1 Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, 505 Parnassus Avenue, San Francisco, CA 94143-0521, USA
2 Department of Neurological Surgery, University of California, 1001 Potrero Avenue, San Francisco, CA 94110, USA
3 Academic Neurosurgery Unit, St George's University of London, Cranmer Terrace, London, SW17 0RE, UK
Author for correspondence (e-mail: verkman{at}itsa.ucsf.edu)
Accepted 8 September 2005
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Summary |
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Key words: AQP4, Astrocyte, Chemotaxis, Reactive gliosis, Water channel
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Introduction |
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Reactive astroglia overexpress several proteins, including glial fibrillary acidic protein (GFAP), which is often used to identify them (Eng et al., 2000), and the water channel protein aquaporin-4 (AQP4) (Saadoun et al., 2002
; Saadoun et al., 2003
; Vizuete et al., 1999
). AQP4, the main water channel of mammalian brain, is normally expressed in astroglia at the border between brain parenchyma and major fluid compartments (cerebrospinal fluid, blood). Phenotype data from AQP4-knockout mice have indicated that AQP4 facilitates water fluxes into and out of the brain parenchyma, and is involved in the pathophysiology of brain edema in stroke, water intoxication, brain tumor, focal cortical freeze injury, acute bacterial meningitis and brain abscess (Bloch et al., 2005
; Manley et al., 2000
; Papadopoulos et al., 2004
; Papadopoulos and Verkman, 2005
; Saadoun et al., 2002
; Saadoun et al., 2003
).
Here, we provide evidence for a new role of AQP4 in astroglia that is unrelated to its role in brain edema. This work was motivated by our recent finding that a different water-selective transporter, AQP1, facilitates tumor angiogenesis by accelerating endothelial cell migration (Saadoun et al., 2005). AQP4 knockout or knockdown in astroglial cell cultures was found to greatly impair their migration, probably by reducing membrane water fluxes that occur during cell migration. Experimental evidence for an in vivo role of AQP4-dependent astroglial cell migration was obtained from analysis of glial scar formation after stab injury. We propose that the AQP4 upregulation found in reactive astroglia after a variety of insults is an adaptive response that facilitates astroglial cell migration and glial scar formation. Our results also suggest the possibility of modulation of AQP4 expression/function as a novel strategy to enhance or disrupt glial scar formation for the treatment of a variety of CNS pathologies.
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Materials and Methods |
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Astroglial cultures
Astroglia were cultured from neocortex of wild-type and AQP4-null neonatal mice as previously described (Solenov et al., 2004). Briefly, the cerebral hemispheres were isolated, minced with forceps and incubated in minimal essential medium (MEM) plus 0.25% trypsin and 0.01% DNase. Dissociated cells were centrifuged, resuspended in MEM containing 10% fetal bovine serum (FBS), seeded on poly-L-lysine-coated flasks and grown at 37°C in a 5% CO2 incubator with a change of medium twice a week. At confluence (days 12-15), cultures were treated with 10 µM cytosine arabinoside for 48 hours to prevent proliferation of other cell types, and the medium was replaced with MEM containing 3% FBS and 0.15 mM dibutyryl cAMP to induce differentiation. Cultures were maintained for up to two more weeks with a change of medium twice a week.
RNA inhibition (RNAi) experiments
Custom SMART pool® RNAi duplexes (more than four sequences) for AQP4 and AQP9 were chemically synthesized by Dharmacon Research (Lafayette, CO). After 10 days in culture, primary astroglia were shaken at 200 rpm for 18 hours at 37°C to remove microglia. Astroglia were recovered in normal medium for 2 days and replated in six-well or 12-well plates. Double-stranded RNAs for RNAi (100 nM, AQP4 or AQP9) were mixed with Oligofectamine (Invitrogen, Carlsbad, CA), diluted 1:50 in Opti-MEM (Invitrogen), and incubated for 20 minutes at room temperature for complex formation. The mixture was added to the cells and AQP4 protein expression was determined by immunoblotting after 6 days.
Osmotic water permeability
Cell membrane water permeability was measured by a calcein-quenching method as described previously (Solenov et al., 2004). The time course of cytoplasmic calcein fluorescence was measured in response to cell swelling produced by the twofold dilution of the extracellular bathing solution with water.
Immunocytochemistry
Cultured astroglia were fixed in 10% formalin and incubated with 1:200 rabbit anti-AQP4 or 1:1000 rabbit anti-GFAP antibody (Chemicon, Temecula, CA), followed by FITC- or Texas-Red-conjugated anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) at 1:80. Nuclei were counterstained blue with DAPI. In some experiments, the plasma membrane was stained with Alexa-Fluor-linked wheatgerm agglutinin (WGA, Molecular Probes, Carlsbad, CA).
