Retinitis Pigmentosa represents a group of genetic disorders that cause progressive vision loss via degeneration of photoreceptors, but there is in principle no treatment available. For any therapy development, a deeper comprehension of the disease-leading mechanism(s) at the molecular level is needed. Here we focused on the cGMP-PKG system, which has been suggested to be a driver in several models of the disease. To gain insights in its downstream signaling we manipulated the cGMP-PKG system with the aid of organotypic retinal explant cultures from either a mouse-based disease model, i.e. the rd1 mouse, or its healthy wild-type counterpart (wt), by adding different types of cGMP analogues to either inhibit or activate PKG in retinal explants from rd1 and wt, respectively. An RNA sequencing was then performed to study the cGMP-PKG dependent transcriptome. Expression changes of gene sets related to specific pathways or functions, that fulfilled criteria involving that the changes should match PKG activation and inhibition, were determined via bioinformatics. The analyses highlighted that several gene sets linked to oxidative phosphorylation and mitochondrial pathways were regulated by this enzyme system. Specifically, the expression of such pathway components was upregulated in the rd1 treated with PKG inhibitor and downregulated in the wt with PKG activator treatment, suggesting that cGMP-PKG act as a negative regulator in this context. Downregulation of energy production pathways may thus play an integral part in the mechanism behind the degeneration for at least several RP mutations.

The hereditary disease Retinitis pigmentosa results in severe vision loss due to photoreceptor degeneration by unclear mechanisms. In several disease models, the second messenger cGMP accumulates in the degenerating photoreceptors, where it may over‐activate specific cGMP‐interacting proteins, like cGMP‐dependent protein kinase. Moreover, interventions that counteract the activity of these proteins lead to reduced photoreceptor cell death. Yet there is little or no information whether other than such regular cGMP‐interactors are present in the retina, which we, therefore, investigated in wild‐type and retinal degeneration (rd1, rd10, and rd2) mouse models. An affinity chromatography based proteomics approach that utilized immobilized cGMP analogs was applied to enrich and select for regular and potentially new cGMP‐interacting proteins as identified by mass spectrometry. This approach revealed 12 regular and ten potentially new retinal cGMP‐interacting proteins (e.g., EPAC2 and CaMKIIα). Several of the latter were found to be expressed in the photoreceptors and to have proximity to cGMP and may thus be of interest when defining prospective therapeutic targets or biomarkers for retinal degeneration.

α-synucleinopathy is driven by an imbalance of synthesis and degradation of α-synuclein (αSyn), causing a build up of αSyn aggregates and post-translationally modified species, which not only interfere with normal cellular metabolism but also by their secretion propagates the disease. Therefore, a better understanding of αSyn degradation pathways is needed to address α-synucleinopathy. Here, we used the nerve growth factor–differentiated catecholaminergic PC12 neuronal cell line, which was conferred α-synucleinopathy by inducible expression of αSyn and tubulin polymerization-promoting protein p25α. p25α aggregates αSyn, and imposes a partial autophagosome–lysosome block to mimic aspects of lysosomal deficiency common in neurodegenerative disease. Under basal conditions, αSyn was degraded by multiple pathways but most prominently by macroautophagy and Nedd4/Ndfip1-mediated degradation. We found that expression of p25α induced strong p38MAPK activity. Remarkably, when opposed by inhibitor SB203580 or p38MAPK shRNA knockdown, endolysosomal localization and degradation of αSyn increased, and αSyn secretion and cytotoxicity decreased. This effect was specifically dependent on Hsc70 and the endosomal sorting complex required for transport machinery, but different from classical microautophagy, as the αSyn Hsc70 binding motif was unnecessary. Furthermore, in a primary neuronal (h)-αSyn seeding model, p38MAPK inhibition decreased pathological accumulation of phosphorylated serine-129-αSyn and cytotoxicity. In conclusion, p38MAPK inhibition shifts αSyn degradation from various forms of autophagy to an endosomal sorting complex required for transport–dependent uptake mechanism, resulting in increased αSyn turnover and cell viability in p25α-expressing cells. More generally, our results suggest that under conditions of autophagolysosomal malfunction, the uninterrupted endosomal pathway offers a possibility to achieve disease-associated protein degradation.

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.

The inherited eye disease retinitis pigmentosa (RP) causes the loss of photoreceptors by a still unknown cell death mechanism. During this degeneration, cyclic guanosine‐3′,5′‐monophosphate (cGMP) levels become elevated, leading to over‐activation of the cGMP‐binding protein cGMP‐dependent protein kinase (PKG). cGMP analogs selectively modified to have inhibitory actions on PKG have aided in impeding photoreceptor death, and one such cGMP analog is Rp‐8‐Br‐PET‐cGMPS. However, cGMP analogs have previously been shown to interact with numerous targets, so to better understand the therapeutic action of Rp‐8‐Br‐PET‐cGMPS, it is necessary to elucidate its target‐selectivity and hence what potential cellular mechanism(s) it may affect within the photoreceptors. Here, we, therefore, applied affinity chromatography together with mass spectrometry to isolate and identify Rp‐8‐Br‐PET‐cGMPS interactors from retinas derived from three different murine RP models (i.e., rd1, rd2, and rd10 mice). Our findings revealed that Rp‐8‐Br‐PET‐cGMPS bound seven known cGMP‐binding proteins, including PKG1β, PDE1β, PDE1c, PDE6α, and PKA1α. Furthermore, an additional 28 proteins were found to be associated with Rp‐8‐Br‐PET‐cGMPS. This latter group included MAPK1/3, which is known to connect with cGMP/PKG in other systems. However, in organotypic retinal cultures, Rp‐8‐Br‐PET‐cGMPS had no effect on photoreceptor MAPK1/3 expression or activity. To summarize, Rp‐8‐Br‐PET‐cGMPS is more target specific compared to regular cGMP.