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Tanya Tolmachova, Diogo A. Feleciano, Clare E. Futter, Miguel C Seabra; Middle age-associated lysosomal upregulation in the healthy mouse retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2321. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelium (RPE) cells exist in a close spatial and functional connection with the light-sensitive cells of the retina (photoreceptors). As part of the blood-retinal barrier, RPE cells provide photoreceptors with nutrients and remove and recycle waste products of visual cycle. A considerable number of ocular diseases start in middle age, including age-related macular degeneration (AMD). Age-related changes in the normal RPE and AMD mechanisms are not fully understood. Lysosomes are organelles that are involved in degradation and recycling in animal cells. The purpose of the study is to identify age-related changes in the lysosomal function in freshly isolated healthy mouse RPE.
RPE cells were isolated from the eyes of 6, 12 and 18-month old C57Bl6 mice. Gene and protein expression were analysed in freshly isolated RPE cells by real-time PCR, flow cytometry and Western blotting. RPE cell size was assessed as a forward scatter (FSC) parameter by flow cytometry in unfixed cells. Lysosomal function was tested by labelling with fluorescent dyes LysoTracker Red (DND99) and LysoSensor Green (DND153).
We detected a significant increase in expression of LAMP-1 and LAMP2 and lysosomal proteases (cathepsins) in 12-month old (n=6) in comparison to 6 month old (n=6) C57Bl6 mice. Significant increase in loading with fluorescent dyes (LysoTracker Red and LysoSensor Green) was detected in 12-month old animals (n=5) in comparison to 6-month old mice (n=5). Comparison of 12-month old (n=6) and 18-month old (n=6) C57Bl6 mice did not reveal significant difference in lysosomal activity. The size parameter of RPE cells was significantly increased in 12-month old animals (n=5) in comparison to 6-month old (n=5) animals and was similar in 12- and 18-month old animals (n=5 for each group).
We identified lysosomal upregulation in middle-age healthy wild-type mice, which is consistent with activation of CLEAR network in RPE. Our data suggest that activation of CLEAR network in middle age might be a beneficial physiological defence mechanism against accumulation of increased waste load. Impairment of this mechanism might be a cause of age-related diseases in the RPE.
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