Astrocytic WNK-SPAK-NKCC1 Cascade in White Matter Astrogliosis and Injury

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PROJECT SUMMARY Vascular contributions to cognitive impairment and dementia (VCID) are currently considered as one of the leading causes of dementing illness. The key feature of VCID is diffuse white mater lesions (WML) and hippocampal damage, including myelin loss, axonal disruption, and astrogliosis. However, the underlying molecular and cellular mechanisms for WML, hippocampal damage, and cognitive impairment are not well understood. Hypertension and atherosclerosis are the most significant risk factors for dementia epidemics. Using two mouse models of VCID with chronic bilateral carotid artery stenosis (BCAS), we detected progressive activation of the WNK-SPAK-NKCC1 protein complex in white matter tracts and hippocampus, which is associated with brain lesion and cognitive deficits. Brain Na+-K+-Cl- cotransporter isoform 1 (NKCC1) contributes to intracellular Na+ and Cl- overload, cytotoxic edema, and excitotoxic ischemic neuronal damage. The serine- threonine WNK kinase family [with no lysine (K)], and its downstream kinase SPAK (the STE20/SPS1-related proline/alanine-rich kinase) activate NKCC1 activity via protein phosphorylation. In our pilot study, we detected that BCAS triggered a time-dependent activation of the SPAK-NKCC1 protein complex, specifically in corpus collosum (CC) and hippocampal GFAP+ reactive astrocytes. How the WNK-SPAK-NKCC1 protein complex is stimulated and its role in the pathogenesis of BCAS-induced WML and hippocampal lesion remains unknown. We detected BCAS-induced elevation of interleukin-18 receptor 1 (IL-18R1) expression and nuclear translocation of pNF-κB in GFAP+ reactive astrocytes, which are correlated with increased NF-kB recruitment on the Wnk/Spak/Nkcc1 gene promoters. Importantly, blocking SPAK function with a novel, selective SPAK inhibitor ZT-1a significantly reduced WML, hippocampal CA1 region neurodegeneration, and attenuated cognitive function impairment. Therefore, we propose that activation of the WNK-SPAK-NKCC1 complex plays an important role in the BCAS-induced pathogenesis. We hypothesize that (1) BCAS-induced hypoperfusion stimulates the IL-18/IL-18R-MyD88-NF-κB cascade to upregulate the WNK-SPAK-NKCC1 complex in reactive astrocytes; (2) elevated astrocytic WNK-SPAK-NKCC1 signaling and astrogliosis contribute to cell death of oligodendrocytes (OLs), demyelination, CA1 neuronal loss, and cognitive deficits; and (3) Post-BCAS administration of the novel SPAK kinase inhibitor ZT-1a reduces brain lesions and cognitive deficits by attenuating astrogliosis and degeneration of OLs and CA1 neurons. These hypotheses will be tested in the following specific aims: Aim 1. Identify molecular mechanisms that stimulate WNK-SPAK-NKCC1 cascade activation and astrogliosis in white matter tracts and hippocampus after BCAS. Aim 2. Determine causative roles of BCAS-induced astrocytic WNK-SPAK-NKCC1 cascade activation in brain lesion and cognitive function impairment. Aim 3. Investigate the efficacy of the novel SPAK inhibitor ZT-1a in reducing astrogliosis, brain lesion, and cognitive function impairment in the BCAS mice.