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Fernanda Balem, Priscilla S. Akamine, Gabriela L. Ioshimoto, Balázs V. Nagy, Dora F. Ventura, Judith Klein-Seetharaman, Dania Hamassaki; Effects of Chlorin e6 on Retinitis Pigmentosa Rhodopsin Mutants in vivo. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6458.
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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 shown to cause misfolding of rhodopsin, but this knowledge has not yet been leveraged in developing treatments for RP. We have recently demonstrated [Balem et al. (2009) Photochem Photobiol. 85, 471-8] that the chlorophyll-derivative chlorin e6 (Ce6) changes and stabilizes the structure of rhodopsin in vitro. To test whether stabilization of rhodopsin by Ce6 may impact and possibly prevent misfolding of rhodopsin carrying RP mutations, we studied the effect of Ce6 in vitro using stable cell lines of the rhodopsin mutants, P23H and N15S, and in vivo using the rat models of RP, P23H and S334ter.
We created stable cell lines expressing the rhodopsin RP mutants P23H and N15S under a tetracycline inducible promoter. At first, the effect of the presence of added 11-cis retinal during expression of the protein was investigated. The purified mutants were then analyzed with respect to their structure and stability in vitro in the presence and absence of Ce6. To test the effects of Ce6 on RP progression, P23H and S334ter rats were treated by intraperitoneal injections with Ce6 (2mg/kg) or PBS. Treatment started at P20 and continued until P120. Rats were electrophysiologically monitored and their retinas characterized by imunohistochemistry using markers for rod bipolar cells (anti-PKC) and Müller glial cells (anti-vimentin), as well as by measurements of the outer nuclear layer thickness at different intervals of time.
In the presence of added 11-cis retinal during expression, both mutants are stabilized and form wild-type like chromophore. Ce6 exerted an effect on the stability of the mutants in vitro. ERG and retinal tissue analysis indicate that Ce6 exerts a positive functional effect on P23H in vivo, slowing the rate of photoreceptor degeneration, as suggested by the tendency of increased ERG responses, especially the b wave. In addition, the retinas from Ce6-treated rats were more preserved than non-treated ones as indicated by the analysis of the outer nuclear layer thickness, as well as the dendritic arborizations of rod bipolar cells. No significant changes were observed on Müller cells visualized with anti-vimentin. Interestingly, Ce6 enhanced photoreceptor degeneration of the S334ter rat in vivo, as observed by the decrease of the outer nuclear layer thickness.
Our studies indicate that Ce6 affects RP progression in vivo which are likely linked to the binding of this molecule to rhodopsin as demonstrated in vitro. This observation may ultimately lead to new approaches to treat or prevent RP in patients.
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