Orthogonal Arrays Of Particles Research Articles

Aquaporin-4 and transient receptor potential vanilloid 4 balance in early postnatal neurodevelopment.

In the adult brain, the water channel aquaporin-4 (AQP4) is expressed in astrocyte endfoot, in supramolecular assemblies, called "Orthogonal Arrays of Particles" (OAPs) together with the transient receptor potential vanilloid 4 (TRPV4), finely regulating the cell volume. The present study aimed at investigating the contribution of AQP4 and TRPV4 to CNS early postnatal development using WT and AQP4 KO brain and retina and neuronal stem cells (NSCs), as an in vitro model of astrocyte differentiation. Western blot analysis showed that, differently from AQP4 and the glial cell markers, TRPV4 was downregulated during CNS development and NSC differentiation. Blue native/SDS-PAGE revealed that AQP4 progressively organized into OAPs throughout the entire differentiation process. Fluorescence quenching assay indicated that the speed of cell volume changes was time-related to NSC differentiation and functional to their migratory ability. Calcium imaging showed that the amplitude of TRPV4 Ca2+ transient is lower, and the dynamics are changed during differentiation and suppressed in AQP4 KO NSCs. Overall, these findings suggest that early postnatal neurodevelopment is subjected to temporally modulated water and Ca2+ dynamics likely to be those sustaining the biochemical and physiological mechanisms responsible for astrocyte differentiation during brain and retinal development.

Thermodynamics and S-Palmitoylation Dependence of Interactions between Human Aquaporin-4 M1 Tetramers in Model Membranes.

Aquaporin-4 (AQP4) is a water channel protein found primarily in the central nervous system (CNS) that helps to regulate water-ion homeostasis. AQP4 exists in two major isoforms: M1 and M23. While both isoforms have a homotetrameric quaternary structure and are functionally identical when transporting water, the M23 isoform forms large protein aggregates known as orthogonal arrays of particles (OAPs). In contrast, the M1 isoform creates a peripheral layer around the outside of these OAPs, suggesting a thermodynamically stable interaction between the two. Structurally, the M1 isoform has an N-terminal tail that is 22 amino acids longer than the M23 isoform and contains two solvent-accessible cysteines available for S-palmitoylation at cysteine-13 (Cys-13) and cysteine-17 (Cys-17) in the amino acid sequence. Earlier work suggests that the palmitoylation of these cysteines might aid in regulating AQP4 assemblies. This work discusses the thermodynamic driving forces for M1 protein-protein interactions and how the palmitoylation state of M1 affects them. Using temperature-dependent single-particle tracking, the standard state free energies, enthalpies, and entropies were measured for these interactions. Furthermore, we present a binding model based on measured thermodynamics and a structural modeling study. The results of this study demonstrate that the M1 isoform will associate with itself according to the following expressions: 2[AQP4-M1]4 ↔ [[AQP4-M1]4]2 when palmitoylated and 3[AQP4-M1]4 ↔ [AQP4-M1]4 + [[AQP4-M1]4]2 ↔ [[AQP4-M1]4]3 when depalmitoylated. This is primarily due to a conformational change induced by adding the palmitic acid groups at Cys-13 and Cys-17 in the N-terminal tails of the homotetramers. In addition, a statistical mechanical model was developed to estimate the Gibbs free energy, enthalpy, and entropy for forming dimers and trimers. These results were in good agreement with experimental values.

S-palmitoylation dependent self assembly of human aquaporin-4 orthogonal arrays of particles in biomimetic membranes.

