TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging and electrophysiology. Anisosmotic conditions elicited proportional changes in TM cell volume, with swelling, but not shrinking, evoking elevations in intracellular calcium concentration [Ca2+]TM. Hypotonicity-evoked calcium signals were sensitive to HC067047, a selective blocker of TRPV4 channels, whereas the agonist GSK1016790A promoted swelling under isotonic conditions. TRPV4 inhibition partially suppressed hypotonicity-induced volume increases and reduced the magnitude of the swelling-induced membrane current, with a substantial fraction of the swelling-evoked current abrogated by Cl- channel antagonists DIDS and niflumic acid. The transcriptome of volume-sensing chloride channel candidates in primary human was dominated by ANO6 transcripts, with moderate expression of ANO3, ANO7, ANO10 transcripts and low expression of LTTRC genes that encode constituents of the volume-activated anion channel. Imposition of 190 mOsm but not 285 mOsm hypotonic gradients increased conventional outflow in mouse eyes. TRPV4-mediated cation influx thus works with Cl- efflux to sense and respond to osmotic stress, potentially contributing to pathological swelling, calcium overload and intracellular signaling that could exacerbate functional disturbances in inflammatory disease and glaucoma.

TRPV4 expression in the renal tubule is necessary for maintaining whole body K+ homeostasis.

The Ca2+-permeable transient receptor potential vanilloid type 4 (TRPV4) channel serves as the sensor of tubular flow, thus being well suited to govern mechanosensitive K+ transport in the distal renal tubule. Here, we directly tested whether the TRPV4 function is significant in affecting K+ balance. We used balance metabolic cage experiments and systemic measurements with different K+ feeding regimens [high (5% K+), regular (0.9% K+), and low (<0.01% K+)] in newly created transgenic mice with selective TRPV4 deletion in the renal tubule (TRPV4fl/fl-Pax8Cre) and their littermate controls (TRPV4fl/fl). Deletion was verified by the absence of TRPV4 protein expression and lack of TRPV4-dependent Ca2+ influx. There were no differences in plasma electrolytes, urinary volume, and K+ levels at baseline. In contrast, plasma K+ levels were significantly elevated in TRPV4fl/fl-Pax8Cre mice on high K+ intake. K+-loaded knockout mice exhibited lower urinary K+ levels than TRPV4fl/fl mice, which was accompanied by higher aldosterone levels by day 7. Moreover, TRPV4fl/fl-Pax8Cre mice had more efficient renal K+ conservation and higher plasma K+ levels in the state of dietary K+ deficiency. H+-K+-ATPase levels were significantly increased in TRPV4fl/fl-Pax8Cre mice on a regular diet and especially on a low-K+ diet, pointing to augmented K+ reabsorption in the collecting duct. Consistently, we found a significantly faster intracellular pH recovery after intracellular acidification, as an index of H+-K+-ATPase activity, in split-opened collecting ducts from TRPV4fl/fl-Pax8Cre mice. In summary, our results demonstrate an indispensable prokaliuretic role of TRPV4 in the renal tubule in controlling K+ balance and urinary K+ excretion during variations in dietary K+ intake. NEW & NOTEWORTHY The mechanoactivated transient receptor potential vanilloid type 4 (TRPV4) channel is expressed in distal tubule segments, where it controls flow-dependent K+ transport. Global TRPV4 deficiency causes impaired adaptation to variations in dietary K+ intake. Here, we demonstrate that renal tubule-specific TRPV4 deletion is sufficient to recapitulate the phenotype by causing antikaliuresis and higher plasma K+ levels in both states of K+ load and deficiency.

TRPV4: Cell type-specific activation, regulation and function in the vertebrate eye.

