B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Glymphatic system clears extracellular tau and protects from tau aggregation and neurodegeneration.

Accumulation of tau has been implicated in various neurodegenerative diseases termed tauopathies. Tau is a microtubule-associated protein but is also actively released into the extracellular fluids including brain interstitial fluid and cerebrospinal fluid (CSF). However, it remains elusive whether clearance of extracellular tau impacts tau-associated neurodegeneration. Here, we show that aquaporin-4 (AQP4), a major driver of the glymphatic clearance system, facilitates the elimination of extracellular tau from the brain to CSF and subsequently to deep cervical lymph nodes. Strikingly, deletion of AQP4 not only elevated tau in CSF but also markedly exacerbated phosphorylated tau deposition and the associated neurodegeneration in the brains of transgenic mice expressing P301S mutant tau. The current study identified the clearance pathway of extracellular tau in the central nervous system, suggesting that glymphatic clearance of extracellular tau is a novel regulatory mechanism whose impairment contributes to tau aggregation and neurodegeneration.

Protective Effects of Aquaporin-4 Deficiency on Longer-term Neurological Outcomes in a Mouse Model.

Traumatic brain injury (TBI) has been a crucial health problem, with more than 50 million patients worldwide each year. Glymphatic system is a fluid exchange system that relies on the polarized water channel aquaporin-4 (AQP4) at the astrocytes, accounting for the clearance of abnormal proteins and metabolites from brain tissues. However, the dysfunction of glymphatic system and alteration of AQP4 polarization during the progression of TBI remain unclear. AQP4-/- and Wild Type (WT) mice were used to establish the TBI mouse model respectively. Brain edema and Evans blue extravasation were conducted 24 h post-injury to evaluate the acute TBI. Morris water maze (MWM) was used to establish the long-term cognitive functions of AQP4-/- and WT mice post TBI. Western-blot and qRT-PCR assays were performed to demonstrate protective effects of AQP4 deficiency to blood-brain barrier (BBB) integrity and amyloid-ß clearance. The inflammation of cerebral tissues post TBI was estimated by ELISA assay. AQP4 deficiency alleviated the brain edema and neurological deficit in TBI mice. AQP4-knockout led to improved cognitive outcomes in mice post TBI. The BBB integrity and cerebral amyloid-ß clearance were protected by AQP4 deficiency in TBI mice. AQP4 deficiency ameliorated the TBI-induced inflammation. AQP4 deficiency improved longer-term neurological outcomes in a mouse model of TBI.

Aquaporin-4 Reduces Post-Traumatic Seizure Susceptibility by Promoting Astrocytic Glial Scar Formation in Mice.

Seizures are important neurological complications after traumatic brain injury (TBI) and are reported for up to 50% of patients with TBI. Despite several studies, no drug strategy has been able to alter the biological events leading to epileptogenesis. The glial water channel, aquaporin-4 (AQP4), was shown to facilitate cytotoxic cell swelling in ischemia and glial scar formation after stab wound injury. In this study, we examined post-traumatic seizure susceptibility of AQP4-deficient mice (AQP4-/-) after injection of pentylenetetrazole (PTZ) 1 month after controlled cortical impact (CCI) and compared them to wild-type sham injury controls. After PTZ injection, AQP4-/- mice demonstrated dramatically shortened seizure latency (120 ± 40 vs. 300 ± 70 sec; p < 0.001) and increased seizure severity (grade 7.5 ± 0.4 vs. 5.8 ± 0.4; p < 0.001) compared to their wild-type counterparts. Morphometric analysis demonstrated a significant 2-fold reduction in astrocytosis, with a concomitant increase in microgliosis in injured AQP4-null mice compared to their injured wild-type counterparts (44 ± 2 vs. 24 ± 3 cells per high power field [cells/hpf], respectively; p < 0.0001). Minocycline, an inhibitor of microglia, reversed the post-TBI epilepsy phenotype of AQP4-null mice. After minocycline treatment, AQP4-/- mice demonstrated similar latency of seizures evoked by PTZ (723 ± 35 vs. 696 ± 38 sec; p > 0.05) and severity of seizures evoked by PTZ (grade 4.0 ± 0.5 vs. 3.81 ± 0.30; p > 0.05) compared to wild-type counterparts. Immunohistochemical analysis demonstrated decreased immunostaining of microglia to levels comparable to wild-type (12 ± 2 vs. 11 ± 4 cells/hpf, respectively; p > 0.05). Taken together, these results suggest a protective role of AQP4 in post-traumatic seizure susceptibility by promoting astrogliosis, formation of a glial scar, and preventing microgliosis.

