AQP4ex is crucial for the anchoring of AQP4 at the astrocyte end-feet and for neuromyelitis optica antibody binding

Abstract

Brain water homeostasis is essential for the appropriate control of neuronal activity. Furthermore, the encasement of the central nervous system (CNS) by a hard structure, greatly limits its tolerance for the volume changes occurring with acute brain edema, which quickly leads to severe damage or death. The recent discovery of the extended isoform of AQP4 (AQP4ex), generated by translational readthrough, revealed a potential new mechanism of water transport regulation and polarization at the blood-brain-barrier level. In the present study we used CRISPR/Cas9 technology to generate an AQP4ex −/− mouse model and evaluate the effect on the overall AQP4 expression, polarization, supramolecular organization in orthogonal arrays of particles (OAPs) and neuromyelitis optica (NMO-IgG) autoantibodies binding. AQP4ex removal did not cause a decrease in total AQP4 protein expression but completely suppressed the specific location of AQP4 at the astrocyte endfeet. Without AQP4ex, AQP4 was mislocalized and α-syntrophin expression, the selective partner for AQP4 localization, was partially altered. The supramolecular organization of AQP4 in OAPs was subtly altered. Indeed, the absence of AQP4ex reduced the size of AQP4-OAPs but the number of AQP4-OAP pools remained largely the same. More importantly, AQP4ex resulted critical for the binding of pathogenic human NMO-IgG autoantibodies to the brain. Indeed, the absence of AQP4ex completely abolished the binding of NMO-IgG at the perivascular astrocyte endfeet. This study provides the first direct evidence in vivo on the specific role of AQP4ex in AQP4 perivascular OAPs assembly and confinement and reveals AQP4ex as new and important player in neuromyelitis optica.

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

“…In the absence of the extended isoforms, the filamentous staining in the spinal cord white matter was still intensely observable; furthermore, the perivascular signal from AQP4ex observed in WT tissues was totally abolished. These data confirm that suppressing AQP4ex expression totally eliminates the perivascular staining of AQP4, confirming, as found in other CNS areas [16], that AQP4ex expression is fundamental for the expression of AQP4 at the perivascular interface. Immunoblot experiments were performed ( Figure 6) to evaluate quantitatively the expression levels of AQP4ex.…”

“…We also evaluated the distribution of AQP4ex and AQP4 in the CNS, analyzing cerebrum, cerebellum and spinal cord ( Figure 5). Most of the AQP4ex staining was confined, as recently reported [16], to the pericapillary astrocyte endfeet of the cerebral cortex ( Figure 5A) and at the perivascular astrocyte processes in the granule cell layer (gcl), with an additional staining of the dense network of astrocyte processes of the cerebellum gcl ( Figure 5E). In the spinal cord, AQP4ex was highly expressed in both white and gray matter ( Figure 5I).…”

“…Immunoblot experiments were performed ( Figure 6) to evaluate quantitatively the expression levels of AQP4ex. A major band of~35 kDa, corresponding to AQP4-M23ex, was detected in WT samples ( Figure 6A) but not in the AQP4ex knockout mice extracts, as reported in the CNS [16]. The results were confirmed by probing the blot membranes with the commercial AQP4 antibody that recognizes all AQP4 isoforms.…”

“…Moreover, phosphorylation of residues Ser331 and Ser335 of the extended isoforms seems to play an important role in the short-term regulation of channel gating and water permeability [14]. Recently, to evaluate the functional role of AQP4ex we generated a transgenic mouse model in which AQP4ex is completely abolished using the CRISPR-Cas9 technique [16]. AQP4ex-KO mice revealed that AQP4ex is essential for the localization of AQP4 at the perivascular astrocytic membrane domains, even if large OAPs made of M1 and M23 canonical isoforms are still expressed.…”

Upregulation of large myelin protein zero leads to Charcot–Marie–Tooth disease-like neuropathy in mice

“…Thus, translational readthrough may be a common physiological mechanism for translational regulation in mammals including humans 20 . Interestingly, VEGF-Ax has anti-angiogenic or weaker angiogenic activity compared with canonical VEGF 18,21 , while AQP4ex displays a crucial role for the anchoring of canonical AQP4 at the astrocyte end-feet and potential regulation of water permeability 19,22 . Together, these findings suggest that quantitative changes of translational readthrough isoforms affect the function of canonical molecules.…”