Dysfunction of the blood-brain barrier is a major hallmark of neuroinflammatory diseases. Interestingly, several studies have shown that increased blood-brain barrier permeability in many of these CNS immune disorders is associated with a decreased expression of a number of tight junction proteins in brain endothelial cells at the protein and/or mRNA level. It is thus likely that expression of tight junctional proteins by cerebral endothelium is transcriptionally suppressed by neuroinflammatory mediators.
In this study, we propose to investigate the transcriptional environment in cultured human cerebral endothelial cells that regulates expression of three integral tight junctional proteins (occludin, claudin-5 and ZO-1) involved in the maintenance of blood-brain barrier permeability. We will then try to establish a link between diverse inflammatory signals from the CNS (either cell- or viral-derived) and endothelial permeability at the level of transcriptional regulation of occludin, claudin-5, and ZO-1.
The Specific Aims are:
1. We will measure the transcriptional activity of genes coding for three tight junctional proteins, occludin, claudin-5 and ZO-1, in human CECs in response to inflammatory mediators. We will initially use reporter gene constructs with cloned non-coding DNA fragments of the selected genes to analyze transcriptional activity. An important indicator of the level of RNA production from a particular gene is the protein content of the supporting chromosomal region, the chromatin, and the presence of polymerases that synthesize the RNA chains. We also propose to detect the presence of RNA polymerase II by chromatin immunoprecipitation (ChIP) techniques as an indirect measure of gene activity.
2. We will identify critical transcription factors (TFs) and/or changes in chromatin structure that are involved in transcriptional regulation of TJ proteins in human CECs under different inflammatory conditions. We will use two approaches to determine the transcriptional regulation of these genes: ChIP techniques to determine the levels of histone modifications affecting gene expression, and DNA footprinting assays to identify putative binding partners to the DNA promoter regions.
3. We shall inhibit the TF activity and/or expression and histone posttranslational modifications (either using pharmacological inhibition or siRNA techniques) identified in Aim 2 and assess its consequences on endothelial permeability using an in vitro human blood-brain barrier system.
It is clearly essential to learn about the specific transcriptional environment that controls expression of tight junctional proteins by human cerebral endothelium, since modulation of transcriptional activity may constitute an important strategy to prevent impairment of the blood-brain barrier during inflammation.