Mahmood Amiry-Moghaddam

The molecular basis of water transport in the brain

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.

An α-syntrophin-dependent pool of AQP4 in astroglial end-feet confers bidirectional water flow between blood and brain

The water channel AQP4 is concentrated in perivascular and subpial membrane domains of brain astrocytes. These membranes form the interface between the neuropil and extracerebral liquid spaces. AQP4 is anchored at these membranes by its carboxyl terminus to α-syntrophin, an adapter protein associated with dystrophin. To test functions of the perivascular AQP4 pool, we studied mice homozygous for targeted disruption of the gene encoding α-syntrophin (α-Syn−/−). These animals show a marked loss of AQP4 from perivascular and subpial membranes but no decrease in other membrane domains, as judged by quantitative immunogold electron microscopy. In the basal state, perivascular and subpial astroglial end-feet were swollen in brains of α-Syn−/− mice compared to WT mice, suggesting reduced clearance of water generated by brain metabolism. When stressed by transient cerebral ischemia, brain edema was attenuated in α-Syn−/− mice, indicative of reduced water influx. Surprisingly, AQP4 was strongly reduced but α-syntrophin was retained in perivascular astroglial end-feet in WT mice examined 23 h after transient cerebral ischemia. Thus α-syntrophin-dependent anchoring of AQP4 is sensitive to ischemia, and loss of AQP4 from this site may retard the dissipation of postischemic brain edema. These studies identify a specific, syntrophin-dependent AQP4 pool that is expressed at distinct membrane domains and which mediates bidirectional transport of water across the brain–blood interface. The anchoring of AQP4 to α-syntrophin may be a target for treatment of brain edema, but therapeutic manipulations of AQP4 must consider the bidirectional water flux through this molecule.

Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, β-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking α-syntrophin (α-Syn−/−). The total level of AQP4 expression appears normal in brains of α-Syn−/− mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of α-Syn−/− mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in α-Syn−/− mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse–chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires α-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.

Delayed K+ clearance associated with aquaporin-4 mislocalization: Phenotypic defects in brains of α-syntrophin-null mice

Recovery from neuronal activation requires rapid clearance of potassium ions (K+) and restoration of osmotic equilibrium. The predominant water channel protein in brain, aquaporin-4 (AQP4), is concentrated in the astrocyte end-feet membranes adjacent to blood vessels in neocortex and cerebellum by association with α-syntrophin protein. Although AQP4 has been implicated in the pathogenesis of brain edema, its functions in normal brain physiology are uncertain. In this study, we used immunogold electron microscopy to compare hippocampus of WT and α-syntrophin-null mice (α-Syn-/-). We found that <10% of AQP4 immunogold labeling is retained in the perivascular astrocyte end-feet membranes of the α-Syn-/- mice, whereas labeling of the inwardly rectifying K+ channel, Kir4.1, is largely unchanged. Activity-dependent changes in K+ clearance were studied in hippocampal slices to test whether AQP4 and K+ channels work in concert to achieve isosmotic clearance of K+ after neuronal activation. Microelectrode recordings of extracellular K+ ([K+]o) from the target zones of Schaffer collaterals and perforant path were obtained after 5-, 10-, and 20-Hz orthodromic stimulations. K+ clearance was prolonged up to 2-fold in α-Syn-/- mice compared with WT mice. Furthermore, the intensity of hyperthermia-induced epileptic seizures was increased in approximately half of the α-Syn-/-mice. These studies lead us to propose that water flux through perivascular AQP4 is needed to sustain efficient removal of K+ after neuronal activation.

