Fluoxetine Requires the Endfeet Protein Aquaporin-4 to Enhance Plasticity of Astrocyte Processes

Abstract

Morphological alterations in astrocytes are characteristic for post mortem brains of patients affected by major depressive disorder (MDD). Recently, a significant reduction in the coverage of blood vessels (BVs) by aquaporin-4 (AQP-4)-positive astrocyte endfeet has been shown in the prefrontal cortex (PFC) of MDD patients, suggesting that either alterations in the morphology of endfeet or in AQP-4 distribution might be responsible for the disease phenotype or constitute a consequence of its progress. Antidepressant drugs (ADs) regulate the expression of several proteins, including astrocyte-specific ones. Thus, they may target AQP-4 to induce morphological changes in astrocytes and restore their proper shape or relocate AQP-4 to endfeet. Using an animal model of depression, rats selectively bred for high anxiety-like behavior (HAB), we confirmed a reduced coverage of BVs in the adult PFC by AQP-4-immunoreactive (AQP-4-IR) astrocyte processes with respect to non-selected Wistar rats (NAB), thereby validating it for our study. A further evaluation of the morphology of astrocyte in brain slices (ex vivo) and in vitro using an antibody against the astrocyte-specific cytoskeletal protein glial fibrillary acidic protein (GFAP) revealed that HAB astrocytes extended less processes than NAB cells. Furthermore, short-term drug treatment in vitro with the AD fluoxetine (FLX) was sufficient to increase the plasticity of astrocyte processes, enhancing their number in NAB-derived cells and recovering their basal number in HAB-derived cells. This enhanced FLX-dependent plasticity occurred, however, only in the presence of intact AQP-4, as demonstrated by the lack of effect after the downregulation of AQP-4 with RNAi in both NAB and HAB cells. Nonetheless, a similar short-term treatment did neither modulate the coverage of BVs with AQP-4-positive astrocyte endfeet in NAB nor in HAB rats, although dosage and time of treatment were sufficient to fully recover GFAP expression in HAB brains. Thus, we suggest that longer treatment regimes may be needed to properly restore the coverage of BVs or to relocate AQP-4 to astrocyte endfeet. In conclusion, FLX requires AQP-4 to modulate the plasticity of astrocyte processes and this effect might be essential to re-establish a functional glia-vasculature interface necessary for a physiological communication between bloodstream and brain parenchyma.

Stress‐induced structural and functional modifications of astrocytes—Further implicating glia in the central response to stress

“…Similar data were obtained by selectively breeding rats for depressive phenotypes, demonstrating that depression induces distinct changes in astrocyte morphology (Di Benedetto et al, 2016). These stress-induced morphological modifications which largely impacted the association of astrocyte endfeet at the blood vessel, could be completely reversed with selective serotonin reuptake inhibitor (SSRI) antidepressant (fluoxetine) treatment, in a mechanism requiring AQP4 (Di Benedetto et al, 2016). To date, despite the indication that astrocyte endfeet lining the cerebrovasculature are modified as a result of stress disorder in humans (Rajkowska et al, 2013) as well as in rodent models (Di Benedetto et al, 2016), evidence for dysfunction at the vascular interface is sparse.…”

“…Furthermore, animals treated with an antidepressant during the chronic stress protocol were resistant to the stress‐induced modifications in astrocyte number and structure (Czéh et al, ). Similar data were obtained by selectively breeding rats for depressive phenotypes, demonstrating that depression induces distinct changes in astrocyte morphology (Di Benedetto et al, ). These stress‐induced morphological modifications which largely impacted the association of astrocyte endfeet at the blood vessel, could be completely reversed with selective serotonin reuptake inhibitor (SSRI) antidepressant (fluoxetine) treatment, in a mechanism requiring AQP4 (Di Benedetto et al, ).…”

“…These stress-induced morphological modifications which largely impacted the association of astrocyte endfeet at the blood vessel, could be completely reversed with selective serotonin reuptake inhibitor (SSRI) antidepressant (fluoxetine) treatment, in a mechanism requiring AQP4 (Di Benedetto et al, 2016). To date, despite the indication that astrocyte endfeet lining the cerebrovasculature are modified as a result of stress disorder in humans (Rajkowska et al, 2013) as well as in rodent models (Di Benedetto et al, 2016), evidence for dysfunction at the vascular interface is sparse. Under physiological conditions astrocytes integrate synaptic, behavioral and vascular information to mediate an appropriate blood vessel response (Tran, Peringod, & Gordon, 2018).…”

GDF15 promotes simultaneous astrocyte remodeling and tight junction strengthening at the blood–brain barrier

“…Recent work revealed a disrupted glia cell morphology, marked by reduced expression of aquaporin‐4 in the endfeet of perivascular astrocyte processes (PAP), in the prefrontal cortex (PFC) of major depressive disorder (MDD) patients (Rajkowska, Hughes, Stockmeier, Javier Miguel‐Hidalgo, & Maciag, 2013). We confirmed those findings in rats selectively bred for high anxiety‐related behavior (HAB), a model of innate depression (Wegener, Mathe, & Neumann, 2012), which additionally showed a remarkable shrinkage of entire astrocyte processes (Barbara Di Benedetto et al, 2016). Our observations suggested that the delayed clinical efficacy of pharmacotherapies might depend on a perturbed permeability of the BBB to treatment drugs possibly caused by an altered astrocyte morphology around BVs.…”

Involvement of aquaporin‐4 in laminin‐enhanced process formation of mouse astrocytes in 2D culture: Roles of dystroglycan and α‐syntrophin in aquaporin‐4 expression

“…Interestingly, a recent paper showed that knockdown of AQP4 decreased the total number of astrocyte processes in rat astrocytes, confirming a critical role of AQP4 in process formation (Di Benedetto et al . ). However, the effects of knockdown of AQP4 on the morphology were different between mouse and rat/human astrocytes (Nicchia et al .…”