促进GAPDH/siah-1相互作用和可能的复合物形成。然后，含有siah-1的NLS在与GAPDH的复合物中易位至细胞核。这个概念是有吸引力的，因为LPS是促炎细胞因子产生的主要诱导剂，例如白细胞介素-1 β（IL-1 β）的诱导剂，并且我们的研究已经表明IL-1 β信号传导介导高葡萄糖诱导的GAPDH体外核积累。高葡萄糖导致IL-1 β介导的GAPDH和siah-1的复合物形成以及该复合物易位到细胞核是一个有趣的想法。因此，本项目将评估（1）在高葡萄糖条件下在视网膜Muller细胞中可能由GAPDH的翻译后修饰介导的GAPDH/siah-1复合物的形成，以及（2）IL-1 β是否介导体外和体内高葡萄糖诱导的GAPDH/siah-1复合物的形成。对于这些研究，在高葡萄糖和细胞因子处理后，将使用蛋白质印迹和免疫荧光分析来评估细胞溶质和细胞核siah-1。我们还将利用免疫共沉淀测定来鉴定GAPDH/siah-1复合物的形成，并利用蛋白质组学分析来测试GAPDH的可能的翻译后修饰，其可能促进与siah-1的复合物形成。我们还将使用糖尿病caspase-1和IL-1受体敲除小鼠来评估IL-1-3对体内视网膜Muller细胞中高糖诱导的GAPDH核积累的贡献。

DESCRIPTION (provided by applicant): Diabetic retinopathy is a leading cause of blindness world wide. Currently there are no therapies for the disease. Therefore, understanding mechanisms underlying the development of diabetic retinopathy will help to facilitate the development of effective therapies. Retinal cell death which occurs through an unknown high glucose-induced mechanism contributes to the disease. Nuclear accumulation of the glycolytic enzyme, glyceraldehyde-3- phosphate dehydrogenase (GAPDH), a cytosolic enzyme is an early pro-apoptotic event. Previously, our laboratory has shown that GAPDH nuclear accumulation occurs in retinal Muller (glial) cells under hyperglycemic conditions in vitro and in vivo. Since GAPDH lacks a nuclear localization signal (NLS), the exact mechanism for GAPDH nuclear accumulation is indefinite. However, recent studies have proposed a role for the NLS containing E3 ubiquitin ligase seven in absentia homolog-1 (siah-1) during lipopolysaccharide (LPS)-induced pro-apoptotic GAPDH nuclear accumulation in neurons and macrophages. These studies have suggested that IPS induced post-translational modifications of GAPDH, such as S-nitrosylation, thereby promoting GAPDH/siah-1 interaction and possible complex formation. NLS containing siah-1 then translocates in a complex with GAPDH to the nucleus. This notion is appealing since LPS is a major inducer of pro- inflammatory cytokine production, such as that of interleukin-1 p (IL-1p), and our studies have shown that IL- 1p signaling mediates high glucose-induced GAPDH nuclear accumulation in vitro. It is an intriguing idea that high glucose leads to IL-1 p mediated complex formation of GAPDH and siah-1 and translocation of this complex to the nucleus. Therefore, this project will assess (1) GAPDH/siah-1 complex formation under high glucose conditions in retinal Muller cells possibly mediated by post-translational modifications of GAPDH, and (2) whether IL-1 p mediates high glucose-induced GAPDH/siah-1 complex formation in vitro and in vivo. For theses studies cytosolic and nuclear siah-1 will be evaluated using Western blot and immunofluorescence analysis following high glucose and cytokine treatment. We will also utilize co- immunoprecipitation assays to identify GAPDH/siah-1 complex formation and proteomics analysis to test for possible post-translational modification(s) of GAPDH, which might facilitate complex formation with siah-1. We will also use diabetic caspase-1 and IL-1 receptor knock-out mice to assess the contribution of IL-1-3 to high glucose-induced GAPDH nuclear accumulation in retinal Muller cells in vivo.