Immunohistochemistry
Mice were anaesthetized using 2,2,2-tribromoethanol and perfusion-fixed with 5 ml of 10% formalin injected i.v. Brains were removed, fixed for another 24 hours in 10% formalin, and processed in paraffin. Coronal sections (7 µm) were cut, deparaffinized, rehydrated and immunolabeled using 1:200 rabbit anti-AQP4 or 1:1000 rabbit anti-GFAP antibody followed by anti-rabbit HRP-linked secondary antibody (1:1000). Immunolabeling was visualized in brown using DAB/H2O2. Distances of reactive astroglia from the wound edge were estimated using Image J (NIH freeware).
Western blotting
Astroglial cell cultures were trypsinized and plasma membrane proteins were extracted (Yang and Verkman, 1997), separated on a 4-12% SDS-PAGE minigel (3 µg protein/lane) and transferred onto a PVDF membrane. The membrane was exposed to rabbit anti-AQP4 antibody (1:1000) followed by HRP-linked anti-rabbit IgG antibody (1:1500) and visualized using enhanced chemiluminescence. AQP4 protein was quantified by scanning densitometry using as standards serial dilutions of protein from wild-type astrocytes.
Adhesion and proliferation assays
Confluent cultured astroglia were trypsinized and suspended in MEM with 3% FBS. After measurement of cell density using a hemocytometer, 2.8x104 cells per cm2 were plated in 24-well plates coated with poly-L-lysine. The medium was exchanged 4 hours after plating. Adhesion was defined as the percentage of plated cells remaining immediately after medium exchange. To assess proliferation, the number of cells per well was determined every 2 days by trypsinization and cell counting.
In vitro migration
Migration was assayed using a modified Boyden chamber (Corning Costar, Fisher Scientific, Pittsburgh, PA) containing a polycarbonate membrane filter (6.5 mm diameter, 8 µm pore size) coated with poly-L-lysine (Saadoun et al., 2005). The upper chamber contained cells in DMEM plus 1% FBS, and the lower chamber contained DMEM plus 10% FBS (chemoattractant) or 1% FBS (control). Cells were incubated for 10 hours at 37°C in 5% CO2/95% air. Non-migrated cells were scraped off the upper surface of the membrane with a cotton swab. Migrated cells remaining on the bottom surface were counted after staining with Coomassie Blue.
In some experiments, the assay was performed in the presence of an osmotic gradient produced by addition of raffinose to the top or bottom chambers. Because pilot experiments showed that the osmotic gradient dissipates by 50% in 2 hours (not shown), the medium in both chambers was exchanged hourly to restore the initial osmotic gradient.
In vitro wound healing
Cells were cultured as confluent monolayers, synchronized in 1% fetal bovine serum for 24 hours, and wounded by removing a 1 mm strip of cells across the well with a standard 200 µl pipette tip (Saadoun et al., 2005
). The wounded monolayers were washed twice to remove non-adherent cells. Phase-contrast light micrographs were taken immediately after cell removal, and at 24 and 48 hours for analysis using Image J. Wound healing was quantified as the average linear speed of the wound edges over 48 hours. Phase-contrast micrographs of the wound edges were also taken 6 hours post-wounding and the leading edge of individual migrating astroglia was outlined. Irregularity (ruffling) of the leading edge was quantified as the fractal dimension of the outline, calculated using the `box-counting method' (Image J) as previously described (Smith et al., 1996
). The fractal dimension is a number between 1 and 2, which increases with the extent of ruffling.
In vivo stab
Anesthetized mice were mounted on a stereotactic frame. After incising the skin, a 5-mm-long stab was performed through the skull in the sagittal plane, 1 mm lateral to the sagittal suture, 2 mm deep from the cerebral cortex. The blade was removed and the skin was sutured closed.
Statistics
Results were analyzed using the two-tailed Student's t-test, Student-Newman-Keuls test or linear regression.
