Endogenous elevation of homocysteine induces retinal neuron death in the cystathionine-beta-synthase mutant mouse.

PURPOSE: To determine the effects of endogenous elevation of homocysteine on the retina using the cystathionine beta-synthase (cbs) mutant mouse. METHODS: Retinal homocysteine in cbs mutant mice was measured by high-performance liquid chromatography (HPLC). Retinal cryosections from cbs(-/-) mice and cbs(+/-) mice were examined for histologic changes by light and electron microscopy. Morphometric analysis was performed on retinas of cbs(+/-) mice maintained on a high-methionine diet (cbs(+/-) HM). Changes in retinal gene expression were screened by microarray. RESULTS: HPLC analysis revealed an approximate twofold elevation in retinal homocysteine in cbs(+/-) mice and an approximate sevenfold elevation in cbs(-/-) mice. Distinct alterations in the ganglion, inner plexiform, inner nuclear, and epithelial layers were observed in retinas of cbs(-/-) and 1-year-old cbs(+/-) mice. Retinas of cbs(+/-) HM mice demonstrated an approximate 20% decrease in cells of the ganglion cell layer (GCL), which occurred as early as 5-weeks after onset of the HM diet. Microarray analysis revealed alterations in expression of several genes, including increased expression of Aven, Egr1, and Bat3 in retinas of cbs(+/-) HM mice. CONCLUSIONS: This study provides the first analysis of morphologic and molecular effects of endogenous elevations of retinal homocysteine in an in vivo model. Increased retinal homocysteine alters inner and outer retinal layers in cbs homozygous mice and older cbs heterozygous mice, and it primarily affects the cells of the GCL in younger heterozygous mice. Elevated retinal homocysteine alters expression of genes involved in endoplasmic reticular stress, N-methyl-d-aspartate (NMDA) receptor activation, cell cycle, and apoptosis.

Diabetes Accelerates Retinal Neuronal Cell Death In A Mouse Model of Endogenous Hyperhomocysteinemia.

Hyperhomocysteinemia has been implicated in visual dysfunction. We reported recently that mice with endogenous hyperhomocysteinemia, due to mutation of the cystathionine-ß-synthase (cbs) gene, demonstrate loss of neurons in the retinal ganglion cell (RGC) layer and other retinal layers as homocysteine levels increase. Some clinical studies implicate hyperhomocysteinemia in the pathogenesis of diabetic retinopathy, which is also characterized by RGC loss. The present study used cbs(+/-) mice to determine whether modest elevation of plasma homocysteine, in the presence of diabetes, accelerates neuronal cell loss. Diabetes (DB) was induced in 3 wk old cbs(+/-) and wildtype mice using streptozotocin; four groups of mice were studied: DB cbs(+/-); non-DB cbs(+/-); DB cbs(+/+); non-DB cbs(+/+). One group of diabetic cbs(+/-) mice was maintained on a high methionine diet (HMD, 0.5% methionine drinking water) to increase plasma homocysteine slightly. Eyes were harvested at 5, 10 and 15 weeks post-onset of diabetes; retinal cryosections were examined by light microscopy and subjected to systematic morphometric analysis. Diabetic cbs(+/-) had significantly fewer RGCs at 5 weeks compared to age-matched, non-diabetic cbs(+/-) and wildtype controls (10.0 ± 0.5 versus 14.9 ± 0.5 and 15.8 ± 0.6 cells/100 µm retina length, respectively). Significant differences in retinas of DB/high homocysteine versus controls were obtained 15 wks post-onset of diabetes including fewer RGCS and decreased thickness of inner nuclear and plexiform layers. Moderate increases in plasma homocysteine coupled with diabetes cause a more dramatic alteration of retinal phenotype than elevated homocysteine or diabetes alone and suggest that diabetes accelerates the retinal neuronal death in hyperhomocysteinemic mice.