The architecture of the vertebrate eye is optimized for efficient delivery and transduction of photons and processing of signaling cascades downstream from phototransduction. The cornea, lens, retina, vasculature, ciliary body, ciliary muscle, iris and sclera have specialized functions in ocular protection, transparency, accommodation, fluid regulation, metabolism and inflammatory signaling, which are required to enable function of the retina-light sensitive tissue in the posterior eye that transmits visual signals to relay centers in the midbrain. This process can be profoundly impacted by non-visual stimuli such as mechanical (tension, compression, shear), thermal, nociceptive, immune and chemical stimuli, which target these eye regions to induce pain and precipitate vision loss in glaucoma, diabetic retinopathy, retinal dystrophies, retinal detachment, cataract, corneal dysfunction, ocular trauma and dry eye disease. TRPV4, a polymodal nonselective cation channel, integrate non-visual inputs with homeostatic and signaling functions of the eye. The TRPV4 gene is expressed in most if not all ocular tissues, which vary widely with respect to the mechanisms of TRPV4 channel activation, modulation, oligomerization, and participation in protein- and lipid interactions. Under- and overactivation of TRPV4 may affect intraocular pressure, maintenance of blood-retina barriers, lens accommodation, neuronal function and neuroinflammation. Because TRPV4 dysregulation precipitates many pathologies across the anterior and posterior eye, the channel could be targeted to mitigate vision loss.

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 TRPC1 channels mediate the response to tensile strain in mouse Müller cells.

Müller glia, a pillar of metabolic, volume regulatory and immune/inflammatory signaling in the mammalian retina, are among the earliest responders to mechanical stressors in the eye. Ocular trauma, edema, detachment and glaucoma evoke early inflammatory activation of Müller cells yet the identity of their mechanotransducers and signaling mechanisms downstream remains unknown. Here, we investigate expression of genes that encode putative stretch-activated calcium channels (SACs) in mouse Müller cells and study their responses to dynamical tensile loading in cells loaded with a calcium indicator dye. Transcript levels in purified glia were Trpc1>Piezo1>Trpv2>Trpv4>>Trpv1>Trpa1. Cyclic radial deformation of matrix-coated substrates produced dose-dependent increases in [Ca2+]i that were suppressed by the TRPV4 channel antagonist HC-067047 and by ablation of the Trpv4 gene. Stretch-evoked calcium responses were also reduced by knockdown and pharmacological inhibition of TRPC1 channels whereas the TRPV2 inhibitor tranilast had no effect. These data demonstrate that Müller cells are intrinsically mechanosensitive, with the response to tensile loading mediated through synergistic activation of TRPV4 and TRPC1 channels. Coupling between mechanical stress and Müller Ca2+ homeostasis has treatment implications, since many neuronal injury paradigms in the retina involve calcium dysregulation associated with inflammatory and immune signaling.

The RNA-binding protein and stress granule component ATAXIN-2 is expressed in mouse and human tissues associated with glaucoma pathogenesis.

Polyglutamine repeat expansions in the Ataxin-2 (ATXN2) gene were first implicated in Spinocerebellar Ataxia Type 2, a disease associated with degeneration of motor neurons and Purkinje cells. Recent studies linked single nucleotide polymorphisms in the gene to elevated intraocular pressure in primary open angle glaucoma (POAG); yet, the localization of ATXN2 across glaucoma-relevant tissues of the vertebrate eye has not been thoroughly examined. This study characterizes ATXN2 expression in the mouse and human retina, and anterior eye, using an antibody validated in ATXN2-/- retinas. ATXN2-ir was localized to cytosolic sub compartments in retinal ganglion cell (RGC) somata and proximal dendrites in addition to GABAergic, glycinergic, and cholinergic amacrine cells in the inner plexiform layer (IPL) and displaced amacrine cells. Human, but not mouse retinas showed modest immunolabeling of bipolar cells. ATXN2 immunofluorescence was prominent in the trabecular meshwork and pigmented and nonpigmented cells of the ciliary body, with analyses of primary human trabecular meshwork cells confirming the finding. The expression of ATXN2 in key POAG-relevant ocular tissues supports the potential role in autophagy and stress granule formation in response to ocular hypertension.

Membrane cholesterol regulates TRPV4 function, cytoskeletal expression, and the cellular response to tension.

