Aquaporin-4 (AQP4) is critically involved in brain water and volume homeostasis and has been implicated in a wide range of pathological conditions. Notably, evidence has been accrued to suggest that AQP4 plays a proinflammatory role by promoting release of astrocytic cytokines that activate microglia and other astrocytes. Neuroinflammation is a hallmark of Parkinson’s disease (PD), and we have previously shown that astrocytes in substantia nigra (SN) are enriched in AQP4 relative to cortical astrocytes, and that their complement of AQP4 is further increased following treatment with the parkinsonogenic toxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Here, we investigated the effect of Aqp4 deletion on microglial activation in mice subjected to unilateral intrastriatal injection of 1-methyl-4-phenylpyridinium (MPP+, the toxic metabolite of MPTP). Our results show that MPP+ injections lead to a pronounced increase in the expression level of microglial activating genes in the ventral mesencephalon of wild type (WT) mice, but not Aqp4−/− mice. We also show, in WT mice, that MPP+ injections cause an upregulation of nigral AQP4 and swelling of astrocytic endfeet. These findings are consistent with the idea that AQP4 plays a pro-inflammatory role in Parkinson’s disease, secondary to the dysregulation of astrocytic volume homeostasis.

The water channel protein aquaporin-4 (AQP4) and the gap junction forming proteins connexin-43 (Cx43) and connexin-30 (Cx30) are astrocytic proteins critically involved in brain water and ion homeostasis. While AQP4 is mainly involved in water flux across the astrocytic endfeet membranes, astrocytic gap junctions provide syncytial coupling allowing intercellular exchange of water, ions, and other molecules. We have previously shown that mice with targeted deletion of Aqp4 display enhanced gap junctional coupling between astrocytes. Here, we investigate whether uncoupling of the astrocytic syncytium by deletion of the astrocytic connexins Cx43 and Cx30 affects AQP4 membrane localization and expression. By using quantitative immunogold cytochemistry, we show that deletion of astrocytic connexins leads to a substantial reduction of perivascular AQP4, concomitant with a down-regulation of total AQP4 protein and mRNA. Isoform expression analysis shows that while the level of the predominant AQP4 M23 isoform is reduced in Cx43/Cx30 double deficient hippocampal astrocytes, the levels of M1, and the alternative translation AQP4ex isoform protein levels are increased. These findings reveal a complex interdependence between AQP4 and connexins, which are both significantly involved in homeostatic functions and astrogliopathologies.

Evidence suggests that extracellular matrix molecules of perivascular basal laminae help orchestrate the molecular assemblies at the gliovascular interface. Specifically, laminin and agrin are thought to tether the dystrophin-associated protein (DAP) complex to the astrocytic basal lamina. This complex includes α-syntrophin (α-Syn), which is believed to anchor aquaporin-4 (AQP4) to astrocytic endfoot membrane domains. We have previously shown that the size of the perivascular AQP4 pool differs considerably between brain regions in an α-Syn-dependent manner. Also, both AQP4 and α-Syn occur at higher densities in endfoot membrane domains facing pericytes than in endfoot membrane domains facing endothelial cells. The heterogeneous distribution of AQP4 at the regional and capillary level has been attributed to a direct interaction between AQP4 and α-Syn. This would be challenged (1) if the microdistributions of laminin and agrin fail to align with those of DAP and AQP4 and (2) if targeted deletion of α-Syn leads to a loss of laminin and/or agrin. Here, we provide the first detailed and quantitative analysis of laminin and agrin in brain basal laminae of mice. We show that the microdistributions of these molecules vary in a fashion that is well aligned with the previously reported microdistribution of AQP4. We also demonstrate that the expression patterns of laminin and agrin are insensitive to targeted deletion of α-Syn, suggesting that α-Syn deletion affects AQP4 directly and not indirectly via laminin or agrin. These data fill remaining voids in the current model of how key molecules are assembled and tethered at the gliovascular interface.

More than 20 years have passed since the first demonstration of Aquaporin-9 (AQP9) in the brain. Yet its precise localization and function in brain tissue remain unresolved. In peripheral tissues, AQP9 is expressed in leukocytes where it is involved in systemic inflammation processes. In this study, we hypothesized that AQP9 plays a proinflammatory role in the brain, analogous to its role in the periphery. We also explored whether Aqp9 is expressed in microglial cells, which would be supportive of this hypothesis. Our results show that targeted deletion of Aqp9 significantly suppressed the inflammatory response to the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP+). This toxin induces a strong inflammatory response in brain. After intrastriatal injections of MPP+, the increase in transcript levels of proinflammatory genes was less pronounced in AQP9−/− mice compared with wild-type controls. Further, in isolated cell subsets, validated by flow cytometry we demonstrated that Aqp9 transcripts are expressed in microglial cells, albeit at lower concentrations than in astrocytes. The present analysis provides novel insight into the role of AQP9 in the brain and opens new avenues for research in the field of neuroinflammation and chronic neurodegenerative disease.