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Results |
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Astroglial cell migration in vitro
Cell migration was measured using a modified Boyden TranswellTM assay. The fraction of cells that migrated across 8 µm diameter pores over 10 hours was determined by counting stained cells on Transwell filters before and after scraping off non-migrated cells on the upper filter surface (Fig. 2A, left). There was remarkably impaired migration of AQP4-null compared with wild-type astroglia (18±2% vs 58±4% migrated cells after 8 hours, P<0.001) towards 10% FBS (Fig. 2A, right). Differences in cell size cannot account for the differences in migration because wild-type and AQP4-null astroglia have similar sizes (respective cell surface areas, 1261±333 vs 1291±316 µm2, P=0.9; area of cell projections, 861±219 vs 803±184 µm2, P=0.8; mean±s.e.m.) as measured using Image J from light micrographs of poly-L-lysine-adhered astroglial cells. Background Transwell migration (in the absence of a serum gradient) was 9-10%; with no difference between wild-type and AQP4-null astroglia.
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RNAi studies were done to confirm that the reduced migration in AQP4-null astroglia is the primary effect of AQP4 deficiency. Treatment conditions (concentration and time) were established to strongly reduce AQP4 protein expression without toxic effects. Under these conditions AQP4 RNAi treatment did not produce a noticeable effect on cell morphology (Fig. 3A), but reduced AQP4 protein expression by 94% in wild-type astroglia (Fig. 3B). AQP4 protein expression was not significantly reduced by treatment with AQP9 RNAi or oligofectamine alone. Plasma membrane osmotic water permeability was measured by a calcein fluorescence-quenching method. As reported previously (Solenov et al., 2004
), water permeability was much lower in AQP4-deficient cells (Fig. 3C). Treatment with RNAi (under the same conditions as in immunoblotting) reduced water permeability substantially in the wild-type astrocytes, but had no effect on water permeability in the AQP4-deficient astrocytes.
The cell migration data from Transwell assays are summarized in Fig. 3D. AQP4 knockdown using RNAi significantly impaired astroglial cell migration to approximately that of AQP4-null cells, whereas treatment with AQP9 siRNA under the same conditions did not impair migration. RNAi treatment did not influence the migration of AQP4-null astroglia, or the background migration (toward 1% FBS) of AQP4-RNAi compared with AQP9-RNAi-treated astroglia.
Mechanistic studies
Membrane proteins that facilitate cell migration are sometimes polarized to the leading edge of the plasma membrane (Huttenlocher, 2005; Klein et al., 2000
). AQP4 has the potential to polarize because in normal brain AQP4 is expressed primarily in astroglial foot processes (Nielsen et al., 1997
). We investigated whether AQP4 polarizes in migrating astroglial cells and found increased AQP4 expression at the leading edge of migrating astroglia (Fig. 4A). The increased green AQP4 immunostaining at the leading edge was not due to folding of the plasma membrane evidenced by the lack of increased staining of the leading edge with the plasma membrane marker wheatgerm agglutinin (WGA).
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We hypothesized that AQP4-facilitated astroglial cell migration probably involves increased plasma membrane osmotic water permeability, which enhances water transport into the cell at its leading edge (where membrane protrusions form). To test this hypothesis, we carried out the Transwell assay in wild-type astroglia, as in Fig. 2A, except that raffinose (20 mM) was added to the upper or lower chambers to create small osmotic gradients from cell back-to-front or front-to-back, resulting in osmotic water transport into or out of the front of the cell, respectively. Fig. 4C shows accelerated cell migration when water flow was induced from cell front-to-back (opposite to the direction of migration), supporting the conclusion that migration involves water movement into the cell at its front edge.
Reactive gliosis in vivo
To determine whether the AQP4-dependent astroglial cell migration in culture models might occur in the brain in vivo, we studied reactive gliosis and glial scar formation using a well-established cortical-stab-injury model (Bush et al., 1999; Hampton et al., 2004
; Rhodes et al., 2003
; Wang et al., 2004
). The stab-injury mouse model, unlike other brain-injury models (tumor, infection, trauma), is not associated with significant brain swelling, which could confound interpretation of results. Intracranial pressure was normal in two wild-type and two AQP4-null mice, measured at day 1 and day 3 after the stab injury.
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Discussion |
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There are at present no selective AQP4 channel blockers for study of AQP4 function. Therefore, we used AQP4-null cells and RNA inhibition to investigate the role of AQP4 in astroglial cell migration. The AQP4 (but not the AQP9) siRNA treatment as used here strongly inhibited AQP4 protein expression and reduced plasma membrane water permeability, without altering cell morphology. This finding contradicts a previous report that AQP4 inhibition with siRNA-altered astroglial cell morphology and growth (Nicchia et al., 2003). In the earlier study, the upregulation of stress genes in all siRNA-treated cells, including the controls, suggested that the astroglia were damaged under the experimental conditions. Our siRNA data support the conclusion that the reduced migration of astroglia cultured from AQP4-null mice was not due to compensatory changes in these cells. The fact that a structurally different aquaporin (AQP1) can also accelerate cell migration (Saadoun et al., 2005
) suggests that the water-transporting function of aquaporins is important in cell migration.
