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Javier Sancho-Pelluz, Winston Lee, Yiting Tsai, Iyas Washington, Stephen Tsang, Chyuan-Sheng Lin; Constitutive Rhodopsin Activation rather than Mistrafficking Results in Photoreceptor Death in the D190N Preclinical Model of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4180.
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© ARVO (1962-2015); The Authors (2016-present)
D190N, a missense mutation in rhodopsin, causes autosomal dominant retinal degeneration (adRP) in humans. Affected patients present hyperfluorescent retinal rings, decreases in a- and b-waves, and progressive rod photoreceptor degeneration. Two hypotheses had been developed to explain this degeneration: (a) defective rhodopsin trafficking prevents proteins from correctly exiting the endoplasmic reticulum, or (b) elevated rate of constitutive mutant rhodopsin signaling causes excitotoxicity. A knock-in mouse model with D190N mutation was used to address the controversy. We approached the question not only by observing the physiology and histology of the retina, but also by checking the rhodopsin glycosylation state, and its thermal decay.
D190N animals were the object of electrophysiological and autofluorescence studies. Rhodopsin localization was studied by immunohistochemistry. To investigate the glycosylation status of D190N rhodopsin, retinal homogenates of wt and D190N animals were treated with Endo H and PNGase F deglycosylation enzymes. Thermal decay rates of retinal extracts from D190N mice and wt controls were studied by measuring the absorbance at 500 nm.
Glycosylated rhodopsin was found at ~35 kD in both mutant and wt homogenates. After Endo H treatment, protein was found also ~35 kD in both homogenates. Rhodopsin from both wt and mutant mice shifted to a lower molecular weight (~32 kD) after PNGase F treatment. Rhodopsin from mutant mice appeared to behave similarly to wt rhodopsin after deglycosylation, which is consistent with correct trafficking to the OS. The half-life of the decay process was 1.18 ± 0.02 min for heterozygous D190N/+ retinal extract; however, the wt extract did not sufficiently decay within the duration of the test (20 minutes) to calculate a half-life.
Our results suggests that the D190N mutation resulted in thermally unstable rhodopsin causing constitutive signaling, not defective trafficking. Rhodopsin glycosylation state in the mutant retina appeared to be similar to that in the wt animal. Furthermore, increased thermal isomerization of rhodopsin 11-cis retinal to all-trans retinal was noted in the mutant.
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