Abstract
Rod and cone phosphodiesterase 6 (PDE6) are key effector enzymes of the vertebrate phototransduction pathway. Rod PDE6 consists of two catalytic subunits PDE6α and PDE6β and two identical inhibitory PDE6γ subunits, while cone PDE6 is composed of two identical PDE6α’ catalytic subunits and two identical cone-specific PDE6γ’ inhibitory subunits. Despite their prominent function in regulating cGMP levels and therefore rod and cone light response properties, it is not known how each subunit contributes to the functional differences between rods and cones. In this study, we generated an rd10/cpfl1 mouse model lacking rod PDE6β and cone PDE6α’ subunits. Both rod and cone photoreceptor cells are degenerated with age and all PDE6 subunits degrade in rd10/cpfl1 mice. We expressed cone PDE6α’ in both rods and cones of rd10/cpfl1 mice by adeno-associated virus (AAV)-mediated delivery driven by the ubiquitous, constitutive small chicken β-actin promoter. We show that expression of PDE6α’ rescues rod function in rd10/cpfl1 mice, and the restoration of rod light sensitivity is attained through restoration of endogenous rod PDE6γ and formation of a functional PDE6α’γ complex. However, improved photopic cone responses were achieved only after supplementation of both cone PDE6α’ and PDE6γ’ subunits but not by PDE6α’ treatment alone. We observed a two fold increase of PDE6α’ levels in the eyes injected with both PDE6α’ plus PDE6γ’ relative to eyes receiving PDE6α’ alone. Despite the presence of both PDE6γ’ and PDE6γ, the majority of PDE6α’ formed functional complexes with PDE6γ’, suggesting that PDE6α’ has a higher association affinity for PDE6γ’ than for PDE6γ. These results suggest that the presence of PDE6γ’ augments cone PDE6 assembly and enhances its stability. Our finding has important implication for gene therapy of PDE6α’-associated achromatopsia.
Highlights
Cyclic nucleotide phosphodiesterases 6 (PDE6) belongs to the highly conserved mammalian PDEs composed of 11 different families that modulate cellular levels of the second messengers, cAMP and cGMP, by controlling their rates of degradation (Beavo, 1995; Francis et al, 2001)
We have shown previously that rods of rd10 mice expressing cone PDE6α’ delivered by an adeno-associated virus (AAV) vector are more sensitive to light than wild type rods, most likely due to the slower shutoff of their light responses (Deng et al, 2013)
Histological examination of hematoxylin and eosin (H&E) stained retinal sections revealed progressive outer nuclear layer (ONL) thinning in rd10/cpfl1 retinas mirroring the retinal degeneration seen in rd10 mice (Figure 1A)
Summary
Cyclic nucleotide phosphodiesterases 6 (PDE6) belongs to the highly conserved mammalian PDEs composed of 11 different families that modulate cellular levels of the second messengers, cAMP and cGMP, by controlling their rates of degradation (Beavo, 1995; Francis et al, 2001). Electron microscopic analysis of purified rod PDE6 catalytic dimers showed that the catalytic domains are located at the C-terminus, and are highly conserved among all known members of PDE family (Beavo, 1995). Functional mapping of PDE6γ with the catalytic subunits showed that the C-terminus of PDE6γ is a key inhibitory domain. It interacts directly with the active site of PDE6 and blocks access of substrate to the catalytic pocket and controls cGMP hydrolysis (Granovsky et al, 1997; Barren et al, 2009; Zhang and Artemyev, 2010). The middle region of PDE6γ stabilizes the overall binding affinity of PDE6γ with the catalytic subunits (Zhang et al, 2012)
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