Expression of the Gi-coupled RASSL Ro1 in GFAP-postive cells: A novel model of hydrocephalus

Hydrocephalus is a disorder of cerebrospinal fluid dynamics within the central nervous system. Although symptoms of hydrocephalus are often relieved by shunt implantation, complications are common and underlying pathology remains. Understanding the molecular mechanisms of hydrocephalus is critical for development of new therapeutics; however few experimental models allow early events to be studied. Originally designed to study astrocytic Gi signaling, we developed a transgenic mouse line expressing the Gi-coupled RASSL (receptor activated solely by synthetic ligand) Ro1 in GFAP-positive cells by crossing the hGFAP-tTA (tet transactivator behind the human glial fibrillary acidic protein promoter) mouse line with the tetO-Ro1/tetO-LacZ mouse line. Surprisingly, we found that all double-transgenic mice developed hydrocephalus by postnatal day 15, while single-transgenic littermate controls appeared normal.;Hydrocephalic Ro1 mice had enlarged lateral and third ventricles, thinned cortex, partial denudation of the ependymal cell layer, abnormal subcommissural organ, and obliteration of the cerebral aqueduct. Severely affected mice had increased phosphoErk and GFAP expression. Giving breeding pairs doxycycline prevented the expression of Ro1 and onset of hydrocephalus in double-transgenic offspring. Double transgenic mice taken off dox at weaning developed enlarged ventricles within 7 weeks, indicating that Ro1 expression also induces hydrocephalus in adults. Double-transgenic mice injected with a Ro1 inverse agonist when taken off dox did not develop enlarged ventricles or have ependymal detachment, demonstrating that signaling through Ro1 is required for hydrocephalus. We have discovered a new model that allows onset of hydrocephalus to be controlled, providing the unique ability to study the earliest events in juvenile and adult-onset hydrocephalus.;Ependymal denudation is a common feature of hydrocephalus that appears to occur early in the disease. Positive GFAP and Ro1 staining was detected in a subset of ependymal cells from double transgenic mice off dox. Timed studies showed that Ro1 expression began within three days of dox removal and ependymal denudation began within ten days. Affymetrix gene arrays were utilized to screen for changes in ependymal gene expression at five and nine days after dox removal. No differentially expressed genes were detected between double-transgenic mice and controls at either timepoint; RT-PCR also failed to detect significant changes.