Critical role of Gα12 and Gα13 proteins in TGF-ß-induced myofibroblast differentiation.

Myofibroblast differentiation, characterized by accumulation of cytoskeletal and extracellular matrix proteins by fibroblasts, is a key process in wound healing and pathogenesis of tissue fibrosis. Transforming growth factor-ß (TGF-ß) is the most powerful known driver of myofibroblast differentiation. TGF-ß signals through transmembrane receptor serine/threonine kinases that phosphorylate Smad transcription factors (Smad2/3) leading to activation of transcription of target genes. Heterotrimeric G proteins mediate a distinct signaling from seven-transmembrane G protein coupled receptors, not commonly linked to Smad activation. We asked if G protein signaling plays any role in TGF-ß-induced myofibroblast differentiation, using primary cultured human lung fibroblasts. Activation of Gαs by cholera toxin blocked TGF-ß-induced myofibroblast differentiation without affecting Smad2/3 phosphorylation. Inhibition of Gαi by pertussis toxin, or siRNA-mediated combined knockdown of Gαq and Gα11 had no significant effect on TGF-ß-induced myofibroblast differentiation. A combined knockdown of Gα12 and Gα13 resulted in a drastic inhibition of TGF-ß-stimulated expression of myofibroblast marker proteins (collagen-1, fibronectin, smooth-muscle α-actin), with siGα12 being significantly more potent than siGα13. Mechanistically, a combined knockdown of Gα12 and Gα13 resulted in a substantially reduced phosphorylation of Smad2 and Smad3 in response to TGF-ß, which was accompanied by a significant decrease in the expression of TGFß receptors (TGFBR1, TGFBR2) and of Smad3 under siGα12/13 conditions. In conclusion, our study uncovers a novel role of Gα12/13 proteins in the control of TGF-ß signaling and myofibroblast differentiation.

Anoctamin-1 is induced by TGF-ß and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-ß (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and 2) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

Anoctamin-1 is induced by TGF-beta and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-beta (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel Anoctamin-1 (ANO1) in human lung fibroblasts (HLF) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle alpha-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1 and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate pro-fibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLF results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that (i) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and (ii) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein, but independent of WNK1 kinase activity.

Conditional deletion of LRRC8A in the brain reduces stroke damage independently of swelling-activated glutamate release.

The ubiquitous volume-regulated anion channels (VRACs) facilitate cell volume control and contribute to many other physiological processes. Treatment with non-specific VRAC blockers or brain-specific deletion of the essential VRAC subunit LRRC8A is highly protective in rodent models of stroke. Here, we tested the widely accepted idea that the harmful effects of VRACs are mediated by release of the excitatory neurotransmitter glutamate. We produced conditional LRRC8A knockout either exclusively in astrocytes or in the majority of brain cells. Genetically modified mice were subjected to an experimental stroke (middle cerebral artery occlusion). The astrocytic LRRC8A knockout yielded no protection. Conversely, the brain-wide LRRC8A deletion strongly reduced cerebral infarction in both heterozygous (Het) and full KO mice. Yet, despite identical protection, Het mice had full swelling-activated glutamate release, whereas KO animals showed its virtual absence. These findings suggest that LRRC8A contributes to ischemic brain injury via a mechanism other than VRAC-mediated glutamate release.

Late adolescence mortality in mice with brain-specific deletion of the volume-regulated anion channel subunit LRRC8A.

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brain-wide conditional LRRC8A knockout mice (LRRC8A bKO) using NestinCre -driven Lrrc8aflox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase. Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.

xCT/SLC7A11 antiporter function inhibits HIV-1 infection.

Human macrophages are protected by intrinsic antiviral defenses that provide moderate protection against HIV-1 infection. Macrophages that do become infected can serve as long-lived reservoirs, to disseminate HIV-1 to CD4+ T cells. Infection of macrophages with HIV-1 and HIV-2 is inhibited by constitutive mobilization of antioxidant response master transcription regulator Nrf2. The downstream mediator of this restriction was not identified. Among the tens of genes controlled directly by Nrf2 in macrophages, we found that xCT/SLC7A11, a 12-transmembrane, cystine-glutamate antiporter promotes antiretroviral activity. We show here that depletion of xCT mRNA increases HIV-1 infection. Reconstitution of xCT knock out cells with wild-type xCT but not a transport-deficient mutant restores anti-HIV-1 activity. Pharmacological inhibitors of xCT amino acid transport also increase infection. The block is independent of known restriction factors and acts against HIV-1 and HIV-2. Like the block triggered through Nrf2, xCT function impedes infection immediately before 2-LTR circle formation.

