the molecular basis of water transport in the brain - Nature

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THE MOLECULAR BASIS OF WATER TRANSPORT IN THE BRAIN Mahmood Amiry-Moghaddam and Ole P. Ottersen Brain function is inextricably coupled to water homeostasis. The fact that most of the volume between neurons is occupied by glial cells, leaving only a narrow extracellular space, represents an important challenge, as even small extracellular volume changes will affect ion concentrations and therefore neuronal excitability. Further, the ionic transmembrane shifts that are required to maintain ion homeostasis during neuronal activity must be accompanied by water. It follows that the mechanisms for water transport across plasma membranes must have a central part in brain physiology. These mechanisms are also likely to be of pathophysiological importance in brain oedema, which represents a net accumulation of water in brain tissue. Recent studies have shed light on the molecular basis for brain water transport and have identified a class of specialized water channels in the brain that might be crucial to the physiological and pathophysiological handling of water.

Centre for Molecular Biology and Neuroscience, Institute of Basic Medical Sciences, University of Oslo, POB 1105 Blindern, N-0317 Oslo, Norway. Correspondence to O.P.O. e-mail: [email protected] doi:10.1038/nrn1252

How is water transported across cell membranes? Given that water constitutes more than 90% of the molecules in the body and that water transport is involved in all secretory and absorptive processes, this is one of the most fundamental questions in biology. Water can pass directly through the lipid bilayer of the plasma membrane, but as this form of water flux is slow and not subject to regulation, it has long been hypothesized that there must be specialized water channels that control rapid transmembrane water fluxes. This hypothesis was not borne out until 1992, when Peter Agre and colleagues identified the first member of the aquaporin family of water channels1. To date, this family has 11 members in mammals2 (TABLE 1) and more than 150 in plants, microbes, invertebrates and nonmammal vertebrates3,4. Aquaporins have been found to facilitate regulated and constitutive transmembrane transport of water in a number of organs, such as the kidney and secretory glands5–8. Together with the isolation of the first aquaporins, the question of whether aquaporins occur in brain arose. It was soon found that the brain contains Aqp1 (REFS 6,8). This aquaporin is restricted to the choroid plexus and is thought to be involved in the secretion of

NATURE REVIEWS | NEUROSCIENCE

cerebrospinal fluid (CSF; see later section). Its role is probably analogous to that of aquaporins in other secretory organs that permit osmotically driven water flux through the epithelial lining7,8. In 1994 came the first hint that the brain contains an additional aquaporin. Two independent studies reported that brain tissue expresses messenger RNA encoding Aqp4 (REFS 9,10). Soon afterwards, the corresponding protein was detected by selective antibodies and found to be widely expressed in brain neuropil, where it is localized to astrocytes and ependymal cells11. This finding spawned a series of studies that aimed to determine the physiological and pathophysiological roles of this aquaporin. The fact that brain Aqp4 is functional was recently confirmed in an analysis of astrocytes isolated from Aqp4-null mice12. The third and final aquaporin to be detected in the central nervous system was Aqp9, whose function is yet to be explored. This review will deal with those aquaporins that are expressed in the brain (Aqp1, Aqp4 and Aqp9) and will focus on Aqp4, which has recently been implicated in the development of brain oedema, and in the regulation of ion and water homeostasis at the synaptic level. VOLUME 4 | DECEMBER 2003 | 9 9 1

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Structure of aquaporins

Table 1 | Mammalian aquaporins Aquaporin

Size

Permeability

Distribution

Aqp0

26 kD, 263 aa

Water (low)

Lens epithelium

Aqp1

28 kD , 269 aa

Water

Kidney, capillary endothelia (except brain), red blood cells, cornea, choroid plexus

Aqp2

29 kD, 271 aa

Water

Kidney collecting duct cells (intracellular and apical membranes)

Aqp3

31 kD, 292 aa

Water, glycerol, urea

Kidney, colon

Aqp4

M1: 32 kD, 301 aa Water (Hg++ insensitive) M23: 34 kD, 323 aa

CNS, skeletal muscle, lung, kidney, inner ear, gastric parietal cells

Aqp5

29 kD, 265 aa

Water

Lung, salivary glands, lacrimal glands, trachea, cornea

Aqp6

28–30 kD, 276 aa

Water (low), anions (HNO3–; high)

Intracellular vesicles in kidney intercalated cells, proximal tubules

Aqp7

26 kD, 269 aa

Water, glycerol

Adipose tissue, testis, kidney

Aqp8

27 kD, 269 aa

Water

Testis, liver, pancreas

Aqp9

32 kD, 342 aa

Water, glycerol, urea

Liver, testis, brain

Aqp10

28 kD, 301 aa

Water (low), glycerol, urea

Small intestine

aa, amino acid; Aqp, aquaporin; CNS, central nervous system; M1/M23, two different isoforms of Aqp4.

The discovery of aquaporins has profoundly altered our understanding of water transport in brain. The idea that water moves solely by diffusion through the lipid bilayer is no longer tenable. Rather, water fluxes in brain are directed by a highly differentiated network of membrane proteins with integral, water-selective pores. Here, we provide an update on the expression, function and pathophysiological roles of these channels.