Modulation of posttraumatic epileptogenesis in aquaporin-4 knockout mice.

OBJECTIVE: To determine the role of aquaporin-4 (AQP4) in posttraumatic epileptogenesis using long-term video-electroencephalographic (vEEG) recordings. Here, differences in EEG were analyzed between wild-type (WT) and AQP4 knockout (KO) mice and between mice with and without posttraumatic epilepsy (PTE). METHODS: WT and AQP4 KO mice were subjected to a single controlled cortical impact traumatic brain injury (TBI) in the frontal cortex, and vEEG was recorded in the ipsilateral hippocampus at 14, 30, 60, and 90 days postinjury (dpi). Intrahippocampal electrical stimulation was also used to assess electrographic seizure threshold and electrographic seizure duration (ESD). RESULTS: The mean seizure frequency per day for WT mice was 0.07 ± 0.07, 0.11 ± 0.07, 0.26 ± 0.13, and 0.12 ± 0.10 at 14, 30, 60, and 90 dpi, respectively. The mean seizure frequency per day for AQP4 KO mice was 0.45 ± 0.27, 0.29 ± 0.12, and 0.26 ± 0.19 at 14, 30, and 60 dpi, respectively. The mean seizure duration was 15 ± 2 seconds and 24 ± 3 seconds for WT and AQP4 KO mice, respectively. The percentage of mice that developed PTE were 28% and 37% for WT and AQP4 KO mice, respectively. Power spectral density (PSD) analysis revealed alterations in EEG frequency bands between sham and TBI in both genotypes. Additionally, PSD analysis of spontaneous recurrent seizures revealed alterations in delta power between genotypes. Morlet wavelet analysis detected heterogeneity in EEG seizure subtypes and dynamic EEG power patterns after TBI. Compared with AQP4 KO mice, a significant increase in ESD was observed in WT mice at 14 dpi. SIGNIFICANCE: Posttraumatic seizures (PTSs) may be modulated by the astrocyte water channel AQP4. Absence of AQP4 increases the number of spontaneous seizures, increases seizure duration, and alters EEG power patterns of PTSs.

AQP4ex is crucial for the anchoring of AQP4 at the astrocyte end-feet and for neuromyelitis optica antibody binding.

Brain water homeostasis is essential for the appropriate control of neuronal activity. Furthermore, the encasement of the central nervous system (CNS) by a hard structure, greatly limits its tolerance for the volume changes occurring with acute brain edema, which quickly leads to severe damage or death.The recent discovery of the extended isoform of AQP4 (AQP4ex), generated by translational readthrough, revealed a potential new mechanism of water transport regulation and polarization at the blood-brain-barrier level.In the present study we used CRISPR/Cas9 technology to generate an AQP4ex-/- mouse model and evaluate the effect on the overall AQP4 expression, polarization, supramolecular organization in orthogonal arrays of particles (OAPs) and neuromyelitis optica (NMO-IgG) autoantibodies binding.AQP4ex removal did not cause a decrease in total AQP4 protein expression but completely suppressed the specific location of AQP4 at the astrocyte endfeet. Without AQP4ex, AQP4 was mislocalized and α-syntrophin expression, the selective partner for AQP4 localization, was partially altered. The supramolecular organization of AQP4 in OAPs was subtly altered. Indeed, the absence of AQP4ex reduced the size of AQP4-OAPs but the number of AQP4-OAP pools remained largely the same. More importantly, AQP4ex resulted critical for the binding of pathogenic human NMO-IgG autoantibodies to the brain. Indeed, the absence of AQP4ex completely abolished the binding of NMO-IgG at the perivascular astrocyte endfeet.This study provides the first direct evidence in vivo on the specific role of AQP4ex in AQP4 perivascular OAPs assembly and confinement and reveals AQP4ex as new and important player in neuromyelitis optica.

