Activation of Unfolded Protein Response Signaling Pathways During Retinal Degeneration in P23H Rhodopsin Transgenic Rats

Abstract

Purpose: : Protein misfolding in the endoplasmic reticulum activates a set of signaling pathways, collectively termed the Unfolded Protein Response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if protein misfolding persists. How the UPR integrates its protective and proapoptotic outputs to select between life and death cell fates is unclear. We have explored the role of the three branches of the UPR in cell culture and in P23H rhodopsin transgenic rats. We have also examined whether cell survival would be sustained if activity of the cell survival branch of the UPR were artificially sustained.

Methods: : Retinas from transgenic rats expressing P23H rhodopsin were collected at multiple timepoints prior to and during retinal photoreceptor degeneration (postnatal days 6, 8, 10, 12, 30 and 60). RNA was extracted and quantitative PCR was performed to measure the levels of specific UPR gene targets. Chemical-genetic activation of IRE1 in cell culture was performed by site-directed mutagenesis of the human IRE1 gene, followed by flippase-mediated insertion of the allele into HEK293 cells.

Results: : Cytoprotective UPR mRNA levels of the IRE1 branch, including the BiP/grp78 chaperone, selectively dropped in the retinas of transgenic rhodopsin animals compared to wild-type Sprague-Dawley controls after postnatal day 12. Proapoptotic UPR mRNA levels of the PERK branch, such as the Chop gene, concomitantly increased in transgenic rhodopsin animals but remained low in wild-type animals at equivalent timepoints. The timecourse of UPR protective mRNA decline and UPR proapoptotic mRNA rise corresponded tightly with the rate of retinal degeneration in the P23H rhodopsin animals. When IRE1 activity was artificially sustained, cell survival in culture was enhanced.

Conclusions: : UPR signaling is activated by protein misfolding in cell culture and by P23H rhodopsin expression in transgenic models of autosomal dominant retinitis pigmentosa. Protective UPR signaling decays while proapoptotic UPR signaling is maintained in vitro and in vivo after persistent protein misfolding. Artificial activation of UPR protective output may provide a new means of rescuing photoreceptor degeneration due to rhodopsin misfolding.

Keywords: retinal degenerations: cell biology • retinal degenerations: hereditary • stress response