Metabolic constraints of swelling-activated glutamate release in astrocytes and their implication for ischemic tissue damage.

Volume-regulated anion channel (VRAC) is a glutamate-permeable channel that is activated by physiological and pathological cell swelling and promotes ischemic brain damage. However, because VRAC opening requires cytosolic ATP, it is not clear if and how its activity is sustained in the metabolically compromised CNS. In the present study, we used cultured astrocytes - the cell type which shows prominent swelling in stroke - to model how metabolic stress and changes in gene expression may impact VRAC function in the ischemic and post-ischemic brain. The metabolic state of primary rat astrocytes was modified with chemical inhibitors and examined using luciferin-luciferase ATP assays and a Seahorse analyzer. Swelling-activated glutamate release was quantified with the radiotracer D-[3 H]aspartate. The specific contribution of VRAC to swelling-activated glutamate efflux was validated by RNAi knockdown of the essential subunit, leucine-rich repeat-containing 8A (LRRC8A); expression levels of VRAC components were measured with qRT-PCR. Using this methodology, we found that complete metabolic inhibition with the glycolysis blocker 2-deoxy-D-glucose and the mitochondrial poison sodium cyanide reduced astrocytic ATP levels by > 90% and abolished glutamate release from swollen cells (via VRAC). When only mitochondrial respiration was inhibited by cyanide or rotenone, the intracellular ATP levels and VRAC activity were largely preserved. Bypassing glycolysis by providing the mitochondrial substrates pyruvate and/or glutamine led to partial recovery of ATP levels and VRAC activity. Unexpectedly, the metabolic block of VRAC was overridden when ATP-depleted cells were exposed to extreme cell swelling (≥ 50% reduction in medium osmolarity). Twenty-four hour anoxic adaptation caused a moderate reduction in the expression levels of the VRAC component LRRC8A, but no significant changes in VRAC activity. Overall, our findings suggest that (i) astrocytic VRAC activity and metabolism can be sustained by low levels of glucose and (ii) the inhibitory influence of diminishing ATP levels and the stimulatory effect of cellular swelling are the two major factors that govern VRAC activity in the ischemic brain.

The signaling role for chloride in the bidirectional communication between neurons and astrocytes.

It is well known that the electrical signaling in neuronal networks is modulated by chloride (Cl-) fluxes via the inhibitory GABAA and glycine receptors. Here, we discuss the putative contribution of Cl- fluxes and intracellular Cl- to other forms of information transfer in the CNS, namely the bidirectional communication between neurons and astrocytes. The manuscript (i) summarizes the generic functions of Cl- in cellular physiology, (ii) recaps molecular identities and properties of Cl- transporters and channels in neurons and astrocytes, and (iii) analyzes emerging studies implicating Cl- in the modulation of neuroglial communication. The existing literature suggests that neurons can alter astrocytic Cl- levels in a number of ways; via (a) the release of neurotransmitters and activation of glial transporters that have intrinsic Cl- conductance, (b) the metabotropic receptor-driven changes in activity of the electroneutral cation-Cl- cotransporter NKCC1, and (c) the transient, activity-dependent changes in glial cell volume which open the volume-regulated Cl-/anion channel VRAC. Reciprocally, astrocytes are thought to alter neuronal [Cl-]i through either (a) VRAC-mediated release of the inhibitory gliotransmitters, GABA and taurine, which open neuronal GABAA and glycine receptor/Cl- channels, or (b) the gliotransmitter-driven stimulation of NKCC1. The most important recent developments in this area are the identification of the molecular composition and functional heterogeneity of brain VRAC channels, and the discovery of a new cytosolic [Cl-] sensor - the Wnk family protein kinases. With new work in the field, our understanding of the role of Cl- in information processing within the CNS is expected to be significantly updated.

Cell Volume Control in Healthy Brain and Neuropathologies.

