Astrocyte Volume Regulation Research Articles

Astrocyte regulation of extracellular space parameters across the sleep-wake cycle.

Multiple subfields of neuroscience research are beginning to incorporate astrocytes into current frameworks of understanding overall brain physiology, neuronal circuitry, and disease etiology that underlie sleep and sleep-related disorders. Astrocytes have emerged as a dynamic regulator of neuronal activity through control of extracellular space (ECS) volume and composition, both of which can vary dramatically during different levels of sleep and arousal. Astrocytes are also an attractive target of sleep research due to their prominent role in the glymphatic system, a method by which toxic metabolites generated during wakefulness are cleared away. In this review we assess the literature surrounding glial influences on fluctuations in ECS volume and composition across the sleep-wake cycle. We also examine mechanisms of astrocyte volume regulation in glymphatic solute clearance and their role in sleep and wake states. Overall, findings highlight the importance of astrocytes in sleep and sleep research.

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema.

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema.

Contribution of inwardly rectifying K+ channel 4.1 of supraoptic astrocytes to the regulation of vasopressin neuronal activity by hypotonicity.

Astrocytic morphological plasticity and its modulation of adjacent neuronal activity are largely determined by astrocytic volume regulation, in which glial fibrillary acidic protein (GFAP), aquaporin 4 (AQP4), and potassium channels including inwardly rectifying K+ channel 4.1 (Kir4.1) are essential. However, associations of astrocyte-dominant Kir4.1 with other molecules in astrocytic volume regulation and the subsequent influence on neuronal activity remain unclear. Here, we report our study on these issues using primary cultures of rat pups' hypothalamic astrocytes and male adult rat brain slices. In astrocyte culture, hyposmotic challenge (HOC) significantly decreased GFAP monomer expression and astrocytic volume at 1.5 min and increased Kir4.1 expression and inwardly rectifying currents (IRCs) at 10 min. BaCl2 (100 μmol/l) suppressed the HOC-increased IRCs, which was simulated by VU0134992 (2μmol/l), a Kir4.1 blocker. Preincubation of the astrocyte culture with TGN-020 (10μmol/l, a specific AQP4 blocker) made the HOC-increased Kir4.1 currents insignificant. In hypothalamic brain slices, HOC initially decreased and then increased the firing rate of vasopressin (VP) neurons in the supraoptic nucleus. In the presence of BaCl2 or VU0134992, HOC-elicited rebound increase in VP neuronal activity was blocked. GFAP was molecularly associated with Kir4.1, which was increased by HOC at 20 min; this increase was blocked by BaCl2 . These results suggest that HOC-evoked astrocytic retraction or decrease in the volume and length of its processes is associated with increased Kir4.1 activity. Kir4.1 involvement in HOC-elicited astrocytic retraction is associated with AQP4 activity and GFAP plasticity, which together determines the rebound excitation of VP neurons.

Channels and Transporters in Astrocyte Volume Regulation in Health and Disease.

Astrocytes are the second most abundant cell type in the central nervous system and serve various functions, many of which maintain homeostasis of the intracellular milieu in the face of constant change. In order to accomplish these important functions, astrocytes must regulate their cell volume. In astrocytes, cell volume regulation involves multiple channels and transporters, including AQP4, TRPV4, TRPM4, VRAC, Na+/K+ ATPase, NKCC1 and Kir4.1. AQP4 is a bidirectional water channel directly involved in astrocyte cell volume regulation. AQP4 also forms heteromultimeric complexes with other channels and transporters involved in cell volume regulation. TRPV4, a mechanosensitive channel in involved in osmotic regulation in various cell types, forms a complex with AQP4 to decrease cell volume in response to cell swelling. TRPM4 also forms a complex with AQP4 and SUR1 in response to injury resulting in cell swelling. Another complex forms between Na+/K+ ATPase, AQP4, and mGluR5 to regulate the perisynaptic space. NKCC1 is a co-transporter involved in cell volume increases either independently through cotransport of water or a functional interaction with AQPs. VRAC is implicated in regulatory volume decreases and may also functionally interact with AQP4. Although Kir4.1 colocalizes with AQP4, its role in cell volume regulation is debated. In diseases where fluid/electrolyte homeostasis is disturbed such as stroke, ischemic injury, inflammation, traumatic brain injury and hydrocephalus, cell volume regulation is challenged, sometimes past the point of recovery. Thus, a greater understanding of signaling pathways which regulate transport proteins as well as the functional and physical interactions that exist between transporters will provide a basis for the development of pharmaceutical targets to treat these prevalent and often devastating diseases.

