Publications by authors named "Antonio Frigeri"

57 Publications

Orchestrating aquaporin-4 and connexin-43 expression in brain: Differential roles of α1- and β1-syntrophin.

Biochim Biophys Acta Biomembr 2021 Apr 16:183616. Epub 2021 Apr 16.

Laboratory of Molecular Neuroscience, Division of Anatomy, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Post box 1105, Blindern, 0317 Oslo, Norway. Electronic address:

Aquaporin-4 (AQP4) water channels and gap junction proteins (connexins) are two classes of astrocytic membrane proteins critically involved in brain water and ion homeostasis. AQP4 channels are anchored by α1-syntrophin to the perivascular astrocytic endfoot membrane domains where they control water flux at the blood-brain interface while connexins cluster at the lateral aspects of the astrocytic endfeet forming gap junctions that allow water and ions to dissipate through the astrocyte syncytium. Recent studies have pointed to an interdependence between astrocytic AQP4 and astrocytic gap junctions but the underlying mechanism remains to be explored. Here we use a novel transgenic mouse line to unravel whether β1-syntrophin (coexpressed with α1-syntrophin in astrocytic plasma membranes) is implicated in the expression of AQP4 isoforms and formation of gap junctions in brain. Our results show that while the effect of β1-syntrophin deletion is rather limited, double knockout of α1- and β1-syntrophin causes a downregulation of the novel AQP4 isoform AQP4ex and an increase in the number of astrocytic gap junctions. The present study highlight the importance of syntrophins in orchestrating specialized functional domains of brain astrocytes.
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http://dx.doi.org/10.1016/j.bbamem.2021.183616DOI Listing
April 2021

Longitudinal Evaluation of Serum MOG-IgG and AQP4-IgG Antibodies in NMOSD by a Semiquantitative Ratiometric Method.

Front Neurol 2021 8;12:633115. Epub 2021 Mar 8.

Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari "Aldo Moro", Bari, Italy.

Immunoadsorption (IA) is an antibody-depleting therapy used to treat neuromyelitis optica spectrum disorder (NMOSD) associated to antiaquaporin 4 (anti-AQP4-IgG) and antimyelin oligodendrocyte glycoprotein (anti-MOG-IgG) serum autoantibodies. Our aim was to evaluate longitudinal changes of serum MOG-IgG and AQP4-IgG antibody titer and to correlate it with the clinical status. Autoantibody titer and clinical features of two MOG-IgG+/AQP4-IgG- and two AQP4-IgG+/MOG-IgG- patients with NMOSD were collected at baseline (T0), after 6 IA courses (T1), and then 2 weeks (T2) and 6 months after treatment (T3). A fluorescent ratiometric assay was used for a quantitative detection of MOG and AQP4 antibodies, based on HEK-293 cells transfected with the full-length hMOG fused to GFP or h-AQP4-M23 isoform fused to m-cherry, respectively. We defined the antibody titer as MOG quantitative ratio (MOGqr) and AQP4 quantitative ratio (AQP4qr). In Case 1, the MOGqr dropped from 0.98 at T0 to 0.14 at T3, and in Case 2, it decreased from 0.96 at T0 to undetectable at T3. In Case3, the AQP4qr remained high: 0.90 at T0 and 0.92 at T3. In Case 4, the AQP4qr decreased from 0.50 at T0 to undetectable at T3. Complete recovery was found in Cases 1, 2, and 4. Semiquantitative ratiometric method accurately detects even slight variation of MOG-IgG and AQP4-IgG titer, suggesting it may be useful to monitor the antibody titer during the disease course and maintenance immunotherapy.
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http://dx.doi.org/10.3389/fneur.2021.633115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7982799PMC
March 2021

Orthogonal arrays of particle assembly are essential for normal aquaporin-4 expression level in the brain.

Glia 2021 Feb 18;69(2):473-488. Epub 2020 Sep 18.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy.

Astrocyte endfeet are endowed with aquaporin-4 (AQP4)-based assemblies called orthogonal arrays of particles (OAPs) whose function is still unclear. To investigate the function of OAPs and of AQP4 tetramers, we have generated a novel "OAP-null" mouse model selectively lacking the OAP forming M23-AQP4 isoform. We demonstrated that AQP4 transcript levels were not reduced by using qPCR. Blue native (BN)/SDS-PAGE and Western blot performed on OAP-null brain and primary astrocyte cultures showed the complete depletion of AQP4 assemblies, the selective expression of M1-AQP4-based tetramers, and a substantial reduction in AQP4 total expression level. Fluorescence quenching and super-resolution microscopy experiments showed that AQP4 tetramers were functionally expressed in astrocyte plasma membrane and their dimensions were reduced compared to wild-type assemblies. Finally, as shown by light and electron microscopy, OAP depletion resulted in a massive reduction in AQP4 expression and a loss of perivascular AQP4 staining at astrocyte endfeet, with only sparse labeling throughout the brain areas analyzed. Our study relies on the unique property of AQP4 to form OAPs, using a novel OAP-null mouse model for the first time, to show that (a) AQP4 assembly is essential for normal AQP4 expression level in the brain and (b) most of AQP4 is organized into OAPs under physiological conditions. Therefore, AQP4 tetramers cannot be used by astrocytes as an alternative to OAPs without affecting AQP4 expression levels, which is important in the physiological and pathological conditions in which OAP aggregation/disaggregation dynamics have been implicated.
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http://dx.doi.org/10.1002/glia.23909DOI Listing
February 2021

Effect of Cladribine on Neuronal Apoptosis: New Insight of In Vitro Study in Multiple Sclerosis Therapy.

