Two major isoforms of aquaporin-4 (AQP4) have been described in human tissue. Here we report the identification and functional analysis of an alternatively spliced transcript of human AQP4, AQP4-Δ4, that lacks exon 4. In transfected cells AQP4-Δ4 is mainly retained in the endoplasmic reticulum and shows no water transport properties. When AQP4-Δ4 is transfected into cells stably expressing functional AQP4, the surface expression of the full-length protein is reduced. Furthermore, the water transport activity of the cotransfectants is diminished in comparison to transfectants expressing only AQP4. The observed down-regulation of both the expression and water channel activity of AQP4 is likely to originate from a dominant-negative effect caused by heterodimerization between AQP4 and AQP4-Δ4, which was detected in coimmunoprecipitation studies. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein and is physiologically associated with different types of skeletal muscles. The expression of AQP4-Δ4 may represent a new regulatory mechanism through which the cell-surface expression and therefore the activity of AQP4 can be physiologically modulated.

We screened human kidney-derived multipotent CD133+/CD24+ ARPCs for the possible expression of all 13 aquaporin isoforms cloned in humans. Interestingly, we found that ARPCs expressed both AQP5 mRNA and mature protein. This novel finding prompted us to investigate the presence of AQP5 in situ in kidney. We report here the novel finding that AQP5 is expressed in human, rat and mouse kidney at the apical membrane of type-B intercalated cells. AQP5 is expressed in the renal cortex and completely absent from the medulla. Immunocytochemical analysis using segment- and cell type-specific markers unambiguously indicated that AQP5 is expressed throughout the collecting system at the apical membrane of type-B intercalated cells, where it co-localizes with pendrin. No basolateral AQPs were detected in type-B intercalated cells, suggesting that AQP5 is unlikely to be involved in the net trans-epithelial water reabsorption occurring in the distal tubule. An intriguing hypothesis is that AQP5 may serve an osmosensor for the composition of the fluid coming from the thick ascending limb. Future studies will unravel the physiological role of AQP5 in the kidney.

In this study we assess the functional role of Aquaporin-4 (AQP4) in the skeletal muscle by analyzing whether physical activity modulates AQP4 expression and whether the absence of AQP4 has an effect on osmotic behavior, muscle contractile properties, and physical activity. To this purpose, rats and mice were trained on the treadmill for 10 (D10) and 30 (D30) days and tested with exercise to exhaustion, and muscles were used for immunoblotting, RT-PCR, and fiber-type distribution analysis. Taking advantage of the AQP4 KO murine model, functional analysis of AQP4 was performed on dissected muscle fibers and sarcolemma vesicles. Moreover, WT and AQP4 KO mice were subjected to both voluntary and forced activity. Rat fast-twitch muscles showed a twofold increase in AQP4 protein in D10 and D30 rats compared to sedentary rats. Such increase positively correlated with the animal performance, since highest level of AQP4 protein was found in high runner rats. Interestingly, no shift in muscle fiber composition nor an increase in AQP4-positive fibers was found. Furthermore, no changes in AQP4 mRNA after exercise were detected, suggesting that post-translational events are likely to be responsible for AQP4 modulation. Experiments performed on AQP4 KO mice revealed a strong impairment in osmotic responses as well as in forced and voluntary activities compared to WT mice, even though force development amplitude and contractile properties were unvaried. Our findings definitively demonstrate the physiological role of AQP4 in supporting muscle contractile activity and metabolic changes that occur in fast-twitch skeletal muscle during prolonged exercise.

