Implication of N-Methyl-d-Aspartate Receptor in Homocysteine-Induced Age-Related Macular Degeneration

Abstract

Age-related macular degeneration (AMD) is a leading cause of vision loss. Elevated homocysteine (Hcy) (Hyperhomocysteinemia) (HHcy) has been reported in AMD. We previously reported that HHcy induces AMD-like features. This study suggests that N-Methyl-d-aspartate receptor (NMDAR) activation in the retinal pigment epithelium (RPE) is a mechanism for HHcy-induced AMD. Serum Hcy and cystathionine-β-synthase (CBS) were assessed by ELISA. The involvement of NMDAR in Hcy-induced AMD features was evaluated (1) in vitro using ARPE-19 cells, primary RPE isolated from HHcy mice (CBS), and mouse choroidal endothelial cells (MCEC); (2) in vivo using wild-type mice and mice deficient in RPE NMDAR (NMDARR-/-) with/without Hcy injection. Isolectin-B4, Ki67, HIF-1α, VEGF, NMDAR1, and albumin were assessed by immunofluorescence (IF), Western blot (WB), Optical coherence tomography (OCT), and fluorescein angiography (FA) to evaluate retinal structure, fluorescein leakage, and choroidal neovascularization (CNV). A neovascular AMD patient’s serum showed a significant increase in Hcy and a decrease in CBS. Hcy significantly increased HIF-1α, VEGF, and NMDAR in RPE cells, and Ki67 in MCEC. Hcy-injected WT mice showed disrupted retina and CNV. Knocking down RPE NMDAR improved retinal structure and CNV. Our findings underscore the role of RPE NMDAR in Hcy-induced AMD features; thus, NMDAR inhibition could serve as a promising therapeutic target for AMD.

Warburg Effect as a Novel Mechanism for Homocysteine-Induced Features of Age-Related Macular Degeneration

“…The OCT and FA evaluations were performed 72 h after intravitreal injection of Hcy. FA examination ( Figure 4 A, upper panel) showed increased fluorescein leakage and disrupted retinal morphology in Hcy-injected wild-type mice compared to wild-type control mice, suggesting decreased integrity of retinal blood vessels and BRB impairment by Hcy, and confirming our previous publications [ 9 , 11 , 16 , 17 , 37 ]. While OCT evaluation ( Figure 4 A, lower panel) showed normal retinal appearance in wild-type mice but evident disruption at the RPE layer and CNV induction in the retinas of Hcy-injected wild-type mice.…”

“…While OCT evaluation ( Figure 4 A, lower panel) showed normal retinal appearance in wild-type mice but evident disruption at the RPE layer and CNV induction in the retinas of Hcy-injected wild-type mice. Blocking GLUT1 by WZB117 markedly improved retinal structure and CNV induction after Hcy injection, and vascular leakage was further confirmed by determining the albumin leakage in the retinas using western blot analysis following perfusion with PBS solution and as previously described in our publications [ 9 , 11 , 16 , 17 , 37 ]. Western blot data analysis and quantification showed a significant increase in albumin leakage in Hcy-injected mice retina compared to the non-injected mice retina, and the leakage was decreased by WZB117 ( Figure 4 B), suggesting that blocking GLUT1 could rescue RPE cells, restore the BRB, prevent retinal leakage induced by HHcy, and reduce CNV induction.…”

“…Recently, we reported that NMDAR activation is an underlying mechanism for Hcy in the AMD retina and that blocking NMDAR both by pharmacologic inhibition (MK801) or genetic inhibition of NMDAR in RPE cells ( NMDAR R−/− mouse) restored RPE barrier function and protected the retina from the HHcy-induced choroidal neovascularization [ 11 ]. To further understand whether Hcy activation of GLUT1 and metabolic shift to glycolysis is due to the direct effect of Hcy or via activation of the NMDAR, we performed another set of in vivo experiments using three groups of mice at age ~6–8 weeks old (wild-type mice without injection compared to wild-type and NMDAR R−/− mice after 72 h of intravitreal injection of Hcy (200 μM)).…”

“…Furthermore, Over the past several years, we have conducted experiments aimed to address this issue. Earlier studies reported a direct impact of excess homocysteine (Hcy) on (retinal pigment epithelium) RPE structure, barrier function, and induced choroidal neovascularization (CNV) in mice [ 9 ], and subsequent experiments reported that HHcy not only increased hypoxia-inducible factor 1 (HIF-1α) and VEGF levels in retinas [ 10 , 11 ], but also angiogenic potential of retinal endothelial cells in vitro and in vivo [ 10 , 12 , 13 ]. These findings were followed by many mechanistic studies that showed that HHcy activates oxidative stress [ 12 ], endoplasmic reticulum (ER) stress [ 14 ], inflammation [ 15 ], and epigenetic modifications [ 16 ].…”

“…These findings were followed by many mechanistic studies that showed that HHcy activates oxidative stress [ 12 ], endoplasmic reticulum (ER) stress [ 14 ], inflammation [ 15 ], and epigenetic modifications [ 16 ]. Recently, we reported activation of the N-methyl-d-aspartate (NMDA) receptor in retinal endothelial cells [ 17 ] and in RPE [ 11 ] cells as a suggested mechanism of HHcy-induced retinal dysfunction. Our goal is to understand the exact mechanism by which HHcy participates in the pathogenesis of AMD; accordingly, we can propose Hcy as a marker and therapeutic target for AMD.…”