Brain Sci 2020 Aug 13;10(8). Epub 2020 Aug 13.

Department of Basic Medical Science, Neuroscience and Sense Organs, School of Medicine, University of Bari Aldo Moro, 70124 Bari, Italy.

Background: Cladribine (2-CdA) can cross the blood-brain barrier, resulting in inhibition of DNA synthesis and repair and disruption of cellular proliferation in actively dividing lymphocytes. No data on effect on neurons are available.

Aim: To study "in vitro" 2-CdA apoptotic effects on neurons in healthy donor and multiple sclerosis patient lymphocytes.

Methods: Neuroblastoma cells were co-cultured with lymphocytes, with and without 2-CdA.

Results: Apoptosis increased in lymphocytes with 2-CdA; increase was also observed when lymphocytes were cultured with neuronal cells. However, neurons were not affected by 2-CdA for apoptosis.

Conclusions: 2-CdA causes peripheral and central lymphocyte death preserving neurons, with a reasonable impact on inflammation and neuroprotection.
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http://dx.doi.org/10.3390/brainsci10080548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464206PMC
August 2020

Stimulation of water and calcium dynamics in astrocytes with pulsed infrared light.

FASEB J 2020 05 23;34(5):6539-6553. Epub 2020 Mar 23.

Istituto per la Sintesi Organica e la Fotoreattività, Consiglio Nazionale delle Ricerche, Bologna, Italy.

Astrocytes are non-neuronal cells that govern the homeostatic regulation of the brain through ions and water transport, and Ca -mediated signaling. As they are tightly integrated into neural networks, label-free tools that can modulate cell function are needed to evaluate the role of astrocytes in brain physiology and dysfunction. Using live-cell fluorescence imaging, pharmacology, electrophysiology, and genetic manipulation, we show that pulsed infrared light can modulate astrocyte function through changes in intracellular Ca and water dynamics, providing unique mechanistic insight into the effect of pulsed infrared laser light on astroglial cells. Water transport is activated and, IP R, TRPA1, TRPV4, and Aquaporin-4 are all involved in shaping the dynamics of infrared pulse-evoked intracellular calcium signal. These results demonstrate that astrocyte function can be modulated with infrared light. We expect that targeted control over calcium dynamics and water transport will help to study the crucial role of astrocytes in edema, ischemia, glioma progression, stroke, and epilepsy.
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http://dx.doi.org/10.1096/fj.201903049RDOI Listing
May 2020

Tissue Distribution of the Readthrough Isoform of AQP4 Reveals a Dual Role of AQP4ex Limited to CNS.

Int J Mol Sci 2020 Feb 24;21(4). Epub 2020 Feb 24.

Department of Basic Medical Sciences, Neurosciences and Sense Organs, School of Medicine, University of Bari Aldo Moro, 70124 Bari, Italy.

Translational readthrough (TRT) of aquaporin-4 (AQP4) has remarkably expanded the importance of this new post-transcriptional mechanism, as well as the regulation potential of AQP4. The TRT isoform of AQP4, named AQP4ex, is central for both AQP4 polarization and water channel activity in the central nervous system (CNS). Here we evaluate the relevance of the TRT mechanism by analyzing whether AQP4ex is also expressed in peripheral tissues and whether the expression of AQP4ex is necessary for its polarized expression as it occurs in perivascular astrocyte processes. To this purpose, AQP4ex null mice were used, and analysis was performed by immunolocalization and immunoblot. The results demonstrate that AQP4ex is expressed in kidney, stomach, trachea and skeletal muscle with the same localization pattern as the canonical AQP4 isoforms. AQP4ex protein levels vary from 6% to about 13% of the total AQP4 protein levels in peripheral tissues. Immunogold electron microscopy experiments demonstrated the localization of AQP4ex at the astrocytic endfeet, and experiments conducted on AQP4ex null mice CNS confirmed that the expression of AQP4ex is necessary for anchoring of the perivascular AQP4. Without the readthrough isoform, AQP4 assemblies are mis-localized, being uniformly distributed on the astrocyte processes facing the neuropile. No alteration of AQP4 polarization was found in AQP4ex null kidney, stomach, trachea or skeletal muscle, suggesting that AQP4ex does not have a role for proper membrane localization of AQP4 in peripheral tissues. We conclude that a dual role for AQP4ex is limited to the CNS.
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http://dx.doi.org/10.3390/ijms21041531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073200PMC
February 2020

Uncoupling of the Astrocyte Syncytium Differentially Affects AQP4 Isoforms.

Cells 2020 02 7;9(2). Epub 2020 Feb 7.

Division of Anatomy, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, 0315 Oslo, Norway.

