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

Abstract

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.

Regulation of aquaporin‐4 expression in the central nervous system investigated using M23‐AQP4 null mouse

“…We have already shown that total AQP4 mRNA level is unchanged between WT and M23-null brain (de Bellis et al, 2020). Those data were obtained using qRT-PCR primers specific for the exon-junction II-III and for Exon IV.…”

“…The animal model used here was already published (de Bellis et al, 2020 ). OAP‐null mice harboring the M23I point mutation were generated on the C57BL/6J background by Cyagen Biosciences (Santa Clara, CA) using CRISPR/Cas9‐based targeting and homology‐directed repair.…”

“…The primary antibodies used were: goat polyclonal anti‐AQP4 (C‐19) (sc‐9888) and rabbit polyclonal anti‐AQP4 (H‐80) (sc‐20812) (Santa Cruz Biotechnology) both diluted 1:400 for immunofluorescence and 1:500 for immunoblot analysis; rat monoclonal anti‐CD31 at 1:100 (BD Bioscience, 550274) for immunofluorescence (GFAP); mouse monoclonal anti‐DDX17 (C‐9) (Santa Cruz Biotechnology, sc‐271112) diluted 1:100 for immunofluorescence and immunoblot analysis; rabbit anti‐DDX17 (Sigma Aldrich, SAB2100550) 1:500 in immunoblotting, mouse anti‐GAPDH (Merck Millipore, MAB374) at 1:2000; rabbit polyclonal anti‐actin (Sigma Aldrich, A2066) at 1:500; Goat anti‐polypyrimidine tract binding protein 1 (PTBP1) (Sigma Aldrich, SAB2500834) at 1:1000. An affinity‐purified rabbit polyclonal antibody to a M1‐AQP4 specific sequence was prepared by Gen‐Script and diluted 1:1000 for immunoblot analysis (de Bellis et al, 2020 ). The following secondary antibodies were used for Western blot diluted to 1:5000: donkey anti‐goat IgG‐horse‐radish peroxidase (HRP); goat anti‐rabbit IgG‐HRP (Santa Cruz Biotechnology) and goat anti‐mouse IgG‐HRP (Bio‐Rad).…”

“…Calcein fluorescence signal was recorded on a Flex Station3 plate reader equipped with a liquid handling module (Molecular Devices, San Jose, CA) able to analyze real‐time fluorescence kinetic data in the 96‐well format (de Bellis et al, 2020 ). Fluorescence was excited at 490 nm and detected at 520 nm using dual monochromators.…”

“…We have reported that in M23-null mice OAPs are absent, the tetrameric form of AQP4 is upregulated and the M1-AQP4 protein expression is strongly downregulated. This demonstrates that M23-AQP4 is pivotal to preserve the normal AQP4 expression level in the CNS (de Bellis et al, 2020). In the present work, we have analyzed the same animal model with the aim of exploring the mechanism by which M23-AQP4 deletion controls AQP4 expression.…”