The cGMP system in normal and degenerating mouse neuroretina: New proteins with cGMP interaction potential identified by a proteomics approach

Abstract

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 10 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.

Keywords: Chemical proteomics; Photoreceptors; Retinal degeneration; cGMP; cGMP-interacting proteins.

FIGURE 1

Enriched cGMP‐interacting proteins in the various RD models and wt. (a) Schematic overview of the sequential elution protocol used for the enrichment of potential cGMP binding‐proteins, (eye from BioRender.com) (b) Chemical structures of different cGMP‐immobilized beads used in this study, (c) Silver stained SDS‐PAGE indicating a successful decrease in unspecific proteins. From left, lane 1: Raw retinal wt tissue (1 µg). Lane 2: Proteins eluted with cGMP from control (Ctrl) beads (EtOH). Lane 3–5: cGMP‐interacting proteins eluted with cGMP from cGMP‐immobilized beads (8‐AET, 2‐AH, 2‐AHC). Red arrowheads indicate protein bands at various sizes, which are unique for the cGMP‐immobilized beads compared to Ctrl beads

FIGURE 2

cGMP interactions in the retina. Proximity ligation assay showing (a) cGMP proximity to PKG, a known cGMP interactor. (b and c) cGMP proximity to EPAC2 and p‐CaMKIIα, respectively, either via direct interaction, or indirectly in a complex. (d) The negative control, where no primary antibodies were added to the sections, showing background staining from the PLA kit which is virtually non‐existing. Black (inverted pictures) punctuations are a positive outcome from the PLA, suggesting interaction. Scale bars: 50 µm. Biologically independent wt retinas n = 9

FIGURE 3

Overview of the ratios of the counted PLA punctuations in the retinas. The PLA punctuations were counted in a blinded fashion and the ratio of ONL/INL displayed in histograms. The statistical method Kruskal‐Wallis was applied to calculate whether some of the proteins had more proximity with cGMP in the respective RD and wt models and revealed that the probability to have more proximity between the cGMP:EPAC2 was higher in the rd10 compared to the rd2 model. Data is based on three biologically independent RD retinas (n = 3) and nine biologically independent wt retinas (n = 9). Error bars indicate standard deviation. Note the different scales of the y‐axes

FIGURE 4

Illustration of the potential cGMP interaction proteins, and corresponding pathways, identified by the proteomics approach. cGMP has previously been experimentally verified to interact directly with PKG (Huang et al., 2014), whereas p‐GSK3β has previously been indicated to interact with PKG (Das et al., ; Hong et al., 2012). cGMP is an accepted EPAC2 interactor (Rehmann et al., 2008) and is suggested to be involved in the MAPK1/3 cascade (Maymó et al., 2012) as well is CaMKIIα (Caunt et al., ; McKay & Morrison, 2007). The present study revealed that EPAC2, MAPK1/3, p‐CaMKIIα and p‐GSK3β are in proximity to cGMP in situ in the retina. Created with Biorender.com