Aquaporin-4 Levels Research Articles

Atorvastatin ameliorates early brain injury through inhibition of apoptosis and ER stress in a rat model of subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage (SAH) is a severe cerebrovascular disease with very poor prognosis. The aim of the present study was to evaluate the protective effects of atorvastatin on early brain injury (EBI) after SAH using a perforation SAH model. Male Sprague–Dawley rats were randomly divided into four groups: the sham group, the SAH group (model group), SAH + 10 mg.kg−1.day−1 atorvastatin (low atorvastatin group), and SAH + 20 mg.kg−1.day−1 atorvastatin (high atorvastatin group). Atorvastatin was administered orally by gastric gavage for 15 days before operation. At 24 h after SAH, we evaluated the effects of atorvastatin on brain water content, apoptosis by TUNEL assay and scanning electron microscope (SEM), and the expression of apoptosis-related proteins by immunofluorescence and Western blotting analysis. Compared with the sham group, we observed increased brain water content, significant apoptosis, and elevated levels of apoptosis-related proteins including caspase-3, CCAAT enhancer-binding protein homologous protein (CHOP), the 78-kDa glucose-regulated protein (GRP78), and aquaporin-4 (AQP4) in the SAH group. Atorvastatin administration under all doses could significantly reduce brain water content, apoptosis, and the expression levels of caspase-3, CHOP, GRP78, and AQP4 at 24 h after SAH. Our data show that early treatment with atorvastatin effectively ameliorates EBI after SAH through anti-apoptotic effects and the effects might be associated inhibition of caspase-3 and endoplasmic reticulum (ER) stress related proteins CHOP and GRP78.

The expression of aquaporin-4 is regulated based on innervation in skeletal muscles.

Aquaporin-4 (AQP4) is a selective water channel, which expresses on the plasma membrane of myofibers and regulates the osmotic pressure, energy metabolism and morphological changes in myofibers by modulating water transport across sarcolemma in skeletal muscles. Although the physiological roles of AQP4 have been gradually clarified in skeletal muscles, the regulatory mechanisms of AQP4 expression have been poorly understood in skeletal muscles. Recently, it was reported that the expression of AQP4 decreased in atrophied skeletal muscles following sciatic nerve transection, but not tail-suspension. Therefore, expecting that the nerve supply to myofibers would be one of the major regulatory factors regulating AQP4 expression in skeletal muscles, we investigated whether the expression patterns of AQP4 were changed in skeletal muscles by denervation and subsequent reinnervation. As a result, while the APQ4 expression levels were significantly decreased by sciatic nerve freezing-induced denervation, subsequently the expression levels of AQP4 were fully restored during reinnervation in skeletal muscles (p < 0.05, respectively). On the other hand, the expression levels of α1-syntrophin and AQP1, which are respectively structural and functional related AQP4 factors, were stably maintained during the denervation and subsequent reinnervation. Therefore, the present study demonstrated that the expression of AQP4 may be regulated depending on the innervation to skeletal muscles. Moreover, AQP4 regulatory mechanisms may be fundamentally different to those of AQP1 in skeletal muscles.

Renal aquaporin-4 associated pathology in TG-26 mice

Human immunodeficiency virus-associated nephropathy (HIVAN) is a leading cause of end-stage renal disease in HIV patients, which is characterized by glomerulosclerosis and renal tubular dysfunction. Aquaporin-4 (AQP-4) is a membrane bound water channel protein that plays a distinct role in water reabsorption from renal tubular fluid. It has been proven that failure of AQP-4 insertion into the renal tubular membrane leads to renal dysfunction. However, the role of AQP-4 in HIVAN is unclear. We hypothesize that impaired water reabsorption leads to renal injury in HIVAN, where AQP-4 plays a crucial role. Renal function is assessed by urinary protein and serum blood urea nitrogen (BUN). Kidneys from HIV Transgenic (TG26) mice (HIVAN animal model) were compared to wild type mice by immunostaining, immunoblotting and quantitative RT-PCR. TG26 mice had increased proteinuria and BUN. We found decreased AQP-4 levels in the renal medulla, increased endothelin-1, endothelin receptor A and reduced Sirtuin1 (SIRT-1) levels in TG26 mice. Also, oxidative and endoplasmic reticulum stress was enhanced in kidneys of TG26 mice. We provide the first evidence that AQP-4 is inhibited due to induction of HIV associated stress in the kidneys of TG26 mice which limits water reabsorption in the kidney which may be one of the cause associated with HIVAN, impairing kidney physiology. AQP-4 dysregulation in TG26 mice suggests that similar changes may occur in HIVAN patients. This work may identify new therapeutic targets to be evaluated in HIVAN.

