Function Of Aquaporin-4 Research Articles

Loss of aquaporin-4 impairs cerebrospinal fluid solute clearance through cerebrospinal fluid drainage pathways

AbstractThe aquaporin-4 (AQP4) water channel is essential in neurofluid dynamics. AQP4 loss impairs solute exchange between the cerebrospinal fluid (CSF) and interstitial fluid (ISF). However, whether AQP4 expression affects solute clearance from the CSF space to the extracranial space remains unclear. This study aimed to investigate this using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) following the intrathecal administration of gadolinium-based contrast agents (GBCAs) to AQP4 knockout (KO) rats. AQP4 KO rats showed reduced efflux of intrathecal GBCAs to the extracranial spaces through CSF drainage pathways and increased retention of intrathecal GBCAs in the CSF space compared with the controls. These results suggest that AQP4 loss impairs solute clearance from the CSF space to the extracranial spaces via the CSF drainage pathways. This study revealed a close relationship between AQP4 expression and CSF solute clearance, contributing to a better understanding of the function of AQP4 in neurofluid dynamics.

AQP4 regulates ferroptosis and oxidative stress of Muller cells in diabetic retinopathy by regulating TRPV4

Diabetic retinopathy (DR) is a common microvascular complication that causes visual impairment or loss. Aquaporin 4 (AQP4) is a regulatory protein involved in water transport and metabolism. In previous studies, we found that AQP4 is related to hypoxia injury in Muller cells. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a non-selective cation channel protein involved in the regulation of a variety of ophthalmic diseases. However, the effects of AQP4 and TRPV4 on ferroptosis and oxidative stress in high glucose (HG)-treated Muller cells are unclear. In this study, we investigated the functions of AQP4 and TRPV4 in DR. HG was used to treat mouse Muller cells. Reverse transcription quantitative polymerase chain reaction was used to measure AQP4 mRNA expression. Western blotting was used to detect the protein levels of AQP4, PTGS2, GPX4, and TRPV4. Cell count kit-8, flow cytometry, 5,5′,6,6′-tetrachloro-1,1,3,3′-tetraethylbenzimidazolyl carbocyanine iodide staining, and glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits were used to evaluate the function of the Muller cells. Streptozotocin was used to induce DR in rats. Haematoxylin and eosin staining was performed to stain the retina of rats. GSH, SOD, and MDA detection kits, immunofluorescence, and flow cytometry assays were performed to study the function of AQP4 and TRPV4 in DR rats. Results found that AQP4 and TRPV4 were overexpressed in HG-induced Muller cells and streptozotocin-induced DR rats. AQP4 inhibition promoted proliferation and cell cycle progression, repressed cell apoptosis, ferroptosis, and oxidative stress, and alleviated retinal injury in DR rats. Mechanistically, AQP4 positively regulated TRPV4 expression. Overexpression of TRPV4 enhanced ferroptosis and oxidative stress in HG-treated Muller cells, and inhibition of TRPV4 had a protective effect on DR-induced retinal injury in rats. In conclusion, inhibition of AQP4 inhibits the ferroptosis and oxidative stress in Muller cells by downregulating TRPV4, which may be a potential target for DR therapy.

Abstract WP27: Tissue Kallikrein Attenuates Ischemic Edema by Increasing Water Transport Function of AQP4 in Glymphatic System in pMCAO Mice

Aquaporin-4 (AQP4) is an essential water channel protein in GS (glymphatic system), and is widely distributed in astrocytes at the brain/cerebrospinal fluid interface, where it facilitates water movement. Altered AQP4 polarization and expression are evident in cytotoxic edema in pMCAO mice in our previous study, glymphatic MRI of which also shows reduced CSF tracer entry and clearance. Tissue Kallikrein treatment provides cerebral infarction patients with vasodilatory activity, which may facilitate the dynamics of CSF in GS. We hypothesized that TK could attenuate depolarization of AQP4 to confer benefit in brain edema by boosting the kinetics in GS. In this study, we evaluated the effects of TK on mice acute ischemic edema in both wild and AQP4-/- pMCAO models. A fluorescent tracer was injected into the striatum of both mice, and the infusion pattern of the fluorescent tracer in the brain parenchyma was detected to confirm the function of AQP4 in alleviating edema by TK. Concurrently, immunohistochemistry analysis was performed to explore the altered AQP4 polarization and expression in TK treatment animals. Our results indicated that TK attenuated brain edema was associated with improved transport function of AQP4 to CSF in pMCAO mice, which might relate to the alteration of polarization and expression of AQP4.

