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

Abstract

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.

Posthemorrhagic hydrocephalus associates with elevated inflammation and CSF hypersecretion via activation of choroidal transporters

“…Various molecules related to hemolysis and clot formation such as hemoglobin, iron, hemin, thrombin, and peroxiredoxin 2 have been implicated in PHH development, in addition to reactive oxygen species in the preterm infants [ 40 – 45 ] and may contribute to depression of CSF drainage leading to undetectable micro-blockage or scarring of the CSF drainage pathways aided by the subsequent inflammatory response [ 9 ]. Alternatively, as here observed, the early phase of PHH may, in part, arise subsequent to IVH-induced hypersecretion of CSF [ 9 , 14 ], as also observed with ischemic stroke-induced hyperactivation of NKCC1 in choroid plexus [ 46 ] and aligned with inhibition of NKCC1 ameliorating IVH-mediated ventriculomegaly in rats [ 14 , 47 ]. These findings align with the CSF hypersecretion leading to the ventriculomegaly observed in patients with choroidal hyperplasia and choroid plexus papilloma [ 17 , 18 ], and can be simulated by experimental mimicry of CSF hypersecretion in rats [ 14 , 48 ].…”

Ptpn20 deletion in H-Tx rats enhances phosphorylation of the NKCC1 cotransporter in the choroid plexus: an evidence of genetic risk for hydrocephalus in an experimental study

“…However, we must note that other potential functions of the CP besides CSF secretion should not be neglected. In particular, NKCC1, the focus of attention in this study, has been shown to play a key role in regulating cell volume and maintaining ionic homeostasis in the CNS [ 75 , 76 ]. Furthermore, the CSF produced from the CP is also thought to play additional role.…”

Confluence and tight junction dependence of volume regulation in epithelial tissue

“…The details of the relationship between NKCC activity and tight junctions have been linked to dysfunction and inflammation in both the gut ( Koumangoye et al. , 2020 ) and the brain ( Wang et al. , 2022 ), where activating NKCC1-related pathways was found to disrupt the tight junctions of the blood–brain barrier.…”