The water channel protein aquaporin-4 (AQP4) and the gap junction forming proteins connexin-43 (Cx43) and connexin-30 (Cx30) are astrocytic proteins critically involved in brain water and ion homeostasis. While AQP4 is mainly involved in water flux across the astrocytic endfeet membranes, astrocytic gap junctions provide syncytial coupling allowing intercellular exchange of water, ions, and other molecules. We have previously shown that mice with targeted deletion of Aqp4 display enhanced gap junctional coupling between astrocytes. Here, we investigate whether uncoupling of the astrocytic syncytium by deletion of the astrocytic connexins Cx43 and Cx30 affects AQP4 membrane localization and expression. By using quantitative immunogold cytochemistry, we show that deletion of astrocytic connexins leads to a substantial reduction of perivascular AQP4, concomitant with a down-regulation of total AQP4 protein and mRNA. Isoform expression analysis shows that while the level of the predominant AQP4 M23 isoform is reduced in Cx43/Cx30 double deficient hippocampal astrocytes, the levels of M1, and the alternative translation AQP4ex isoform protein levels are increased. These findings reveal a complex interdependence between AQP4 and connexins, which are both significantly involved in homeostatic functions and astrogliopathologies.
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http://dx.doi.org/10.3390/cells9020382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072498PMC
February 2020

AQP4ex is crucial for the anchoring of AQP4 at the astrocyte end-feet and for neuromyelitis optica antibody binding.

Acta Neuropathol Commun 2019 04 1;7(1):51. Epub 2019 Apr 1.

Department of Basic Medical Sciences, Neurosciences and Sense Organs, School of Medicine, University of Bari Aldo Moro, Bari, Italy.

Brain water homeostasis is essential for the appropriate control of neuronal activity. Furthermore, the encasement of the central nervous system (CNS) by a hard structure, greatly limits its tolerance for the volume changes occurring with acute brain edema, which quickly leads to severe damage or death.The recent discovery of the extended isoform of AQP4 (AQP4ex), generated by translational readthrough, revealed a potential new mechanism of water transport regulation and polarization at the blood-brain-barrier level.In the present study we used CRISPR/Cas9 technology to generate an AQP4ex mouse model and evaluate the effect on the overall AQP4 expression, polarization, supramolecular organization in orthogonal arrays of particles (OAPs) and neuromyelitis optica (NMO-IgG) autoantibodies binding.AQP4ex removal did not cause a decrease in total AQP4 protein expression but completely suppressed the specific location of AQP4 at the astrocyte endfeet. Without AQP4ex, AQP4 was mislocalized and α-syntrophin expression, the selective partner for AQP4 localization, was partially altered. The supramolecular organization of AQP4 in OAPs was subtly altered. Indeed, the absence of AQP4ex reduced the size of AQP4-OAPs but the number of AQP4-OAP pools remained largely the same. More importantly, AQP4ex resulted critical for the binding of pathogenic human NMO-IgG autoantibodies to the brain. Indeed, the absence of AQP4ex completely abolished the binding of NMO-IgG at the perivascular astrocyte endfeet.This study provides the first direct evidence in vivo on the specific role of AQP4ex in AQP4 perivascular OAPs assembly and confinement and reveals AQP4ex as new and important player in neuromyelitis optica.
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http://dx.doi.org/10.1186/s40478-019-0707-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444679PMC
April 2019

AQP4 Aggregation State Is a Determinant for Glioma Cell Fate.

Cancer Res 2019 05 15;79(9):2182-2194. Epub 2019 Mar 15.

Department of Bioscience, Biotechnology and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari Aldo Moro, Bari, Italy.

The glial water channel protein aquaporin-4 (AQP4) forms heterotetramers in the plasma membrane made of the M23-AQP4 and M1-AQP4 isoforms. The isoform ratio controls AQP4 aggregation into supramolecular structures called orthogonal arrays of particles (AQP4-OAP). The role of AQP4 aggregation into OAP in malignant gliomas is still unclear. In this study, we demonstrate that AQP4 aggregation/disaggregation into OAP influences the biology of glioma cells. Selective expression of the OAP-forming isoform M23-AQP4 (AQP4-OAP) triggered cell shape changes in glioma cells associated with alterations to the F-actin cytoskeleton that affected apoptosis. By contrast, expression of M1-AQP4 (AQP4-tetramers), which is unable to aggregate into OAP, ameliorated glioma cell invasiveness, improved cell migration, and increased methalloproteinase-9 activity. Two prolines (254 and 296) at the C-terminus tail were shown to be important in mediating the relationship between the actin cytoskeleton and AQP4-OAP and AQP4-tetramers. In conclusion, this study demonstrates that AQP4 aggregation state might be an important determinant in orienting glioma cells to persist or perish. AQP4 disaggregation may potentiate invasiveness potential, whereas AQP4 aggregation may activate the apoptotic path. This study shows a new perspective on the role of AQP4 in brain tumors not necessarily associated with edema formation but with AQP4 aggregation/disaggregation dynamics and their link with the actin cytoskeleton. SIGNIFICANCE: This study demonstrates how AQP4 aggregation influences plasma membrane dynamics to alter cell proliferation, invasiveness, migration, and apoptotic potential in glioma cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-2015DOI Listing
May 2019

Host-Cell Type Dependent Features of Recombinant Human Aquaporin-4 Orthogonal Arrays of Particles-New Insights for Structural and Functional Studies.

