Pharmacology and Therapeutics - Research Publications
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ItemThe inner CSF-brain barrier: developmentally controlled access to the brain via intercellular junctions(FRONTIERS MEDIA SA, 2015-02-12)In the adult the interface between the cerebrospinal fluid and the brain is lined by the ependymal cells, which are joined by gap junctions. These intercellular connections do not provide a diffusional restrain between the two compartments. However, during development this interface, initially consisting of neuroepithelial cells and later radial glial cells, is characterized by "strap" junctions, which limit the exchange of different sized molecules between cerebrospinal fluid and the brain parenchyma. Here we provide a systematic study of permeability properties of this inner cerebrospinal fluid-brain barrier during mouse development from embryonic day, E17 until adult. Results show that at fetal stages exchange across this barrier is restricted to the smallest molecules (286Da) and the diffusional restraint is progressively removed as the brain develops. By postnatal day P20, molecules the size of plasma proteins (70 kDa) diffuse freely. Transcriptomic analysis of junctional proteins present in the cerebrospinal fluid-brain interface showed expression of adherens junctional proteins, actins, cadherins and catenins changing in a development manner consistent with the observed changes in the permeability studies. Gap junction proteins were only identified in the adult as was claudin-11. Immunohistochemistry was used to localize at the cellular level some of the adherens junctional proteins of genes identified from transcriptomic analysis. N-cadherin, β - and α-catenin immunoreactivity was detected outlining the inner CSF-brain interface from E16; most of these markers were not present in the adult ependyma. Claudin-5 was present in the apical-most part of radial glial cells and in endothelial cells in embryos, but only in endothelial cells including plexus endothelial cells in adults. Claudin-11 was only immunopositive in the adult, consistent with results obtained from transcriptomic analysis. These results provide information about physiological, molecular and morphological-related permeability changes occurring at the inner cerebrospinal fluid-brain barrier during brain development.
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ItemAge-Dependent Transcriptome and Proteome Following Transection of Neonatal Spinal Cord of Monodelphis domestica (South American Grey Short-Tailed Opossum)(PUBLIC LIBRARY SCIENCE, 2014-06-10)This study describes a combined transcriptome and proteome analysis of Monodelphis domestica response to spinal cord injury at two different postnatal ages. Previously we showed that complete transection at postnatal day 7 (P7) is followed by profuse axon growth across the lesion with near-normal locomotion and swimming when adult. In contrast, at P28 there is no axon growth across the lesion, the animals exhibit weight-bearing locomotion, but cannot use hind limbs when swimming. Here we examined changes in gene and protein expression in the segment of spinal cord rostral to the lesion at 24 h after transection at P7 and at P28. Following injury at P7 only forty genes changed (all increased expression); most were immune/inflammatory genes. Following injury at P28 many more genes changed their expression and the magnitude of change for some genes was strikingly greater. Again many were associated with the immune/inflammation response. In functional groups known to be inhibitory to regeneration in adult cords the expression changes were generally muted, in some cases opposite to that required to account for neurite inhibition. For example myelin basic protein expression was reduced following injury at P28 both at the gene and protein levels. Only four genes from families with extracellular matrix functions thought to influence neurite outgrowth in adult injured cords showed substantial changes in expression following injury at P28: Olfactomedin 4 (Olfm4, 480 fold compared to controls), matrix metallopeptidase (Mmp1, 104 fold), papilin (Papln, 152 fold) and integrin α4 (Itga4, 57 fold). These data provide a resource for investigation of a priori hypotheses in future studies of mechanisms of spinal cord regeneration in immature animals compared to lack of regeneration at more mature stages.
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ItemMechanisms That Determine the Internal Environment of the Developing Brain: A Transcriptomic, Functional and Ultrastructural Approach(PUBLIC LIBRARY SCIENCE, 2013-07-02)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.