Modulating the water channel AQP4 alters miRNA expression, astrocyte connectivity and water diffusion in the rodent brain

Aquaporins (AQPs) facilitate water diffusion through the plasma membrane. Brain aquaporin-4 (AQP4) is present in astrocytes and has critical roles in normal and disease physiology. We previously showed that a 24.9% decrease in AQP4 expression after in vivo silencing resulted in a 45.8% decrease in tissue water mobility as interpreted from magnetic resonance imaging apparent diffusion coefficients (ADC). Similar to previous in vitro studies we show decreased expression of the gap junction protein connexin 43 (Cx43) in vivo after intracortical injection of siAQP4 in the rat. Moreover, siAQP4 induced a loss of dye-coupling between astrocytes in vitro, further demonstrating its effect on gap junctions. In contrast, silencing of Cx43 did not alter the level of AQP4 or water mobility (ADC) in the brain. We hypothesized that siAQP4 has off-target effects on Cx43 expression via modification of miRNA expression. The decreased expression of Cx43 in siAQP4-treated animals was associated with up-regulation of miR224, which is known to target AQP4 and Cx43 expression. This could be one potential molecular mechanism responsible for the effect of siAQP4 on Cx43 expression, and the resultant decrease in astrocyte connectivity and dramatic effects on ADC values and water mobility.

Effects of aquaporin 4 and inward rectifier potassium channel 4.1 on medullospinal edema after methylprednisolone treatment to suppress acute spinal cord injury in rats.

To investigate the effects of aquaporin 4 (AQP4) and inward rectifier potassium channel 4.1 (Kir4.1) on medullospinal edema after treatment with methylprednisolone (MP) to suppress acute spinal cord injury (ASCI) in rats. Sprague Dawley rats were randomly divided into control, sham, ASCI, and MP-treated ASCI groups. After the induction of ASCI, we injected 30 mg/kg MP via the tail vein at various time points. The Tarlov scoring method was applied to evaluate neurological symptoms, and the wet-dry weights method was applied to measure the water content of the spinal cord. The motor function score of the ASCI group was significantly lower than that of the sham group, and the spinal water content was significantly increased. In addition, the levels of AQP4 and Kir4.1 were significantly increased, as was their degree of coexpression. Compared with that in the ASCI group, the motor function score and the water content were significantly increased in the MP group; in addition, the expression and coexpression of AQP4 and Kir4.1 were significantly reduced. Methylprednisolone inhibited medullospinal edema in rats with acute spinal cord injury, possibly by reducing the coexpression of aquaporin 4 and Kir4.1 in medullospinal tissues.

Goreisan Prevents Brain Edema after Cerebral Ischemic Stroke by Inhibiting Aquaporin 4 Upregulation in Mice

Aquaporin 4 (AQP4) is a water-selective transport protein expressed in astrocytes throughout the central nervous system. AQP4 level increases after cerebral ischemia and results in ischemic brain edema. Brain edema markedly influences mortality and motor function by elevating intracranial pressure that leads to secondary brain damage. Therefore, AQP4 is an important target to improve brain edema after cerebral ischemia. The Japanese herbal Kampo medicine, goreisan, is known to inhibit AQP4 activity. Here, we investigated whether goreisan prevents induction of brain edema by cerebral ischemia via AQP4 using 4-hour middle cerebral artery occlusion (4h MCAO) mice. Goreisan was orally administered at a dose of 500 mg/kg twice a day for 5 days before MCAO. AQP4 expression and motor coordination were measured by Western blotting and rotarod test, respectively. Brain water content of 4h MCAO mice was significantly increased at 24 hours after MCAO. Treatment with goreisan significantly decreased both brain water content and AQP4 expression in the ischemic brain at 24 hours after MCAO. In addition, treatment with goreisan alleviated motor coordination deficits at 24 hours after MCAO. The results of this study suggested that goreisan may be a useful new therapeutic option for ischemic brain edema.

