New insights into water transport and edema in the central nervous system from phenotype analysis of aquaporin-4 null mice

Aquaporin-4 (AQP4) is the major water channel in the CNS. Its expression at fluid-tissue barriers (blood-brain and brain-cerebrospinal fluid barriers) throughout the brain and spinal cord suggests a role in water transport under normal and pathological conditions. Phenotype studies of transgenic mice lacking AQP4 have provided evidence for a role of AQP4 in cerebral water balance and neural signal transduction. Primary cultures of astrocytes from AQP4-null mice have greatly reduced osmotic water permeability compared with wild-type astrocytes, indicating that AQP4 is the principal water channel in these cells. AQP4-null mice have reduced brain swelling and improved neurological outcome following water intoxication and focal cerebral ischemia, establishing a role of AQP4 in the development of cytotoxic (cellular) cerebral edema. In contrast, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema caused by freeze-injury and brain tumor, probably due to impaired AQP4-dependent brain water clearance. AQP4-null mice also have markedly reduced acoustic brainstem response potentials and significantly increased seizure threshold in response to chemical convulsants, implicating AQP4 in modulation of neural signal transduction. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the CNS associated with altered brain water balance.     

Lead induces increased water permeability in astrocytes expressing aquaporin 4

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we report that lead increases AQP4 water permeability (P(f)) in astrocytes. A rat astrocyte cell line that does not express aquaporin 4 was transiently transfected with aquaporin 4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability in these cells and in AQP4-negative cells located on the same plate. AQP4-expressing astrocytes had a three-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 40%, increase in water permeability in astrocytes expressing AQP4, but had no effect on P(f) in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout, while treatment with a lead chelator, meso-2,3-dimercaptosuccinic acid, abolished the lead-induced increase in P(f). The effect of lead was attenuated in the presence of a calcium (Ca(2+))/calmodulin-dependent protein kinase II (CaMKII) inhibitor, but not in the presence of a protein kinase C inhibitor. In cells expressing AQP4 where the consensus site for CaMKII phosphorylation was mutated, lead failed to increase water permeability. Lead exposure also increased P(f) in rat astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on P(f) in astrocytes transfected with aquaporin 3. In situ hybridization studies on rat brain after oral lead intake for three days showed no change in distribution of AQP4 mRNA. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may contribute to the pathology of acute lead intoxication.     

Functionality of aquaporin-2 missense mutants in recessive nephrogenic diabetes insipidus

Aquaporin-2 (AQP2) missense mutants in recessive nephrogenic diabetes insipidus (NDI) are all retained in the endoplasmic reticulum (ER), but some could function as water channels. No conclusions could be drawn about the water permeability (Pf) of others, because there was no method for quantifying AQP2 expression in the plasma membrane. We recently developed such a method, which has allowed us to study the functionality of these AQP2 mutants. Immunoblot analysis of membranes of injected oocytes revealed that all mutants (AQP2-G64R, AQP2-N68S, AQP2 T126M, AQP2-A147T, AQP2-R187C, AQP2-S216P) are expressed as unglycosylated and high-mannose glycosylated AQP2. The level of the high-mannose form of AQP2-A147T in the plasma membranes was low, indicating that this mutation has a less severe effect on proper folding. Analysis of Pf values and plasma membrane expression levels reveals that AQP2-N68S, AQP2-R187C and AQP2-S216P are non-functional, AQP2-A147T is as functional as wt-AQP2, while AQP2-T126M and AQP2-G64R retain 20% of the permeability of wt-AQP2. Since G64 is highly conserved between AQPs and expected to form essential interactions with other amino acids within AQP1, the residual functionality of AQP2-G64R is surprising. Our data furthermore indicate that an eventual therapy with chemical chaperones that restores the routing of AQP2 mutants to the apical membrane of collecting ducts cells might relieve NDI in patients encoding AQP2-A147T, and to a lesser extent AQP2-T126M and AQP2-G64R, but not in patients encoding AQP2-N68S, AQP2-R187C or AQP2-S216P.     