Despite the association of cholesterol with debilitating pressure-related diseases such as glaucoma, heart disease, and diabetes, its role in mechanotransduction is not well understood. We investigated the relationship between mechanical strain, free membrane cholesterol, actin cytoskeleton, and the stretch-activated transient receptor potential vanilloid isoform 4 (TRPV4) channel in human trabecular meshwork (TM) cells. Physiological levels of cyclic stretch resulted in time-dependent decreases in membrane cholesterol/phosphatidylcholine ratio and upregulation of stress fibers. Depleting free membrane cholesterol with m-ß-cyclodextrin (MßCD) augmented TRPV4 activation by the agonist GSK1016790A, swelling and strain, with the effects reversed by cholesterol supplementation. MßCD increased membrane expression of TRPV4, caveolin-1, and flotillin. TRPV4 did not colocalize or interact with caveolae or lipid rafts, apart from a truncated ∼75 kDa variant partially precipitated by a caveolin-1 antibody. MßCD induced currents in TRPV4-expressing Xenopus laevis oocytes. Thus, membrane cholesterol regulates trabecular transduction of mechanical information, with TRPV4 channels mainly located outside the cholesterol-enriched membrane domains. Moreover, the biomechanical milieu itself shapes the lipid content of TM membranes. Diet, cholesterol metabolism, and mechanical stress might modulate the conventional outflow pathway and intraocular pressure in glaucoma and diabetes in part by modulating TM mechanosensing.

TRPV4-Rho signaling drives cytoskeletal and focal adhesion remodeling in trabecular meshwork cells.

Intraocular pressure (IOP) is dynamically regulated by the trabecular meshwork (TM), a mechanosensitive tissue that protects the eye from injury through dynamic regulation of aqueous humor flow. TM compensates for mechanical stress impelled by chronic IOP elevations through increased actin polymerization, tissue stiffness, and contractility. This process has been associated with open angle glaucoma; however, the mechanisms that link mechanical stress to pathological cytoskeletal remodeling downstream from the mechanotransducers remain poorly understood. We used fluorescence imaging and biochemical analyses to investigate cytoskeletal and focal adhesion remodeling in human TM cells stimulated with physiological strains. Mechanical stretch promoted F-actin polymerization, increased the number and size of focal adhesions, and stimulated the activation of the Rho-associated protein kinase (ROCK). Stretch-induced activation of the small GTPase Ras homolog family member A (RhoA), and tyrosine phosphorylations of focal adhesion proteins paxillin, focal adhesion kinase (FAK), vinculin, and zyxin were time dependently inhibited by ROCK inhibitor trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Y-27632), and by HC-067047, an antagonist of transient receptor potential vanilloid 4 (TRPV4) channels. Both TRPV4 and ROCK activation were required for zyxin translocation and increase in the number/size of focal adhesions in stretched cells. Y-27632 blocked actin polymerization without affecting calcium influx induced by membrane stretch and the TRPV4 agonist GSK1016790A. These results reveal that mechanical tuning of TM cells requires parallel activation of TRPV4, integrins, and ROCK, with chronic stress leading to sustained remodeling of the cytoskeleton and focal complexes.

TRPV4 channels mediate the mechanoresponse in retinal microglia.

The physiological and neurological correlates of plummeting brain osmolality during edema, traumatic CNS injury, and severe ischemia are compounded by neuroinflammation. Using multiple approaches, we investigated how retinal microglia respond to challenges mediated by increases in strain, osmotic gradients, and agonists of the stretch-activated cation channel TRPV4. Dissociated and intact microglia were TRPV4-immunoreactive and responded to the selective agonist GSK1016790A and substrate stretch with altered motility and elevations in intracellular calcium ([Ca2+ ]i ). Agonist- and hypotonicity-induced swelling was associated with a nonselective outwardly rectifying cation current, increased [Ca2+ ]i , and retraction of higher-order processes. The antagonist HC067047 reduced the extent of hypotonicity-induced microglial swelling and inhibited the suppressive effects of GSK1016790A and hypotonicity on microglial branching. Microglial TRPV4 signaling required intermediary activation of phospholipase A2 (PLA2), cytochrome P450, and epoxyeicosatrienoic acid production (EETs). The expression pattern of vanilloid thermoTrp genes in retinal microglia was markedly different from retinal neurons, astrocytes, and cortical microglia. These results suggest that TRPV4 represents a primary retinal microglial sensor of osmochallenges under physiological and pathological conditions. Its activation, associated with PLA2, modulates calcium signaling and cell architecture. TRPV4 inhibition might be a useful strategy to suppress microglial overactivation in the swollen and edematous CNS.