Regulation of cellular volume is a critical homeostatic process that is intimately linked to ionic and osmotic balance in the brain tissue. Because the brain is encased in the rigid skull and has a very complex cellular architecture, even minute changes in the volume of extracellular and intracellular compartments have a very strong impact on tissue excitability and function. The failure of cell volume control is a major feature of several neuropathologies, such as hyponatremia, stroke, epilepsy, hyperammonemia, and others. There is strong evidence that such dysregulation, especially uncontrolled cell swelling, plays a major role in adverse pathological outcomes. To protect themselves, brain cells utilize a variety of mechanisms to maintain their optimal volume, primarily by releasing or taking in ions and small organic molecules through diverse volume-sensitive ion channels and transporters. In principle, the mechanisms of cell volume regulation are not unique to the brain and share many commonalities with other tissues. However, because ions and some organic osmolytes (e.g., major amino acid neurotransmitters) have a strong impact on neuronal excitability, cell volume regulation in the brain is a surprisingly treacherous process, which may cause more harm than good. This topical review covers the established and emerging information in this rapidly developing area of physiology.

Downregulation of Leucine-Rich Repeat-Containing 8A Limits Proliferation and Increases Sensitivity of Glioblastoma to Temozolomide and Carmustine.

BACKGROUND: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Ubiquitously expressed volume-regulated anion channels (VRAC) are thought to play a role in cell proliferation, migration, and apoptosis. VRAC are heteromeric channel complexes assembled from proteins belonging to the leucine-rich repeat-containing 8A (LRRC8A through E), among which LRRC8A plays an indispensable role. In the present work, we used an RNAi approach to test potential significance of VRAC and LRRC8A in GBM survival and sensitivity to chemotherapeutic agents. METHODS: Primary GBM cells were derived from a human surgical tissue sample. LRRC8A expression was determined with quantitative RT-PCR and downregulated using siRNA. The effects of LRRC8A knockdown on GBM cell viability, proliferation, and sensitivity to chemotherapeutic agents were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and Coulter counter assays. Cell cycle progression was further explored using fluorescence-activated cell sorting analysis of propidium iodide-stained cells. RESULTS: Temozolomide (TMZ), carmustine, and cisplatin reduced GBM cell survival with the IC50 values of ~1,250, 320, and 30 µM, respectively. Two of three tested gene-specific siRNA constructs, siLRRC8A_3 and siLRRC8A_6, downregulated LRRC8A expression by >80% and significantly reduced GBM cell numbers. The most potent siLRRC8A_3 itself reduced viable cell numbers by ≥50%, and significantly increased toxicity of the sub-IC50 concentrations of TMZ (570 µM) and carmustine (167 µM). In contrast, the effects of siLRRC8A_3 and cisplatin (32 µM) were not additive, most likely because cisplatin uptake is VRAC-dependent. The results obtained in primary GBM cells were qualitatively recapitulated in U251 human GBM cell line. CONCLUSION: Downregulation of LRRC8A expression reduces GBM cell proliferation and increases sensitivity to the clinically used TMZ and carmustine. These findings indicate that VRAC represents a potential target for the treatment of GBM, alone or in combination with the current standard-of-care.

Molecular composition and heterogeneity of the LRRC8-containing swelling-activated osmolyte channels in primary rat astrocytes.