Cell Volume Regulation Mechanisms in Differentiated Astrocytes.

The ability of astrocytes to control extracellular volume homeostasis is critical for brain function and pathology. Uncovering the mechanisms of cell volume regulation by astrocytes will be important for identifying novel therapeutic targets for neurological conditions, such as those characterized by imbalances to hydro saline challenges (as in edema) or by altered cell volume regulation (as in glioma). One major challenge in studying the astroglial membrane channels involved in volume homeostasis in cell culture model systems is that the expression patterns of these membrane channels do not resemble those observed in vivo. In our previous study, we demonstrated that rat primary astrocytes grown on nanostructured interfaces based on hydrotalcite-like compounds (HTlc) in vitro are differentiated and display molecular and functional properties of in vivo astrocytes, such as the functional expression of inwardly rectifying K+ channel (Kir 4.1) and Aquaporin-4 (AQP4) at the astrocytic microdomain. Here, we take advantage of the properties of differentiated primary astrocytes in vitro to provide an insight into the mechanism underpinning astrocytic cell volume regulation and its correlation with the expression and function of AQP4, Transient Receptor Potential Vanilloid 4 (TRPV4), and Volume Regulated Anion Channel (VRAC). The calcein quenching method was used to study water transport and cell volume regulation. Calcium imaging and electrophysiology (patch-clamp) were used for functional analyses of calcium dynamics and chloride currents. Western blot and immunofluorescence were used to analyse the expression and localization of the channel proteins of interest. We found that the increase in water permeability, previously observed in differentiated astrocytes, occurs simultaneously with more efficient regulatory volume increase and regulatory volume decrease. Accordingly, the magnitude of the hypotonic induced intracellular calcium response, typically mediated by TRPV4, as well as the hypotonic induced VRAC current, was almost twice as high in differentiated astrocytes. Interestingly, while we confirmed increased AQP4 expression in the membrane of differentiated astrocytes, the expression of the channels TRPV4 and Leucine-Rich Repeats-Containing 8-A (LRRC8-A) were comparable between differentiated and non-differentiated astrocytes. The reported results indicate that AQP4 up-regulation observed in differentiated astrocytes might promote higher sensitivity of the cell to osmotic changes, resulting in increased magnitude of calcium signaling and faster kinetics of the RVD and RVI processes. The implications for cell physiology and the mechanisms underlying astrocytic interaction with nanostructured interfaces are discussed.

Modulation of Cerebrospinal Fluid Production by Transient Receptor Potential Vanilloid 4: Implications for Disorders of Brain Fluid Volume

