Tissue Distribution of the Readthrough Isoform of AQP4 Reveals a Dual Role of AQP4ex Limited to CNS

Claudia Palazzo,Maria Trojano,Pasqua Abbrescia,Antonio Frigeri,Onofrio Valente,Grazia Paola Nicchia,Shervin Banitalebi,Mahmood Amiry-Moghaddam

doi:10.3390/ijms21041531

Abstract

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.

Highlights

Aquaporins (AQPs) are a family of membrane water transporting proteins that facilitate water movement across the cell plasma membrane in the presence of hydrostatic and/or osmotic gradients [1]
The recent discovery of AQP4ex as a translational readthrough isoform of AQP4 has remarkably expanded the importance of this new post-transcriptional mechanism, as well as the regulation capabilities of AQP4
We have investigated whether the readthrough is a generic mechanism or restricted to the central nervous system (CNS)

Summary

Introduction

Aquaporins (AQPs) are a family of membrane water transporting proteins that facilitate water movement across the cell plasma membrane in the presence of hydrostatic and/or osmotic gradients [1]. AQPs generally form homotetramers in the cell plasma membrane, whereas in the case of AQP4, the M1- and M23 isoforms form heterotetramers [6], which further aggregate in the plasma membrane to form well-ordered two-dimensional supramolecular structures, called orthogonal arrays of particles (OAPs), observable by freeze fracture electron microscopy (FFEM) [7,8]. Both AQP4 isoforms function as water channels, their relative abundance is tissue-specific and affects binding of pathogenic neuromyelitis optica antibodies [9]. AQP4ex is necessary to generate the NMO-IgG epitope in mice

Methods

Results

Conclusion