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Retinitis pigmentosa (RP) is a frequent cause of blindness among the working population in industrial countries due to the inheritable death of photoreceptors. Though gene therapy was recently approved for mutations in the RPE65 gene, there is in general no effective treatment presently. Previously, abnormally high levels of cGMP and overactivation of its dependent protein kinase (PKG) have been suggested as causative for the fatal effects on photoreceptors, making it meaningful to explore the cGMP-PKG downstream signaling for more pathological insights and novel therapeutic target development purposes. Here, we manipulated the cGMP-PKG system in degenerating retinas from the rd1 mouse model pharmacologically via adding a PKG inhibitory cGMP-analogue to organotypic retinal explant cultures. A combination of phosphorylated peptide enrichment and mass spectrometry was then applied to study the cGMP-PKG-dependent phosphoproteome. We identified a host of novel potential cGMP-PKG downstream substrates and related kinases using this approach and selected the RAF1 protein, which may act as both a substrate and a kinase, for further validation. This showed that the RAS/RAF1/MAPK/ERK pathway may be involved in retinal degeneration in a yet unclarified mechanism, thus deserving further investigation in the future.

The disease retinitis pigmentosa (RP) leads to photoreceptor degeneration by a yet undefined mechanism(s). In several RP mouse models (i.e., rd mice), a high cyclic GMP (cGMP) level within photoreceptors is detected, suggesting that cGMP plays a role in degeneration. The rap guanine exchange factor 4 (EPAC2) is activated by cyclic AMP (cAMP) and is an accepted cGMP-interacting protein. It is unclear whether and how cGMP interacts with EPAC2 in degenerating photoreceptors; we therefore investigated EPAC2 expression and interactions with cGMP and cAMP in retinas of the rd1 and rd10 models for retinal degeneration. EPAC2 expression in the photoreceptor layer increased significantly during rd1 and rd10 degeneration, and an increase in EPAC2 interactions with cGMP but not cAMP in the rd1 was also seen via a proximity ligation assay on histological sections. Retinal explant cultures revealed that pharmacological inhibition of the EPAC2 activity reduced the photoreceptor layer thickness in the rd10 retina, suggesting that EPAC2 inhibition promotes degeneration. Taken together, our results support the hypothesis that high degeneration-related cGMP leads to increased EPAC2 and cGMP interactions, inhibiting EPAC2. By inference, EPAC2 could have neuroprotective capacities that may be exploited in the future.

Cyclin dependent kinase 1 (CDK1) has long been known to drive the cell cycle and to regulate the division and differentiation of cells. Apart from its role in mitosis regulation, it also exerts multiple functions as a protein kinase, including engagement in cell death, possibly via a cell cycle-independent mechanism. The latter is suggested, since CDK1 re-expression can be found in non-dividing and terminally differentiated neurons in several neurodegeneration models. However, the details of if and how CDK1 might be involved in the neurodegenerative condition, retinitis pigmentosa (RP), which displays progressive vision loss, are unclear. In the present study, we investigated CDK1 in degenerating RP photoreceptors of the rd1 RP model, including whether there is a link between this kinase and the cGMP-PKG system, which is regarded as a disease driver. With experiments performed using either in vivo retinal tissue or in vitro material, via organotypic retinal explants, our results showed that CDK1 appears in the photoreceptors at a late stage of their degeneration, and in such a position, it may be associated with the cGMP-PKG network.

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