Purpose Aquaporin 4 (AQP4) is the most abundant water channel in the retina and participates in the formation of blood-retinal barrier (BRB). In several retinal pathologies accompanied by BRB dysfunction the expression of AQP4 is altered. In the present study, we investigated the effects of AQP4 deletion in the vascular retinal response to hypoxia. Methods We used wild type (WT) and AQP4 knockout (KO) mice. The retinal angiogenic response to hypoxia was studied in a mouse model of oxygen-induced retinopathy (OIR) using real time RT-PCR, Western blot, ELISA and immunohistochemistry. Results In WT mice, retinal levels of AQP4 were increased in response to hypoxia. In OIR mice, the BRB dysfunction was more pronounced in KO than in WT mice. BRB tight junction proteins, occludin, ZO-1 and JAM-C, were analyzed and significant alteration was found for JAM-C in KO mice, indicating a role for this protein in the observed dysfunctions. The formation of neovascular tufts, which are characteristic of OIR, was completely prevented in KO mice, possibly as a result of the decrease in hypoxia-induced up-regulation of VEGF mRNA and protein. Unexpectedly, this reduced increase in VEGF levels was paralleled by a dramatic increase in the activity of transcription factors which regulate VEGF expression indicating that in KO retinas mechanisms of VEGF transcription, such as promoter methylation, may be altered. Conclusion Together, the present results indicate a role for AQP4 in the formation of angiogenic processes in the retina and suggest that inhibiting AQP4 function may be a strategy to reduce pathologic angiogenesis in proliferative retinopathies.

Aquaporin-4 (AQP4) is expressed in skeletal muscle fast-twitch fibers. The purpose of this study was to determine whether AQP4 has a physiological role during muscle activity. Six-weeks old Wistar male rats were subjected to 5 (D5), 10 (D10) and 30 (D30) consecutive days of exhaustive exercise on a treadmill without electrical stimulation. Furthermore, CD1 AQP4-null mice were used to test whether the absence of the water channel affects voluntary and forced activity, and muscles were further used to study contractile kinetics by in vivo and ex vivo analysis. Immunoblotting and Real-Time PCR analysis performed on rat fast-twitch muscles showed a significant increase in AQP4 protein and transcripts levels in D10 and D30 rats compared to their sedentary controls, without variation in myosin heavy chain distribution and in AQP4-positive fibers percentage. In contrast, no differences were observed in D5 rats compared to the sedentaries. In particular, AQP4 up-regulation was observed in rats which ran more than 15 minutes/day, suggesting that a threshold of daily activity during a prolonged exercise (at least 10 days) must be overcome so that AQP4 over-expression occurs. Furthermore, AQP4 KO mice had significant attenuation of daily mean distances in both voluntary (-50% at D30) and forced activities (-25% at D30), even though contractile kinetics measured by ex vivo and in vivo activity were unvaried when compared to WT mice. These findings confirm that AQP4 plays a pivotal role during adaptive processes that confers the metabolic phenotype leading to improved fatigue resistance during prolonged exercise.

Aquaporin‐4 (AQP4) is the neuromuscular water channel that is also expressed at the basolateral membranes of other cell types in kidney, stomach, and lung. In skeletal muscle, AQP4 is found at the sarcolemma of fast‐twitch fibers and its function is strictly correlated with the glycolytic metabolism. In the central nervous system, AQP4 is expressed at the basolateral membranes of ependymal cells, and is highly concentrated at the glial end‐foot processes surrounding blood vessels and forming the glia limitans, as well as at the nonend‐foot glial processes of the granule cell layer in the cerebellum. AQP4 plasma membrane organization is different from other aquaporins (AQPs). AQP4 is expressed as two major polypeptides called M1 and M23. These two isoforms form heterotetramers appearing in the plasma membrane as intramembrane particles (IMPs) observable by freeze‐fracture electron microscopy. Such tetrameric organization is common to all other AQPs. In the case of AQP4, however, multiple IMPs further aggregate to form structures called orthogonal arrays of particles (OAPs). The relative abundance of M23 and M1 in vivo is the major determinant for the formation of OAPs of different sizes. The function of AQP4 aggregation into OAPs under normal conditions is still not completely understood. Interestingly, there are several reports indicating that OAPs are involved in different neuromuscular diseases. In particular, the OAP‐related diseases that have attracted more attention are Duchenne muscular distrophy and, more recently, neuromyelitis optica, the two pathological conditions in which OAPs are involved in completely different ways.

