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Sarah Redmon, Monika Lakk, Oleg Yarishkin, David Krizaj; Activation of the cAMP signaling pathway sensitizes retinal microglia response to TRPV4 activation. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4005.
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© ARVO (1962-2015); The Authors (2016-present)
Mounting evidence suggest neuroinflammation plays a critical role in the pathology of glaucoma. Glaucoma patients show elevation of several neuroinflammatory molecules including prostaglandins, endothelins, and ATP. These substances can activate GPCRs to increase the levels of cAMP. However, the effects of elevated cAMP are cell-type specific. Furthermore, it is unclear how PKA signaling interacts with transient receptor potential vanilloid channel 4 (TRPV4), a channel activated by the high intraocular pressure (IOP) in glaucoma. Using biophysical, electrophysiological and physiological tools we characterize changes in TRPV4 activity during modulation of cAMP levels.
IOP was elevated by injection of microbeads into the anterior chamber of CX3CR1GFP/+ mice. Cryosections/wholemounts were stained with antibodies against Iba1, P2Y12, and CHOP. qPCR was used to quantify changes in mRNA levels in the same proteins. Retinas were acutely dissociated and loaded with Fura-2 for monitoring [Ca2+]i levels in the presence of a selective TRPV4 agonist, GSK1016790A with and without forskolin (FSK). Whole cell patch clamp experiments were performed using the same experimental paradigm as the calcium imaging experiments.
In a mouse model of glaucoma we observed increased levels of the cAMP-dependent transcription factor, CHOP as well as downregulation of the Gi-coupled P2Y12 receptor. Retinal microglia exhibit relatively low levels of TRPV4; however application of a GSK101 results in robust elevation of intracellular calcium ([Ca2+])i. Microglial TRPV4 channels rapidly desensitize, with sequential applications of GSK101 failing to elevate [Ca2+]i. In contrast, rises in [Ca2+]i in response to sequential GSK101 exposures were seen with treatment with the adenylate cyclase activator FSK, and were modulated by SpcAMP, an agonist for cAMP-dependent proteins kinases(PKs) and RpcAMP, an inhibitor of cAMP-dependent PKs. TRPV4 resistance to inactivation was only observed if FSK was applied before the first GSK101 treatment.
Our data suggests that time-dependent phosphorylation associated with increased cAMP/PKA signaling may sensitize microglial mechanosensitive channels to respond to increases in IOP, pathological swelling, retinal detachment and other forms of biomechanical stress. The augmented microglial sensitivity to forces might promote neuroinflammation and degeneration.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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