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. 2010 Mar 30;107(13):5961-6.
doi: 10.1073/pnas.0911991107. Epub 2010 Mar 15.

Restoration of visual function in P23H rhodopsin transgenic rats by gene delivery of BiP/Grp78

Marina S Gorbatyuk  1 , Tessa Knox, Matthew M LaVail, Oleg S Gorbatyuk, Syed M Noorwez, William W Hauswirth, Jonathan H Lin, Nicholas Muzyczka, Alfred S Lewin
Affiliations

Restoration of visual function in P23H rhodopsin transgenic rats by gene delivery of BiP/Grp78

Marina S Gorbatyuk et al. Proc Natl Acad Sci U S A. .

Abstract

The P23H mutation within the rhodopsin gene (RHO) causes rhodopsin misfolding, endoplasmic reticulum (ER) stress, and activates the unfolded protein response (UPR), leading to rod photoreceptor degeneration and autosomal dominant retinitis pigmentosa (ADRP). Grp78/BiP is an ER-localized chaperone that is induced by UPR signaling in response to ER stress. We have previously demonstrated that BiP mRNA levels are selectively reduced in animal models of ADRP arising from P23H rhodopsin expression at ages that precede photoreceptor degeneration. We have now overexpressed BiP to test the hypothesis that this chaperone promotes the trafficking of P23H rhodopsin to the cell membrane, reprograms the UPR favoring the survival of photoreceptors, blocks apoptosis, and, ultimately, preserves vision in ADRP rats. In cell culture, increasing levels of BiP had no impact on the localization of P23H rhodopsin. However, BiP overexpression alleviated ER stress by reducing levels of cleaved pATF6 protein, phosphorylated eIF2alpha and the proapoptotic protein CHOP. In P23H rats, photoreceptor levels of cleaved ATF6, pEIF2alpha, CHOP, and caspase-7 were much higher than those of wild-type rats. Subretinal delivery of AAV5 expressing BiP to transgenic rats led to reduction in CHOP and photoreceptor apoptosis and to a sustained increase in electroretinogram amplitudes. We detected complexes between BiP, caspase-12, and the BH3-only protein BiK that may contribute to the antiapoptotic activity of BiP. Thus, the preservation of photoreceptor function resulting from elevated levels of BiP is due to suppression of apoptosis rather than to a promotion of rhodopsin folding.

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Conflict of interest statement

Conflict of interest statement: W.W.H. and the University of Florida have a financial interest in the use of AAV therapies and own equity in a company (AGTC Inc.) that might, in the future, commercialize some aspects of this work. All other authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Immunostaining of HeLa cells cotransfected with wild-type RHO, P23H RHO, and BiP-Flag. (A) Immunostaining analysis demonstrated the localization of wild-type RHO to the cell membrane (green). (B) In contrast, P23H RHO was not able to traffic to the cell membrane and was localized in the cytoplasm (green). (C) Overexpression of BiP did not affect the trafficking of wild-type RHO, which was still associated with the plasma membrane in the presence of excess BiP–Flag (red). (D) The distribution of the P23H RHO was not affected by extra BiP (red). P23H RHO was still localized in the cytoplasm and did not traffic to the cell membrane. The average transfection efficiency was 75%.
Fig. 2.
Fig. 2.
Increased level of BiP in HeLa cells expressing mutant RHO leads to modulation of the ER stress pathways and the apoptotic signal. (A) HeLa cells were cotransfected with plasmids expressing P23H RHO or P23H RHO and BiP proteins. The level of total BiP was increased by 62% (P < 0.01) leading to decrease in CHOP/GADD153 protein by 27% (P < 0.0002), phosphorylated ATF6 protein p50 by 22% (P < 0.04), pEIF2α by 30% (P < 0.006), and cleaved caspase-7 by 33% (P < 0.04). (B) HeLa cells were transfected with either wild-type RHO, P23H RHO, both wild-type RHO, or P23H RHO plus BiP. At 48 h cell lysates were obtained to perform a nucleosome release assay. Increased expression of P23H rhodopsin led to elevation of the apoptotic signal by 48% (P < 0.007) although wild-type RHO did not induce apoptosis. The overexpression of BiP led to down-regulation of the apoptotic signal by over 30% (P < 0.003) Transfection of cells with BiP alone elevated the apoptotic signal only slightly (P = 0.47). Data were normalized to the “Mock, BiP” mean. Experiments were performed in quadruplicate. The bars show the average ± SEM.
Fig. 3.
Fig. 3.
Study of the UPR in P23H-3 RHO transgenic (white) and wild-type (black) rats at P30. (A) At P30 in P23H-3 RHO rats, we observed up to 2.7-fold increase in the cleaved pATF6 50kD protein (P<0.03), a 51% increase in peIF2α (P<0.01), a 26% increase in the- level of CHOP protein (P<0.003), and an almost fourfold increase in activated caspase-7 (P < 0.016) (n = 5). (B) At P30 we also observed persistence IRE1 pathway in transgenic retinas. The level of spliced Xbp1 mRNA was fourfold higher in P23H-3 RHO rats compared to wild-type rats, P < 0.012 (n = 6). The bars show the average ± SEM.
Fig. 4.
Fig. 4.
Subretinal delivery of AAV-BiP preserves function of P23H-3 RHO photoreceptors. (A) Expression of BiP in the right retinas of transgenic rats (n = 10) carrying P23H RHO delays the decline of ERG a- and b-wave amplitudes compared with the left eyes expressing AAV5 GFP. The a-wave amplitudes of the scotopic ERG in treated eye at 2.68 cd-s/m2 intensity increased by 43% (P < 0.048) at 1 month, 44% (P < 0.16) at 2 months, and 115% (P < 0.014) at 3 months, relative to control eyes. The b-wave amplitudes were also increased by 47% (P < 0.015), 51% (P < 0.011), and 97% (P < 0.028), at 1, 2, and 3 months, respectively. (B) Two examples of wave forms of scotopic ERGs obtained by analyzing the BiP-treated rats at 3 months. (C) Increased BiP expression in P23H-3 RHO eyes (n = 8) preserved retinal integrity of photoreceptors in inferior and superior hemispheres. Modest increase (18%; P = 0.0043) in the thickness of the ONL was observed in the central inferior hemisphere. The bars show the average ± SEM.
Fig. 5.
Fig. 5.
Increased expression of BiP protein in P23H-3 RHO retinas reduced apoptosis and diminished expression of CHOP 1 month after AAV-BiP delivery. (A) AAV-BiP reduced nucleosome release by 42% (n = 6), P = 0.006 compared to control (AAV-GFP) treated retina. (B) Quantification of CHOP protein. Immunoblot analysis revealed that AAV-BiP reduced the levels of proapoptotic CHOP protein by 30% (P = 0.0052) (n = 8).
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