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

INTRODUCTION: The glymphatic system, a brain waste clearance pathway, is impaired during ischemic stroke-induced edema, although the underlying mechanisms remain unclear. OBJECTIVES: This 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. METHODS: Using 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. RESULTS: Cerebrospinal 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. CONCLUSION: Cerebral 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.