Rescue of the Stargardt phenotype in Abca4 knockout mice through inhibition of vitamin A dimerization

Abstract

Stargardt disease, an ATP-binding cassette, subfamily A, member 4 (ABCA4)-related retinopathy, is a genetic condition characterized by the accelerated accumulation of lipofuscin in the retinal pigment epithelium, degeneration of the neuroretina, and loss of vision. No approved treatment exists. Here, using a murine model of Stargardt disease, we show that the propensity of vitamin A to dimerize is responsible for triggering the formation of the majority of lipofuscin and transcriptional dysregulation of genes associated with inflammation. Data further demonstrate that replacing vitamin A with vitamin A deuterated at the carbon 20 position (C20-D3-vitamin A) impedes the dimerization rate of vitamin A--by approximately fivefold for the vitamin A dimer A2E--and subsequent lipofuscinogenesis and normalizes the aberrant transcription of complement genes without impairing retinal function. Phenotypic rescue by C20-D3-vitamin A was also observed noninvasively by quantitative autofluorescence, an imaging technique used clinically, in as little as 3 months after the initiation of treatment, whereas upon interruption of treatment, the age-related increase in autofluorescence resumed. Data suggest that C20-D3-vitamin A is a clinically amiable tool to inhibit vitamin A dimerization, which can be used to determine whether slowing the dimerization of vitamin A can prevent vision loss caused by Stargardt disease and other retinopathies associated with the accumulation of lipofuscin in the retina.

Keywords: A2E; ALK-001; C20-D3-vitamin A; age-related macular degeneration; bisretinoid.

Conflict of interest statement

Conflict of interest statement: I.W. is listed as an inventor on a patent related to treating macular degeneration.

Figures

Fig. 1.

C20-D3-vitamin administration reduces lipofuscin-related fundus AF. Representative fundus AF images taken with 488-nm (A) or 790-nm (C) excitation from 9-mo-old wild-type (WT) and Abca4−/− mice. Animals were fed with either a standard diet (control) or a diet in which vitamin A was replaced by C20-D3-vitamin A from weaning (treated). The bar charts illustrate average fundus AF intensities at 488 nm (B) and 790 nm (D), with SDs for each cohort of animals (n = 10 eyes of 10 animals). Unless noted with a bar, asterisks represent significant deviations (P < 0.05) from WT control animals fed a standard diet.

Fig. 2.

Inhibiting vitamin A dimerization prevents lipofuscin granule accumulation in the RPE. Representative transmission electron micrographs of a RPE cell taken from 9-mo-old Abca4−/− mutant mice administered either vitamin A (control, A) or C20-D3-vitamin A mice (treated, B). Lipofuscin granules were reconstructed from 20 serial sections taken at 100-nm intervals and overlaid in red on a representative section of the RPE shown in light gray. (C) Average lipofuscin volume per cytoplasmic volume, with 95% confidence intervals from animals described in A and B. −/−, Abca4−/− mutant mice; WT, wild-type, Abca4+/+, controls. Unless noted with a bar, asterisks represent significant deviations (P < 0.05, as determined by one-way analysis of variance) from wild-type animals given vitamin A (WT Control). (Scale bar, approximately 1 μm.)

Fig. 3.

C20-D3-vitamin A administration prevents vitamin A dimerization. (A) Alcohol extracts of retinas from 9-mo-old, Abca4−/− mutant mice administered vitamin A (control) or C20-D3-vitamin A from weaning (treated). Extracts from control eyes are less orange in color. (B) Representative UPLC chromatograms of eye extracts from A, along with that from wild-type (WT) mice administered vitamin A (control). Each peak represents an orange ocular pigment derived from vitamin A. (C) Average amounts of total orange ocular pigments and the A2E vitamin A dimer with SDs from the 9-mo-old animals described in the text, as determined by UPLC. Unless noted with a bar, asterisks represent significant deviations (P < 0.05) from wild-type animals given vitamin A (WT Control). (D) Average amounts of orange ocular pigment and SDs measured over time in the above groups of animals. (E) Average amounts of the A2E vitamin A dimer with SDs measured over time in the above groups of animals. Ten left eyes were averaged for each bar or point in the above panels (n = 10). −/−, Abca4−/− mutant mice; RAU, relative absorbance units; WT, wild-type.

Fig. 4.

C20-D3-vitamin normalizes transcription of complement genes. Quantification of mRNA from 9-mo-old wild-type and Abca4−/− mice, administered either vitamin A (control) or C20-D3-vitamin A from weaning (treated). To generate each bar, total RNA from 5–10 eyes was extracted. −/−, Abca4−/− mutant mice; WT, wild-type. Unless noted with a bar, asterisks represent significant deviations (P < 0.05 by two-tailed, unpaired t test) from wild-type animals given vitamin A (black bars).

Fig. 5.

Long-term inhibition of vitamin A dimerization by C20-D3-vitamin A does not compromise retinal function. (A) Average dark-adapted a- and b-wave amplitudes in response to flashes of light of increasing intensity. (B) Average light-adapted, b-wave amplitudes with amplitudes elicited by light flickering at 20 Hz. Mice were given the control or C20-D3-vitamin A (treated) diet from weaning to 9 mo of age. All curves represent averages and SDs for 10 eyes (n = 10). −/−, Abca4−/− mutant mice; WT: wild-type. All ERG responses were identical between the three cohorts.

Fig. 6.

C20-D3-vitamin administration rapidly prevents vitamin A dimerization and lipofuscin accumulation. (A) Abca4−/− mutant mice were divided into six experimental cohorts, and each cohort was administered a diet containing C20-D3-vitamin A (treated) or vitamin A (control) over time, according to the panel. (B) Relative average 488-nm fundus AF intensities with SDs from animals described in A. Ten eyes were averaged for each data point (n = 10). The red lines correspond to the periods during which animals were administered C20-D3-vitamin A, as shown in A, whereas the blue lines correspond to the control diet. Treatment crossovers are highlighted by black arrows. Data were normalized so that untreated controls had a relative AF intensity of 1 at 3 mo. Data points at weaning and 1.5 mo are extrapolated from historical controls. (C) Relative average fundus fluorescence intensities with SDs, at 9 mo of age, at the conclusion of treatment regimens described in A and B. (D) Average amounts of total orange ocular pigments and of the A2E dimer, with SDs, in 9-mo-old animals, at the end of treatment, as determined by UPLC. *P < 0.05. P values compare significant deviations from wild-type animals fed vitamin A.