Abstract
Purpose: :
Cumulative oxidative stress is thought to be a key pathogenic mechanism in age-related macular degeneration (AMD). Accordingly, mice deficient in Cu-, Zn- superoxide dismutase (Sod1) develop an AMD-like phenotype beyond the age of 10 months (PNAS 103:11282). One source of oxidative stress in AMD is lipofuscin. A2E is a well-known photo-oxidative toxin in lipofuscin. To explore the role of A2E in oxidative AMD pathogenesis, Sod1-/- mice were characterized for ocular A2E content and compared with abcr-/- mice.
Methods: :
A 96-well format, LC/MS/MS-based A2E assay was developed. A2E was synthesized and purified by flash chromatography and reverse-phase HPLC. The assay was calibrated with purified A2E spiked into naïve eye extracts. The assay was linear from 1 to 300 pmol of A2E/eye. Purified A2-hexanolamine, generated by the same methodology as for A2E, was used as an internal standard. A2E was extracted from enucleated eyeballs with organic solvent and quantified in pmol/eye +/- SEM. At least 3 animals were used per data point.
Results: :
Among 7-month-old animals, A2E content was significantly higher (p = 0.04) in Sod1-/- (20.1 +/- 1.3 pmol/eye) compared with that in Sod1+/+ (13.2 +/- 0.4 pmol/eye); Sod1+/- were not significantly different from Sod1+/+. Five-month-old abcr-/- mice, although younger, had several fold higher A2E level (73.5 +/- 2.6 pmol/eye). A2E level in abcr-/- rose almost linearly between 1 and 8 months from 2.7 to102 pmol/eye; there was relatively little increase by 16 months (116.6 pmol/eye).
Conclusions: :
Elevated A2E level is associated with and precedes the AMD phenotype in Sod1-/-. Although this A2E level was substantially lower than that in abcr-/-, it may have become pathogenic in Sod1-/- due to anti-oxidant deficiency. The A2E plateau in senescent abcr-/-, which presumably have a functioning Sod1 enzyme, may explain why there is no gross retinal degeneration in these animals. We are currently generating Sod1:abcr double knockouts to develop an animal model with an earlier AMD phenotype and to explore the role of A2E in AMD pathogenesis.
Keywords: age-related macular degeneration • retinal degenerations: hereditary • ipofuscin