Piezo1 channels mediate trabecular meshwork mechanotransduction and promote aqueous fluid outflow.

KEY POINTS: Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye. ABSTRACT: Chronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure-induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1-deficient cells. The majority of TM cells exhibited a delayed stretch-activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell-matrix contacts. Analysis of TM-dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells.

Purpose: Contact lenses, osmotic stressors, and chemical burns may trigger severe discomfort and vision loss by damaging the cornea, but the signaling mechanisms used by corneal epithelial cells (CECs) to sense extrinsic stressors are not well understood. We therefore investigated the mechanisms of swelling, temperature, strain, and chemical transduction in mouse CECs. Methods: Intracellular calcium imaging in conjunction with electrophysiology, pharmacology, transcript analysis, immunohistochemistry, and bioluminescence assays of adenosine triphosphate (ATP) release were used to track mechanotransduction in dissociated CECs and epithelial sheets isolated from the mouse cornea. Results: The transient receptor potential vanilloid (TRPV) transcriptome in the mouse corneal epithelium is dominated by Trpv4, followed by Trpv2, Trpv3, and low levels of Trpv1 mRNAs. TRPV4 protein was localized to basal and intermediate epithelial strata, keratocytes, and the endothelium in contrast to the cognate TRPV1, which was confined to intraepithelial afferents and a sparse subset of CECs. The TRPV4 agonist GSK1016790A induced cation influx and calcium elevations, which were abolished by the selective blocker HC067047. Hypotonic solutions, membrane strain, and moderate heat elevated [Ca2+]CEC with swelling- and temperature-, but not strain-evoked signals, sensitive to HC067047. GSK1016790A and swelling evoked calcium-dependent ATP release, which was suppressed by HC067027 and the hemichannel blocker probenecid. Conclusions: These results demonstrate that cation influx via TRPV4 transduces osmotic and thermal but not strain inputs to CECs and promotes hemichannel-dependent ATP release. The TRPV4-hemichannel-ATP signaling axis might modulate corneal pain induced by excessive mechanical, osmotic, and chemical stimulation.

The Neuroprotective Peptide PACAP1-38 Contributes to Horizontal Cell Development in Postnatal Rat Retina.

Purpose: PACAP1-38, a member of the secretin/glucagon superfamily, is expressed in the developing retina with documented neuroprotective effects. However, its function in retinal cell differentiation has yet to be elucidated. Our goals, therefore, were to identify PAC1 expressing cells morphologically, investigate the PACAP1-38 action functionally, and establish PACAP1-38 regulated events developmentally during the first postnatal week in rat retina. Methods: P1 retinal sections or whole mounts of Wistar rats were used to reveal PAC1 and calbindin immunoreactive structures. P1, P3, or P7 pups were injected intravitreally with 100 pmol PACAP1-38. Tissues were harvested 24 hours post-treatment, then processed for calbindin immunohistochemistry to determine horizontal cell number, or 6, 12, 24 hours post-treatment for real-time PCR and immunoblots to detect PCNA expression. To localize proliferating cells, anti-PCNA antibody was applied. Results: We showed various PAC1 expressing cells in RPE, NBL, and GCL in P1 retina including calbindin positive horizontal cells. We found that PACAP1-38 induced a marked cell number increase at P3 and P7 and showed upregulated cell proliferation as its mechanism; however, it was ineffective at P1. PACAP1-38 induced proliferative cells localized in the NBL, and double-marker studies demonstrated that the induced proliferative cells were horizontal cells. Conclusions: PACAP1-38 appears to act in retinal differentiation by inducing mitosis selectively in a time and cell specific manner through PAC1. The control of horizontal cell proliferation raises the novel possibilities that (1) PACAP1-38 may be a major player in retinal patterning and (2) PACAP signaling may be critical in retinoblastoma.

Polymodal TRPV1 and TRPV4 Sensors Colocalize but Do Not Functionally Interact in a Subpopulation of Mouse Retinal Ganglion Cells.

