Molecular scaffolds underpinning macroglial polarization: An analysis of retinal Müller cells and brain astrocytes in mouse

Key roles of macroglia are inextricably coupled to specialized membrane domains. The perivascular endfoot membrane has drawn particular attention, as this domain contains a unique complement of aquaporin‐4 (AQP4) and other channel proteins that distinguishes it from perisynaptic membranes. Recent studies indicate that the polarization of macroglia is lost in a number of diseases, including temporal lobe epilepsy and Alzheimer's disease. A better understanding is required of the molecular underpinning of astroglial polarization, particularly when it comes to the significance of the dystrophin associated protein complex (DAPC). Here, we employ immunofluorescence and immunogold cytochemistry to analyze the molecular scaffolding in perivascular endfeet in macroglia of retina and three regions of brain (cortex, dentate gyrus, and cerebellum), using AQP4 as a marker. Compared with brain astrocytes, Müller cells (a class of retinal macroglia) exhibit lower densities of the scaffold proteins dystrophin and α‐syntrophin (a DAPC protein), but higher levels of AQP4. In agreement, depletion of dystrophin or α‐syntrophin—while causing a dramatic loss of AQP4 from endfoot membranes of brain astrocytes—had only modest or insignificant effect, respectively, on the AQP4 pool in endfoot membranes of Müller cells. In addition, while polarization of brain macroglia was less affected by dystrophin depletion than by targeted deletion of α‐syntrophin, the reverse was true for retinal macroglia. These data indicate that the molecular scaffolding in perivascular endfeet is more complex than previously assumed and that macroglia are heterogeneous with respect to the mechanisms that dictate their polarization. © 2012 Wiley Periodicals, Inc.     

Anti‐aquaporin‐4 antibodies in the context of assorted immune‐mediated diseases

Background and purposes: Anti‐aquaporin 4 antibodies are specific markers for Devic‘s disease. This study aimed to test if this high specificity holds in the context of a large spectrum of systemic autoimmune and non‐autoimmune diseases. Methods: Anti‐aquaporin‐4 antibodies (NMO‐IgG) were determined by indirect immunofluorescence (IIF) on mouse cerebellum in 673 samples, as follows: group I (clinically defined Devic’s disease, n = 47); group II [inflammatory/demyelinating central nervous system (CNS) diseases, n = 41]; group III (systemic and organ‐specific autoimmune diseases, n = 250); group IV (chronic or acute viral diseases, n = 35); and group V (randomly selected samples from a general clinical laboratory, n = 300). Results: MNO‐IgG was present in 40/47 patients with classic Devic’s disease (85.1% sensitivity) and in 13/22 (59.1%) patients with disorders related to Devic’s disease. The latter 13 positive samples had diagnosis of longitudinally extensive transverse myelitis (n = 10) and isolated idiopathic optic neuritis (n = 3). One patient with multiple sclerosis and none of the remaining 602 samples with autoimmune and miscellaneous diseases presented NMO‐IgG (99.8% specificity). The autoimmune disease subset included five systemic lupus erythematosus individuals with isolated or combined optic neuritis and myelitis and four primary Sjögren’s syndrome (SS) patients with cranial/peripheral neuropathy. Conclusions: The available data clearly point to the high specificity of anti‐aquaporin‐4 antibodies for Devic’s disease and related syndromes also in the context of miscellaneous non‐neurologic autoimmune and non‐autoimmune disorders.     

Deletion of aquaporin-4 changes the perivascular glial protein scaffold without disrupting the brain endothelial barrier

Expression of the water channel aquaporin-4 (AQP4) at the blood-brain interface is dependent upon the dystrophin associated protein complex. Here we investigated whether deletion of the Aqp4 gene affects the molecular composition of this protein scaffold and the integrity of the blood-brain barrier. High-resolution immunogold cytochemistry revealed that perivascular expression of α-syntrophin was reduced by 60% in Aqp4(-/-) mice. Additionally, perivascular AQP4 expression was reduced by 88% in α-syn(-/-) mice, in accordance with earlier reports. Immunofluorescence showed that Aqp4 deletion also caused a modest reduction in perivascular dystrophin, whereas β-dystroglycan labeling was unaltered. Perivascular microglia were devoid of AQP4 immunoreactivity. Deletion of Aqp4 did not alter the ultrastructure of capillary endothelial cells, the expression of tight junction proteins (claudin-5, occludin, and zonula occludens 1), or the vascular permeability to horseradish peroxidase and Evans blue albumin dye. We conclude that Aqp4 deletion reduces the expression of perivascular glial scaffolding proteins without affecting the endothelial barrier. Our data also indicate that AQP4 and α-syntrophin are mutually dependent upon each other for proper perivascular expression.     

