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

Abstract

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.

Keywords: aquaporin 4; astrocytes; atomic force microscopy; glia; orthogonal arrays of particles; structured illumination microscopy.

Figure 1.

Measurements of AQP4 microdomains (MiDs) in astrocytes. A, The DIC image of a cultured rat astrocyte. The white line delineates the cell. Scale bar, A (for A, B), 10 μm. B, Fluorescence micrograph of the same astrocyte as in A, recorded with SIM, showing a punctiform pattern of MiDs labeled with AQP4 antibodies that labeled intracellular and plasma membrane AQP4 channels (AQP4 MiD). The arrow denotes an MiD (197 nm in diameter) enlarged in the framed inset. C, MiD diameters were determined by measuring the full-width at half-maximum (FWHM) of the fluorescence intensity profile along the equatorial line (the length of the line, shown in B, is 500 nm) in the horizontal and vertical directions (here only the horizontal direction is displayed). D, Histograms show diameter distributions for AQP4 MiDs (594 MiDs from 7 cells) and AQP4e MiDs (intracellular and plasma membrane MiDs in astrocytes expressing the recombinant AQP4e; 676 MiDs from 8 cells). E, AQP4e-expressing MiDs were on average wider (237 ± 1 nm) than AQP4-labeled MiDs (166 ± 1 nm). ***p < 0.001. F, G, The DIC image of a cultured rat astrocyte and corresponding fluorescent micrograph displaying recombinant AQP4e. Scale bar, F (for F, G), 10 μm.

Figure 2.

AQP4e is localized in OAPs, and it promotes their formation in hypoosmotic conditions. A, Schematically depicted double labeling of AQP4 channels at the plasma membrane OAPs. Primary NMO antibodies label the extracellular site of AQP4s (secondary antibodies are conjugated to a red fluorescent dye) and are thus markers of OAPs, whereas primary commercial antibodies label OAPs at the intracellular domain of the AQP4 channel (secondary antibodies are conjugated to a green fluorescent dye). Recombinant AQP4e isoforms are tagged with GFP. Of note, commercial pan antibodies also label intracellular MiDs containing AQP4s (see E_iii). B, Fluorescent micrographs of astrocytes labeled with NMO primary antibodies and delineated with white lines. B_i shows an equatorial plane of NMO-labeled impermeabilized astrocytes, where NMO labeling is restricted to the cell surface. We used this type of labeling to assess OAPs. B_ii shows an equatorial plane of NMO-labeled formaldehyde-permeabilized astrocytes, where NMO labeling is observed also in the cytoplasm. C, DIC image of a cultured rat astrocyte. The cell is delineated with the white line. D, SIM fluorescence micrograph of the same astrocyte as in B, labeled as schematically shown in A. The squared area is enlarged in E, displaying OAPs extracellularly labeled with NMO antibodies (E_i), intracellularly labeled with commercial AQP4 antibodies (E_ii), and the overlay of both signals (E_iii). The white line represents the edge of the astrocyte, as determined on the DIC image. F, Section of an astrocyte showing OAPs extracellularly labeled with NMO antibodies (F_i), the intracellular signal of AQP4e-GFP (F_ii), and the overlay of both signals (F_iii). The arrowheads point to colocalized fluorescent signals of OAPs, labeled by NMO antibodies, that were either colabeled by AQP4 antibodies (E_iii) or AQP4e-GFP (F_iii). G, OAPs represented 3.4 ± 0.3% of all antibody-labeled AQP4 microdomains in untransfected cells. Stimulation with Hypo solution increased the percentage of OAPs to 4.7 ± 0.3% (2 min) and to 4.5 ± 0.5% (10 min), respectively (G_i). Of all recombinant AQP4e-GFP MiDs in the transfected cells, 7.6 ± 1.3% were detected in OAPs. G_ii, Stimulation with Hypo increased the percentages to 12.3 ± 2.2% (2 min) and to 15.9 ± 2.0% (10 min). Numbers in the bars denote the number of cells analyzed. **p < 0.01.

Figure 3.

