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Jason Lim, Wennan Lu, Alan M Laties, Claire H Mitchell; Age-dependent increases in lysosomal pH, lysosomal gene expression and autofluorescence of mouse RPE cells; parallels with the ABCA4-/- mice suggest causal factors in age-dependent pathophysiology. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2957.
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RPE cells change in many ways as they age, but identifying which changes have a causal impact on the development of diseases like age-related macular degeneration is difficult. Parallels between aging mice and genetic disease models may identify key pathological changes, and thus possible targets for intervention. As lysosomes are emerging as an important factor in RPE pathophysiology, we compared changes in RPE lysosomes of aged mice and the ABCA4-/- model of Stargardt’s early-onset retinal degeneration.
Lysosomal pH was measured directly from 6 and 24 month old mice using the Lysosensor assay. Gene expression was determined using qPCR. RPE autofluorescence was measured from sections using the spectral detector function of the Nikon A1 confocal microscope using 406, 488 and 561 nm lasers.
As ABCA4-/- mice have an elevated lysosomal pH, this pH was determined in young and old control mice. Lysosomal pH was elevated in older mice and the rise in pH was proportional to mouse age. RPE cells from aged mice had a reduced expression of genes for the vesicular proton pump vHATPase and of the lysosome/autophagy transcription factor TFEB, consistent with the reduction in lysosomal pH. As lysosomal alkalinization is predicted to impair enzymatic degradation and lead to accumulation of partially digested lipids and proteins, levels of lipofuscin-like autofluorescence in aged RPE cells were determined. Spectral analysis of the autofluorescence of aged RPE cells showed a difference in emission at 650-660 nm as compared to younger mice. The emission for ABCA4-/- mice was also increased at this wavelength, but the magnitude was much greater than that measured from aged control mice.
In summary, aging of mouse RPE cells induces changes in lysosomal pH and autofluorescence similar to those found in ABCA4-/- mice. Whether treatment to restore lysosomal pH found effective in RPE cells from ABCA4-/- mice prevents age-dependent pathological changes remains to be determined.
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