Translational readthrough (TRT) of aquaporin-4 (AQP4) has remarkably expanded the importance of this new post-transcriptional mechanism, as well as the regulation potential of AQP4. The TRT isoform of AQP4, named AQP4ex, is central for both AQP4 polarization and water channel activity in the central nervous system (CNS). Here we evaluate the relevance of the TRT mechanism by analyzing whether AQP4ex is also expressed in peripheral tissues and whether the expression of AQP4ex is necessary for its polarized expression as it occurs in perivascular astrocyte processes. To this purpose, AQP4ex null mice were used, and analysis was performed by immunolocalization and immunoblot. The results demonstrate that AQP4ex is expressed in kidney, stomach, trachea and skeletal muscle with the same localization pattern as the canonical AQP4 isoforms. AQP4ex protein levels vary from 6% to about 13% of the total AQP4 protein levels in peripheral tissues. Immunogold electron microscopy experiments demonstrated the localization of AQP4ex at the astrocytic endfeet, and experiments conducted on AQP4ex null mice CNS confirmed that the expression of AQP4ex is necessary for anchoring of the perivascular AQP4. Without the readthrough isoform, AQP4 assemblies are mis-localized, being uniformly distributed on the astrocyte processes facing the neuropile. No alteration of AQP4 polarization was found in AQP4ex null kidney, stomach, trachea or skeletal muscle, suggesting that AQP4ex does not have a role for proper membrane localization of AQP4 in peripheral tissues. We conclude that a dual role for AQP4ex is limited to the CNS.

AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical isoforms M1 and M23 of AQP4. Previous site-directed mutagenesis experiments suggested the potential role of serine³³¹ and serine³³⁵, located in the extended peptide of AQP4ex, in water channel activity by phosphorylation. In the present study we evaluated the effective phosphorylation of human AQP4ex. A small scale bioinformatic analysis indicated that only Ser³³⁵ is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser³³⁵ was assessed through generation of phospho-specific antibodies in rabbits. Antibody specificity was first evaluated in binding phosphorylated peptide versus its unphosphorylated analog by ELISA, which was further confirmed by site-directed mutagenesis experiments. Western blot and immunofluorescence experiments revealed strong expression of phosphorylated AQP4ex (p-AQP4ex) in human brain and localization at the perivascular astrocyte endfeet in supramolecular assemblies identified by BN/PAGE experiments. All together, these data reveal, for the first time, the existence of a phosphorylated form of AQP4, at Ser³³⁵ in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of p-AQP4ex in human brain water homeostasis.

The choroid plexus (ChP) is a complex ventricular structure that forms a semi-permeable barrier between the blood and cerebrospinal fluid (CSF). It is responsible for CSF secretion and clearance, contains macrophages, and is one of the few sites within the central nervous system (CNS) where T cells are present. Additionally, the ChP plays a role in detecting peripheral inflammation, which leads to the modulation of its epithelial cell function. Despite its critical importance in maintaining brain homeostasis, the ChP is often overlooked, particularly concerning the changes it undergoes with aging, such as reduced barrier function, impaired CSF production, and altered immunosurveillance. These age-related alterations may contribute to several harmful effects, including neuroinflammation and oxidative damage, potentially predisposing individuals to neurodegenerative conditions. Although knowledge is still limited, gut dysbiosis and decreased Klotho levels—of which the ChP is one of the main sources—appear to be significant contributors to ChP impairments. This narrative review will examine the impact of age-related gut dysbiosis on the CNS, focusing on the ChP, and explore the effects of reduced Klotho levels in this brain structure. We will also propose the hypothesis that combining the administration of probiotics capable of restoring gut microbiota eubiosis with gene therapy to upregulate Klotho in the ChP could help preserve the structural and functional integrity of the aging brain. Finally, we will provide a technical overview to ensure that vectors encoding Klotho cDNA achieve maximum specificity for the ChP, thereby avoiding off-target effects. Graphical abstract

Water deprivation triggers coordinated physiological responses to preserve body fluid balance, yet the molecular mechanisms that regulate aquaporin-mediated water transport under dehydration remain incompletely understood. Aquaporin-4 (AQP4), the main water channel in the brain and a basolateral water pathway in the kidney collecting duct, exists in multiple isoforms, including the translational readthrough variant AQP4ex, whose regulatory role is only beginning to be defined. Here, we investigated the effects of acute water deprivation (6–12 h) on AQP4 isoform expression and phosphorylation in a mouse kidney and brain. While total AQP4 and AQP4ex protein levels remained largely unchanged in both tissues, dehydration induced a marked and divergent regulation of the phosphorylated form of AQP4ex. Levels increased in the kidney medulla, consistent with enhanced antidiuretic water transport, but decreased in the cerebral cortex, suggesting a protective reduction in perivascular water permeability. No changes were detected in the cerebellum. These findings identify phosphorylation of AQP4ex as a rapid, tissue-specific regulatory mechanism that adjusts water flux according to the physiological needs of each organ, revealing an additional layer of control in systemic water homeostasis and highlighting AQP4ex as a potential target in dehydration-related and osmotic disorders. Future studies could explore the signaling pathways regulating AQP4ex phosphorylation and investigate its potential involvement in pathological conditions, such as diabetes insipidus or cerebral edema.