Retinal ganglion cells (RGCs) are projection neurons that transmit the visual signal from the retina to the brain. Their excitability and survival can be strongly influenced by mechanical stressors, temperature, lipid metabolites, and inflammatory mediators but the transduction mechanisms for these non-synaptic sensory inputs are not well characterized. Here, we investigate the distribution, functional expression, and localization of two polymodal transducers of mechanical, lipid, and inflammatory signals, TRPV1 and TRPV4 cation channels, in mouse RGCs. The most abundant vanilloid mRNA species was Trpv4, followed by Trpv2 and residual expression of Trpv3 and Trpv1. Immunohistochemical and functional analyses showed that TRPV1 and TRPV4 channels are expressed as separate molecular entities, with TRPV1-only (∼10%), TRPV4-only (∼40%), and TRPV1 + TRPV4 (∼10%) expressing RGC subpopulations. The TRPV1 + TRPV4 cohort included SMI-32-immunopositive alpha RGCs, suggesting potential roles for polymodal signal transduction in modulation of fast visual signaling. Arguing against obligatory heteromerization, optical imaging showed that activation and desensitization of TRPV1 and TRPV4 responses evoked by capsaicin and GSK1016790A are independent of each other. Overall, these data predict that RGC subpopulations will be differentially sensitive to mechanical and inflammatory stressors.

TREK-1 channels regulate pressure sensitivity and calcium signaling in trabecular meshwork cells.

Mechanotransduction by the trabecular meshwork (TM) is an essential component of intraocular pressure regulation in the vertebrate eye. This process is compromised in glaucoma but is poorly understood. In this study, we identify transient receptor potential vanilloid isoform 4 (TRPV4) and TWIK-related potassium channel-1 (TREK-1) as key molecular determinants of TM membrane potential, pressure sensitivity, calcium homeostasis, and transcellular permeability. We show that resting membrane potential in human TM cells is unaffected by "classical" inhibitors of voltage-activated, calcium-activated, and inwardly rectifying potassium channels but is depolarized by blockers of tandem-pore K+ channels. Using gene profiling, we reveal the presence of TREK-1, TASK-1, TWIK-2, and THIK transcripts in TM cells. Pressure stimuli, arachidonic acid, and TREK-1 activators hyperpolarize these cells, effects that are antagonized by quinine, amlodipine, spadin, and short-hairpin RNA-mediated knockdown of TREK-1 but not TASK-1. Activation and inhibition of TREK-1 modulates [Ca2+]TM and lowers the impedance of cell monolayers. Together, these results suggest that tensile homeostasis in the TM may be regulated by balanced, pressure-dependent activation of TRPV4 and TREK-1 mechanotransducers.

TRPV4 Does Not Regulate the Distal Retinal Light Response.

The transient receptor potential vanilloid isoform 4 (TRPV4) functions as polymodal transducer of swelling, heat, stretch, and lipid metabolites, is widely expressed across sensory tissues, and has been implicated in pressure sensing in vertebrate retinas. Although TRPV4 knockout mice exhibit a variety of mechanosensory, nociceptive, and thermo- and osmoregulatory phenotypes, it is not known whether the transmission of light-induced signals in the eye is affected by the loss of TRPV4. We utilized field potentials, a measure of rod and cone signaling, to determine whether TRPV4 impacts on the generation and/or transmission of the photoreceptor light response and neurotransmission. Luminance intensity-response relationships were acquired in anesthetized wild-type and TRPV4-/- mice and evaluated for peak amplitude and implicit time under scotopic and photopic conditions. We found that the morphology of the outer retina is unaffected by the ablation of the Trpv4 gene. Calcium imaging of dissociated Müller glia showed that selective TRPV4 stimulation induces oscillatory calcium signals in adjacent rods. However, no differences in scotopic or photopic light-evoked signaling in the distal retina were observed in TRPV4-/- eyes, suggesting that TRPV4 signaling in healthy Müller cells does not modulate the transmission of light-evoked signals at rod and cone synapses.

Dyslipidemia modulates Müller glial sensing and transduction of ambient information.