Aquaporins Aqp2

Aqp4

Aqp5

Aqp6 Aqp1 Aqp0

Aqp8

Aqp10

Aqp9 0.1

PROTEOLIPOSOME

A liposome into which a specific protein, or group of proteins, has been incorporated.

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Aquaporins assemble in membranes as homotetramers. Each monomer contains a water channel and consists of six membrane-spanning α-helical domains (demonstrated experimentally for Aqp1, 2 and 4) with intracellular carboxyl (C) and amino (N) termini13–16. The molecular weights of the monomers range from 26 kDa (Aqp7) to 34 kDa (Aqp4) and the percent amino-acid identity varies from 19 to 52. A characteristic of the aquaporins is the extensive homology between the N- and C-terminal halves of the molecules17. Aqp4 is unique in having two different initiation sites giving functional polypeptides of 301 (M1 isoform) and 323 (M23 isoform) amino acids, respectively10. The aquaporin family comprises two subfamilies: aquaporins and aquaglyceroporins. As the names imply, the first subfamily is permeable only to water whereas the second subfamily is permeable also to glycerol and other solutes (TABLE 1; FIG. 1). Aquaporin-mediated water permeability has been assessed in transfected Xenopus oocytes and cell lines18,19, and in reconstituted PROTEOLIPOSOMES containing purified aquaporin proteins20,21. According to one study, Aqp4 has the highest water permeability coefficient, followed by Aqp1, Aqp2 and Aqp5; Aqp3 and Aqp0 have the lowest water permeability coefficients22. In another study, Aqp1 and the two Aqp4 isoforms (M1 and M23) produced an identical increase in osmotic water flux when expressed in Xenopus oocytes10. A separate study showed that Aqp9 has the same water permeability coefficient as Aqp1 (REF. 23). So far, crystallographic data are available only for Aqp1 (REFS 24–26). Each Aqp1 subunit contains a pore which narrows to a diameter of 2.8 Å at about 8 Å above the midpoint of the channel. So the narrowest portion of the channel corresponds to the size of a water molecule. Two additional factors contribute to water selectivity. An arginine residue in the pore region is a barrier to protonated water and other cations, whereas positively charged dipoles at the midpoint of the channel reorient water molecules and prevent protons from flowing through the channel (FIG. 2). Pending crystallographic data on the aquaglyceroporins, our understanding of how glycerol permeates the channel is derived from studies of the bacterial glycerol facilitator (GlpF)27. GlpF channels have constriction sites that are significantly wider than those of Aqp1 (4.8 versus 2.8 Å), thereby explaining why larger molecules like glycerol can pass through. It is also relevant that a histidine residue (His180) in Aqp1 is replaced by glycine in GlpF and in the aquaglyceroporins (except Aqp9, which has an Ala in lieu of a Gly), thereby providing a more hydrophobic pore lining28.

Aqp7 Aqp3

Aquaglyceroporins

Figure 1 | Diagram showing members of the aquaporin (Aqp) family expressed in mammals. The aquaglyceroporins are permeable for glycerol as well as water. The scale bar represents genetic distance between homologues (in number of nucleotide substitutions per site). Modified, with permission, from REF. 2  (2002) The Physiological Society.

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Regulation of aquaporins

Most aquaporins are inhibited by HgCl2, which interacts with free sulfhydryls. Site-directed mutagenesis of Aqp1 has identified Cys189 as the mercury-sensitive site29. Aqp4 is the only mercury-insensitive aquaporin that has been identified so far and is unique in lacking the cysteine that corresponds to Cys189 in Aqp1 (REF. 10).

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Extracellular –

Size restriction 180H

Electrostatic repulsion

+

R195

192N

+ Water dipole reorientation

+ N76

– Intracellular

Figure 2 | Architecture of the aquaporin-1 (Aqp1) water channel (longitudinal section through an individual subunit). The geometry of the aqueous pore (blue) is calculated from data on bovine Aqp1 (REF. 26). Four water molecules are shown interacting transiently with specific porelining residues. Selectivity for water depends on the following features: 1) at the narrowest the pore has a diameter of 2.8 Å, which corresponds to size of a water molecule; 2) a positively charged residue (Arg-195) prevents cation flux, and 3) near the channel midpoint there are two positively charged dipoles that disrupt hydrogen bonds and hinder passage of protons. Modified, with permission, from REF. 129  (2002) American Society for Clinical Investigation.

Substitution of cysteine for alanine at this site (position 210) does not confer mercury sensitivity10, indicating that there are additional differences between Aqp4 and other aquaporins that underlie their differential sensitivities. Aqp6 is unusual in that it is activated by HgCl2 (REFS 30,31). It is also the only aquaporin that conducts ions and is pH sensitive. Both water and ion conductance are increased by mercury; this effect depends on Cys155 and Cys190 (REF. 31). The mechanisms of Aqp2 regulation are understood in some detail. Vasopressin increases water reabsorption in the kidney by causing translocation of intracellular Aqp2 to the apical membrane of collecting duct cells. This effect is mediated through V2 receptors and cyclic AMP-dependent protein kinase A (PKA)-mediated phosphorylation of Aqp2 (REFS 32,33). Additional regulatory pathways have been demonstrated in vitro34. Superimposed on short-term regulation by translocation, the activation of V2 receptors also leads to sustained upregulation of Aqp2 expression35.


