Activation of multiple pathways during photoreceptor apoptosis in the rd mouse

PURPOSE. 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. METHODS. 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. RESULTS. 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. CONCLUSIONS. 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.