These findings demonstrate the vitamin A-dependent nature of A2E biosynthesis and validate a novel therapeutic approach with potential to halt the accumulation of lipofuscin fluorophores in the eye.

The findings of this study and the established safety profile of fenretinide in chronic dosing regimens warrant further study of fenretinide in the treatment of geographic atrophy.

Mutations in the photoreceptor-specific ABCA4 gene are associated with several inherited retinal and macular degenerations. A prominent phenotype of these diseases is the accumulation of cytotoxic lipofuscin fluorophores such as A2E within the retinal pigment epithelium. Another compound, dihydro-N-retinylidene-Nretinylphosphatidyl-ethanolamine (A2PE-H 2 ), also accumulates in retinas of mice and humans harboring ABCA4 mutations and was proposed to be a precursor of A2E. The role of A2PE-H 2 in the biogenesis of A2E and its relationship to other retinal fluorophores has not been previously investigated. We report spectral properties and structural relationships of the principal retinal fluorophores that accumulate in retina and retinal pigment epithelium of abca4 ؊/؊ mice.A long wavelength fluorescence emission intrinsic to abca4 ؊/؊ retinal explants is shown to emanate from A2PE-H 2 . All-trans retinal dimer conjugates, which were also identified in the retinal explants, possessed distinct fluorescence and structural properties and, unlike A2PE-H 2 , did not accumulate in an age-dependent manner. Derivative absorbance and fluorescence spectroscopy revealed that A2PE-H 2 , A2E, and N-retinylidene-N-retinyl-phosphatidylethanolamine (A2PE), a known precursor of A2E, share common electronic and resonant structures. Importantly, collision-induced dissociation of A2PE-H 2 produced daughter ions that were identical to authentic A2E and its daughter ions. Finally, intravitreal administration of A2PE-H 2 to wild-type mice resulted in the formation of A2PE and A2E. These data validate a previously hypothesized biosynthetic pathway for A2E and implicate A2PE-H 2 as a precursor in this pathway. Fluorescence properties of A2PE-H 2 and other related fluorophores characterized in this report have significance for evaluation of human retinal diseases characterized by aberrant fundus autofluorescence.

The principles of fluorescence resonance energy transfer have been utilized to develop a high-throughput assay which detects compounds that interfere with interaction between retinol-binding protein (RBP) and transthyretin (TTR). In this assay, the intrinsic fluorescence from the RBP-retinol complex excites a probe molecule which is covalently coupled to TTR. Generation of an emission signal from the TTR probe indicates interaction between RBP-retinol and TTR. Importantly, the inclusion of retinol in the assay allows discrimination of test compounds which bind RBP versus those which bind to TTR. Thus, compounds which bind to RBP must compete with retinol in order to affect RBP-TTR interaction. This feature of the assay will be useful to identify test compounds which are more likely to have an effect in vivo.