Fig 1: Hypoxia Induces AQP4 Subcellular Relocalization in Primary Cortical Astrocytes(A) Mean fold change in AQP4 surface expression (±SEM), measured by cell-surface biotinylation in primary cortical astrocytes. Cells were treated with 5% oxygen for 6 h (hypoxia) or 85 mOsm/kg H2O (hypotonicity) compared with untreated normoxic astrocytes (control). The CaM inhibitor (CaMi) was 127 µM trifluoperazine (TFP). The TRPV4 inhibitor (TRPV4i) was 4.8 µM HC-067047, and the intracellular Ca2+ chelator was 5 µM EGTA-AM. The TRPV4 channel agonist (TRPV4a) was 2.1 µM GSK1016790A. Kruskal-Wallis with Conover-Inman post hoc tests were used to identify significant differences between samples. *p < 0.05; ns represents p > 0.05 compared with the untreated control (Table S2; n = 4).(B) Mean fold change in AQP4 surface expression (±SEM) with time under hypoxia. Rat primary cortical astrocytes were exposed to 5% oxygen, and AQP4 surface expression was measured by cell-surface biotinylation after 1, 3, and 6 h and compared with untreated normoxic astrocytes (normoxia). Cells were returned to normoxic conditions (21% oxygen), and AQP4 surface expression was measured at 1, 3, and 6 h. *p < 0.05 by ANOVA followed by t test with Bonferroni correction (Table S2; n = 3).(C) Calcein fluorescence quenching in response to elevation of extracellular osmolality to 600 mOsm with mannitol was used to quantify astrocyte plasma membrane water permeability following the same hypoxia and inhibitor regimen described in (A). *p < 0.05; ns indicates p > 0.05 by ANOVA followed by t test with Bonferroni correction (Table S2; n = 3).(D) Normalized membrane water permeability of hypoxic rat primary cortical astrocytes over 6 h. Cells were returned to normoxic conditions (21% oxygen), and permeability was measured over the subsequent 6 h. *p < 0.05 compared with the t = 0 normoxic control (Table S2; n = 3).(E) Representative calcein fluorescence quenching traces for hypoxia and normoxia.(F) Intracellular cAMP accumulation in rat primary cortical astrocytes (±SEM) in response to forskolin, hypotonicity (with or without extracellular Ca2+ [the intracellular Ca2+ chelator was 5 µM EGTA-AM] or the CaMi W-7 or TFP), and the CaMa peptide CALP-3. PKA activity in lysates made from rat astrocytes subjected to the same treatments. Activity is normalized to the average of the untreated control. *p < 0.05. ns represents p > 0.05 by ANOVA followed by t test with Bonferroni correction (Table S2; n = 3).(G) Phosphorylation of glutathione S-transferase (GST)-recAQP4ct, analyzed by Phos-tag SDS-PAGE. The phosphorylation stoichiometry was judged to be complete (~1 mol phosphate/mol GST-recAQP4ct, 1P) after 2 h.