Cerebrospinal fluid (CSF) secretion into the brain ventricular system is an active process via the choroid plexus (CP) - a specialized convoluted epithelial sheet surrounding a fenestrated capillary bed which is fed from the cerebral arterioles. The final CSF composition differs from a simple plasma filtrate implicating the CP as an important regulatory site controlling CSF composition and volume. Flux across this blood-CSF barrier is tightly controlled by ion and water transport proteins. Transient Receptor Potential Vanilloid 4 (TRPV4) is a polymodal, non-selective cation channel which functions as a regulatory hub protein in epithelia, including the CP. Our previous studies have shown that TRPV4-selective antagonists were effective at reducing ventriculomegaly in a genetic rat model of severe postnatal hydrocephalus. In CP, we found that activation of TRPV4 caused the influx of both Ca2+ and Na+ ions, and entry of both of these ions was selectively blocked by a TRPV4 antagonist. Systemic treatment with TRPV4 antagonists reduced TRPV4 mRNA expression in the CP in hydrocephalic rats, but not in normal rats, indicating a role for TRPV4 as part of a regulatory pathway in CSF production. To study the mechanism of CSF secretion by the CP, we utilized two continuous, high resistance CP cell lines: the porcine choroid plexus – Reims (PCP-R) and the human choroid plexus papilloma (HIBCPP) cells. The current experiments utilize Ussing chamber electrophysiological techniques, western blotting, qRT-PCR, and fluorescent immunolabeling. Immunolabeling and mRNA expression demonstrate a robust abundance for TRPV4 and other well-characterized CP membrane proteins in both cell lines. In both lines, TRPV4 activation stimulates a large change in short circuit current (ISC), a measure of net electrogenic ion flux. This change in ISCis coupled with a substantial change in transepithelial membrane conductance, a measure of cellular permeability. The transepithelial membrane transport changes due to TRPV4 activation in human and porcine cells are similar in overall effect, but differ in baseline, duration, and magnitude, indicating species-specific CP transport mechanisms. In both cases, these responses can be inhibited by either pre- or post-incubation with TRPV4 antagonists, substantiating the specificity to TRPV4 activation. The human cells, but not the porcine cells, demonstrate a substantial baseline transepithelial transport which is dependent on K+, Na+, and Cl- gradients. Furthermore, in both the porcine and human cells, the TRPV4 response is dependent upon K+ secretion, Cl- absorption, and Na+ secretion. These data confirm a role for TRPV4 in regulating ionic gradients, modulating both electrogenic and electroneutral transporters, and controlling CSF production. The promising results of these studies, coupled with published data from other investigators suggesting a role for TRPV4 in blood-brain-barrier permeability and astrocyte volume regulation, shows the importance of TRPV4 as a regulatory hub protein in brain fluid volume regulation.

The Contribution of TRPV4 Channels to Astrocyte Volume Regulation and Brain Edema Formation

Transient receptor potential vanilloid type 4 (TRPV4) channels are involved in astrocyte volume regulation; however, only limited data exist about its mechanism in astrocytes in situ. We performed middle cerebral artery occlusion in adult mice, where we found twice larger edema 1 day after the insult in trpv4−/− mice compared to the controls, which was quantified using magnetic resonance imaging. This result suggests disrupted volume regulation in the brain cells in trpv4−/− mice leading to increased edema formation. The aim of our study was to elucidate whether TRPV4 channel-based volume regulation occurs in astrocytes in situ and whether the disrupted volume regulation in trpv4−/− mice might lead to higher edema formation after brain ischemia. For our experiments, we used trpv4−/− mice crossed with transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the glial fibrillary acidic protein promoter, which leads to astrocyte visualization by EGFP expression. For quantification of astrocyte volume changes, we used two-dimensional (2D) and three-dimensional (3D) morphometrical approaches and a quantification algorithm based on fluorescence intensity changes during volume alterations induced by hypotonicity or by oxygen-glucose deprivation. In contrast to in vitro experiments, we found little evidence of the contribution of TRPV4 channels to volume regulation in astrocytes in situ in adult mice. Moreover, we only found a rare expression of TRPV4 channels in adult mouse astrocytes. Our data suggest that TRPV4 channels are not involved in astrocyte volume regulation in situ; however, they play a protective role during the ischemia-induced brain edema formation.

LRRC8A is essential for swelling-activated chloride current and for regulatory volume decrease in astrocytes.