Water channel aquaporin 4 is required for T cell receptor mediated lymphocyte activation.

Aquaporins are a family of ubiquitously expressed transmembrane water channels implicated in a broad range of physiological functions. We have previously reported that aquaporin 4 (AQP4) is expressed on T cells and that treatment with a small molecule AQP4 inhibitor significantly delays T cell mediated heart allograft rejection. Using either genetic deletion or small molecule inhibitor, we show that AQP4 supports T cell receptor mediated activation of both mouse and human T cells. Intact AQP4 is required for optimal T cell receptor (TCR)-related signaling events, including nuclear translocation of transcription factors and phosphorylation of proximal TCR signaling molecules. AQP4 deficiency or inhibition impairs actin cytoskeleton rearrangements following TCR crosslinking, causing inferior TCR polarization and a loss of TCR signaling. Our findings reveal a novel function of AQP4 in T lymphocytes and identify AQP4 as a potential therapeutic target for preventing TCR-mediated T cell activation.

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.

Aquaporin-4 Expression during Toxic and Autoimmune Demyelination.

The water channel protein aquaporin-4 (AQP4) is required for a normal rate of water exchange across the blood–brain interface. Following the discovery that AQP4 is a possible autoantigen in neuromyelitis optica, the function of AQP4 in health and disease has become a research focus. While several studies have addressed the expression and function of AQP4 during inflammatory demyelination, relatively little is known about its expression during non-autoimmune-mediated myelin damage. In this study, we used the toxin-induced demyelination model cuprizone as well as a combination of metabolic and autoimmune myelin injury (i.e., Cup/EAE) to investigate AQP4 pathology. We show that during toxin-induced demyelination, diffuse AQP4 expression increases, while polarized AQP4 expression at the astrocyte endfeet decreases. The diffuse increased expression of AQP4 was verified in chronic-active multiple sclerosis lesions. Around inflammatory brain lesions, AQP4 expression dramatically decreased, especially at sites where peripheral immune cells penetrate the brain parenchyma. Humoral immune responses appear not to be involved in this process since no anti-AQP4 antibodies were detected in the serum of the experimental mice. We provide strong evidence that the diffuse increase in anti-AQP4 staining intensity is due to a metabolic injury to the brain, whereas the focal, perivascular loss of anti-AQP4 immunoreactivity is mediated by peripheral immune cells.

Inhibition of Aquaporin 4 Decreases Amyloid A\u03b240 Drainage Around Cerebral Vessels

Aquaporin-4 (AQP4) is located mainly in the astrocytic end-feet around cerebral blood vessels and regulates ion and water homeostasis in the brain. While deletion of AQP4 is shown to reduce amyloid-β (Aβ) clearance and exacerbate Aβ peptide accumulation in plaques and vessels of Alzheimer’s disease mouse models, the mechanism and clearing pathways involved are debated. Here, we investigated how inhibiting the function of AQP4 in healthy male C57BL/6 J mice impacts clearance of Aβ40, the more soluble Aβ isoform. Using two-photon in vivo imaging and visualizing vessels with Sulfurodamine 101 (SR101), we first showed that Aβ40 injected as a ≤ 0.5-μl volume in the cerebral cortex diffused rapidly in parenchyma and accumulated around blood vessels. In animals treated with the AQP4 inhibitor TGN-020, the perivascular Aβ40 accumulation was significantly (P < 0.001) intensified by involving four times more vessels, thus suggesting a generalized clearance defect associated with vessels. Increasing the injecting volume to ≥ 0.5 ≤ 1 μl decreased the difference of Aβ40-positive vessels observed in non-treated and AQP4 inhibitor-treated animals, although the difference was still significant (P = 0.001), suggesting that larger injection volumes could overwhelm intramural vascular clearance mechanisms. While both small and large vessels accumulated Aβ40, for the ≤ 0.5-μl volume group, the average diameter of the Aβ40-positive vessels tended to be larger in control animals compared with TGN-020-treated animals, although the difference was non-significant (P = 0.066). Using histopathology and ultrastructural microscopy, no vascular structural change was observed after a single massive dose of TGN-020. These data suggest that AQP4 deficiency is directly involved in impaired Aβ brain clearance via the peri-/para-vascular routes, and AQP4-mediated vascular clearance might counteract blood-brain barrier abnormalities and age-related vascular amyloidopathy.

