The present study was performed to investigate changes of cytosolic and mitochondrial
calpain activities, and effects of intravitreously injected
calpain inhibitor on photoreceptor apoptosis in Royal College of Surgeon's (RCS) rats. Time courses of activities for both cytosolic and mitochondrial calpains and amount of
calpastatin in RCS rat retina were analyzed by subcellular fractionation,
calpain assay and western blotting.
Calpain assay was colorimetrically performed using Suc-LLVY-Glo as substrate. Effects of intravitreously injected
calpain inhibitor (ALLN and
PD150606) on RCS rat
retinal degeneration were analyzed by TUNEL staining. Effects of mitochondrial
calpain activity on activation and translocation of
apoptosis-inducing factor (AIF) were analyzed by western blotting. Mitochondrial
calpain started to be significantly activated at postnatal (p) 28 days in RCS rat retina, whereas cytosolic micro-
calpain was activated at p 35 days, although specific activity of mitochondrial
calpain was 13% compared to cytosolic micro-
calpain. Intravitreously injected ALLN and
PD150606 effectively inhibited photoreceptor apoptosis only when injected at p 25 days, but did not inhibit photoreceptor apoptosis when injected at p 32 days. Parts of AIF were truncated/activated by mitochondrial calpains and translocated to the nucleus. These results suggest that 1),
calpain presents not only in the cytosolic fraction but also in the mitochondrial fraction in RCS rat retina; 2), mitochondrial
calpain is activated earlier than cytosolic
calpain during
retinal degeneration in RCS rats; 3), photoreceptor apoptosis may be regulated by not only
calpain systems but also other mechanisms; 4), mitochondrial
calpain may activate AIF to induce apoptosis; and 5),
calpain inhibitors may be partially effective to inhibit photoreceptor apoptosis in RCS rats. The present study provides new insights into the molecular basis for photoreceptor apoptosis in RCS rats and the future possibility of new
pharmaceutical treatments for
retinitis pigmentosa.