Aquaporin 4 deficiency alleviates experimental colitis in mice.

Aquaporin (AQP) 4 is expressed in the basolateral membrane of colonic epithelial cells, and the purpose of this study was to explore the mechanistic role of AQP4 in experimental colitis. Experimental colitis was induced in AQP4 knockout (AQP4-/-) CD-1 mice and AQP4 wild-type (AQP4wt) mice by oral administration of dextran sulfate sodium (DSS). Experimental colitis was clinically established. Compared with AQP4wt mice, AQP4-/- mice showed increased tolerance to DSS-induced experimental colitis, including lesser degree of weight loss, diarrhea and bleeding, lower disease activity index scores, longer colon lengths, and lesser histologic scores. DSS-treated AQP4-/- mice had lower serum levels of IL-6 and TNF, higher IL-10 level, and lesser inflammatory cell infiltration. DSS-treated AQP4-/- mice also had lower immunostaining of NF-κB p65 as well as nuclear levels of p65 and phosphorylated p65. Sequencing of 16S rRNA indicated that DSS-treated AQP4-/- mice maintained intestinal microbial diversity and had higher Firmicutes/Bacteroidetes ratios and greater relative abundance of Erysipelotrichaceae species. These results suggested for the first time that AQP4 deficiency alleviates experimental colitis in mice. Our study helps to understand the pathogenesis of inflammatory bowel diseases, and blocking AQP4 may represent a novel therapeutic approach for ulcerative colitis.-Wang, L., Tang, H., Wang, C., Hu, Y., Wang, S., Shen, L. Aquaporin 4 deficiency alleviates experimental colitis in mice.

Aquaporin-4-independent volume dynamics of astroglial endfeet during cortical spreading depression.

Cortical spreading depression (CSD) is a slowly propagating wave of depolarization of gray matter. This phenomenon is believed to underlie the migraine aura and similar waves of depolarization may exacerbate injury in a number of neurological disease states. CSD is characterized by massive ion dyshomeostasis, cell swelling, and multiphasic blood flow changes. Recently, it was shown that CSD is associated with a closure of the paravascular space (PVS), a proposed exit route for brain interstitial fluid and solutes, including excitatory and inflammatory substances that increase in the wake of CSD. The PVS closure was hypothesized to rely on swelling of astrocytic endfeet due to their high expression of aquaporin-4 (AQP4) water channels. We investigated whether CSD is associated with swelling of endfeet around penetrating arterioles in the cortex of living mice. Endfoot cross-sectional area was assessed by two-photon microscopy of mice expressing enhanced green fluorescent protein in astrocytes and related to the degree of arteriolar constriction. In anesthetized mice CSD triggered pronounced endfoot swelling that was short-lasting and coincided with the initial arteriolar constriction. Mice lacking AQP4 displayed volume changes of similar magnitude. CSD-induced endfoot swelling and arteriolar constriction also occurred in awake mice, albeit with faster kinetics than in anesthetized mice. We conclude that swelling of astrocytic endfeet is a robust event in CSD. The early onset and magnitude of the endfoot swelling is such that it may significantly delay perivascular drainage of interstitial solutes in neurological conditions where CSD plays a pathophysiological role.

The role of aquaporin-4 and transient receptor potential vaniloid isoform 4 channels in the development of cytotoxic edema and associated extracellular diffusion parameter changes.