Differences in distribution and regulation of astrocytic aquaporin‐4 in human and rat hydrocephalic brain

A. D. Skjolding, A. V. Holst, H. Broholm, H. Laursen and M. Juhler (2013) Neuropathology and Applied Neurobiology 39, 179–191 Differences in distribution and regulation of astrocytic aquaporin‐4 in human and rat hydrocephalic brain Aims: Aquaporin‐4 (AQP4) is the most abundant cellular water channel in brain and could be a molecular basis for a cerebrospinal fluid absorption route additional to the arachnoid villi. In the search for ‘alternative’ cerebrospinal fluid absorption pathways it is important to compare experimental findings with human pathophysiology. This study compares expression of AQP4 in hydrocephalic human brain with human controls and hydrocephalic rat brain. Methods: Cortical biopsies from patients with chronic hydrocephalus (n = 29) were sampled secondary to planned surgical intervention. AQP4 in human hydrocephalic cortex relative to controls was quantified by Western blotting (n = 28). A second biopsy (n = 13) was processed for immunohistochemistry [glial fibrillary acidic protein (GFAP), CD68, CD34 and AQP4] and double immunofluorescence (AQP4 + GFAP and AQP4 + CD34). Brain tissue from human controls and kaolin‐induced hydrocephalic rats was processed in parallel. Immunohistochemistry and immunofluorescence were assessed qualitatively. Results: Western blotting showed that AQP4 abundance was significantly increased (P < 0.05) in hydrocephalic human brain compared with controls. AQP4 immunoreactivity was present in both white and grey matter. In human brain (hydrocephalic and controls) AQP4 immunoreactivity was found on the entire astrocyte membrane, unlike hydrocephalic rat brain where pronounced endfeet polarization was present. Endothelial AQP4 immunoreactivity was not observed. Conclusions: This study shows a significant increase in astrocytic AQP4 in human hydrocephalic cortex compared with control. Cell type specific expression in astrocytes is conserved between rat and human, although differences of expression in specific membrane domains are seen. This study addresses direct translational aspects from rat to human, hereby emphasizing the relevance and use of models in hydrocephalus research.     

Phosphorylation of rat aquaporin‐4 at Ser111 is not required for channel gating

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. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)‐dependent phosphorylation of Ser¹¹¹ has been reported to increase the water permeability of AQP4 expressed in an astrocytic cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser¹¹¹ was indeed a site involved in phosphorylation‐mediated gating of AQP4. The water permeability of AQP4‐expressing Xenopus oocytes was not altered by a range of activators and inhibitors of PKG and PKA. Mutation of Ser¹¹¹ to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of AQP4. PKG activation had no effect on the water permeability of AQP4 in primary cultures of rat astrocytes. Molecular dynamics simulations of a phosphorylation of AQP4.Ser¹¹¹ recorded no phosphorylation‐induced change in water permeability. A phospho‐specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser¹¹¹, failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. Thus, our data indicate a lack of phosphorylation of Ser¹¹¹ and of phosphorylation‐dependent gating of AQP4.     

Aquaporin‐4 regulates the velocity and frequency of cortical spreading depression in mice

The astrocyte water channel aquaporin‐4 (AQP4) regulates extracellular space (ECS) K⁺ concentration ([K⁺]_e) and volume dynamics following neuronal activation. Here, we investigated how AQP4‐mediated changes in [K⁺]_e and ECS volume affect the velocity, frequency, and amplitude of cortical spreading depression (CSD) depolarizations produced by surface KCl application in wild‐type (AQP4^+/+) and AQP4‐deficient (AQP4^?/?) mice. In contrast to initial expectations, both the velocity and the frequency of CSD were significantly reduced in AQP4^?/? mice when compared with AQP4^+/+ mice, by 22% and 32%, respectively. Measurement of [K⁺]_e with K⁺‐selective microelectrodes demonstrated an increase to ～35 mM during spreading depolarizations in both AQP4^+/+ and AQP4^?/? mice, but the rates of [K⁺]_e increase (3.5 vs. 1.5 mM/s) and reuptake (t_1/2 33 vs. 61 s) were significantly reduced in AQP4^?/? mice. ECS volume fraction measured by tetramethylammonium iontophoresis was greatly reduced during depolarizations from 0.18 to 0.053 in AQP4^+/+ mice, and 0.23 to 0.063 in AQP4^?/? mice. Analysis of the experimental data using a mathematical model of CSD propagation suggested that the reduced velocity of CSD depolarizations in AQP4^?/? mice was primarily a consequence of the slowed increase in [K⁺]_e during neuronal depolarization. These results demonstrate that AQP4 effects on [K⁺]_e and ECS volume dynamics accelerate CSD propagation. GLIA 2015;63:1860–1869     

