Emergent temporal signaling in human trabecular meshwork cells: role of TRPV4-TRPM4 interactions

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway

TRPV4 and AQP4 Channels Synergistically Regulate Cell Volume and Calcium Homeostasis in Retinal Müller Glia

Brain edema formation occurs after dysfunctional control of extracellular volume partly through impaired astrocytic ion and water transport. Here, we show that such processes might involve synergistic cooperation between the glial water channel aquaporin 4 (AQP4) and the transient receptor potential isoform 4 (TRPV4), a polymodal swelling-sensitive cation channel. In mouse retinas, TRPV4 colocalized with AQP4 in the end feet and radial processes of Müller astroglia. Genetic ablation of TRPV4 did not affect the distribution of AQP4 and vice versa. However, retinas from Trpv4(−/−) and Aqp4(−/−) mice exhibited suppressed transcription of genes encoding Trpv4, Aqp4, and the Kir4.1 subunit of inwardly rectifying potassium channels. Swelling and [Ca(2+)](i) elevations evoked in Müller cells by hypotonic stimulation were antagonized by the selective TRPV4 antagonist HC-067047 (2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide) or Trpv4 ablation. Elimination of Aqp4 suppressed swelling-induced [Ca(2+)](i) elevations but only modestly attenuated the amplitude of Ca(2+) signals evoked by the TRPV4 agonist GSK1016790A [(N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide]. Glial cells lacking TRPV4 but not AQP4 showed deficits in hypotonic swelling and regulatory volume decrease. Functional synergy between TRPV4 and AQP4 during cell swelling was confirmed in the heterologously expressing Xenopus oocyte model. Importantly, when the swelling rate was osmotically matched for AQP4-positive and AQP4-negative oocytes, TRPV4 activation became independent of AQP4. We conclude that AQP4-mediated water fluxes promote the activation of the swelling sensor, whereas Ca(2+) entry through TRPV4 channels reciprocally modulates volume regulation, swelling, and Aqp4 gene expression. Therefore, TRPV4–AQP4 interactions constitute a molecular system that fine-tunes astroglial volume regulation by integrating osmosensing, calcium signaling, and water transport and, when overactivated, triggers pathological swelling. SIGNIFICANCE STATEMENT We characterize the physiological features of interactions between the astroglial swelling sensor transient receptor potential isoform 4 (TRPV4) and the aquaporin 4 (AQP4) water channel in retinal Müller cells. Our data reveal an elegant and complex set of mechanisms involving reciprocal interactions at the level of glial gene expression, calcium homeostasis, swelling, and volume regulation. Specifically, water influx through AQP4 drives calcium influx via TRPV4 in the glial end foot, which regulates expression of Aqp4 and Kir4.1 genes and facilitates the time course and amplitude of hypotonicity-induced swelling and regulatory volume decrease. We confirm the crucial facets of the signaling mechanism in heterologously expressing oocytes. These results identify the molecular mechanism that contributes to dynamic regulation of glial volume but also provide new insights into the pathophysiology of glial reactivity and edema formation

Newly developed reversible MAO-B inhibitor circumvents the shortcomings of irreversible inhibitors in Alzheimer’s disease

Monoamine oxidase–B (MAO-B) has recently emerged as a potential therapeutic target for Alzheimer’s disease (AD) because of its association with aberrant -aminobutyric acid (GABA) production in reactive astrocytes. Although short-term treatment with irreversible MAO-B inhibitors, such as selegiline, improves cognitive deficits in AD patients, long-term treatments have shown disappointing results. We show that prolonged treatment with selegiline fails to reduce aberrant astrocytic GABA levels and rescue memory impairment in APP/PS1 mice, an animal model of AD, because of increased activity in compensatory genes for a GABA-synthesizing enzyme, diamine oxidase (DAO). We have developed a potent, highly selective, and reversible MAO-B inhibitor, KDS2010 (IC 50 = 7.6 nM; 12,500-fold selectivity over MAO-A), which overcomes the disadvantages of the irreversible MAO-B inhibitor. Long-term treatment with KDS2010 does not induce compensatory mechanisms, thereby significantly attenuating increased astrocytic GABA levels and astrogliosis, enhancing synaptic transmission, and rescuing learning and memory impairments in APP/PS1 mice. Copyright © 2019 The Authors, some rights reserved11Nsci