Activation of Pannexin1 channel induces ganglion cell loss in retinal ischemia

Abstract

Purpose: Membrane channels formed by pannexin1 (Panx1) permeate the cell to small molecules and are activated by purinergic P2 receptor stimulation, mechanical stress and ischemia. Previous in vitro and in vivo studies showed that such permeation is an essential contributor to lethal neuronal injury, and is likely to play a role in retinal pathologies. Here, we investigated a causative relationship between susceptibility of retinal ganglion cells (RGCs) to injury and Panx1 activity or expression level.

Methods: RGC susceptibility to ischemia was tested in models of oxygen/glucose deprivation (OGD) and retinal ischemia-reperfusion (IR). Cell death was measured using Annexin V/propidium iodide labeling in primary RGCs. The sub-population of ganglion cell layer neurons most vulnerable to Panx1-induced degeneration was identified using patch-clamp whole cell recording in freshly isolated wholemount mouse retinas, followed by morhometric phenotyping. In situ detection of Panx1 transcripts in retinal cell types was performed using high-resolution RNAscope technology.

Results: Distinct RGC subpopulations with differential Panx1 expression and activity levels were detected in the mouse retina. These RGC types differ significantly in both Panx1 mRNA levels and Panx1-mediated currents. Patch-clamp recordings indicated that only ~ 30% RGCs possessed robust Panx1-mediated currents in response to bzATP, 20 mM KCl and 0.5 mM glucose. These currents were detectable only in WT, but not in Panx1 knockout or inhibitor-treated tissue. The fraction of RGCs roughly corresponds to the proportion of cells that typically succumb to injury 7 days following IR. Amacrine cells known to be resistant to IR injury, lacked Panx1 activity altogether.

Conclusions: The activation of Panx1 channels is an essential trigger of ischemic injury affecting primarily RGCs. Our results suggest that this mechanism underlies high vulnerability of these neurons to degenerative diseases induced by ischemia, intraocular pressure and increased ATP.

Keywords: 531 ganglion cells • 532 gap junctions/coupling • 562 inner retina dysfunction: biochemistry and cell biology