Identification of geraldol as an inhibitor of aquaporin‑4 binding by NMO‑IgG.

Neuromyelitis optica (NMO) is a severe neurological demyelinating autoimmune disease that affects the optic nerves and spinal cord. There is currently no effective cure or therapy. Aquaporin-4 (AQP4) is a known target of the autoimmune antibody NMO-IgG. Therefore, binding of NMO-IgG to AQP4, and subsequent activation of antibody-mediated and complement-dependent cytotoxicity (CDC), are thought to underlie the pathogenesis of NMO. In the present study, a cell-based high-throughput screening approach was developed to identify molecular inhibitors of NMO-IgG binding to AQP4. Using this approach, extracts from the herb Petroselinum crispum were shown to have inhibitory effects on NMO-IgG binding to AQP4, and the natural compound geraldol was purified from the herb extracts. Analytical high performance liquid chromatography, electrospray ionization-mass spectrometry and nuclear magnetic resonance analyses confirmed the identity of the isolated compound as geraldol, a flavonoid. Geraldol effectively blocked binding of NMO-IgG to AQP4 in immunofluorescence assays and decreased CDC in NMO-IgG/complement-treated FRTL-AQP4 cells and primary astrocytes. Geraldol exhibited low cytotoxicity, with no effect on proliferation or apoptosis of FRTL-AQP4 cells and primary astrocytes. Permeability assays indicated that geraldol did not alter the water transport function of AQP4 in either cell system. The present study suggests the potential therapeutic value of geraldol for NMO drug development.

Evolutionary Adaptation of Aquaporin-4 in Yak (Bos grunniens) Brain to High-Altitude Hypoxia of Qinghai-Tibetan Plateau.

Background: In high-altitude animals, brain cell resilience against hypoxia stress is one critical evolutionary step that has promoted individual survival and species adaptation to the environment. Aquaporin-4 (AQP4) is implicated in a number of physiopathological processes, particularly in the development of brain edema, and other functions such as the regulation of extracellular space volume, potassium buffering, waste clearance, and calcium signaling. Still, the role of AQP4 in the adaptation to high-altitude hypoxia remains unknown. The yak (Bos grunniens) is the only large mammal that is currently known to have adapted to the high-altitude hypoxic environment of the Qinghai-Tibet Plateau (>4000 m above sea level). Methods: In this study, we cloned the complementary DNA (cDNA) for yak AQP4 and analyzed structural differences of AQP4 between yak and cattle. We used reverse transcription quantitative polymerase chain reaction and western blot to investigate whether the expression of AQP4 mRNA and protein was different in brain of yak and cattle. In addition, immunohistochemistry was use to analyze the localization and expression of AQP4 in brain of yak and cattle. Results: Immunohistochemical results have shown that AQP4 is expressed in many regions of the yak brain, and both protein and messenger RNA (mRNA) levels are significantly lower than those of low-altitude cattle (Bos taurus). Phylogenetic analysis revealed that yak AQP4 is evolutionarily conserved. Interestingly, a substitution of Ala (cattle) to Ser in position 82, and eight additional amino acid residues composing an α-helix region are present in yak AQP4 protein. These sequence modifications potentially modulate the function of AQP4 in distinct environments. Conclusions: Our findings suggest that AQP4 may have an important role in the resistance to cerebral edema through low expression and maintenance of normal physiological function in the yak brain.

Agrin plays a major role in the coalescence of the aquaporin-4 clusters induced by gamma-1-containing laminin.

