Increase of aquaporin 9 expression in astrocytes participates in astrogliosis
Astrocytes are known to be recruited in glial scar formation after brain injuries (Sofroniew and Vinters, 2010). Recent work has shown that the response of astrocytes to brain lesion is heterogeneous, with cell division and proliferation occurring in a juxtavascular subpopulation of astrocytes, and formation and extension of long processes towards the lesion site in a second subpopulation, while a third subpopulation retains its morphology (Bardehle et al., 2013). In various conditions including physiological responses such as osmotic stress, parturition, and lactation, it has been well established that astrocyte processes are retracted.
AQP9 expression in astrocytes increases with time after stroke onset in mice, with a significant rise at 24 hr (de Castro Ribeiro et al., 2006b), in contrast to AQP4, whose expression is correlated with edema formation after brain injuries including stroke (Badaut et al., 2014). To date, the exact role of the increase in AQP9 expression in astrocytes in the brain after stroke has never been investigated. AQP4 is also involved in astrocyte migration after injury by facilitation of water entry (Saadoun et al., 2005). The formation of protrusions such as filopodia and lamellipodia plays a key role in the cell migration process (Loitto et al., 2002). In this action, water flux through membrane water channels was shown to be a key element in peripheral cells such as leukocytes (Loitto et al., 2002). In fact, AQP1 and AQP4 were the first water channels shown to contribute to cell movement by facilitating water entrance into the protrusions (Verkman, 2005). More recently, AQP9 was also proposed to have a role in leukocyte transmigration through facilitating filopodia formation by acting on the entrance of water and thereby inducing polymerization of actin filaments (Loitto et al., 2002). In leukocytes and other peripheral cells, the increase in plasmatic AQP9 and associated morphological changes with a higher number of filopodia critically depend on AQP9 phosphorylation at serine residues in positions 11 and 222. Phosphorylation at these sites involves protein kinase C (PKC) zeta (PKCζ) (Loitto et al., 2007).
Astrocyte morphology changes after brain injury, exhibiting various morphology patterns and the gradual extension of astrocyte processes, with the loss of individual astrocyte domains directly correlated to the severity of the injury or the distance from the site of the primary lesion (Sofroniew and Vinters, 2010). We also previously observed an increase in AQP9 expression after stroke in the areas bordering the lesion (de Castro Ribeiro et al., 2006b). However, so far the link between the level of expression and possible phosphorylation of AQP9 and astrocyte morphology after stroke has never been investigated. Therefore, we wanted to determine whether increased AQP9 expression affects the morphology of astrocytes by generating a larger number of processes and to investigate how this might influence outcome. We examined this in vitro after transfection into cultured astrocytes of a plasmid‐containing green fluorescent protein (GFP) fused to wild‐type AQP9 (Loitto et al., 2007). In vivo, we used a model of neuroprotection induced by thrombin preconditioning (TPC). Early induction or downregulation of brain AQP4 has been observed in preconditioning experiments (Dirnagl et al., 2009; Hirt et al., 2009; Hoshi et al., 2011).