KEY POINTS: The volume-regulated anion channel (VRAC) is a swelling-activated chloride channel that is permeable to inorganic anions and a variety of small organic molecules. VRAC is formed via heteromerization of LRRC8 proteins, among which LRRC8A is essential, while LRRC8B/C/D/E serve as exchangeable complementary partners. We used an RNAi approach and radiotracer assays to explore which LRRC8 isoforms contribute to swelling-activated release of diverse organic osmolytes in rat astrocytes. Efflux of uncharged osmolytes (myo-inositol and taurine) was suppressed by deletion of LRRC8A or LRRC8D, but not by deletion of LRRC8C+LRRC8E. Conversely, release of charged osmolytes (d-aspartate) was strongly reduced by deletion of LRRC8A or LRRC8C+LRRC8E, but largely unaffected by downregulation of LRRC8D. Our findings point to the existence of multiple heteromeric VRACs in the same cell type: LRRC8A/D-containing heteromers appear to dominate release of uncharged osmolytes, while LRRC8A/C/E, with the additional contribution of LRRC8D, creates a conduit for movement of charged molecules. ABSTRACT: The volume-regulated anion channel (VRAC) is the ubiquitously expressed vertebrate Cl- /anion channel that is composed of proteins belonging to the LRRC8 family and activated by cell swelling. In the brain, VRAC contributes to physiological and pathological release of a variety of small organic molecules, including the amino acid neurotransmitters glutamate, aspartate and taurine. In the present work, we explored the role of all five LRRC8 family members in the release of organic osmolytes from primary rat astrocytes. Expression of LRRC8 proteins was modified using an RNAi approach, and amino acid fluxes via VRAC were quantified by radiotracer assays in cells challenged with hypoosmotic medium (30% reduction in osmolarity). Consistent with our prior work, knockdown of LRRC8A potently and equally suppressed the release of radiolabelled d-[14 C]aspartate and [3 H]taurine. Among other LRRC8 subunits, downregulation of LRRC8D strongly inhibited release of the uncharged osmolytes [3 H]taurine and myo-[3 H]inositol, without major impact on the simultaneously measured efflux of the charged d-[14 C]aspartate. In contrast, the release of d-[14 C]aspartate was preferentially sensitive to deletion of LRRC8C+LRRC8E, but unaffected by downregulation of LRRC8D. Finally, siRNA knockdown of LRRC8C+LRRC8D strongly inhibited the release of all osmolytes. Overall, our findings suggest the existence of at least two distinct heteromeric VRACs in astroglial cells. The LRRC8A/D-containing permeability pathway appears to dominate the release of uncharged osmolytes, while an alternative channel (or channels) is composed of LRRC8A/C/D/E and responsible for the loss of charged molecules.

Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo.

Recombinant adeno-associated virus vectors are an increasingly popular tool for gene delivery to the CNS because of their non-pathological nature, low immunogenicity, and ability to stably transduce dividing and non-dividing cells. One of the limitations of rAAVs is their preferential tropism for neuronal cells. Glial cells, specifically astrocytes, appear to be infected at low rates. To overcome this limitation, previous studies utilized rAAVs with astrocyte-specific promoters or assorted rAAV serotypes and pseudotypes with purported selectivity for astrocytes. Yet, the reported glial infection rates are not consistent from study to study. In the present work, we tested seven commercially available recombinant serotypes- rAAV1, 2, and 5 through 9, for their ability to transduce primary rat astrocytes [visualized via viral expression of green fluorescent protein (GFP)]. In cell cultures, rAAV6 consistently demonstrated the highest infection rates, while rAAV2 showed astrocytic transduction in some, but not all, of the tested viral batches. To verify that all rAAV constructs utilized by us were viable and effective, we confirmed high infectivity rates in retinal pigmented epithelial cells (ARPE-19), which are known to be transduced by numerous rAAV serotypes. Based on the in vitro results, we next tested the cell type tropism of rAAV6 and rAAV2 in vivo, which were both injected in the barrel cortex at approximately equal doses. Three weeks later, the brains were sectioned and immunostained for viral GFP and the neuronal marker NeuN or the astrocytic marker GFAP. We found that rAAV6 strongly and preferentially transduced astrocytes (>90% of cells in the virus-infected areas), but not neurons (∼10% infection rate). On the contrary, rAAV2 preferentially infected neurons (∼65%), but not astrocytes (∼20%). Overall, our results suggest that rAAV6 can be used as a tool for manipulating gene expression (either delivery or knockdown) in rat astrocytes in vivo.

