Abstract
Purpose :
Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness in the elderly. Translocator protein (TSPO), a protein present in the outer membrane of mitochondria, is known to perform functions such as mitochondrial respiration, cholesterol binding, steroidogenesis, and microglia activation, and is particularly abundant in the retinal pigment epithelium (RPE) layer in the retina. We investigated the effects of TSPO on the mitochondrial and lysosomal structural and functional regulation of RPE and compared those changes with aged RPE cells.
Methods :
To investigate the function of TSPO in RPE cells, TSPO was knock-down. After treatment with TSPO siRNA for 24 hours, the accumulation of A2E and the level of oxidative stress were compared with those of the control group, and the morphological and functional changes of mitochondria and lysosomes were examined in the absence of TSPO. In addition, the changes in autophagy flux weres investigated using TSPO knock down mRFP-GFP-LC3-expressing H4 glioma cells, and the expression level of autophagy proteins was measured in RPE cells under the same conditions.
Results :
The absence of TSPO increased the accumulation of A2E in RPE cells and exacerbated cellular oxidative stress. Mitochondria lacking TSPO condensed around the nucleus, and the mitochondrial membrane potential decreased slightly. In meanwhile, lysosomal pH and cathepsin B activity of lysosomes did not significantly change, however, lysosomes are expanded in morphologically. In addition, the absence of TSPO increased the amount of intracellular calcium and translocated the TPEB protein from the cytosol into the nucleus. When TSPO was knock-down in RFP-GFP-LC3-H4 cells, the size of LC3 puncta was markedly increased. Consistent with this, we observed that TSPO knock down using siRNA accumulated p62 protein.
Conclusions :
Our study indicates that TSPO is an important factor that regulates intracellular organelles, and the changes induced by the absence of TSPO affect the normal function of RPE cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.