Vasopressin-dependent short-term regulation of aquaporin 4 expressed in Xenopus oocytes

Aquaporin 4 (AQP4) is abundantly expressed in the perivascular glial endfeet in the central nervous system (CNS), where it is involved in the exchange of fluids between blood and brain. At this location, AQP4 contributes to the formation and/or the absorption of the brain edema that may arise following pathologies such as brain injuries, brain tumours, and cerebral ischemia. As vasopressin and its G-protein-coupled receptor (V1_aR) have been shown to affect the outcome of brain edema, we have investigated the regulatory interaction between AQP4 and V1_aR by heterologous expression in Xenopus laevis oocytes. The water permeability of AQP4/V1_aR-expressing oocytes was reduced in a vasopressin-dependent manner, as a result of V1_aR-dependent internalization of AQP4. Vasopressin-dependent internalization was not observed in AQP9/V1_aR-expressing oocytes. The regulatory interaction between AQP4 and V1_aR involves protein kinase C (PKC) activation and is reduced upon mutation of Ser¹⁸⁰ on AQP4 to an alanine. Thus, the present study demonstrates at the molecular level a functional link between the vasopressin receptor V1_aR and AQP4. This functional interaction between AQP4 and V1_aR may prove to be a potential therapeutic target in the prevention and treatment of brain edema.     

Time-Dependent Expression Patterns of Cardiac Aquaporins Following Myocardial Infarction

Aquaporins (AQPs) are expressed in myocardium and the implication of AQPs in myocardial water balance has been suggested. We investigated the expression patterns of AQP subtypes in normal myocardium and their changes in the process of edema formation and cardiac dysfunction following myocardial infarction (MI). Immunostaining demonstrated abundant expression of AQP1, AQP4, and AQP6 in normal mouse heart; AQP1 in blood vessels and cardiac myocytes, AQP4 exclusively on the intercalated discs between cardiac myocytes and AQP6 inside the myocytes. However, neither AQP7 nor AQP9 proteins were expressed in CD1 mouse myocardium. Echocardiography revealed that cardiac function was reduced at 1 week and recovered at 4 weeks after MI, whereas myocardial water content determined by wet-to-dry weight ratio increased at 1 week and rather reduced below the normal at 4 weeks. The expression of cardiac AQPs was up-regulated in MI-induced groups compared with sham-operated control group, but their time-dependent patterns were different. The time course of AQP4 expression coincided with that of myocardial edema and cardiac dysfunction following MI. However, expression of both AQP1 and AQP6 increased persistently up to 4 weeks. Our findings suggest a different role for cardiac AQPs in the formation and reabsorption of myocardial edema after MI.     

Bayesian approach to discovering pathogenic SNPs in conserved protein domains

The success rate of association studies can be improved by selecting better genetic markers for genotyping or by providing better leads for identifying pathogenic single nucleotide polymorphisms (SNPs) in the regions of linkage disequilibrium with positive disease associations. We have developed a novel algorithm to predict pathogenic single amino acid changes, either nonsynonymous SNPs (nsSNPs) or missense mutations, in conserved protein domains. Using a Bayesian framework, we found that the probability of a microbial missense mutation causing a significant change in phenotype depended on how much difference it made in several phylogenetic, biochemical, and structural features related to the single amino acid substitution. We tested our model on pathogenic allelic variants (missense mutations or nsSNPs) included in OMIM, and on the other nsSNPs in the same genes (from dbSNP) as the nonpathogenic variants. As a result, our model predicted pathogenic variants with a 10% false-positive rate. The high specificity of our prediction algorithm should make it valuable in genetic association studies aimed at identifying pathogenic SNPs.     