Consolidated evidence indicates that astroglial cells are critical in the homeostatic regulation of cellular volume by means of ion channels and aquaporin-4. Volume-regulated anion channel (VRAC) is the chloride channel that is activated upon cell swelling and critically contributes to cell volume regulation in astrocytes. The molecular identity of VRAC has been recently defined, revealing that it belongs to the leucine-rich repeat-containing 8 (LRRC8) protein family. However, there is a lack of evidence demonstrating that LRRC8A underpins VRAC currents in astrocyte. Nonetheless, direct evidence of the role of LRRC8A in astrocytic regulatory volume decrease remains to be proved. Here, we aim to bridge this gap in knowledge by combining RNA interference specific for LRRC8A with patch-clamp analyses and a water-permeability assay. We demonstrated that LRRC8A molecular expression is essential for swelling-activated chloride current via VRAC in primary-cultured cortical astrocytes. The knockdown of LRRC8A with a specific short interference RNA abolished the recovery of the cell volume after swelling induced by hypotonic challenge. In addition, immunoblotting, immunofluorescence, confocal imaging, and immunogold electron microscopy demonstrated that LRRC8A is expressed in the plasma membrane of primary cortical astrocytes and in situ in astrocytes at the perivascular interface with endothelial cells. Collectively, our results suggest that LRRC8A is an essential subunit of VRAC and a key factor for astroglial volume homeostasis.-Formaggio, F., Saracino, E., Mola, M. G., Rao, S. B., Amiry-Moghaddam, M., Muccini, M., Zamboni, R., Nicchia, G. P., Caprini, M., Benfenati, V. LRRC8A is essential for swelling-activated chloride current and for regulatory volume decrease in astrocytes.

Seizures and disturbed brain potassium dynamics in the leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts.

ObjectiveLoss of function of the astrocyte‐specific protein MLC1 leads to the childhood‐onset leukodystrophy “megalencephalic leukoencephalopathy with subcortical cysts” (MLC). Studies on isolated cells show a role for MLC1 in astrocyte volume regulation and suggest that disturbed brain ion and water homeostasis is central to the disease. Excitability of neuronal networks is particularly sensitive to ion and water homeostasis. In line with this, reports of seizures and epilepsy in MLC patients exist. However, systematic assessment and mechanistic understanding of seizures in MLC are lacking.MethodsWe analyzed an MLC patient inventory to study occurrence of seizures in MLC. We used two distinct genetic mouse models of MLC to further study epileptiform activity and seizure threshold through wireless extracellular field potential recordings. Whole‐cell patch‐clamp recordings and K+‐sensitive electrode recordings in mouse brain slices were used to explore the underlying mechanisms of epilepsy in MLC.ResultsAn early onset of seizures is common in MLC. Similarly, in MLC mice, we uncovered spontaneous epileptiform brain activity and a lowered threshold for induced seizures. At the cellular level, we found that although passive and active properties of individual pyramidal neurons are unchanged, extracellular K+ dynamics and neuronal network activity are abnormal in MLC mice.InterpretationDisturbed astrocyte regulation of ion and water homeostasis in MLC causes hyperexcitability of neuronal networks and seizures. These findings suggest a role for defective astrocyte volume regulation in epilepsy. Ann Neurol 2018;83:636–649

Megalencephalic leukoencephalopathy with cysts: theGlialcam‐null mouse model

ObjectiveMegalencephalic leukoencephalopathy with cysts (MLC) is a genetic infantile‐onset disease characterized by macrocephaly and white matter edema due to loss of MLC1 function. Recessive mutations in either MLC1 or GLIALCAM cause the disease. MLC1 is involved in astrocytic volume regulation; GlialCAM ensures the correct membrane localization of MLC1. Their exact role in brain ion‐water homeostasis is only partly defined. We characterized Glialcam‐null mice for further studies.MethodsWe investigated the consequences of loss of GlialCAM in Glialcam‐null mice and compared GlialCAM developmental expression in mice and men.Results Glialcam‐null mice had early‐onset megalencephaly and increased brain water content. From 3 weeks, astrocytes were abnormal with swollen processes abutting blood vessels. Concomitantly, progressive white matter vacuolization developed due to intramyelinic edema. Glialcam‐null astrocytes showed abolished expression of MLC1, reduced expression of the chloride channel ClC‐2 and increased expression and redistribution of the water channel aquaporin4. Expression of other MLC1‐interacting proteins and the volume regulated anion channel LRRC8A was unchanged. In mice, GlialCAM expression increased until 3 weeks and then stabilized. In humans, GlialCAM expression was highest in the first 3 years to then decrease and stabilize from approximately 5 years.Interpretation Glialcam‐null mice replicate the early stages of the human disease with early‐onset intramyelinic edema. The earliest change is astrocytic swelling, further substantiating that a defect in astrocytic volume regulation is the primary cellular defect in MLC. GlialCAM expression affects expression of MLC1, ClC‐2 and aquaporin4, indicating that abnormal interplay between these proteins is a disease mechanism in megalencephalic leukoencephalopathy with cysts.

