Glia-derived tumor necrosis factor-alpha promotes retinal ganglion cell death through overexpression of philantotoxin-sensitive calcium permeable AMPA receptors

Abstract

Purpose: Tumor Necrosis Factor alpha (TNFα) has been proposed to mediate retinal ganglion cell (RGC) death in glaucoma, but its mechanism of action is currently unknown. TNFα regulates synaptic function by controlling neurotransmitter receptor trafficking. Here we tested whether TNFα increases cell surface expression of Ca⁺² - permeable AMPA receptors (CP-AMPAR) in retinal neurons, thus increasing their vulnerability to glaucomatous damage.

Methods: Ocular hypertension (OHT) was induced by injection of hypertonic saline solution into an episcleral vein in Brown Norway rats. Retinal TNFα, TNFR1/2, and AMPAR GluA2 subunit expression was examined by RT-PCR, western blot analysis and immunohistochemistry. Cell surface expression of CP-AMPAR was assessed by cobalt (Co⁺²) staining assays. GluA2 RNA editing, which regulates calcium permeability through AMPARs, was evaluated by nested PCR. Electrophysiological responses to light stimulation were examined by whole-cell patch-clamping. RGC soma or axon density was assessed on flat-mounted retinas or optic nerve cross sections, respectively.

Results: Our data demonstrate that retinal TNFα and TNFR1/2 levels increased as early as 1 week after OHT induction and remained elevated thereafter. TNFα was detected in microglia and Müller cells in experimental glaucoma. Co⁺² uptake, which occurs only via CP-AMPAR, was observed in RGCs of glaucomatous eyes and was blocked by the selective inhibitor philantotoxin. While the GluA2 subunit was fully edited at the Q/R site, the level of GluA2 was dramatically reduced in RGCs, which may account for increased Ca⁺² permeability through AMPAR in glaucoma. Electrophysiological data confirmed that RGCs in hypertensive eyes are more sensitive to philantotoxin than those in control eyes, suggesting CP-AMPA upregulation. Robust protection of RGC soma and axons was observed with TNFα and CP-AMPAR inhibitors following OHT induction.

Conclusions: Our data support a model in which TNFα-induced upregulation of CP-AMPAR plays a key role in RGC damage in experimental glaucoma.

Keywords: 615 neuroprotection • 568 intraocular pressure