Cystathionine beta-synthase deficiency in Georgia (USA): correlation of clinical and biochemical phenotype with genotype

Cystathionine beta-synthase (CBS) deficiency is a rare autosomal recessive disorder that is the most frequent cause of clinical homocystinuria. Patients not treated in infancy have multi-systems disorders including dislocated lenses, mental deficiency, osteoporosis, premature arteriosclerosis, and thrombosis. In this paper, we examine the relationship of the clinical and biochemical phenotypes with the genotypes of 12 CBS deficient patients from 11 families from the state of Georgia, USA. By DNA sequencing of all of the coding exons we identified mutations in the CBS genes in 21 of the 22 possible mutant alleles. Ten different missense mutations were identified and one novel splice-site mutation was found. Five of the missense mutations were previously described (G307S, I278T, V320A, T353M, and L101P), while five were novel (A226T, N228S, A231L, D376N, Q526K). Each missense mutation was tested for function by expression in S. cerevisiae and all were found to cause decreased growth rate and to have significantly decreased levels of CBS enzyme activity. The I278T and T353M mutations accounted for 45% of the mutant alleles in this patient cohort. The T353M mutation, found exclusively in four African American patients, was associated with a B(6)-nonresponsive phenotype and detection by newborn screening for hypermethioninemia. The I278T mutation was found exclusively in Caucasian patients and was associated with a B(6)-responsive phenotype. We conclude that these two mutations occurred after ethnic socialization and that the CBS genotype is predictive of phenotype.     

Studies of mdx mice

Cerebral water accumulation-clinically denoted as brain edema-is a potentially life threatening complication of almost every intracranial neuropathological state. The molecular membrane water channel aquaporin-4 (AQP4) has been shown to be present at the blood-brain barrier (BBB) where it plays pivotal role in the transport of water between the tissue water compartments of the brain. Accumulating evidence indicates that the blockade of AQP4 function at the BBB would be a new therapeutic approach to the treatment and prevention of brain swelling. The cytoskeletal protein dystrophin has been shown to be involved in the maintenance of the polarized expression of AQP4 at the BBB. In order to further elucidate the mechanisms responsible for the highly polarized AQP4 expression, we studied brain tissue water accumulation during induction of brain edema in dystrophin-null transgenic mice (mdx-bgeo) and control mice. Immunofluorescence and immunoelectron microscopic analyses of dystrophin-null brains revealed a dramatic reduction of AQP4 in astroglial end-feet surrounding capillaries (BBB) and at the glia limitans (cerebrospinal fluid-brain interface). The AQP4 protein is mislocalized, because immunoblotting showed that the total AQP4 protein abundance was unaltered. Brain edema was induced by i.p. injection of distilled water and 8-deamino-arginine vasopressin. Changes in cerebral water compartments were assessed by diffusion-weighted MRI (DWI) with determination of the apparent diffusion coefficient (ADC). In dystrophin-null mice and control mice, ADC gradually decreased by 5-6% from baseline levels during the first 35 min, indicating the initial phase of intracellular water accumulation is similar in the two groups. At this point, the control mice sustained an abrupt, rapid decline in ADC to 58%+/-2.2% of the baseline at 52.5 min, and all of the animals were dead by 56 min. After a consistent delay, the dystrophin-null mice sustained a similar decline in ADC to 55%+/-3.4% at 66.5 min, when all of the mice were dead. These results demonstrate that dystrophin is necessary for polarized distribution of AQP4 protein in brain where facilitated movements of water occur across the BBB and cerebrospinal fluid-brain interface. Moreover, these results predict that interference with the subcellular localization of AQP4 may have therapeutic potential for delaying the onset of impending brain edema.     

Can variation in aquaporin 4 gene be associated with different outcomes in traumatic brain edema?