Glutamate transporter-associated anion channels adjust intracellular chloride concentrations during glial maturation.

Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride-sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl- ]int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na+ -K+ -2Cl- cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl- ]int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion-selective channels during glutamate transport, and thus represent a class of ligand-gated anion channels. Age-dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT-1 increased [Cl- ]int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl- ]int . Other tested chloride channels or chloride transporters do not contribute to [Cl- ]int under our experimental conditions. Neither genetic removal of ClC-2 nor pharmacological block of K+ -Cl- cotransporter change resting Bergmann glial [Cl- ]int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388-400.

Neurotoxicity of Ammonia.

Abnormal liver function has dramatic effects on brain functions. Hyperammonemia interferes profoundly with brain metabolism, astrocyte volume regulation, and in particular mitochondrial functions. Gene expression in the brain and excitatory and inhibitory neurotransmission circuits are also affected. Experiments with a number of pertinent animal models have revealed several potential mechanisms which could underlie the pathological phenomena occurring in hepatic encephalopathy.

The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins.

Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes.

Modulation of neural activation following treatment of hepatic encephalopathy

To measure changes in psychometric state, neural activation, brain volume (BV), and cerebral metabolite concentrations during treatment of minimal hepatic encephalopathy. As proof of principle, 22 patients with well-compensated, biopsy-proven cirrhosis of differing etiology and previous minimal hepatic encephalopathy were treated with oral l-ornithine l-aspartate for 4 weeks. Baseline and 4-week clinical review, blood chemistry, and psychometric evaluation (Psychometric Hepatic Encephalopathy Score and Cognitive Drug Research Score) were performed. Whole-brain volumetric and functional MRI was conducted using a highly simplistic visuomotor task, together with proton magnetic resonance spectroscopy of the basal ganglia. Treatment-related changes in regional BV and neural activation change (blood oxygenation level dependent) were assessed. Although there was no change in clinical, biochemical state, basal ganglia magnetic resonance spectroscopy, or in regional BV, there were significant improvements in Cognitive Drug Research Score (+1.2, p = 0.003) and Psychometric Hepatic Encephalopathy Score (+1.5, p = 0.003) with treatment. This cognitive amelioration was accompanied by changes in blood oxygenation level-dependent activation in the posterior cingulate and ventral medial prefrontal cortex, 2 regions that form part of the brain's structural and metabolic core. In addition, there was evidence of greater visual cortex activation. These structurally interconnected regions all showed increased function after successful encephalopathy treatment. Because no regional change in BV was observed, this implies that mechanisms unrelated to astrocyte volume regulation were involved in the significant improvement in cognitive performance.

Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice.

