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Valery I Shestopalov, Alexey N. Pronin, Vladlen Z. Slepak, Hailey D. Rooney, Anddre Valdivia; P2RX4 facilitates Panx1-mediated neurotoxicity in ischemic and ocular hypertension injuries. Invest. Ophthalmol. Vis. Sci. 2017;58(8):852.
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© ARVO (1962-2015); The Authors (2016-present)
A hemichannel-mediated ATP release is essential for glial and neuronal functionality and purinergic signaling in the retina. Molecular complex, comprised of P2X7 receptor (P2RX7) and pannexin-1 (Panx1) was postulated as the key component of purinergic signaling pathway in the retinal ganglion cells (RGCs). Compared to P2RX7, P2RX4 is much more sensitive to ATP and was shown to be co-activated with Panx1 in homeostatic brain, as well as in different injury conditions. In this work we explored whether a P2X4 receptor, is involved in this signaling and whether it is activated in ischemia and retinal injuries, induced by ocular hypertension.
Quantitative gene expression analysis, RNA scope in situ hybridization and immunofluorescence imaging were used to study gene expression and protein localization. Pharmacological inhibitors were applied to test the involvement of individual P2X receptors. Induced ocular hypertension injury was used in vivo and oxygen-glucose deprivation in vitro to model pathological conditions. Cell survival was assayed by measuring EGFP release in vitro and RGC density changes in vivo. Cell permeation tests were performed in stable Neuro2A cell lines expressing EGFP and different levels of the Panx1 protein.
We showed that the P2X4 receptor is expressed in the retina at much higher levels, relative to P2RX7. In the inner retina, RNAscope in situ RNA hybridization and immunofluorescence imaging localized P2RX4 to neurons, particularly RGCs. P2RX4 blockade by 5-BDBD antagonist suppressed cell death from acute ischemic injury in vitro and from ocular hypertension injury in vivo. Significantly, the blockade of P2RX7 was only protective at high concentrations of extracellular ATP.
Our results show that P2RX4 is expressed in RGCs and is involved in extracellular ATP-induced and Panx1-mediated signaling in RGCs in physiological and mild pathological conditions.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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