Insulin promotes RGC dendrite regeneration through ribosomal protein S6 kinase activation leading to restoration of neuronal function in glaucoma

Abstract

Purpose : We previously demonstrated that insulin promotes RGC dendrite regeneration through activation of the mTOR pathway. However, the precise mechanisms of insulin-mediated regeneration and the effect of insulin on vision restoration are not well understood. Here, we asked: 1) what are the mTOR downstream effectors responsible for insulin-induced dendritic regrowth? 2) does insulin restore RGC function and visual responses in glaucoma?

Methods : Ocular hypertension (OHT) was induced by injection of magnetic microbeads in Thy1-YFP mice. Daily insulin or saline eye drops started at 2-weeks after OHT induction, when there is marked dendritic retraction, and dendrites were imaged and reconstructed 1 or 4 weeks later. The role of the mTORC1 downstream effectors, S6K and 4EBP1, was assessed by loss of function using targeted siRNAs. RGC survival and function were evaluated using complementary methods: i) quantification of RBPMS-positive neurons, ii) single-RGC calcium dynamics using two-photon microscopy live imaging in transgenic mice carrying the calcium indicator GCaMP6f, and iii) optomotor reflex assays.

Results : Insulin promoted a substantial increase in RGC dendritic length and complexity in glaucomatous eyes (n=6 mice/group, ANOVA, p<0.001). siRNA-based knockdown of S6K impaired insulin-mediated RGC dendrite regeneration, while 4EBP1 silencing had no effect. Intriguingly, S6K increased mTORC2 activity through phosphorylation of mSIN1 enhancing RGC dendrite regeneration. Insulin promoted robust RGC survival at 3 and 6 weeks of OHT induction relative to saline (OHT-3wks, insulin: 2057±34 RGC/mm², vehicle: 1679±66 RGC/mm², N=3-6 mice/group, ANOVA, p<0.001). Importantly, insulin restored light-evoked RGC calcium dynamics (n=6 mice/group, p<0.01) and improved visual acuity (N=5-8 mice/group, ANOVA, p<0.01) in glaucoma

Conclusions : Our data show that S6K is a key signaling component required for insulin-mediated RGC dendrite regeneration, an effect that is enhanced by cross-talk with mTORC2 through mSIN1 activation. Importantly, insulin prevents RGC loss while restoring light-evoked responses and visual acuity. These findings support a critical role for insulin as a pro-regenerative therapy and identify downstream targets to restore RGC connectivity and function in glaucoma

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.