Hypotonicity increases the average OAP diameter in astrocytes. A_i, The average diameter of OAPs (in nanometers) in rat astrocytes intracellularly colabeled with AQP4 antibodies was 170 ± 1 in isoosmotic conditions. After 2 min of Hypo stimulation, it remained virtually unchanged (171 ± 1) and increased to 188 ± 1 after 10 min of Hypo stimulation. The corresponding average fluorescence intensity in arbitrary units (A.U.) is shown in A_ii; it decreased from 13.8 ± 0.6 in isoosmotic conditions to 13.1 ± 0.6 and 10.9 ± 0.5 after 2 min and after 10 min of Hypo, respectively. B_i, The average diameter of OAPs (in nm) containing GFP-tagged AQP4e isoform was 178 ± 1 in isoosmotic conditions. After 2 min of Hypo stimulation, the average diameter did not change (178 ± 2), while it increased to 183 ± 1 after 10 min of Hypo stimulation. B_ii, The average fluorescence intensity (in A.U.) of the same OAPs as in B_i was 16.6 ± 0.8 in isoosmotic conditions, transiently decreased to 12.3 ± 0.8 after 2 min of Hypo stimulation, and then increased to 16.1 ± 0.5 after 10 min. C, The AQP4 density was measured as the ratio between the average fluorescence intensity of OAP and the OAP diameter. The density of AQP4 (in A.U.) in OAPs (C_i) was 83.8 ± 3.5 in controls and decreased to 77.5 ± 2.9 (after 2 min Hypo stimulation) and to 56.6 ± 1.9 (after 10 min of Hypo stimulation). C_ii, The decrease in the density of AQP4e in OAPs was transient, from 92.5 ± 4.5 in controls, to 69.5 ± 4.6 (after 2 min of Hypo stimulation), and to 88.5 ± 2.7 (after 10 min of Hypo stimulation). Numbers in the bars or brackets represent the number of OAPs analyzed. *p < 0.05.

Figure 4.

Astrocytes overexpressing AQP4e exhibit a faster but smaller increase in cell volume in hypoosmotic conditions. A, An astrocyte labeled by SR101, before (t = 22 s) and after stimulation with hypotonic milieu (at t = 46 s and t = 90 s, respectively). Graphs adjacent to the micrographs represent fluorescence intensity profiles obtained along the line denoted in respective micrographs. Gray line represents fluorescence intensity profile at t = 22 s. B, The relative volume changes, obtained by measuring the average fluorescence intensity of SR101 of the whole cell, in control untransfected (Ctr) and in AQP4e-overexpressing (AQP4e) astrocytes in isoosmotic (Iso; B_i) and Hypo conditions (B_ii). C, Normalized time-dependent changes in the volume shown in B_ii. B_ii, C, Note the smaller peak of the response (i.e., swelling; B_ii) and the better recovery (of the cell volume in RVD) in cells overexpressing AQP4e compared with untransfected controls (C). The kinetics of the volume change was faster in cells overexpressing AQP4e. The dashed line represents the application of hypotonic solution. D, Time constants (in seconds) of the swelling phase (D_i) were on average significantly smaller in cells overexpressing AQP4e (4.9 ± 0.5) than in control untransfected cells (7.5 ± 0.4). The RVD phase (D_ii) occurred faster in transfected cells (11.4 ± 0.7) than in control untransfected cells (19.2 ± 2.0). E, Maximal amplitude of the volume increase (ΔV swelling) was smaller in cells overexpressing AQP4e (0.16 ± 0.01 A.U.) than in controls (0.22 ± 0.02 A.U.; p = 0.02). F, Recovery of the cell volume in the RVD phase was higher in cells overexpressing AQP4e (Ctr, 31.6 ± 2.8%; AQP4e, 46.8 ± 4.6%; p = 0.02). Numbers in the bars or brackets represent the number of cells analyzed. Student's t test was used for statistical comparison: *p < 0.05; ***p < 0.001.

Figure 5.

Prolonged hypoosmotic stimulation induces a slow increase in astrocyte volume monitored by AFM. A_i, A_ii, Linearized height images of an astrocyte before (A_i) and 21 min after (A_ii) the application of a hypotonic solution and corresponding height profiles. A_iii, A subtraction image (hypotonic − control) indicating cell swelling. B, Time-dependent changes in the total cell volume of astrocytes treated in isoosomotic solution (controls, black diamonds) or in hypotonic solution (empty circles), and astrocytes transfected with AQP4e and treated with hypotonic milieu (gray triangles). Numbers in the brackets represent the number of cells analyzed. One-way ANOVA with the Holm–Sidak post hoc test versus control was used for statistical comparison.

Figure 6.

A schematic drawing of rapid AQP4e-dependent changes in cell swelling and RVD in astrocytes. Astrocytes in hypoosmotic conditions (marked by light gray) respond by rapid swelling and by subsequent RVD, both of which are faster in cells overexpressing AQP4e. The maximal volume increase at the end of the swelling phase is smaller in cells overexpressing AQP4e compared with nontransfected cells (ΔV2 < ΔV1; changes in cell volume in different phases can also be observed by comparing the cell area in dark gray compared with the initial cell volume in white). In addition, the recovery of the cell volume during the RVD phase was higher in cells overexpressing AQP4e compared with nontransfected cells.