Recently, we have identified two astrocytic subpopulations in the cortex of GFAP-EGFP mice, in which the astrocytes are visualized by the enhanced green–fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promotor. These astrocytic subpopulations, termed high response- (HR-) and low response- (LR-) astrocytes, differed in the extent of their swelling during oxygen-glucose deprivation (OGD). In the present study we focused on identifying the ion channels or transporters that might underlie the different capabilities of these two astrocytic subpopulations to regulate their volume during OGD. Using three-dimensional confocal morphometry, which enables quantification of the total astrocytic volume, the effects of selected inhibitors of K+ and Cl− channels/transporters or glutamate transporters on astrocyte volume changes were determined during 20 minute-OGD in situ. The inhibition of volume regulated anion channels (VRACs) and two-pore domain potassium channels (K2P) highlighted their distinct contributions to volume regulation in HR-/LR-astrocytes. While the inhibition of VRACs or K2P channels revealed their contribution to the swelling of HR-astrocytes, in LR-astrocytes they were both involved in anion/K+ effluxes. Additionally, the inhibition of Na+-K+-Cl− co-transporters in HR-astrocytes led to a reduction of cell swelling, but it had no effect on LR-astrocyte volume. Moreover, employing real-time single-cell quantitative polymerase chain reaction (PCR), we characterized the expression profiles of EGFP-positive astrocytes with a focus on those ion channels and transporters participating in astrocyte swelling and volume regulation. The PCR data revealed the existence of two astrocytic subpopulations markedly differing in their gene expression levels for inwardly rectifying K+ channels (Kir4.1), K2P channels (TREK-1 and TWIK-1) and Cl− channels (ClC2). Thus, we propose that the diverse volume changes displayed by cortical astrocytes during OGD mainly result from their distinct expression patterns of ClC2 and K2P channels.

The β1 subunit of the Na,K-ATPase pump interacts with megalencephalic leucoencephalopathy with subcortical cysts protein 1 (MLC1) in brain astrocytes: new insights into MLC pathogenesis

Megalencephalic leucoencephalopathy with subcortical cysts (MLC) is a rare congenital leucodystrophy caused by mutations in MLC1, a membrane protein of unknown function. MLC1 expression in astrocyte end-feet contacting blood vessels and meninges, along with brain swelling, fluid cysts and myelin vacuolation observed in MLC patients, suggests a possible role for MLC1 in the regulation of fluid and ion homeostasis and cellular volume changes. To identify MLC1 direct interactors and dissect the molecular pathways in which MLC1 is involved, we used NH2-MLC1 domain as a bait to screen a human brain library in a yeast two-hybrid assay. We identified the β1 subunit of the Na,K-ATPase pump as one of the interacting clones and confirmed it by pull-downs, co-fractionation assays and immunofluorescence stainings in human and rat astrocytes in vitro and in brain tissue. By performing ouabain-affinity chromatography on astrocyte and brain extracts, we isolated MLC1 and the whole Na,K-ATPase enzyme in a multiprotein complex that included Kir4.1, syntrophin and dystrobrevin. Because Na,K-ATPase is involved in intracellular osmotic control and volume regulation, we investigated the effect of hypo-osmotic stress on MLC1/Na,K-ATPase relationship in astrocytes. We found that hypo-osmotic conditions increased MLC1 membrane expression and favoured MLC1/Na,K-ATPase-β1 association. Moreover, hypo-osmosis induced astrocyte swelling and the reversible formation of endosome-derived vacuoles, where the two proteins co-localized. These data suggest that through its interaction with Na,K-ATPase, MLC1 is involved in the control of intracellular osmotic conditions and volume regulation in astrocytes, opening new perspectives for understanding the pathological mechanisms of MLC disease.

Ammonia and proinflammatory cytokines modify expression of genes coding for astrocytic proteins implicated in brain edema in acute liver failure

There is evidence to suggest that, in acute liver failure (ALF), brain ammonia and proinflammatory cytokines may act synergistically to cause brain edema and its complications (intracranial hypertension, brain herniation). However, the molecular mechanisms involved remain to be established. In order to address this issue, semi-quantitative RT-PCR was used to measure the expression of genes coding for astrocytic proteins with an established role in cell volume regulation in cerebral cortical astrocytes exposed to toxic agents previously identified in experimental and clinical ALF. Such agents include ammonia, the proinflammatory cytokine interleukin-1beta (IL-1beta) and combinations of the two. Exposure of cultured astrocytes to recombinant IL-1beta (but not ammonia) resulted in increased expression of aquaporin-4 (AQP-4). Both ammonia and proinflammatory mediators led to decreased expression of glial fibrillary acidic protein (GFAP), a cytoskeletal protein, but these effects were not additive. On the other hand, heme oxygenase-1 (HO-1) and inducible nitric oxide synthase (iNOS) expression were significantly increased by exposure to both ammonia and proinflammatory mediators and although modest, these effects were additive suggestive of a synergistic mechanism. These findings suggest that worsening of brain edema and its complications in ALF due to proinflammatory mechanisms may result from exacerbation of oxidative stress-related mechanisms rather than upregulation of AQP-4 or decreases in expression of the astrocytic structural protein GFAP.