The phosphorylation site that is involved in vasopressin regulation of Aqp2 is Ser256 (REF. 33). Consensus sequences for phosphorylation by protein kinase C (PKC) or PKA are also present in other aquaporins, but the functional relevance of these consensus sites is yet to be established. For Aqp4, phorbol ester has been shown to cause phosphorylation and reduced permeability to water36,but this finding has not been confirmed. Activation or inhibition of PKC affects activitydependent radial water flux in neocortical slices37. This might reflect regulation of flow through astroglial Aqp4, but other target molecules might be involved. Unlike Aqp2, Aqp4 is not present on internal membranes, implying that its activity cannot be regulated by translocation. It is possible that its activity depends on the mode of expression in the plasma membrane. Plasmalemmal Aqp4 occurrs either as large assemblies of proteins — orthogonal arrays of particles — or as individual molecules38. If the mode of expression does affect water permeability it will be important to resolve how array formation is controlled39. The expression level of Aqp4 might be subject to regulation. Up- and downregulation of Aqp4 have been described in a number of pathological conditions40–45, but data are still fragmentary. Further, the mechanisms that govern Aqp4 expression are largely unknown although recent evidence indicates that PKC and mitogen-activated protein kinase (MAPK) might be involved46–48. In one study48, hyperosmolar mannitol was found to stimulate expression of Aqp4 and Aqp9 through a p38 MAPK-dependent pathway in rat astrocytes. Taniguchi et al. found an increased level of Aqp4 mRNA in the ipsilateral cortex following focal ischaemia in rats. This increase reached a maximum level after three days44. In the same species, Kiening reported a decrease in Aqp4 protein following controlled cortical impact injury41, whereas Ke et al., in another injury model, found a decrease in Aqp4 mRNA in the oedematous regions with impaired blood–brain barrier40. No decrease was observed where the blood–brain barrier was intact. Changes in AQP4 expression have also been demonstrated in oedematous human brain tumours42. Expression and physiological roles

Aqp1. Aqp1 is expressed in the epithelium of the choroid plexus where it is believed to have a role in the production of CSF8,49,50. Pending direct experimental data, this assumption is based on extrapolation from studies of aquaporin function in other secretory organs. Notably, knockout of Aqp5, an aquaporin that is known to be strongly expressed in the epithelium of sweat glands, severely depresses sweat secretion51. Animals lacking Aqp1 show reduced urinary concentration ability and impaired proximal tubular fluid reabsorption52, but the ability of Aqp1-null animals to produce CSF has not been investigated. The osmotic gradient that drives water through the choroid epithelium is generated by a Na+/K+ATPase53–55, and the secretory process involves an apical Na+/K+/2Cl– co-transporter, as well as apical HCO3– and VOLUME 4 | DECEMBER 2003 | 9 9 3


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c

b a

Choroid plexus Ependymal cells Perivascular membranes

Supraoptic nucleus

d

Figure 3 | Aquaporins in the brain. Distribution in brain of aquaporin-1 (AQP1, blue) and AQP4 (orange), schematically illustrated on a sagittal section of a human brain. The brain aquaporins show four different expression patterns (a–d). a | AQP4 occurs in the basolateral membrane of ependymal cells. b | AQP1 is expressed at the apical membrane of choroid plexus epithelial cells. c | AQP4 is concentrated in astrocytic end-feet, specifically in those membrane domains that abut on brain capillaries or on pia (see also FIG. 6). d | AQP4 is expressed in glial lamellae of the supraoptic nucleus and other osmosensitive regions (see FIG. 7). This is the only site where AQP4 occurs in a nonpolarized manner. AQP4 also occurs in non-endfeet membranes of astrocytes, but at comparatively low concentrations. In the neocortex, AQP4 expression in non-endfeet membranes increases from deep to superficial layers. The cerebellum shows the opposite gradient, with higher concentrations in the granule cell layer than in the molecular layer.

RETINAL MÜLLER CELL

The main glial elements of the retina that assume many of the functions that are carried out by astrocytes, oligodendrocytes and ependymal cells in other central nervous system regions. PDZ DOMAIN

A peptide-binding domain that is important for the organization of membrane proteins, particularly at cell–cell junctions, including synapses. It can bind to the carboxyl termini of proteins or can form dimers with other PDZ domains. PDZ domains are named after the proteins in which these sequence motifs were originally identified (PSD95, discs large, zona occludens 1).

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K+ channels49,56–58. The basolateral membranes contain Cl–/HCO3– and Na+/H+ exchangers49,59,60, which are needed to establish the electrochemical gradient that drives CSF production. Aqp1 is restricted to the apical membrane of the choroid epithelium (FIG. 3). The mechanism for water transport across the basolateral membranes remains to be established, as aquaporins have yet to be identified at this site. However, the choroid epithelial cells have been shown to contain Aqp4 mRNA, indicating that the basolateral membranes might express an Aqp4 splice variant that is not recognized by available antibodies50. Aqp1 occurs in capillary endothelial cells throughout the body, except for those in the brain8. The mechanisms for water flux through brain endothelial cells are discussed later.


