C20-D3-vitamin A slows lipofuscin accumulation and electrophysiological retinal degeneration in a mouse model of Stargardt disease

Abstract

Stargardt disease, also known as juvenile macular degeneration, occurs in approximately one in 10,000 people and results from genetic defects in the ABCA4 gene. The disease is characterized by premature accumulation of lipofuscin in the retinal pigment epithelium (RPE) of the eye and by vision loss. No cure or treatment is available. Although lipofuscin is considered a hallmark of Stargardt disease, its mechanism of formation and its role in disease pathogenesis are poorly understood. In this work we investigated the effects of long-term administration of deuterium-enriched vitamin A, C20-D(3)-vitamin A, on RPE lipofuscin deposition and eye function in a mouse model of Stargardt's disease. Results support the notion that lipofuscin forms partly as a result of the aberrant reactivity of vitamin A through the formation of vitamin A dimers, provide evidence that preventing vitamin A dimerization may slow disease related, retinal physiological changes and perhaps vision loss and suggest that administration of C20-D(3)-vitamin A may be a potential clinical strategy to ameliorate clinical symptoms resulting from ABCA4 genetic defects.

FIGURE 1.

C20-D₃-vitamin A impedes A2E- and ATR-dimer biosynthesis in a mouse model of Stargardt disease. A and B, representative HPLC traces of retinoids and vitamin A dimers extracted from the eyes of ABCA4^−/− mice raised on vitamin A. C–E, UV-Vis spectra of the represented peaks. In this report, A2E refers to both A2E and its geometric isomer, iso-A2E, and ATR-dimer refers to peaks between 16.8 and 17.4 min, those with characteristic absorbance spectra of ATR dimer. Relative concentrations of A2E at 3 and 6 months (F) and ATR-dimer at 3 months (G) in control (vitamin A) and treated (C20-D₃-vitamin A) animals. Each smaller open circle represents a measurement for 10-pooled eyecups for a total of n = 15 animals per bar. ***: p < 0.001.

FIGURE 2.

Vitamin A is swapped for C20-D₃-vitamin A in treated animals. Representative MS of retina (top) and liver (bottom) extracts from animals raised on naturally occurring vitamin A (control group, panels A and C) or on C20-D₃-vitamin A (treated group, panels B and D). Numbers above lines are m/z ratios.

FIGURE 3.

C20-D₃-vitamin A reduces RPE autofluorescence in the Stargardt mouse. A, average retinaldehyde autofluorescence from RPE cells (eyecups), from 5 three-month-old mice administered vitamin A (control, left bar) and 5 three-month-old mice administered C20-D₃-vitamin A (treatment, right bar). Circles represent individual pixel intensity measurements for each flat mount image. Standard deviations are shown. ***: p < 0.001. B and C, representative confocal fluorescence microscopy images taken under identical conditions of an eyecup from a treated (C) and control animal (B).

FIGURE 4.

C20-D₃-vitamin A reduces lipofuscin deposits. Representative electron micrographs of RPE layer from 6-month-old ABCA4^−/− mice raised on a diet containing either vitamin A (panel A), revealing electron-dense bodies (dark spots, total relative area of 8147; average relative size 24, area fraction 5.1); and C20-D₃-vitamin A (panel B), revealing ∼50% fewer electron-dense bodies, distributed throughout the cytoplasm (total relative area of 4164, average relative size 13, area fraction 2.6). BM: Bruch's membrane. Same magnification level between the two micrographs.

FIGURE 5.

Animals raised on C20-D₃-vitamin A have altered ocular gene expression. Volcano plot of p values correlated to fold change in gene expression in 1-year-old treated animals relative to controls, as measured using qRT-PCR. Experiments were done in triplicate with a sample size of n = 15 animals per group or 30 eyes.

FIGURE 6.

C20-D₃-vitamin A delays electrophysiological retinal deterioration in a mouse model of Stargardt disease. A, average flicker ERG curves in response to 635 ± 25 nm light flickering at different frequencies, at one year. n = 10 animals were used for the vitamin A group (control) and n = 15 for the C20-D₃-vitamin A group (treatment). ***: p < 0.001. B, change in flicker ERG amplitude upon aging for control and treated mice. The first bar of each grouping represents 3-month-olds and the second bar 1-year-olds (data from panel A). At 3 months, the difference between the control and treated groups were not statistically significant. Standard errors are shown. C, average ERG curves in response to a single flash of white light for 1-year-old animals raised on the two diets. D, ERG intensity response curves for the a- (lower curves) and b-waves (top curves) corresponding to ERG curves shown in panel C. The numbers above the curves are p values.

FIGURE 7.

Hypothesized model of lipofuscin prevention by C20-D₃-vitamin A. Pathways for retinaldehyde/phosphatidylethanolamine Schiff base (Retinaldehyde-PE) clearance from the disk lumen. The shaded area represents the disk lumen. Clearance pathways are marked 1–5. Arrows going outside the gray area represent movement out of the disk lumen. In theory, the retinaldehyde-PE Schiff base that forms in the rod outer segment after photon isomerization may follow several pathways: 1) it can react to form A2E leading to lipofuscin; 2) it may be actively (ABCA4 mediated) or passively (diffusion) transported out of the disk lumen; 3) it may dissociate back to retinaldehyde and PE and retinaldehyde may then be actively or passively transported out of the disk lumen (79); 4) it may isomerize, dissociate and the generated 11-cis-retinaldehyde may recombine with opsin to form rhodopsin (66) or 11-cis-retinaldehyde and its other isomers may be actively or passively transported out of the disk lumen; and lastly 5) it may be delivered to the RPE directly when the outer segment is phagocytosed. In pathways 2–5 retinaldehyde can presumably be reincorporated back into the visual cycle. Once outside the disk lumen retinaldehyde can be reduced to retinol. In the case of ABCA4 defects leading to Stargardt disease pathway two is slowed resulting in, presumably, an increase in pathway one leading initially to vitamin A dimerization, lipofuscin formation and ultimately vision loss. By the incorporation of deuterium atoms at the C20 position of vitamin A, we have shown in prior work that pathway one becomes higher in energy, and as a result is slowed down. This should allow more time for retinaldehyde to be re-incorporated into the visual cycle via pathways 2–5. Through this mechanism, the use of C20-deuterated vitamin A may be an effective methodology toward mitigating the clinical consequences of alterations in the ABCA4 gene.