The energy sensor AMP-activated protein kinase (AMPK) signals neuronal death in glaucoma through inhibition of the mammalian target of rapamycin (mTOR).

Abstract

Purpose: The AMP-activated protein kinase (AMPK) is a key intracellular energy sensor that is activated when endogenous ATP levels decline and AMP increases, as in nutrient deprivation and hypoxia. Active AMPK inhibits the mammalian target of rapamycin (mTOR), a key regulator of cell growth and protein synthesis. We recently showed that mTOR activity in retinal ganglion cells (RGCs) is markedly reduced after acute optic nerve lesion. Here, we tested the hypothesis that ocular hypertension (OHT) increases AMPK activity leading to loss of mTOR function and RGC death in glaucoma.

Methods: C57BL/6 mice were subjected to OHT by injection of polystyrene magnetic microbeads (8 x 10⁸ beads/ml) in the anterior chamber. AMPK and mTOR activity were assessed by retinal immunohistochemistry using antibodies against phospho-AMPK^(Thr172) or phospho-S6 (a target of mTOR), respectively, in combination with RGC-specific markers. AMPK was inhibited in vivo by intraperitoneal administration of compound C (20 mg/kg), a cell-permeable and selective AMPK blocker, while mTORC1 was inhibited with rapamycin (6 mg/kg). RGC soma or axons were quantified by Brn3a immunostaining on flat-mounted retinas or toluidine blue-stained optic nerve cross sections, respectively. RGC dendrites were analyzed in Thy1-YFP mice followed by three-dimensional reconstruction of dendritic arbors.

Results: Our data demonstrate marked upregulation of AMPK activity at one and two weeks after induction of OHT, prior to RGC death, which correlated closely with decreased mTOR function in RGCs. Blockade of AMPK activity with compound C effectively restored mTOR function and promoted robust protection of RGC soma, axons and dendrites. For example, 95% of RGCs (2983 ± 258 RGCs/mm², mean ± S.E.M.) survived with compound C compared to 77% found in vehicle-treated eyes (2430 ± 233 RGCs/mm²) (ANOVA, p<0.001) at three weeks after OHT induction (N=8-10/group). Administration of rapamycin completely inhibited compound C-mediated neuroprotection indicating that this response was mTORC1-dependent

Conclusions: Our study provides evidence that early activation of the energy sensor AMPK leading to loss of mTOR function contributes to OHT-induced RGC degeneration. We conclude that the AMPK-mTOR pathway plays a crucial role in RGC loss caused by metabolic stress in glaucoma.