Fig 2: Binding of CaM to the AQP4 Carboxyl Terminus(A) Microscale thermophoresis (MST) data showing that full-length AQP4 interacts directly with CaM. The binding curve, obtained by plotting ?Fnorm against [AQP4], could be fitted to a one-to-one binding model (estimated Kd of 29 ± 5.6 µM). Addition of 5 mM EDTA demonstrated that binding was Ca2+ dependent. The interaction was also inhibited by addition of 1 mM TFP. Truncation of the AQP4 carboxyl terminus before the predicted CBD (AQP4-?256) resulted in a construct that did not interact with CaM. AQP4 F258/262/266A did not bind CaM. The phospho-mimetic mutant AQP4-S276E bound CaM with approximately 2-fold higher affinity (Kd = 17 ± 3.1 µM) than WT AQP4 (p = 0.031), suggesting that phosphorylation of S276 affects the interaction with CaM.(B) Response curve showing that TFP inhibits the interaction between AQP4 and CaM in a concentration-dependent manner. The concentration of TFP needed for 50% inhibition (IC50) was estimated to be 790 ± 2 µM.(C) Left: 1H, 15N-HSQC NMR data showing the interaction of the recombinant carboxyl terminus of AQP4 (recAQP4ct; residues 254–323) with CaM at 30°C. Chemical shift perturbations (CSPs) were observed by titrating CaM into 0.5 mM 13C, 15N-labeled recAQP4ct with 0 (black) and 2 (green) molar equivalents of CaM in the presence of 6 mM Ca2+. Top right: CSPs induced by CaM binding to recAQP4ct were plotted as a function of the residue number (X indicates that data are not available). Bottom right: chemical shift index (CSI) values of the Ca and C' atoms of recAQPct. The sequence of the CBD is underlined. The region with consecutive positive CSI values (red) represents an a-helical conformation.(D) Anti-CaM immunoblotting following nickel affinity chromatography (IMAC) from 1% Triton X-100 lysates (input) of HEK293 cells transfected with AQP4-His6 (AQP4) or AQP4 F258/262/266A-His6 (AQP4 CBDmut) demonstrates that the F258A/F262A/F266A mutation abrogates AQP4-CaM binding.(E) Cell-surface biotinylation followed by neutravidin/anti-AQP4 ELISA demonstrates a reduced rate of AQP4 plasma membrane accumulation in AQP4-transfected HEK293 cells upon F258A/F262A/F266A mutation (AQP4 CBDmut) compared with the wild-type control (AQP-WT). Normalized data were fitted to functions of the form 1-e-kt, and t1/2 was calculated as -ln(0.5)/k.See also Figures S4 and S5.
Fig 3: Targeted Inhibition of CaM or PKA Reduces Acute Brain Water Content after an In Vivo Brain Stab Injury Model of Cytotoxic Edema; Increases in AQP4 Expression and AQP4 Translocation to the Blood-Spinal-Cord Barrier are Localized to the Injury Site In Vivo.a, Water content of the ipsilateral and contralateral brain cortex 3 days after stab injury and treatment with CaM, PKA or PKC inhibitors (CaMi or PKAi). Sham = craniotomy only; Stab injury+Vehicle = 3 mm cortical stab injury + PBS; Stab injury+CaMi = 3 mm cortical stab injury + intra-lesion injection of 41 mM trifluoperazine (TFP); Stab injury + PKAi = 3 mm cortical stab injury + intra-lesion injection of 10 mM H89; Stab injury+PKCi = 3 mm cortical stab injury + intra-lesion injection of 9.94 µM Gö 6983; n = 18 rats per treatment group, * represents p < 0.05, ns represents p > 0.05 (see Table S2 for p values). Central bars represent median, crosses represent the mean, outer bars represent upper and lower quartiles. Outliers (data points more than 1.5 × IQR from the median) are shown as separate points; b, Increases in AQP4 expression and AQP4 translocation to the blood-spinal-cord barrier are localized to the injury site in vivo. Representative images showing AQP4 expression (green) with endothelial cells labeled with RECA1 (red) in Sham animals; c, 3 days after DC crush (3 dpi), an increase in total AQP4 expression and translocation to the blood-spinal-cord barrier (BSCB) is observed at the injury site; d, At a minimum of 3 mm away from the lesion, total AQP4 expression and translocation to the BSCB are indistinguishable from that of sham animals; e, Normalized relative perivascular fluorescence (A.U. ± SD 3 days after DC crush injury; sham = laminectomy only; DC+vehicle = T8 DC crush + PBS at the injury site; DC+vehicle (away) = T8 DC crush + PBS at sites at a minimum of 3 mm away from the lesion site; f, Normalized total AQP4 fluorescence (A.U. ± SD) following the same experimental conditions described in panel e. * represents p < 0.05, ns represents p > 0.05 (see Table S2 for p values). Related to Figure 2.
