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Vishal Shinde, Olga Sizova, Marina Gorbatyuk; Modulation of Cellular signaling Pathways in P23H Rhodopsin Rat Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):664.
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© ARVO (1962-2015); The Authors (2016-present)
The activation of the unfolded protein response (UPR) in P23H rhodopsin (RHO) retinas with autosomal dominant Retinitis Pigmentosa (ADRP) has been reported previously by our lab. In this study we examined the modulation in autophagy, mTOR/AKT pathway, Bcl2 family proteins, Ca2+-based toxicity and mitochondria associated apoptosis in ADRP retina.
The RNA and protein extracts were obtained from P21, P30 and P40 P23H RHO (line 3) and Sprague Dawley rat retinas. qRT-PCR and western blot analysis were performed to analyze the modulation of cellular signaling. Calpain m, µ activity assay was conducted to determine the level of active calpains in degenerating retina. Mitochondria and cytosolic fractions of ADRP retina were separated to detect release of the cytochrome C and AIF and translocation of pro-apoptotic BAX protein to the mitochondria.
Our results showed that the autophagy- associated proteins ATG5 and Lamp2 were significantly down-regulated by 33% and 31% in P30 P23H RHO retina, while the mTOR signaling was highly activated by 290% and 430% at P30 and P40 respectively. The level of pro-survival pAKT was found to be significantly down-regulated by 75% and 44% at P30 and P40, respectively. Our data also revealed that the Ca2+-induced caspase-12 cleavage was significantly increased by 2.6-fold in P30 P23H RHO retinas. At same time point we also observed that the level of anti-apoptotic Bcl-xl protein was down-regulated by 0.6 fold and the level of pro-apoptotic Bax protein was up-regulated by 136 fold. Another pro-apoptotic protein Puma was elevated by 1.7-fold in P40 P23H RHO retina. Release of the cytochrome C and AIF to the cytosol and translocation of the BAX protein to the mitochondria of P23H RHO photoreceptors was observed in P21 and P40 ADRP retina, respectively which led to elevation of activated capspase-3, 7 and 9 by 1.5 fold, 2 fold and 1.49 fold.
This study gives important insight to the activation and alteration of cellular signaling pathways in P23H RHO photoreceptors and validated the mTor and calpains as therapeutic targets for ADRP treatment.
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