Faculty Bibliography

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.

BACKGROUND: The choroid plexuses are the interface between the blood and the cerebrospinal fluid (CSF) contained within the ventricular spaces of the central nervous system. The tight junctions linking adjacent cells of the choroidal epithelium create a physical barrier to paracellular movement of molecules. Multispecific efflux transporters as well as drug-metabolizing and antioxidant enzymes functioning in these cells contribute to a metabolic barrier. These barrier properties reflect a neuroprotective function of the choroid plexus. The choroid plexuses develop early during embryogenesis and provide pivotal control of the internal environment throughout development when the brain is especially vulnerable to toxic insults. Perinatal injuries like hypoxia and trauma, and exposure to drugs or toxic xenobiotics can have serious consequences on neurogenesis and long-term development. The present study describes the developmental expression pattern of genes involved in the neuroprotective functions of the blood-CSF barrier. METHODS: The transcriptome of rat lateral ventricular choroid plexuses isolated from fifteen-day-old embryos, nineteen-day old fetuses, two-day old pups, and adults was analyzed by a combination of Affymetrix microarrays, Illumina RNA-Sequencing, and quantitative RT-PCR. RESULTS: Genes coding for proteins involved in junction formation are expressed early during development. Overall perinatal expression levels of genes involved in drug metabolism and antioxidant mechanisms are similar to, or higher than levels measured in adults. A similar developmental pattern was observed for multispecific efflux transporter genes of the Abc and Slc superfamilies. Expression of all these genes was more variable in choroid plexus from fifteen-day-old embryos. A large panel of transcription factors involved in the xenobiotic- or cell stress-mediated induction of detoxifying enzymes and transporters is also expressed throughout development. CONCLUSIONS: This transcriptomic analysis suggests relatively well-established neuroprotective mechanisms at the blood-CSF barrier throughout development of the rat. The expression of many transcription factors early in development raises the possibility of additional protection for the vulnerable developing brain, should the fetus or newborn be exposed to drugs or other xenobiotics.

The protective barriers of the brain provide a complex series of physical and chemical obstacles to movement of macromolecules from the periphery into the central nervous system. Studies on these barriers have been focused on two main research areas: (i) anatomical and physiological descriptions of their properties, including during development where functioning barriers are likely to be important for normal neuronal growth; and (ii), investigations of these barriers during disease and attempts at overcoming their defenses in order to deliver drugs to the central nervous system. Both fields are now advanced by the application of molecular gene expression studies of cerebral endothelia (blood vasculature, site of the blood-brain barrier) and choroid plexus epithelia (site of the blood-cerebrospinal fluid barrier) from developing and adult brains, particularly with respect to solute-linked carriers and other transporters. These new techniques provide a wealth of information on the changing nature of transporters at barrier interfaces during normal development and following disease. This review outlines published findings from transcriptome and qPCR studies of expression of genes coding for transporters in these barriers, with a focus on developing brain. The findings clearly support earlier published physiological data describing specific transport mechanisms across barrier interfaces both in the adult and in particular in the developing brain.

We provide comprehensive identification of embryonic (E15) and adult rat lateral ventricular choroid plexus transcriptome, with focus on junction-associated proteins, ionic influx transporters and channels. Additionally, these data are related to new structural and previously published permeability studies. Results reveal that most genes associated with intercellular junctions are expressed at similar levels at both ages. In total, 32 molecules known to be associated with brain barrier interfaces were identified. Nine claudins showed unaltered expression, while two claudins (6 and 8) were expressed at higher levels in the embryo. Expression levels for most cytoplasmic/regulatory adaptors (10 of 12) were similar at the two ages. A few junctional genes displayed lower expression in embryos, including 5 claudins, occludin and one junctional adhesion molecule. Three gap junction genes were enriched in the embryo. The functional effectiveness of these junctions was assessed using blood-delivered water-soluble tracers at both the light and electron microscopic level: embryo and adult junctions halted movement of both 286Da and 3kDa molecules into the cerebrospinal fluid (CSF). The molecular identities of many ion channel and transporter genes previously reported as important for CSF formation and secretion in the adult were demonstrated in the embryonic choroid plexus (and validated with immunohistochemistry of protein products), but with some major age-related differences in expression. In addition, a large number of previously unidentified ion channel and transporter genes were identified for the first time in plexus epithelium. These results, in addition to data obtained from electron microscopical and physiological permeability experiments in immature brains, indicate that exchange between blood and CSF is mainly transcellular, as well-formed tight junctions restrict movement of small water-soluble molecules from early in development. These data strongly indicate the brain develops within a well-protected internal environment and the exchange between the blood, brain and CSF is transcellular and not through incomplete barriers.