In traumatic brain injury (TBI), cerebral edema and hemorrhage are factors involved in the determination of the clinical presentation and outcome. The aquaporin 4 (AQP4) water channel is abundant in mammalian brain and there is a growing body of evidence suggesting that this protein plays a major role in the control of water flow within the central nervous system. Previous studies examined the influence of genetic variants in cerebral edema of TBI. However, to our knowledge, there are no previous studies of molecular variations of the AQP4 gene and its association with TBI. Thus, we sought to investigate if the clinical presentation and outcome of TBI could be influenced by the presence of mutations on exon 4 of the AQP4 gene. One hundred and two patients were enrolled in this study. A neurologist assessed the clinical severity at admission according to the GCS followed by a brain computer tomography (CT) scan. Then, DNA was extracted from blood cells and exon 4 of the AQP4 gene amplified by the polymerase chain reaction and directly sequenced. On discharge, GOS was assigned by a neurologist blind to the CGS on admission. We did not find any variation in exon 4 of the AQP4 gene in our considerable large sample. Despite this negative result, there is a strong biological rationale for the involvement of AQP4 gene in brain edema regulation and, as consequence, in TBI. Therefore, further studies should be performed, including the assessment of the other three exons of the AQP4 gene.     

Dominant-negative suppression of big brain ion channel activity by mutation of a conserved glutamate in the first transmembrane domain

The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negative effect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila. The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negative effect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila.     

SIRT1对早期脑缺血大鼠水通道蛋白4表达的影响

目的:检测水通道蛋白4(AQP4)及沉默信息调节因子1(SIRT1)在脑缺血大鼠脑组织中的表达变化,探究SIRT1对AQP4的调节作用,明确二者在脑缺血及脑水肿发生发展中的作用。方法:雄性SD大鼠随机分成假手术组(sham组)和脑缺血模型组(MCAO组),其中MCAO组又分为6 h、12 h、24 h和48 h 4个时点组别。采用线栓法阻塞大脑中动脉建立局灶性脑缺血模型,于相应时点进行神经症状评分,Morris水迷宫检测大鼠学习认知功能,TTC染色观察脑梗死体积,干湿重法检测脑含水量的变化,HE染色观察大脑皮层周围组织神经细胞形态学变化,Western blot检测AQP4、SIRT1和基质金属蛋白酶9(MMP-9)的表达情况。结果:与sham组相比较,随着再灌注时间增加,MCAO组大鼠神经功能评分、脑梗死体积、脑组织通透性和脑组织含水量均持续增加,而大鼠学习认知功能则降低显著,HE染色显示脑缺血大脑皮层周围组织神经元细胞形态不规则,数目减少;Western blot实验结果显示AQP4表达水平呈增高趋势,SIRT1表达降低,MMP-9表达增高,MCAO-48 h组与sham组比较差异最明显(P0.01)。结论:脑缺血后,伴随脑组织损伤的加剧,SIRT1和MMP-9信号通路的表达激活影响了AQP4的表达活化,共同参与了脑水肿的形成。     

Identification and characterization of aquaporin-2 water channel mutations causing nephrogenic diabetes insipidus with partial vasopressin response

Congenital nephrogenic diabetes insipidus (NDI) is a rare disease caused most often by mutations in the vasopressin V2 receptor (AVPR2). We studied a family which included a female patient with NDI with symptoms dating from infancy. The patient responded to large doses of desmopressin (dDAVP) which decreased urine volume from 10 to 4 I/day. Neither the parents nor the three sisters were polyuric. The patient was found to be a compound heterozygote for two novel recessive point mutations in the aquaporin-2 (AQP2) gene: L22V in exon 1 and C181W in exon 3. Residue Cys181 in AQP2 is the site for inhibition of water permeation by mercurial compounds and is located near to the NPA motif conserved in all aquaporins. Osmotic water permeability (Pf) in Xenopus oocytes injected with cRNA encoding C181W-AQP2 was not increased over water control, while expression of L22V cRNA increased the Pf to approximately 60% of that for wild-type AQP2. Co-injection of the mutant cRNAs with the wild-type cRNA did not affect the function of the wild-type AQP2. Immunolocalization of AQP2-transfected CHO cells showed that the C181W mutant had an endoplasmic reticulum-like intracellular distribution, whereas L22V and wild-type AQP2 showed endosome and plasma membrane staining. Water permeability assays showed a high Pf in cells expressing wild-type and L22V AQP2. This study indicates that AQP2 mutations can confer partially responsive NDI.      

