Changes in TRPV4, AQP1, and AQP4 in a Genetic Model of Hydrocephalus Treated with a TRPV4 Antagonist

Abstract

Hydrocephalus occurs as a result of cerebrospinal fluid (CSF) accumulating in the brain’s ventricles causing ventriculomegaly. Hydrocephalus can affect various cell types in the brain, and this study focuses on choroid plexus epithelial cells (CPe) and astrocytes. The choroid plexus is a tight epithelial structure within the ventricular system, responsible for producing CSF. Astrocytes are supportive cells that serve in blood‐brain barrier maintenance and brain fluid/electrolyte regulation. The CP and astrocytes contain channels and transporters that are important in regulating both CSF and brain interstitial fluid. These include transient receptor potential vanilloid 4 (TRPV4), aquaporin 1 (AQP1), and aquaporin 4 (AQP4). TRPV4 is a mechanosensitive channel and has been implicated in osmotic regulation. AQPs are a family of membrane proteins that facilitate water transport in various tissues. The CPe expresses AQP1 and TRPV4 which may be important in CSF production. Astrocytes express TRPV4 and AQP4 on their endfeet to regulate cell volume and vascular permeability. Previous studies done by the Blazer‐Yost laboratory have shown increases in AQP1, but not TRPV4 mRNA of CPe from hydrocephalic animals compared to wildtype. This study further examined changes in localization and expression of TRPV4, AQP1, and AQP4 in a rodent model of hydrocephalus.A single missense point mutation in the Transmembrane 67 (TMEM67) protein, that causes a ciliopathy resulting in hydrocephalus and polycystic kidney disease (PKD) in our rodent model, is orthologous to the human Meckel Gruber syndrome type 3 (MKS3). Treatment with the TRPV4 antagonist, RN1734, ameliorates hydrocephalus in our genetic model of hydrocephalus. Using immunohistochemical techniques, TRPV4, AQP1, and AQP4 antibodies were used to examine localization in untreated and RN1734 treated hydrocephalic animals at P15. Changes of AQP4 and TRPV4 were observed in the subventricular zone of homozygous animals. RTqPCR was used to quantitate mRNA from the CPe and subventricular region of the cortex. Preliminary RTqPCR showed increased AQP1, decreased TRPV4 and no change in AQP4 mRNA in the cortex and CPe of P15 untreated and RN1734 treated hydrocephalic animals compared to wild‐type. These data suggests that the changes occurring may be posttranslational. To observe this, Western blots were utilized to look at changes in protein. The protein expression of AQP1 and AQP4 may be increased in untreated cortical tissue from hydrocephalic animals, while total TRPV4 remaining unchanged. AQP1 and AQP4 may be still increased, while total TRPV4 decreased in animals treated with RN1734. These results provide further characterization of the role of several channels and transporters in the pathophysiology of hydrocephalus. Future studies will explore post‐translational changes in protein expression and membrane localization to compliment these studies. Examining channels and transporters can elucidate how brain fluid regulation may be altered in hydrocephalus and produce targets for pharmacological treatment in the future.