cGMP Accumulation Causes Cone Degeneration in CNG Channel Deficiency

Abstract

 

Purpose

 

Cone phototransduction mediated by cyclic nucleotide-gated (CNG) channels is essential for central and color vision and for visual acuity. Naturally occurring mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia, progressive cone dystrophy, and some forms of maculopathies. Cyclic guanosine monophosphate (cGMP) is the native ligand of photoreceptor CNG channel and its accumulation has been associated with cytotoxicity. This work investigated the role of cGMP accumulation in cone degeneration.

 

Methods

 

Retinal cGMP levels in Cnga3^-/-/Nrl^-/- mice at varying ages were examined by ELISA. Expression levels of retinal guanylyl cyclases (GC), guanylyl cyclase activating proteins (GCAP), cone phosphodiesterase (PDE), and protein kinase G in the channel-deficient retina were examined by immunoblotting. The activities of GC, PDE and PKG were examined by chromatographic and colorimetric analyses. The role of cGMP accumulation in cone death was evaluated by using the GC inhibitor 1-H-[1,2,4]oxadiaxolo[4,3-a] quinolalin-1-one (ODQ) treatment and by using the Cnga3^-/-/Gucy2e^-/-mouse line (Gucy2e^-/- mice lack the expression of retinal GC1). Cone death/survival was evaluated by examining cone density and expression levels of cone specific proteins using immunohistochemical and biochemical approaches.

 

Results

 

The time-course study showed that the retinal cGMP level in Cnga3^-/- Nrl^-/- mice was sharply increased at postnatal day 8 (P8), peaked around P10-15, stayed high through P30-60, and returned to near control level at P90. This elevation pattern correlated well with photoreceptor apoptotic death in these mice, which was peaked around P15-20. The expression levels of retinal GC and cone PDE were increased in Cnga3^-/-/Nrl^-/- mice though the activities and regulation of these enzymes in vitro were unchanged, suggesting that an over-production due to lowered Ca²⁺ levels may contribute to the cGMP accumulation. Treatment with ODQ improved cone survival in Cnga3^-/- mice. Moreover, cone density and expression levels of M-opsin, S-opsin and Gnat2 were significantly increased in Cnga3^-/-/Gucy2e^-/- mice, compared to the age-matched Cnga3^-/- controls.

 

Conclusions

 

This work demonstrates a role of cGMP accumulation in cone degeneration and sheds light on the mechanism of cone defects in CNG channel deficiency.

 

Keywords: 426 apoptosis/cell death • 569 ion channels • 648 photoreceptors