The basement membrane that seperates the endothelial cells and astrocytic endfeet that comprise the blood-brain barrier is rich in collagen, laminin, agrin, and perlecan. Previous studies have demonstrated that the proper recruitment of the water-permeable channel aquaporin-4 (AQP4) to astrocytic endfeet is dependent on interactions between laminin and the receptor dystroglycan. In this study, we conducted a deeper investigation into how the basement membrane might further regulate the expression, localization, and function of AQP4, using primary astrocytes as a model system. We found that treating these cells with laminin causes endogenous agrin to localize to the cell surface, where it co-clusters with β-dystroglycan (β-DG). Conversely, agrin sliencing profoundly disrupts β-DG clustering. As in the case of laminin111, Matrigel™, a complete basement membrane analog, also causes the clustering of AQP4 and β-DG. This clustering, whether induced by laminin111 or Matrigel™ is inhibited when the astrocytes are first incubated with an antibody against the γ1 subunit of laminin, suggesting that the latter is crucial to the process. Finally, we showed that laminin111 appears to negatively regulate AQP4-mediated water transport in astrocytes, suppressing the cell swelling that occurs following a hypoosmotic challenge. This suppression is abolished if DG expression is silenced, again demonstrating the central role of this receptor in relaying the effects of laminin.

Retinal and optic nerve degeneration in liver X receptor β knockout mice

The retina is an extension of the brain. Like the brain, neurodegeneration of the retina occurs with age and is the cause of several retinal diseases including optic neuritis, macular degeneration, and glaucoma. Liver X receptors (LXRs) are expressed in the brain where they play a key role in maintenance of cerebrospinal fluid and the health of dopaminergic neurons. Herein, we report that LXRs are expressed in the retina and optic nerve and that loss of LXRβ, but not LXRα, leads to loss of ganglion cells in the retina. In the retina of LXRβ-/- mice, there is an increase in amyloid A4 and deposition of beta-amyloid (Aβ) aggregates but no change in the level of apoptosis or autophagy in the ganglion cells and no activation of microglia or astrocytes. However, in the optic nerve there is a loss of aquaporin 4 (AQP4) in astrocytes and an increase in activation of microglia. Since loss of AQP4 and microglial activation in the optic nerve precedes the loss of ganglion cells, and accumulation of Aβ in the retina, the cause of the neuronal loss appears to be optic nerve degeneration. In patients with optic neuritis there are frequently AQP4 autoantibodies which block the function of AQP4. LXRβ-/- mouse is another model of optic neuritis in which AQP4 antibodies are not detectable, but AQP4 function is lost because of reduction in its expression.

Research progress in the role of aquaproin-4 and inward rectifying potassium channel 4.1 in spinal cord edema

Spinal edema is a very important pathophysiological basis for secondary spinal cord injury, which affects the repair and prognosis of spinal cord injury. Aquaporin-4 is widely distributed in various organs of the body, and is highly expressed in the brain and spinal cord. Inward rectifying potassium channel 4.1 is a protein found in astrocytes of central nervous system. It interacts with aquaporins in function. Aquaporin-4 and inward rectifying potassium channel 4.1 play an important role in the formation and elimination of spinal cord edema, inhibition of glial scar formation and promotion of excitotoxic agents exclusion. The distribution and function of aquaporin-4 and inward rectifying potassium channel 4.1 in the central nervous system and their expression after spinal cord injury have multiple effects on spinal edema. Studies of aquaporin-4 and inward rectifying potassium channel 4.1 in the spinal cord may provide new ideas for the elimination and treatment of spinal edema.

Insights into the aquaporin 4 of zebrafish (Danio rerio) through evolutionary analysis, molecular modeling and structural dynamics

Aquaporins (AQPs) constitute a super family of major intrinsic proteins (MIPs) and aquaporin 4 (AQP4) is one of the most popular protein, participating in different types of bio-physiological pathways in animal, plant and even in bacteria. Zebrafish, considered as a novel model organism, possess an Aquaporine 4 protein (zAQP4) that shares a high degree of homology with mammalian orthologs which was mostly found to be express in brain, ovary, testis, and low levels in other tissues. To focus more light on AQP4 medical related therapeutic applications, the structure of zAQP4 as a drug target protein is extremely essential and there is no such structural information available in protein data bank (PDB). Herein, for the first time we generate the 3D structure of zAQP4 was modeled through a Homology modeling approach. The root-mean-square deviation (RMSD) of atomic positions between the generated model structure and human AQP4, revealed 69.2% sequence identity over align region with RMSD value 2.723 Å. Structural integrity of the model was assessed using molecular dynamics simulation studies to achieve a better understanding of the progress, structure, and function of AQP4. Finally residual involvements for druggable pocket identification were made and some important residues such as VAL-237, PRO-272, GLN-274, PRO-277, TYR-278, VAL-118, LEU-122 and LEU-126 which shows highest druggability probability (1.0) that can emphasize the importance of aquaporin-4 as a target protein in the field of drug research in near feature.