AMPA-Kainate Receptor Inhibition Promotes Neurologic Recovery in Premature Rabbits with Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) in preterm infants leads to cerebral inflammation, reduced myelination of the white matter, and neurological deficits. No therapeutic strategy exists against the IVH-induced white matter injury. AMPA-kainate receptor induced excitotoxicity contributes to oligodendrocyte precursor cell (OPC) damage and hypomyelination in both neonatal and adult models of brain injury. Here, we hypothesized that IVH damages white matter via AMPA receptor activation, and that AMPA-kainate receptor inhibition suppresses inflammation and restores OPC maturation, myelination, and neurologic recovery in preterm newborns with IVH. We tested these hypotheses in a rabbit model of glycerol-induced IVH and evaluated the expression of AMPA receptors in autopsy samples from human preterm infants. GluR1-GluR4 expressions were comparable between preterm humans and rabbits with and without IVH. However, GluR1 and GluR2 levels were significantly lower in the embryonic white matter and germinal matrix relative to the neocortex in both infants with and without IVH. Pharmacological blockade of AMPA-kainate receptors with systemic NBQX, or selective AMPA receptor inhibition by intramuscular perampanel restored myelination and neurologic recovery in rabbits with IVH. NBQX administration also reduced the population of apoptotic OPCs, levels of several cytokines (TNFα, IL-ß, IL-6, LIF), and the density of Iba1(+) microglia in pups with IVH. Additionally, NBQX treatment inhibited STAT-3 phosphorylation, but not astrogliosis or transcription factors regulating gliosis. Our data suggest that AMPA-kainate receptor inhibition alleviates OPC loss and IVH-induced inflammation and restores myelination and neurologic recovery in preterm rabbits with IVH. Therapeutic use of FDA-approved perampanel treatment might enhance neurologic outcome in premature infants with IVH. SIGNIFICANCE STATEMENT: Intraventricular hemorrhage (IVH) is a major complication of prematurity and a large number of survivors with IVH develop cerebral palsy and cognitive deficits. The development of IVH leads to inflammation of the periventricular white matter, apoptosis and arrested maturation of oligodendrocyte precursor cells, and hypomyelination. Here, we show that AMPA-kainate receptor inhibition by NBQX suppresses inflammation, attenuates apoptosis of oligodendrocyte precursor cells, and promotes myelination as well as clinical recovery in preterm rabbits with IVH. Importantly, AMPA-specific inhibition by the FDA-approved perampanel, which unlike NBQX has a low side-effect profile, also enhances myelination and neurological recovery in rabbits with IVH. Hence, the present study highlights the role of AMPA-kainate receptor in IVH-induced white matter injury and identifies a novel strategy of neuroprotection, which might improve the neurological outcome for premature infants with IVH.

Volume-regulated anion channel--a frenemy within the brain.

The volume-regulated anion channel (VRAC) is a ubiquitously expressed yet highly enigmatic member of the superfamily of chloride/anion channels. It is activated by cellular swelling and mediates regulatory cell volume decrease in a majority of vertebrate cells, including those in the central nervous system (CNS). In the brain, besides its crucial role in cellular volume regulation, VRAC is thought to play a part in cell proliferation, apoptosis, migration, and release of physiologically active molecules. Although these roles are not exclusive to the CNS, the relative significance of VRAC in the brain is amplified by several unique aspects of its physiology. One important example is the contribution of VRAC to the release of the excitatory amino acid neurotransmitters glutamate and aspartate. This latter process is thought to have impact on both normal brain functioning (such as astrocyte-neuron signaling) and neuropathology (via promoting the excitotoxic death of neuronal cells in stroke and traumatic brain injury). In spite of much work in the field, the molecular nature of VRAC remained unknown until less than 2 years ago. Two pioneer publications identified VRAC as the heterohexamer formed by the leucine-rich repeat-containing 8 (LRRC8) proteins. These findings galvanized the field and are likely to result in dramatic revisions to our understanding of the place and role of VRAC in the brain, as well as other organs and tissues. The present review briefly recapitulates critical findings in the CNS and focuses on anticipated impact on the LRRC8 discovery on further progress in neuroscience research.

Intracellular levels of glutamate in swollen astrocytes are preserved via neurotransmitter reuptake and de novo synthesis: implications for hyponatremia.

