Abstract
Purpose: :
Macular degeneration is a complex disorder wherein initial defects trigger secondary pathologies. The autofluorescent retinoid A2E accumulates in the lysosomes of RPE cells as lipofuscin. RPE cells from the ABCA4-/- mouse model of Stargardt’s degeneration have enhanced A2E levels. A2E alkalinizes RPE lysosomes, and the lysosomal pH of RPE cells from ABCA4-/- mice is elevated. As lysosomal alkalinization can impair acid lipase activity, alkalinization itself may lead to partially degraded autofluorescent lipids, another component of lipofuscin. Here we examined the ability of lysosomal alkalinization to increase autofluorescence as part of a detailed analysis of autofluorescence in wildtype and ABCA4 mice-/-.
Methods: :
Autofluorescence excited at 488nm in ARPE-19 cells was determined after 2 wks in chloroquine with FACS analysis. Levels of autophagy marker LC3BII were assayed with Western blots. TFEN mRNA was quantified using qPCR. Autofluorescent emission was measured from fixed sections of RPE cells from wildtype C57 and ABCA4-/- mice on samples from 6, 16 and 24 months old mice. Emission was determined in 2.5 nm widths using the spectral detector in the Nikon A1N microscope.
Results: :
Chloroquine increased the autofluorescence in cultured RPE cells by 60%.Chloroquine also increased levels of LC3BII, consistent with impaired autophagy leading to autofluorescence. Autofluorescence was detected at multiple wavelengths in RPE cells from both control C57 and ABCA4-/- mice. Peaks were Ex (excite) 405 nm; Em (emission) 490, 571, and 651 nm; Ex 488 nm Em 571 and 651 nm; Ex 561 nm, Em 612 and 663 nm; Ex 639 nm, Em 663 and 702 nm. When excited at 405 nm the largest emission peak in ABCA4-/- mice was at 571nm; this is very close to the emission for A2E in fixed RPE cells. The 571nm emission peak was present, but not as prominent, in control mice. Preliminary results suggest that the autophagy transcription factor TFEN is upregulated in ABCA4-/- mice, consistent with secondary impairment of autophagy.
Conclusions: :
Autofluorescence in RPE cells can be triggered either by A2E or other means of lysosomal alkalinization. Future studies will tell if lysosomal acidification can reduce one or both of these sources of lipofuscin.
Keywords: age-related macular degeneration • pH regulation/protons • retinal pigment epithelium