Endfoot targeting of AQP4ex is the determining factor of the anchoring of AQP4 water channel molecules at the brain-blood interface

AQP4 plays a central role in the preservation of the CNS water homeostasis, essential for the maintenance of osmotic composition and volume within the glial and neuronal compartments. The recent discovery of the extended isoform of AQP4 (AQP4ex), generated by translational readthrough, revealed a potential new mechanism of water transport regulation and polarization at the brain-blood interface. We employed CRISPR/Cas9 technology to generate an AQP4ex-KO mouse model and evaluate the effect on the overall AQP4 expression, polarization, supramolecular organization in orthogonal arrays of particles (OAPs) and neuromyelitis optica (NMO-IgG) autoantibodies binding. In WT mouse, AQP4ex, representing about 10% of all AQP4 isoforms, showed a polarized distribution in the cerebrum mostly confined to the pericapillary astrocyte endfeet. AQP4ex removal completely suppressed the specific location of AQP4 at the astrocyte endfeet and was compensated by an increased expression of the canonical isoforms (M1 and M23) indicating that the KI stop codons tightly work. Without AQP4ex, AQP4 was mislocalized in the brain parenchima, and α-syntrophin expression, the selective partner for AQP4 localization, was partially altered. The supramolecular organization of AQP4 in OAPs was subtly altered. Indeed, the absence of AQP4ex slightly reduced the size of AQP4-OAPs but the number of AQP4-OAPs pools remained largely the same. The absence of AQP4 at the perivascular pole completely abolished the binding of pathogenic human neuromyelitis optica autoantibodies to the brain. This study provides the first direct evidence in vivo on the specific role of AQP4ex in AQP4 perivascular OAP assembly and confinement, as well as its involvement as a structural component of the glial endfoot membrane protein functional unit.