The importance of water fluxes across the plasma membrane in causing localized swelling of lamellipodia has been discussed extensively in the early literature on cell migration (Condeelis et al., 1990; Oster and Perelson, 1987
). However, the idea that these water fluxes are primarily mediated by aquaporins is new (Saadoun et al., 2005
). Theoretical calculations show that transmembrane water fluxes induced by small extracellular osmotic gradients can generate enough force to propel vesicles (Anderson, 1983
; Zinemanas and Nir, 1995
) and even cells (Jaeger et al., 1999
) through extracellular solutions. During this process, termed osmophoresis, the vesicles/cells move towards hypo-osmolality as water enters into the front end and leaves from the rear end of the cell. To determine whether water fluxes across the plasma membrane play a role in astroglial cell migration, we altered the osmolalities of both chambers in the Transwell assay (Fig. 4C). This maneuver produces a steep osmotic gradient across the pore length (10 µm), which is maintained by hourly exchange of media in both chambers. During migration through the pore, the front end of astroglia becomes exposed to the osmolality of the bottom chamber while the rear end is exposed to the osmolality of the top chamber. In agreement with previous findings (Anderson, 1983
; Jaeger et al., 1999
; Zinemanas and Nir, 1995
), our experiments showed that the speed of astroglial cell migration can be altered by a small osmotic gradient in the extracellular medium, resulting in accelerated astroglia migration towards hypo-osmolality.
We suggest that AQP4 accelerates astroglial cell migration by increasing plasma membrane water permeability, which in turn increases the transmembrane water fluxes that take place during cell movement. This hypothesis could explain the observation (Fig. 4) that AQP4 deletion slows the rapid changes in cell shape that take place at the leading end of migrating astroglia. It has been suggested that the generation of osmoles produced by rapid actin depolymerization drives the entry of water into the leading end of migrating cells (Oster and Perelson, 1987), possibly in concert with transmembrane ionic movements (Huttenlocher, 2005
; Klein et al., 2000
). This osmotic influx of water across the plasma membrane expands the leading end of the cell, which is followed by actin polymerization to stabilize the membrane protrusion (Oster and Perelson, 1987
).
Unlike normal brain, where AQP4 expression is polarized to astroglial foot processes and the basolateral plasma membranes of ependymal cells (Nielsen et al., 1997), in confluent cultured astroglia AQP4 is expressed throughout the plasma membrane (Fig. 1). An interesting observation is that in migrating cultured astroglia, AQP4 polarizes to the leading edge of the plasma membrane (Fig. 4), where rapid transmembrane water movement occurs. This is consistent with reports that several ion channels and ion transporters, which play a role in cell migration, also polarize to the front end of migrating cells (Huttenlocher, 2005
; Klein et al., 2000
). These ion movements might contribute to the osmotic gradient that drives water influx during cell movement. The molecular mechanisms responsible for AQP4 polarization in migrating astroglia are not known, but might involve interactions between AQP4, the
-syntrophin complex and the actin cytoskeleton.
In agreement with prior reports (Bloch et al., 2005; Papadopoulos and Verkman, 2005
; Saadoun et al., 2002
; Saadoun et al., 2003
; Vizuete et al., 1999
), we found upregulation of AQP4 within the glial scar in response to brain injury in vivo (Fig. 6). Increased AQP4 expression, in addition to its polarization to the leading edge, may further augment the contribution of AQP4 to cell migration. The glial scar, where AQP4 expression is increased, consists primarily of reactive astroglia, but also contains activated microglia (Fawcett and Asher, 1999
; Rhodes et al., 2003
). Interestingly, activated microglia have recently been reported to express AQP4 (Tomas-Camardiel et al., 2004
), which may serve to increase their migration speed during glial scar formation.
In conclusion, we have discovered a new function for AQP4 in the brain: the enhancement of astroglial cell migration. This property of AQP4 appears unrelated to the well-established role of AQP4 in brain edema formation (Manley et al., 2000) and absorption (Papadopoulos et al., 2004
), and suggests novel therapeutic possibilities. AQP4 inhibitors might reduce glial scarring, which could enhance neuronal regeneration after injury and improve the efficiency of incorporation of neural grafts into CNS tissue. AQP4 inhibitors might also limit the spread of the AQP4-overexpressing, malignant astroglial cells into the surrounding brain.
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Acknowledgments |
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Footnotes |
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