Simvastatin improves intracerebral hemorrhage through NF-κB-mediated apoptosis via the MyD88/TRIF signaling pathway.

The aim was to investigate the neuroprotective effects and potential mechanism mediated by simvastatin in a mouse model of intracerebral hemorrhage. CD-1 mice were subjected to infusion of collagenase type IV into the left striatum in order to induce intracerebral hemorrhage. Western blot analysis, the TUNEL assay and the modified neurological severity score were used in the present study to analyze the efficacy of simvastatin for intracerebral hemorrhage. The results demonstrated that simvastatin treatment improved the cerebral water content and blood-brain barrier disruption in the intracerebral hemorrhage animals. Intracerebral hemorrhage-induced neuronal cell death was downregulated by simvastatin treatment compared with the vehicle-treated model group. In addition, the expression levels of aquaporin-4, matrix metallopeptidase 9 and caspase-3 were downregulated and B-cell lymphoma-2 was upregulated by simvastatin treatment compared with the vehicle-treated model. Simvastatin treatment also significantly reduced the Evans blue leakage into the injured hemispheres and improved motor function. Mechanism analysis further indicated that simvastatin treatment downregulated nuclear factor (NF)-κB expression, and upregulated the myeloid differentiation primary response 88 (MyD88) and TIR domain-containing adaptor protein inducing interferon-β (TRIF) expression levels in neuronal cells in experimental mice. Furthermore, the results revealed that NF-κB overexpression abolished the simvastatin-downregulated MyD88 and TRIF expression levels, as well as the apoptosis of neuronal cells. In conclusion, these results indicated that simvastatin was able to attenuate brain edema and reduce cellular apoptosis by suppressing the NF-κB-mediated MyD88/TRIF signaling pathway subsequent to the induction of intracerebral hemorrhage in mice.

Neuroprotective effects and mechanism of saikosaponin A on acute spinal cord injury in rats

To investigate the effect of saikosaponin a (SSa) on the levels of immune inflammation in rats with acute spinal cord injury and its possible mechanism. Seventy-two Sprague Dawley rats (weighing, 220-250 g) were randomly divided into sham operation group (group A), spinal cord injury group (group B), and SSa treatment group (group C) respectively, 24 rats in each group. The spinal cord injury model was induced by using the Allen's method in groups B and C; the spinous process and vertebral plate at both sides were cut off by lamina excision to expose the spinal cord in group A. The rats were given intraperitoneal injection of 10 mg/kg SSa in group C and equal volume of normal saline in group B at immediate after injury. The spinal cord tissue was harvested from 18 rats of each group at 24 hours after operation to measure the levels of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) by ELISA, to detect the expressions of nuclear factor κB (NF-κB) P65, NF-κB P-P65, and aquaporin 4 (AQP4) by Western blot and to observe the morphology of spinal cord by HE staining. The motor function of the lower limbs was evaluated by BBB score and tiltboard experiment in 6 rats at 1, 3, 7, 14, 21, and 28 days after injury. The BBB score and tiltboard experiment maximum angle were significantly higher in group A than groups B and C at each time point ( P<0.05) and in group C than group B at 14, 21, and 28 days after operation ( P<0.05). ELISA test showed that the concentrations of TNF-α and IL-6 were significantly lower in group A than groups B and C, and in group C than group B ( P<0.05). Western blot results showed that the protein expression levels of NF-κB P65, NF-κB P-P65, and AQP4 were significantly lower in group A than groups B and C, and in group C than group B ( P<0.05). HE staining demonstrated normal neurons of the spinal cord and no obvious lesion in group A; neuronal cells were observed in the injured area of group B, with hemorrhage, neutrophil infiltration, and nerve cell edema in the injured area; the neuronal cells were visible in the spinal cord of group C, with microglia mild hyperplasia, and the pathological changes were improved when compared with group B. SSa has neuroprotective effects on acute spinal cord injury in rats by inhibiting NF-κB signaling pathway and AQP4 protein expression and reducing inflammation response and edema.

