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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 hyponatremia¹

MAHMOOD AMIRY-MOGHADDAM^*, RONG XUE, FINN-MOGENS HAUG^*, JOHN D. NEELY^¶, ANISH BHARDWAJ, PETER AGRE^¶, MARVIN E. ADAMS, STANLEY C. FROEHNER, SUSUMU MORI and OLE P. OTTERSEN^*,2

^* Centre for Molecular Biology and Neuroscience, and Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo, Norway; Departments of
Physiology and Biophysics, University of Washington, Seattle, Washington, USA; and Departments of
Radiology,
Neurology, and
^¶ Biological Chemistry and Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

²Correspondence: Centre for Molecular Biology and Neuroscience, University of Oslo, P.O. Box 1105, Blindern, N-0317 Oslo, Norway. E-mail: o.p.ottersen{at}basalmed.uio.no

SPECIFIC AIMS

There is consensus that rapid water transport across cell membranes is mediated by a family of membrane proteins called aquaporins. The predominant aquaporin in the brain is AQP4, and several studies suggest that this aquaporin provides an access route for water influx during brain edema formation. The extent of brain edema after an experimental stroke is reduced in AQP4 knockout animals.

The primary aim of this study was to resolve how AQP4 is expressed at the interface between blood and brain (the likely site of water entry in pathological conditions) and to identify the pool of AQP4 that is rate limiting for water influx in a well-established model of brain edema (acute hyponatremia). Knowledge of these issues is essential for the development of novel and more rational therapies. Current treatment for brain edema is much the same as 70 years ago, and new principles for treatment are needed.

If water entry in brain occurs through specialized water channels rather than by mere diffusion through the plasma membrane, the prediction would be that the time course of edema development differs between different brain regions. Thus, previous studies suggest that the predominant brain aquaporin, AQP4, is heterogeneously distributed. A secondary aim of the present study was to address this prediction by recording apparent diffusion coefficients in three separate brain structures: the cerebral cortex, striatum, and cerebellum. To our knowledge, this is the first attempt to resolve whether edema develops at different rates in different parts of the brain—an issue of clinical significance.

PRINCIPAL FINDINGS

1. Immunogold analysis confirms -syntrophin-dependent enrichment of AQP4 in perivascular endfeet and demonstrates a novel pool of AQP4 in endothelial cells
Quantitative immunogold analysis of wild-type animals revealed a high concentration of AQP4 in perivascular endfeet. Consistent with earlier reports, labeling for AQP4 was much stronger in the endfoot membrane abutting on the basal lamina than in the endfoot membrane facing the neuropil. In animals lacking -syntrophin (-syn-/- mice), most of the AQP4 was lost from the former membrane whereas the latter membrane displayed a slightly increased AQP4 immunogold signal.

Gold particles signaling AQP4 were associated with the adluminal and abluminal membranes of the endothelial cells. The latter membrane contained about twice as many particles per unit length than the former. Nevertheless, labeling intensity of the abluminal membrane was only 20% of that in the perivascular membranes of astrocytes, which probably explains why the endothelial pool of AQP4 has escaped detection in previous studies. The endothelial pool of AQP4 was resistant to -syntrophin deletion (Fig. 1 ).

View larger version (163K): [in this window] [in a new window]   Figure 1. Electron micrographs showing immunogold labeling for AQP4 around vessels in the molecular (A, B) and granule cell layers (C, D) of the cerebellum. A, C) wild-type animals; B, D) -syntrophin deletion. Perivascular membranes in the granule cell layer (C) contain a higher density of AQP4 immunogold particles than perivascular membranes in the molecular layer (A) and retain a substantial amount of immunoreactivity after deletion of -syntrophin (D). The deletion causes an enhanced labeling of nonperivascular membranes that is apparent only in the granule cell layer (D). AQP4 immunogold particles occur in endothelial cells, with higher densities in the abluminal membrane than adluminally. Some particles decorate intracellular membrane domains. Endothelial labeling is insensitive to -syntrophin deletion. Twin arrows, perivascular membrane; single arrows, abluminal membrane of the endothelial cell; arrowheads, adluminal (apical) membrane of the endothelial cell; P, pericyte; E, endothelial cells; L, vessel lumen; *basal lamina on the luminal side of the pericytes; **basal lamina interposed between pericyte and perivascular endfeet. Bars: 500 nm.