Mutations in the lamin A/C gene (LMNA) were associated with dilated cardiomyopathy (DCM) and, recently, were related to severe forms of arrhythmogenic right ventricular cardiomyopathy (ARVC). Both genetic and phenotypic overlap between DCM and ARVC was observed; molecular pathomechanisms leading to the cardiac phenotypes caused by LMNA mutations are not yet fully elucidated. This study involved a large Italian family, spanning 4 generations, with arrhythmogenic cardiomyopathy of different phenotypes, including ARVC, DCM, system conduction defects, ventricular arrhythmias, and sudden cardiac death. Mutation screening of LMNA and ARVC-related genes PKP2, DSP, DSG2, DSC2, JUP, and CTNNA3 was performed. We identified a novel heterozygous mutation (c.418_438dup) in LMNA gene exon 2, occurring in a highly conserved protein domain across several species. This newly identified variant was not found in 250 ethnically-matched control subjects. Genotype-phenotype correlation studies suggested a co-segregation of the LMNA mutation with the disease phenotype and an incomplete and age-related penetrance. Based on clinical, pedigree, and molecular genetic data, this mutation was considered likely disease-causing. To clarify its potential pathophysiologic impact, functional characterization of this LMNA mutant was performed in cultured cardiomyocytes expressing EGFP-tagged wild-type and mutated LMNA constructs, and indicated an increased nuclear envelope fragility, leading to stress-induced apoptosis as the main pathogenetic mechanism. This study further expands the role of the LMNA gene in the pathogenesis of cardiac laminopathies, suggesting that LMNA should be included in mutation screening of patients with suspected arrhythmogenic cardiomyopathy, particularly when they have ECG evidence for conduction defects. The combination of clinical, genetic, and functional data contribute insights into the pathogenesis of this form of life-threatening arrhythmogenic cardiac laminopathy.

Aquaporins (AQPs) play a physiological role in several organs and tissues, and their alteration is associated with disorders of water regulation. The identification of molecular interactions, which are crucial in determining the rate of water flux through the channel, is of pivotal role for the discovery of molecules able to target those interactions and therefore to be used for pathologies ascribable to an altered AQP-dependent water balance. In the present study, a mutational screening of human aquaporin-4 (AQP4) gene was performed on subjects with variable degrees of hearing loss. One heterozygous missense mutation was identified in a Spanish sporadic case, leading to an Asp/Glu amino acid substitution at position 184 (D184E). A BLAST analysis revealed that the amino acid D184 is conserved across species, consistently with a crucial role in the structure/function of AQP4 water channels. The mutation induces a significant reduction in water permeability as measured by the Xenopus laevis oocytes swelling assay and by the use of mammalian cells by total internal reflection microscopy. By Western blot, immunofluorescence and 2D Blue Native/SDS-PAGE we show that the reduction in water permeability is not ascribable to a reduced expression of AQP4 mutant protein or to its incorrect plasma membrane targeting and aggregation into orthogonal arrays of particles. Molecular dynamics simulation provided a molecular explanation of the mechanism whereby the mutation induces a loss of function of the channel. Substituting glutamate for aspartate affects the mobility of the D loop, which acquires a higher propensity to equilibrate in a "closed conformation", thus affecting the rate of water flux. We speculate that this mutation, combined with other genetic defects or concurrently with certain environmental stimuli, could confer a higher susceptibility to deafness. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

In this study, we recruited 10 neuromyelitis optica patients, two multiple sclerosis patients and two myelitis patients. Chinese hamster lung fibroblast (V79) cells transfected with a human aquaporin-4-mCherry fusion protein gene were used to detect anti-aquaporin-4 antibody in neuromyelitis optica patient sera by immunofluorescence. Anti-aquaporin-4 autoantibody was stably detected by immunofluorescence in neuromyelitis optica patient sera exclusively. The sensitivity of the assay for neuromyelitis optica was 90% and the specificity for neuromyelitis optica was 100%. The anti-aquaporin-4 antibody titers in sera were tested with serial dilutions until the signal disappeared. A positive correlation was detected between Expanded Disability Status Scale scores and serum anti-aquaporin-4 antibody titers. The anti-aquaporin-4 antibody assay is highly sensitive and specific in the sera of Chinese neuromyelitis optica patients. Detection of aquaporin-4 autoantibody is important for the diagnosis and treatment of neuromyelitis optica.

