Homocysteine's role in Age-Related Macular Degeneration

总结/摘要 视网膜相关性黄斑变性（AMD）是老年人群中视力丧失的主要原因。 据报道，患有高同型半胱氨酸（HCy）的患者会出现高同型半胱氨酸血症（HHCy） AMD;从而提示HHcy与AMD风险之间的关联。最近，我们报道视网膜 在缺乏胱硫醚-β-合酶（cbs+/-）或接受了抗HHcy治疗的HHcy小鼠模型中， 玻璃体内注射Hcy。这些模型显示出显著的视网膜色素上皮（RPE）功能障碍， 脉络膜新生血管（CNV）然而，缺乏对CNV的分子/细胞机制的了解， 这些变化是提出Hcy作为AMD治疗靶点的关键障碍。我们的初步数据显示 HHcy诱导的RPE功能障碍与N-甲基-D-天冬氨酸（NMDAr）的上调有关， 和GLUT 1受体以及糖酵解增加。因此，我们假设HHcy有助于 AMD的发病机制通过激活NMDAr和GLUT 1信号通路诱导代谢 从氧化磷酸化转变为糖酵解。因此，通过 药物或遗传干预可能有益于AMD的治疗。为了验证我们的假设，我们 将使用RPE和脉络膜内皮细胞（CEC）进行体外实验，并使用cbs+/-、野生型cbs +/-和野生型cbs+/-进行体内实验。 型小鼠和缺乏内皮或RPE NMDAr（NMDAr-/-E或 分别为NMDAr-/-R）。本研究的具体目的包括：1、验证HHcy诱导HHcy表达的假说。 通过激活RPE细胞中的GLUT 1从线粒体呼吸到糖酵解的代谢转换：我们 将检查视网膜GLUT 1表达和定位的变化，线粒体呼吸， 糖酵解和限速糖酵解酶。此外，我们将确定GLUT 1的作用 抑制HHcy诱导的RPE功能障碍和CNV。目的2：检验抑制 NMDAr保留RPE功能并减少HHcy下CNV的发展。我们会研究 NMDAr的药物抑制或遗传操作对HHcy诱导的RPE功能障碍的影响 CNV将在NMDAr-/-E或NMDAr-/-R中评价玻璃体内注射Hcy的效果，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 RPE和CEC在有或没有NMDAr抑制剂的情况下经受Hcy，随后评估RPE功能， CEC的血管生成潜力。目的3：验证通过饮食消除过量Hcy的假设 补充或遗传/药理学修饰可预防AMD的进展。Hcy 在高同型半胱氨酸模型中，将通过两种方法提高清除率，然后评估RPE功能 CEC的血管生成潜力：（a）使用维生素增强Hcy代谢的再甲基化途径 补充维生素B6、B12和叶酸。(b)CBS增强同型半胱氨酸代谢的转硫途径 过度表达过量Hcy的成功清除在AMD的治疗中具有巨大的前景。

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.