Laminar and subcellular heterogeneity of GLAST and GLT‐1 immunoreactivity in the developing postnatal mouse hippocampus

Astrocytes express two sodium‐coupled transporters, glutamate–aspartate transporter (GLAST) and glutamate transporter‐1 (GLT‐1), which are essential for the maintenance of low extracellular glutamate levels. We performed a comparative analysis of the laminar and subcellular expression profile of GLAST and GLT‐1 in the developing postnatal mouse hippocampus by using immunohistochemistry and western blotting and employing high‐resolution fluorescence microscopy. Astrocytes were identified by costaining with glial fibrillary acidic protein (GFAP) or S100β. In CA1, the density of GFAP‐positive cells and GFAP expression rose during the first 2 weeks after birth, paralleled by a steady increase in GLAST immunoreactivity and protein content. Upregulation of GLT‐1 was completed only at postnatal days (P) P20–25 and was thus delayed by about 10 days. GLAST staining was highest along the stratum pyramidale and was especially prominent in astrocytes at P3–5. GLAST immunoreactivity indicated no preferential localization to a specific cellular compartment. GLT‐1 exhibited a laminar expression pattern from P10–15 on, with the highest immunoreactivity in the stratum lacunosum‐moleculare. At the cellular level, GLT‐1 immunoreactivity did not entirely cover astrocyte somata and exhibited clusters at processes. In neonatal and juvenile animals, discrete clusters of GLT‐1 were also detected at perivascular endfeet. From these results, we conclude there is a remarkable subcellular heterogeneity of GLAST and GLT‐1 expression in the developing hippocampus. The clustering of GLT‐1 at astrocyte endfeet indicates that it might serve a specialized functional role at the blood–brain barrier during formation of the hippocampal network. J. Comp. Neurol. 522:204–224, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

Neuromyelitis optica IgG does not alter aquaporin‐4 water permeability,plasma membrane M1/M23 isoform content,or supramolecular assembly

Neuromyelitis optica (NMO) is thought to be caused by immunoglobulin G autoantibodies (NMO‐IgG) against astrocyte water channel aquaporin‐4 (AQP4). A recent study (Hinson et al. (2012) Proc Natl Acad Sci USA 109:1245‐1250) reported that NMO‐IgG inhibits AQP4 water permeability directly and causes rapid cellular internalization of the M1 but not M23 isoform of AQP4, resulting in AQP4 clustering, enhanced complement‐dependent cytotoxicity, and tissue swelling. Here, we report evidence challenging this proposed mechanism of NMO‐IgG‐mediated pathology. We measured osmotic water permeability by stopped‐flow light scattering on plasma membrane vesicles isolated from AQP4‐expressing CHO cells, an approach that can detect changes in water permeability as small as 5% and is not confounded by internalization effects. We found similar single‐molecule water permeability for M1‐AQP4 tetramers and M23‐AQP4 clusters (orthogonal arrays of particles, OAPs). Exposure of AQP4 to high concentrations of NMO‐IgG from six seropositive NMO patients, and to high‐affinity recombinant monoclonal NMO antibodies, did not reduce AQP4 water permeability. Also, NMO‐IgG did not reduce water permeability in AQP4‐reconstituted proteoliposomes. In transfected cells expressing M1‐ or M23‐AQP4 individually, NMO‐IgG caused more rapid internalization of M23‐ than M1‐AQP4. In cells coexpressing both isoforms, M1‐ and M23‐AQP4 comingled in OAPs that were internalized together in response to NMO‐IgG. Super‐resolution imaging and native gel electrophoresis showed that the size of AQP4 OAPs was not altered by NMO sera or recombinant NMO antibodies. We conclude that NMO‐IgG does not: (i) inhibit AQP4 water permeability, (ii) cause preferential internalization of M1‐AQP4, or (iii) cause intramembrane AQP4 clustering. © 2012 Wiley Periodicals, Inc.     

