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Daniel Higbee, Danielle Gutierrez, Peter H. Tang, Yiannis Koutalos, Rosalie K. Crouch, Zsolt Ablonczy; Accumulation Of A2E With 9-cis-Retinal Supplementation In Rpe65-/- Mice. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3647.
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Lipofuscin, a fluorescent lysosomal pigment made up of partially degraded lipophilic molecules, is associated with age related pathophysiological processes in the retinal pigment epithelium (RPE). The best characterized component of lipofuscin is the bis-retinoid A2E; however, a direct correlation with lipofuscin has not previously been possible. Moreover, little is known about the rate of A2E generation in the RPE.
A2E was extracted from cyclic light reared wild type and Rpe 65-/- mice of various ages (0, 2, 5, and 7 weeks) with and without 9-cis-retinal treatment. Extractions were analyzed by nano-LC-MS/MS on an LTQ ion-trap mass spectrometer. Additionally, whole eyecups with retina removed were flattened onto ITO coated slides, dried, and sprayed with 10 mg/mL DHA matrix in 80% ethanol and then analyzed in a Bruker Autoflex III MALDI TOF-TOF mass spectrometer. For MALDI imaging, mass spectral information was obtained across the entire sample surface in 50 micron steps in the 250-2500 m/z range.
Untreated Rpe 65-/- mice showed no detectable levels of A2E over the entire time course of the experiments. 9-cis-Retinal treated Rpe 65-/- mice at 0 through 2 weeks also showed no traces of A2E. However, by 5 weeks, the level of A2E was detectable and by 7 weeks, there was remarkable accumulation of A2E (5 pmol/eye). Wild type mice were used as positive controls. Our studies also revealed the presence of various A2E adduct species in the mass range 500-1200 m/z. These masses were consistent with the presence of various phosphorylated A2E species and agree with the generation of A2E from A2PE. Interestingly, imaging studies have revealed that there is a geographic segregation between A2E and lipid-linked bis-retinoids, which had higher accumulation in the periphery.
9-cis-Retinal treated Rpe 65-/- mice are an excellent model for studying A2E accumulation. Our studies reinforced the hypothesis that A2E is generated from the visual chromophore. However, in addition to quantitative information about A2E, our data also provide new evidence on the rate of generation of A2E from A2PE and on the distribution of the possible intermediate species in the RPE. The segregation of A2E from the lipid-linked bis-retinoids is likely to reflect different photoreceptor numbers as well as rates of RPE metabolism in the central and peripheral retina.
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