Phosphoproteomic Insights into TRPV4 - AMPK Signaling Axis in the Choroid Plexus Epithelium: Implications for Therapeutic Targeting in Hydrocephalus

BackgroundHydrocephalus is characterized by abnormal accumulation of cerebrospinal fluid (CSF) due to disrupted secretion, circulation, or reabsorption. CSF homeostasis is regulated by ion and water channels on choroid plexus epithelial (CPe) cells, including the mechanosensitive cation channel, transient receptor potential vanilloid 4 (TRPV4). Although TRPV4 antagonism prevents hydrocephalus progression in rats, how TRPV4 activity modulates CSF production remains unclear. MethodsBecause TRPV4 function is phosphorylation-dependent, we examined its activation (GSK1016790A) and inhibition (RN1734) and the downstream signaling alterations in human choroid plexus papilloma cells (HIBCPP) using phosphoproteomic mass spectrometry. ResultsOur phosphoproteomic analysis revealed significant changes in kinases and tight junction (TJ) proteins regulating epithelial barrier integrity. TRPV4 activation altered TJ proteins such as Zonula Occludens-1 (ZO-1), and Claudin-7 (CLDN7) and AMP-activated protein kinase (AMPK), phosphorylation, with a notable foldchange increase of AMPK inhibitory phospho form (Ser496). Further our electrophysiological and biochemical analyses demonstrated that AMPK activation followed by TRPV4 stimulation increased AMPK Ser496 phosphorylation, and epithelial permeability was subsequently elevated, although it was statistically not significant. In contrast, AMPK inhibition prior to TRPV4 activation resulted in the opposite effect with substantial epithelial barrier tightness which was evident from the ZO-1 expression. Moreover, a pharmacologically available anti-diabetic drug, metformin exhibited a similar trend like Compound C in reducing the barrier epithelial permeability after TRPV4 agonist treatment, highlighting that metformin promotes barrier tightness via an AMPK-dependent pathway coupled to TRPV4 signaling. ConclusionsOverall, these findings identify an AMPK-TRPV4 signaling axis that modulates epithelial permeability and may influence CSF regulation, highlighting a potential therapeutic target for hydrocephalus.