Aquaporin 4 and its isoforms regulation ameliorate AQP4 Mis-localization-induced glymphatic dysfunction in ischemic stroke.

introductionThe glymphatic system, a brain waste clearance pathway, is impaired during ischemic stroke-induced edema, although the underlying mechanisms remain unclear.

objectivesThis study investigates the temporal dynamics of glymphatic dysfunction post-stroke and the roles of aquaporin 4 (AQP4), its isoforms, and syntrophin alpha 1 (SNTA1) in AQP4 polarization.

methodsUsing a transient middle cerebral artery occlusion (tMCAO) mouse model, glymphatic function was assessed via cisterna magna contrast injection and magnetic resonance imaging. The AQP4 antagonist TGN-020 was administered to elucidate edema's role in glymphatic dysfunction. AQP4 isoforms viral vectors and SNTA1 modulation were used to study AQP4 polarization and glymphatic function. Techniques included western blotting, q-PCR, immunofluorescence, TEM and behavioral tests. Transcriptomic and metabolomic analyses were performed to assess gene expression and metabolic changes.

resultsCerebrospinal fluid (CSF) flow decreased during the hyperacute phase, recovering with edema resolution. By administering TGN-020 to reduce edema, distinct alterations in the localization of AQP4 were observed. Specifically, there was a notable increase in AQP4 localization within the astrocyte end-feet. Consequently, CSF inflow and interstitial fluid (ISF) drainage were restored. Transcriptomic sequencing was used to analyze ubiquitination-related channels in tMCAO mice. Metabolic sequencing showed that TGN-020 therapy protected the metabolic stability. Our findings highlight the critical role of AQP4 isoforms in the polarized distribution of AQP4. The upregulation of the AQP4-M1 isoform exacerbated edema and motor dysfunction, whereas the AQP4-M23 isoform corrected the mis-localization of AQP4. Inhibition of AQP4 not only restored the polarized integrity of AQP4 in astrocyte end-feet but also alleviated the metabolic disruptions caused by tMCAO. Furthermore, overexpression of SNTA1 enhanced AQP4 polarity by modulating the expression of AQP4 isoforms.

conclusionCerebral edema disrupts AQP4 localization and glymphatic function following stroke. TGN-020 modulates AQP4 polarization through regulation of AQP4 isoforms and restores glymphatic dysfunction. AQP4-M23 isoform emerges as a key regulator of AQP4 polarization, providing new insights into ischemic stroke pathophysiology.