sCD44 Is Neurotoxic to Retinal Ganglion Cells

Abstract

Abstract: : Purpose: Retinal ganglion cells (RGC) are susceptible to a variety of insults, and current glaucoma research targets neurotrophic and/or neuroprotective therapies for RGC in primary open angle-glaucoma (POAG). RGC survival is dependent upon trophic signal transduction pathways, particularly those involving tyrosine kinase receptors, which if disrupted, may result in cell death. Soluble CD44 (sCD44), a molecule found to be increased in the aqueous humor in POAG, interferes with CD44-mediated growth factor receptor signal transduction in susceptible cells in culture. The purpose of this study was to determine if exogenous sCD44 affects RGC survival. Methods: RGC-5, a transformed rat RGC line, was grown in Dulbecco's minimum essential medium (DMEM) containing 10% fetal calf serum (FCS) until 80% confluent. The cells were washed twice with PBS, and incubated in minimum essential medium Eagle (MEM) containing 0.1% FCS with 1 or 40 ng/ml purified sCD44, 10 ng/ml heat-inactivated sCD44, or PBS for 6, 12, and 24 hours. RGC viability was assessed by trypan blue staining, total cell number was determined by cell counting, and morphological changes were observed using a phase-contrast microscope. Results: Within 12 hours of incubation with sCD44, RGC displayed morphological changes that included cell rounding, detachment, aggregation, and swelling. There was a statistically significant time-dependent (n=9, p<0.0001) and dose-dependent (n=9, p<0.03) decrease in cell number and cell viability in RGC treated with sCD44. sCD44-induced cell death was cell-specific. Cultured rat aortic smooth muscle cells were resistant to 10 ng/ml sCD44 treatment, whereas human cortical neurons were susceptible to sCD44 after 24 hours (n=6, p<0.001) but recovered and were resistant after 48 hours and were not significantly different from control (n=6, p<0.55). RGC treated with 1 ng/ml sCD44 for 24 hours were significantly less susceptible to cell death than human trabecular meshwork cells treated under the same conditions (n=6, p<0.002). Number and viability of cells treated with the heat-inactivated sCD44 preparation were not significantly different from the PBS-treated cells. Conclusions: The results indicate that exogenous sCD44 adversely affects RGC survival. Disruption of membrane CD44-specific signaling pathways by sCD44 may be responsible for the loss of RGC that occurs in POAG.

Keywords: ganglion cells • growth factors/growth factor receptors • protein structure/function