Down-Regulation of AQP4 Expression via p38 MAPK Signaling in Temozolomide-Induced Glioma Cells Growth Inhibition and Invasion Impairment.

Glioma is the most common and lethal central nervous system tumors. Temozolomide (TMZ) is an effective drug for malignant glioma, however, the intracellular and molecular mechanisms behind this anti-cancer effect have yet to be fully understood. The aim of the present study was to determine whether TMZ inhibits proliferation, invasion of glioma cells in vitro and whether these effects can be mediated through modulation of aquaporin 4 (AQP4) and phosphorylation of the MAPK pathway. The viability of U87 and U251 human glioma cells was evaluated using MTT assay. The cell cycle distribution was detected with flow cytometry. Migration ability and invasion ability were tested by scratch assays and transwell assays, respectively. The levels of AQP4 and MAPK were measured using immunoblot analyses. Our results showed that TMZ inhibited proliferation, migration and invasion, and induced G2/M arrest in U87 and U251 glioma cell lines. These changes were associated with a decrease in the levels of AQP4 expression as well as activation phosphorylated level of p38. Treatment with a p38 chemical activator (anisomycin) resulted in similar effects as TMZ treatment on glioma cells. And p38 chemical inhibitor (SB203580) could block these effects in glioma treated with TMZ, suggesting a direct up-regulation of the p38 signaling pathway. Therefore, we identified that TMZ might have therapeutic potential for controlling proliferation, invasion of malignant glioma by inhibiting AQP4 expression through activation of p38 signal transduction pathway. J. Cell. Biochem. 118: 4905-4913, 2017. © 2017 Wiley Periodicals, Inc.

Astrocytic water channel aquaporin-4 modulates brain plasticity in both mice and humans: a potential gliogenetic mechanism underlying language-associated learning.

The role of astrocytes in brain plasticity has not been extensively studied compared with that of neurons. Here we adopted integrative translational and reverse-translational approaches to explore the role of an astrocyte-specific major water channel in the brain, aquaporin-4 (AQP4), in brain plasticity and learning. We initially identified the most prevalent genetic variant of AQP4 (single nucleotide polymorphism of rs162008 with C or T variation, which has a minor allele frequency of 0.21) from a human database (n=60 706) and examined its functionality in modulating the expression level of AQP4 in an in vitro luciferase reporter assay. In the following experiments, AQP4 knock-down in mice not only impaired hippocampal volumetric plasticity after exposure to enriched environment but also caused loss of long-term potentiation after theta-burst stimulation. In humans, there was a cross-sectional association of rs162008 with gray matter (GM) volume variation in cortices, including the vicinity of the Perisylvian heteromodal language area (Sample 1, n=650). GM volume variation in these brain regions was positively associated with the semantic verbal fluency. In a prospective follow-up study (Sample 2, n=45), the effects of an intensive 5-week foreign language (English) learning experience on regional GM volume increase were modulated by this AQP4 variant, which was also associated with verbal learning capacity change. We then delineated in mice mechanisms that included AQP4-dependent transient astrocytic volume changes and astrocytic structural elaboration. We believe our study provides the first integrative evidence for a gliogenetic basis that involves AQP4, underlying language-associated brain plasticity.

Cortical Aquaporin-4 in relation to brain oedema and neurological function of cortical cryo-injured mice