Genetic variation in human aquaporins and effects on phenotypes of water homeostasis

Aquaporins (AQPs) are critical for the transport of water and small solutes. The 13 known human AQPs are divided into those that transport only water molecules, the "orthodox" AQPs, and those that transport glycerol and small solutes in addition to water, the aquaglyceroporins. In humans, genetic variation in AQPs can cause phenotypes of abnormal water homeostasis. Cellular and human studies of naturally-occurring and synthetic mutations have provided insight into the biology and phenotypes of these variants. Many AQPs have not been well-characterized in terms of the effect of genetic variation on protein function and clinical phenotype. In this review, we discuss functional features in human AQPs and summarize previous studies of naturally-occurring variants. We focus on nonsynonymous mutations since they typically have the greatest effect on function. We develop a map of AQP variation and functional features and examine uncharacterized variants by sequence and structure analysis. We find that variation has been studied relative to the AQP pore, terminal domains, and sites critical to posttranslational modifications. Finally, we propose possible variant-based phenotypes for further research. Other open questions relate to the discovery of novel AQP gene variants as well as their functions and phenotypes.     

Freeze-fracture and immunogold analysis of aquaporin-4 (AQP4) square arrays, with models of AQP4 lattice assembly

Each day, approximately 0.5-0.9 l of water diffuses through (primarily) aquaporin-1 (AQP1) channels in the human choroid plexus, into the cerebrospinal fluid of the brain ventricles and spinal cord central canal, through the ependymal cell lining, and into the parenchyma of the CNS. Additional water is also derived from metabolism of glucose within the CNS parenchyma. To maintain osmotic homeostasis, an equivalent amount of water exits the CNS parenchyma by diffusion into interstitial capillaries and into the subarachnoid space that surrounds the brain and spinal cord. Most of that efflux is through AQP4 water channels concentrated in astrocyte endfeet that surround capillaries and form the glia limitans. This report extends the ultrastructural and immunocytochemical characterizations of the crystalline aggregates of intramembrane proteins that comprise the AQP4 "square arrays" of astrocyte and ependymocyte plasma membranes. We elaborate on recent demonstrations in Chinese hamster ovary cells of the effects on AQP4 array assembly resulting from separate vs. combined expression of M1 and M23 AQP4, which are two alternatively spliced variants of the AQP4 gene. Using improved shadowing methods, we demonstrate sub-molecular cross-bridges that link the constituent intramembrane particles (IMPs) into regular square lattices of AQP4 arrays. We show that the AQP4 core particle is 4.5 nm in diameter, which appears to be too small to accommodate four monomeric proteins in a tetrameric IMP. Several structural models are considered that incorporate freeze-fracture data for submolecular "cross-bridges" linking IMPs into the classical square lattices that characterize, in particular, naturally occurring AQP4.     

Water permeability through aquaporin-4 is regulated by protein kinase C and becomes rate-limiting for glioma invasion

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K⁺–Cl⁻ cotransporter 1 (KCC1), believed to provide the pathways for Cl⁻ and K⁺ secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.     