Cells 2019 02 2;8(2). Epub 2019 Feb 2.

School of Medicine, Basic Medical Sciences, Neuroscience and Sense Organs, Univ. of Bari "Aldo Moro", 70124 Bari, Italy.

The CNS plasma-membrane water channel aquaporin-4 (AQP4) is expressed as two major isoforms able to aggregate into supramolecular assemblies known as 'orthogonal arrays of particles' (OAPs). OAP subnanometric features are largely unknown mainly because a method for the expression, isolation, and crystallization of integral human OAPs has not been developed. Here, the human OAP-forming isoform M23-AQP4 was expressed in insect and mammalian cell lines and AQP4 and OAP features evaluated. Native size exclusion chromatography was employed to isolate and analyze authentically folded OAPs, and neuromyelitis optica (NMO)-specific sandwich ELISA was developed to test OAP-integrity. The results demonstrate that in insect cells most AQP4 remains intracellular and unfolded and that OAPs are largely disassembled after the detergent extraction step. In mammalian cells, AQP4 showed regular plasma membrane targeting and OAPs exhibited strong post-extraction stability. Starting from the mammalian cell expression system, we isolated authentically folded OAPs. Together these data suggest a new strategy for expressing and isolating integral recombinant human OAPs and providing new insights into the cell-type dependent OAP-assembly and post-extraction stability, potentially useful to design new approaches for structural and functional studies of OAP and for other plasma membrane proteins organized into supramolecular structures.
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http://dx.doi.org/10.3390/cells8020119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406603PMC
February 2019

Response to 'The evolving mystery of why skeletal muscle is spared in seropositive neuromyelitis optica'.

J Cell Mol Med 2018 03 31;22(3):2041-2042. Epub 2018 Jan 31.

Department of Bioscience, Biotechnology and Biopharmaceutics, University of Bari "Aldo Moro", Bari, Italy.

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http://dx.doi.org/10.1111/jcmm.13513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824411PMC
March 2018

Supramolecular aggregation of aquaporin-4 is different in muscle and brain: correlation with tissue susceptibility in neuromyelitis optica.

J Cell Mol Med 2018 02 20;22(2):1236-1246. Epub 2017 Oct 20.

Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA.

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system (CNS) caused by autoantibodies (NMO-IgG) against the water channel aquaporin-4 (AQP4). Though AQP4 is also expressed outside the CNS, for example in skeletal muscle, patients with NMO generally do not show clinical/diagnostic evidence of skeletal muscle damage. Here, we have evaluated whether AQP4 supramolecular organization is at the basis of the different tissue susceptibility. Using immunofluorescence we found that while the sera of our cohort of patients with NMO gave typical perivascular staining in the CNS, they were largely negative in the skeletal muscle. This conclusion was obtained using human, rat and mouse skeletal muscle including the AQP4-KO mouse. A biochemical analysis using a new size exclusion chromatography approach for AQP4 suprastructure fractionation revealed substantial differences in supramolecular AQP4 assemblies and isoform abundance between brain and skeletal muscle matching a lower binding affinity of NMO-IgG to muscle compared to the brain. Super-resolution microscopy analysis with g-STED revealed different AQP4 organization in native tissues, while in the brain perivascular astrocyte endfoot membrane AQP4 was mainly organized in large interconnected and raft-like clusters, in the sarcolemma of fast-twitch fibres AQP4 aggregates often appeared as small, relatively isolated linear entities. In conclusion, our results provide evidence that AQP4 supramolecular structure is different in brain and skeletal muscle, which is likely to result in different tissues susceptibility to the NMO disease.
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http://dx.doi.org/10.1111/jcmm.13401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5783885PMC
February 2018

Aquaporin-1 inhibition reduces metastatic formation in a mouse model of melanoma.

J Cell Mol Med 2018 02 17;22(2):904-912. Epub 2017 Oct 17.

Department of Bioscience, Biotechnologies and Biopharmaceutics, Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, BA, Italy.

Aquaporin-1 (AQP1) is a proangiogenic water channel protein promoting endothelial cell migration. We previously reported that AQP1 silencing by RNA interference reduces angiogenesis-dependent primary tumour growth in a mouse model of melanoma. In this study, we tested the hypothesis that AQP1 inhibition also affects animal survival and lung nodule formation. Melanoma was induced by injecting B16F10 cells into the back of C57BL6J mice. Intratumoural injection of AQP1 siRNA and CTRL siRNA was performed 10 days after tumour cell implantation. Lung nodule formation was analysed after the death of the mice. Western blot was used to quantify HIF-1α, caspase-3 (CASP3) and metalloproteinase-2 (MMP2) protein levels. We found that AQP1 knock-down (KD) strongly inhibited metastatic lung nodule formation. Moreover, AQP1 siRNA-treated mice showed a twofold survival advantage compared to mice receiving CTRL siRNAs. The reduced AQP1-dependent tumour angiogenesis caused a hypoxic condition, evaluated by HIF-1α significant increase, in turn causing an increased level of apoptosis in AQP1 KD tumours, assessed by CASP3 quantification and DNA fragmentation. Importantly, a decreased level of MMP2 after AQP1 KD indicated a decreased activity against extracellular matrix associated with reduced vascularization and metastatic formation. In conclusion, these findings highlight an additional role for AQP1 as an important determinant of tumour dissemination by facilitating tumour cell extravasation and metastatic formation. This study adds knowledge on the role played by AQP1 in tumour biology and supports the view of AQP1 as a potential drug target for cancer therapy.
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http://dx.doi.org/10.1111/jcmm.13378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5783831PMC
February 2018

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen-induced retinopathy: beneficial effect of the absence of AQP4.