To estimate the spatial and temporal expression of Aquaporin-4 (AQP-4) in a murine model of automated cerebral cryoinjury and correlate AQP-4 expression with development of brain oedema and neurological function. AQP-4 levels were determined quantitatively by Western blots at site of injury and at sites adjacent to and distant from injury in brains of cryoinjured (experimental) (n=18), sham injured (n=18) & normal mice at 24, 48, 72h post injury. AQP-4 expression was correlated with percentage water content of brain, Neurological Severity Score (NSS) and rotarod scores. We found a 1.4-fold increase in expression of AQP-4 at the site of injury and at sites distant from injury at 24h when compared to normal mice (p=0.05). The increase in expression of AQP-4 24h post injury was significantly higher in experimental group at the site of injury and at the site adjacent to the injury in the ipsilateral hemisphere when compared to the sham injured mice (p=0.05). At 24h post injury the median NSS score in the experimental group was 9 (interquartile range 7.25–10) and that in the sham group was 0.5 (interquartile range 0.0–1.0) (p<0.001).At 48 and 72h, AQP-4 expression remained elevated in the experimental group when compared to normal brain, but the levels were not significantly different from that in sham group. AQP-4 expression was significantly elevated in the ipsilateral hemisphere in the first 24h following cerebral cortical injury in mice and this could be correlated with worsening of neurological function. Over the next 48h, there was a trend towards decrease in AQP-4 expression that was associated with partial recovery of neurological function.

Orientin Attenuates Cerebral Ischemia/Reperfusion Injury in Rat Model through the AQP-4 and TLR4/NF-κB/TNF-α Signaling Pathway

Orientin has been reported to have extensive pharmaceutical effects of antioxidant, anti-inflammatory, antithrombosis, antiapoptosis, and so on. In the present study, we tried to investigate the protective effects of orientin on cerebral ischemia-reperfusion (I/R) injury and explored the possible mechanisms. Middle cerebral artery occlusion rat model was established and then treated with low, middle, and high concentrations of orientin, respectively, with edaravone as a positive control. The treatment effect of orientin was evaluated by measuring the neurological deficit score, cerebral infarction, brain edema, oxidative stress, excitatory amino acids release, the expression levels of aquaporin-4 (AQP-4), and related inflammatory molecules using different methods including immunohistochemistry, enzyme-linked immunosorbent assay, real-time PCR, and western blot. Moreover, morphological and structural changes were also observed by hematoxylin-eosin staining and transmission electron microscope. Orientin provided a significant reduction on neurological deficits, cerebral infarction, cerebral edema, oxidative damage, and neurotoxicity of excitatory amino acids compared to model group (P < .05) in a dose-dependent manner. In addition, orientin substantially downregulated AQP-4 and inflammatory factors expression (P < .05) and improved cell morphology and structure in rats following I/R injury. Orientin was able to mediate noticeable protection against cerebral I/R injury through the attenuation of oxidative stress and neurotoxicity of amino acids and inhibiting the upregulation of AQP-4 and inflammatory cytokines.

Lentivirus-mediated inhibition of AQP4 accelerates motor function recovery associated with NGF in spinal cord contusion rats

Aquaporin-4 (AQP4) is a water channel protein in spinal cord and plays a critical role in the pathophysiological process of spinal cord injury (SCI). However, little is known about the molecular mechanism of AQP4 involved in SCI. The present study was performed to investigate the possible molecules regulated by AQP4 after SCI by use of lentivirus-mediated RNA interference (RNAi). First, the motor function was evaluated by Basso, Beattie, Bresnahan (BBB) scale and the expression of AQP4 was measured by western blot, immunohistochemical staining and immunofluorescence in rats after contusion spinal cord injury (cSCI). After cSCI, the rats exhibited a gradual motor function recovery from 3dpo to 28dpo. And the expression and localization of AQP4 changed with different post-injury stages. At 3d after SCI, AQP4 located mainly in vascular endothelial cells (VECs) in the anterior horn of spinal cord, which was similar to the sham rats. At 7d and 28d after SCI, AQP4 was expressed both in VECs and the position of neuron membranes. The protein level of AQP4 increased significantly at 12h, 1d and 3d after SCI, and decreased slightly at 7d after SCI. Then lentivirus-mediated AQP4 RNA interference (AQP4-RNAi) was constructed and used to inhibit AQP4 expression in cSCI rats. The results from real-time quantitative polymerase chain reactions (RT-qPCR) and immunohistochemical staining suggested that the expression of nerve growth factor (NGF) was up-regulated by lentivirus-mediated AQP4 inhibition in cSCI rats, while the motor function recovery was accelerated. The results suggested that the acceleration of motor function recovery by AQP4 inhibition was associated with NGF up-regulation. This is the first report on the relationship between AQP4 and NGF in SCI, which may shed light on illustrating the role of AQP4 in SCI. These findings may also provide strategies of the clinical treatment for SCI in the future.

