Abstract
Purpose:
Retinitis pigmentosa (RP) is a group of genetically heterogeneous diseases causing photoreceptor degeneration and blindness. In the rd1 mouse model, a loss of function mutation in the gene for the rod photoreceptor-specific phosphodiesterase-6 beta subunit (Pde6b) causes accumulation of cGMP leading to photoreceptor cell death. So far two pathways have been identified: the first linked to opening of CNG channels, increased calcium influx and calpain activity; the second linked to increased Protein Kinase G (PKG) activity. We tested cGMP analogs that were modified to inhibit one or both of the pathways on photoreceptor-like cells derived from rd1 mutant retinal stem cells, to identify compounds that might be used for treating patients with a mutation in the PDE6B gene. Such treatments could be promising approaches for pharmacological interventions.
Methods:
We used retinal stem cells derived from rd1 mice that, after differentiation into photoreceptors, were exposed to compounds at different concentrations. To analyze their protective effect, we examined cell death using ethidium homodimer intercalation. To study the effect on the two pathways in detail, we measured calcium influx with Fluo4, calpain activity with a fluorescent substrate and phosphorylation of VASP as an indicator for PKG activity. Several new cGMP analogs were selected based on the chemical properties as inhibitors/activators of CNG channels and/or PKG.
Results:
We tested the protective effect of seven different cGMP analogs and found that one of them with activating properties on CNG channels and PKG was not protective on rd1 and even toxic on wild type photoreceptors. At the peak of cell death, treatment with the analogs that inhibited one or both pathways significantly decreased cell death when compared to untreated cells. The best results were achieved with analogs that inhibited both pathways and we observed a decrease of calcium accumulation and calpain activity with analogs that inhibited the opening of the CNG channel.
Conclusions:
In this work, we characterized compounds that had a protective effect on rd1 photoreceptor-like cells. Inhibition of CNG channels and/or reduction of PKG activity might be a promising strategy to protect from rod photoreceptor degeneration and loss of vision in patients.
Keywords: 648 photoreceptors •
615 neuroprotection •
503 drug toxicity/drug effects