Kinase activity profiling identifies putative downstream targets of cGMP/PKG signaling in inherited retinal neurodegeneration

Akanksha Roy,John Groten,Riet Hilhorst,Arianna Tolone,Tushar Tomar,François Paquet-Durand

doi:10.1038/s41420-022-00897-7

Abstract

Inherited retinal diseases (IRDs) are a group of neurodegenerative disorders that lead to photoreceptor cell death and eventually blindness. IRDs are characterised by a high genetic heterogeneity, making it imperative to design mutation-independent therapies. Mutations in a number of IRD disease genes have been associated with a rise of cyclic 3’,5’-guanosine monophosphate (cGMP) levels in photoreceptors. Accordingly, the cGMP-dependent protein kinase (PKG) has emerged as a new potential target for the mutation-independent treatment of IRDs. However, the substrates of PKG and the downstream degenerative pathways triggered by its activity have yet to be determined. Here, we performed kinome activity profiling of different murine organotypic retinal explant cultures (diseased rd1 and wild-type controls) using multiplex peptide microarrays to identify proteins whose phosphorylation was significantly altered by PKG activity. In addition, we tested the downstream effect of a known PKG inhibitor CN03 in these organotypic retina cultures. Among the PKG substrates were potassium channels belonging to the Kv1 family (KCNA3, KCNA6), cyclic AMP-responsive element-binding protein 1 (CREB1), DNA topoisomerase 2-α (TOP2A), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (F263), and the glutamate ionotropic receptor kainate 2 (GRIK2). The retinal expression of these PKG targets was further confirmed by immunofluorescence and could be assigned to various neuronal cell types, including photoreceptors, horizontal cells, and ganglion cells. Taken together, this study confirmed the key role of PKG in photoreceptor cell death and identified new downstream targets of cGMP/PKG signalling that will improve the understanding of the degenerative mechanisms underlying IRDs.

Highlights

Inherited retinal degeneration (IRD) relates to a genetically highly heterogeneous group of neurodegenerative diseases causing photoreceptor cell death and eventually blindness [1, 2]
Serine/Threonine Kinase (STK) activity in rd1 retinal explants treated with PKG inhibitor CN03 As we found a higher phosphorylation of peptides in rd1 explants, when compared to WT explants, and with a clear role for PKGs observed, we sought to investigate the effect of the PKG inhibitor CN03 on the STK activity
These recent findings corresponded well with our results might be linked to increased inflammation, which is an early where we identified these same kinases as activated in rd1 phenomenon observed for degenerative retinal disorders such as diseased retinal explants and repressed by the PKG inhibitor CN03

Summary

Introduction

Inherited retinal degeneration (IRD) relates to a genetically highly heterogeneous group of neurodegenerative diseases causing photoreceptor cell death and eventually blindness [1, 2]. To this day, in almost all cases, these diseases are untreatable. A key effector of cGMP-signalling is cGMPdependent protein kinase (PKG), the overactivation of which may trigger photoreceptor cell death [5, 6]. Activated PKG phosphorylates numerous cellular proteins at serine/threonine amino acid positions, which in turn regulates numerous cellular pathways. PKG1 regulates smooth muscle contraction [8], platelet activation and adhesion [9], cardiac function [10], feedback of the NO-signalling pathways [11], and various processes in the central nervous system, such as hippocampal and cerebellar learning [12]. PKG2 is involved in translocation of CFTR channels in jejunum [13] and regulation of bone growth by activation of kinases such as MAPK3/ERK1 and MAPK1/ERK2 in mechanically stimulated osteoblasts [14]

Methods

Findings

Discussion

Conclusion