The proper function of the nervous system is dependent on the balance of ions and water between the intracellular and extracellular space (ECS). It has been suggested that the interaction of aquaporin-4 (AQP4) and the transient receptor potential vaniloid isoform 4 (TRPV4) channels play a role in water balance and cell volume regulation, and indirectly, of the ECS volume. Using the real-time iontophoretic method, we studied the changes of the ECS diffusion parameters: ECS volume fraction α (α = ECS volume fraction/total tissue volume) and tortuosity λ (λ2  = free/apparent diffusion coefficient) in mice with a genetic deficiency of AQP4 or TRPV4 channels, and in control animals. The used models of cytotoxic edema included: mild and severe hypotonic stress or oxygen-glucose deprivation (OGD) in situ and terminal ischemia/anoxia in vivo. This study shows that an AQP4 or TRPV4 deficit slows down the ECS volume shrinkage during severe ischemia in vivo. We further demonstrate that a TRPV4 deficit slows down the velocity and attenuates an extent of the ECS volume decrease during OGD treatment in situ. However, in any of the cytotoxic edema models in situ (OGD, mild or severe hypotonic stress), we did not detect any alterations in the cell swelling or volume regulation caused by AQP4 deficiency. Overall, our results indicate that the AQP4 and TRPV4 channels may play a crucial role in severe pathological states associated with their overexpression and enhanced cell swelling. However, detailed interplay between AQP4 and TRPV4 channels requires further studies and additional research.

Aquaporin-4 deficiency reduces TGF-ß1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease.

Aquaporin-4 (AQP4), the main water-selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) model, AQP4-deficient (AQP4-/- ) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH+ )-positive neurons than did wild-type AQP4 (AQP4+/+ ) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor-ß1 (TGF-ß1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4-/- mice after MPTP treatment. Furthermore, the lower level of TGF-ß1 in AQP4-/- mice partially resulted from impairment of its generation by astrocytes; reduced TGF-ß1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH+ neurons in AQP4-/- mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4-mediated immune regulation.

Deletion of aquaporin-4 aggravates brain pathology after blocking of the meningeal lymphatic drainage.

The glymphatic pathway and meningeal lymphatic vessels are involved in clearance of metabolic macromolecules from the brain. However, the functional interaction between the two systems in the maintenance of brain homeostasis remains unclear. Here we reported that deletion of aquaporin-4 (AQP4), a functional regulator of glymphatic clearance, aggravated brain pathology of 3 month-old mice after blocking of the meningeal lymphatic drainage for 2 weeks via ligation of the deep cervical lymphatic nodes (LdcLNs). LdcLNs increased total and phosphorylated Tau protein levels in the hippocampus of both genotype mice, but increased hippocampal amyloid beta 1-40 and 1-42 levels only in AQP4 null mice, with up-regulation of beta-site amyloid precursor protein-cleaving enzyme 1 and down-regulation of insulin degrading enzyme. Consistently, LdcLNs caused microglial reactivity and activation of nod-like receptor protein-3 inflammasomes in the AQP4 null hippocampus. These mice also showed hippocampal neuronal apoptosis and declines in exploring and cognitive abilities. Deletion of AQP4, but not LdcLNs, increased brain water content. Together, these findings have revealed respective and interactive roles of the glymphatic system and the dural lymphatic system in maintaining amyloid beta, Tau proteins and water homeostasis in the brain, helping to understand the pathogenesis of neurological diseases associated with mis-accumulation of brain macromolecules.

Complement-dependent bystander injury to neurons in AQP4-IgG seropositive neuromyelitis optica.

