Decreased expression of the glial water channel aquaporin-4 in the intrahippocampal kainic acid model of epileptogenesis

Recent evidence suggests that astrocytes may be a potential new target for the treatment of epilepsy. The glial water channel aquaporin-4 (AQP4) is expressed in astrocytes, and along with the inwardly-rectifying K(+) channel K(ir)4.1 is thought to underlie the reuptake of H(2)O and K(+) into glial cells during neural activity. Previous studies have demonstrated increased seizure duration and slowed potassium kinetics in AQP4(-/-) mice, and redistribution of AQP4 in hippocampal specimens from patients with chronic epilepsy. However, the regulation and role of AQP4 during epileptogenesis remain to be defined. In this study, we examined the expression of AQP4 and other glial molecules (GFAP, K(ir)4.1, glutamine synthetase) in the intrahippocampal kainic acid (KA) model of epilepsy and compared behavioral and histologic outcomes in wild-type mice vs. AQP4(-/-) mice. Marked and prolonged reduction in AQP4 immunoreactivity on both astrocytic fine processes and endfeet was observed following KA status epilepticus in multiple hippocampal layers. In addition, AQP4(-/-) mice had more spontaneous recurrent seizures than wild-type mice during the first week after KA SE as assessed by chronic video-EEG monitoring and blinded EEG analysis. While both genotypes exhibited similar reactive astrocytic changes, granule cell dispersion and CA1 pyramidal neuron loss, there were an increased number of fluorojade-positive cells early after KA SE in AQP4(-/-) mice. These results indicate a marked reduction of AQP4 following KA SE and suggest that dysregulation of water and potassium homeostasis occurs during early epileptogenesis. Restoration of astrocytic water and ion homeostasis may represent a novel therapeutic strategy.     

Functional changes in astroglial cells in epilepsy

Epilepsy comprises a group of disorders characterized by the periodic occurrence of seizures, and pathologic specimens from patients with temporal lobe epilepsy demonstrate marked reactive gliosis. Since recent studies have implicated glial cells in novel physiological roles in the CNS, such as modulation of synaptic transmission, it is plausible that glial cells may have a functional role in the hyperexcitability characteristic of epilepsy. Indeed, alterations in distinct astrocyte membrane channels, receptors and transporters have all been associated with the epileptic state. This review integrates the current evidence regarding astroglial dysfunction in epilepsy and the potential underlying mechanisms of hyperexcitability. Functional understanding of the cellular and molecular alterations of astroglia-dependent hyperexcitability will help to clarify the physiological role of astrocytes in neural function as well as lead to the identification of novel therapeutic targets.     

Unique expression and localization of aquaporin-4 and aquaporin-9 in murine and human neural stem cells and in their glial progeny

Aquaporins (AQP) are water channel proteins that play important roles in the regulation of water homeostasis in physiological and pathological conditions. AQP4 and AQP9, the main aquaporin subtypes in the brain, are expressed in the adult forebrain subventricular zone (SVZ), where neural stem cells (NSCs) reside, but little is known about their expression and role in the NSC population, either in vivo or in vitro. Also, no reports are available on the presence of these proteins in human NSCs. We performed a detailed molecular and phenotypical characterization of different AQPs, and particularly AQP4 and AQP9, in murine and human NSC cultures at predetermined stages of differentiation. We demonstrated that AQP4 and AQP9 are expressed in adult murine SVZ-derived NSCs (ANSCs) and that their levels of expression and cellular localization are differentially regulated upon ANSC differentiation into neurons and glia. AQP4 (but not AQP9) is expressed in human NSCs and their progeny. The presence of AQP4 and AQP9 in different subsets of ANSC-derived glial cells and in different cellular compartments suggests different roles of the two proteins in these cells, indicating that ANSC-derived astrocytes might maintain in vitro the heterogeneity that characterize the astrocyte-like cell populations in the SVZ in vivo. The development of therapeutic strategies based on modulation of AQP function relies on a better knowledge of the functional role of these channels in brain cells. We provide a reliable and standardized in vitro experimental model to perform functional studies as well as toxicological and pharmacological screenings.     

