Disordered P2Y6 receptor signaling causes age-dependent pathogenesis of glaucoma

[Speaker] Youichi Shinozaki:1
[Co-author] Kenji Kashiwagi:2, Kazuhiko Namekata:3, Nobuhiko Ohno:4, Akiko Takeda:1, Takayuki Harada:3, Takeshi Iwata:5, Schuichi Koizumi:1
1:Neuropharmacology, University of Yamanashi, Japan, 2:Dept Ophthalmol, Grad Sch Med, Univ Yamanashi, Japan, 3:Vis Res Project, Tokyo Metro Inst Med Sci, Japan, 4:Div Neurobiol Bioinfo, NIPS, Japan, 5:Div Mol Cell Biol, NISO, Natl Hosp Org Tokyo Med Cent, Japan

Background: Glaucoma is a progressive optic neuropathy causing death of retinal ganglion cells (RGCs) and blindness. Although the highest risk factor for glaucoma is elevated intraocular pressure (IOP), detailed mechanisms of IOP regulation are poorly understood. Extracellular nucleotides such as ATP have been shown to exist in aqueous humor and be released in response to IOP. ATP level in aqueous humor of glaucoma patients is elevated, but causal relationship of nucleotide signaling and pathogenesis of glaucoma is totally unrevealed.
Methods: We measured IOP using rebound-type tonometer (TonoLab). The effects of nucleotides were estimated by instillation. Dynamics of aqueous humor were measured by fluorophotometry. Thinning of ganglion cell layer and inner plexiform layer (GCL/IPL) was estimated using optical coherence tomography. Ultrastructure of optic nerve was visualized with serial block-face scanning electron microscopy. Degeneration of RGCs was estimated by immunohistochemistry with anti-Brn3a antibody on whole-mounted retinae.
Results: We found that physiological P2Y6 receptor (P2Y6R) is essential for lowering IOP and knocking out of P2ry6 gene (P2Y6KO) induces sustained elevation in IOP. Uridine diphosphate (UDP), an endogenous agonist for P2Y6R, reduced IOP in wild type mouse but not in P2Y6KO mice. P2Y6R was expressed in non-pigmented epithelial cells of ciliary body and contributed to control production of aqueous humor. P2Y6KO mice showed accelerated production of aqueous humor and elevated IOP. Young-adult P2Y6KO mice (3 months old) showed elevated IOP but no histological or functional abnormalities of the eye. Middle-aged P2Y6KO mice (6-12 months old) showed significant structural and functional abnormalities including optic nerve atrophy, thinning of GCL/IPL, degeneration of RGCs and disordered visual functions. These changes were attenuated by an IOP lowering agent.
Conclusion: Dysfunctional P2Y6R signaling causes IOP elevation, resulting in glaucomatous optic neuropathy.

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