J Cell Mol Med 2018 01 22;22(1):613-627. Epub 2017 Sep 22.

Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.

Hypoxia-dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin-4 (AQP4) is involved in the hypoxia-dependent VEGF upregulation in the retina of a mouse model of oxygen-induced retinopathy (OIR). The expression levels of VEGF, the hypoxia-inducible factor-1α (HIF-1α) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF-1 binding site (HBS) in the VEGF gene promoter, the binding of HIF-1α to the HBS, the retinal vascularization and function have been determined in the retina of wild-type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF-1α, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF-1α. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1α to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF-1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF-induced retinal damage in response to hypoxia.
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http://dx.doi.org/10.1111/jcmm.13348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742711PMC
January 2018

Translational readthrough generates new astrocyte AQP4 isoforms that modulate supramolecular clustering, glial endfeet localization, and water transport.

Glia 2017 05 16;65(5):790-803. Epub 2017 Feb 16.

Department of Basic Medical Sciences, Neurosciences and Sense Organs, University of Bari Aldo Moro, Bari, Italy.

Regulation of water homeostasis is a central feature of central nervous system pathophysiology. In this context, several lines of evidence suggest a crucial role for the water channel aquaporin-4 (AQP4) and its plasma membrane supramolecular organization as the key element. Here, we demonstrate the expression in tissues of additional isoforms of AQP4 characterized by a C-terminal extension generated by programmed translational readthrough. These extended isoforms (AQP4ex) display a perivascular polarization and expression in dystrophin-dependent pools. AQP4ex reduces supramolecular clustering tendency and allows AQP4 interactions with syntrophin. Furthermore, site-directed mutagenesis of two serines in the extended C-terminus of AQP4ex showed potential regulation of water permeability by phosphorylation. Finally, AQP4ex expression can be positively modulated by gentamicin treatment, demonstrating the possibility of regulating the AQP4 translational readthrough frequency. This novel regulatory mechanism could have important pathophysiological implications for conditions in which alternations have been reported in AQP4 structure.
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http://dx.doi.org/10.1002/glia.23126DOI Listing
May 2017

Role of the H-bond between L53 and T56 for Aquaporin-4 epitope in Neuromyelitis Optica.

Biochim Biophys Acta Biomembr 2017 03 24;1859(3):368-376. Epub 2016 Dec 24.

Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy; Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA. Electronic address:

Aquaporin-4 (AQP4) is the CNS water channel organized into well-ordered protein aggregates called Orthogonal Arrays of Particles (OAPs). Neuromyelitis Optica (NMO) is an autoimmune disease caused by anti-OAP autoantibodies (AQP4-IgG). Molecular Dynamics (MD) simulations have identified an H-bond between L53 and T56 as the key for AQP4 epitope and therefore of potential interest for drug design in NMO field. In the present study, we have experimentally tested this MD-prediction using the classic mutagenesis approach. We substituted T56 with V56 and tested this mutant for AQP4 aggregates and AQP4-IgG binding. gSTED super-resolution microscopy showed that the mutation does not affect AQP4 aggregate dimension; immunofluorescence and cytofluorimetric analysis demonstrated its unaltered AQP4-IgG binding, therefore invalidating the MD-prediction. We later investigated whether AQP4, expressed in Sf9 insect and HEK-293F cells, is able to correctly aggregate before and after the purification steps usually applied to obtain AQP4 crystal. The results demonstrated that AQP4-IgG recognizes AQP4 expressed in Sf9 and HEK-293F cells by immunofluorescence even though BN-PAGE analysis showed that AQP4 forms smaller aggregates when expressed in insect cells compared to mammalian cell lines. Notably, after AQP4 purification, from both insect and HEK-293F cells, no aggregates are detectable by BN-PAGE and AQP4-IgG binding is impaired in sandwich ELISA assays. All together these results indicate that 1) the MD prediction under analysis is not supported by experimental data and 2) the procedure to obtain AQP4 crystals might affect its native architecture and, as a consequence, MD simulations. In conclusion, given the complex nature of the AQP4 epitope, MD might not be the suitable for molecular medicine advances in NMO.
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http://dx.doi.org/10.1016/j.bbamem.2016.12.018DOI Listing
March 2017

Glio-vascular modifications caused by Aquaporin-4 deletion in the mouse retina.

Exp Eye Res 2016 05 24;146:259-268. Epub 2016 Mar 24.

Department of Bioscience, Biotechnologies and Biopharmaceutics and Centre of Excellence in Comparative Genomics, University of Bari "Aldo Moro", 70126 Bari, Italy; Institute of Biomembranes and Bioenergetics, National Research Council, Via Amendola 165/A, 70126 Bari, Italy; National Institute of Biostructures and Biosystems (INBB), Viale Medaglie D'Oro 305, 00136 Rome, Italy.

