Abstract
Purpose: :
The Y99C GCAP1 reduces Ca2+–sensitivity of retinal guanylyl cyclase (retGC), elevates the free intracellular cGMP and Ca2+ concentrations, and causes degeneration of photoreceptors in transgenic mice [1]. We hypothesize that activation of PDE6 in the dark can slow retinal degeneration in the Y99C mice by lowering the free cGMP and Ca2+concentrations.
Methods: :
A mutation, G90D, in rhodopsin that activates the PDE6 cascade in the dark [2], was introduced into a fast degenerating mouse line, L53 [1], expressing the Y99C GCAP1 (Y99C+); a single allele for wild type rhodopsin (R) remained [2]. The Y99C+;G90D+;R+/– mice were analyzed for ERG, retinal morphology, Ca2+–sensitivity of retGC, and the intracellular free Ca2+concentrations [3].
Results: :
Ca2+ sensitivity of retGC in the L53 mice (both Y99C+;R+/+ and Y99C+;R+/–) was shifted toward higher than normal dark–adapted free Ca2+ concentrations. They rapidly lost ERG responses and nearly all photoreceptor nuclei by the age of 6 months. The maximal amplitude of the ERG a–wave in the Y99C+;R+/– mice after 120 days was less than 5% of normal (15 µV ±15 SD, n=27), often with no measurable a–wave at saturating flash strength. In the Y99C+;G90D+;R+/– retinas, retGC also remained active at high free Ca2+ concentrations in vitro, but the dark adapted free intracellular Ca2+ level was closure to the wild type. The Y99C+;G90D+;R+/– mice had clear ERG–responses at > 8 months of age (the oldest age group tested). The light–sensitivity of ERG was reduced, but the maximal a–wave reached half the normal amplitude (227 µV±76 SD, n=58) and half of the rod nuclei remained in their retinas after 6 months of age; the morphology of the photoreceptors was similar to the wild type.
Conclusions: :
Activation of PDE6 in the dark by G90D rhodopsin can effectively restore near–normal levels of cGMP and Ca2+ in Y99C rods thus rescuing them from degeneration caused by abnormal cGMP synthesis.
References: :
[1]. Olshevskaya et al., (2004) J. Neuroscience 24, 6078; [2] Sieving et al., (2001) J. Neuroscience 21, 5449; [3] Woodruff et al., (2002) J.Physiol (London) 542, 843.
Keywords: proteins encoded by disease genes • photoreceptors • retinal degenerations: cell biology