Aquaporin-4 independent Kir4.1 K+ channel function in brain glial cells

Functional interaction of glial water channel aquaporin-4 (AQP4) and inwardly rectifying K+ channel Kir4.1 has been suggested from their apparent colocalization and biochemical interaction, and from the slowed glial cell K+ uptake in AQP4-deficient brain. Here, we report multiple lines of evidence against functionally significant AQP4-Kir4.1 interactions. Whole-cell patch-clamp of freshly isolated glial cells from brains of wild-type and AQP4 null mice showed no significant differences in membrane potential, barium-sensitive Kir4.1 K+ current or current-voltage curves. Single-channel patch-clamp showed no differences in Kir4.1 unitary conductance, voltage-dependent open probability or current-voltage relationship. Also, Kir4.1 protein expression and distribution were similar in wild-type and AQP4 null mouse brain and in the freshly isolated glial cells. Functional inhibition of Kir4.1 by barium or RNAi knock-down in primary glial cell cultures from mouse brain did not significantly alter AQP4 water permeability, as assayed by calcein fluorescence quenching following osmotic challenge. These studies provide direct evidence against functionally significant AQP4-Kir4.1 interactions in mouse glial cells, indicating the need to identify new mechanism(s) to account for altered seizure dynamics and extracellular space K+ buffering in AQP4 deficiency.     

Impact of aquaporin-4 channels on K+ buffering and gap junction coupling in the hippocampus

Aquaporin-4 (AQP4) is the main water channel in the brain and primarily localized to astrocytes where the channels are thought to contribute to water and K(+) homeostasis. The close apposition of AQP4 and inward rectifier K(+) channels (Kir4.1) led to the hypothesis of direct functional interactions between both channels. We investigated the impact of AQP4 on stimulus-induced alterations of the extracellular K(+) concentration ([K(+)](o)) in murine hippocampal slices. Recordings with K(+)-selective microelectrodes combined with field potential analyses were compared in wild type (wt) and AQP4 knockout (AQP4(-/-)) mice. Astrocyte gap junction coupling was assessed with tracer filling during patch clamp recording. Antidromic fiber stimulation in the alveus evoked smaller increases and slower recovery of [K(+)](o) in the stratum pyramidale of AQP4(-/-) mice indicating reduced glial swelling and a larger extracellular space when compared with control tissue. Moreover, the data hint at an impairment of the glial Na(+)/K(+) ATPase in AQP4-deficient astrocytes. In a next step, we investigated the laminar profile of [K(+)](o) by moving the recording electrode from the stratum pyramidale toward the hippocampal fissure. At distances beyond 300 μm from the pyramidal layer, the stimulation-induced, normalized increases of [K(+)](o) in AQP4(-/-) mice exceeded the corresponding values of wt mice, indicating facilitated spatial buffering. Astrocytes in AQP4(-/-) mice also displayed enhanced tracer coupling, which might underlie the improved spatial re- distribution of [K(+)](o) in the hippocampus. These findings highlight the role of AQP4 channels in the regulation of K(+) homeostasis.     

Glial Pathology in Bipolar Disorder: Potential Therapeutic Implications

Bipolar disorder (BD) is a chronic and severe mental disorder with recurrent episodes of mania and depression. In addition to neuronal alterations, accumulating evidences have revealed the importance of glial system in pathophysiology and phenotype of the illness. Postmortem studies have repeatedly demonstrated the alterations in glial cells and its functions in patients with BD. The activated microglia and inflammatory cytokines are proposed to be the potential biomarkers that may help to predict disease exacerbation in BD. On the other hand, anti‐BD drugs have been shown to produce profound effects on glial activity, which not only contributes to the therapeutic efficacy, but may also provide a potential target for the drug development of BD. We will focus on the recent development of glial abnormalities and potential therapeutic benefits targeted to glial modulation in BD.     

Blood-brain barrier disruption highly induces aquaporin-4 mRNA and protein in perivascular and parenchymal astrocytes: protective effect by estradiol treatment in ovariectomized animals

Strong evidence involves aquaporin-4 (AQP4) in the physiopathology of brain edema. Two major points remain unsolved: (1) the capacity of perivascular glial cells to regulate AQP4 in response to disruption of the blood-brain barrier (BBB); and (2) the potential beneficial role of AQP4 in the clearance of brain edema. We used intraparenchymal injection of lipopolysaccharide (LPS) as an efficient model to induce BBB disruption. This was monitored by IgG extravasation and AQP4 was studied at the mRNA and protein level. The first signs of BBB disruption coincided with strong induction of AQP4 mRNA in perivascular glial cells. At the early phase, estradiol treatment highly prevented the LPS-induced disruption of the BBB and the induction of AQP4. Efficient clearance of vasogenic edema is supposed to occur once BBB is restored. This phase coincided with high induction of AQP4 mRNA in parenchymal reactive astrocytes and perivascular glial processes. High levels of AQP4 mRNA may be beneficial under these conditions. Our data may clarify why estradiol treatment reduces mortality in conditions typically associated with edema formation, like stroke.     