Unesterified cholesterol controls the fluidity, permeability and electrical properties of eukaryotic cell membranes. Consequently, cholesterol levels in the retina and the brain are tightly regulated whereas depletion or oversupply caused by diet or heredity contribute to neurodegenerative diseases and vision loss. Astroglia play a central role in the biosynthesis, uptake and transport of cholesterol and also drive inflammatory signaling under hypercholesterolemic conditions associated with high-fat diet (diabetes) and neurodegenerative disease. A growing body of evidence shows that unesterified membrane cholesterol modulates the ability of glia to sense and transduce ambient information. Cholesterol-dependence of Müller glia - which function as retinal sentinels for metabolic, mechanical, osmotic and inflammatory signals - is mediated in part by transient receptor potential V4 (TRPV4) channels. Cholesterol supplementation facilitates, whereas depletion suppresses, TRPV4-mediated transduction of temperature and lipid agonists in Müller cells. Acute effects of cholesterol supplementation/depletion on plasma membrane ion channels and calcium homeostasis differ markedly from the effects of chronic dyslipidemia, possibly due to differential modulation of modality-dependent energy barriers associated with the functionality of polymodal channels embedded within lipid rafts. Understanding of cholesterol-dependence of TRP channels is thus providing insight into dyslipidemic pathologies associated with diabetic retinopathy, glaucoma and macular degeneration.

Cholesterol regulates polymodal sensory transduction in Müller glia.

Over- and underexposure to cholesterol activates glia in neurodegenerative brain and retinal diseases but the molecular targets of cholesterol in glial cells are not known. Here, we report that disruption of unesterified membrane cholesterol content modulates the transduction of chemical, mechanical and temperature stimuli in mouse Müller cells. Activation of TRPV4 (transient receptor potential vanilloid type 4), a nonselective polymodal cation channel was studied following the removal or supplementation of cholesterol using the methyl-beta cyclodextrin (MßCD) delivery vehicle. Cholesterol extraction disrupted lipid rafts and caveolae without affecting TRPV4 trafficking or membrane localization protein. However, MßCD suppressed agonist (GSK1016790A)- and temperature-evoked elevations in [Ca2+ ]i , and suppressed transcellular propagation of Ca2+ waves. Lowering the free membrane cholesterol content markedly prolonged the time-course of the glial swelling response, whereas MßCD:cholesterol supplementation enhanced agonist- and temperature-induced Ca2+ signals and shortened the swelling response. Taken together, these data show that membrane cholesterol modulates polymodal transduction of agonists, swelling and temperature stimuli in retinal radial glia and suggest that dyslipidemic retinas might be associated with abnormal glial transduction of ambient sensory inputs.

Mouse retinal ganglion cell signalling is dynamically modulated through parallel anterograde activation of cannabinoid and vanilloid pathways.

KEY POINTS: Retinal cells use vanilloid transient receptor potential (TRP) channels to integrate light-evoked signals with ambient mechanical, chemical and temperature information. Localization and function of the polymodal non-selective cation channel TRPV1 (transient receptor potential vanilloid isoform 1) remains elusive. TRPV1 is expressed in a subset of mouse retinal ganglion cells (RGCs) with peak expression in the mid-peripheral retina. Endocannabinoids directly activate TRPV1 and inhibit it through cannabinoid type 1 receptors (CB1Rs) and cAMP pathways. Activity-dependent endocannabinoid release may modulate signal gain in RGCs through simultaneous manipulation of calcium and cAMP signals mediated by TRPV1 and CB1R. ABSTRACT: How retinal ganglion cells (RGCs) process and integrate synaptic, mechanical, swelling stimuli with light inputs is an area of intense debate. The nociceptive cation channel TRPV1 (transient receptor potential vanilloid type 1) modulates RGC Ca2+ signals and excitability yet the proportion of RGCs that express it remains unclear. Furthermore, TRPV1's response to endocannabinoids (eCBs), the putative endogenous retinal activators, is unknown, as is the potential modulation by cannabinoid receptors (CBRs). The density of TRPV1-expressing RGCs in the Ai9:Trpv1 reporter mouse peaked in the mid-peripheral retina. TRPV1 agonists including capsaicin (CAP) and the eCBs anandamide and N-arachidonoyl-dopamine elevated [Ca2+ ]i in 30-40% of wild-type RGCs, with effects suppressed by TRPV1 antagonists capsazepine (CPZ) and BCTC ((4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide), and lacking in Trpv1-/- cells. The cannabinoid receptor type 1 (CB1R) colocalized with TRPV1:tdTomato expression. Its agonists 2-arachidonoylglycerol (2-AG) and WIN55,122 inhibited CAP-induced [Ca2+ ]i signals in adult, but not early postnatal, RGCs. The suppressive effect of 2-AG on TRPV1 activation was emulated by positive modulators of the protein kinase A (PKA) pathway, inhibited by the CB1R antagonist rimonabant and Gi uncoupler pertussis toxin, and absent in Cnr1-/- RGCs. We conclude that TRPV1 is a modulator of Ca2+ homeostasis in a subset of RGCs that show non-uniform distribution across the mouse retina. Non-retrograde eCB-mediated modulation of RGC signalling involves a dynamic push-pull between direct TRPV1 activation and PKA-dependent regulation of channel inactivation, with potential functions in setting the bandwidth of postsynaptic responses, sensitivity to mechanical/excitotoxic stress and neuroprotection.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP), a Novel Secretagogue, Regulates Secreted Morphogens in Newborn Rat Retina.

