Orthogonal arrays of particle assembly are essential for normal aquaporin-4 expression level in the brain

Astrocyte endfeet are endowed with aquaporin-4 (AQP4)-based assemblies called orthogonal arrays of particles (OAPs) whose function is still unclear. To investigate the function of OAPs and of AQP4 tetramers, we have generated a novel “OAP-null” mouse model selectively lacking the OAP forming M23-AQP4 isoform. We demonstrated that AQP4 transcript levels were not reduced by using qPCR. Blue native (BN)/SDS-PAGE and Western blot performed on OAP-null brain and primary astrocyte cultures showed the complete depletion of AQP4 assemblies, the selective expression of M1-AQP4-based tetramers, and a substantial reduction in AQP4 total expression level. Fluorescence quenching and super-resolution microscopy experiments showed that AQP4 tetramers were functionally expressed in astrocyte plasma membrane and their dimensions were reduced compared to wild-type assemblies. Finally, as shown by light and electron microscopy, OAP depletion resulted in a massive reduction in AQP4 expression and a loss of perivascular AQP4 staining at astrocyte endfeet, with only sparse labeling throughout the brain areas analyzed. Our study relies on the unique property of AQP4 to form OAPs, using a novel OAP-null mouse model for the first time, to show that (a) AQP4 assembly is essential for normal AQP4 expression level in the brain and (b) most of AQP4 is organized into OAPs under physiological conditions. Therefore, AQP4 tetramers cannot be used by astrocytes as an alternative to OAPs without affecting AQP4 expression levels, which is important in the physiological and pathological conditions in which OAP aggregation/disaggregation dynamics have been implicated.