Inflammasome activation in the inner retina contributes to ganglion cell loss in acute ocular hypertension

Abstract

Purpose : Retinal ganglion cell (RGC) loss in glaucoma is commonly associated with neuroinflammation and IL-1β cytokine production in the retina. However, cellular identity of inflammasome-active cells remains poorly explored. Here, we examined cellular localization of the inflammasome and tested whether pannexin1/P2X receptor signaling contributes to RGC loss in acute ocular hypertension (aOHT).

Methods : All experiment were performed in compliance with the NIH Guidelines and ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. A unilateral aOHT was induced by transient intraocular pressure elevation with contralateral and naïve eyes as controls. Bioindicator mice expressing a fluorescent ASC-citrin protein were used to identify ASC-speck in inflammasome-active cells by confocal microscopy. NLRP1/3 expression was examined by RNAscope in situ RNA hybridization and immunohistochemistry. Mice deficient for Panx1 and/or caspase-1 (Casp1) were used to study inflammasome neurotoxicity. Probenecid and RNAi treatments were applied to block the Panx1/P2X7 signaling. RGC loss was assessed by direct cell counts in retinal flat mounts.

Results : In the aOHT injured inner retina, the ASC-speck marker co-localized predominantly with astroglia, Mueller glia and RGCs, but not the microglial cells. Abundant expression of NLRP1/3 proteins was observed in neurons and glia in the inner retina and optic nerve. Relative to wild type, mice lacking Panx1 and/or Casp1 possessed significantly attenuated inflammasome activity and significant RGC protection. Similar protection was observed in mice treated with a Panx1 inhibitor probenecid, where Casp1, Casp11, and IL-1β were significantly decreased relative to wild type controls.

Conclusions : Our results show that both macroglia and RGCs activate the NLRP1/3 inflammasome in response to aOHT injury. Robust inflammasome induction is mediated by Panx1/P2X signaling and contributes to aOHT-induced RGC loss, supporting feasibility of targeting Panx1/P2X signaling and inflammasome as part of neuroprotective strategy in ischemic and glaucomatous neuropathies.

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

 

(A) Inflammasome biomarker ASC specks (green) are sparse in naïve retinas of ASC-citrin mice and do not co-localize with glia or neurons; n=3, bar, 25µm. (B) Increased density and high grade ASC speck co-localized with GFAP⁺ glia and TUJ1⁺ RGCs (panels C) at 3 and 7 days (dpi) post-aOHT injury. Bar, 10 µm