Aquaporin-4 (AQP4) is the Central Nervous System water channel highly expressed at the perivascular glial domain. In the retina, two types of AQP4 expressing glial cells take part in the blood-retinal barrier (BRB), astrocytes and Müller cells. The aim of the present study is to investigate the effect of AQP4 deletion on the retinal vasculature by looking at typical pathological hallmark such as BRB dysfunction and gliotic condition. AQP4 dependent BRB properties were evaluated by measuring the number of extravasations in WT and AQP4 KO retinas by Evans blue injection assay. AQP4 deletion did not affect the retinal vasculature, as assessed by Isolectin B4 staining, but caused BRB impairment to the deep plexus capillaries while the superficial and intermediate capillaries were not compromised. To investigate for gliotic responses caused by AQP4 deletion, Müller cells and astrocytes were analysed by immunofluorescence and western blot, using the Müller cell marker Glutamine Synthetase (GS) and the astrocyte marker GFAP. While GS expression was not altered in AQP4 KO retinas, a strong GFAP upregulation was found at the level of AQP4 KO astrocytes at the superficial plexus and not at Müller cells at the intermediate and deep plexi. These data, together with the upregulation of inflammatory markers (TNF-α, IL-6, IL-1β and ICAM-1) in AQP4 KO retinas indicated AQP4 deletion as responsible for a gliotic phenotype. Interestingly, no GFAP altered expression was found in AQP4 siRNA treated astrocyte primary cultures. All together these results indicate that AQP4 deletion is directly responsible for BRB dysfunction and gliotic condition in the mouse retina. The selective activation of glial cells at the primary plexus suggests that different regulatory elements control the reaction of astrocytes and Müller cells. Finally, GFAP upregulation is strictly linked to gliovascular crosstalk, as it is absent in astrocytes in culture. This study is useful to understand the role of AQP4 in the perivascular domain in the retina and its possible implications in the pathogenesis of retinal vascular diseases and of Neuromyelitis Optica, a human disease characterized by anti-AQP4 auto-antibodies.
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http://dx.doi.org/10.1016/j.exer.2016.03.019DOI Listing
May 2016

Development of an Aquaporin-4 Orthogonal Array of Particle-Based ELISA for Neuromyelitis Optica Autoantibodies Detection.

PLoS One 2015 24;10(11):e0143679. Epub 2015 Nov 24.

Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", 70126, Bari, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143679PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4658006PMC
June 2016

The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins.

Glia 2016 Jan 28;64(1):139-54. Epub 2015 Sep 28.

Department of Bioscience, Biotechnology, and Biopharmaceutics, University of Bari "Aldo Moro,", Bari, Italy.

Regulatory volume decrease (RVD) is a process by which cells restore their original volume in response to swelling. In this study, we have focused on the role played by two different Aquaporins (AQPs), Aquaporin-4 (AQP4), and Aquaporin-1 (AQP1), in triggering RVD and in mediating calcium signaling in astrocytes under hypotonic stimulus. Using biophysical techniques to measure water flux through the plasma membrane of wild-type (WT) and AQP4 knockout (KO) astrocytes and of an astrocyte cell line (DI TNC1) transfected with AQP4 or AQP1, we here show that AQP-mediated fast swelling kinetics play a key role in triggering and accelerating RVD. Using calcium imaging, we show that AQP-mediated fast swelling kinetics also significantly increases the amplitude of calcium transients inhibited by Gadolinium and Ruthenium Red, two inhibitors of the transient receptor potential vanilloid 4 (TRPV4) channels, and prevented by removing extracellular calcium. Finally, inhibition of TRPV4 or removal of extracellular calcium does not affect RVD. All together our study provides evidence that (1) AQP influenced swelling kinetics is the main trigger for RVD and in mediating calcium signaling after hypotonic stimulus together with TRPV4, and (2) calcium influx from the extracellular space and/or TRPV4 are not essential for RVD to occur in astrocytes.
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http://dx.doi.org/10.1002/glia.22921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905710PMC
January 2016

Identification of a point mutation impairing the binding between aquaporin-4 and neuromyelitis optica autoantibodies.

J Biol Chem 2014 Oct 19;289(44):30578-30589. Epub 2014 Sep 19.

Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro," 70126 Bari,. Electronic address:

Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp(69)) of TM2 for NMO-IgG epitope assembly. Mutation of Asp(69) to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp(69) mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D(69)H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp(69) is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis.
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http://dx.doi.org/10.1074/jbc.M114.582221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215237PMC
October 2014

A new gating site in human aquaporin-4: Insights from molecular dynamics simulations.

Biochim Biophys Acta 2014 Dec 19;1838(12):3052-60. Epub 2014 Aug 19.