Translational regulation mechanisms of aquaporin-4 supramolecular organization in astrocytes

The two predominant isoforms of Aquaporin-4 (AQP4), AQP4-M23 and AQP4-M1, assemble in the plasma membrane to form supramolecular structures called Orthogonal Array of Particles (OAPs) whose dimension is tightly associated to the M1/M23 ratio. Here, we explore translational regulation contribution to M1/M23 expression in primary cultures of rat astrocytes, and analyze the role of M1 mRNA 5'untranslated region (5'UTR) in this mechanism. Using isoform-specific RNAi we found that in rat astrocytes primary cultures a large proportion of M23 protein derives from M1 mRNA translation. Furthermore, site-specific mutagenesis of the 5'UTR sequence of AQP4-M1 mRNA indicates that a multiple-site leaky scanning mechanism, an out-of-frame upstream ORF (uORF), and a reinitiation mechanism are able to modulate the M1/M23 ratio and consequently, OAPs formation. These mechanisms are likely to be shared by different species, including human, and they can also be assumed to play a role in those pathophysiological situations where the organization of AQP4 in supramolecular structures (OAPs) is involved. Finally, we report that, when transfected in Hela cells, the longer rat AQP4 isoform, called Mz, which is not present in human impairs OAPs formation.     

Actin cytoskeleton remodeling governs aquaporin-4 localization in astrocytes

Aquaporin-4 (AQP4) is constitutively concentrated in the plasma membrane of the perivascular glial processes, and its expression is altered in certain pathological conditions associated with brain edema or altered glial migration. When astrocytes are grown in culture, they lose their characteristic star-like shape and AQP4 continuous plasma membrane localization observed in vivo. In this study, we differentiated primary astrocyte cultures with cAMP and lovastatin, both able to induce glial stellation through a reorganization of F-actin cytoskeleton, and obtained AQP4 selectively localized on the cell plasma membrane associated with an increase in the plasma membrane water transport level, but only cAMP induced an increase in AQP4 total protein expression. Phosphorylation experiments indicated that AQP4 in astrocytes is neither phosphorylated nor a substrate of PKA. Depolymerization of F-actin cytoskeleton performed by cytochalasin-D suggested that F-actin cytoskeleton plays a primary role for AQP4 plasma membrane localization and during cell adhesion. Finally, AQP4 knockdown does not compromise the ability of astrocytes to stellate in the presence of cAMP, indicating that astrocyte stellation is independent of AQP4.     

Region‐specific expression of a water channel protein,aquaporin 4, on brain astrocytes

Cellular activities within the brain display regional specificity and a neuronal and glia interdependence. Components characterizing the regional specificity of neurons have been identified. However, characterization of the astrocyte remains in question. To identify region specific features of astrocytes, we have characterized the molecular phenotype of cells derived from regions with different levels of neuronal excitability, the cortex and striatum. Astrocytes were identified in cryostat sections of adult rat brain by rapid immunostaining for glial fibrillary acidic protein (GFAP), and individual cells were collected from each region by using laser microdissection (LMD). Total RNA was isolated and subjected to DNA microarray analysis. At least eight genes showed a differential expression level. Among them, aquaporin 4 (AQP4), a water channel protein, was expressed at higher levels within the cortex compared with the striatum, as confirmed by immunohistochemistry. Primary cultured astrocytes isolated from rat cortex or striatum also showed a differential expression of AQP4. These data may reflect unique properties of astrocytes across different brain regions. However, they may also reflect the interactive demands of neurons with different activity levels. Further examination of the heterogeneous astrocyte populations within each region will lend additional support to the regional specificity of neuronal functions and neuronal–glial interactions. © 2012 Wiley Periodicals, Inc.     