Automated Cell-Based Assay for Screening of Aquaporin Inhibitors

Aquaporins form water channels that play major roles in a variety of physiological processes so that altered expression or function may underlie pathological conditions. In order to identify compounds that modulate aquaporin function, we have implemented a functional assay based on rapid measurement of osmotically induced cell volume changes to screen several libraries of diverse drugs. The time course of fluorescence changes in calcein-loaded cells was analyzed during an osmotic challenge using a 96-multiwell fluorescence plate reader. This system was validated using astrocyte primary cultures and fibroblasts that strongly express endogenous AQP4 and AQP1 proteins, respectively, as well as AQP4-transfected cells. We screened 3575 compounds, including 418 FDA-approved and commercially available drugs, for their effect on AQP-mediated water transport. Primary screening yielded 10 compounds that affected water transport activity in both astrocytes and AQP4-transfected cells and 42 compounds that altered cell volume regulation in astrocytes. Selected drugs were then analyzed on AQP1-expressing erythrocytes and AQP4-expressing membrane vesicles by stopped-flow light scattering. Four molecules of the National Cancer Institute's chemical library (NSC164914, NSC670229, NSC168597, NSC301460) were identified that differentially affected both AQP4 and AQP1 mediated water transport, with EC50 values between 20 and 50 microM. This fluorescence microplate reader-based assay may, thus, provide a platform for high-throughput screening which, when coupled to a secondary evaluation to confirm target specificity, should allow discovery of AQP-specific compounds for novel therapeutic strategies in the treatment of water balance disorders.

Molecular Mechanisms of Ischemic Cerebral Edema: Role of Electroneutral Ion Transport

The brain achieves homeostasis of its intracellular and extracellular fluids by precisely regulating the transport of solute and water across its major cellular barriers: endothelia of the blood-brain barrier (BBB), choroid plexus epithelia, and neuroglial cell membranes. Cerebral edema, the pathological accumulation of fluid in the brain's intracellular and extracellular spaces, is a major cause of morbidity and mortality following stroke and other forms of ischemic brain injury. Until recently, mechanisms of cerebral edema formation have been obscure; consequently, its treatment has been empiric and suboptimal. Here, we provide a paradigm for understanding ischemic cerebral edema, showing that its molecular pathogenesis is a complex yet step-wise process that results largely from impaired astrocytic cell volume regulation and permeability alterations in the cerebral microvasculature, both of which arise from pathological changes in the activities of specific ion channels and transporters. Recent data has implicated the bumetanide-sensitive NKCC1, an electroneutral cotransporter expressed in astrocytes and the BBB, in cerebral edema formation in several different rodent models of stroke. Pharmacological inhibition or genetic deficiency of NKCC1 decreases ischemia-induced cell swelling, BBB breakdown, cerebral edema, and neurotoxicity. Combination pharmacological strategies that include NKCC1 as a target might thus prove beneficial for the treatment of ischemic, and potentially other types of, cerebral edema.

Expression and functional role of potassium-chloride cotransporters (KCC) in astrocytes and C6 glioma cells

Brain edema formation following brain injury is a serious but still poorly treatable medical condition. The understanding of volume regulation in astrocytes, the main cells involved in the formation of cytotoxic brain edema, is key for the development of novel treatment strategies. This study investigates the role of potassium-chloride cotransporters (KCC) for cell volume regulation in glial cells. PCR revealed the expression of KCC isoforms in a glial cell line (C6) and primary cultured astrocytes. Specific inhibition of KCCs caused glial cell swelling and resulted in a complete inhibition of regulatory volume decrease upon hypotonic medium-induced cell swelling. Therefore, our results show that KCCs play an important role in the maintenance and regulation of cell volume in astrocytes.