The above findings distinguish the brain endothelial cells from all other endothelial cells in the body. Only the brain endothelial cells have been found to contain AQP4; other endothelial cells express AQP1.

2. Distribution of AQP4 and rate of edema development show regional heterogeneities
The general level of AQP4 was higher in the cerebellum than in the neocortex or striatum. The neocortex and cerebellum also displayed very different distributions of this water channel. The former structure showed higher concentrations superficially just underneath the pia than in the deep layers, whereas the cerebellum displayed the inverse gradient with more AQP4 in the granule cell layer than in the molecular layer. The intracortical gradients reflected differences in the labeling intensities of the perivascular as well as of the non-endfeet membranes.

Regional differences in AQP4 levels and distribution matched differences in the rate of development of brain edema as assessed by diffusion weighted MR. The apparent diffusion coefficient (ADC) showed a more abrupt decrease in the cerebellum than in the neocortex or striatum. This suggests that water influx occurs more rapidly in the cerebellum than in the other structures investigated (Fig. 2 ).

View larger version (44K): [in this window] [in a new window]   Figure 2. Schematic diagram showing effect of -syntrophin deletion on subcellular distribution of AQP4 at the blood–brain interface (left) and on the time course of edema development after induction of acute hyponatremia (right). Edema development was assessed by diffusion-weighted MRI and the signal recorded was the apparent diffusion coefficient (ADC). Hatched line indicates time when the homeostatic mechanisms break down, giving way to a fulminant edema. Judged by these criteria, the onset of edema is delayed by 15 min in animals that lack perivascular AQP4 but retain endothelial AQP4. Data on the right are obtained from 3 wild-type animals and 3 -syn–/– animals. WT, wild-type animals; SYN–/–, -syntrophin null mice.

3. Deletion of -syntrophin abolishes regional differences in edema development and uniformly protracts the time course of water accumulation
In the -syn-/- animals the ADC signal developed with the same time course in all regions. Compared with the wild-type animals, the plateau phase of the ADC signal was significantly prolonged (by 15 min), suggesting a reduced speed of water influx (Fig. 2) .

CONCLUSIONS AND SIGNIFICANCE

The present study has shown there are three distinct and serially coupled pools of AQP4 at the brain–blood interface. The largest of these pools has been demonstrated before: it resides in the perivascular membrane of astrocytes and is dependent on -syntrophin. The remaining two pools are expressed by endothelial cells and occur in the ad- and abluminal membranes. These pools are resistant to -syntrophin deletion. The finding that edema development is delayed in -syn-/- animals indicates that the perivascular membrane limits water influx in these animals. Thus, water influx is gated by a membrane distinct from the blood–brain barrier.

The data reported here have served to identify the AQP4 pools and membranes involved in water uptake in pathological conditions and suggest that the perivascular endfeet may assume rate-limiting properties even when the endothelial pool of AQP4 is intact. This calls for a more diversified concept of the blood–brain barrier and points to the important role of astrocytes for regulating not only the neuronal microenvironment, but also the large-scale exchange of water between blood and brain.

The novel observation, that there are substantial regional differences in the time course of edema development, may be attributed to regional differences in AQP4 levels and distribution. That the regional differences in ADC pattern were abolished in -syn-/- animals (which are depleted of perivascular AQP4) supports this view.

The present findings hold promise for the development of new therapies against brain edema. For example, delivery to the perivascular endfeet of peptides that interfere with AQP4 anchoring to -syntrophin could significantly curtail the influx of water in pathological conditions. Such treatment is within reach, given the recent development of techniques for transendothelial gene transfer to brain astrocytes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0869fje;

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