Translational readthrough generates new astrocyte AQP4 isoforms that modulate supramolecular clustering, glial endfeet localization, and water transport

Abstract

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.

Cell type specific profiling of alternative translation identifies novel protein isoforms in the mouse brain

“…S3B, C). Similar results were also recently reported by others with an independently raised antibody in rats 28 . Thus, readthrough confers a conserved perivascular localization signal to AQP4.…”

“…We next wanted to test if readthrough is of functional significance. We focused on AQP4, an astrocyte membrane protein shown to have a readthrough version in other species by previous studies 28,44 . First, to determine if a stable readthrough-extended AQP4 protein (designated AQP4X hereafter) is indeed detectable in the mouse brain, we developed a rabbit polyclonal antibody against AQP4X using a peptide from the readthrough region as an epitope.…”

Response to ‘The evolving mystery of why skeletal muscle is spared in seropositive neuromyelitis optica’

“…We believe that the unquestionable advantage of our study is that it has been performed on tissues expressing endogenous AQP4 with all the players (known and unknown) for AQP4 clustering. One of these players is the recently identified AQP4ex isoform [6], which is strongly expressed in skeletal muscle. As AQP4ex modulates AQP4 cluster size, [6] it may, for example, have a role in the different supramolecular organisation.…”

“…One of these players is the recently identified AQP4ex isoform [6], which is strongly expressed in skeletal muscle. As AQP4ex modulates AQP4 cluster size, [6] it may, for example, have a role in the different supramolecular organisation.…”

“…We believe that the unquestionable advantage of our study is that it has been performed on tissues expressing endogenous AQP4 with all the players (known and unknown) for AQP4 clustering. One of these players is the recently identified AQP4ex isoform [6], which is strongly expressed in skeletal muscle. As AQP4ex modulates AQP4 cluster size, [6] it may, for example, have a role in the different supramolecular organisation.With regard to the concern about the use of non-fixed frozen tissues in our study, it is well-established that the use of unfixed tissue for immunofluorescence is crucial to preserve the conformational epitopes necessary for AQP4-IgG binding [5].While the fascinating mystery of why skeletal muscle is spared in seropositive neuromyelitis optica will certainly benefit from further studies, we believe that a very small piece has been added in this direction here.We would like to thank all involved for the opportunity to continue this fascinating discussion and look forward to hearing from you.Yours sincerely,…”