David Križaj

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 [Ca2+]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 [Ca2+]i elevations but only modestly attenuated the amplitude of Ca2+ 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 Ca2+ 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.

From Mechanosensitivity to Inflammatory Responses: New Players in the Pathology of Glaucoma

Abstract Purpose of the study: Many blinding diseases of the inner retina are associated with degeneration and loss of retinal ganglion cells (RGCs). Recent evidence implicates several new signaling mechanisms as causal agents associated with RGC injury and remodeling of the optic nerve head. Ion channels such as Transient receptor potential vanilloid isoform 4 (TRPV4), pannexin-1 (Panx1) and P2X7 receptor are localized to RGCs and act as potential sensors and effectors of mechanical strain, ischemia and inflammatory responses. Under normal conditions, TRPV4 may function as an osmosensor and a polymodal molecular integrator of diverse mechanical and chemical stimuli, whereas P2X7R and Panx1 respond to stretch- and/or swelling-induced adenosine triphosphate release from neurons and glia. Ca2+ influx, induced by stimulation of mechanosensitive ion channels in glaucoma, is proposed to influence dendritic and axonal remodeling that may lead to RGC death while (at least initially) sparing other classes of retinal neuron. The secondary phase of the retinal glaucoma response is associated with microglial activation and an inflammatory response involving Toll-like receptors (TLRs), cluster of differentiation 3 (CD3) immune recognition molecules associated with the T-cell antigen receptor, complement molecules and cell type-specific release of neuroactive cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The retinal response to mechanical stress thus involves a diversity of signaling pathways that sense and transduce mechanical strain and orchestrate both protective and destructive secondary responses. Conclusions: Mechanistic understanding of the interaction between pressure-dependent and independent pathways is only beginning to emerge. This review focuses on the molecular basis of mechanical strain transduction as a primary mechanism that can damage RGCs. The damage occurs through Ca2+-dependent cellular remodeling and is associated with parallel activation of secondary ischemic and inflammatory signaling pathways. Molecules that mediate these mechanosensory and immune responses represent plausible targets for protecting ganglion cells in glaucoma, optic neuritis and retinal ischemia.

Anatomical and Neurochemical Characterization of Dopaminergic Interplexiform Processes in Mouse and Rat Retinas

Dopaminergic (DA) neurons of mouse and rat retinas are of the interplexiform subtype (DA‐IPC), i.e., they send processes distally toward the outer retina, exhibiting numerous varicosities along their course. The primary question we addressed was whether distally located DA‐IPC varicosities, identified by tyrosine hydroxylase (TH) immunoreactivity, had the characteristic presynaptic proteins associated with calcium‐dependent vesicular release of neurotransmitter. We found that TH immunoreactive varicosities in the outer retina possessed vesicular monoamine transporter 2 and vesicular GABA transporter, but they lacked immunostaining for any of nine subtypes of voltage‐dependent calcium channel. Immunoreactivity for other channels that may permit calcium influx such as certain ionotropic glutamate receptors and canonical transient receptor potential channels (TRPCs) was similarly absent, although DA‐IPC varicosities did show ryanodine receptor immunoreactivity, indicating the presence of intracellular calcium stores. The synaptic vesicle proteins sv2a and sv2b and certain other proteins associated with the presynaptic membrane were absent from DA‐IPC varicosities, but the vesicular SNARE protein, vamp2, was present in a fraction of those varicosities. We identified a presumed second class of IPC that is GABAergic but not dopaminergic. Outer retinal varicosities of this putative GABAergic IPC did colocalize synaptic vesicle protein 2a, suggesting they possessed a conventional vesicular release mechanism. J. Comp. Neurol. 510:158–174, 2008.

Swelling and Eicosanoid Metabolites Differentially Gate TRPV4 Channels in Retinal Neurons and Glia

Activity-dependent shifts in ionic concentrations and water that accompany neuronal and glial activity can generate osmotic forces with biological consequences for brain physiology. Active regulation of osmotic gradients and cellular volume requires volume-sensitive ion channels. In the vertebrate retina, critical support to volume regulation is provided by Müller astroglia, but the identity of their osmosensor is unknown. Here, we identify TRPV4 channels as transducers of mouse Müller cell volume increases into physiological responses. Hypotonic stimuli induced sustained [Ca2+]i elevations that were inhibited by TRPV4 antagonists and absent in TRPV4−/− Müller cells. Glial TRPV4 signals were phospholipase A2- and cytochrome P450-dependent, characterized by slow-onset and Ca2+ waves, and, in excess, were sufficient to induce reactive gliosis. In contrast, neurons responded to TRPV4 agonists and swelling with fast, inactivating Ca2+ signals that were independent of phospholipase A2. Our results support a model whereby swelling and proinflammatory signals associated with arachidonic acid metabolites differentially gate TRPV4 in retinal neurons and glia, with potentially significant consequences for normal and pathological retinal function.

Role of ELOVL4 and very long-chain polyunsaturated fatty acids in mouse models of Stargardt type 3 retinal degeneration.

