Activation of mTORC1 in RPE leads to RPE cell death by modulating PTEN and BAD

Abstract

Purpose: In multi-cellular organisms, cell death is linked to pathological conditions and is a significant component to approximately half of all medical illness, for many of which, such as age-related macular degeneration (AMD), no adequate therapy exists. The mechanisms underlying the death of retinal pigment epithelium (RPE) in AMD remain poorly understood. This has led us to explore the signaling mediators that affect cell death in RPE. The objective of this study was to determine if downstream effectors of mTORC1 signaling are involved in initiating apoptosis in RPE.

Methods: Cryba1 cKO mice homozygous for deletion of Cryba1 (encodes for βA3/A1-crystallin protein that is localized to the lysosomal lumen of RPE cells) specifically in RPE were generated. mTOR phospho antibody ELISA based array was used on RPE extracts for protein phosphorylation profiling and identification of downstream effectors of mTOR signaling. Immunoblotting was used to detect mechanistic target of rapamycin (mTOR), phospho-mTOR, phospho-Raptor, phospho-PTEN (phosphatase and tensin homolog) and phospho-BAD (Bcl-2 associated death promoter). The extent of cell death was determined by immunostaining Cryba1^fl/fl (age-matched floxed controls) and cKO retinal sections with anti-cleaved caspase-3 antibody.

Results: Our studies show that loss of Cryba1 in RPE activates mTORC1 signaling. Our recent array data shows that increased activation of mTOR pathway is accompanied by phosphorylation and inactivation of PTEN at the C-terminal serine-threonine cluster (Ser380, Thr382, Thr 383). Our data also shows elevated phosphorylation and inhibition of BAD at Ser 136. Furthermore, our results also suggest increased caspase-3 positive cells in Cryba1 cKO RPE compared to floxed controls.

Conclusions: Activation of mTORC1 has been reported in aging RPE cells and implicated in AMD. Our data suggest that phosphorylation and inactivation of PTEN leads to activation of the mTORC1 pathway in the cKO RPE. Activated mTORC1 causes phosphorylation and inactivation of BAD in the cKO RPE. Despite inactivation of a pro-apoptotic protein, BAD, our data shows increased caspase-3 in the cKO RPE. Since, our previous studies suggested impaired autophagy in the Cryba1 cKO RPE, further studies are underway to determine the crosstalk between apoptosis and autophagy pathways in the RPE.<br />