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Calpain Activation Is the Major Cause of Cell Death in Photoreceptors Expressing a Rhodopsin Misfolding Mutation

  • Antonella Comitato
  • Davide Schiroli
  • Monica Montanari
  • Valeria MarigoEmail author
Article
  • 53 Downloads

Abstract

The majority of mutations in rhodopsin (RHO) cause misfolding of the protein and has been linked to degeneration of photoreceptor cells in the retina. A lot of attention has been set on targeting ER stress for the development of new therapies for inherited retinal degeneration caused by mutations in the RHO gene. Nevertheless, the cell death pathway activated by RHO misfolded protein is still debated. In this study, we analyzed the retina of the knock-in mouse expressing the P23H misfolded mutant RHO. We found persistent unfolded protein response (UPR) during degeneration. Interestingly, long-term stimulation of the PERK branch of ER stress had a protective effect by phosphorylating nuclear factor erythroid 2–related factor 2 (NRF2) transcription factor, associated with antioxidant responses. Otherwise, we provide evidence that increased intracellular calcium and activation of calpains strongly correlated with rod photoreceptor cell death. By blocking calpain activity, we significantly decreased the activation of caspase-7 and apoptosis-inducing factor (AIF), two cell death effectors, and cell demise, and effectively protected the retina from degeneration caused by the P23H dominant mutation in RHO.

Keywords

adRP Rod eIF2α Spectrin PD150606 Z-VAD-FMK GSK2606414A 

Notes

Acknowledgments

The authors acknowledge the Cell-lab Facility and CSSI of the University of Modena and Reggio Emilia for the cytofluorimetric analysis and animal husbandry assistance.

Author Contributions

A.C. and D.S. performed experimental procedures and contributed to the writing of the manuscript. M.M. performed flow cytometry analysis. V. M designed and supervised the experiments and wrote the manuscript.

Funding Information

V.M. was supported by the research grant Fondazione Roma (call for proposals 2013 on Retinitis Pigmentosa), European Union (transMed, MSCA-ITN-2017-765441), and Fondazione Telethon (grant numbers GGP11210, GGP14180).

Compliance with Ethical Standards

All procedures on mice were conducted at CSSI (Centro Servizi Stabulario Interdipartimentale), approved by the Ethical Committee of University of Modena and Reggio Emilia and by the Italian Ministero della Salute (346/2015-PR), and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Conflict of Interest

The authors declare that they have no conflict of interest.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

Supplementary material

12035_2019_1723_Fig5_ESM.png (2.7 mb)
Figure S1

Activation of calpains inRhoP23H/+retinas. (a) Controls for the specificity of the calpain assay. Retina cryosections from PN19 RhoP23H/+ mutant mice were processed for the calpain activity assay either in the absence of t CMAC, t-BOC-Leu-Met substrate (No substrate) or after pretreatment of the crysection with the PD150606 calpain inhibitor (Calpain inh. pretreatment) to block calpain activity. No fluorescent signal could be detected in the control experiments. (b) Retina cryosections from PN15, PN19, PN31 and PN60 wild type (WT) and RhoP23H/+ mutant mice were exposed to a calpain substrate that becomes fluorescent (blue) upon cleavage by calpains. White dots in the outer nuclear layer (onl) containing photoreceptor cells identify cells activating calpains and were detected in mutant retinas but not in WT retinas. The same sections were assayed by TUNEL assay (red) to detect cells undergoing cell death. White dots in the outer nuclear layer (onl) containing photoreceptor cells identify dying cells and were detected in mutant retinas but not in WT retinas. The few PN60 photoreceptor cells undergoing cell death and activating calpains are indicated by arrows. Merged images of calpain activity staining and TUNEL are shown on the right-hand side. Scale bars: 50μm. (PNG 2735 kb)

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High Resolution Image (TIF 17727 kb)
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Figure S2

Validation of the specificity of antibodies used in western blotting experiments. Antibodies used for western blotting to detect activation/phosphorylation of ER-stress sensors P-IRE1, P-PERK and P-eIF2α were validated on protein extracts from the NIH3T3 cell line treated with 2 μg/ml Tunicamycin, for 24 h as in Sanges et al [63]. Antibodies used for western blotting to detect activated/cleaved caspase-7 were validated on protein extracts from NIH3T3 cell line treated with 1 μM Staurosporine for 2 h, as in Petit et al [64]. Antibodies used for western blotting to detect nuclear translocation of NRF2 were validated on protein extracts from the NIH3T3 cell line treated with 5 μg/ml Tunicamycin for 30 min, as in Cullinan et al [37]. (PNG 462 kb)

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High Resolution Image (TIF 1542 kb)
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Figure S3

Entire membranes of western blotting experiments shown in Figs. 1, 2, 3 and 4. (PNG 1408 kb)

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High Resolution Image (TIF 4273 kb)
12035_2019_1723_Fig8_ESM.png (470 kb)
Figure S4

Cell death analyses on treatedRhoP23H/+retinas. Sections from RhoP23H/+ mutant PN19 retinas either treated with GSK2606414A (PERK inhibitor) or with PD150606 (calpain inhibitor) or Z-VAD-FMK (Caspase inhibitor) or with vehicle (mock) were analyzed by TUNEL assay (red). Nuclei were stained with DAPI (blue) and show all retinal cells. Red dots indicate dying cells. (PNG 470 kb)

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High Resolution Image (TIF 2713 kb)
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Figure S5

Histological analysis of injected eyes. Examples of eyes stained with Hematoxylin and Eosin from not injected and intravitreally injected animals. (PNG 1487 kb)

12035_2019_1723_MOESM5_ESM.tif (10.8 mb)
High Resolution Image (TIF 11048 kb)

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Authors and Affiliations

  1. 1.Department of Life SciencesUniversity of Modena and Reggio EmiliaModenaItaly

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