Aqp4: expression. Aqp4 is the predominant aquaporin in the brain, and the brain is the primary site of Aqp4 expression10. Other organs that contain Aqp4 include the lung, skeletal muscle, stomach, inner ear and kidney61–63. Aqp4 is distributed throughout the brain but is found predominantly in the plasma membrane of astrocytes. The channel is present along the entire plasma membrane, including the processes that ensheath glutamatergic synapses, but is concentrated in those astrocyte membrane domains that face vessels and pia11. These membrane domains are part of the perivascular and subpial endfeet, respectively (FIGS 3 and 4). Quantitative analyses of RETINAL MÜLLER CELLS (a specialized type of astrocyte) indicated that endfeet membranes have Aqp4 concentrations one order of magnitude higher than non-endfeet membranes64. Aqp4 was also found to be expressed in ependymal cells, but was absent from neurons, oligodendrocytes and microglia. The original studies of Aqp4 distribution did not report any immunolabelling in brain endothelia, but more recent data indicate that endothelial cells are endowed with Aqp4 mRNA65 and express low levels of Aqp4 protein66. What mechanisms are responsible for the anchoring of Aqp4 at perivascular and subpial membranes of astrocytes? As Aqp4 has a terminal Ser-Ser-Val sequence, it was proposed that anchoring could occur through PDZ-DOMAIN-containing proteins. The dystrophin complex was a candidate, as this complex contains syntrophins (known to have PDZ domains) and is co-expressed with Aqp4 in striated muscle and endfeet membranes67–71. Evidence supporting a role of dystrophin in Aqp4 anchoring was provided by Frigeri et al. who showed a loss of Aqp4 from striated muscle in Mdx mice, which lack the dystrophin complex72,73. These animals also displayed a strong reduction in Aqp4 immunolabelling in the ependymal lining, around brain microvessels and subjacent to the pia mater. The Aqp4 pools in kidney, lung and gastrointestinal tract were unaffected by the Mdx mutation. Evidence for the specific involvement of α-syntrophin (α-Syn) in Aqp4 anchoring was obtained by deleting the gene that encodes this member of the dystrophin complex. α-Syn–/– mice exhibited a marked loss of Aqp4 from perivascular membranes (FIG. 5) and only a modest loss from other membrane domains, including ependymal cells and subpial endfeet74,75. Aqp4 could be immunoprecipitated by an antibody to α-Syn, providing additional support for an interaction between these two molecules75. This interaction is probably indirect, as chemical crosslinking is required for coimmunoprecipitation. Further, it has not been possible to detect direct binding between peptides or recombinant fusion proteins representing the Aqp4 C terminus and α-Syn PDZ domains75. The finding that the loss of Aqp4 is more restricted after α-Syn deletion than in Mdx animals indicates that there might be several syntrophins that participate in Aqp4 anchoring and that α-Syn is the predominant syntrophin in the perivascular membranes (BOX 1). The identification of proteins involved in Aqp4 anchoring in astrocytes paved the way for analyses that



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a

Lc

E Endfoot

b Lc

E

*

Figure 4 | Aqp4 expression at the blood—brain interface a | Drawing showing the expression pattern of aquaporin-4 (Aqp4) around brain microvessels. The concentration of Aqp4 is high in those perivascular endfeet membrane domains that face the vessels, and drops to low levels as soon as the membrane loses contact with the basal lamina. Endothelial cells express Aqp4 at their adluminal as well as abluminal membranes but at much lower levels than the astrocytic endfeet. b | Electron micrograph (corresponding to the box in a) shows strong Aqp4 immunogold labelling in the perivascular membrane of an astrocytic endfoot. (The endothelial Aqp4 was not evident at this level of labelling sensitivity.) Asterisk, basal lamina; E, endothelium; Endfoot, astrocyte endfoot; Lc, capillary lumen. Arrow indicates the two membrane domains of the endfoot. Scale bar, 0.5 µm. Reprinted, with permission, from REF. 11  (1997) Society for Neuroscience.

could specifically address the physiological and pathophysiological functions of Aqp4 in the brain. Taken together, these analyses have provided strong evidence in favour of the idea that perivascular and subpial Aqp4 promote the exchange of water between brain and extracerebral liquids (blood and CSF). This is logical, taking into account the strategic expression of Aqp4 at the interface with these liquid spaces. Regarding the developmental profile, Aqp4 appears between one and two weeks postnatally and reaches adult levels at four weeks76. Throughout development, Aqp4 is present in glial cells and absent from neurons. It has been suggested that Aqp4 plays a part in the development of the blood–brain barrier77.


