Abstract
To maintain the precise internal milieu of the mammalian central nervous system, well-controlled transfer of molecules from periphery into brain is required. Recently the soluble and cell-surface albumin-binding glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been implicated in albumin transport into developing brain, however the exact mechanism remains unknown. We postulate that SPARC is a docking site for albumin, mediating its uptake and transfer by choroid plexus epithelial cells from blood into cerebrospinal fluid (CSF). We used in vivo physiological measurements of transfer of endogenous (mouse) and exogenous (human) albumins, in situ Proximity Ligation Assay (in situ PLA), and qRT-PCR experiments to examine the cellular mechanism mediating protein transfer across the blood–CSF interface. We report that at all developmental stages mouse albumin and SPARC gave positive signals with in situ PLAs in plasma, CSF and within individual plexus cells suggesting a possible molecular interaction. In contrast, in situ PLA experiments in brain sections from mice injected with human albumin showed positive signals for human albumin in the vascular compartment that were only rarely identifiable within choroid plexus cells and only at older ages. Concentrations of both endogenous mouse albumin and exogenous (intraperitoneally injected) human albumin were estimated in plasma and CSF and expressed as CSF/plasma concentration ratios. Human albumin was not transferred through the mouse blood–CSF barrier to the same extent as endogenous mouse albumin, confirming results from in situ PLA. During postnatal development Sparc gene expression was higher in early postnatal ages than in the adult and changed in response to altered levels of albumin in blood plasma in a differential and developmentally regulated manner. Here we propose a possible cellular route and mechanism by which albumin is transferred from blood into CSF across a sub-population of specialised choroid plexus epithelial cells.
Highlights
The brain develops and is maintained in a tightly controlled internal environment, protected from fluctuations of constituents of blood plasma by a set of mechanisms referred to as the blood brain barriers [1]
cerebrospinal fluid (CSF)/plasma concentration ratios for endogenous and exogenous albumins It is well known that the level of protein in the circulating plasma increases during development while simultaneously decreasing in the CSF [1,7,26] leading to a decrease of CSF/ plasma concentration ratios – an index traditionally used as a measurement of penetration into the CSF ([26,27] and see Discussion)
The lower CSF/plasma concentration ratio of human albumin compared to endogenous mouse albumin at the earlier age confirms that during development choroid plexus is able to distinguish between different species of albumin and that this recognition mechanism is no longer present at later stages and in the adult [10,13,14,25], where the apparent higher ratio for human albumin is most likely to be due to its clearance from plasma being faster than that from the CSF as has been described previously in other species [18,19,24]
Summary
The brain develops and is maintained in a tightly controlled internal environment, protected from fluctuations of constituents of blood plasma by a set of mechanisms referred to as the blood brain barriers [1] These exchange interfaces are present between brain endothelial cells (the blood–brain barrier proper), choroid plexus epithelial cells (blood–cerebrospinal fluid [CSF] barrier), pial surface (pia–arachnoid barrier) and between neuroependymal cells lining the ventricular system The morphological basis of these barriers is dependent on intercellular junctions that occlude paracellular transfer of lipid insoluble molecules from the very earliest stages of brain development [1,2] In spite of this physical barrier the concentration of protein in fetal CSF is well known to be much higher than in the adult in all animal species studied [3,4,5,6,7,8,9,10,11,12]. We have recently proposed a model in which binding of SPARC to albumin acts as a shuttle, enabling transfer of the protein from the basal (plasma) membrane of albumin-transferring plexus epithelial cells though the blood–CSF barrier and into the CSF of the brain ventricular system (see [16,17])
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