Pharmacological inhibition of SPAK-NKCC1 complex attenuates astrogliosis and restores cerebral blood flow in a mouse model of VCID.

Vascular contributions to cognitive impairment and dementia (VCID) are one of the leading causes of dementia, where reactive astrogliosis, blood-brain barrier (BBB) disruption, and white matter lesions (WML) are the key features. However, the molecular and cellular mechanisms underlying VCID are not well understood. Na-K-Cl cotransporter 1 (NKCC1) activation via its upstream regulatory kinase SPAK (STE20/SPS1-related proline/alanine-rich kinase) causes intracellular Na+ overload, hypertrophy, and astrogliosis, a cascade that has been implicated in VCID. In this study, we investigated whether treatment with the SPAK inhibitor ZT-1a at the symptomatic stage in a VCID mouse model is effective in reducing reactive astrogliosis and BBB breakdown, and in improving cerebral blood flow (CBF). VCID was induced in adult C57BL/6J mice using bilateral carotid artery stenosis (BCAS), and either Vehicle (Veh, DMSO) or ZT-1a was administered from weeks 4 to 8 post-BCAS. CBF was monitored by laser speckle imaging, and cognitive deficits were assessed using the Morris water maze test. BBB integrity, astrocytic endfeet coverage, and demyelination were assessed by immunofluorescence (IF) analysis. BCAS mice exhibited a biphasic reduction of CBF and cognitive impairments, parallel with a significant loss of myelin basic protein (MBP) in white matter tracts. Increased expression and phosphorylation of NKCC1 were detected in GFAP+ astrocytes and Iba1+ microglia/macrophage following BCAS. Reduced ZO-1, Claudin-5, and AQP4 expression in vessels and extravasation of serum albumin into the brain parenchyma in BCAS mice indicate the loss of BBB integrity. Importantly, ZT-1a treatment of the BCAS mice significantly improved CBF recovery and prevented the learning and memory deficit. These mice displayed reduced astrogliosis, microglial activation, MMP-2/9 expression, BBB damage, and axonal demyelination. Our results strongly suggest that hypoperfusion-induced SPAK-NKCC1 activation contributes to reactive astrogliosis, BBB disruption, CBF reduction, and cognitive impairment. The SPAK-NKCC1 complex represents a modifiable therapeutic target for counteracting VCID progression.