SPAK-NKCC1 complex and the blood-CSF barrier dysregulation in VCID - Project Summary The choroid plexus (ChP) modulates brain homeostasis through its multifunctional roles, including secretion of the cerebrospinal fluid (CSF) and forming the blood-CSF barrier via tight junctions (TJs) and adheren junctions (AJs) at the choroid plexus epithelial cells for gating circulating immune cell entry into the CSF. The Ste20-related serine-proline-alanine rich kinase SPAK in the ChP epithelial cells emerged as a central player in mediating immune cell signaling as well as modulating the secretion of CSF [via phosphorylation of the secretory ion transporter Na-K-Cl cotransporter protein (NKCC1)]. Activation of SPAK-NKCC1 protein complex in the ChP has been reported to contribute to infectious hydrocephalus, hemorrhagic hydrocephalus and brain inflammation. Moreover, ChP dysfunction is suggested as a key contributor to Alzheimer's disease (AD) pathogenesis. Breakdown of the blood-CSF barrier in AD brains has been associated with more pronounced changes in proinflammatory phenotype of myeloid cells, which were detected in both the ChP and brain parenchyma. Suppressed CSF secretion and turnover have also been observed in AD brains. However, the pathological mechanism of blood-CSF barrier dysfunction in AD/VCID brains remains unknown. The potential role of SPAK signaling complex in the ChP structural impairment and neuroinflammation in non-infectious, chronic sterile brain disease conditions has not been defined. To fill the knowledge gap, we conducted initial study using a mouse vascular contribution to cognitive impairment and dementia (VCID) model induced by the bilateral carotid artery stenosis (BCAS). We observed the following new findings (Prelim data): 1). BCAS-mediated chronic cerebral hypoperfusion caused abnormal accumulation of TJ proteins, which was accompanied with phosphorylatory stimulation of SPAK, NKCC and NF-κB in the ChP epithelial cells; 2). These changes are associated with impaired blood-CSF barrier integrity, as evidenced by ChP infiltration of immune cells; 3). Pharmacological blockade of SPAK with its potent inhibitor ZT1a in the BCAS mice attenuated the ChP damage and improved neurological function. These findings led us to hypothesize that BCAS-induced pathological stimulation of the epithelial SPAK signaling cascade plays a role in disruption of the blood-CSF barrier, choroidal route of leukocyte infiltration, and ChP dysfunction. We will investigate underlying cellular mechanisms in three Specific Aims: 1. Investigate whether BCAS causes disruption of the ChP epithelial cytoarchitecture and polarity. 2. Investigate whether SPAK- and NF-κB-dependent transcriptome changes drive dysregulation of the ChP epithelial cytoarchitecture and polarity. 3. Determine whether BCAS alters the ChP volume and CSF secretion. In summary: completion of this study will provide new knowledge about the cellular and molecular mechanisms underlying ChP dysfunction and leukocyte invasion in VCID and will shed light on ChP SPAK signaling cascade as a novel therapeutic target for blood-CSF barrier impairment.
