Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca2+-imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general.
Retinal Degeneration , Mice , Animals , Retinal Degeneration/drug therapy , Protein Kinase Inhibitors/pharmacology , Cyclic GMP-Dependent Protein Kinases/metabolism , Photoreceptor Cells/metabolism , Retina/metabolism , Disease Models, Animal , Mice, Inbred C57BL
Proteins of the 14-3-3 family are well known modulators of the leucine-rich repeat kinase 2 (LRRK2) regulating kinase activity, cellular localization, and ubiquitylation. Although binding between those proteins has been investigated, a comparative study of all human 14-3-3 isoforms interacting with LRRK2 is lacking so far. In a comprehensive approach, we quantitatively analyzed the interaction between the seven human 14-3-3 isoforms and LRRK2-derived peptides covering both, reported and putative 14-3-3 binding sites. We observed that phosphorylation is an absolute prerequisite for 14-3-3 binding and generated binding patterns of 14-3-3 isoforms to interact with peptides derived from the N-terminal phosphorylation cluster (S910 and S935), the Roc domain (S1444) and the C-terminus. The tested 14-3-3 binding sites in LRRK2 preferentially were recognized by the isoforms γ and η, whereas the isoforms ϵ and especially σ showed the weakest or no binding. Interestingly, the possible pathogenic mutation Q930R in LRRK2 drastically increases binding affinity to a peptide encompassing pS935. We then identified the autophosphorylation site T2524 as a so far not described 14-3-3 binding site at the very C-terminus of LRRK2. Binding affinities of all seven 14-3-3 isoforms were quantified for all three binding regions with pS1444 displaying the highest affinity of all measured singly phosphorylated peptides. The strongest binding was detected for the combined phosphosites S910 and S935, suggesting that avidity effects are important for high affinity interaction between 14-3-3 proteins and LRRK2.
