Alteration of the translational readthrough isoform AQP4ex induces redistribution and downregulation of AQP4 in human glioblastoma

Abstract

Glioblastoma multiforme (GBM) is characterized by a remarkable cellular and molecular heterogeneity that make the behavior of this tumor highly variable and resistant to therapy. In addition, the most serious clinical complication of GBM and other brain tumors is the development of vasogenic edema which dramatically increase the intracranial pressure. In the present study we evaluate the expression, supramolecular organization and spatial distribution of AQP4 and AQP4ex, the new readthrough isoform of AQP4, in relationship with the degree of vasogenic brain edema and tumor progression. To this purpose, tissue samples from regions of tumor core, peritumoral and non-infiltrated tissues of each GBM patient (n = 31) were analyzed. Immunofluorescence experiments revealed that the expression of AQP4ex was almost absent in tumoral regions while the canonical AQP4 isoforms appear mostly delocalized. In peritumoral tissues, AQP4 expression was found altered in those perivascular astrocyte processes where AQP4ex appeared reduced and partially delocalized. Protein expression levels measured by immunoblot showed that global AQP4 was reduced mainly in the tumor core. Notably, the relative amount of AQP4ex was more severely reduced starting from the peritumoral region. BN-PAGE experiments showed that the supramolecular organization of AQP4 is only partially affected in GBM. Edema assessment by magnetic resonance imaging revealed that the level of AQP4ex downregulation correlated with edema severity. Finally, the degree of BBB alteration, measured with sodium fluorescein content in GBM biopsies, correlated with the edema index and AQP4ex downregulation. Altogether these data suggest that the AQP4ex isoform is critical in the triggering event of progressive downregulation and mislocalization of AQP4 in GBM, which may affect the integrity of the BBB and contributes to accumulation of edema in the peritumoral tissue. Thus, AQP4ex could be considered as a potential early biomarker of GBM progression.

Identification of the prognostic and immunological roles of aquaporin 4: A potential target for survival and immunotherapy in glioma patients

“…AQP4 protein is expressed as a particular morphological feature called orthogonal array of particles (OAPs). These structures are aggregates of the tetrameric unit ( Valente et al, 2022 ). AQP4 protein is expressed as two major isoforms that differ in regard to methionine (M) starting codon.…”

“…Indeed, large OAPs made of M1 and M23 canonical isoforms, still abundantly expressed in the AQP4ex mouse, are delocalized and confined at the astrocytic processes facing the brain neuropile. Thus AQP4ex may be supposed to be involved in the AQP4 alteration observed in the GBM ( Valente et al, 2022 ). In the study of Valente et al (2022) , they evaluated the difference of expression and spatial distribution of AQP4 in grossly tumoral, peritumoral or non-tumoral brain regions.…”

“…The authors ( Simone et al, 2019 ) found that AQP4 protein aggregation/disaggregation into OAP influences the biology of glioma cells, demonstrating that AQP4 protein disaggregation may potentiate invasiveness potential, whereas AQP4 protein aggregation may activate the apoptotic path. Besides, Valente et al (2022) found that AQP4ex, the new read through isoform of AQP4, could be associated with the degree of vasogenic brain edema and GBM cell progression. They found that the reduction in AQP4ex, leading to reduction and delocalization of AQP4, could be likely to undermine the integrity of the BBB, indicating that AQP4ex could be considered as a potential new early biomarker of GBM progression and a target for AQP4 modulation ( Frigeri et al, 2007 ).…”

“…They found that the reduction in AQP4ex, leading to reduction and delocalization of AQP4, could be likely to undermine the integrity of the BBB, indicating that AQP4ex could be considered as a potential new early biomarker of GBM progression and a target for AQP4 modulation ( Frigeri et al, 2007 ). Various studies have confirmed that AQP4 plays a vital role in GBM’s malignant progression and drug resistance ( Valente et al, 2022 ), but its molecular mechanism still needs more in-depth and comprehensive research ( Peng et al, 2020 ; Behnam et al, 2022 ).…”

AQP4-dependent glioma cell features affect the phenotype of surrounding cells via extracellular vesicles

“…Using a readthrough mechanism, about 10–20% of AQP4 can be expressed with a longer C-terminus (AQP4ex), which is important to correctly anchor the OAPs to the perivascular side of the glial endfeet and to allow AQP4 phopsphorylation, the function of which is still under investigation [ 13 , 10 ]. Interestingly, AQP4ex is critical in the triggering event of AQP4 alterations in GBM, and it has been proposed as a potential early biomarker of GBM progression [ 14 ].…”