Changes in the astrocytic aquaporin‐4 and inwardly rectifying potassium channel expression in the brain of the amyotrophic lateral sclerosis SOD1G93A rat model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting upper and lower motor neurons. Dysfunction and death of motor neurons are closely related to the modified astrocytic environment. Astrocytic endfeet, lining the blood–brain barrier (BBB), are enriched in two proteins, aquaporin‐4 (AQP4) and inwardly rectifying potassium channel (Kir) 4.1. Both channels are important for the maintainance of a functional BBB astrocytic lining. In this study, expression levels of AQP4 and Kir4.1 were for the first time examined in the brainstem and cortex, along with the functional properties of Kir channels in cultured cortical astrocytes of the SOD1^G93A rat model of ALS. Western blot analysis showed increased expression of AQP4 and decreased expression of Kir4.1 in the brainstem and cortex of the ALS rat. In addition, higher immunoreactivity of AQP4 and reduced immunolabeling of Kir4.1 in facial and trigeminal nuclei as well as in the motor cortex were also observed. Particularly, the observed changes in the expression of both channels were retained in cultured astrocytes. Furthermore, whole‐cell patch‐clamp recordings from cultured ALS cortical astrocytes showed a significantly lower Kir current density. Importantly, the potassium uptake current in ALS astrocytes was significantly reduced at all extracellular potassium concentrations. Consequently, the Kir‐specific Cs⁺‐ and Ba²⁺‐sensitive currents were also decreased. The changes in the studied channels, notably at the upper CNS level, could underline the hampered ability of astrocytes to maintain water and potassium homeostasis, thus affecting the BBB, disturbing the neuronal microenvironment, and causing motoneuronal dysfunction and death. © 2012 Wiley Periodicals, Inc.     

Decrease of aquaporin‐4 and excitatory amino acid transporter‐2 indicate astrocyte dysfunction for pathogenesis of cortical degeneration in HIV‐associated neurocognitive disorders

Human immunodeficiency virus (HIV) encephalitis and degeneration of cerebral cortex are established histopathologies of HIV‐associated neurocognitive disorders (HAND). We previously reported decreased excitatory amino acid transporter‐2 (EAAT‐2) and astrocytic apoptosis in cortical degeneration using SIVmac239 and simian‐human immunodeficiency virus (SHIV)‐infected macaques and human AIDS autopsy cases. In the present study, we added highly pathogenic SIVsm543‐3‐infected macaques. These animals showed similar degenerative changes in the frontal cortex. Using 11 SIV‐infected macaques, three SIVsm543‐3, five SIVmac239 and three SHIV, we compared brain pathology caused by three different viruses and further analyzed the pathogenic process of HAND. We noticed vacuolar changes in perivascular processes of astrocytes by electron microscopy, and examined expression of astrocyte‐specific protein aquaporin‐4 (AQP4) by immunohistochemistry. APQ4 was diffusely positive in the neuropil and perivascular area in control brains. There was patchy or diffuse decrease of AQP4 staining in the neuropil of SIV‐infected macaques, which was associated with EAAT‐2 staining by double immunostaining. A quantitative analysis demonstrated significant positive correlation between areas of AQP4 and EAAT‐2. Some astrocytes express EAAT‐2 but not AQP4, and decrease of EAAT‐2 expression tended to be less than the decrease of AQP4. Active‐caspase‐3 immunostaining demonstrated apoptosis of neurons and astrocytes in the area of AQP4/EAAT‐2 reduction. These results suggest that AQP4 is damaged first and decrease of EAAT‐2 may follow in pathogenesis of cortical degeneration. This is the first demonstration of decrease of AQP4 and its association with EAAT‐2 decrease in AIDS brain, suggesting a role in the pathogenesis of HAND.     

Aquaporin‐4 in manganese‐treated cultured astrocytes

Manganese in excess is neurotoxic and causes CNS injury resembling that of Parkinson's disease. In brain, astrocytes predominantly take up and accumulate manganese and are thus vulnerable to its toxicity. Manganese was shown to induce cell swelling in cultured astrocytes, and oxidative/nitrosative stress (ONS) mediates such swelling. As aquaporin‐4 (AQP4) is important in the mechanism of astrocyte swelling, we examined the effect of manganese on AQP4 protein levels in cultured astrocytes. Treatment of cultures with manganese increased AQP4 protein in the plasma membrane (PM), whereas total cellular AQP4 protein and mRNA levels were unchanged, suggesting that increased AQP4 levels is due to its increased stability and/or increased trafficking to the PM and not to its neosynthesis. AQP4 gene silencing by small interfering ribonucleic acid resulted in a marked reduction in astrocyte swelling by manganese. Antioxidants, as well as an inhibitor of nitric oxide synthase, diminished the increase in AQP4 protein expression, suggesting a role of ONS in the mechanism of AQP4 increase. As ONS is known to activate mitogen‐activated protein kinases (MAPKs) and MAPK activation has been implicated in astrocyte swelling, we examined the effect of manganese on the activation of MAPKs and found an increased phosphorylation of extracellular signal‐regulated kinase (ERK)1/2, C‐Jun amino‐terminal kinase (JNK)1/2/3, and p38‐MAPK. Inhibitors of ERK1/2 and p38‐MAPK (but not of JNK) blocked (40–60%) the manganese‐induced increase in AQP4 protein content and astrocyte swelling, suggesting the involvement of these kinases in the increased AQP4 content. Inhibition of oxidative stress or MAPKs may represent potential strategies for counteracting AQP4‐related neurological complications associated with manganese toxicity. © 2010 Wiley‐Liss, Inc.     

