The role of mTORC1 signaling in retinal bipolar cells

Abstract

Purpose : To investigate the role of mTORC1 signaling in mouse retinal bipolar cell proliferation and function.

Methods : Chx2-Cre, floxed Tsc1 and Rosa-tdTomato mice were used to trace cells affected by Chx2-Cre and to obtain retinal bipolar cell-specific Tsc1 gene ablation (Chx2-Cre/Tsc1^fx/fx, cKO). Rapamycin at 5 µg/g body weight was injected intraperitoneally for 30 continuous days to reduce the activation of mTORC1 in cKO mice. 5-ethynyl-2-deoxyuridine (EdU) was injected intraperitoneally to label dividing cells in the retina. Cellular organization of the retina was analyzed by hematoxylin-eosin and immunofluorescent staining. Bipolar cell function was analyzed by electroretinography (ERG).

Results : Bipolar-specific ablation of Tsc1 was achieved in cKO mice. Western blot analysis of the cKO retina showed increased phosphorylation of S6 kinase and 4EBP1, suggesting the activation of mTORC1 signaling. At postnatal day 3.5 and 6.5, more EdU-positive bipolar cells (Chx-Cre/Rosa-tdTomato) were found in cKO retina. Consistently, the adult cKO mouse also had more PKCα positive cells than controls, suggesting increased bipolar cell proliferation in the absence of Tsc1. However, these cells had irregular shaped axonal projections in the INL layer. No retinal hamartoma was found. The adult cKO mouse also showed progressively thickened inner nuclear layer (INL) and inner plexiform layer (IPL). At the age of 7-months, the INL and IPL layers of the cKO mouse were 60% and 80% thicker than controls, respectively. Displaced cells positive of CRALBP or calretinin staining were found in IPL layer of the adult cKO mouse retina. ERG analysis revealed that light-provoked b-wave of the dark-adapted cKO mouse progressively reduced with age. By the age of 7-months, the b-wave amplitude of cKO retina was less than 40% of the controls. Rapamycin successfully reduced the thickness of INL and IPL in the adult cKO mouse to normal levels. However, the displaced amacrine and Müller cells remained in the IPL layer. Furthermore, no significant improvement in ERG was observed in rapamycin-treated cKO mice.

Conclusions : Upregulation of mTORC1 signaling by the ablation of Tsc1 in mouse bipolar cells led to increased bipolar cell proliferation with irregular morphology. However, bipolar cells without Tsc1 were defective in relaying visual signals from photoreceptor cells to gangalion cells.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.