Aqp4: physiological roles. The first clues to the roles of astrocytic Aqp4 were derived from immunolocalization experiments11,61. Quantitative immunogold analyses of retinal Müller cells revealed a strict co-localization between Aqp4 and Kir4.1 — a potassium channel that is known to be involved in the clearance of extracellular K+ (REF. 64). Specifically, both molecules were about tenfold enriched in endfeet membranes facing vessels and the corpus vitreum. As endfeet membranes represent only about 10% of the total membrane surface, this observation implies that the complement of Aqp4 and Kir4.1 in endfeet is equivalent to the complement of these molecules in non-endfeet membranes. It is known that the retinal Müller cells are engaged in the clearance of excess K+ from active neuropil78–80. Notably, K+ is taken up through the plasma membrane facing the neuropil, and siphoned into the corpus vitreum and blood vessels through the endfeet. Does the strict co-expression of Aqp4 and Kir4.1 indicate that these two molecules act in concert, and that activity dependent K+ fluxes are accompanied by water? This would agree with earlier studies demonstrating that high neuronal activity is associated with a shrinkage of extracellular space, indicative of water transport away from active neuropil81–83. These volume changes occur so rapidly that they probably depend on specialized water channels rather than on slow diffusion through the lipid bilayer. The hypothesis that K+ and water transport are functionally coupled was tested in an acute cortical slice model that allowed monitoring of water and K+ flux after evoked activity37. Water flux could be assessed indirectly by taking advantage of the optical signals that are generated when the extracellular space increases or decreases. The parallel use of an impermeant ion to record volume changes attested to the validity of this methodological approach. High neuronal activity was evoked in layer IV of the cortical slice by electrical stimulation of the afferent fibres from the thalamus. Immediately after the stimulating current was turned on, there was a distinct optical signal in layer IV, indicative of a shrinkage of the extracellular space and a centrifugal transport of water through glial cells. Simultaneously, an optical signal emerged in the superficial cortical layers, indicative of an increase in the extracellular space there. The optical signals were assumed to reflect a net flux of water along the radial axis of the cortex, directed towards the subarachnoidal space (which normally serves as a water sink). By use of ion-selective microelectrodes, it could be confirmed that the activity dependent water flux was coupled to a K+ flux. The above data show that the neocortex can sustain radial water fluxes analogous to those that occur through kidney collecting tubule cells. This begged the question of whether similar regulatory mechanisms could be involved. The water flux that exists in collecting tubule cells is known to be regulated by vasopressin (antiduretic hormone), acting through V2 receptors and Aqp2 translocation5.

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Lv

E E Lv

α -Syn–/– Aqp4 Ab

Wild type Aqp4 Ab

α-Syn) deletion on aquaporin-4 (Aqp4) distribution. Compared with wild-type animals, animals Figure 5 | Effect of α-syntrophin (α lacking α-Syn–/– show an almost complete loss of Aqp4 from perivascular membranes (arrowheads) and a moderate increase in Aqp4 at the abluminal membrane domain of the astrocytic endfeet. The gene deletion is also associated with a swelling of the perivascular endfeet (double arrows). Ab, antibody; E, endothelium; Lv, vessel lumen. Scale bar, 0.5 µm. Reprinted, with permission, from REF. 74  (2003) National Academy of Sciences.

Astrocytes express vasopressin 1 (V1) receptors84. In the acute cortical slice model, the radial water flux was enhanced by adding vasopressin to the medium and was reduced by V1 antagonists37. This finding supported the idea that radial water transport occurs by way of the astrocyte syncytium that spans the entire thickness of the cortex. The analogy between kidney and brain (organs that show radial water transport regulated by vasopressin) does not extend to the downstream mechanisms. As pointed out above, Aqp4 seems to be constitutively expressed in the astrocyte plasma membrane, implying that the V1 receptor-mediated regulation of water transport cannot involve Aqp4 translocation. The mechanisms involved remain to be identified. Spatial K+ buffering, in the strict sense, is not assumed to be associated with significant water movement. However, K+ clearance involves a number of mechanisms that generate osmotic gradients and water flux81. An important question is whether there is an obligatory coupling between water and K+ fluxes, in the sense that K+ clearance will be impaired if the water transport is interfered with85–87. To address this issue, ion selective microelectrodes were used to assess the clearance rate of K+ after orthodromic stimulation of two main fibre pathways in the hippocampus — the perforant path and Schaffer commissural/associational pathway. It was found that the recovery of extracellular K+ concentration was significantly delayed in α-Syn–/– mice, which lack perivascular Aqp4 (REF. 88). This indicates that efficient clearance of K+ is contingent on parallel water transport and that the latter might serve to short-circuit the osmotic gradients that are set up by the K+ fluxes. However, it cannot be ruled out that the deletion of α-Syn causes additional changes, which contribute to deficient K+ clearance.

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The most parsimonious explanation of the data discussed above is that activity-dependent water uptake through the Aqp4 pool in non-endfeet membranes is coupled to water efflux through the Aqp4 pool in the endfeet. Most probably, the former pool mediates the water uptake that is responsible for the shrinkage of extracellular space around active synapses. Aqp4 has been shown to be expressed on astrocytic processes abutting on glutamate synapses in the cerebellum (FIG. 6), but it remains to be determined whether the Aqp4 density differs between synapses with different transmitter phenotype. One would predict that the degree to which the extracellular space shrinks in response to synaptic activity is determined by the density of Aqp4 molecules in adjacent glial membranes. So, a regulated expression of Aqp4 along astrocyte membranes could help fine-tune the extracellular ion concentrations and modify extracellular space tortuosity and transmitter spillover. This would constitute a subtle regulatory system that is superimposed on the robust point-to-point transmission in central synapses. Observations in α-Syn–/– mice raise the possibility that perivascular Aqp4 serves another important function, in addition to having a permissive role in clearing K+. In basic conditions, α-Syn–/– mice consistently exhibited a swelling of the perivascular endfeet74. This finding was taken to indicate that the perivascular Aqp4 pool normally mediates efflux of metabolically generated water. The brain is a water-secreting organ, because glucose, its main energy substrate, yields substantial amounts of water when broken down through the oxidative pathway89. One could argue that the lack of a distinct phenotype in Aqp4 knockouts90 speaks against an important role of Aqp4 in brain physiology. However, what many of the aquaporins seem to have in common is that their