脑出血大鼠大脑颞顶叶皮质水通道蛋白-4的表达

目的：研究脑出血脑水肿时，健侧大脑半球颞顶叶皮质水通道蛋白-4(AQP-4)表达变化及机制。 方法： 采用立体定向注射无肝素自体血制作大鼠脑出血模型,用MRI T2加权和病理学检查测定双侧半球颞顶叶皮质的水肿程度，RT-PCR和Western blotting检测两个部位AQP-4 mRNA和蛋白质的表达变化，最后从超微结构的角度探讨健侧半球颞顶叶皮质AQP-4改变的原因。 结果： 脑出血模型组大鼠出血侧和健侧颞顶叶皮质AQP-4表达均高于假手术组（P<0.05），脑出血1 d AQP-4 mRNA和蛋白即明显加强,第3 d达峰值,其后逐渐有所下降,但持续1周仍高于假手术组。健侧AQP-4表达弱于出血侧，但仍高于假手术组。电镜观察结果：出血侧颞顶叶皮质出现神经元水样变性，健侧颞顶叶皮质神经细胞核膜间隙无扩张，核周体无明显空泡形成，但线粒体明显肿胀呈球形,部分嵴断裂或溶解消失,透明成空泡;粗面内质网扩张,表面核糖体出现脱落，提示该部位细胞水肿不明显，但是出现了缺氧性改变。 结论： 脑出血后健侧颞顶叶皮质AQP-4在水肿不明显的情况下出现表达增强，其增高可能与脑缺氧有关。     

Molecular mechanisms of brain tumor edema

Despite their diverse histological types, most brain tumours cause brain oedema, which is a significant cause of patient morbidity and mortality. Brain tumour oedema occurs when plasma-like fluid enters the brain extracellular space through impaired capillary endothelial tight junctions in tumours. Under-expression of the tight junction proteins occludin, claudin-1 and claudin-5 are key molecular abnormalities responsible for the increased permeability of tumour endothelial tight junctions. Recent evidence suggests that the membrane water channel protein aquaporin-4 (AQP4) also plays a role in brain tumour oedema. AQP4-deficient mice show remarkably altered brain water balance after various insults, including brain tumour implantation. AQP4 expression is strongly upregulated around malignant human brain tumours in association with reduced extracellular volume, which may restrict the flow of extracellular fluid from the tumour bed into the brain parenchyma. Elimination of excess fluid leaking into brain parenchyma requires passage across three AQP4-rich barriers: a) the glia limitans externa, b) the glia limitans interna/ependyma, and c) the blood-brain barrier. Modulation of the expression and/or function of endothelial tight junction proteins and aquaporins may provide novel therapeutic options for reducing brain tumour oedema.     

Effect of mild hypothermia on the expression of aquaporin family in cultured rat astrocytes under hypoxic condition

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens, that increase plasma membrane water permeability in secretory and absorptive cells. In this study, we investigated the effect of mild hypothermia on the expression of AQP4, AQP5 and AQP9 in rat astrocytes cultured under hypoxic conditions. At 37 degrees C, a marked decrease in the expression of AQP4, AQP5 and AQP9 mRNAs was observed. However, at 32 degrees C (mild hypothermia), the expression of AQP5 mRNA was restored to its basal level. Interestingly, under mild hypothermia AQP4 mRNA expression transiently decreased and then increased about two-fold; while AQP9 mRNA expression decreased the same as at 37 degrees C. The changes in the expression of AQP4 and AQP9 proteins were confirmed by Western blot analysis. The restoration of the AQP4 and AQP5 expression at 32 degrees C from the hypoxia-induced decrease at 37 degrees C may play an important role in the reduction of brain edema under hypothermic conditions.     

Accounting for human polymorphisms predicted to affect protein function

A major interest in human genetics is to determine whether a nonsynonymous single-base nucleotide polymorphism (nsSNP) in a gene affects its protein product and, consequently, impacts the carrier's health. We used the SIFT (Sorting Intolerant From Tolerant) program to predict that 25% of 3084 nsSNPs from dbSNP, a public SNP database, would affect protein function. Some of the nsSNPs predicted to affect function were variants known to be associated with disease. Others were artifacts of SNP discovery. Two reports have indicated that there are thousands of damaging nsSNPs in an individual's human genome; we find the number is likely to be much lower.