Aquaporin-4 and ischemic brain edema

OBJECTIVE： To investigate the relationship of aquaporin 4 （AQP4） and brain edema. DATA SOURCES： Using the terms of ＂aquaporin-4, brain edema＂, we searched PubMed database to identify studies published from January 1997 to April 2006 in the English languages. Meanwhile, we also searched China National Knowledge Infrastructure （CNKI） for related studies. STUDY SELECTION： The collected data were selected firstly. Studies on AQP4 and brain edema were chosen and their full-texts were searched for, and those with repetitive or review studies were excluded. DATA EXTRACTION： Totally 146 related studies were collected, 42 of them were involved and the other 104 studies were used for reading reference data. DATA SYNTHESIS： AQP4 is a selective water permeable integral membrane protein. It is mainly expressed in astrocytes and ependymocyte, and is the important structural basis for water regulation and transportation between glial cells and cerebrospinal fluid or vessels. Phosphorylation is involved in the regulation of AQP4. AQP4 participates in the formation of brain edema caused by various factors. Studies on the structure and pathological changes of AQP4 are still in the initial stage, and the role and mechanism of AQP4 in the formation of brain edema is very unclear. CONCLUSION： AQP4 plays a critical regulating role in the formation of ischemic brain edema, but whether it is regulated by drugs lacks reliable evidence.     

Immunological function of aquaporin-4 in stab-wounded mouse brain in concert with a pro-inflammatory cytokine inducer,osteopontin

During injury to the central nervous system (CNS), astrocytes and microglia proliferate and migrate around the lesion sites. Recently, it has been reported that one of the water channels, aquaporin-4 (AQP4) is seemed to have a role in astroglial migration and glial scar formation caused by brain injury, although its molecular mechanism is largely unknown. In the present study, we examined the expression profiles in wild-type (WT) and AQP4-deficient (AQP4/KO) mice after a stab wound to the cerebral cortex. Three days after the stab wound, AQP4 expression was observed in activated microglia around the lesion site as well as in astrocytes. A microarray analysis revealed that 444 genes around the lesion site were upregulated 3 days after the wounding in WT mice. Surprisingly, most of these up-regulations were significantly attenuated in AQP4/KO mice. Real-time RT-PCR and immunofluorescence showed that osteopontin (OPN) expression around the lesion site was much lower in AQP4/KO mice than in WT mice. Moreover, the up-regulation of pro-inflammatory cytokines was significantly attenuated in AQP4/KO mice. Taken together, these results suggest that AQP4 plays an important role in immunological function in concert with OPN under pathological conditions in the CNS.     

Aquaporin-4 and spinal cord injury

Edema formation is a major problem following traumatic spinal cord injury (SCI) that acts to exacerbate secondary damage. Severity of edema correlates with reduced neurological outcome in human patients. To date, there are no effective treatments to directly resolve edema within the spinal cord. The aquaporin-4 (AQP4) water channel is found on membranes of astrocytic endfeet in direct contact with blood vessels, the glia limitans in contact with the cerebrospinal fluid and ependyma around the central canal. Being so locally expressed at the interface between fluid and tissue allow AQP4 channels to play an important role in the bidirectional regulation of water homeostasis under normal conditions and following trauma. With the need to better understand the pathophysiology underlying the devastating cellular events in SCI, animal models have become an integral part of exploration. Inevitably, several injury models have been developed (contusion, compression, transection) resulting in difficult interpretation between studies with conflicting results. This is true in the case of understanding the role of AQP4 in the progression and resolution of edema following SCI, whose role is still not completely understood and is highly dependent on the type of edema present (vasogenic vs cytotoxic). Here, we discuss regulation of AQP4 in varying injury models and the effects of potential therapeutic interventions on expression, edema formation and functional recovery. Better understanding of the precise role of AQP4 following a wide range of injuries will help to understand optimal treatment timing following human SCI for prime therapeutic benefit and enhanced neurological outcome.     

