Resumo

Aim: Retinitis Pigmentosa (RP) is an inherited disease that progressively leads to blindness. Mutations in the rod photoreceptor and prototypical G protein coupled receptor rhodopsin associated with RP have been shown to cause misfolding of rhodopsin, but this knowledge has not yet been leveraged in developing treatments for RP. Studies in deep-sea fish suggested that the chlorophyll-derivative, chlorin e6 (Ce6), enhances the sensitivity of rhodopsin to red light. Here, we propose that Ce6 alters rhodopsin structure and stability and may prevent misfolding and RP progression in vivo. Methodology: To test this hypothesis, we investigated if Ce6 binds to rhodopsin and modulates rhodopsin structure and stability by circular dichroism (CD) and fluorescence spectroscopy analysis of secondary and tertiary structure in vitro. The temperature was varied between 5oC and 100oC, or maintained at 55oC. In vivo experiments were carried out through electroretinogram recordings (ERG), where the ERG responses of the rats model of RP, P23H and S334ter, were recorded after treatment with Ce6 at different concentration (2, 10 and 20 mg/Kg), and monitored at different intervals of time (from 30 to 150 days). Results: Computational docking and NMR studies support binding of Ce6 to rhodopsin. Thermal denaturation at 55oC shows that the secondary and tertiary structure of rhodopsin is preserved in presence of Ce6. Temperature titration studies of Ce6 on rhodopsin have shown to stabilize the helical content of rhodopsin, where a decrease in helicity of about 30 percent less than rhodopsin alone was observed at 100oC. ERG measurements indicate that Ce6 exerts a positive functional effect in vivo, as suggested by the observed increase in the ERG waves. Conclusions: Thermal denaturation and stability studies using circular dichroism and fluorescence spectroscopy show that the molecule Ce6 exerts an effect on rhodopsin structure and stability in vitro. Treatment with Ce6 confirms the hypothesis in vivo, indicating thought the electrophysiology of the retina that Ce6 is capable of slow down the degeneration effects of the photoreceptors. It may be possible to exploit this observation to treat or prevent retinal dysfunctions by administering Ce6 as a supplement to RP patients.