Regulation of water homeostasis is a central feature of central nervous system pathophysiology. In this context, several lines of evidence suggest a crucial role for the water channel aquaporin-4 (AQP4) and its plasma membrane supramolecular organization as the key element. Here, we demonstrate the expression in tissues of additional isoforms of AQP4 characterized by a C-terminal extension generated by programmed translational readthrough. These extended isoforms (AQP4ex) display a perivascular polarization and expression in dystrophin-dependent pools. AQP4ex reduces supramolecular clustering tendency and allows AQP4 interactions with syntrophin. Furthermore, site-directed mutagenesis of two serines in the extended C-terminus of AQP4ex showed potential regulation of water permeability by phosphorylation. Finally, AQP4ex expression can be positively modulated by gentamicin treatment, demonstrating the possibility of regulating the AQP4 translational readthrough frequency. This novel regulatory mechanism could have important pathophysiological implications for conditions in which alternations have been reported in AQP4 structure.

The glial water channel protein aquaporin-4 (AQP4) forms heterotetramers in the plasma membrane made of the M23-AQP4 and M1-AQP4 isoforms. The isoform ratio controls AQP4 aggregation into supramolecular structures called orthogonal arrays of particles (AQP4-OAP). The role of AQP4 aggregation into OAP in malignant gliomas is still unclear. In this study, we demonstrate that AQP4 aggregation/disaggregation into OAP influences the biology of glioma cells. Selective expression of the OAP-forming isoform M23-AQP4 (AQP4-OAP) triggered cell shape changes in glioma cells associated with alterations to the F-actin cytoskeleton that affected apoptosis. By contrast, expression of M1-AQP4 (AQP4-tetramers), which is unable to aggregate into OAP, ameliorated glioma cell invasiveness, improved cell migration, and increased methalloproteinase-9 activity. Two prolines (254 and 296) at the C-terminus tail were shown to be important in mediating the relationship between the actin cytoskeleton and AQP4-OAP and AQP4-tetramers. In conclusion, this study demonstrates that AQP4 aggregation state might be an important determinant in orienting glioma cells to persist or perish. AQP4 disaggregation may potentiate invasiveness potential, whereas AQP4 aggregation may activate the apoptotic path. This study shows a new perspective on the role of AQP4 in brain tumors not necessarily associated with edema formation but with AQP4 aggregation/disaggregation dynamics and their link with the actin cytoskeleton.Significance: This study demonstrates how AQP4 aggregation influences plasma membrane dynamics to alter cell proliferation, invasiveness, migration, and apoptotic potential in glioma cells.

An alternatively spliced transcript of human AQP4 that lacks exon 4 is identified. In transfected cells, AQP4-Δ4 shows no water transport properties, is retained in the ER, and has a dominant-negative effect on full-length AQP4. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein in different muscles.

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.

Astrocytes are non‐neuronal cells that govern the homeostatic regulation of the brain through ions and water transport, and Ca2+‐mediated signaling. As they are tightly integrated into neural networks, label‐free tools that can modulate cell function are needed to evaluate the role of astrocytes in brain physiology and dysfunction. Using live‐cell fluorescence imaging, pharmacology, electrophysiology, and genetic manipulation, we show that pulsed infrared light can modulate astrocyte function through changes in intracellular Ca2+ and water dynamics, providing unique mechanistic insight into the effect of pulsed infrared laser light on astroglial cells. Water transport is activated and, IP3R, TRPA1, TRPV4, and Aquaporin‐4 are all involved in shaping the dynamics of infrared pulse‐evoked intracellular calcium signal. These results demonstrate that astrocyte function can be modulated with infrared light. We expect that targeted control over calcium dynamics and water transport will help to study the crucial role of astrocytes in edema, ischemia, glioma progression, stroke, and epilepsy.