Hyponatremia and several other CNS pathologies are associated with substantial astrocytic swelling. To counteract cell swelling, astrocytes lose intracellular osmolytes, including l-glutamate and taurine, through volume-regulated anion channel. In vitro, when swollen by exposure to hypo-osmotic medium, astrocytes lose endogenous taurine faster, paradoxically, than l-glutamate or l-aspartate. Here, we explored the mechanisms responsible for differences between the rates of osmolyte release in primary rat astrocyte cultures. In radiotracer assays, hypo-osmotic efflux of preloaded [(14) C]taurine was indistinguishable from d-[(3) H]aspartate and only 30-40% faster than l-[(3) H]glutamate. However, when we used HPLC to measure the endogenous intracellular amino acid content, hypo-osmotic loss of taurine was approximately fivefold greater than l-glutamate, and no loss of l-aspartate was detected. The dramatic difference between loss of endogenous taurine and glutamate was eliminated after inhibition of both glutamate reuptake [with 300 µM dl-threo-ß-benzyloxyaspartic acid (TBOA)] and glutamate synthesis by aminotransferases [with 1 mM aminooxyacetic acid (AOA)]. Treatment with TBOA+AOA made reductions in the intracellular taurine and l-glutamate levels approximately equal. Taken together, these data suggest that swollen astrocytes actively conserve intracellular glutamate via reuptake and de novo synthesis. Our findings likely also explain why in animal models of acute hyponatremia, extracellular levels of taurine are dramatically elevated with minimal impact on extracellular l-glutamate. We identified mechanisms that allow astrocytes to conserve intracellular l-glutamate (Glu) upon exposure to hypo-osmotic environment. Cell swelling activates volume-regulated anion channel (VRAC) and triggers loss of Glu, taurine (Tau), and other cytosolic amino acids. Glu is conserved via reuptake by Na(+) -dependent transporters and de novo synthesis in the reactions of mitochondrial transamination (TA). These findings explain why, in acute hyponatremia, extracellular levels of Tau can be dramatically elevated with minimal changes in extracellular Glu.

Critical role of the α1-Na(+), K(+)-ATPase subunit in insensitivity of rodent cells to cytotoxic action of ouabain.

In rodents, ubiquitous α1-Na(+), K(+)-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~10(3)-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS-sensitive α1S- and CTS-resistant α1R-Na(+), K(+)-ATPase isoforms, we compared the effects of ouabain on intracellular Na(+) and K(+) content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. 6-h exposure of HASMC and HUVEC to 3 µM ouabain dramatically increased the intracellular [Na(+)]/[K(+)] ratio to the same extend as in RASMC and RAEC treated with 3000 µM ouabain. In 24, 3 µM ouabain triggered the death of all types of human cells used in this study. Unlike human cells, we did not detect any effect of 3000-5000 µM ouabain on the survival of rat cells, or smooth muscle cells from mouse aorta (MASMC). Unlike in the wild-type α1(R/R) mouse, ouabain triggered death of MASMC from α1(S/S) mouse expressing human α1-Na(+), K(+)-ATPase. Furthermore, transfection of HUVEC with rat α1R-Na(+), K(+)-ATPase protected them from the ouabain-induced death. In HUVEC, ouabain led to phosphorylation of p38 MAPK, whereas in RAEC it stimulated phosphorylation of ERK1/2. Overall, our results, demonstrate that the drastic differences in cytotoxic action of ouabain on human and rodent cells are caused by unique features of α1S/α1R-Na(+), K(+)-ATPase, rather than by any downstream CTS-sensitive/resistant components of the cell death machinery.

The neuroprotective properties of the superoxide dismutase mimetic tempol correlate with its ability to reduce pathological glutamate release in a rodent model of stroke.

The contribution of oxidative stress to ischemic brain damage is well established. Nevertheless, for unknown reasons, several clinically tested antioxidant therapies have failed to show benefits in human stroke. Based on our previous in vitro work, we hypothesized that the neuroprotective potency of antioxidants is related to their ability to limit the release of the excitotoxic amino acids glutamate and aspartate. We explored the effects of two antioxidants, tempol and edaravone, on amino acid release in the brain cortex, in a rat model of transient occlusion of the middle cerebral artery (MCAo). Amino acid levels were quantified using a microdialysis approach, with the probe positioned in the ischemic penumbra as verified by a laser Doppler technique. Two-hour MCAo triggered a dramatic increase in the levels of glutamate, aspartate, taurine, and alanine. Microdialysate delivery of 10mM tempol reduced the amino acid release by 60-80%, whereas matching levels of edaravone had no effect. In line with these data, an intracerebroventricular injection of tempol but not edaravone (500 nmol each, 15 min before MCAo) reduced infarction volumes by ~50% and improved neurobehavioral outcomes. In vitro assays showed that tempol was superior at removing superoxide anion, whereas edaravone was more potent at scavenging hydrogen peroxide, hydroxyl radical, and peroxynitrite. Overall, our data suggest that the neuroprotective properties of tempol are probably related to its ability to reduce tissue levels of the superoxide anion and pathological glutamate release and, in such a way, limit progression of brain infarction within ischemic penumbra. These new findings may be instrumental in developing new antioxidant therapies for treatment of stroke.