Severe alterations of endothelial and glial cells in the blood-brain barrier of dystrophic mdx mice

In this study, we investigated the involvement of the blood-brain barrier (BBB) in the brain of the dystrophin-deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO-1) and claudin-1 proteins, and a glial marker, the aquaporin-4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron-dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO-1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO-1 immunocytochemistry and claudin-1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO-1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO-1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of alpha-actin and ZO-1 revealed colocalization of these proteins. As in ZO-1 staining, the pattern of immunolabeling with anti-alpha-actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. alpha-actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas alpha-actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti-AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4-, CD8-, CD20-, and CD68-positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in alpha-actin cytoskeleton, ZO-1, claudin-1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD.     

Altered blood-brain barrier integrity in adult aquaporin-4 knockout mice

To investigate the role of astroglial water channel aquaporin-4 (AQP4) in maintaining blood-brain barrier integrity, structure and permeability of the brain microvessels were investigated in adult AQP4 knockout mice. Altered ultrastructure of brain microvessels, including open tight junctions and swollen perivascular astrocytic endfeet, were frequently observed in the AQP4 null mice. Likewise, AQP4 deficiency significantly downregulated expression of glial fibrillary acidic protein in perivascular processes of astrocytes. Furthermore, the horseradish peroxidase analysis demonstrated hyperpermeability of the blood-brain barrier in AQP4 knockout mice. These findings provide direct evidence that AQP4 is essential for the maintenance of blood-brain barrier integrity.     

New ideas in epilepsy genetics: novel epilepsy genes, copy number alterations, and gene regulation

The majority of genes associated with epilepsy syndromes to date are ion channel genes. Selection bias may have allowed us to establish their role in epilepsy based on a priori knowledge of the significance of these proteins in regulating neuronal excitability. There are, however, more than 3000 genes expressed at the synapse, as well as many other genes expressed nearby in supporting cells and glia that can likewise regulate excitability. Identification of new genes involved in epilepsy may arise from studying the targets of anticonvulsant medications, ascertainment of an epileptic phenotype in mice, or as a result of positional cloning efforts. There are several loci for idiopathic focal and generalized epilepsies that lie in chromosomal regions that are devoid of known ion channels; therefore, the number of novel genes involved in epilepsy is likely to increase. Establishing the role of these novel genes in the pathogenesis of epilepsy has not been an easy task compared with the relative ease with which ion channel mutations can be studied. This review will describe several novel epilepsy genes and will then discuss other genetic causes of epilepsy, including alterations of chromosomal copy number and gene regulatory elements.     

AQP4 expression in striatal primary cultures is regulated by dopamine – implications for proliferation of astrocytes

Proliferation of astrocytes plays an essential role during ontogeny and in the adult brain, where it occurs following trauma and in inflammation and neurodegenerative diseases as well as in normal, healthy mammals. The cellular mechanisms underlying glial proliferation remain poorly understood. As dopamine is known to modulate proliferation in different cell populations, we investigated the effects of dopamine on the proliferation of striatal astrocytes in vitro. We found that dopamine reduced proliferation. As proliferation involves, among other things, a change in cell volume, which normally comes with water movement across the membrane, water channels might represent a molecular target of the dopamine effect. Therefore we studied the effect of dopamine on aquaporin 4 (AQP4) expression, the main aquaporin subtype expressed in glial cells, and observed a down‐regulation of the AQP4‐M23 isoform. This down‐regulation was the cause of the dopamine‐induced decrease in proliferation as knockdown of AQP4 using siRNA techniques mimicked the effects of dopamine on proliferation. Furthermore, stimulation of glial proliferation by basic fibroblast growth factor was also abolished by knocking down AQP4. In addition, blocking of AQP4 with 10 μm tetraethylammonium inhibited osmotically induced cell swelling and stimulation of glial cell proliferation by basic fibroblast growth factor. These results demonstrate a clear‐cut involvement of AQP4 in the regulation of proliferation and implicate that modulation of AQP4 could be used therapeutically in the treatment of neurodegenerative diseases as well as in the regulation of reactive astrogliosis by preventing or reducing the glia scar formation, thus improving regeneration following ischemia or other trauma.     

