Modulation of Rac1/PAK1/connexin43-Mediated ATP Release from Astrocytes Contributes to Retinal Ganglion Cell Survival in Experimental Glaucoma

Abstract Background: Connexin43 (Cx43) is one of major gap junction proteins in glial cells. Mutation in the gap junction protein alpha 1 gene of Cx43 has been detected in human glaucomatous retinas, suggestive of involvement of Cx43 in pathogenesis of glaucoma. However, the role of Cx43 in glaucoma has not been clearly elucidated. Methods: A mouse model of chronic ocular hypertension (COH) was produced by injecting magnetic microbeads into the eye anterior chamber. To explore the role of Rac1 in regulating Cx43 function, Rac1 conditional knockout in astrocytes was generated by subretinal injection of AAV-GFAP-Cre in Rac1 flox/flox mice. The hemichannel activity was assayed by ethidium bromide uptake. The level and source of ATP were assayed by a commercial ATP assay kit, ATP sensors and removing microglia. Results: In this study, we showed that Cx43 were mainly expressed in retinal astrocytes. Intraocular pressure (IOP) elevation induced astrocyte activation, as evidenced by increased expressions of c-Fos and glial fibrillary acidic protein (GFAP), which results in downregulation of Cx43 and changes of Cx43 phosphorylation at Ser373 and Ser368 sites. In the optic nerve head of COH mice, Cx43 expression in Gap43 (an activity-dependent plasticity protein) positive astrocytes was reduced. In COH retinas, Rac1, a member of the Rho family, was activated, which was consistent with the decrease of Cx43 expression in a time course. Pharmacological inhibition of Rac1 inhibited the activity of its downstream molecule PAK1, and reversed the reduction of Cx43 expression and astrocyte activation induced by IOP elevation. Co-immunoprecipitation experiments further demonstrated the interactions between Cx43 and active Rac1 or p-PAK1. Inhibition of Rac1 or conditional knockout of Rac1 in macroglial cells increased the ATP release through Cx43 hemichannels in astrocytes of COH retinas. Additionally, Rac1 deletion in astrocytes upregulated the expression of adenosine A3 receptor in retinal ganglion cells (RGCs) and promoted RGC survival, at least at early stage of IOP elevation through activating adenosine receptors. Conclusions: Our results showed that Rac1 in astrocytes regulated glaucomatous RGC survival through Cx43-mediated ATP release. These findings suggest that modulation of Rac1/PAK1/Cx43 pathway in astrocytes may be a potential strategy of neuroprotection in glaucoma.