Effects of erythropoietin and methylprednisolone on AQP4 expression in astrocytes.

Methylprednisolone sodium succinate (MPSS) has been suggested as a treatment for spinal cord injury (SCI), but its use has been limited due to its adverse effects. Erythropoietin (EPO) has been suggested as a promising candidate for limiting SCI in mammals. The aim of the present study was to investigate the effects of EPO in combination with MPSS on astrocytes following ischemic injury in vitro. Astrocytes were isolated from the cerebral cortex of postnatal day 3 Sprague-Dawley rats and cultured in vitro. Astrocyte ischemic injury was induced by oxygen and glucose deprivation for 4 h, and reperfusion was simulated by subsequent culture under normoxic conditions. The effects of EPO and MPSS on the expression of aquaporin-4 (AQP4) were investigated. Ischemic astrocytes were treated with EPO (10 U/ml), MPSS (10 µg/ml), or EPO (10 U/ml) in combination with MPSS (10 µg/ml) during reperfusion. The cell viability of astrocytes was assessed using an MTT assay. The mRNA and protein expression levels of AQP4 were determined using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The role of the protein kinase C (PKC) signaling pathway in the molecular mechanisms underlying the effects of EPO and MPSS was also investigated. The present results demonstrated that following treatment with EPO and MPSS, the mRNA expression levels of AQP4 were upregulated and cell viability was enhanced. EPO and MPSS effectively inhibited the oxygen and glucose deprivation-mediated downregulation of AQP4 following reperfusion. In addition, the combined treatment with EPO and MPSS exhibited higher AQP4 expression levels and cell viability compared with each treatment alone. Finally, the effects of EPO and MPSS on AQP4 expression were partially reversed by pretreatment with the PKC inhibitor Ro 31–8220. The present study indicated that EPO and MPSS had a synergistic effect on AQP4 expression following reperfusion, and suggest that they may be combined in the treatment of SCI.

Apelin-13 Protects against Ischemic Blood-Brain Barrier Damage through the Effects of Aquaporin-4

Background: Apelin-13 has been found to have protective effects on many neurological diseases, including cerebral ischemia. However, whether Apelin-13 acts on blood-brain barrier (BBB) disruption following cerebral ischemia is largely unknown. Aquaporin-4 (AQP4) has a close link with BBB due to the high concentration in astrocyte foot processes and regulation of astrocytes function. Here, we aimed to test Apelin-13′s effects on ischemic BBB injury and examine whether the effects were dependent on AQP4. Methods: We detected the expression of AQP4 induced by Apelin-13 injection at 1, 3, and 7 days after middle cerebral artery occlusion. Meanwhile, we examined the effects of Apelin-13 on neurological function, infarct volume, and BBB disruption owing to cerebral ischemia in wild type mice, and tested whether such effects were AQP4 dependent by using AQP4 knock-out mice. Furthermore, we assessed the possible signal transduction pathways activated by Apelin-13 to regulate AQP4 expression via astrocyte cultures. Results: It was found that Apelin-13 highly increased AQP4 expression as well as reduced neurological scores and infarct volume. Importantly, Apelin-13 played a role of BBB protection in both types of mice by reducing BBB permeability, increased vascular endothelial growth factor, upregulated endothelial nitric oxide synthase, and downregulated inducible NOS. In morphology, we demonstrated Apelin-13 suppressed tight junction opening and endothelial cell swelling via electron microscopy detection. Meanwhile, Apelin-13 also alleviated apoptosis of astrocytes and promoted angiogenesis. Interestingly, effects of AQP4 on neurological function and infarct volume varied with time course, while AQP4 elicited protective effects on BBB at all time points. Statistical analysis of 2-way analysis of variance with replication indicated that AQP4 was required for these effects. In addition, Apelin-13 upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and Akt as well as AQP4 protein in cultured astrocytes. The latter was inhibited by ERK and phosphatidylinositol 3′-kinase (PI3K) inhibitors. Conclusion: Our data suggest that Apelin-13 protects BBB from disruption after cerebral ischemia both morphologically and functionally, which is highly associated with the increased levels of AQP4, possibly through the activation of ERK and PI3K/Akt pathways. This study provides double targets to protection of ischemic BBB damage, which can present new insights to drugs development.

