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全世界失明的主要原因。目前还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机制将有助于开发有效的治疗方法。视网膜细胞死亡发生通过未知的高糖诱导机制有助于疾病。核积累的糖酵解酶，甘油醛-3-磷酸脱氢酶（GAPDH），细胞质酶是一个早期的促凋亡事件。在此之前，我们的实验室已经证明，GAPDH核积累发生在体外和体内高血糖条件下的视网膜Muller（胶质）细胞中。由于GAPDH缺乏核定位信号（NLS）， GAPDH核积累的确切机制尚不明确。然而，最近的研究提出了含有E3泛素连接酶7缺失同源物-1 （siah-1）的NLS在脂多糖（LPS）诱导的神经元和巨噬细胞中促凋亡的GAPDH核积累中的作用。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相互作用并可能形成复合物。含有siah-1的NLS随后与GAPDH复合物一起转运到细胞核。这一观点很有吸引力，因为脂多糖是促炎细胞因子产生的主要诱导剂，如白细胞介素-1p (IL-1p)，我们的研究表明，IL-1p信号传导介导高糖诱导的体外GAPDH核积累。这是一个有趣的想法，高葡萄糖导致IL-1 p介导的GAPDH和siah-1复合物的形成，并将该复合物转运到细胞核。因此，本项目将评估(1)高糖条件下视网膜Muller细胞中GAPDH/siah-1复合物的形成可能是由GAPDH翻译后修饰介导的，(2)IL-1 p是否介导高糖诱导的GAPDH/siah-1复合物的体外和体内形成。在这些研究中，在高糖和细胞因子治疗后，细胞质和细胞核siah-1将使用Western blot和免疫荧光分析进行评估。我们还将利用共免疫沉淀法来鉴定GAPDH/siah-1复合物的形成，并利用蛋白质组学分析来检测GAPDH可能的翻译后修饰(s)，这可能有助于与siah-1形成复合物。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对体内高糖诱导的视网膜Muller细胞中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.