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Box 1 | The dystrophin-associated protein complex The dystrophin-associated protein complex (DAP) is a large Laminin membrane assembly that connects the cytoskeleton to the Basal lamina extracellular matrix123. Analyses of the dystrophin gene Aqp4 revealed the presence of eight different promoters, spread α-DG throughout the gene, that give rise to a tissue-specific Out distribution of full-length dystrophins (Dp427), as well as 1 2 3 4 5 6 several shorter isoforms. Along with Dp427, three shorter In carboxy (C)-terminal transcripts have been discovered in SXV COOH β-DG brain. These are Dp140, Dp116 and the main brain isoform, NH2 Dp71. With the exception of Dp71, all dystrophins form a PD PD Z Sy Z bridge between filamentous actin and the transmembrane S yn n protein β-dystroglycan (β-DG). Dp71 lacks the actin-binding Sy DP71 n PD n domain of other dystrophins124–126. β-DG is associated with a Sy Z Z PD Dystrobrevin laminin/agrin-binding protein, α-DG. On the cytoplasmic side of the complex, dystrophin (or utrophin, which can NH2 replace dystrophin) binds to α-dystrobrevin. Each of these proteins (dystrophin or utrophin on one side and α-DG) can NH2 bind up to two syntrophins68,127. Syntrophins are a family of COOH COOH five proteins (α, β1, β2, γ1 and γ2) containing two PLECKSTRIN 128 HOMOLOGY DOMAINS, a PDZ domain and a C-terminal syntrophin-unique region . The PDZ domain serves as an adaptor for the recruitment of membrane channels, receptors, kinases and other signalling proteins. Tissue-specific variations in the dystrophin complex are, in part, due to different syntrophins. α-Syn is the main syntrophin expressed in the astrocyte endfeet. It is thought to be responsible for the polarized expression of Aqp4 at this site through direct or indirect interaction of the Aqp4 sequence Ser-X-Val with its PDZ domain67,75.

PLECKSTRIN HOMOLOGY DOMAIN

A sequence of about 100 amino acids that is present in many signalling molecules. Pleckstrin is a protein of unknown function that was originally identified in platelets. It is a principal substrate of protein kinase C. TANYCYTE

A type of ependymal cell found principally in the walls of the third ventricle of the brain. The tanycytes might have branched or unbranched processes, some of which end on capillaries or neurons.

functions are clearly revealed only when the organs are subjected to stress. A case in point is the finding that in Aqp1-null mice, their severely reduced ability to concentrate urine becomes obvious only after 36 hours of water deprivation52. Similarly, whereas the brain might normally be able to sustain ion and water homeostasis even in the absence of Aqp4, Aqp4 might be required to cope with ion and water shifts that occur during high neural activity. One also has to bear in mind that Aqp4 activity might be essential for a number of brain functions that remain to be tested in Aqp4-deficient animals. Only one set of brain structures — the osmosensitive organs — shows non-polarized distribution of glial Aqp4 (REF. 11) (FIG. 7). These organs, which include the supraoptic nuclei and circumventricular organs, contain glial lamellae with uniformly high Aqp4 expression along their plasma membranes. These glial lamellae also express high levels of taurine, an amino acid that acts as an endogenous ligand for glycine receptors91,92. Hypoosmotic stress causes a release of taurine onto the glycine receptors of osmosensitive neurons93. Activation of these glycine receptors causes reduced secretion of antidiuretic hormone, which leads to reduced water retention in the kidney collecting ducts. In this way, the Aqp4-bearing glial lamellae might act as transducers in the osmosensory response93. Aqp9. Immunoblots have shown that the brain expresses Aqp9 (REF. 94). Together with Aqp3, 7, 9 and 10 this aquaporin belongs to a subfamily of aquaporins that facilitates the flux of glycerol as well as water2,95,96 (the aquaglyceroporins; FIG. 1). Light microscopy has revealed the presence of Aqp9 in the ependymal lining of the third ventricle, including the TANYCYTES, whereas


in situ hybridization indicates expression of Aqp9 mRNA also in astrocytes and endothelial cells97. The functional role of Aqp9 in the brain is still obscure, but its permeability to glycerol indicates that it might have a role in energy metabolism. Pathophysiological roles of brain aquaporins

Among the brain aquaporins, only Aqp4 has been subjected to careful analyses with respect to possible pathophysiological roles. Summing up the studies that we have discussed here, there is now ample evidence that Aqp4 is coupled functionally and spatially to proteins that are responsible for K+ buffering. So, it is likely that Aqp4 primarily serves to mediate activity-dependent water fluxes that are required to sustain ion and volume homeostasis at central synapses. Moving in parallel with K+, water must be siphoned into blood or CSF during periods of high neuronal activity. This siphoning of water occurs through the Aqp4 pools that are strategically placed in those astrocyte membranes that abut vessels or pia. Superimposed on this activity-dependent water flux is a constitutive efflux of water derived from the metabolic breakdown of brain glucose. As water flux through Aqp4 is bidirectional and driven solely by osmotic gradients, perivascular Aqp4 might have negative effects in pathophysiological conditions that involve water accumulation in the brain. Water accumulation is the hallmark of brain oedema, a potentially lethal condition. The first evidence linking Aqp4 to brain oedema came with the seminal study of Manley et al. who reported that knocking out Aqp4 reduced the extent of oedema after experimental stroke or hypo-osmotic stress90. As Aqp4 is expressed not only in brain but also in the kidney, lungs



