Uncoupling of the Astrocyte Syncytium Differentially Affects AQP4 Isoforms

Abstract

The water channel protein aquaporin-4 (AQP4) and the gap junction forming proteins connexin-43 (Cx43) and connexin-30 (Cx30) are astrocytic proteins critically involved in brain water and ion homeostasis. While AQP4 is mainly involved in water flux across the astrocytic endfeet membranes, astrocytic gap junctions provide syncytial coupling allowing intercellular exchange of water, ions, and other molecules. We have previously shown that mice with targeted deletion of Aqp4 display enhanced gap junctional coupling between astrocytes. Here, we investigate whether uncoupling of the astrocytic syncytium by deletion of the astrocytic connexins Cx43 and Cx30 affects AQP4 membrane localization and expression. By using quantitative immunogold cytochemistry, we show that deletion of astrocytic connexins leads to a substantial reduction of perivascular AQP4, concomitant with a down-regulation of total AQP4 protein and mRNA. Isoform expression analysis shows that while the level of the predominant AQP4 M23 isoform is reduced in Cx43/Cx30 double deficient hippocampal astrocytes, the levels of M1, and the alternative translation AQP4ex isoform protein levels are increased. These findings reveal a complex interdependence between AQP4 and connexins, which are both significantly involved in homeostatic functions and astrogliopathologies.

Blockade of 67-kDa Laminin Receptor Facilitates AQP4 Down-Regulation and BBB Disruption via ERK1/2-and p38 MAPK-Mediated PI3K/AKT Activations

“…AQP4ex also regulates water transport at the BBB level, and binds with human neuromyelitis optica autoantibody that is associated with autoimmune demyelinating diseases in the central nervous system [67]. In addition, the deletion of gap junction forming proteins connexin-43 (Cx43) and connexin-30 (Cx30) increases AQP4a (M1) and AQP4ex isoform levels, but reduces AQP4c (M23) isoform in astrocytes [68]. Considering these reports, further studies are needed to validate AQP4 isoforms affected by 67LR functions in astroglio-vascular interactions.…”

Astrocytes in the regulation of cerebrovascular functions

“…Dual knock‐out of Cx30 and Cx43 uncouples astrocytes in the hippocampus, reduces water transport across astrocytic plasma membranes, and increases the leakiness of the BBB upon shear stress (Ezan et al, 2012; Lutz et al, 2009). This dual knock‐out is accompanied with the partial loss of β‐dystroglycan and Aqp4M23 (but not Aqp4M1 and Aqp4ex) (Ezan et al, 2012; Katoozi et al, 2020). Taken individually, each astroglial connexin has very distinct functions at the gliovascular interface.…”

Generation and Characterization of Immortalized Mouse Cortical Astrocytes From Wildtype and Connexin43 Knockout Mice

“…In addition to the effect of endothelial cell expression on astrocyte expression, the finding in Figures 1 , 2 indicating decreasing expression of AQP4 across passage number, mean that verification of AQP4 expression level will be important for future use of the immortalized astrocyte lines described here. Future studies that examine the effect of Cx43 expression and functional outcomes will need to test the level and cellular localization of AQP4 because a direct interaction between Cx43 and AQP4 has been shown in past research ( Rash and Yasumura, 1999 ; Nicchia et al, 2005 ; Katoozi et al, 2017 , 2020 ). Prior studies have shown that IL-1β, IL-1α, NFκB, and other factors produce important changes to gene expression, morphology and GJ connectivity in vivo and in primary astrocyte cultures.…”