For a long time the brain has been considered an immune-privileged site due to a muted inflammatory response and the presence of protective brain barriers. It is now recognized that neuroinflammation may play an important role in almost all neurological disorders and that the brain barriers may be contributing through either normal immune signaling or disruption of their basic physiological mechanisms. The distinction between normal function and dysfunction at the barriers is difficult to dissect, partly due to a lack of understanding of normal barrier function and partly because of physiological changes that occur as part of normal development and ageing. Brain barriers consist of a number of interacting structural and physiological elements including tight junctions between adjacent barrier cells and an array of influx and efflux transporters. Despite these protective mechanisms, the capacity for immune-surveillance of the brain is maintained, and there is evidence of inflammatory signaling at the brain barriers that may be an important part of the body's response to damage or infection. This signaling system appears to change both with normal ageing, and during disease. Changes may affect diapedesis of immune cells and active molecular transfer, or cause rearrangement of the tight junctions and an increase in passive permeability across barrier interfaces. Here we review the many elements that contribute to brain barrier functions and how they respond to inflammation, particularly during development and aging. The implications of inflammation-induced barrier dysfunction for brain development and subsequent neurological function are also discussed.

Exchange mechanisms across the blood-cerebrospinal fluid (CSF) barrier in the choroid plexuses within the cerebral ventricles control access of molecules to the central nervous system, especially in early development when the brain is poorly vascularised. However, little is known about their molecular or developmental characteristics. We examined the transcriptome of lateral ventricular choroid plexus in embryonic day 15 (E15) and adult mice. Numerous genes identified in the adult were expressed at similar levels at E15, indicating substantial plexus maturity early in development. Some genes coding for key functions (intercellular/tight junctions, influx/efflux transporters) changed expression during development and their expression patterns are discussed in the context of available physiological/permeability results in the developing brain. Three genes: Secreted protein acidic and rich in cysteine (Sparc), Glycophorin A (Gypa) and C (Gypc), were identified as those whose gene products are candidates to target plasma proteins to choroid plexus cells. These were investigated using quantitative- and single-cell-PCR on plexus epithelial cells that were albumin- or total plasma protein-immunopositive. Results showed a significant degree of concordance between plasma protein/albumin immunoreactivity and expression of the putative transporters. Immunohistochemistry identified SPARC and GYPA in choroid plexus epithelial cells in the embryo with a subcellular distribution that was consistent with transport of albumin from blood to cerebrospinal fluid. In adult plexus this pattern of immunostaining was absent. We propose a model of the cellular mechanism in which SPARC and GYPA, together with identified vesicle-associated membrane proteins (VAMPs) may act as receptors/transporters in developmentally regulated transfer of plasma proteins at the blood-CSF interface.

The adult brain functions within a well-controlled stable environment, the properties of which are determined by cellular exchange mechanisms superimposed on the diffusion restraint provided by tight junctions at interfaces between blood, brain and cerebrospinal fluid (CSF). These interfaces are referred to as "the" blood-brain barrier. It is widely believed that in embryos and newborns, this barrier is immature or "leaky," rendering the developing brain more vulnerable to drugs or toxins entering the fetal circulation from the mother. New evidence shows that many adult mechanisms, including functionally effective tight junctions are present in embryonic brain and some transporters are more active during development than in the adult. Additionally, some mechanisms present in embryos are not present in adults, e.g., specific transport of plasma proteins across the blood-CSF barrier and embryo-specific intercellular junctions between neuroependymal cells lining the ventricles. However developing cerebral vessels appear to be more fragile than in the adult. Together these properties may render developing brains more vulnerable to drugs, toxins, and pathological conditions, contributing to cerebral damage and later neurological disorders. In addition, after birth loss of protection by efflux transporters in placenta may also render the neonatal brain more vulnerable than in the fetus.