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a

b

*

Presynaptic

Kir4.1

Aqp4

Endfoot

K+ H2O Glutamate

Pf

S

Postsynaptic specialization

GluR

Postsynaptic

Eaat1/ Eaat2

Astrocyte

Figure 6 | Homeostatic functions of astrocytes. a | Expression pattern of aquaporin-4 (Aqp4) in the superficial part of the cerebellar molecular layer. Gold particles labelling Aqp4 occur in astrocytic lamellae (arrowheads) ensheathing synapses between parallel fibres (Pf) and Purkinje cell spines (S), and, at higher density, in endfeet membranes facing the pia and the subarachnoidal space (asterisk). The double arrow indicates subpial endfoot. b | Drawing showing a simplified glutamatergic synapse along with an astrocyte with different molecules (inwardly rectifying K+ channel (Kir) 4.1 and glutamate transporters Eaat1 and 2) that are believed to take part in the clearance of K+ and glutamate. Scale bar, 0.5 µm. Part a reprinted, with permission, from REF. 11  (1997) Society for Neuroscience. GluR, glutamate receptors.

and gastrointestinal tract, an Aqp4 knockout mouse could have many effects on the body’s water homeostasis. Therefore, in recent studies it was deemed important to selectively remove aquaporins at the brain–liquid interfaces to more accurately assess the functions of these pools. The concentration of Aqp4 around vessels and subjacent to the pia mater reflects the highly polarized expression of Aqp4 in the endfeet of brain astrocytes. The crucial roles of the dystrophin complex and α-Syn in Aqp4 anchoring at brain–liquid interfaces were discussed above. Mdx mice, which lack dystrophin, were found to be more resistant to hypo-osmotic stress than wild-type mice98, supporting the idea that brain Aqp4 contributes to water uptake under conditions that favour the development of brain oedema. A delay in the development of post-ischaemic and hypo-osmotic brain oedema was also observed in mice lacking α-Syn7,66. As α-Syn deletion primarily removes perivascular Aqp4 (FIG. 5), the latter finding indicated that the perivascular membrane domain is an influx route for water during the development of oedema. The Aqp4 pools along the other brain–liquid interfaces (pial surface and ependyma) might act as water sinks in the acute phase of oedema development99–101. Taken together, data from the transgenic models indicate that gating of water influx occurs at a site that is distinct from the blood–brain barrier, which resides at the level of the endothelial cells. It is important in this regard to recall that removal of perivascular Aqp4 offers only partial protection against oedema. During hypoosmotic stress, as well as after brain ischaemia, the volume of the oedema is reduced by about 50% after deleting

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perivascular Aqp4 (REFS 74,90). The residual oedema probably reflects water fluxes through the lipid bilayer and through the clefts between the perivascular endfeet. With respect to ischaemia, it is important to note that Aqp4 is lost from perivascular membranes in the post-ischaemic phase74. This might be regarded as a mechanism to limit water influx, but will inevitably slow down the resolution of oedema. The α-Syn pool remains intact despite the loss of Aqp4, indicating that the binding between these two molecules is sensitive to ischaemia. The mechanisms underlying this sensitivity are unknown, as the binding of Aqp4 to α-Syn is unlikely to be direct and probably involves intermediate proteins. The post-ischaemic downregulation of Aqp4 might explain why oedema persists for such a long time after stroke, as efflux of water will be impeded. Interestingly, long before the discovery of aquaporins, it was reported that orthogonal arrays of protein, characteristic of endfeet membranes, disappear after ischaemia102. Now we know that these protein assemblies represent Aqp4 (REF. 38). For water to reach the astrocytic endfeet it must pass through the endothelial cells. As noted earlier, these cells are endowed with Aqp4 mRNA and express low levels of Aqp4 protein. The endothelial pool of Aqp4 is resistant to α-Syn deletion66. It is possible that plasma membrane co-transporters contribute to transendothelial water passage in physiological and pathophysiological conditions103,104 (see later in text). Water passage between the endothelial cells is restricted by tight junctions, which form an essential part of the blood–brain barrier105. In addition to facilitating the formation of brain oedema, Aqp4 could also be implicated in pathological



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Figure 7 | Expression pattern of aquaporin-4 (Aqp4) in the subfornical organ of rat. This pattern is representative of all osmosensitive regions and is characterized by a non-polarized distribution of Aqp4 in glial lamellae ensheathing dendrites of local neurons (De in inset). The latter neurons are invariably immunonegative. In glial lamellae, only those membrane patches that are engaged in gap junctions (arrows) are devoid of Aqp4. Arrowheads in inset, synaptic specializations; double arrow in inset, glial lamella. Co, collagen fibrils; Fi; fibrocyte; Gf, glial filaments; Lv, vessel lumen; PVS, perivascular space. Asterisks, basal lamina. Scale bar, 1 µm; Scale bar in inset, 0.5 µm. Reprinted, with permission, from REF. 11  (1997) Society for Neuroscience.

