Abstract
By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24–72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6–8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation.
Highlights
Traumatic brain injury (TBI) is often associated with poor clinical outcomes because an over exuberant inflammatory response can cause unintended damage to both injured and non-injured central nervous system (CNS) tissue [1]
Augurin is a cell membrane protein Immunohistochemical analyses of augurin in periventricular regions of the brain (Figure 1A, red) showed that, in many instances, the greatest intensity of augurin immunolabeling was polarized with significant staining localizing to the ventricular side of choroid plexus epithelial (CPe) cells
To determine whether augurin is released into cerebrospinal fluid (CSF) in vivo, we processed rat CSF for immunoblotting and detected a 14 kDa band (Figure 1B) that corresponded to the predicted molecular weight of augurin, the processed 118 amino acid hormone-like peptide encoded by ECRG4(31–148) [18]
Summary
Traumatic brain injury (TBI) is often associated with poor clinical outcomes because an over exuberant inflammatory response can cause unintended damage to both injured and non-injured central nervous system (CNS) tissue [1]. Edema may disrupt blood-brain (BBB) and blood-cerebrospinal fluid (BCSFB) barriers, compress brain parenchyma [4] and compromise endothelial, choroid plexus epithelial (CPe), ventricular ependymal (Ve) and subarachnoid (SA) cells that normally filter toxins from interstitial fluid, maintain the ionic balance of CSF, and secrete hormones and growth factors to maintain CNS homeostasis [5]. The CPe in particular suffers unique metabolic and structural stresses during the acute stages of CNS injury, which restrict functions that would otherwise restore homeostasis during repair. The resulting mechanical stress [6] and neutrophil invasion [7] increase leakiness of BCSFB tight junctions and sloughing of apical cilia from CPe and Ve cells. Disrupted tight junctions impair protein ultrafiltration and lead to increased bulk and osmotic flow of water into CSF [8,9,10]
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