Purpose: Pituitary adenylate cyclase-activating peptide (PACAP)1-38 has been reported to be responsible for regulation of a disparate array of developmental processes in the central nervous system, and its antiapoptotic effect has been revealed in numerous models, pointing to its relevance in the etiology of neurodegenerative disorders. However, its function in retinal development remains unclear. Here, we aimed to point out that versatility can be achieved through interaction with other regulators, in which PACAP can act indirectly on the retinal microenvironment. Methods: Wistar rats at age postnatal day 1 were injected intravitreally with PACAP or PAC1 receptor antagonist (PACAP6-38, M65) or VPAC1 antagonist (PG97-269) alone or in combination. Retinas were removed at 3, 6, 12, or 24 hours after injection. Changes in mRNA level were assessed using quantitative PCR, whereas changes in protein levels were measured by Western blot. Results: Intravitreal injection of PACAP or PAC1 receptor antagonists or the VPAC1 antagonist showed that PACAP receptors regulate the expression of five key secreted molecules (i.e., Fgf1, Bmp4, Wnt1, Gdf3, and Ihh), wherease other crucial morphogens (i.e., Fgf2, Fgf4, Fgf8, Fgf9, Shh, and Bmp9) were not affected. Pharmacologic dissection revealed that both PAC1 and VPAC1 induced downstream signaling and could cause upregulation of Fgf1, Bmp4, and Wnt1, whereas expression of Gdf3 might be mediated through the VPAC2 receptor. Conclusions: Our data are the first to shed light on PACAP as a secretagogue regulating a sustained production of morphogens, which in turn could enable PACAP to serve as a mitogen for retinal cells, to induce ganglion cell differentiation, and to contribute to RPE development.

TRPV4 regulates calcium homeostasis, cytoskeletal remodeling, conventional outflow and intraocular pressure in the mammalian eye.

An intractable challenge in glaucoma treatment has been to identify druggable targets within the conventional aqueous humor outflow pathway, which is thought to be regulated/dysregulated by elusive mechanosensitive protein(s). Here, biochemical and functional analyses localized the putative mechanosensitive cation channel TRPV4 to the plasma membrane of primary and immortalized human TM (hTM) cells, and to human and mouse TM tissue. Selective TRPV4 agonists and substrate stretch evoked TRPV4-dependent cation/Ca(2+) influx, thickening of F-actin stress fibers and reinforcement of focal adhesion contacts. TRPV4 inhibition enhanced the outflow facility and lowered perfusate pressure in biomimetic TM scaffolds populated with primary hTM cells. Systemic delivery, intraocular injection or topical application of putative TRPV4 antagonist prodrug analogs lowered IOP in glaucomatous mouse eyes and protected retinal neurons from IOP-induced death. Together, these findings indicate that TRPV4 channels function as a critical component of mechanosensitive, Ca(2+)-signaling machinery within the TM, and that TRPV4-dependent cytoskeletal remodeling regulates TM stiffness and outflow. Thus, TRPV4 is a potential IOP sensor within the conventional outflow pathway and a novel target for treating ocular hypertension.