Aquaporin-4 (AQP4) is a water channel protein located in the plasma membrane of astrocytes in the central nervous system (CNS) and helps to regulate water-ion homeostasis. Isoforms of AQP4 further order themselves into aggregates known as orthogonal arrays of particles (OAPs). AQP4 exists in two main isoforms, M1 and M23, with M23 being the isoform that favors stabilization by aggregation into OAPs. It has been hypothesized that the aggregation size is regulated by the amount of M1 isoform present in the membrane. In turn, the palmitoylation state of two cysteine residues on the N-terminal tail of the M1 isoform greatly affects its mobility and ability to inhibit OAP formation. In this study, M1 was chemically cleaved and palmitoylated in vitro, and inserted in biomimetic lipid bilayers to study kinetics and aggregation behavior using single-protein fluorescence tracking. This study showed that when M1 was palmitoylated, it displays fast diffusion through the membrane with short lived protein-protein interactions and no evidence of aggregation. The depalmitoylated form of M1 showed evidence of extensive aggregation and long-lived protein-protein interactions. This study is the first of its kind to investigate human AQP4 in vitro and to extract the kinetic rate constants for protein-protein interactions as well as estimates of their equilibrium constants.

AQP4-A25Q Point Mutation in Mice Depolymerizes Orthogonal Arrays of Particles and Decreases Polarized Expression of AQP4 Protein in Astrocytic Endfeet at the Blood-Brain Barrier.

Aquaporin-4 (AQP4) is characterized by the formation of orthogonal arrays of particles (OAPs) comprising its M1 and M23 isoforms in the plasma membrane. However, the biological importance of OAP formation is obscure. Here, we developed an OAP depolymerization male mouse model by transgenic knock-in of an AQP4-A25Q mutation. Analyses of the mutant brain tissue using blue native polyacrylamide gel electrophoresis, super-resolution imaging, and immunogold electron microscopy revealed remarkably reduced OAP structures and glial endfeet localization of the AQP4-A25Q mutant protein without effects on its overall mRNA and protein expression. AQP4A25Q/A25Q mice showed better survival and neurologic deficit scores when cerebral edema was induced by water intoxication or middle cerebral artery occlusion/reperfusion. The brain water content and swelling of pericapillary astrocytic endfeet processes in AQP4A25Q/A25Q mice were significantly reduced, functionally supporting decreased AQP4 protein expression at the blood-brain barrier. The infarct volume and neuronal damage were also reduced in AQP4A25Q/A25Q mice in the middle cerebral artery occlusion/reperfusion model. Astrocyte activation in the brain was alleviated in AQP4A25Q/A25Q mice, which may be associated with decreased cell swelling. We conclude that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.SIGNIFICANCE STATEMENT Aquaporin-4 (AQP4) is characterized by orthogonal arrays of particles (OAPs) comprising the M1 and M23 isoforms in the membrane. Here, an OAP depolymerization male mouse model induced by AQP4-A25Q mutation was first established, and the functions of OAP depolymerization in cerebral edema have been studied. The results revealed that AQP4 lost its OAP structure without affecting AQP4 mRNA and protein levels in AQP4-A25Q mice. AQP4-A25Q mutation mice has neuroprotective effects on cerebral edema induced by water intoxication and middle cerebral artery occlusion/reperfusion through relieving the activation of astrocytes and suppressed microglia-mediated neuroinflammation. We concluded that the OAP structure of AQP4 plays a key role in its polarized expression in astrocytic endfeet processes at the blood-brain barrier. Therefore, our study provided new insights into intervention of cerebral cellular edema caused by stroke and traumatic brain injury through regulating AQP4 OAP formation.

Aquaporin-4 in Neuromyelitis Optica Spectrum Disorders: A Target of Autoimmunity in the Central Nervous System.

Since the discovery of a specific autoantibody in patients with neuromyelitis optica spectrum disorder (NMOSD) in 2004, the water channel aquaporin-4 (AQP4) has attracted attention as a target of autoimmune diseases of the central nervous system. In NMOSD, the autoantibody (NMO-IgG) binds to the extracellular loops of AQP4 as expressed in perivascular astrocytic end-feet and disrupts astrocytes in a complement-dependent manner. NMO-IgG is an excellent marker for distinguishing the disease from other inflammatory demyelinating diseases, such as multiple sclerosis. The unique higher-order structure of AQP4—called orthogonal arrays of particles (OAPs)—as well as its subcellular localization may play a crucial role in the pathogenesis of the disease. Recent studies have also demonstrated complement-independent cytotoxic effects of NMO-IgG. Antibody-induced endocytosis of AQP4 has been suggested to be involved in this mechanism. This review focuses on the binding properties of antibodies that recognize the extracellular region of AQP4 and the characteristics of AQP4 that are implicated in the pathogenesis of NMOSD.