Blood-brain barrier integrity in the pathogenesis of Alzheimer’s disease

The blood–brain barrier (BBB) tightly controls the molecular exchange between the brain parenchyma and blood. Accumulated evidence from transgenic animal Alzheimer’s disease (AD) models and human AD patients have demonstrated that BBB dysfunction is a major player in AD pathology. In this review, we discuss the role of the BBB in maintaining brain integrity and how this is mediated by crosstalk between BBB-associated cells within the neurovascular unit (NVU). We then discuss the role of the NVU, in particular its endothelial cell, pericyte, and glial cell constituents, in AD pathogenesis. The effect of substances released by the neuroendocrine system in modulating BBB function and AD pathogenesis is also discussed. We perform a systematic review of currently available AD treatments specifically targeting pericytes and BBB glial cells. In summary, this review provides a comprehensive overview of BBB dysfunction in AD and a new perspective on the development of therapeutics for AD.     

The relevance of individual genetic background and its role in animal models of epilepsy

Growing evidence has indicated that genetic factors contribute to the etiology of seizure disorders. Most epilepsies are multifactorial, involving a combination of additive and epistatic genetic variables. However, the genetic factors underlying epilepsy have remained unclear, partially due to epilepsy being a clinically and genetically heterogeneous syndrome. Similar to the human situation, genetic background also plays an important role in modulating both seizure susceptibility and its neuropathological consequences in animal models of epilepsy, which has too often been ignored or not been paid enough attention to in published studies. Genetic homogeneity within inbred strains and their general amenability to genetic manipulation have made them an ideal resource for dissecting the physiological function(s) of individual genes. However, the inbreeding that makes inbred mice so useful also results in genetic divergence between them. This genetic divergence is often unaccounted for but may be a confounding factor when comparing studies that have utilized distinct inbred strains. The purpose of this review is to discuss the effects of genetic background strain on epilepsy phenotypes of mice, to remind researchers that the background genetics of a knockout strain can have a profound influence on any observed phenotype, and outline the means by which to overcome potential genetic background effects in experimental models of epilepsy.     

Increased seizure threshold in mice lacking aquaporin-4 water channels

Mice deficient in the glial water channel aquaporin-4 (AQP4) show decreased cerebral edema and improved neurological outcome following water intoxication or ischemic challenge. In this report, we tested seizure susceptibility in AQP4 mice. AQP4 mice and wild-type controls were given the chemoconvulsant pentylenetetrazol (PTZ) and monitored for seizure activity. At 40 mg/kg PTZ, all wild-type mice exhibited seizure activity, whereas six of seven AQP4 mice did not exhibit seizure activity. At 50 mg/kg PTZ, both groups exhibited seizure activity; however, the latency to generalized (tonic-clonic) seizures was significantly lower in wild-type than AQP4 mice. These results suggest that glial water channels may modulate brain excitability and the initiation and generalization of seizure activity.     

Aquaporin 4‐Dependent expression of glial fibrillary acidic protein and tenascin‐C in activated astrocytes in stab wound mouse brain and in primary culture

We previously reported that aquaporin 4 (AQP4) has a neuroimmunological function via astrocytes and microglial cells involving osteopontin. AQP4 is a water channel localized in the endofoot of astrocytes in the brain, and its expression is upregulated after a stab wound to the mouse brain or the injection of methylmercury in common marmosets. In this study, the correlation between the expression of AQP4 and the expression of glial fibrillary acidic protein (GFAP) or tenascin‐C (TN‐C) in reactive astrocytes was examined in primary cultures and brain tissues of AQP4‐deficient mice (AQP4/KO). In the absence of a stab wound to the brain or of any stimulation of the cells, the expressions of both GFAP and TN‐C were lower in astrocytes from AQP4/KO mice than in those from wild‐type (WT) mice. High levels of GFAP and TN‐C expression were observed in activated astrocytes after a stab wound to the brain in WT mice; however, the expressions of GFAP and TN‐C were insignificant in AQP4/KO mice. Furthermore, lipopolysaccharide (LPS) stimulation activated primary culture of astrocytes and upregulated GFAP and TN‐C expression in cells from WT mice, whereas the expressions of GFAP and TN‐C were slightly upregulated in cells from AQP4/KO mice. Moreover, the stimulation of primary culture of astrocytes with LPS also upregulated inflammatory cytokines in cells from WT mice, whereas modest increases were observed in cells from AQP4/KO mice. These results suggest that AQP4 expression accelerates GFAP and TN‐C expression in activated astrocytes induced by a stab wound in the mouse brain and LPS‐stimulated primary culture of astrocytes. © 2014 Wiley Periodicals, Inc.