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Activation of multiple pathways during photoreceptor apoptosis in the rd mouse
Francesca Doonan1, Maryanne Donovan, Thomas G Cotter
1Tumour Biology Laboratory, Department of Biochemistry, Biosciences Institute, University College Cork, Ireland.
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.
Insights
Photoreceptor apoptosis in rd mice involves calcium overload and protease activation, but inhibiting these pathways alone does not prevent cell death, suggesting multiple therapeutic targets for retinal degeneration.
Area of Science:
- Cell Biology
- Molecular Biology
- Ophthalmology
Background:
- Apoptosis is a key mechanism in retinal degeneration, including retinitis pigmentosa (RP).
- Understanding the molecular events of photoreceptor apoptosis is crucial for developing therapeutic interventions.
- The rd mouse model provides a platform to study these degenerative processes.
Purpose of the Study:
- To characterize the process of photoreceptor apoptosis in the rd mouse.
- To identify molecular targets for potential therapies against retinal degeneration.
- To investigate the role of calcium influx and proteases in photoreceptor cell death.
Main Methods:
- Flow cytometry was utilized to analyze photoreceptor cell characteristics.
- Western blotting was employed to detect the cleavage of calpain substrates.
- Retinal explant cultures were used to test the efficacy of calpain inhibitors.
Main Results:
- Calcium overload was observed in both the cytosol and mitochondria of photoreceptor cells.
- Mitochondrial membrane depolarization and reactive oxygen species (ROS) production occurred during peak cell death.
- Calpain activation was detected early, but inhibition with ALLN did not prevent photoreceptor degeneration.
- Increased levels of both precursor and processed forms of cathepsin D were observed.
Conclusions:
- Excessive calcium influx initiates the activation of calcium-activated proteases.
- These proteases are not the sole cause of photoreceptor apoptosis, as their inhibition did not prevent cell death.
- Multiple pathways contribute to photoreceptor cell death, necessitating a multi-targeted approach for therapeutic intervention in retinal degeneration.
Related Concept Videos
01:22Photoreceptors and Visual Pathways
01:31The Intrinsic Apoptotic Pathway