Human Aquaporin-4 orthogonal array of particles in biomimetic membranes: aggregation, protein tracking, and STED

Aquaporin-4 (AQP4) is an integral water channel protein located in the plasma membrane of astrocyte cells in the central nervous system (CNS) and helps to regulate water-ion homeostasis. Some isoforms of AQP4 further order themselves into aggregates known as orthogonal arrays of particles (OAPs). AQP4 exists in two main isoforms, M1 and M23, with M23 being the isoform that favors stabilization by aggregation into OAPs. What role these OAPs play in the membrane of cells is still largely unknown. This study takes these AQP4 aggregates and incorporates them in a supported CNS biomimetic and images them using stimulated emission depletion (STED) superresolution microscopy.

Aquaporin-4 expression in the human choroid plexus

The choroid plexus (CP) consists of specialized ependymal cells and underlying blood vessels and stroma producing the bulk of the cerebrospinal fluid (CSF). CP epithelial cells are considered the site of the internal blood-cerebrospinal fluid barrier, show epithelial characteristics (basal lamina, tight junctions), and express aquaporin-1 (AQP1) apically. In this study, we analyzed the expression of aquaporins in the human CP using immunofluorescence and qPCR. As previously reported, AQP1 was expressed apically in CP epithelial cells. Surprisingly, and previously unknown, many cells in the CP epithelium were also positive for aquaporin-4 (AQP4), normally restricted to ventricle-lining ependymal cells and astrocytes in the brain. Expression of AQP1 and AQP4 was found in the CP of all eight body donors investigated (3 males, 5 females; age 74–91). These results were confirmed by qPCR, and by electron microscopy detecting orthogonal arrays of particles. To find out whether AQP4 expression correlated with the expression pattern of relevant transport-related proteins we also investigated expression of NKCC1, and Na/K-ATPase. Immunostaining with NKCC1 was similar to AQP1 and revealed no particular pattern related to AQP4. Co-staining of AQP4 and Na/K-ATPase indicated a trend for an inverse correlation of their expression. We hypothesized that AQP4 expression in the CP was caused by age-related changes. To address this, we investigated mouse brains from young (2 months), adult (12 months) and old (30 months) mice. We found a significant increase of AQP4 on the mRNA level in old mice compared to young and adult animals. Taken together, we provide evidence for AQP4 expression in the CP of the aging brain which likely contributes to the water flow through the CP epithelium and CSF production. In two alternative hypotheses, we discuss this as a beneficial compensatory, or a detrimental mechanism influencing the previously observed CSF changes during aging.

Regulation of aquaporin-4 expression in the central nervous system investigated using M23-AQP4 null mouse.

In astrocytes, unknown mechanisms regulate the expression of M1 and M23 isoforms of water channel aquaporin‐4 (M1‐AQP4 and M23‐AQP4). The ratio between these two isoforms controls the AQP4 assembly state in the plasma membrane known as orthogonal arrays of particles (OAPs). To give new insights into these mechanisms, here, we explore the regulation of AQP4 expression in the spinal cord of a CRISPR/Cas9 M23‐null mouse model (M23‐null). In the M23‐null spinal cord OAP assembly, the perivascular localization of AQP4 and M1‐AQP4 protein were drastically reduced. In heterozygous, M1‐AQP4 was proportionally reduced with M23‐AQP4, maintaining the isoform ratio unaffected. We hypothesize a role of the M23‐AQP4 in the regulation of M1‐AQP4 expression. M1‐AQP4 transcription, splicing and M1‐AQP4 protein degradation were found to be unaffected in M23‐null spinal cord and in M23‐null astrocyte primary culture. The translational control was investigated by mRNA‐protein pull down and quantitative mass spectrometry, to isolate and quantify AQP4 mRNA binding proteins (AQP4‐RBPs). Compared to WT, in M23‐null spinal cord, the interaction between AQP4 mRNA and polypyrimidine tract binding protein 1, a positive regulator of AQP4 translation, was higher, while interaction with the RNA helicase DDX17 was lower. In astrocyte primary cultures, DDX17 knockdown upregulated AQP4 protein expression and increased cell swelling, leaving AQP4 mRNA levels unchanged. Here, we identify AQP4‐RBPs and provide evidence that in mouse spinal cord M23‐AQP4 deletion changes the interaction between AQP4 mRNA and some RBPs involved in AQP4 translation. We describe for the first time the RNA helicase DDX17 as a regulator of AQP4 expression in astrocytes.

