Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: gene transfer/gene therapy • photoreceptors • chaperones