Mechanism of depression as a risk factor in the development of Alzheimer's disease: the function of AQP4 and the glymphatic system.

Many studies have indicated that a history of depression increases the risk of developing Alzheimer's disease (AD); however, the potential pathogenestic mechanism by which depression functions as a high risk factor for AD remains unknown. Recently, a "cerebral lymphatic system" referred to as "glymphatic system" has been demonstrated to be responsible for neuronal extracellular waste protein clearance via a paravascular pathway. However, the function of glymphatic pathway has not been determined in depressive disorders. The present study used an animal model of chronic unpredictable mild stress (CUMS) to determine the function of glymphatic pathway by using fluorescence tracers. Immunohistochemistry was used to assess the accumulation of endogenous mouse and exogenous human amyloid beta 42 (Aβ42) in CUMS-treated mice with or without treatment with antidepressant fluoxetine. Glymphatic pathway circulation was impaired in mice treated with CUMS; moreover, glymphatic pathway dysfunction suppressed Aβ42 metabolism, because the accumulation of endogenous and exogenous Aβ42 was increased in the brains of the CUMS-treated mice. However, treatment with fluoxetine reversed these destructive effects of CUMS on glymphatic system. In anhedonic mice, the expression of the water channel aquaporin 4 (AQP4), a factor in glymphatic pathway dysfunction, was down-regulated in cortex and hippocampus. The dysfunction of glymphatic system suggested why a history of depression may be a strong risk factor for AD in anhedonic mice. We hope our study will contribute to an understanding of the risk mechanism of depressive disorder in the development of AD and the mechanisms of antidepressant therapies in AD.

Aquaporin-4 deficiency facilitates fear memory extinction in the hippocampus through excessive activation of extrasynaptic GluN2B-containing NMDA receptors

Aquaporin-4 (AQP-4) is the predominant water channel in the brain and primarily expressed in astrocytes. Astrocytes have been generally believed to play important roles in regulating synaptic plasticity and information processing. A growing number of evidence shows that AQP-4 plays a potential role in the regulation of astrocyte function. However, little is known about the function of AQP-4 for synaptic plasticity in the hippocampus. Therefore, we evaluated long-term depression (LTD) in the hippocampus and the extinction of fear memory of AQP-4 knockout (KO) and wild-type (WT) mice. We found that AQP-4 deficiency facilitated fear memory extinction and NMDA receptors (NMDARs)-dependent LTD in the CA3-CA1 pathway. Furthermore, AQP-4 deficiency selectively increased GluN2B-NMDAR-mediated excitatory postsynaptic currents (EPSCs). The excessive activation of extrasynaptic GluN2B-NMDAR contributed to the facilitation of NMDAR-dependent LTD and enhancement of fear memory extinction in AQP-4 KO mice. Thus, it appears that AQP-4 may be a potential target for intervention in fear memory extinction.This article is part of the Special Issue entitled ‘Ionotropic glutamate receptors’.

The study of the function of AQP4 in cerebral ischaemia-reperfusion injury using poroelastic theory.

Brain oedema is thought to form and to clear through the use of water-protein channels, aquaporin-4 (AQP4), which are found in the astrocyte endfeet. The model developed here is used to study the function of AQP4 in the formation and elimination of oedema fluid in ischaemia-reperfusion injury. The cerebral space is assumed to be made of four fluid compartments: astrocyte, neuron, ECS and blood microvessels, and a solid matrix for the tissue, and this is modelled using multiple-network poroelastic theory. AQP4 allows the movement of water between astrocyte and the ECS and the microvessels. It is found that the presence of AQP4 may help in reducing vasogenic oedema shown by a decrease in brain tissue extracellular pressure. However, the astrocyte pressure will increase to compensate for this decrease, which may lead to cytotoxic oedema. In addition, the swelling will also depend on the ionic concentrations in the astrocyte and extracellular space, which may change after ischaemic stroke. Understanding the role of AQP4 in oedema may thus help the development of a treatment plan in reducing brain swelling after ischaemia-reperfusion.

