Elevated Endogenous Homocysteine (HCY) Leads to Ganglion Cell Death and Disruption of Retinal Inner Nuclear and Plexiform Layers

Abstract

Abstract: : Purpose: HCY is a non–protein–forming sulfur amino acid whose metabolism lies at the intersection of the remethylation and transsulfuration metabolic pathways. Recent clinical studies implicate HCY in age–related maculopathy, retinal vein occlusion, open–angle glaucoma, pseudoexfoliation glaucoma, and diabetic retinopathy. Previously, we showed that intravitreal exposure to high levels of HCY in mice leads to dramatic loss of retinal ganglion cells (RGCs). There have been no systematic studies, however, of the impact of endogenous elevation of HCY on retinal structure or function. To assess the impact of elevated HCY on retinal structure, we utilized a mouse with targeted disruption of the cystathionine–ß–synthase (cbs) gene, which develops a 2–4 fold increase in plasma HCY levels in cbs +/– mice, a 7–fold increase in cbs +/– mice fed a high methionine diet (HMD), and a 40–fold increase in cbs –/– mice. Methods: cbs –/– mice were analyzed only at 2–4 wks owing to early mortality in these mice; cbs +/– and cbs +/+ mice were fed the HMD from weaning and were compared to cbs +/+ fed a normal diet. Mice were analyzed at 13 and 18 wks postnatally. Eyes were embedded in OCT compound and cryosectioned or were fixed in 2% glutaraldehyde, embedded in Embed 812 and were examined by EM. Systematic morphometric analyses were performed using Axiovision software. TUNEL analysis was used to detect apoptotic cells. Results: Retinas of cbs –/– mice showed marked dropout of RGCs, a somewhat "foamy" appearance of the nerve fiber layer and apparent hypertrophy of the RPE layer. TUNEL analysis detected marked apoptosis in cells of the ganglion, inner and outer nuclear layers. Retinas of cbs +/– mice fed the HMD showed striking disruption of the inner retina, including a marked reduction of RGCs (∼10 cells/100 µm compared to ∼14–15 cells/100 µm in cbs +/+ mice). There was ∼25% decrease in the thickness of the inner nuclear and plexiform layers compared to controls. Conclusions: Sustained endogenous elevation of HCY alters the inner retina dramatically. Substantial decreases in the numbers of RGCs and reduced numbers of cells in the inner nuclear layer lead us to predict that retinal function will be severely compromised in these mice. Future studies will determine the effects of HCY on retinal function and the molecular/biochemical basis of the disruption.

Keywords: retinal degenerations: cell biology • apoptosis/cell death • retina: proximal (bipolar, amacrine, and ganglion cells)