The glial water channel protein aquaporin-4 (AQP4) forms heterotetramers in the plasma membrane made of the M23-AQP4 and M1-AQP4 isoforms. The isoform ratio controls AQP4 aggregation into supramolecular structures called orthogonal arrays of particles (AQP4-OAP). The role of AQP4 aggregation into OAP in malignant gliomas is still unclear. In this study, we demonstrate that AQP4 aggregation/disaggregation into OAP influences the biology of glioma cells. Selective expression of the OAP-forming isoform M23-AQP4 (AQP4-OAP) triggered cell shape changes in glioma cells associated with alterations to the F-actin cytoskeleton that affected apoptosis. By contrast, expression of M1-AQP4 (AQP4-tetramers), which is unable to aggregate into OAP, ameliorated glioma cell invasiveness, improved cell migration, and increased methalloproteinase-9 activity. Two prolines (254 and 296) at the C-terminus tail were shown to be important in mediating the relationship between the actin cytoskeleton and AQP4-OAP and AQP4-tetramers. In conclusion, this study demonstrates that AQP4 aggregation state might be an important determinant in orienting glioma cells to persist or perish. AQP4 disaggregation may potentiate invasiveness potential, whereas AQP4 aggregation may activate the apoptotic path. This study shows a new perspective on the role of AQP4 in brain tumors not necessarily associated with edema formation but with AQP4 aggregation/disaggregation dynamics and their link with the actin cytoskeleton.Significance: This study demonstrates how AQP4 aggregation influences plasma membrane dynamics to alter cell proliferation, invasiveness, migration, and apoptotic potential in glioma cells.

Astrocyte proliferation and migration toward injured Central Nervous System (CNS) areas are key features of astrogliosis and glial scar formation. Even though it is known that intracellular and environmental Reactive Oxygen and Nitrogen Species (RONS) affect astrocyte behaviour in physiological and pathophysiological conditions, their effects on the migration and growth of astrocytes are still unclear. Plasma-technologies are emerging in medicine as a tool to generate RONS for treating cells directly or through Plasma Activated Liquid Media (PALM). In this paper, we show for the first time how the use of PALM can modulate both astrocyte growth and migration as a function of active species produced by plasma in liquids. Our results show that PALM, generated by means of cold atmospheric pressure plasmas fed with N_2, air or O₂, can modulate astrocyte behaviour depending on the content of hydrogen peroxide and nitrite in the liquid. In particular, H₂O₂ enriched PALM induced a negative effect on cell growth associated with the mild wound healing improvement of primary astrocytes, in a scratch assay. Nitrite enriched PALM induced a selective effect on the wound healing without affecting cell growth. PALM containing a more balanced level of H₂O₂ and NO₂⁻ were able to affect cell growth, as well as significantly ameliorate wound healing. None of the PALM investigated induced upregulation of the gliotic inflammatory marker glial fibrillary acidic protein (GFAP), or of the astrocyte markers Aquaporin-4 (AQP4) and Connexin-43 (Cx-43) analysed by Western blot. Finally, immunofluorescence analysis revealed the presence of NO₂^- able to induce elongated protrusions at the front end of wounded astrocytes in the direction of cell migration. With our study we believe to have shown that PALM offer a novel tool to modulate astrocyte behaviour and that they are promising candidates for controlling astrogliosis in the case of CNS injuries.