Abstract
Purpose :
Dark-adapted Rpe65-/- mice have been known to synthesize 9-cis-retinal that confers light sensitivity to the iso-rhodopsin of rods. In the previous studies we have shown that deletion of Fatty Acid Transport Protein-4 (FATP4) significantly improves the cone-mediated vision and the syntheses of both 11-cis- and 9-cis-retinal chromophores in the RPE65 R91W knock-in mouse model of Leber congenital amaurosis. The purpose of this study was to analyze the synthesis of 9-cis-retinal and the visual function of cones in rd12 (RPE65-null) mice lacking FATP4.
Methods :
Using rd12 and Fatp4-/-;Ivl-Fatp4tg/+ (shown as Fatp4-/- hereafter) mice with the 129S2/Sv genetic background, we generated rd12;Fatp4-/- mice. The 129S2/Sv strain mice are used as WT controls. The genotypes of these mice are confirmed by PCR, DNA sequencing and immunoblot analysis detecting RPE65, LRAT and FATP4 in the RPE. Retinoids in dark adapted eyes were analyzed by HPLC. Retinal degeneration was assessed by immunoblot and immunohistochemical analyses of rod and cone specific proteins. Visual functions of rods and cones were evaluated by scotopic and photopic ERG evoked with various flash intensities of white, 530-nm green or 360-nm UV light.
Results :
Dark-adapted WT and Fatp4-/- mice exhibited similar profiles of retinoids: undetectable 9-cis-retinal (9cRAL), 394~410 pmol 11-cis-retinal (11cRAL), and 45~50 pmol all-trans retinylesters (atREs) per eye. 11cRAL was undetectable and atREs were largely accumulated (819~1001 pmol/eye) in rd12 and rd12;Fatp4-/- mice. Although both rd12 and rd12;Fatp4-/- mice had 9cRAL, its amount in rd12;Fatp4-/- mice was approximately 43% greater than those in rd12. Both immunoblotting and immunohistochemistry showed similar expression levels of rhodopsin in 2- and 4-month-old rd12 and rd12;Fatp4-/- mice whereas the expression levels of M-opsin and cone arrestin, but not S-opsin, in 2- and 4-month-old rd12;Fatp4-/- retinas were significantly higher than those in the age matched rd12 retinas. Consistent with these results, rd12 and rd12;Fatp4-/- mice exhibited comparable scotopic ERG responses whereas M-cone ERGs, but not S-cone ERGs, were clearly greater in rd12;Fatp4-/- mice as compared to rd12 mice.
Conclusions :
1) 9cRAL synthesized in a RPE65-independent mechanism plays an important role in M-cone survival and function of LCA mice; 2) FATP4 negatively regulates the synthesis of 9cRAL.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.