Alpha-syntrophin deletion removes the perivascular but not endothelial pool of aquaporin-4 at the blood–brain barrier and delays the development of brain edema in an experimental model of acute hyponatremia

The formation of brain edema, commonly occurring as a potentially lethal complication of acute hyponatremia, is delayed following knockout of the water channel aquaporin‐4 (AQP4). Here we show by high‐resolution immunogold analysis of the blood–brain‐barrier that AQP4 is expressed in brain endothelial cells as well as in the perivascular membranes of astrocyte endfeet. A selective removal of perivascular AQP4 by α‐syntrophin deletion delays the buildup of brain edema (assessed by Diffusion‐weighted MRI) following water intoxication, despite the presence of a normal complement of endothelial AQP4. This indicates that the perivascular membrane domain, which is peripheral to the endothelial blood–brain barrier, may control the rate of osmotically driven water entry. This study is also the first to demonstrate that the time course of edema development differs among brain regions, probably reflecting differences in aquaporin‐4 distribution. The resolution of the molecular basis and subcellular site of osmotically driven brain water uptake should help design new therapies for acute brain edema.

Anchoring of aquaporin-4 in brain: Molecular mechanisms and implications for the physiology and pathophysiology of water transport

Astrocytes show an enrichment of aquaporin-4 (AQP4) in those parts of the plasma membrane that are apposed to pial or perivascular basal laminae. This observation begged the following questions: 1, What are the molecular mechanisms that are responsible for the site specific anchoring of AQP4? 2, What are the physiological and pathophysiological roles of the AQP4 pools at these specialized membrane domains? Recent studies suggest that the site specific anchoring depends on the dystrophin complex. Further, alpha-syntrophin (a member of the dystrophin complex) is required to maintain a polarized expression of AQP4 in the perivascular membranes. Hence transgenic mice deficient in alpha-syntrophin provided a model where the perivascular pool of AQP4 could be removed for assessment of its functional roles. Data suggest that the perivascular pool of AQP4 plays a role in edema formation and that this pool (through its serial coupling with the AQP4 pools in other astrocyte membranes) is involved in K(+) siphoning. In the cerebral cortex, the astrocyte membrane domain contacting the pial basal lamina differs from the perivascular membrane domain in regard to the mechanisms for AQP anchoring. Thus deletion of alpha-syntrophin causes only a 50% loss of AQP4 from the former membrane (compared with a 90% loss in the latter), pointing to the existence of additional anchoring proteins. We will also discuss the subcellular distribution and anchoring of AQP4 in the other cell types that express this protein: endothelial cells, ependymal cells, and the specialized astrocytes of the osmosensitive organs.

An aquaporin-4/transient receptor potential vanilloid 4 (AQP4/TRPV4) complex is essential for cell-volume control in astrocytes

Regulatory volume decrease (RVD) is a key mechanism for volume control that serves to prevent detrimental swelling in response to hypo-osmotic stress. The molecular basis of RVD is not understood. Here we show that a complex containing aquaporin-4 (AQP4) and transient receptor potential vanilloid 4 (TRPV4) is essential for RVD in astrocytes. Astrocytes from AQP4-KO mice and astrocytes treated with TRPV4 siRNA fail to respond to hypotonic stress by increased intracellular Ca2+ and RVD. Coimmunoprecipitation and immunohistochemistry analyses show that AQP4 and TRPV4 interact and colocalize. Functional analysis of an astrocyte-derived cell line expressing TRPV4 but not AQP4 shows that RVD and intracellular Ca2+ response can be reconstituted by transfection with AQP4 but not with aquaporin-1. Our data indicate that astrocytes contain a TRPV4/AQP4 complex that constitutes a key element in the brains volume homeostasis by acting as an osmosensor that couples osmotic stress to downstream signaling cascades.

Ontogeny of water transport in rat brain: postnatal expression of the aquaporin-4 water channel.

