Hydrocephalus is characterized by the abnormal accumulation of cerebrospinal fluid (CSF) within the brain ventricles, disrupting its normal secretion, circulation or reabsorption. CSF production and homeostasis are tightly regulated by ion and water channels on the choroid plexus epithelial (CPe) membrane. Among these, transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive cation channel, may be important in stress situations like hydrocephalus. Previous studies highlighted that inhibiting TRPV4 activity can halt disease development of congenital hydrocephalus in rats. However, the mechanistic details of how TRPV4 activity enhances CSF production remains unclear. TRPV4 function is dynamically regulated by phosphorylation and TRPV4 activation can also trigger downstream phosphoproteomic changes. Therefore, to investigate the molecular role of TRPV4 in hydrocephalus, we examined TRPV4 regulation upon activation with a TRPV4 agonist (GSK1016790A) and inhibition with an antagonist (RN1734), in human choroid plexus papilloma cells (HIBCPP), a well characterized model of the choroid plexus epithelial cells. Interestingly, our study identified significant upregulation of phosphorylated kinases and tight junction proteins involved in maintaining epithelial barrier integrity. Specifically, treatment with both a TRPV4 antagonist and subsequent agonist caused notable changes in Zonula Occludens-1 (ZO-1) while agonist treatment alone caused changes in ZO-1, AMP-activated protein kinase (AMPK), Claudin7 (CLDN7). AMPK demonstrated increased inhibitory phosphorylation at Ser496 following TRPV4 agonist treatment, suggesting that TRPV4 activation modulates epithelial barrier function through AMPK modulation. This finding was further supported by electrophysiological experiments, which showed that activating AMPK with AICAR led to elevated inhibitory phosphorylation at Ser496, resulting in feedback suppression of AMPK activity (decreasing phosphorylation on canonical activation site on Thr172) and increased epithelial membrane permeability. Conversely, inhibition of AMPK with compound C decreased Ser496 phosphorylation, which markedly reduced membrane permeability and helped preserve barrier integrity. Moreover, AMPK, in active form, indirectly regulates ZO-1 by phosphorylating adherent junction proteins such as afadin and cingulin, and thereby promoting tight junction assembly. Consistent with this mechanism, our study identified phosphorylated ZO-1 following TRPV4 activation, suggesting that phospho-ZO-1 may instead contribute to tight junction disassembly which, in turn, could explain the increased membrane permeability we observed in parallel with AMPK inhibitory phosphorylation at Ser496. Collectively, our phosphoproteomics results reveal a functional coupling between AMPK activity and TRPV4-mediated ion transport, demonstrating that this interaction modulates epithelial barrier permeability and, potentially, CSF regulation. Thus, our findings position AMPK–TRPV4 signaling axis as a promising target for therapeutic intervention in hydrocephalus. This study was supported by DoD Congressionally Directed Medical Research Program Grants #HT94252310296 and #HT94252310401 (BBY) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Mahendran et al. (Fri,) studied this question.