| dc.description.abstract | The blood-brain barrier (BBB) is comprised of the vasculature surrounding the central nervous system (CNS), and most importantly consists of endothelial cells. These brain endothelial cells have unique properties that protect the neurons which are integral for CNS function. These specialized endothelial cell functions allow for modulation of the flow of solutes from the bloodstream to the CNS, and include reduced flow across endothelial cells mediated via transporters (transcellular pathway), and reduced flow between cells (paracellular transport), which is mediated by specialized tight junctions (TJs). While this is beneficial for protecting the neurons from harmful blood-borne pathogens, this also poses a problem for drug delivery across the barrier, as neurotherapeutics which are large and hydrophilic in nature are not able to pass through the TJs through paracellular transport. Other cell types such as pericytes, microglia, astrocytes, etc. form crosstalk interactions with the endothelial cells at the BBB, creating an overarching neurovascular unit that maintains proper BBB function. However, the focus of this investigation is on endothelial cells, with respect to tight junctions and their associated mediation of BBB integrity. As the BBB is dynamic, variations in TJ expression mechanisms can affect BBB permeability through various regulatory pathways which we are interested in studying in this investigation, namely circadian mediated regulation, and 3 UTR mediated post-transcriptional and post-translational regulation of the key TJ component claudin-5.
It was previously shown by Hudson et al., 2019, that claudin-5 (which is the most enriched tight junction component) is associated with Bmal1 (a circadian clock component), and cycles in tissues in a circadian manner, with other tight junction proteins such as ZO-1 and occludin cycling in a temporal manner. Serum shock studies as shown by Balsalobre et al., 1998 can recapitulate a circadian rhythm in cell lines, and these experiments were done for a short period of time in Hudson et al., 2019. The goal of this investigation was to model the circadian rhythm in endothelial cell lines for an extended period of time (72 hours), and characterize protein and transcript expression patterns, as this could have an impact on BBB integrity. Temporal changes were observed for all TJ components and clock components analyzed (claudin-5, occludin, ZO-1, Bmal1). In the future, changes in BBB permeability should be looked at, with particular focus on points when claudin-5 expression is high and low. These changes can be taken advantage of, and drug delivery could be designed for administration at points when BBB permeability is higher, as revealed by TJ protein expression patterns.
The 3 UTR is a non-coding region on the mRNA transcript that has been known to mediate RNA-protein interactions via cis elements, about which not much is known. This has an effect on post-transcriptional and post-translational modifications for the overall protein. It was found that a 3 UTR single nucleotide polymorphisms (SNP) on claudin-5, rs10314, was associated with schizophrenia in certain populations, and a study by Greene et al., 2018 showed that rs10314 results in a 50% downregulation of claudin-5, and up to 75% of claudin-5 is downregulated in patients with both the 22q11 deletion syndrome as well as presence of this SNP. This has a significant effect on tight junction formation and BBB integrity. Our study focused on expanding these findings, and assessing the effect that various other SNPs in the 3 UTR of claudin-5 have on its overall expression. Various plasmid constructs were made using site- directed mutagenesis to express various SNPs in the 3 UTR, and were assayed for claudin-5 expression levels. Several 3 UTR claudin-5 SNPs resulted in abrogated protein expression, which indicates that the 3 UTR plays a significant role in the overall protein structure and function, and should be explored further, particularly with relation to the associated RNA-binding protein (RBP) interactions, that impact post-transcriptional and post-translational outcomes. Overall, the results are consistent with the general findings in the field that suggest that tight junction expression at the BBB is not static, but rather constantly changing through various regulatory mechanisms in order to maintain homeostasis and protect the CNS. As BBB breakdown is a hallmark of neurological diseases, it is important to understand these phenomena. | en |
