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M. S. Gorbatyuk, T. J. Knox, J. H. Lin, M. M. LaVail, W. W. Hauswirth, A. S. Lewin; AAV-Delivered BiP/Grp78 Suppresses Photoreceptor Apoptosis in P23H RHO Transgenic Rats, a Model of ADRP. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1736.
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The P23H Rhodopsin (Rho) mutation is thought to form a misfolded protein resulting in endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), which ultimately triggering apoptosis. We have studied these phenomena in a rat model of autosomal dominant retinitis pigmentosa (ADRP) caused by a P23H Rho transgene. We previously reported that the over-expression of BiP/Grp78 ameliorates retinal degeneration in P23H-3 Rho retinas. The goal of this project is to find the mechanism by which this over-expression leads to a therapeutic effect in degenerating retinas.
HeLa cells were co-trasfected with plasmids expressing P23H Rho or wild-type Rho and human BiP/Grp78 cDNAs under control of human cytomegalovirus immediate-early promoter (CMV) promoter. At 48 hours post-transfection, cells were harvested and the level of apoptosis was analyzed by measuring nucleosome release using the Cell Death Detection Kit (Roche). For in vivo experiments, we inserted BiP/Grp78 gene under control of the hybrid CMV-beta-actin (CBA) promoter in AAV plasmid (TRs-2) and packaged in serotype 5 capsid. Injection of P23H line-3 transgenic rat pups in their right eyes was done at postnatal day 15. AAV5-GFP served as a control and was injected in the left eyes. Animals were analyzed by electroretinography (ERG) in one month after injection to record the a- and b-wave amplitudes. Rats were then sacrificed and retinas were removed to perform the detection of apoptotic signal by using the Nucleosome release assay.
Transfection of HeLa cells with the P23H Rho plasmid led to significant increase of apoptotic signal by 48% (P<0.002) compared to cells transfected with wild-type Rho. We also found that the co-expression of both P23H Rho and BiP/Grp78 plasmids reduces the apoptotic signal by 30% (P<0.004). In rats, treatment with AAV5-BiP/Grp78 reduced the level of apoptosis by 42% (P<0.05). ERG of a-wave and b-wave amplitudes were elevated in BiP/Grp78 treated eyes by 43 and 47%, respectively, compared to control eyes.
The over-expression of BiP/Grp78 in photoreceptors appears to reduce progression of the ADRP associated with P23H Rho. Preventing apoptosis is one possible mechanism by which BiP/Grp78 ameliorates retinal degeneration in P23H-3 Rho animals.
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