Stargardt type 3 (STGD3) disease is a juvenile macular dystrophy caused by mutations in the ELOVL4 (Elongation of very long chain fatty acids 4) gene. Its protein product, ELOVL4, is an elongase required for the biosynthesis of very long-chain polyunsaturated fatty acids (VLC-PUFAs). It is unclear whether photoreceptor degeneration in STGD3 is caused by loss of VLC-PUFAs or by mutated ELOVL4 protein trafficking/aggregation. We therefore generated conditional knockout (cKO) mice with Elovl4 ablated in rods or cones and compared their phenotypes to transgenic (TG) animals that express the human STGD3-causing ELOVL4STGD3 allele. Gas chromatography–mass spectrometry was used to assess C30–C34 VLC-PUFA and N-retinylidene-N-retinylethanolamine content; electroretinography was used to measure phototransduction and outer retinal function; electron microscopy was used for retinal ultrastructure; and the optomotor tracking response was used to test scotopic and photopic visual performance. Elovl4 transcription and biosynthesis of C30–C34 VLC-PUFAs in rod cKO and TG retinas were reduced up to 98%, whereas the content of docosahexaenoic acid was diminished in TG, but not rod cKO, retinas. Despite the near-total loss of the retinal VLC-PUFA content, rod and cone cKO animals exhibited no electrophysiological or behavioral deficits, whereas the typical rod–cone dystrophic pattern was observed in TG animals. Our data suggest that photoreceptor-specific VLC-PUFA depletion is not sufficient to induce the STGD3 phenotype, because depletion alone had little effect on photoreceptor survival, phototransduction, synaptic transmission, and visual behavior.

Store-operated channels regulate intracellular calcium in mammalian rods

•  Light closes cyclic nucleotide‐gated and voltage operated calcium channels in vertebrate rod photoreceptors, resulting in a decrease in the intracellular calcium concentration ([Ca2+]i). A protracted decrease in [Ca2+]i experienced under saturating illuminations is toxic for these cells. •  Eukaryotic cells express voltage‐independent plasma membrane ion channels that protect against pathological [Ca2+]i decreases and can be activated by depletion of intracellular calcium stores. An invertebrate homologue of canonical transient receptor potential (TRPC) channels that have been implicated in store‐operated calcium entry (SOCE) in vertebrates is expressed in photoreceptors. •  We show that mouse rods express a potent SOCE mechanism that gates cation entry which subsequently modulates activation of L‐type calcium channels. Furthermore, we show what the majority of the retinal Trpc1 signal is localized to rod photoreceptors. •  We found, using knockout animal models, that neither TRPC1 nor TRPC3 channels contribute to SOCE in mouse rod perikarya, or regulate light‐evoked responses in the outer segment and the synaptic terminal, suggesting that the channels are receptor operated. •  We conclude that mammalian rods express two new calcium signalling mechanisms associated with SOCE and TRPC1 signalling which modulate calcium homeostasis and may protect against prolonged [Ca2+]i decreases in saturating light.

Intracellular organelles and calcium homeostasis in rods and cones

The role of intracellular organelles in Ca2+ homeostasis was studied in salamander rod and cone photoreceptors under conditions that simulate photoreceptor activation by darkness and light. Sustained depolarization evoked a Ca2+ gradient between the cell body and ellipsoid regions of the inner segment (IS). The standing pattern of calcium fluxes was created by interactions between the plasma membrane, endoplasmic reticulum (ER), and mitochondria. Pharmacological experiments suggested that mitochondria modulate both baseline [Ca2+]i in hyperpolarized cells as well as kinetics of Ca2+ entry via L type Ca2+ channels in cell bodies and ellipsoids of depolarized rods and cones. Inhibition of mitochondrial Ca2+ sequestration by antimycin/oligomycin caused a three-fold reduction in the amount of Ca2+ accumulated into intracellular organelles in both cell bodies and ellipsoids. A further 50% decrease in intracellular Ca2+ content within cell bodies, but not ellipsoids, was observed after suppression of SERCA-mediated Ca2+ uptake into the ER. Inhibition of Ca2+ sequestration into the endoplasmic reticulum by thapsigargin or cyclopiazonic acid decreased the magnitude and kinetics of depolarization-evoked Ca2+ signals in cell bodies of rods and cones and decreased the amount of Ca2+ accumulated into internal stores. These results suggest that steady-state [Ca2+]i in photoreceptors is regulated in a region-specific manner, with the ER contribution predominant in the cell body and mitochondrial buffering [Ca2+] the ellipsoid. Local [Ca2+]i levels are set by interactions between the plasma membrane Ca2+ channels and transporters, ER and mitochondria. Mitochondria are likely to play an essential role in temporal and spatial buffering of photoreceptor Ca2+.

