leupeptins and Retinal-Degeneration

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.

Topics: Acrylates; Animals; Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Cytosol; Electrophoresis, Polyacrylamide Gel; Immunoenzyme Techniques; In Situ Nick-End Labeling; Leupeptins; Mitochondria; Photoreceptor Cells, Vertebrate; Rats; Rats, Mutant Strains; Retinal Degeneration

The primary purpose of this study was to characterize photoreceptor apoptosis in the rd mouse. Given that apoptosis is the final common pathway in many cases of retinal degeneration, the ability to retard or even arrest this process may ameliorate retinal disorders such as retinitis pigmentosa (RP). The absence of any recognized therapy emphasizes the fact that a detailed knowledge of the molecular events involved is necessary to identify rational targets for therapeutic intervention.. Flow cytometry was used to measure physical and chemical characteristics in the photoreceptor population. Individual cells flow in suspension past one or more lasers, scattering light and emitting fluorescence. Western blot techniques demonstrated cleavage of calpain-specific substrates. Retinal explant cultures were used for inhibitor studies. Postnatal day 10 (P(10)) rd retinas were cultured without retinal pigment epithelium (RPE) attached up to P(17).. This study demonstrated calcium overload in the cytosol and subsequently in mitochondria. Mitochondrial membrane depolarization and reactive oxygen species (ROS) were detected later, during the peak of cell death. Analysis of downstream events indicated early activation of calcium-activated calpains. Treatment of rd retinal explants with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO (ALLN) successfully inhibited calpain-induced alpha-fodrin cleavage, yet it did not protect against photoreceptor degeneration. Finally, the results demonstrate an increase in the levels of both precursor and processed forms of the aspartate protease cathepsin D.. Excessive calcium influx is an early event that initiates the activation of calcium-activated proteases. However, these proteases are not singularly the cause of death, because their inhibition does not prevent apoptosis. Indeed, the results presented herein suggest that multiple pathways are involved and that each of these components may have to be addressed for cell death to be successfully inhibited.

Topics: Animals; Apoptosis; Blotting, Western; Calcium; Calpain; Cathepsin D; Cell-Free System; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Immunoenzyme Techniques; In Situ Nick-End Labeling; Leupeptins; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Organ Culture Techniques; Photoreceptor Cells, Vertebrate; Reactive Oxygen Species; Retinal Degeneration; Signal Transduction