BACKGROUND: Aquaporin-4-immunoglobulin G (AQP4-IgG) seropositive neuromyelitis optica spectrum disorder (herein called NMO) is an autoimmune disease of the central nervous system in which AQP4-IgG binding to AQP4 on astrocytes results in complement-dependent astrocyte injury and secondary inflammation, demyelination, and neuron loss. We previously reported evidence for a complement bystander mechanism for early oligodendrocyte injury in NMO. Herein, we tested the hypothesis that complement bystander injury, which involves diffusion to nearby cells of activated soluble complement components from complement-injured astrocytes, is a general phenomenon that may contribute to neuronal injury in NMO. METHODS: Primary cocultures of rat astrocytes and cortical neurons were established to study complement-dependent cell death after exposure to AQP4-IgG and complement. In animal experiments, AQP4-IgG was delivered to adult rats by intracerebral injection. Cell cultures and rat brain were studied by immunofluorescence. RESULTS: In primary astrocyte-neuron cocultures, addition of AQP4-IgG and complement resulted in death of neurons nearby astrocytes. Deposition of complement membrane attack complex C5b-9 was seen on neurons nearby astrocytes, whereas C1q, the initiating protein in the complement pathway, was seen only on astrocytes. Neuron death was not seen with a complement inhibitor, with C1q- or C6-depleted complement, in pure neuron cultures exposed to AQP4-IgG and complement or in cocultures exposed to an astrocyte toxin. Intracerebral injection in rats of AQP4-IgG and a fixable dead cell fluorescent marker produced death of neurons near astrocytes, with C5b-9 deposition. Neuron death was not seen in rats receiving a complement inhibitor or in AQP4-IgG-injected AQP4 knockout rats. CONCLUSION: These results support a novel mechanism for early neuron injury in NMO and provide evidence that complement bystander injury may be a general phenomenon for brain cell injury following AQP4-IgG-targeted astrocyte death.

Aquaporin-4 knockout mice exhibit increased hypnotic susceptibility to ketamine.

PURPOSE: This study examines anesthetic/hypnotic effects of ketamine in AQP4 knockout (KO) and wild-type (WT) mice with the particular focus on neurotransmission. MATERIALS AND METHODS: Ketamine (100 mg/kg) was intraperitoneally injected in 16 WT and 16 KO mice. The hypnotic potencies were evaluated by the loss of the righting reflex (LORR). The amino acids neurotransmitter levels in prefrontal cortex were measured by microdialysis. RESULTS: This study demonstrated that AQP4 knockout significantly shortened the latency compared with WT mice (98.0 ± 4.2 vs. 138.1 ± 15.0 s, p < .05) and prolonged duration of LORR (884.7 ± 58.6 vs. 562.0 ± 51.7 s, p < .05) compared with WT mice in LORR induced by ketamine. Microdialysis showed that lack of AQP4 markedly decreased glutamate level within 20 min (p < .05) and increased γ-aminobutyric acid (GABA) level within 30-40 min (p < .05) after use of ketamine. Moreover, the levels of taurine were remarkably higher in KO mice than in WT mice, but no obvious differences in aspartate were observed between two genotypes. CONCLUSION: AQP4 deficiency led to more susceptibility of mice to ketamine, which is probably due to the modulation of specific neurotransmitters, hinting an essential maintenance of synaptic activity mediated by AQP4 in the action of ketamine.

Aquaporin-4 deletion ameliorates hypoglycemia-induced BBB permeability by inhibiting inflammatory responses.

BACKGROUND: Severe hypoglycemia induces brain edema by upregulating aquaporin-4 (AQP4) expression and by degrading tight junctions. Acute severe hypoglycemia induces a proinflammatory environment that may contribute to a disruption in the epithelial barrier by decreasing tight junction protein expression. Interestingly, the altered AQP4 expression has been considered to play a critical role in neuroinflammation during acute brain injury. It has been shown that AQP4 deletion reduces brain inflammation in AQP4-null mice after intracerebral LPS injection. However, the effect of AQP4 deletion regarding protection against hypoglycemia-induced blood-brain barrier (BBB) breakdown is unknown. METHODS: An acute severe hypoglycemic stress model was established via injection of 4 unit/kg body weight of insulin. Evans blue (EB) staining and water measurement were used to assess BBB permeability. Western blot, reverse transcription polymerase chain reaction, and immunofluorescence were used to detect the expression of related proteins. The production of cytokines was assessed via enzyme-linked immunosorbent assay. RESULTS: Hypoglycemia-induced brain edema and BBB leakage were reduced in AQP4-/- mice. AQP4 deletion upregulated PPAR-γ and inhibited proinflammatory responses. Moreover, knockdown of aquaporin-4 by small interfering RNA in astrocytes co-cultured with endothelial cells effectively reduced transendothelial permeability and degradation of tight junctions. Treatment with PPAR-γ inhibitors showed that upregulation of PPAR-γ was responsible for the protective effect of AQP4 deletion under hypoglycemic conditions. CONCLUSIONS: Our data suggest that AQP4 deletion protects BBB integrity by reducing inflammatory responses due to the upregulation of PPAR-γ expression and attenuation of proinflammatory cytokine release. Reduction in AQP4 may be protective in acute severe hypoglycemia.