Fig 4: Knockdown of AQP4 Suppresses Acute Cytotoxic Edema and Improves Functional Recovery in Rats at 3 dpi but Not at 28 dpi following DC Crush Injurya, ELISA for AQP4 showing no increase in total AQP4 protein in primary astrocytes after 6 h under hypoxia (5% O2; n = 6); b, Representative immunoblot and c, densitometry to confirm 60% and 75% knockdown of AQP4 using in vivo JetPEI-delivered shRNA to AQP4 (shAQP4) at 3 dpi and 28 dpi following DC crush injury (n = 3 independent repeats from 3 pooled rat spinal cords/experiment (total n = 9 rats/condition)); d, Spinal cord water content was significantly suppressed at 3 dpi, but at 28 dpi the water content in DC + shAQP4-treated animals was significantly higher than DC + Vehicle-treated controls, despite 75% AQP4 protein knockdown (n = 3-4 rats/condition, 3 independent repeats (total n = 10 rats/condition)); e, Knockdown of AQP4 improved tape sensing and removal time up to 1 week after DC crush injury, but the sensing and removal time gradually increased to above DC + vehicle-treated controls at 4 weeks after DC crush injury (n = 3-4 rats/condition, 3 independent repeats (total n = 10 rats/condition)); f, The early improvement in ladder crossing ability of rats gradually worsened 1 week after and at 28dpi is higher than DC+vehicle-treated controls (n = 3-4 rats/condition, 3 independent repeats (total n = 10 rats/condition)). * represents p < 0.05, ns represents p > 0.05 (see Table S2 for p values). Animals were euthanized at 28dpi due to episodes of vomiting, gait problems, lethargy and convulsions, possibly reflecting their inability to regulate water in the CNS. Related to Figure 2.
Fig 5: MST and CAP Experimental Controlsa, Typical MST traces. The relative fluorescence is plotted against the experiment time. Each trace corresponds to a sample with a different concentration of AQP4 whereas calmodulin (CaM) concentration remained constant, except for the trifluoperazine (TFP) titration experiment (bottom right) in which TFP was varied and both AQP4 and CaM were kept constant. The difference in relative fluorescence before (blue column) and after (red column) heating is used to calculate ?Fnorm. The position of the red column was determined by the M.O. Affinity Analysis software (Nanotemper) as the time interval that gave the best signal to noise ratio; b, Recombinant AQP4 is a functional water channel. Fluorescence traces from a proteoliposome shrinking assay showing that liposomes containing purified AQP4 (blue) have significantly higher water permeability than empty liposomes (gray). The increase in fluorescence corresponds to the fluorophore ((5)6-carboxyfluorescein) present on the inside of the liposomes becoming more fluorescent as the liposomes shrinks when mixed with hyperosmotic solution. The data were fitted to a double exponential function (solid blue and black lines for AQP4-containing and empty liposomes, respectively) and the rate constant (k) was used to calculate the osmotic water permeability (Pf). For AQP4-containing liposomes Pf = 5.9 ± 0.5 × 10-2 cm/s and for control liposomes Pf = 1.2 ± 0.2 × 10-2 cm/s, corresponding to an AQP4 single channel permeability of 1.1 ± 0.1 × 10-13 cm3/s. Analysis by t test showed a statistically-significant difference between AQP4-containing liposomes and empty liposomes (p = 0.0015; Table S2). Related to Figure 3; c, Representative Spike 2 software processed CAP traces. A normal short-latency negative-positive CAP trace was observed in sham animals which was ablated in DC+vehicle- and DC+PKCi-treated rats, but partially restored in DC + CaMi and DC + PKAi-treated rats. Following a dorsal hemi-section in the same animals at the end of the experiment, CAP traces were ablated in all animals, indicating DC axon regeneration. Related to Figure 4.
Supplier Page from OriGene Technologies for Aqp4 Rat shRNA Plasmid (Locus ID 25293)