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Decrease of ATP by mitochondrial m-calpain inhibitory peptide in the rat retinas
Taku Ozaki1, Mitsuru Nakazawa, Tetsuro Yamashita
1Department of Biochemistry and Molecular Biology, Faculty of Agriculture and Life Science, Hirosaki University.
Abstract:
Activations of mitochondrial calpains cause apoptosis-inducing factor-dependent apoptosis of retinal photoreceptor cells in the Royal College of Surgeons (RCS) rat, an animal model of retinitis pigmentosa. In the present study, we attempted to develop specific inhibitors of mitochondrial calpains that would prevent the retinal degeneration. We examined the inhibitory potency of 20-mer peptides of the m-calpain for mitochondrial calpains activity, determined the inhibitory regions, and conjugated the cell-penetrating peptides (CPP). The cytotoxicity and delivery of the peptide was evaluated using mouse photoreceptor-derived 661W cells. After intravitreal injection of the peptide in RCS rats, we examined the peptide delivery to the retina, photoreceptor cell death numbers, responses of the electroretinogram (ERG), concentrations of intracellular ATP, and changes of retinal morphology. Results showed that one of the peptides inhibited the activity of the mitochondrial m-calpain. The HIV-1 tat-conjugated m-calpain peptide, HIV-Nm, could preserve the inhibitory potency of the mitochondrial m-calpain, and penetrate into the 661W cells. While intravitreal injection of HIV-Nm made it possible to deliver to the retina, it did not prevent photoreceptor cell death. Furthermore, it caused the ERG attenuation and the decrease in the intracellular ATP only a day after the injection. Although HIV-Nm did not cause histological change of the retina after 1 or 2 days of the administration, the morphological abnormality of the retina was observed after 3-14 days. Our results demonstrated that HIV-Nm failed to prevent the photoreceptor cell death, but rather caused the attenuation of ERG response and the decrease of ATP.
Insights
Researchers developed a peptide inhibitor for mitochondrial calpains to treat retinitis pigmentosa. However, the conjugated peptide, HIV-Nm, failed to prevent photoreceptor cell death and worsened retinal function.
Area of Science:
- Ophthalmology
- Neuroscience
- Molecular Biology
Background:
- Mitochondrial calpain activation leads to photoreceptor cell apoptosis in Royal College of Surgeons (RCS) rats, a model for retinitis pigmentosa.
- Developing specific inhibitors for mitochondrial calpains is crucial for preventing retinal degeneration.
Purpose of the Study:
- To develop specific inhibitors of mitochondrial calpains to prevent retinal degeneration in a retinitis pigmentosa animal model.
- To evaluate the efficacy and safety of a novel peptide inhibitor conjugated with a cell-penetrating peptide (CPP).
Main Methods:
- Examined the inhibitory potency of 20-mer peptides against m-calpain activity.
- Conjugated cell-penetrating peptides (CPPs) to the inhibitory peptides.
- Evaluated peptide cytotoxicity and delivery in 661W cells and intravitreally injected into RCS rats.
- Assessed retinal delivery, photoreceptor cell death, electroretinogram (ERG) responses, intracellular ATP levels, and retinal morphology.
Main Results:
- Identified a peptide that inhibited mitochondrial m-calpain activity.
- The HIV-1 tat-conjugated peptide (HIV-Nm) retained inhibitory potency and penetrated 661W cells.
- Intravitreal injection delivered HIV-Nm to the retina but did not prevent photoreceptor cell death.
- HIV-Nm administration led to ERG attenuation and decreased intracellular ATP levels within 24 hours.
- Retinal morphological abnormalities were observed 3-14 days post-injection.
Conclusions:
- The developed peptide inhibitor HIV-Nm failed to prevent photoreceptor cell death in the RCS rat model.
- HIV-Nm administration resulted in adverse effects, including ERG attenuation and ATP depletion.
- Further research is needed to develop effective mitochondrial calpain inhibitors for retinitis pigmentosa treatment.