Dipartimento di Farmacia, Scienze del Farmaco, Università di Bari "Aldo Moro," Via Orabona 4, Bari 70126, Italy. Electronic address:

Aquaporin-4 (AQP4) is the predominant water channel in different organs and tissues. An alteration of its physiological functioning is responsible for several disorders of water regulation and, thus, is considered an attractive target with a promising therapeutic and diagnostic potential. Molecular dynamics (MD) simulations performed on the AQP4 tetramer embedded in a bilayer of lipid molecules allowed us to analyze the role of spontaneous fluctuations occurring inside the pore. Following the approach by Hashido et al. [Hashido M, Kidera A, Ikeguchi M (2007) Biophys J 93: 373-385], our analysis on 200ns trajectory discloses three domains inside the pore as key elements for water permeation. Herein, we describe the gating mechanism associated with the well-known selectivity filter on the extracellular side of the pore and the crucial regulation ensured by the NPA motifs (asparagine, proline, alanine). Notably, on the cytoplasmic side, we find a putative gate formed by two residues, namely, a cysteine belonging to the loop D (C178) and a histidine from loop B (H95). We observed that the spontaneous reorientation of the imidazole ring of H95 acts as a molecular switch enabling H-bond interaction with C178. The occurrence of such local interaction seems to be responsible for the narrowing of the pore and thus of a remarkable decrease in water flux rate. Our results are in agreement with recent experimental observations and may represent a promising starting point to pave the way for the discovery of chemical modulators of AQP4 water permeability.
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http://dx.doi.org/10.1016/j.bbamem.2014.08.015DOI Listing
December 2014

A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability.

Mol Biol Cell 2014 Feb 19;25(4):470-80. Epub 2013 Dec 19.

Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro," 70126 Bari, Italy Center for Integrative Genomics, University of Lausanne, 1015 Lausanne, Switzerland M. Sarcone Hospital, 70038 Terlizzi, Bari, Italy.

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.
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http://dx.doi.org/10.1091/mbc.E13-06-0331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923639PMC
February 2014

Aquaporin-4 autoantibodies in Neuromyelitis Optica: AQP4 isoform-dependent sensitivity and specificity.

PLoS One 2013 15;8(11):e79185. Epub 2013 Nov 15.

Department of Bioscience, Biotechnologies and Biopharmaceutic and Center of Excellence in Comparative Genomics, University of Bari "Aldo Moro", Bari, Italy.

Neuromyelitis Optica (NMO) is an autoimmune demyelinating disease, characterized by the presence of autoantibody (NMO-IgG) to Aquaporin-4 (AQP4). NMO-IgG identification supports NMO diagnosis and several diagnostic tests have been developed, but their sensitivity is too variable, and some assay show low sensitivity. This impairs correct diagnosis of NMO. By cell based assay (CBA) we here evaluate the efficacy of different strategies to express AQP4 in mammalian cells in terms of: a) AQP4 translation initiation signals; b) AQP4 isoforms (M1 and M23) and fluorescent tag position; c) NMO serum concentration and AQP4 degradation. Our results demonstrate that when using AQP4-M1, the nucleotide in position -3 of the AUG greatly affects the AQP4-M1/M23 protein ratio, NMO-IgG binding, and consequently test sensitivity. Test sensitivity was highest with M23 expressing cells (97.5%) and only 27.5% with AQP4-M1. The fluorescent tag added to the N-terminus of AQP4-M23 considerably affected the NMO-IgG binding, and test sensitivity, due to disruption of AQP4 suprastructures. Furthermore, sera used at high concentration resulted in AQP4 degradation which affected test sensitivity. To further evaluate the reliability of the M23 based CBA test, samples of one NMO patient collected during about 2 years clinical follow-up were tested. The results of serum titer correlated with disease activity and treatment response. In conclusion, we provide a molecular explanation for the contrasting CBA test data reported and suggest the use of M23 with a C-terminus fluorescent tag as the proper test for NMO diagnosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079185PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829826PMC
July 2014

AQP4-dependent water transport plays a functional role in exercise-induced skeletal muscle adaptations.

PLoS One 2013 8;8(3):e58712. Epub 2013 Mar 8.

Department of Bioscience, Biotechnologies and Biopharmaceutics and Center of Excellence in Comparative Genomics (CEGBA), University of Bari, Bari, Italy.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058712PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592820PMC
September 2013

Detection of anti-aquaporin-4 autoantibodies in the sera of Chinese neuromyelitis optica patients.

Neural Regen Res 2013 Mar;8(8):708-13

Membrane Channel Research Laboratory, Northeast Normal University, Changchun 130024, Jilin Province, China.

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.
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http://dx.doi.org/10.3969/j.issn.1673-5374.2013.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146080PMC
March 2013

Inhibition of aquaporin-1 dependent angiogenesis impairs tumour growth in a mouse model of melanoma.

J Mol Med (Berl) 2013 May 30;91(5):613-23. Epub 2012 Nov 30.

Department of Bioscience, Biotechnology and Pharmacological Sciences and Centre of Excellence in Comparative Genomics (CEGBA), University of Bari, Via Amendola 165/A, 70126, Bari, Italy.