The Role of Astrocytic Aquaporin-4 in Synaptic Plasticity and Learning and Memory.

Aquaporin-4 (AQP4) is the predominant water channel expressed by astrocytes in the central nervous system (CNS). AQP4 is widely expressed throughout the brain, especially at the blood-brain barrier where AQP4 is highly polarized to astrocytic foot processes in contact with blood vessels. The bidirectional water transport function of AQP4 suggests its role in cerebral water balance in the CNS. The regulation of AQP4 has been extensively investigated in various neuropathological conditions such as cerebral edema, epilepsy, and ischemia, however, the functional role of AQP4 in synaptic plasticity, learning, and memory is only beginning to be elucidated. In this review, we explore the current literature on AQP4 and its influence on long term potentiation (LTP) and long term depression (LTD) in the hippocampus as well as the potential relationship between AQP4 and in learning and memory. We begin by discussing recent in vitro and in vivo studies using AQP4-null and wild-type mice, in particular, the impairment of LTP and LTD observed in the hippocampus. Early evidence using AQP4-null mice have suggested that impaired LTP and LTD is brain-derived neurotrophic factor dependent. Others have indicated a possible link between defective LTP and the downregulation of glutamate transporter-1 which is rescued by chronic treatment of β-lactam antibiotic ceftriaxone. Furthermore, behavioral studies may shed some light into the functional role of AQP4 in learning and memory. AQP4-null mice performances utilizing Morris water maze, object placement tests, and contextual fear conditioning proposed a specific role of AQP4 in memory consolidation. All together, these studies highlight the potential influence AQP4 may have on long term synaptic plasticity and memory.

Regulation and Function of AQP4 in the Central Nervous System.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. Based on studies on AQP4(-/-) mice, AQP4 has been assigned physiological roles in stimulus-induced K(+) clearance, paravascular fluid flow, and brain edema formation. Conflicting data have been presented on the role of AQP4 in K(+) clearance and associated extracellular space shrinkage and on the stroke-induced alterations of AQP4 expression levels during edema formation, raising questions about the functional importance of AQP4 in these (patho)physiological aspects. Phosphorylation-dependent gating of AQP4 has been proposed as a regulatory mechanism for AQP4-mediated osmotic water transport. This paradigm was, however, recently challenged by experimental evidence and molecular dynamics simulations. Regulatory patterns and physiological roles for AQP4 thus remain to be fully explored.

Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark

The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5′ and 3′ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill > intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (Pf) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species.

Expression and function of aquaporin-4 in olfactory system of mice

Objective To investigate the expression and function of aquaporin-4(AQP4) in the olfactory system of mice.Methods The differences of AQP4 expression in olfaction system between wild-type and AQP4-null mice were studied by immunoblotting and immunofluorescence methods. The differences of mouse olfactory functions in the two groups were examined by two olfactory behavioral assays: the buried food pellet test and olfaction maze test,and the odorant-stimulated electroolfactogram(EOG) recording.Results The results of immunoblotting and immunofluorescence showed no AQP4 expression in the olfactory system in AQP4-null mice.Immunofluorescence result also indicated that AQP4 was mainly distributed in the membrane of support cells,duct cells of Bowmann's gland,basal cells of olfactory epithelium,membrane of Bowmann's gland epithelial cells,olfactory sheath cells surrounding the olfactory bundles,and the membrane of cells in the olfactory bundle layer and glomeruler layer.The results of olfactory behavioral assay were significantly different between the two groups at all time points tested in both the olfaction maze test and the buried food pellet test(P0.05).It was showed that the EOGs under different pressures of saturated trimethylamine had a similar shape in both groups,and the amplitude of EOGs increased with the increase of pressure.While under the same pressure,the EOG amplitude of AQP4-null mice was significantly lower than that of wild-type mice(P0.05).Conclusion AQP4 is widely distributed in the olfactory system of mice,including the olfactory mucosa,olfactory nerve,and olfactory bulb,which can protect the olfactory neural bundle and facilitate neural signal transfer.