Augurin production in the mammalian choroid plexus: Implications for CSF and hydrocephalus

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Whereas augurin, a protein encoded by the ecrg4 gene, is highly conserved across species, bioinformatic algorithms predict the existence of several other potential hormonelike peptide products transcribed from the same gene. With gene expression highest in the mammalian choroid plexus (CP) compared to all other tissue types however, we deemed it critical to know which peptide(s) is produced by the CP so as to determine its potential release into, and activity in, cerebrospinal fluid (CSF). Our previous data has shown that gene knockdown in developing zebrafish causes severe and dose-dependent hindbrain edema/hydrocephalus. Accordingly, we suggested a novel function for ecrg4 gene products in CP physiology and implicated them as new hormone(s) regulating CSF and CP function. Immunohistochemical staining showed protein in CP epithelium in vitro and in vivo, and ligandtargeting shows internalization into ependymal cells. Materials and methods Ecrg4 gene products were detected by immunoblotting and immunofluorescence with chicken and rabbit polyclonal or mouse monoclonal antibodies that we raised. DNA sequences for fragments (31-148), (31-70) and (71-148) were cloned into pET15b vector, expressed in BL21DE3pLysS and purified. The human gene was cloned into the pLVx-IRES-ZsGreen vector and virus generated using the LentiX kit (Clontech) while siRNA lentivirus was obtained from Santa Cruz Biotechnology.