Abstract
Apoptosis is the mode of photoreceptor cell death in many retinal dystrophies. Exposure of Balb/c mice to excessive levels of light induces photoreceptor apoptosis and represents an animal model for the study of retinal degenerations. Caspases have emerged as central regulators of apoptosis, executing this tightly controlled death pathway in many cells. Previously we have reported that light-induced photoreceptor apoptosis occurs independently of one the key executioners of apoptosis, caspase-3. This present study extends these results reporting on the lack of activation of other caspases in this model including caspases-8, -9, -7, and -1. Furthermore, photoreceptor apoptosis cannot be inhibited with the broad range caspase inhibitor zVAD-fmk indicating that light-induced retinal degeneration is caspase-independent. We demonstrate that cytochrome c does not translocate from mitochondria to the cytosol during photoreceptor apoptosis. We also show that during retinal development apoptotic protease activating factor (Apaf-1) protein levels are markedly decreased and this is associated with the inability to activate the mitochondrial caspase cascade in the mature retina. In addition, there is also a significant reduction in expression of caspases-3 and -9 during retinal maturation and these levels do not increase following light exposure. Finally, we show that the calcium-dependent proteases calpains are active during light-induced retinal degeneration and establish that the calcium channel blocker D-cis-diltiazem completely inhibits photoreceptor apoptosis.
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Abbreviations
- AcIETD-ρNA:
-
Ac-Ile-Glu-Thr-Asp-p-nitroanilide
- AcYVAD-ρNA:
-
Ac-Tyr-Val-Ala-Asp-p-nitroanilide
- Apaf-1:
-
apoptotic protease activating factor
- CFE:
-
cell free extract
- DEVD-fmk:
-
Asp-Glu-Val-Asp-fluromethylketone
- DMSO:
-
dimethyl sulphoxide
- FITC:
-
fluorescein isothiocyanate
- GAPDH:
-
glyceraldehydes-3-phosphate dehydrogenase
- INL:
-
inner nuclear layer
- ip:
-
intraperitoneal
- ONL:
-
outer nuclear layer
- PI3K:
-
phosphatidylinositol 3-kinase
- PBS:
-
phosphate buffered saline
- RCS:
-
Royal College of Surgeons
- rd:
-
retinal degeneration
- RP:
-
Retinitis Pigmentosa
- TUNEL:
-
Terminal dUTP nick end labelling
- TdT:
-
terminal deoxynucleotidyl transferase
- UV:
-
ultraviolet
- zVAD-fmk:
-
Z-Val-Ala-Asp.fluoromethylketone
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Acknowledgements
The authors wish to acknowledge Dr. Paul Kenna for assistance with sub-retinal injections and Professor Peter Humphries and Dr. Melanie Walsh for useful discussions. This work was supported by Fighting Blindness (Ireland), The Health Research Board of Ireland and Bausch and Lomb, Co. Waterford Ireland.
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Donovan, M., Cotter, T. Caspase-independent photoreceptor apoptosis in vivo and differential expression of apoptotic protease activating factor-1 and caspase-3 during retinal development. Cell Death Differ 9, 1220–1231 (2002). https://doi.org/10.1038/sj.cdd.4401105
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DOI: https://doi.org/10.1038/sj.cdd.4401105
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