Are diurnal variations in glymphatic clearance driven by circadian regulation of Aquaporin-4 expression?

Aquaporin-4 (AQP4) is a key astrocytic water channel protein predominantly localized to perivascular endfeet, where it is believed to play a crucial role in brain fluid homeostasis and glymphatic waste clearance. Previous studies have suggested that the perivascular expression of AQP4 is regulated by circadian rhythms and peaks during the sleep phase, potentially influencing glymphatic function. However, direct evidence supporting circadian regulation of AQP4 remains limited. Here, we investigated AQP4 expression and perivascular distribution in the hippocampus and neocortex of mice at mid rest (day) and mid active phase (night). We employed quantitative immunogold electron microscopy (EM), in addition to immunofluorescence confocal microscopy, Western blotting, and qPCR to assess AQP4 localization and expression at the mRNA and protein levels. Our findings revealed no significant differences in AQP4 perivascular localization, total protein levels, or mRNA expression between day and night. Furthermore, the expression of key dystrophin-associated protein complex (DAPC) components, which anchor AQP4 to astrocytic endfeet, also remained largely unchanged. These results challenge the hypothesis that AQP4 localization is regulated by circadian rhythms and suggest that previously reported circadian differences in glymphatic function are mediated by factors other than AQP4 expression. This study highlights the need for further research into the mechanisms governing glymphatic function.