Brain water transport is poorly understood at the molecular level, and marked changes occur during brain development. As the aquaporin‐4 (AQP4) water channel protein is abundant in brain, the expression levels and subcellular distribution of this protein were examined during postnatal development. This study focused on the cerebellum, which showed the same pattern of AQP4 development as the rest of the brain. Semiquantitative immunoblotting revealed very low levels of AQP4 in the first postnatal week. A pronounced increase was noted in the second week, from 2% of adult level at postnatal day 7 (PN7) to 25% at PN14. At PN1 and PN3 immunofluorescence microscopy revealed weak labelling, mainly in radial processes (Bergmann fibres) and at the pial surface. Between PN7 and PN14 the labelling underneath the pia showed a strong increase, and immunoreactivity also appeared around blood vessels throughout the cerebellum. High‐resolution immunogold electron microscopy revealed that the subpial and perivascular labelling was restricted to glial end feet, notably to those plasma membrane domains that were apposed to the basal laminae. At no stage was there any evidence of neuronal AQP4 labelling, and AQP1, −2, −3 and −5 proteins were not detected in the neuropil. Riboprobes to AQP4 mRNA produced a particularly strong in situ hybridization signal in glial cells between PN7 and PN14, corresponding to the stage of the most rapid increase of AQP4 protein. The time course and pattern of AQP4 expression suggests that this aquaporin plays an important role in brain water and K+ homeostasis from the second week of development.

Expression and functional characterization of transient receptor potential vanilloid-related channel 4 (TRPV4) in rat cortical astrocytes.

Cell-cell communication in astroglial syncytia is mediated by intracellular Ca(2+) ([Ca(2+)](i)) responses elicited by extracellular signaling molecules as well as by diverse physical and chemical stimuli. Despite the evidence that astrocytic swelling promotes [Ca(2+)](i) elevation through Ca(2+) influx, the molecular identity of the channel protein underlying this response is still elusive. Here we report that primary cultured cortical astrocytes express the transient receptor potential vanilloid-related channel 4 (TRPV 4), a Ca(2+)-permeable cation channel gated by a variety of stimuli, including cell swelling. Immunoblot and confocal microscopy analyses confirmed the presence of the channel protein and its localization in the plasma membrane. TRPV4 was functional because the selective TRPV4 agonist 4-alpha-phorbol 12,13-didecanoate (4alphaPDD) activated an outwardly rectifying cation current with biophysical and pharmacological properties that overlapped those of recombinant human TRPV4 expressed in COS cells. Moreover, 4alphaPDD and hypotonic challenge promoted [Ca(2+)](i) elevation mediated by influx of extracellular Ca(2+). This effect was abolished by low micromolar concentration of the TRPV4 inhibitor Ruthenium Red. Immunofluorescence and immunogold electron microscopy of rat brain revealed that TRPV4 was enriched in astrocytic processes of the superficial layers of the neocortex and in astrocyte end feet facing pia and blood vessels. Collectively, these data indicate that cultured cortical astroglia express functional TRPV4 channels. They also demonstrate that TRPV4 is particularly abundant in astrocytic membranes at the interface between brain and extracerebral liquid spaces. Consistent with its roles in other tissues, these results support the view that TRPV4 might participate in astroglial osmosensation and thus play a key role in brain volume homeostasis.

Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice

The aquaporin-4 (AQP4) pool in the perivascular astrocyte membranes has been shown to be critically involved in the formation and dissolution of brain edema. Cerebral edema is a major cause of morbidity and mortality in stroke. It is therefore essential to know whether the perivascular pool of AQP4 is up- or down-regulated after an ischemic insult, because such changes would determine the time course of edema formation. Here we demonstrate by quantitative immunogold cytochemistry that the ischemic striatum and neocortex show distinct patterns of AQP4 expression in the reperfusion phase after 90 min of middle cerebral artery occlusion. The striatal core displays a loss of perivascular AQP4 at 24 hr of reperfusion with no sign of subsequent recovery. The most affected part of the cortex also exhibits loss of perivascular AQP4. This loss is of magnitude similar to that of the striatal core, but it shows a partial recovery toward 72 hr of reperfusion. By freeze fracture we show that the loss of perivascular AQP4 is associated with the disappearance of the square lattices of particles that normally are distinct features of the perivascular astrocyte membrane. The cortical border zone differs from the central part of the ischemic lesion by showing no loss of perivascular AQP4 at 24 hr of reperfusion but rather a slight increase. These data indicate that the size of the AQP4 pool that controls the exchange of fluid between brain and blood during edema formation and dissolution is subject to large and region-specific changes in the reperfusion phase.