conditions associated with perturbed ion homeostasis. Notably, if Aqp4 plays a permissive part in K+ clearance, then the removal of Aqp4 might be predicted to increase susceptibility to epileptic seizures. It is well known that increased [K+]o tends to depolarize neurons and facilitate the development of epileptiform discharges. Using a hyperthermia model, it was found that α-Syn–/– animals had a seizure threshold that was similar to that of wildtype animals. However, the severity of the seizures was greatly enhanced88. This finding indicates that functional integrity of perivascular endfeet is required to handle the excessive amounts of extracellular K+ that are generated during overt seizures. Other molecules that transport water

It would be incorrect to give the impression that water transport is carried out exclusively by aquaporins. Whereas aquaporins constitute the only known family of molecules that selectively admit water, water can be co-transported with ions or organic molecules across the plasma membrane. In fact, water transport seems


to be a general feature of co-transporters that are expressed in the plasma membrane103,106–110. Co-transporters can have one of two modes of water transport. The secondary active mode is defined as translocation of water molecules in association with substrate transport, independently of osmotic gradients. The passive transport mode describes the activity of transporters as water channels, mediating osmotic water flux. For instance, the glutamate transporter Eaat1, which is one of the most efficient water co-transporters, admits 436 ± 55 water molecules per glutamate anion103,108. However, the single-unit water permeability of Eaat1 is (in cm3 sec–1 × 10–14) roughly 0.2 (REF. 111) compared to 6 for Aqp1 and 24 for Aqp4 (REF. 22). Several of the co-transporters that flux significant amounts of water are expressed at the brain–blood interface103,104,112–116. The glucose transporter Glut1, the monocarboxylate transporter Mct1, and the NaKCl2 cotransporter are all localized in brain endothelial cells and might be involved in transendothelial movement of water. Mct1 and Glut1 are also expressed in perivascular membranes103,112–114,117. The anchoring of Mct1 and Glut1 at this site seems to be independent of α-Syn74. The glutamate transporters Eaat1 and Eaat2 are enriched in those membrane domains that ensheath glutamatergic synapses, and are relatively weakly expressed in endfeet membranes118. Despite their significant water fluxing capacity it is doubtful whether they contribute to activity-dependent glial swelling. This is because glutamate uptake seems to be concomitant with extrusion of lactate and H+, which is also associated with water flux119. In conclusion, water transport in the brain is not solely the domain of aquaporins. However, the aquaporins are unsurpassed when it comes to the capacity and selectivity for water transport. Aqp4: a target for new therapies?

The development of brain oedema can no longer be reduced to an issue of osmotic forces acting across a lipid bilayer. It must be explained with reference to the properties and differential distribution of membrane proteins that permit water flux through their hydrophilic interior. As a result, new therapeutic strategies for this common and often lethal condition should evolve. The principles that underlie the treatment of brain oedema are the same today as they were 70 years ago120 — hyperosmotic therapy and surgical decompression. More rational therapy is needed, but several problems must be resolved before Aqp4 can be exploited as a therapeutic target. One outstanding question is how Aqp4 is regulated. Another problem is posed by the fact that Aqp4 facilitates bidirectional water transport2,74. As efflux of water would be expected to be beneficial in the resolution phase of brain oedema, any therapy that entails long-lasting downregulation of Aqp4 would seem less than attractive. Attempts to inhibit Aqp4 to counteract the development of oedema must take into account possible side effects relating to the physiological role of Aqp4 in the maintenance of K+ homeostasis. One must also bear in mind that any treatment that affects all Aqp4 pools in the



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ELECTRORETINOGRAM

Measurement of the retinal response to light, typically using an electrode attached to the cornea.

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organism could produce side effects in the inner ear, eye and kidney, which are sites of Aqp4 expression9,61. Notably, Aqp4 -knockout mice are hearing impaired and show mild changes in the ELECTRORETINOGRAM121,122, Given the scarcity of knowledge on Aqp4 regulation, targeting the Aqp4 anchoring mechanisms might be one way to approach therapeutic modulation of water transport. Recent studies indicate that the binding of Aqp4 to α-Syn is labile, as Aqp4 but not α-Syn disappears from perivascular membranes following ischaemic insult74. Strategies that aim to compete with Aqp4 binding might therefore prove fruitful.

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These problems notwithstanding, Aqp4 should now be explored as a target for the development of new therapies for brain oedema. None of the current therapies are based on a molecular understanding of brain water transport. Modulation of Aqp4 could prove to be therapeutically useful not only in curtailing water uptake, but also in controlling neuronal excitability. Even if clinical applications are a long way into the future, the discovery of brain aquaporins has already had a profound impact on our understanding of how the brain regulates its volume and synaptic microenvironment.

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Acknowledgements We thank C. Knudsen and G. Lothe for help with the illustrations and P. Agre, H. Kimelberg and S. Froehner for invaluable advice. Supported by the Norwegian Research Council, the European Cooperation in Scientific and Technological Research (COST) and the European Union.

Competing interests statement The authors declare that they have no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ aquaporins | Eaat1 | Glut1 | Mct1 | Mdx | α-Syn Access to this interactive links box is free online.

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