Conditional knockout of mTORC1 in retina blocks developmental formation of the astrocyte network and retinal vasculature

Abstract

Purpose : During development the retinal vasculature forms to supply oxygen and nutrients to the growing retina. Formation of an astrocyte network precedes endothelial cell radial migration and serves as a template for vessel formation. Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase central to a nutrient sensing pathway that regulates cell metabolism, growth, proliferation and survival. mTOR forms two major signaling complexes, mTORC1 and mTORC2, containing Raptor and Rictor proteins, respectively. We tested the hypothesis that mTORC1 signaling influences retinal development.

Methods : mTOR^f/f and Raptor^f/f mice were crossed with Six3cre mice to cause conditional knockout (cKO) of mTOR complexes in retinal precursor cells (RPC) as well as in astrocyte precursor cells (APC) within the optic stalk. Six3cre mice were also bred with mT/mG Cre-reporter mice to confirm recombination in the entire neural retina and astrocytes. Retinal APCs, astrocyte network and vasculature were evaluated by immunofluorescence (IF) of flat mounted retinas. Retinal structure was determined by optical coherence tomography (OCT) and histology. Retinal organization and neuron populations were evaluated by IF of retinal sections. Visual acuity (VA) was assessed by optokinetic reflex testing.

Results : At postnatal day 5 (P5), when the astrocyte network is normally nearing completion, mTOR cKO retinas exhibited a nearly total lack of astrocyte network formation and vascular development; the effects of Raptor cKO were similar but less absolute. At P12 mTOR cKO mice exhibited a total lack of retinal vascular development, while 20-100% of retinal area was avascular in Raptor cKO retinas. Retinas of adult mTOR and Raptor cKO mice were correctly organized but significantly reduced in thickness, with approximately 50% reduction in ganglion cell and amacrine cell numbers. Both mTOR and Raptor cKO mice exhibited nearly complete loss of VA.

Conclusions : Lack of mTORC1 function in retinal neurons and astrocytes inhibited astrocyte network formation and subsequent vascular development. Loss of mTORC1 also resulted in thin retinas with fewer inner neurons. These findings reveal a profound role of mTORC1 signaling in retinal development and may have implications for the pathophysiology of adult retinal diseases.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.