Abstract
Purpose :
The mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that regulates cell growth and survival. Previous studies indicate mTOR may play important roles in regulating the functions of retinal pigment epithelial (RPE). To investigate the consequences of genetically stimulating mTOR pathway in the RPE, we mutated tuberous sclerosis complex 1 (Tsc1), a critical negative regulator of mTOR, by using tissue-specific knockout mouse model.
Methods :
To inactivate Tsc1 in the RPE cells, we crossed Tsc1 conditional targeted mice (Tsc1f/f) with a transgenic BEST1-Cre mouse line to generate a mouse model in which Tsc1 was specifically eliminated in RPE cells (Tsc1f/f: BEST1-Cre, cKO). PCR was used for genotyping of Tsc1 gene and Cre transgene. The activation of mTOR pathway was examined by western blotting. Immunofluorescence on cryosection was used for detecting the distribution of Cre. Contour length of outer segments and thickness of the RPE layers were measured by unbiased stereology. Immunofluorescence on RPE flat mounts were performed to assess the cuboidal appearance and the junctional integrity of RPE cells.
Results :
The highest expressing percentage of Cre transgene reached to 70% in RPE cells and displayed a patchy mosaic expression pattern in the cKO mice. Within the eyes of cKO mice, only the RPE cells were immuno-reactive with an anti-cre antibody. The level of Tsc1 expression was almost diminished and mTOR was activated. The cKO mice showed a gradual thinning of the outer nuclear layer and the redistribution of melanosomes in RPE cells. These morphological abnormalities indicated the process of RPE dedifferentiation. Furthermore, in the Tsc1 deficiency RPE cells, the distributions of β-catenin were increased in cytoplasm and decreased on cell boundary, and the phosphorylation of Y489 in β-catenin increased with age.
Conclusions :
The mTOR pathway was successfully activated after the deletion of Tsc1 in RPE cells and the cKO mice displayed some typical phenotypes of AMD disease. This mice model enabled us to investigate physical role of mTOR signaling in RPE cells. Furthermore, this mice model may provide new insights into the pathogenic mechanism of AMD and new therapeutic targets for this disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.