Poldip2 mediates blood‐brain barrier disruption and cerebral edema by inducing AQP4 polarity loss in mouse bacterial meningitis model

BackgroundSpecific highly polarized aquaporin‐4 (AQP4) expression is reported to play a crucial role in blood‐brain barrier (BBB) integrity and brain water transport balance. The upregulation of polymerase δ‐interacting protein 2 (Poldip2) was involved in aggravating BBB disruption following ischemic stroke. This study aimed to investigate whether Poldip2‐mediated BBB disruption and cerebral edema formation in mouse bacterial meningitis (BM) model occur via induction of AQP4 polarity loss.Methods and ResultsMouse BM model was induced by injecting mice with group B hemolytic streptococci via posterior cistern. Recombinant human Poldip2 (rh‐Poldip2) was administered intranasally at 1 hour after BM induction. Small interfering ribonucleic acid (siRNA) targeting Poldip2 was administered by intracerebroventricular (i.c.v) injection at 48 hours before BM induction. A specific inhibitor of matrix metalloproteinases (MMPs), UK383367, was administered intravenously at 0.5 hour before BM induction. Western blotting, immunofluorescence staining, quantitative real‐time PCR, neurobehavioral test, brain water content test, Evans blue (EB) permeability assay, transmission electron microscopy (TEM), and gelatin zymography were carried out. The results showed that Poldip2 was upregulated and AQP4 polarity was lost in mouse BM model. Both Poldip2 siRNA and UK383367 improved neurobehavioral outcomes, alleviated brain edema, preserved the integrity of BBB, and relieved the loss of AQP4 polarity in BM model. Rh‐Poldip2 upregulated the expression of MMPs and glial fibrillary acidic protein (GFAP) and downregulated the expression of β‐dystroglycan (β‐DG), zonula occludens‐1 (ZO‐1), occludin, and claudin‐5; whereas Poldip2 siRNA downregulated the expression of MMPs and GFAP, and upregulated β‐DG, ZO‐1, occludin, and claudin‐5. Similarly, UK383367 downregulated the expression of GFAP and upregulated the expression of β‐DG, ZO‐1, occludin, and claudin‐5.ConclusionPoldip2 inhibition alleviated brain edema and preserved the integrity of BBB partially by relieving the loss of AQP4 polarity via MMPs/β‐DG pathway.     

亚低温对缺氧/复氧后星形胶质细胞AQP4表达的影响

目的 观察缺氧/复氧条件下大脑星形胶质细胞水通道蛋白4(AQP4)的表达变化以及亚低温对其表达的影响,探讨脑缺血再灌注脑水肿与AQP4的关系以及亚低温对脑缺血再灌注损伤的保护机制.方法 利用新生24 h内的SD大鼠,进行原代、传代培养,将星形胶质细胞分为对照组、常温组及亚低温组.用台盼蓝染色法测定37℃及32℃时,缺氧/复氧不同时间点星形胶质细胞的存活率,作为细胞受损指标,用倒置相差显微镜对细胞进行形态学观察,应用细胞免疫化学技术检测星形胶质细胞缺氧/复氧各个时间点AQP4的表达变化及亚低温的干预效果.结果 (1)缺氧4,8h时细胞形态变化不明显,随着复氧时间的延长,可见细化逐渐明显,而亚低温干预的细胞形态及细胞存活力变化均较相应的常温组明显减轻;(2)缺氧及复氧早期AQP4的表达降低,复氧后6h随着时间延长AQP4表达明显增多,在复氧≤8h,常温组及亚低温组的表达均低于对照组(均P＜0.05或0.01),而复氧后10,12h,AQP4蛋白表达均明显高于对照组(均P＜0.05或0.01);(3)在复氧后各时间点亚低温组AQP4的表达均明显低于常温组(均P＜0.05或0.01).结论 星形胶质细胞对缺氧的耐受能力较强,亚低温可以减轻缺氧/复氧后星形胶质细胞的损伤,通过降低AQP4的表达,可能是亚低温减轻缺血性脑水肿的作用机制之一.