Activation of mTORC1 stimulates the ubiquitin-proteasome system in rods

Abstract

Purpose : Inherited retinal degenerations are caused by an unusually large number of genetic mutations. Approaches to delay retinal degeneration in a mutation-independent manner would accelerate the delivery of the treatments to the patients. Previous studies demonstrated that activation of the mTORC1 (mammalian Target of Rapamycin Complex 1) pathway had beneficial effects in a broad range of mouse models of retinal degeneration and attributed these observations to the improved glucose metabolism. mTORC1 activation supported the survival of cones in mouse models of rod degeneration, slowed down the early stages of retinal degeneration in PDE6b mutant mice and increased retinal resistance to the RPE injury. Recent studies performed outside of the vision research field suggest that that activation of mTORC1 could increase the total proteasomal pool and activity in cell cultures and mouse brains. Therefore, here we investigated the stimulation of mTORC1 also as an approach to delay vision loss caused specifically by proteostatic stressors.

Methods : mTORC1 was stimulated genetically in rods by knocking out its negative regulator Tsc2 (Tuberous Sclerosis Complex 2) in Tsc2^flx/flx mice using rod-specific transgenic CRE mouse line. The heterozygote P23H rhodopsin knock-in mouse (P23H^+/-) was used as a model of retinal degeneration caused by protein misfolding and mice expressing reporter of proteasomal activity (Ub^G76V-GFP) were used as a tool to assess the status of ubiquitin-proteasomal system (UPS) in vivo. Proteasomal activity was measured in retinal lysates with fluorogenic substrates. Protein translation and autophagy were probed with puromycin- and chloroquine-based WB methods. Retinal health and function were evaluated with OCT, funduscopy, morphometric studies and ERG.

Results : Genetic activation of mTORC1 delays the late phase of rod photoreceptor loss in P23H^+/- mice by approximately 3 months. This improvement is accompanied by stimulation of proteasomal activity and more efficient protein degradation through the UPS as assessed using the Ub^G76V-GFP reporter mice. Stimulation of mTORC1 in P23H^+/- rods did not result in measurable changes in autophagy and translation.

Conclusions : These findings focus attention on a previously unappreciated ability of the mTORC1 pathway to modulate UPS in degenerating rods and position it as a promising therapeutic target for treating retinal degenerations associated with protein misfolding.

This is a 2021 ARVO Annual Meeting abstract.