Removal of aquaporin-4 from glial and ependymal membranes causes brain water accumulation

Abstract

There is a constitutive production of water in brain. The efflux routes of this excess water remain to be identified. We used basal brain water content as a proxy for the capacity of water exit routes. Basal brain water content was increased in mice with a complete loss of aquaporin-4 (AQP4) water channels (global Aqp4^-/- mice), but not in mice with a selective removal of perivascular AQP4 or in a novel mouse line with a selective deletion of ependymal AQP4 (Foxj1-Cre:Aqp4^flox/flox mice). Unique for the global Aqp4^-/- mice is the loss of the AQP4 pool subjacent to the pial membrane. Our data suggest that water accumulates in brain when subpial AQP4 is missing, pointing to a critical role of this pool of water channels in brain water exit.

Keywords: AQP4; Alpha-syntrophin; Aquaporin; Astrocytes; Brain edema; CSF; Cerebrospinal fluid; Endfeet; Ependyma; Extracellular space; Foxj1; Glia; Glymphatic; Interstitial fluid; Neuron-glial; Paravascular; Water homeostasis.

Figure 1

Effect of gene knockout on AQP4 distribution. AQP4 immunofluorence labeling (red) of the cortex (A-E), periventricular area (F-J; nuclear labeling in blue) and choroid plexus (K-O; nuclear labeling in blue) of wild-type (A, F, K), global Aqp4^−/− mice (B, G, L), α-syntrophin^−/− mice (C, H, M), homozygous Aqp4 floxed mice (“Aqp4^f/f”) (D, I, N) and ependymal-conditional Aqp4^−/− mice (”Foxj1-Cre:Aqp4^f/f mice”) (E, J, O). A, F, K: In wild-type mice AQP4 labeling is most pronounced around cortical blood vessels (arrowhead; same vessel shown at higher magnification in inset) and along the pial surface (double arrowhead). Distinct AQP4 immunolabeling is evident in the basolateral membrane (arrow) of the ependyma covering the third ventricle (asterisk). The choroid plexus epithelium is devoid of AQP4 immunolabeling. B, G, L: Global Aqp4 deletion abolishes AQP4 immunolabeling. Labels as in A, F. C, H, M: In α-syntrophin^−/− mice AQP4 labeling is lost around cortical blood vessels but still prominent along the pial surface and in the basolateral ependymal membrane. Labels as in A, F. D, I, N: AQP4 labeling in Aqp4^f/f mice is indiscriminately from that of wild-types. Labels as in A, F. E, J, O: In Foxj1-Cre:Aqp4^f/f mice the AQP4 immunofluorescence signal is lost from ependymal membranes but otherwise similar to that of litter controls (Aqp4^f/fl mice) and wild-types. Labels as in A,F. Scale bars: 20 µm.

Figure 2

Ependymal-conditional Aqp4^−/− mice do not reveal apparent abnormalities in the ependymal architecture. A–H: Hematoxylin-eosin staining of coronal brain sections revealed comparable morphology of the ependyma (arrowheads) lining the third (3^rd) and lateral (L) ventricles in control (“Aqp4^f/f”; A, C, E and F) and ependymal-conditional Aqp4^−/− (”Foxj1-Cre:Aqp4^f/f”; B, D, G and H) mice. Boxed areas in A, B, E and G are enlarged in C, D, F and H, respectively. CA, commisura anterior. Scale bars: A, B: 100 µm; C, D: 10 µm; E, G: 1 mm; F, H: 30 µm.

Figure 3

Impact of gene deletion on basal brain water content and brain wet weight. A, B: In Aqp4^−/− mice (n=10) basal brain water content and brain wet weight are significantly elevated compared to wild-types (n=10; p values are indicated, unpaired t test). C, D: Neither α-syntrophin^−/− mice (n=12) nor Foxj1-Cre:Aqp4^f/f mice (n=10) differed significantly from their controls (n=10 and n=5, respectively) in basal brain water content (p>0.05 for both comparisons, unpaired t test).

Figure 4

Effect of intracisternal infusion of aCSF on ICP. Infusion of aCSF at 2 µl/min for 5 min (infusion period indicated by bar) elevates ICP in both Aqp4^−/− and wild-types. Neither peak ICP increase nor ICP decay differed between the genotypes (p>0.05, unpaired t test for both comparisons).