Insights into Cell Surface Expression, Supramolecular Organization, and Functions of Aquaporin 4 Isoforms in Astrocytes.

Aquaporin 4 (AQP4) is the most abundant water channel in the central nervous system (CNS). Its expression is confined to non-neuronal glial cells, predominantly to astrocytes that represent a heterogeneous glial cell type in the CNS. The membrane of astrocyte processes, which align brain capillaries and pia, is particularly rich in AQP4. Several isoforms of AQP4 have been described; however, only some (AQP4a (M1), AQP4 c (M23), AQP4e, and AQP4ex) have been identified in the plasma membrane assemblies of astrocytes termed orthogonal arrays of particles (OAPs). Intracellular splicing isoforms (AQP4b, AQP4d, AQP4f, AQP4-Δ4) have been documented, and most of them are postulated to have a role in the cell surface distribution of the plasma membrane isoforms and in the formation of OAPs in murine and human astrocytes. Although OAPs have been proposed to play various roles in the functioning of astrocytes and CNS tissue as a whole, many of these still need to be described. OAPs are studied primarily from the perspective of understanding water permeability regulation through the plasma membrane and of their involvement in cell adhesion and in the dynamics of astrocytic processes. This review describes the cellular distribution of various AQP4 isoforms and their implications in OAP assembly, which is regulated by several intracellular and extracellular proteins.

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

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.

Indirect Role of AQP4b and AQP4d Isoforms in Dynamics of Astrocyte Volume and Orthogonal Arrays of Particles.

Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the central nervous system (CNS). It is predominantly expressed in astrocytes lining blood–brain and blood–liquor boundaries. AQP4a (M1), AQP4c (M23), and AQP4e, present in the plasma membrane, participate in the cell volume regulation of astrocytes. The function of their splicing variants, AQP4b and AQP4d, predicted to be present in the cytoplasm, is unknown. We examined the cellular distribution of AQP4b and AQP4d in primary rat astrocytes and their role in cell volume regulation. The AQP4b and AQP4d isoforms exhibited extensive cytoplasmic localization in early and late endosomes/lysosomes and in the Golgi apparatus. Neither isoform localized to orthogonal arrays of particles (OAPs) in the plasma membrane. The overexpression of AQP4b and AQP4d isoforms in isoosmotic conditions reduced the density of OAPs; in hypoosmotic conditions, they remained absent from OAPs. In hypoosmotic conditions, the AQP4d isoform was significantly redistributed to early endosomes, which correlated with the increased trafficking of AQP4-laden vesicles. The overexpression of AQP4d facilitated the kinetics of cell swelling, without affecting the regulatory volume decrease. Therefore, although they reside in the cytoplasm, AQP4b and AQP4d isoforms may play an indirect role in astrocyte volume changes.

A Single Particle Tracking Study of More Natively Folded Recombinant Human Aquaporin-4 Orthogonal Array of Particles

Aquaporin-4 (AQP4) is a water channel protein located in the plasma membrane of astrocytes in the central nervous system (CNS) and helps to regulate water-ion homeostasis. Some isoforms of AQP4 further order themselves into aggregates known as orthogonal arrays of particles (OAPs). AQP4 exists in two main isoforms, M1 and M23, with M23 being the isoform that favors stabilization by aggregation into OAPs. What role these OAPs play in the membrane of cells is still largely unknown and the dynamics of OAP assembly into the plasma membrane remain elusive. Recently, techniques have been developed to isolate and express the M23-AQP4 isoform in insect cell lines in its authentically folded state. This study takes these natively folded AQP4 aggregates and incorporates them in a supported lipid bilayer CNS biomimetic, which have been characterized using fluorescence recovery after photobleaching (FRAP). The protein-protein and protein-membrane interactions, characterized by single particle tracking experiments, will be discussed within this CNS mimetic environment.