Serological markers of Nuromyelitis Optica (NMO), an autoimmune disorder of the central nervous system, are autoantibodies targeting the astrocytic water channel aquaporin-4 (AQP4). We have previously demonstrated that the main epitopes for these autoantibodies (AQP4-IgG) are generated by the supramolecular arrangement of AQP4 tetramers into an Orthogonal Array of Particles (OAPs). Many tests have been developed to detect AQP4- IgG in patient sera but several procedural issues affect OAP assembly and consequently test sensitivity. To date, the protein based ELISA test shows the lowest sensitivity while representing a valid alternative to the more sensitive cell based assay (CBA), which, however, shows economic, technical and interpretation problems. Here we have developed a high perfomance ELISA in which native OAPs are used as the molecular target. To this aim a native size exclusion chromatography method has been developed to isolate integral, highly pure and AQP4-IgG-recognized OAPs from rat brain. These OAPs were immobilized and oriented on a plastic plate by a sandwich approach and 139 human sera were tested, including 67 sera from NMO patients. The OAP-ELISA showed a 99% specificity and a higher sensitivity (91%) compared to the CBA test. A comparative analysis revealed an end-point titer three orders of magnitude higher than the commercial ELISA and six times higher than our in-house CBA test. We show that CNS-extracted OAPs are crucial elements in order to perform an efficient AQP4-IgG test and the OAP-ELISA developed represents a valid alternative to the CBA currently used. © 2015 Pisani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Aquaporin-4 (AQP4) exists as two major isoforms that differ in the length of the N terminus, the shorter AQP4-M23 and the longer AQP4-M1. Both isoforms form tetramers, which can further aggregate in the plasma membrane to form typical orthogonal arrays of particles (OAPs) whose dimension depends on the ratio of the M1 and M23. In this study, we tested the hypothesis that the M23 isoform can be produced directly by the M1 mRNA. In cells transiently transfected with AQP4-M1 coding sequence we observed besides AQP4-M1 the additional presence of the AQP4-M23 isoform associated with the formation of typical OAPs observable by two-dimensional blue native/SDS-PAGE and total internal reflection microscopy. The mutation of the second in-frame methionine M23 in AQP4-M1 (AQP4-M1(M23I)) prevented the expression of the M23 isoform and the formation of OAPs. We propose "leaky scanning" as a translational mechanism for the expression of AQP4-M23 protein isoform and that the formation of OAPs may occur even in the absence of AQP4-M23 mRNA. This mechanism can have important pathophysiological implications for the cell regulation of the M1/M23 ratio and thus OAP size. In this study we also provide evidence that AQP4-M1 is mobile in the plasma membrane, that it is inserted and not excluded into immobile OAPs, and that it is an important determinant of OAP structure and size

A novel mutation in the Lamin A/C gene (LMNA c.418_438dup) was detected in the index patient and in additional family members with diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) and history of sudden cardiac death. The functional characterization of this LMNA mutant was performed in cultured HL-1 cardiomyocytes expressing EGFP-tagged wild-type and mutated LMNA constructs and subjected to confocal microscopy analysis and hypoxic stress conditions in 100% N2 for 8h. Mutated LMNA was clearly expressed in aggregates of different sizes and not uniformly distributed along the nuclear envelope as WT LMNA. Moreover, the mutated LMNA variant causes perturbation in nuclear shape and Nuclear Pore Complexes organization. Of note, we observed that under hypoxic conditions nuclear envelopes expressing mutated LMNA become leaky, leading a nuclear fluorescent marker to escape into the cytoplasm. This indicates that, under cell stressing conditions, the nucleo-cytoplasmic compartmentalization is affected in cardiomyocytes expressing this LMNA mutation, inducing, as final fatal consequence, cell apoptosis. In conclusion, we not only open new avenues to gain more insights in the pathogenesis of ARVC and to conceive novel therapeutic strategies but we also shed lights on the role of nuclear Lamin A in the physio-pathology of cardiomyocytes.

