Abstract
Purpose :
Appropriate intracellular membrane trafficking is essential for cell to keep its physiological function. Lysosome-associated membrane protein 2 (LAMP2) is reported to play an important role in autophagy and lysosomal function and to be responsible for pathogenesis of Danon disease, which can cause retinal degeneration, though its pathophysiological contribution to retinal dysfunction remains unclear. The purpose of our research is to investigate expression of LAMP2 variants in the mammalian retina and retinal pigment epithelium (RPE) and to reveal how dysfunction of LAMP2 causes retinal diseases.
Methods :
Relative mRNA expression levels of three splicing variants of mouse Lamp2a-2c in the wild type mouse retina and RPE (n=3) or human LAMP2A-2C in ARPE-19 cells (n=3) and photoreceptor cell line 661W (n=5) were quantified by real-time PCR. LAMP2 was knocked down by siRNA in ARPE-19 and its effect on LC3, an autophagy marker, was assessed by Western blotting. Intracellular localization of LAMP2 and LC3 was also analyzed by immunocytochemistry in LAMP2-knocked-down ARPE-19 cells.
Results :
Expression of Lamp2a and Lamp2b was significantly higher in the mouse RPE than that in the mouse retina (ΔCT mean ± S.E. was -2.74±0.62 vs 6.85±0.31, and -1.89±-0.70 vs 6.58±0.65, respectively), while Lamp2c expression was higher in RPE than in retina but the difference was not significant (ΔCT mean ± S.E. 6.77±0.34 vs 4.17±0.77). Expression of Lamp2a and Lamp2b were significantly higher than that of Lamp2c in the mouse RPE, while there was no difference of expression among three Lamp2 variants in the mouse retina. Both ARPE-19 and 661W cells showed significantly higher expression of LAMP2A/Lamp2a and LAMP2B/Lamp2b than that of LAMP2C/Lamp2c (1.34 x 103- and 9.94 x 102-fold in ARPE-19 cells, and 39.3- and 31.6-fold in 661W cells, respectively). LAMP2 knockdown reduced LC3-II amount in whole cell lysate of ARPE-19.
Conclusions :
Our study revealed that the expression profile of LAMP2 is different in the mouse retina and RPE and that ARPE-19 had similar expression pattern to mouse RPE. Furthermore, our results of LAMP2 knockdown showed the role of LAMP2 in autophagosome generation. Collectively, our results imply that LAMP2 plays an essential role in keeping RPE function through autophagic pathway, and ARPE-19 may be useful for further research of its function.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.