The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain

Abstract

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.

Connecting the Dots: The Cerebral Lymphatic System as a Bridge Between the Central Nervous System and Peripheral System in Health and Disease

“…The astrocyte endfeet are special subcellular structures emanating from astrocytes, which are distributed with a large amount of aquaporin-4 [ 59 ]. They play an important role in regulating brain homeostasis, maintaining the integrity of the blood-brain barrier and removing waste solutes [ 60 , 61 ]. Numerous studies have shown that astrocytes are an important component of the cerebral lymphatic system [ 62 , 63 ].…”

“…In fact, numerous studies have shown that AQP-4 on the astrocyte endfeet promotes fluid flow between the cerebrospinal fluid and interstitial fluid [ 3 , 21 , 65 , 68 , 69 ]. Yifan Wang et al found that a decrease in AQP-4 may likewise disrupt the normal structure and function of astrocytes, leading to an impaired establishment of a reactive astrocytic network [ 21 , 61 , 65 , 68 , 70 ]. Nadia Aalling Jessen and Kazuhisa Ishida et al demonstrated that the drainage of cerebrospinal fluid was severely impaired in AQP-4 knockout mice compared to wild group mice.…”

Aquaporin-4 and Parkinson’s Disease

“…There are two main AQP4 isoforms: M23 and M1 [98][99][100]. An alternative translational read-through mechanism provides the possibility of producing two extended AQP4 iso-forms, M23ex and M1ex, in humans, rats, and mice [100][101][102]. These isoforms are thought to be modulators of function and assembly of OAPs [100].…”

In Vitro Modeling of the Blood–Brain Barrier for the Study of Physiological Conditions and Alzheimer’s Disease

“…An important aspect related to the astrocytes which are part of the BBB is their role in the osmotic equilibrium. At the astrocyte’s endfeet, specialized molecules such as aquaporin-4 (AQP-4) are the key players in regulating the water and ionic balance at the CNS level [ 52 ]. This has also diagnostic and therapeutic consequences: the detection of antibodies to AQP-4 is a highly suggestive marker for the autoimmune disorder neuromyelitis optica [ 53 ], while AQP-4 is recently considered a valuable target in treating cerebral edema [ 54 ].…”