Exposure to the complement C5b-9 complex sensitizes 661W photoreceptor cells to both apoptosis and necroptosis.

The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.

Electroretinogram and visual-evoked potential assessment of retinal and central visual function in a rat ocular hypertension model of glaucoma.

PURPOSE/AIM: The aim of the study was to investigate the long-term functional changes that may occur in the retina and visual cortex in a rat ocular hypertension (OHT) model of glaucoma, used in our lab for treatment studies, using electroretinogram (ERG) and visual-evoked potential (VEP) cortical recordings in order to test the hypothesis that experimental glaucoma has differential retinal and central effects. MATERIALS AND METHODS: Experimental glaucoma was induced unilaterally in Dark Agouti rats using hypertonic saline injection into the episcleral veins. After 3, 8, 16 and 26 weeks, ERGs and VEPs were recorded under scotopic conditions using brief full-field white flashes (10 µcd s m(-2) to 10.4 cd s m(-2)) and under photopic conditions using a rod-adapting background and white light flashes (0.13-10.4 cd s m(-2)). RESULTS: At 16 and 26 weeks after OHT induction, there was a significant reduction in the amplitudes of the a- (50% and 30% of unoperated eye values, respectively) and b-waves (55% and 40%, respectively) of the scotopic ERG and the b-waves of the photopic ERG (55% and 45%, respectively) in the glaucomatous eyes. However, no significant changes in the VEPs simultaneously recorded over the visual cortex were seen at any of the time points. CONCLUSIONS: The reductions in ERG amplitudes suggest that this model of glaucoma not only causes retinal ganglion cell (RGC) degeneration but also degeneration of the outer retinal cells, and this was confirmed by histology showing a reduction in the outer retinal layers in the glaucomatous eyes. Cortical VEPs did not show detrimental effects suggesting that the retinal damage in this model was not extensive enough to be detected with the VEP methods used or that there could be central compensation in this model of glaucoma.