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen-induced retinopathy: beneficial effect of the absence of AQP4.

Hypoxia-dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin-4 (AQP4) is involved in the hypoxia-dependent VEGF upregulation in the retina of a mouse model of oxygen-induced retinopathy (OIR). The expression levels of VEGF, the hypoxia-inducible factor-1α (HIF-1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF-1 binding site (HBS) in the VEGF gene promoter, the binding of HIF-1α to the HBS, the retinal vascularization and function have been determined in the retina of wild-type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF-1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF-1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF-1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF-induced retinal damage in response to hypoxia.

Dissociation of blood-brain barrier disruption and disease manifestation in an aquaporin-4-deficient mouse model of amyotrophic lateral sclerosis.

Aquaporin-4 (AQP4) is abundantly expressed in the central nervous system and is involved in the water balance in the cellular environment. Previous studies have reported that AQP4 expression is upregulated in rat models of amyotrophic lateral sclerosis (ALS), a fatal disease that affects motor neurons in the brain and spinal cord. In this study, we report that astrocytic AQP4 overexpression is evident during the course of disease in the spinal cord of an ALS mouse model, as well as in tissue from patients with ALS. AQP4 overexpression appears to be specifically associated with ALS because it was not induced by other experimental manipulations that produced acute or chronic gliosis. In order to examine the contribution of AQP4 to ALS disease development, we crossed AQP4 knockout mice with a mouse model of ALS. Significant improvement in blood-brain barrier (BBB) permeability was observed in the AQP4-deficient ALS mouse model. However, the time to disease onset and total lifespan were reduced in the AQP4-deficient ALS mouse model. The contradictory results suggest that changes in AQP4 may be context-dependent and further studies are required to understand the precise contribution of brain water balance in ALS.

Aquaporin-4 as a New Target against Methamphetamine-Induced Brain Alterations: Focus on the Neurogliovascular Unit and Motivational Behavior.

Methamphetamine (METH) abuse/misuse is a worldwide problem, and despite extensive characterization of its neurotoxicity over the last years, many questions remain unanswered. Recently, it was shown that METH compromises the blood-brain barrier (BBB) and causes a disturbance in the water homeostasis leading to brain edema. Importantly, water transport at BBB is regulated by water channels, aquaporins (AQPs), with AQP4 being expressed in astrocytic end-feet surrounding brain endothelium. Thus, the main goal of this work was to unravel the role of AQP4 under conditions of METH consumption. Our results show that METH (4× 10 mg/kg, 2 h apart, i.p.) interferes with AQP4 protein levels causing brain edema and BBB breakdown in both mice striatum and hippocampus, which culminated in locomotor and motivational impairment. Furthermore, these effects were prevented by pharmacological blockade of AQP4 with a specific inhibitor (TGN-020). Moreover, siRNA knockdown of this water channel protected astrocytes from METH-induced swelling and morphologic alterations. Herein, we unraveled AQP4 as a new therapeutic target to prevent the negative impact of METH.

Test of the 'glymphatic' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma.

