The potential role of retinal cGMP-interaction partners within the degeneration mechanism causing Retinitis Pigmentosa

The eye disease Retinitis Pigmentosa (RP) represents a heterogeneous group of inherited retinal dystrophies, and it has been estimated that 1 in 3000 to 7000 people worldwide has the disease. The symptoms of RP are photoreceptor degeneration that leads to night blindness, followed by a decline in peripheral and central vision, and potential blindness. However, the mechanism(s) behind the photoreceptor degeneration is still not fully understood, although a high photoreceptor cyclic GMP (cGMP) level exists in several mouse models having RP (i.e., rd1, rd2, and rd10 mouse). It is thus very likely that cGMP, as well as its targets, may play a key role in the degeneration. Currently, numerous cGMP targets are known, and one of them is the cGMP-dependent protein kinase (PKG). cGMP can extensively govern PKG activity, and during RP, it over-activates PKG, which may propel the disease progression. However, it is possible that also other targets exist, and exposing those might help comprehend the RP mechanism(s). Therefore, this thesis studied the current cGMP targets and potential new targets. This was done by applying a proteomics approach utilizing affinity chromatography and various cGMP-analogs linked to agaroses to enrich and select for the proteins of interest. In Paper I, five potential new cGMP-interactors showed to be in proximity with cGMP in the photoreceptor layer, supporting that other cGMP-interactors may exist in the retina. Paper II showed that cGMP-interactors have various stereospecific requirements and that cGMP-analogs possess target specificity. Such selectivity could aid in studying functional and physical associations for a given cGMP interactor and provide valid information on the target specificity and drug design in the field of cGMP-analog-based therapies. Paper III investigated the promising selectively modified cGMP-analog (i.e., CN03), which has PKG inhibiting actions and can reduce degeneration. Paper III studied CN03’s target-specificity by applying the newly generated CN03-agarose and found that CN03 is more target-specific when compared to regular cGMP studied in Paper I and thus CN03 is a promising RP therapy. In Paper IV, rd and wt retinal crosssections and organotypic retinal cultures were used. First, it was seen that some rd models might temporally increase the expression of the newly found retinal cGMPinteractor EPAC2 in the photoreceptors. Furthermore, some rd models revealed 12 increased interaction between cGMP and EPAC2 in the photoreceptors over time, whereas such augmentation was not observed between EPAC2 and cAMP. The discrepancy in interactions may be explained by the co-localization of highly expressed EPAC2 and accumulated cGMP and the lack of such between EPAC2 and accumulated cAMP in diseased photoreceptors. Lastly, through pharmacological manipulation in rd10 retinal cultures, it was seen that EPAC2 activity may have neuroprotective abilities. Taken together, this thesis adds to the understanding of the cGMP-system by suggesting potential new cGMP-associated proteins, which may help pave the way for comprehending the cell death mechanism(s) within the photoreceptors as well as reveal new potential therapeutic targets