Retinal degeneration-3 protein promotes photoreceptor survival by suppressing activation of guanylyl cyclase rather than accelerating GMP recycling

Abstract

Retinal degeneration-3 protein (RD3) deficiency causes photoreceptor dysfunction and rapid degeneration in the rd3 mouse strain and in human Leber’s congenital amaurosis, a congenital retinal dystrophy that results in early vision loss . However, the mechanisms responsible for photoreceptor death remain unclear. Here, we tested two hypothesized biochemical events that may underlie photoreceptor death: (i) the failure to prevent aberrant activation of retinal guanylyl cyclase (RetGC) by calcium-sensor proteins (GCAPs) versus (ii) the reduction of GMP phosphorylation rate, preventing its recycling to GDP/GTP. We found that GMP converts to GDP/GTP in the photoreceptor fraction of the retina ∼24-fold faster in WT mice and ∼400-fold faster in rd3 mice than GTP conversion to cGMP by RetGC. Adding purified RD3 to the retinal extracts inhibited RetGC 4-fold but did not affect GMP phosphorylation in wildtype or rd3 retinas. RD3-deficient photoreceptors rapidly degenerated in rd3 mice that were reared in constant darkness to prevent light-activated GTP consumption via RetGC and phosphodiesterase 6. In contrast, rd3 degeneration was alleviated by deletion of GCAPs. After 2.5 months, only ∼40% of photoreceptors remained in rd3 / rd3 retinas. Deletion of GCAP1 or GCAP2 alone preserved 68% and 57% of photoreceptors, respectively, whereas deletion of GCAP1 and GCAP2 together preserved 86%. Taken together, our in vitro and in vivo results support the hypothesis that RD3 prevents photoreceptor death primarily by suppressing activation of RetGC by both GCAP1 and GCAP2 but do not support the hypothesis that RD3 plays a significant role in GMP recycling.

Retinal degeneration-3 protein attenuates photoreceptor degeneration in transgenic mice expressing dominant mutation of human retinal guanylyl cyclase

“…The results of our present study demonstrate that removal of either RetGC isozyme markedly reduces the rate of Rd3 -/photoreceptor degeneration (Fig. 1), similarly to the previously documented deletion of GCAPs (25)(26)(27). Taken together, these results do not support the hypothesis that GCAPs in the absence of RD3 facilitate apoptosis primarily through a process unrelated to activation of RetGC.…”

“…Both changes occur in the R838S + mouse rods in vivo (17). However, in a previous study (16,26), we observed that another, more recently recognized aspect of the RetGC regulationits inhibition by RD3may contribute to the disease (Fig. 3).…”

“…The most interesting alternative possibility that has been suggested is that GCAP2 causes photoreceptor death by activating in the inner segment an apoptotic pathway(s) in a way that is unrelated to RetGC regulation (27). In our recent study (26), we observed that deletion of GCAP2 indeed led to partial rescue, but it was less effective than deletion of GCAP1 and especially less effective than simultaneous deletion of both GCAPs. The results of our present study demonstrate that removal of either RetGC isozyme markedly reduces the rate of Rd3 -/photoreceptor degeneration (Fig.…”

“…9B). Deletion of the two GCAP isoforms dramatically rescued Rd3 -/photoreceptors from degeneration (25)(26)(27). Yet the possibility that the apoptotic effect of GCAPs in Rd3 -/photoreceptors was propagated not via aberrant activation of the guanylyl cyclase but through some other process (27) could not be fully excluded.…”

“…RD3 also strongly inhibits RetGC basal activity and activation by GCAPs (23,24). The in vivo evidence suggests that aberrant guanylyl cyclase activation by GCAPs in the absence of RD3 provokes the apoptotic process (25,26), but this does not fully exclude the possibility that GCAPs mediate the apoptotic process via some target(s) other than RetGC (27). Therefore, we tested here the effects of deleting RetGC1 or RetGC2 in Rd3 -/retinas.…”