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

Abstract

Hydrocephalus has an incidence of 1:1000 in the pediatric population. In hydrocephalus, cerebrospinal fluid (CSF) accumulates in the brain's ventricles causing ventriculomegaly. Hydrocephalus can affect various cell types in the brain, and this study focuses on astrocytes and choroid plexus epithelial cells (CPe). 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 causes a ciliopathy resulting in hydrocephalus and polycystic kidney disease (PKD) in our rodent model. This mutation is orthologous to the human Meckel Gruber syndrome type 3 (MKS3). The TMEM67 gene codes for Mecklin, a ciliary protein found predominately in the brain and kidneys. The phenotypes seen in TMEM67 (-/-) (homozygous) animals are so severe that death will occur by post-natal day 21 (P21). We have previously shown that treatment with TRPV4 antagonists ameliorate hydrocephalus in our genetic model of hydrocephalus. Using immunohistochemical techniques, TRPV4, AQP1, and AQP4 antibodies were used to examine localization in hydrocephalic animals at P15. Changes of AQP4 and TRPV4 were observed in the cortex of homozygous animals. AQP4 appears to move from astrocyte endfeet in hydrocephalic animals and is increased at the brain-CSF barrier. TRPV4 appears to be increased throughout the cortex. qPCR was used to quantitate mRNA from the CPe and cortex. Preliminary qPCR showed increased TRPV4, AQP1, and AQP4 mRNA in the cortex and CPe of P15 hydrocephalic animals compared to wild-type. In summary, in a model of hydrocephalus, channels and transporters show changes in localization and transcription. These results provide further characterization of the role of several channels and transporters in the pathophysiology of hydrocephalus. Futures studies will explore developmental 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.