Marked decrease of aquaporin-4 protein is independent of the changes in α1-syntrophin and TRPV4 levels in response to denervation-induced muscle atrophy in vivo.

Aquaporin-4 (AQP4) is a selective water channel mediating water transport across cell membranes in skeletal muscles. Recently, it was noted that AQP4 is one of the key molecules regulating muscle morphology. Indeed, the AQP4 accumulation level was stably maintained in hypertrophied skeletal muscles. On the other hand, whether the AQP4 accumulation level is stably maintained in atrophied muscles remains poorly understood. The present study investigated the changes in the AQP4 accumulation level in the atrophied muscles at 2 weeks after denervation. As a result, the accumulation level of AQP4 in the atrophied muscle was significantly decreased compared with that in the control muscle (p < 0.05). Interestingly, the accumulation level of α1-syntrophin, which is an essential factor in regulating the stable accumulation level of AQP4, was stably maintained in the atrophied muscles. On the other hand, the accumulation level of the transient receptor potential vanilloid 4 (TRPV4), which contributes to cell volume control via interaction with AQP4, was significantly increased in the atrophied muscles compared with that in the control muscle (p < 0.05). Therefore, the present study suggested that the imbalance between the AQP4 accumulation level and skeletal muscle volume may be induced in the atrophied muscles by denervation, and the decrease in the accumulation level of AQP4 may be accompanied by defects in the functional and structural relationships with α1-syntrophin and TRPV4.

Rearing Light Intensity Affects Inner Retinal Pathology in a Mouse Model of X-Linked Retinoschisis but Does Not Alter Gene Therapy Outcome.

PurposeTo test the effects of rearing light intensity on retinal function and morphology in the retinoschisis knockout (Rs1-KO) mouse model of X-linked retinoschisis, and whether it affects functional outcome of RS1 gene replacement.MethodsSeventy-six Rs1-KO mice were reared in either cyclic low light (LL, 20 lux) or moderate light (ML, 300 lux) and analyzed at 1 and 4 months. Retinal function was assessed by electroretinogram and cavity size by optical coherence tomography. Expression of inward-rectifier K+ channel (Kir4.1), water channel aquaporin-4 (AQP4), and glial fibrillary acidic protein (GFAP) were analyzed by Western blotting. In a separate study, Rs1-KO mice reared in LL (n = 29) or ML (n = 27) received a unilateral intravitreal injection of scAAV8-hRs-IRBP at 21 days, and functional outcome was evaluated at 4 months by electroretinogram.ResultsAt 1 month, no functional or structural differences were found between LL- or ML-reared Rs1-KO mice. At 4 months, ML-reared Rs1-KO mice showed significant reduction of b-wave amplitude and b-/a-wave ratio with no changes in a-wave, and a significant increase in cavity size, compared to LL-reared animals. Moderate light rearing increased Kir4.1 expression in Rs1-KO mice by 4 months, but not AQP4 and GFAP levels. Administration of scAAV8-hRS1-IRBP to Rs1-KO mice showed similar improvement of inner retinal ERG function independent of LL or ML rearing.ConclusionsRearing light conditions affect the development of retinal cavities and post-photoreceptor function in Rs1-KO mice. However, the effect of rearing light intensity does not interact with the efficacy of RS1 gene replacement in Rs1-KO mice.

Role of NADPH Oxidase as a Mediator of Oxidative Damage in Low-Dose Irradiated and Hindlimb-Unloaded Mice.

