Abstract
Abstract: :
Purpose: Regeneration of rhodopsin requires RPE65-dependent generation of 11-cis retinal and dephosophorylation of opsin. RPE65-/- mice do not produce measurable levels of 11-cis and display minimal rod ERG signals. We hypothesized that administering exogenous 11-cis will improve retinal function due to two mechanisms; increase in quantum catch (availability of chromophore) and reduction of equivalent background (dephosphorylation of opsin). Methods: Scotopic single flash and photopic flicker ERGs were recorded from vehicle- and 11-cis-injected (IP at 5 and 25 µg/g bodyweight) mice (dark-adapted wild type and RPE65-/-, 1 month). Rhodopsin (endogenous and maximally recoverable amounts (rhomax) after addition of 11-cis) was measured in dodecyl-maltoside solubilized retinal extracts using absorption spectrophotometry. Opsin phosphorylation (opsin-P) was determined in Asp-N-digested samples using electrospray ionization mass spectrometry. Results: (1) Deletion of the RPE65 gene reduced the sensitivity of both scotopic and photopic ERG by ∼4 log units, eliminated measurable amounts of rhodopsin and resulted in constitutive opsin-P of ∼20%. (2) 11-cis injections improved all measured ERG parameters in a dose-dependent manner: a-wave amplitudes (to 4-12% of wild type levels), b-wave thresholds (1-1.6 log), b-wave maximal amplitudes (400-500%) and photopic flicker sensitivity (0.8-1.6 log). Concomitantly, it restored 4-7% of rhomax and reduced opsin-P by ≷50%. There was no indication of recovery of cone function. In vitro experiments demonstrated the opsin-P is totally reduced with full rhodopsin regeneration. Conclusion: (1) RPE65-/- rods contain mostly opsin, ∼1/5 of which is phosphorylated, and little rhodopsin, leading to impaired rod function. (2) Adding back 11-cis significantly improved rod physiology by regenerating rhodopsin and, most likely, by dephosphorylating opsin, shifting rods functionally into a dark-adapted state.
Keywords: 396 electroretinography: non-clinical • 497 opsins • 515 phosphorylation