Homocysteine's role in Age-Related Macular Degeneration

摘要/摘要 老年性黄斑变性(AMD)是老年人视力丧失的主要原因。 高同型半胱氨酸(Hcy)，也称为高同型半胱氨酸血症(HHcy)，已被报道在以下患者中 因此提示HHcy与AMD的风险之间存在关联。最近，我们报道了视网膜 缺乏胱硫醚-β-合成酶(cbs+/-)的同型半胱氨酸小鼠模型中类似于AMD的变化 玻璃体内注射同型半胱氨酸。这些模型显示出明显的视网膜色素上皮(RPE)功能障碍和 然而，脉络膜新生血管(CNV)的分子/细胞机制缺乏了解 这些变化是提出将同型半胱氨酸作为AMD治疗靶点的关键障碍。我们的初步数据显示 同型半胱氨酸诱导的RPE功能障碍与N-甲基-D-天冬氨酸(NMDAR)上调有关 和GLUT1受体，糖酵解增加。因此，我们假设HHcy对 诱导代谢的NMDAR和GLUT1信号通路激活在AMD发病机制中的作用 从氧化磷酸化转变为糖酵解。因此，通过以下途径消除过剩的Hcy 药物或遗传干预可能有益于AMD的治疗。为了检验我们的假设，我们 将进行体外实验，使用RPE和脉络膜内皮细胞(CEC)，并在体内使用CBS+/-，WARD 接受玻璃体内注射同型半胱氨酸的小鼠和缺乏内皮或RPE NMDAR(NMDAR-/-E或 NMDAR-/-R分别)。我们的具体目标包括：1：检验HHcy诱导 RPE细胞通过激活GLUT1从线粒体呼吸到糖酵解的代谢转换：WE 将检测视网膜GLUT1表达和定位的变化，线粒体呼吸， 同型半胱氨酸模型中的糖酵解和限速糖酵解酶。此外，我们还将确定GLUT1的作用 抑制同型半胱氨酸诱导的RPE功能障碍和CNV。目标2：检验假设，抑制 NMDAR保留了RPE功能，减少了HHcy状态下CNV的发展。我们将研究 NMDAR的药物抑制或基因操作对HHcy诱导的RPE功能障碍的影响 和CNV。玻璃体内注射Hcy的效果将在NMDAR-/-E或NMDAR-/-R中进行评估，与 野生型和CBS+/-小鼠，使用或不使用Namdar抑制剂。平行的体外实验将在 用或不用NMDAR抑制剂对RPE和CEC进行同型半胱氨酸干预，然后评估RPE功能和 CEC的血管生成潜能。目标3：检验通过饮食消除过量Hcy的假设 补充剂或基因/药物修饰可防止AMD的进展。同型半胱氨酸 在HHcy模型中，将通过两种方法增强清除，然后评估RPE功能 和CEC的血管生成潜能：(A)利用维生素增强Hcy代谢的再甲基化途径 补充B6、B12和叶酸。(B)通过CBS增强同型半胱氨酸代谢的转化途径 过度表达。过量同型半胱氨酸的成功清除为AMD的治疗带来了巨大的希望。

Summary/Abstract Age-related macular degeneration (AMD) is the leading cause of vision loss in among elderly populations. Elevated homocysteine (Hcy), also known as hyperhomocysteinemia (HHcy) has been reported in patients with AMD; thereby suggesting an association between HHcy and the risk of AMD. Recently, we reported retinal changes similar to AMD in a mouse model of HHcy which lacks Cystathionine-β-synthase (cbs+/-) or received intravitreal injections of Hcy. These models showed significant retinal pigment epithelium (RPE) dysfunction and choroidal neovascularization (CNV) However, the lack of understanding the molecular/cellular mechanisms of these changes is a critical barrier in proposing Hcy as a therapeutic target in AMD. Our preliminary data show that HHcy-induced RPE dysfunction is associated with the upregulation of the N-methyl-D-aspartate (NMDAr) and GLUT1 receptors and increased glycolysis. Hence, we hypothesize that HHcy contributes to the pathogenesis of AMD via activation of the NMDAr and GLUT1 signaling pathways that induce the metabolic switch from oxidative phosphorylation to glycolysis. Therefore, elimination of excess Hcy through pharmacological or genetic intervention could be beneficial in the treatment of AMD. To test our hypothesis, we will conduct in vitro experiments, using RPE and choroidal endothelial cells (CEC) and in vivo using cbs+/-, wild type mice receiving intravitreal injection of Hcy and mice lacking the endothelial or RPE NMDAr (NMDAr-/-E or NMDAr-/-R respectively). Our specific aims include: 1: Testing the hypothesis that HHcy induces the metabolic switch from mitochondrial respiration to glycolysis via activation of GLUT1 in RPE cells: We will examine the changes in the retinal expression and localization of GLUT1, mitochondrial respiration, glycolysis and rate-limiting glycolytic enzymes in HHcy models. Moreover, we will determine the effect of GLUT1 inhibition on HHcy-induced RPE dysfunction and CNV. Aim 2: Testing the hypothesis that inhibition of NMDAr preserves RPE function and reduces the development of CNV under HHcy. We will examine the effects of pharmacological inhibition or genetic manipulation of the NMDAr on HHcy-induced RPE dysfunction and CNV. The effect of intravitreal injection of Hcy will be evaluated in NMDAr-/-E or NMDAr-/-R as compared to wild type and cbs+/- mice with or without NAMDAr inhibitors. Parallel in vitro experiments will be performed on RPE and CEC subjected to Hcy with or without NMDAr inhibitors followed by assessment of RPE function and angiogenic potential of CEC. Aim 3: Testing the hypothesis that elimination of excess Hcy by dietary supplementation or genetic/ pharmacological modifications prevents the progression of AMD. Hcy clearance will be enhanced in models of HHcy through two approaches, followed by assessment of RPE function and angiogenic potential of CEC: (a) Enhancing the remethylation pathway of Hcy metabolism using vitamins B6, B12 and folic acid supplementation. (b) Enhancing the transsulforation pathway of Hcy metabolism via CBS overexpression. Successful clearance of excess Hcy holds immense promise in the treatment of AMD.