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

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.

Grant Number: 7R01NS119166-05
NIH Institute/Center: NIH
Principal Investigator: Mohammad Iqbal Bhuiyan