TRPV1 and Endocannabinoids: Emerging Molecular Signals that Modulate Mammalian Vision

Transient Receptor Potential Vanilloid 1 (TRPV1) subunits form a polymodal cation channel responsive to capsaicin, heat, acidity and endogenous metabolites of polyunsaturated fatty acids. While originally reported to serve as a pain and heat detector in the peripheral nervous system, TRPV1 has been implicated in the modulation of blood flow and osmoregulation but also neurotransmission, postsynaptic neuronal excitability and synaptic plasticity within the central nervous system. In addition to its central role in nociception, evidence is accumulating that TRPV1 contributes to stimulus transduction and/or processing in other sensory modalities, including thermosensation, mechanotransduction and vision. For example, TRPV1, in conjunction with intrinsic cannabinoid signaling, might contribute to retinal ganglion cell (RGC) axonal transport and excitability, cytokine release from microglial cells and regulation of retinal vasculature. While excessive TRPV1 activity was proposed to induce RGC excitotoxicity, physiological TRPV1 activity might serve a neuroprotective function within the complex context of retinal endocannabinoid signaling. In this review we evaluate the current evidence for localization and function of TRPV1 channels within the mammalian retina and explore the potential interaction of this intriguing nociceptor with endogenous agonists and modulators.

Calcium Stores in Vertebrate Photoreceptors

This review lays out the emerging evidence for the fundamental role of Ca(2+) stores and store-operated channels in the Ca(2+) homeostasis of rods and cones. Calcium-induced calcium release (CICR) is a major contributor to steady-state and light-evoked photoreceptor Ca(2+) homeostasis in the darkness whereas store-operated Ca(2+) channels play a more significant role under sustained illumination conditions. The homeostatic response includes dynamic interactions between the plasma membrane, endoplasmic reticulum (ER), mitochondria and/or outer segment disk organelles which dynamically sequester, accumulate and release Ca(2+). Coordinated activation of SERCA transporters, ryanodine receptors (RyR), inositol triphosphate receptors (IP3Rs) and TRPC channels amplifies cytosolic voltage-operated signals but also provides a memory trace of previous exposures to light. Store-operated channels, activated by the STIM1 sensor, prevent pathological decrease in [Ca(2+)]i mediated by excessive activation of PMCA transporters in saturating light. CICR and SOCE may also modulate the transmission of afferent and efferent signals in the outer retina. Thus, Ca(2+) stores provide additional complexity, adaptability, tuneability and speed to photoreceptor signaling.

Store-Operated Calcium Entry in Müller Glia Is Controlled by Synergistic Activation of TRPC and Orai Channels

The endoplasmic reticulum (ER) is at the epicenter of astrocyte Ca2+ signaling. We sought to identify the molecular mechanism underlying store-operated calcium entry that replenishes ER stores in mouse Müller cells. Store depletion, induced through blockade of sequestration transporters in Ca2+-free saline, induced synergistic activation of canonical transient receptor potential 1 (TRPC1) and Orai channels. Store-operated TRPC1 channels were identified by their electrophysiological properties, pharmacological blockers, and ablation of the Trpc1 gene. Ca2+ release-activated currents (ICRAC) were identified by ion permeability, voltage dependence, and sensitivity to selective Orai antagonists Synta66 and GSK7975A. Depletion-evoked calcium influx was initiated at the Müller end-foot and apical process, triggering centrifugal propagation of Ca2+ waves into the cell body. EM analysis of the end-foot compartment showed high-density ER cisternae that shadow retinal ganglion cell (RGC) somata and axons, protoplasmic astrocytes, vascular endothelial cells, and ER–mitochondrial contacts at the vitreal surface of the end-foot. The mouse retina expresses transcripts encoding both Stim and all known Orai genes; Müller glia predominantly express stromal interacting molecule 1 (STIM1), whereas STIM2 is mainly confined to the outer plexiform and RGC layers. Elimination of TRPC1 facilitated Müller gliosis induced by the elevation of intraocular pressure, suggesting that TRPC channels might play a neuroprotective role during mechanical stress. By characterizing the properties of store-operated signaling pathways in Müller cells, these studies expand the current knowledge about the functional roles these cells play in retinal physiology and pathology while also providing further evidence for the complexity of calcium signaling mechanisms in CNS astroglia. SIGNIFICANCE STATEMENT Store-operated Ca2+ signaling represents a major signaling pathway and source of cytosolic Ca2+ in astrocytes. Here, we show that the store-operated response in Müller cells, radial glia that perform key structural, signaling, osmoregulatory, and mechanosensory functions within the retina, is mediated through synergistic activation of transient receptor potential and Orai channels. The end-foot disproportionately expresses the depletion sensor stromal interacting molecule 1, which contains an extraordinarily high density of endoplasmic reticulum cisternae that shadow neuronal, astrocytic, vascular, and axonal structures; interface with mitochondria; but also originate store-operated Ca2+ entry-induced transcellular Ca2+ waves that propagate glial excitation into the proximal retina. These results identify a molecular mechanism that underlies complex interactions between the plasma membrane and calcium stores, and contributes to astroglial function, regulation, and response to mechanical stress.