Prohibiting angiogenesis is an important therapeutic approach for fighting cancer and other angiogenic related diseases. Research focused on proteins that regulate abnormal angiogenesis has attracted intense interest in both academia and industry. Such proteins are able to target several angiogenic factors concurrently, thereby increasing the possibility of therapeutic success. Aquaporin-1 (AQP1) is a water channel membrane protein that promotes tumour angiogenesis by allowing faster endothelial cell migration. In this study we test the hypothesis that AQP1 inhibition impairs tumour growth in a mouse model of melanoma. After validating the inhibitor efficacy of two different AQP1 specific siRNAs in cell cultures, RNA interference experiments were performed by intratumoural injections of AQP1 siRNAs in mice. After 6 days of treatment, AQP1 siRNA treated tumours showed a 75 % reduction in volume when compared to controls. AQP1 protein level, in AQP1 knockdown tumours, was around 75 % that of the controls and was associated with a significant 40 % reduced expression of the endothelial marker, Factor VIII. Immunofluorescence analysis of AQP1 siRNA treated tumours showed a significantly lower microvessel density. Time course experiments demonstrated that repeated injections of AQP1 siRNA over time are effective in sustaining the inhibition of tumour growth. Finally, we have confirmed the role of AQP1 in sustaining an active endothelium during angiogenesis and we have shown that AQP1 reduction causes an increase in VEGF levels. In conclusion, this study validates AQP1 as a pro-angiogenic protein, relevant for the therapy of cancer and other angiogenic-related diseases such as psoriasis, endometriosis, arthritis and atherosclerosis.
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http://dx.doi.org/10.1007/s00109-012-0977-xDOI Listing
May 2013

Translational regulation mechanisms of aquaporin-4 supramolecular organization in astrocytes.

Glia 2011 Dec 17;59(12):1923-32. Epub 2011 Aug 17.

Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA), University of Bari, Bari, Italy.

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.
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http://dx.doi.org/10.1002/glia.21234DOI Listing
December 2011

Absence of aquaporin-4 in skeletal muscle alters proteins involved in bioenergetic pathways and calcium handling.

PLoS One 2011 Apr 28;6(4):e19225. Epub 2011 Apr 28.

Department of General and Environmental Physiology, University of Bari-Aldo Moro, Bari, Italy.

Aquaporin-4 (AQP4) is a water channel expressed at the sarcolemma of fast-twitch skeletal muscle fibers, whose expression is altered in several forms of muscular dystrophies. However, little is known concerning the physiological role of AQP4 in skeletal muscle and its functional and structural interaction with skeletal muscle proteome. Using AQP4-null mice, we analyzed the effect of the absence of AQP4 on the morphology and protein composition of sarcolemma as well as on the whole skeletal muscle proteome. Immunofluorescence analysis showed that the absence of AQP4 did not perturb the expression and cellular localization of the dystrophin-glycoprotein complex proteins, aside from those belonging to the extracellular matrix, and no alteration was found in sarcolemma integrity by dye extravasation assay. With the use of a 2DE-approach (BN/SDS-PAGE), protein maps revealed that in quadriceps, out of 300 Coomassie-blue detected and matched spots, 19 proteins exhibited changed expression in AQP4(-/-) compared to WT mice. In particular, comparison of the protein profiles revealed 12 up- and 7 down-regulated protein spots in AQP4-/- muscle. Protein identification by MS revealed that the perturbed expression pattern belongs to proteins involved in energy metabolism (i.e. GAPDH, creatine kinase), as well as in Ca(2+) handling (i.e. parvalbumin, SERCA1). Western blot analysis, performed on some significantly changed proteins, validated the 2D results. Together these findings suggest AQP4 as a novel determinant in the regulation of skeletal muscle metabolism and better define the role of this water channel in skeletal muscle physiology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019225PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084271PMC
April 2011

Identification of two major conformational aquaporin-4 epitopes for neuromyelitis optica autoantibody binding.

J Biol Chem 2011 Mar 6;286(11):9216-24. Epub 2011 Jan 6.

Department of General and Environmental Physiology, Centre of Excellence in Comparative Genomics, University of Bari, I-70126 Bari, Italy.

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.
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http://dx.doi.org/10.1074/jbc.M110.123000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059066PMC
March 2011

Analysis by two-dimensional Blue Native/SDS-PAGE of membrane protein alterations in rat soleus muscle after hindlimb unloading.

Eur J Appl Physiol 2010 Dec 24;110(6):1215-24. Epub 2010 Aug 24.

Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA), University of Bari, 70126, Bari, Italy.

Muscle atrophy occurring in several pathophysiological conditions determines decreases in muscle protein synthesis, increases in the rate of proteolysis and changes in muscle fiber composition. To determine the effect of muscle atrophy induced by hindlimb unloading (HU) on membrane proteins from rat soleus, a proteomic approach based on two-dimensional Blue Native/SDS-PAGE was performed. Proteomic analysis of normal and HU soleus muscle demonstrates statistically significant changes in the relative level of 36 proteins. Among the proteins identified by mass spectrometry, most are involved in pathways associated with muscle fuel utilization, indicating a shift in metabolism from oxidative to glycolytic. Moreover, immunoblotting analysis revealed an increase in aquaporin-4 (AQP4) water channel and an alteration of proteins belonging to the dystrophin-glycoprotein complex (DGC). AQP4 and DGC are regulated in soleus muscle subjected to simulated microgravity in response to compensatory mechanisms induced by muscle atrophy, and they parallel the slow-to-fast twitch conversion that occurs in soleus fibers during HU. In conclusion, the alterations of soleus muscle membrane proteome may play a pivotal role in the mechanisms involved in disuse-induced muscle atrophy.
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http://dx.doi.org/10.1007/s00421-010-1592-6DOI Listing
December 2010