Abstract
Purpose: :
Moderate elevations of HCY are implicated in glaucoma. We reported earlier that retinal ganglion cells (RGC) die upon acute intravitreal exposure to HCY [75-200 µM] as do primary RGCs exposed 18 h to 50 µM HCY. To determine consequences of sustained moderate elevations of HCY on retinal gene expression, microarray analysis was performed on retinas of mice deficient in CBS, an enzyme that converts HCY to cystathionine.
Methods: :
Cbs+/- mice were administered 0.5 % methionine in drinking water, which elevates plasma HCY 7-fold. Retinal levels of HCY were analyzed by HPLC. Systematic morphometric analysis was performed on mouse retinal cryosections at 5, 15, and 30 wks. Microarray analysis was performed in triplicate from RNA isolated from neural retinas of cbs+/+ and cbs+/- mice using the FairPlay Microarray labeling kit; probes were labeled with Cy3 or Cy5 reactive dye; Cy3/Cy5 intensity ratios were analyzed to compare gene expression profiles quantitatively.
Results: :
HPLC analysis revealed marked elevation of retinal HCY in cbs+/- mice. Morphometric analysis revealed ~20% loss of RGCs in cbs+/- mice by 5 wks and decreased thickness of inner plexiform and nuclear layers by 15 and 30 wks, respectively. Of 36,212 genes screened, microarray analysis revealed a ≥2-fold change in expression of 1216 categorized into 6 functional groups. Of particular interest: (1) pro-apoptotic: Bat3 (↑2.3-fold), (2) anti-apoptotic: Survivin (↓2.8-fold), (3) cell cycle: Egr1 (↑ 4.7-fold), (4) antioxidant: subunits of GST (↓2.0-5.2-fold) and SOD3 (↓2.6-fold), (5) calcium signaling: Slc24a4 (↑3.4-fold), (6) axon growth/guidance: Efnb3 (↑2.6-fold), Ablim2 (↑2.66-fold), Tsc2 (↑2.5 fold).
Conclusions: :
Cbs+/- mice have elevated retinal HCY, significant loss of RGCs and decreased thickness of inner retinal layers. Altered expression of genes related to apoptosis, axon guidance, cell cycle, and antioxidant levels lay the foundation for future studies to determine which contribute most significantly to the retinal phenotypic changes observed in this model of retinal hyperhomocysteinemia.
Keywords: retina • ganglion cells • apoptosis/cell death