Unlike other mammalian AQPs, multiple tetramers of AQP4 associate in the plasma membrane to form peculiar structures called Orthogonal Arrays of Particles (OAPs), that are observable by freeze-fracture electron microscopy (FFEM). However, FFEM cannot give information about the composition of OAPs of different sizes, and due to its technical complexity is not easily applicable as a routine technique. Recently, we employed the 2D gel electrophoresis BN-SDS/PAGE that for the first time enabled the biochemical isolation of AQP4-OAPs from several tissues. We found that AQP4 protein is present in several higher-order complexes (membrane pools of supra-structures) which contain different ratios of M1/M23 isoforms corresponding to AQP4-OAPs of different size. In this paper, we illustrate in detail the potentiality of 20 BN/SDS-PAGE for analyzing AQP4 supra-structures, their relationship with the dystrophin glycoprotein complex and other membrane proteins, and their role as a specific target of Neuromyelitis Optica autoantibodies.

Neuromyelitis optica (NMO) is an inflammatory autoimmune demyelinating disease of the central nervous system (CNS). NMO autoantibodies (NMO-IgG) recognize the glial water channel Aquaporin-4 which exists as two major isoforms differing in the length of the N terminus, the shorter AQP4-M23 and the longer AQP4-M1. Both isoforms form tetramers, which further aggregate in the plasma membrane to form typical Orthogonal Arrays of Particles (OAPs). We recently demonstrated that NMO-IgG epitope is intrinsic in AQP4 assemblies into OAPs. Other OAP-forming water-channel proteins, such as the lens Aquaporin-0 and the insect Aquaporin-cic, were not recognized by NMO-IgG, indicating an epitope characteristic of AQP4-OAPs. In this study we map the NMO-IgG antigenic determinants in the OAP extracellular surface. To identify the AQP4-OAP extracellular epitope for NMO IgG, we generated a series of AQP4 mutants, based on multi-alignment sequence analysis between AQP4 and other OAP-forming AQPs. Mutations were introduced in the three extracellular loops (A, C and E) and the binding capacity of NMO sera was tested by immunofluorecence and immunoprecipitation. Results indicate that one group of sera was able to recognize a limited portion of loop C containing the amino acid sequence G146VTTV150. It is likely that this conformational epitope is generated by surface associations of the C loops among different tetramers. A second group of sera was characterized by a predominant role of the loop A. Deletion of four AQP4-specific amino acids (G61SEN64) in loop A substantially affected the binding of this group of sera. However, the binding capacity was further reduced when amino acids in loop A were mutated together with those in loop E or when those in loop C were mutated in combination with loop E suggesting that loop C and E contribute together with loop A to generate the conformational epitope. Our data indicate that the NMO-IgG autoantibodies have a polyclonal origin and that the three AQP4 extracellular loops (A, C, and E) participate in the formation of the NMO-IgG epitope. This study identifies two major key immunodominant conformational epitopes and provides crucial information for the generation of a NMO disease model.

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease characterized by the presence of anti-aquaporin-4 (AQP4) antibodies in the patient sera. We recently reported that these autoantibodies are able to bind AQP4 when organized in the supramolecular structure called the orthogonal array of particles (OAP). To map the antigenic determinants, we produced a series of AQP4 mutants based on multiple alignment sequence analysis between AQP4 and other OAP-forming AQPs. Mutations were introduced in the three extracellular loops (A, C, and E), and the binding capacity of NMO sera was tested on AQP4 mutants. Results indicate that one group of sera was able to recognize a limited portion of loop C containing the amino acid sequence (146)GVT(T/M)V(150). A second group of sera was characterized by a predominant role of loop A. Deletion of four AQP4-specific amino acids ((61)G(S/T)E(N/K)(64)) in loop A substantially affected the binding of this group of sera. However, the binding capacity was further reduced when amino acids in loop A were mutated together with those in loop E or when those in loop C were mutated in combination with loop E. Finally, a series of AQP0 mutants were produced in which the extracellular loops were progressively changed to make them identical to AQP4. Results showed that none of the mutants was able to reproduce in AQP0 the NMO-IgG epitopes, indicating that the extracellular loop sequence by itself was not sufficient to determine the rearrangement required to create the epitopes. Although our data highlight the complexity of the disease, this study identifies key immunodominant epitopes and provides direct evidence that the transition from AQP4 tetramers to AQP4-OAPs involves conformational changes of the extracellular loops.

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.