Role of SPAK-NKCC1 signaling cascade in the choroid plexus blood-CSF barrier damage after stroke.

BACKGROUND: The mechanisms underlying dysfunction of choroid plexus (ChP) blood-cerebrospinal fluid (CSF) barrier and lymphocyte invasion in neuroinflammatory responses to stroke are not well understood. In this study, we investigated whether stroke damaged the blood-CSF barrier integrity due to dysregulation of major ChP ion transport system, Na+-K+-Cl- cotransporter 1 (NKCC1), and regulatory Ste20-related proline-alanine-rich kinase (SPAK). METHODS: Sham or ischemic stroke was induced in C57Bl/6J mice. Changes on the SPAK-NKCC1 complex and tight junction proteins (TJs) in the ChP were quantified by immunofluorescence staining and immunoblotting. Immune cell infiltration in the ChP was assessed by flow cytometry and immunostaining. Cultured ChP epithelium cells (CPECs) and cortical neurons were used to evaluate H2O2-mediated oxidative stress in stimulating the SPAK-NKCC1 complex and cellular damage. In vivo or in vitro pharmacological blockade of the ChP SPAK-NKCC1 cascade with SPAK inhibitor ZT-1a or NKCC1 inhibitor bumetanide were examined. RESULTS: Ischemic stroke stimulated activation of the CPECs apical membrane SPAK-NKCC1 complex, NF-κB, and MMP9, which was associated with loss of the blood-CSF barrier integrity and increased immune cell infiltration into the ChP. Oxidative stress directly activated the SPAK-NKCC1 pathway and resulted in apoptosis, neurodegeneration, and NKCC1-mediated ion influx. Pharmacological blockade of the SPAK-NKCC1 pathway protected the ChP barrier integrity, attenuated ChP immune cell infiltration or neuronal death. CONCLUSION: Stroke-induced pathological stimulation of the SPAK-NKCC1 cascade caused CPECs damage and disruption of TJs at the blood-CSF barrier. The ChP SPAK-NKCC1 complex emerged as a therapeutic target for attenuating ChP dysfunction and lymphocyte invasion after stroke.

Microglial-oligodendrocyte interactions in myelination and neurological function recovery after traumatic brain injury.

Differential microglial inflammatory responses play a role in regulation of differentiation and maturation of oligodendrocytes (OLs) in brain white matter. How microglia-OL crosstalk is altered by traumatic brain injury (TBI) and its impact on axonal myelination and neurological function impairment remain poorly understood. In this study, we investigated roles of a Na+/H+ exchanger (NHE1), an essential microglial pH regulatory protein, in microglial proinflammatory activation and OL survival and differentiation in a murine TBI model induced by controlled cortical impact. Similar TBI-induced contusion volumes were detected in the Cx3cr1-CreERT2 control (Ctrl) mice and selective microglial Nhe1 knockout (Cx3cr1-CreERT2;Nhe1flox/flox, Nhe1 cKO) mice. Compared to the Ctrl mice, the Nhe1 cKO mice displayed increased resistance to initial TBI-induced white matter damage and accelerated chronic phase of OL regeneration at 30 days post-TBI. The cKO brains presented increased anti-inflammatory phenotypes of microglia and infiltrated myeloid cells, with reduced proinflammatory transcriptome profiles. Moreover, the cKO mice exhibited accelerated post-TBI sensorimotor and cognitive functional recovery than the Ctrl mice. These phenotypic outcomes in cKO mice were recapitulated in C57BL6J wild-type TBI mice receiving treatment of a potent NHE1 inhibitor HOE642 for 1-7 days post-TBI. Taken together, these findings collectively demonstrated that blocking NHE1 protein stimulates restorative microglial activation in oligodendrogenesis and neuroprotection, which contributes to accelerated brain repair and neurological function recovery after TBI.

Transient ischemic stroke triggers sustained damage of the choroid plexus blood-CSF barrier.