Transport of solutes through brain involves diffusion and convection. The importance of convective flow in the subarachnoid and paravascular spaces has long been recognized; a recently proposed 'glymphatic' clearance mechanism additionally suggests that aquaporin-4 (AQP4) water channels facilitate convective transport through brain parenchyma. Here, the major experimental underpinnings of the glymphatic mechanism were re-examined by measurements of solute movement in mouse brain following intracisternal or intraparenchymal solute injection. We found that: (i) transport of fluorescent dextrans in brain parenchyma depended on dextran size in a manner consistent with diffusive rather than convective transport; (ii) transport of dextrans in the parenchymal extracellular space, measured by 2-photon fluorescence recovery after photobleaching, was not affected just after cardiorespiratory arrest; and (iii) Aqp4 gene deletion did not impair transport of fluorescent solutes from sub-arachnoid space to brain in mice or rats. Our results do not support the proposed glymphatic mechanism of convective solute transport in brain parenchyma.

AQP4-knockout aggravation of isoprenaline-induced myocardial injury is mediated by p66Shc and endoplasmic reticulum stress.

Aquaporin 4 (AQP4) is a type of water channel protein that maintains the water balance of cardiomyocytes. However, the physiological role of AQP4 in cardiovascular disease is poorly understood. We wanted to explore whether p66Shc and endoplasmic reticulum stress participates in AQP4 knockout (KO)-mediated cardiac injury. There were two types of mice: AQP4 knockout and wild-type mice. Each type was randomly divided into three groups: Control group, isoprenaline stimulation group (ISO, 1 mg/kg, s.c., 5 days), and apocynin treatment group (APO, 100 mg/kg, p.o., 3 days). H9c2 rat cardiomyocytes were cultured for RNA interference of AQP4. Results showed increased left ventricular weight index and more severe myocardial inflammation were induced in AQP4 knockout mice relative to wild-type mice, accompanied by significantly increased levels of the oxidative stress biomarkers MDA and NOX4. In addition, the expressions of p66Shc, ER stress markers PERK, GRP78 and CHOP and proinflammatory factors such as ETA , IL6 and TNFα were upregulated in the myocardium of AQP4 knockout mice or AQP4 siRNA treated cardiomyocytes, whereas CASQ2 was downregulated. ISO stimulation aggravated these abnormalities, which were significantly attenuated by apocynin. This study showed that AQP4 knockout mice were susceptible to cardiac injury induced by ISO. The mechanism was closely connected with p66Shc and proinflammatory factors. Endoplasmic reticulum stress was also involved in the pathological process.

Targeted deletion of Aqp4 promotes the formation of astrocytic gap junctions.

Aquaporin-4 (AQP4) is the predominant water channel in the brain and is expressed in high density in astrocytes. By fluxing water along osmotic gradients, AQP4 contributes to brain volume and ion homeostasis. Here we ask whether deletion of Aqp4 leads to upregulation of the gap junctional proteins connexin-43 (Cx43) and connexin-30 (Cx30). These molecules couple adjacent astrocytes to each other and allow water and ions to redistribute within the astrocyte syncytium. Immunogold analysis of parietal cortex and hippocampus showed that the number of gap junctions per capillary profile is increased in AQP4 knockout (AQP4 KO) mice. The most pronounced changes were observed for Cx43 in hippocampus where the number of connexin labeled gap junctions increased by 100% following AQP4 KO. Western blot analysis of whole tissue homogenates showed no change in the amount of Cx43 or Cx30 protein after AQP4 KO. However, AQP4 KO led to a significant increase in the amount of Cx43 in a Triton X-100 insoluble fraction. This fraction is associated with connexin assembly into gap junctional plaques in the plasma membrane. In line with our immunoblot data, RT-qPCR showed no significant increase in Cx43 and Cx30 mRNA levels after AQP4 KO. Our findings suggest that AQP4 KO leads to increased aggregation of Cx43 into gap junctions and provide a putative mechanistic basis for the enhanced tracer coupling in hippocampi of AQP4 KO mice. The increased number of gap junctions in AQP4 deficient mice may explain why Aqp4 deletion has rather modest effects on brain volume and K+ homeostasis.