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

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.

Point Mutation of Aquaporin 4 Impairs its Binding in Neuromyelitis Optica

Aquaporin 4 (AQP4) is the principal aquaporin expressed in astrocytes throughout the central nervous system. An autoantibody, IgG, that binds to AQP4 leads to a rare disease called neuromyelitis optica (NMO), which is characterized by paralysis and loss of vision. Current treatments for NMO are few and involve non‐specific immunosuppression. The Summit Country Day School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of the AQP4 epitope for NMO‐IgG. Understanding the molecular structure of the AQP4 epitope is an important step in explaining its binding to NMO‐IgG. NMO‐IgG primarily interacts with the accessible AQP4 extracellular loops A, C, and E. These loops change their conformation when AQP4 arrange into orthogonal arrays of particles, thus enabling attachment of NMO‐IgG and facilitating complement binding. Studies on the point mutation of aspartate in position 69 (D69) revealed conformational changes such as 1) reorientation of threonine in position 62, 2) weakened hydrogen bond interaction between leucine in position 53 and threonine in position 56, 3) increased mobility of loop A, and 4) disruption of epitope reorganization of AQP4. Overall, D69 substitution and the subsequent conformational changes in loop A leads to impairment of binding between AQP4 and the NMO‐IgG. Determining the role of extracellular loop A between and within AQP4 monomers will aid in understanding how it contributes to antigen recognition. Prevention of the binding of AQP4 by NMO‐IgG is an important strategy for developing targeted therapy in NMO.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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

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.

Aquaporins in brain edema.

Brain edema is a common feature of brain injuries, which leads to increased intracranial pressure (ICP) and ischemia that worsen outcome. Current management of edema focuses on reduction of ICP, but there are no treatments targeting the molecular players directly involved in edema process. The perivascular astrocyte endfeet are critical in maintaining brain homeostasis with ionic and water exchange; in this context, aquaporins (AQPs), astrocyte water channels, have emerged as privileged targets for edema modulation. However, AQPs can facilitate either accumulation or drainage of water, depending on the osmotic gradients between extra-intracellular space; and thus inhibition of AQPs leads to different outcomes depending on specific tissue characteristics and time post-injury. Most of this knowledge has been gathered from the study of AQP4, the best characterized AQP and the one that has the biggest impact on water movement. In addition to the level of expression, the ratio of AQP4 isoforms (m1, m23 or mz), the spatial distribution of AQP4 into orthogonal arrays of particles, and the interaction of AQP4 with neighboring ionic channels and gap junctions could directly impact edema formation. Although there are no specific AQP4 pharmacological blockers, the development of AQP4 siRNA offers a promising therapeutic tool. Given the complex dynamics of AQP4, therapies targeting AQP4 should carefully take into account the particular features of the injury (e.g., hemorrhagic vs. non-hemorrhagic) and different times after injury (e.g., phase of edema formation vs. resolution).

AQP4e-Based Orthogonal Arrays Regulate Rapid Cell Volume Changes in Astrocytes.

Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. It is predominantly expressed in astrocytes at the blood-brain and blood-liquor interfaces. Although several AQP4 isoforms have been identified in the mammalian brain, two, AQP4a (M1) and AQP4c (M23), have been confirmed to cluster into plasma membrane supramolecular structures, termed orthogonal arrays of particles (OAPs) and to enhance water transport through the plasma membrane. However, the role of the newly described water-conductive mammalian isoform AQP4e is unknown. Here, the dynamics of AQP4e aggregation into OAPs and its role in the regulation of astrocyte water homeostasis have been studied. Using super-resolution structured illumination, atomic force, and confocal microscopies, the results revealed that, in female rat astrocytes, AQP4e isoform colocalizes with OAPs, affecting its structural dynamics. In hypoosmotic conditions, which elicit cell edema, OAP formation was considerably enhanced by overexpressed AQP4e. Moreover, the kinetics of the cell swelling and of the regulatory volume decrease was faster in astrocytes overexpressing AQP4e compared with untransfected controls. Furthermore, the increase in maximal cell volume elicited by hypoosmotic stimulation was significantly smaller in AQP4e-overexpressing astrocytes. For the first time, this study demonstrates an active role of AQP4e in the regulation of OAP structural dynamics and in water homeostasis.SIGNIFICANCE STATEMENT Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. To date, only AQP4a and AQP4c isoforms have been confirmed to enhance water transport through plasmalemma and to cluster into orthogonal arrays of particles (OAPs). We here studied the dynamics, aggregation, and role in the regulation of astrocyte water homeostasis of the newly described water-conductive mammalian isoform AQP4e. Our main findings are as follows: brain edema mimicking hypoosmotic conditions stimulates the formation of new OAPs with larger diameters, due to the incorporation of additional cytoplasmic AQP4 channels and the redistribution of AQP4 channels of the existing OAPs; and AQP4e affects the dynamics of cell swelling and regulatory volume decrease in astrocytes exposed to hypoosmotic conditions.

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

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.

Placental adaptive up-regulation of calcium transport in wild-type mice is not driven by parathyroid hormone-related protein (PTHrP)

The main water channel in the brain, aquaporin-4 (AQP4) is involved in maintaining homeostasis and water exchange in the brain. In adult mammalian brains, it is expressed in astrocytes, mainly, and in high densities in the membranes of perivascular and subpial endfeet. Here, we addressed the question how this polarized expression is established during development. We used immunocytochemistry against AQP4, zonula occludens protein-1, glial fibrillary acidic protein, and β-dystroglycan to follow astrocyte development in E15 to P3 NMRI mouse brains, and expression of AQP4. In addition we used freeze-fracture electron microscopy to detect AQP4 in the form of orthogonal arrays of particles (OAPs) on the ultrastructural level.We analyzed ventral, lateral, and dorsal regions in forebrain sections and found AQP4 immunoreactivity to emerge at E16 ventrally before lateral (E17) and dorsal (E18) areas. AQP4 staining was spread over cell processes including radial glial cells in developing cortical areas and became restricted to astroglial endfeet at P1–P3. This was confirmed by double labeling with GFAP. In freeze-fracture replicas OAPs were found with a slight time delay but with a similar ventral to dorsal gradient. Thus, AQP4 is expressed in the embryonic mouse brain starting at E16, earlier than previously reported. However a polarized expression necessary for homeostatic function and water balance emerges at later stages around and after birth.

Is Upregulation of Aquaporin 4-M1 Isoform Responsible for the Loss of Typical Orthogonal Arrays of Particles in Astrocytomas?

The astrocytic endfoot membranes of the healthy blood-brain barrier—contacting the capillary—are covered with a large number of the water channel aquaporin 4 (AQP4). They form orthogonal arrays of particles (OAPs), which consist of AQP4 isoform M1 and M23. Under pathologic conditions, AQP4 is distributed over the whole cell and no or only small OAPs are found. From cell culture experiments, it is known that cells transfected only with AQP4-M1 do not form OAPs or only small ones. We hypothesized that in astrocytomas the situation may be comparable to the in vitro experiments expecting an upregulation of AQP4-M1. Quantitative Real-time PCR (qRT-PCR) of different graded astrocytomas revealed an upregulation of both isoforms AQP4 M1 and M23 in all astrocytomas investigated. In freeze fracture replicas of low-grade malignancy astrocytomas, more OAPs than in high-grade malignancy astrocytomas were found. In vitro, cultured glioma cells did not express AQP4, whereas healthy astrocytes revealed a slight upregulation of both isoforms and only a few OAPs in freeze fracture analysis. Taken together, we found a correlation between the decrease of OAPs and increasing grade of malignancy of astrocytomas but this was not consistent with an upregulation of AQP4-M1 in relation to AQP4 M23.