Neuroinflammation is a pathological event associated with many neurological disorders, including dementia and stroke. The choroid plexus (ChP) is a key structure in the ventricles of the brain that secretes cerebrospinal fluid (CSF), forms a blood-CSF barrier, and responds to disease conditions by recruiting immune cells and maintaining an immune microenvironment in the brain. Despite these critical roles, the exact structural and functional changes to the ChP over post-stroke time remain to be elucidated. We induced ischemic stroke in C57BL/6J mice via transient middle cerebral artery occlusion which led to reduction of cerebral blood flow and infarct stroke. At 1-7 days post-stroke, we detected time-dependent increase in the ChP blood-CSF barrier permeability to albumin, tight-junction damage, and dynamic changes of SPAK-NKCC1 protein complex, a key ion transport regulatory system for CSF production and clearance. A transient loss of SPAK protein complex but increased phosphorylation of the SPAK-NKCC1 complex was observed in both lateral ventricle ChPs. Most interestingly, stroke also triggered elevation of proinflammatory Lcn2 mRNA and its protein as well as infiltration of anti-inflammatory myeloid cells in ChP at day 5 post-stroke. These findings demonstrate that ischemic strokes cause significant damage to the ChP blood-CSF barrier, contributing to neuroinflammation in the subacute stage.

Elevated microglial oxidative phosphorylation and phagocytosis stimulate post-stroke brain remodeling and cognitive function recovery in mice.

New research shows that disease-associated microglia in neurodegenerative brains present features of elevated phagocytosis, lysosomal functions, and lipid metabolism, which benefit brain repair. The underlying mechanisms remain poorly understood. Intracellular pH (pHi) is important for regulating aerobic glycolysis in microglia, where Na/H exchanger (NHE1) is a key pH regulator by extruding H+ in exchange of Na+ influx. We report here that post-stroke Cx3cr1-CreER+/-;Nhe1flox/flox (Nhe1 cKO) brains displayed stimulation of microglial transcriptomes of rate-limiting enzyme genes for glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation. The other upregulated genes included genes for phagocytosis and LXR/RXR pathway activation as well as the disease-associated microglia hallmark genes (Apoe, Trem2, Spp1). The cKO microglia exhibited increased oxidative phosphorylation capacity, and higher phagocytic activity, which likely played a role in enhanced synaptic stripping and remodeling, oligodendrogenesis, and remyelination. This study reveals that genetic blockade of microglial NHE1 stimulated oxidative phosphorylation immunometabolism, and boosted phagocytosis function which is associated with tissue remodeling and post-stroke cognitive function recovery.

Blockade of Cell Volume Regulatory Protein NKCC1 Increases TMZ-Induced Glioma Apoptosis and Reduces Astrogliosis.

Glioma is one of the most common primary malignant tumors of the central nervous system accounting for approximately 40% of all intracranial tumors. Temozolomide is a conventional chemotherapy drug for adjuvant treatment of patients with high-risk gliomas, including grade II to grade IV. Our bioinformatic analysis of The Cancer Genome Atlas and Chinese Glioma Genome Atlas datasets and immunoblotting assay show that SLC12A2 gene and its encoded Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) protein are abundantly expressed in grade II-IV gliomas. NKCC1 regulates cell volume and intracellular Cl- concentration, which promotes glioma cell migration, resistance to temozolomide, and tumor-related epilepsy in experimental glioma models. Using mouse syngeneic glioma models with intracranial transplantation of two different glioma cell lines (GL26 and SB28), we show that NKCC1 protein in glioma tumor cells as well as in tumor-associated reactive astrocytes was significantly upregulated in response to temozolomide monotherapy. Combination therapy of temozolomide with the potent NKCC1 inhibitor bumetanide reduced tumor proliferation, potentiated the cytotoxic effects of temozolomide, decreased tumor-associated reactive astrogliosis, and restored astrocytic GLT-1 and GLAST glutamate transporter expression. The combinatorial therapy also led to suppressed tumor growth and prolonged survival of mice bearing GL26 glioma cells. Taken together, these results demonstrate that NKCC1 protein plays multifaceted roles in the pathogenesis of glioma tumors and presents as a therapeutic target for reducing temozolomide-mediated resistance and tumor-associated astrogliosis.

Modulation of brain cation-Cl- cotransport via the SPAK kinase inhibitor ZT-1a.

The SLC12A cation-Cl- cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.