Date of this Version
To examine the occurrence of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) following acute light damage in the naturally-occurring canine model of RHO-adRP (T4R RHOdog).
The left eyes of T4R RHOdogs were briefly light-exposed and retinas collected 3, 6 and 24 hours later. The contra-lateral eyes were shielded and used as controls. To evaluate the time course of cell death, histology and TUNEL assays were performed. Electron microscopy was used to examine ultrastructural alterations in photoreceptors at 15 min, 1 hour, and 6 hours after light exposure. Gene expression of markers of ER stress and UPR were assessed by RT-PCR, qRT-PCR and western blot at the 6 hour time-point. Calpain and caspase-3 activation were assessed at 1, 3 and 6 hours after exposure.
A brief exposure to clinically-relevant levels of white light causes within minutes acute disruption of the rod outer segment disc membranes, followed by prominent ultrastructural alterations in the inner segments and the initiation of cell death by 6 hours. Activation of the PERK and IRE1 pathways, and downstream targets (BIP, CHOP) of the UPR was not observed. However increased transcription of caspase-12 and hsp70 occurred, as well as calpain activation, but not that of caspase-3.
The UPR is not activated in the early phase of light-induced photoreceptor cell death in the T4R RHO model. Instead, disruption in rods of disc and plasma membranes within minutes after light exposure followed by increase in calpain activity and caspase-12 expression suggests a different mechanism of degeneration.
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Marsilli, S., Genini, S., Sudharsan, R., Gingrich, J., Aguirre, G. D., & Beltran, W. (2015). Exclusion of the Unfolded Protein Response in Light-Induced Retinal Degeneration in the Canine T4R RHO Model of Autosomal Dominant Retinitis Pigmentosa. PLoS ONE, 10 (2), e0115723-. http://dx.doi.org/10.1371/journal.pone.0115723
Additional Filesfigure 1.ppt (519 kB)
Histological alterations and photoreceptor cell death in T4R RHO retinas following acute light exposure
figure 2.ppt (239 kB)
Ultrastructural alterations in rods following acute light exposure in T4R RHO canine retinas
figure 3.ppt (54 kB)
Luminal ER chaperones in T4R RHO and WT canine retinas 6 hours after light exposure
figure 4.ppt (62 kB)
PERK-elF2α-ATF4 pathway in mutant T4R RHO and WT canine retinas 6 hours after light exposure
figure 5.ppt (102 kB)
IRE1-XBP1 pathway in mutant T4R RHO and WT canine retinas 6 hours after light exposure
figure 6.ppt (59 kB)
Downstream targets of the unfolded protein response (BIP and CHOP) in mutant T4R RHO and WT canine retinas 6 hours after light exposure
figure 7.ppt (60 kB)
Cytosolic markers of ER associated stress and ER associated degradation (ERAD) in mutant T4R RHO and WT retinas 6 hours after light exposure
figure 8.ppt (139 kB)
Effect of light exposure on calpain activation in mutant T4R RHO retinas
figure 9.ppt (126 kB)
Schematic representation of the signaling pathways activated during ER stress
table 1.ppt (143 kB)
Summary of the experimental procedures performed in the dogs of this study
table 2.ppt (265 kB)
List of forward (F), reverse (R), or TaqMan expression assay (Applied Biosystems) used for qRT-PCR
table 3.ppt (104 kB)
List of primary antibodies successfully used for western blotting in the current study
table 4.ppt (96 kB)
List of primary antibodies that were tested but failed to detect by western blotting the specific antigen in canine retina lysates when used overnight at the indicated dilution
Date Posted: 20 August 2015
This document has been peer reviewed.