Specialized populations of choroid plexus epithelial cells have previously been shown to be responsible for the transfer of individual plasma proteins from blood to the cerebrospinal fluid (CSF), contributing to their characteristically high concentrations in CSF of the developing brain. The mechanism of this protein transfer remains elusive. Using a marsupial, Monodelphis domestica, we demonstrate that the albumin-binding protein SPARC (osteonectin/BM-40/culture-shock protein) is present in a subset of choroid plexus epithelial cells from its first appearance, throughout development, and into adulthood. The synthesis of SPARC by the lateral ventricular plexus was confirmed with real-time PCR. The expression level of SPARC was higher in plexuses of younger than older animals. Western blot analysis of the gene product confirmed the quantitative PCR results. The co-localization of SPARC and albumin shown by immunocytochemistry and its cellular location indicate that this glycoprotein may act as a recognition site for albumin. In addition, the numbers of SPARC-immunopositive cells and its expression were responsive to experimental changes of albumin concentration in the blood. It is suggested that SPARC may be one of the molecules that govern the uptake and delivery of proteins from blood to the CSF. The results also confirm that protein transfer across the blood-CSF barrier is developmentally and physiologically regulated.

A developmentally regulated protein-specific transfer mechanism across choroid plexus epithelial cells has previously been proposed to contribute to the characteristically high concentration of protein in cerebrospinal fluid (CSF) in the immature brain. Here we demonstrate that this mechanism is sensitive to protein variations in plasma resulting in changed numbers of transferring cells for individual proteins and altered transfer into the CSF. Pups of Monodelphis domestica at postnatal day (P)9, P65 and P110 were injected intraperitoneally with either adult Monodelphis plasma or exogenous bovine fetuin. Samples of CSF, blood and brain were collected from terminally anaesthetized animals 3-48 h later. The concentration of total protein was measured and levels of albumin, hemopexin, alpha-fetoprotein and bovine fetuin were estimated by western blotting. Numbers of lateral ventricular choroid plexus cells positive for total and individual plasma proteins were counted in paraffin sections of brains stained with appropriate antibodies. Following intraperitoneal injections, the content of proteins in the CSF increased at all three ages, but the concentration increased only in the CSF of older animals. The total numbers of plexus cells positive for plasma protein did not change significantly, but cells positive for individual proteins did. Fetuin was detected in all protein-positive cells, but apparently displaced alpha-fetoprotein and, to a lesser degree, hemopexin. The results indicate that protein transfer across the blood/CSF barrier appears to be regulated by a molecular recognition mechanism that is probably saturable but may not be as specific for individual proteins as previously suggested.

Tracing the exact origins of modern science can be a difficult but rewarding pursuit. It is possible for the astute reader to follow the background of any subject through the many important surviving texts from the classical and ancient world. While empirical investigations have been described by many since the time of Aristotle and scientific methods have been employed since the Middle Ages, the beginnings of modern science are generally accepted to have originated during the 'scientific revolution' of the 16th and 17th centuries in Europe. The scientific method is so fundamental to modern science that some philosophers consider earlier investigations as 'pre-science'. Notwithstanding this, the insight that can be gained from the study of the beginnings of a subject can prove important in the understanding of work more recently completed. As this journal undergoes an expansion in focus and nomenclature from cerebrospinal fluid (CSF) into all barriers of the central nervous system (CNS), this review traces the history of both the blood-CSF and blood-brain barriers from as early as it was possible to find references, to the time when modern concepts were established at the beginning of the 20th century.