The purpose of this study was to determine whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived stress can account for unloading- and radiation-induced endothelial damage and neurovascular remodeling in a mouse model. Wild-type (WT, Nox2+/+) C57BL/6 mice or Nox2-/- (B6.129S6-CYBBM) knockout (KO) mice were placed into one of the following groups: age-matched control; hindlimb unloading (HLU); low-dose/low-dose-rate radiation (LDR); or HLU with LDR simultaneously for 21 days. The mice were then sacrificed one month later. Anti-orthostatic tail suspension was used to model the unloading, fluid shift and physiological stress aspects of microgravity. The LDR was delivered using 57Co plates (0.04 Gy at 0.01 cGy/h) to the simulate whole-body irradiation, similar to that experienced while in space. Brains were isolated for characterization of various oxidative stress markers and vascular topology. The level of 4-hydroxynonenal (4-HNE) protein, a specific marker for lipid peroxidation, was measured. Expression of aquaporin-4 (AQP4), a water channel protein expressed in astrocyte end-feet, was quantified. Thirty days after simulated spaceflight, KO mice showed decreased apoptosis (P < 0.05) in the brain compared to WT counterparts. The HLU-dependent increase in apoptosis in WT mice was not observed in KO mice. The level of 4-HNE protein was significantly elevated in the hippocampus of the LDR with HLU treatment group compared to WT controls (P < 0.05). However, there were no significant differences among groups of Nox2-KO mice at the one-month time point. In contrast to findings in the WT animals, superoxide dismutase (SOD) level and expression of AQP4 were similar among all KO groups. In summary, for most of the parameters, the oxidative response to HLU and LDR was suppressed in Nox2-KO mice. This suggests that Nox2-containing NADPH oxidase may contribute to spaceflight environment-induced oxidative stress.

Down-regulated expression of aquaporin-4 in the cerebellum after status epilepticus.

Status epilepticus (SE) is a common neurological condition associated with high rates of mortality and permanent brain injury. SE usually leads to neuronal death which may be accompanied by edema, epileptogenesis and learning impairment. Aquaporin-4 (AQP4), is a transmembrane water channel protein in the neuropil of the central nervous system that has an important role in water transport in the brain; AQP4 expression is altered in many pathological conditions such as changes in the blood- brain barrier and/or astrocytic activation, including seizures. AQP4 was shown to be downregulated in the piriform cortex and the hippocampus after SE. Although it is normally expressed at a high level in the cerebellum, little is known about AQP4 levels in the cerebellum following SE. We addressed this in the present study in a mouse model of pilocarpine-induced SE. We found that AQP4 expression was reduced from 3h to 3days after SE, with the levels recovering on day 7. Moreover, mice in the acute post-SE stages exhibited impaired motor coordination and learning. These results indicate that cerebellar damage following SE involves changes in AQP4 expression.

Aquaporin-4 Cell-Surface Expression and Turnover Are Regulated by Dystroglycan, Dynamin, and the Extracellular Matrix in Astrocytes

The water-permeable channel aquaporin-4 (AQP4) is highly expressed in perivascular astrocytes of the mammalian brain and represents the major conduit for water across the blood-brain barrier. Within these cells, AQP4 is found in great quantities at perivascular endfoot sites but is detected in lesser amounts at the membrane domains within the brain parenchyma. We had previously established that this polarization was regulated by the interaction between dystroglycan (DG), an extracellular matrix receptor that is co-expressed with AQP4, and the laminin that is contained within the perivascular basal lamina. In the present study, we have attempted to describe the mechanisms that underlie this regulation, using primary astrocyte cultures. Via biotinylation, we found that the cell-surface expression of AQP4 is DG-dependent and is potentiated by laminin. We also determined that this laminin-dependent increase occurs not through an upregulation of total AQP4 levels, but rather from a redirection of AQP4 from an intracellular, EEA-1-associated pool to the cell surface. We then demonstrated an association between DG and dynamin and showed that dynamin functioned in conjunction with clathrin to regulate surface AQP4 amounts. Furthermore, we observed that DG preferentially binds to the inactive forms of dynamin, suggesting that this interaction was inhibitory for AQP4 endocytosis. Finally, we showed that laminin selectively upregulates the cell-surface expression of the M23 isoform of AQP4. Our data therefore indicate that the dual interation of DG with laminin and dynamin is involved in the regulation of AQP4 internalization, leading to its asymmetric enrichment at perivascular astrocyte endfeet.