Determining the Transcriptional Environment that Suppresses Expression of Tight Junctional Proteins at the Blood-Brain Barrier in Neuroinflammation

Ignacio Romero, Ph.D.

The Open University

Funded in September, 2006: $200000 for 3 years


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Exploring How Brain Inflammation May Produce a Faulty Blood-Brain Barrier

The investigators will undertake molecular studies of how brain inflammation may weaken the normally impermeable blood-brain barrier in conditions as diverse as autoimmune multiple sclerosis and HIV-related dementia.  The findings may lead to more effective therapies for a wide range of inflammatory brain diseases.

Blood vessels in the brain tightly restrict passage of molecules from the bloodstream into brain tissues and thus constitute the formidable “blood-brain barrier” (BBB).  By keeping foreign molecules out of the extracellular environment in the brain, the BBB ensures that brain cells’ electrical properties are not destabilized. Cells that form the brain’s blood vessel membranes are called “endothelial” cells.  They are joined together through structures, called “tight junctions,” composed of mainly three specific proteins. In many inflammatory brain conditions, however, the tight junctions are weakened, rendering the BBB faulty.  Molecules in the blood, including immune cells that are recruited to the brain’s inflammatory sites, seep into the brain, and amplify the inflammatory process. 

Scientists do not know why the diverse array of inflammatory brain conditions—ranging from autoimmune diseases like MS to HIV-related dementia—results in a faulty BBB.  Recent studies suggest that brain inflammation is associated with lowered levels of the proteins that constitute the BBB’s tight junctions.  The researchers will study, at the molecular level, how the tight junction proteins become depleted.  While degradation may occur once the proteins are formed, the investigators hypothesize that diverse inflammatory substances suppress the proteins’ formation.  Specifically, they suspect that inflammatory substances interfere with the ability of RNA to carry out the directions of DNA to produce the proteins.   They will test this hypothesis in human brain blood vessel endothelial tissue obtained at autopsy or through surgical resection. If this is the case, the investigators will identify the molecular processes involved and then inhibit the molecules responsible to see whether this restores tight junction protein development.

Significance:  This study will help to determine whether brain inflammation suppresses the genetic processes involved in constructing a tight BBB. If this is a common pathway in many diverse inflammatory brain conditions, it could lead to a common approach to preventive and therapeutic interventions.


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Determining the Transcriptional Environment that Suppresses Expression of Tight Junctional Proteins at the Blood-Brain Barrier in Neuroinflammation

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.


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Ignacio Romero, Ph.D.

Ignacio Romero is a Senior Lecturer in Cellular Neuroscience at the Open University (OU), Milton Keynes, United Kingdom. His research is mainly concerned with the roles of cells that form the blood vessels of the brain and spinal cord in immune reactions, in particular how brain cells interact with white blood cells in both health and disease. Prior to taking his position at the OU, he obtained his first degree in Pharmacology (B.Sc.) at the University of Valencia, Spain, in 1987 and his doctoral degree (Ph.D.) at King’s College London, UK, in 1993. His doctoral dissertation was on the toxicity of nitrobenzenes to the blood-brain barrier, an anatomical feature of the blood vessels in the brain and spinal cord that results in the restricted passage of molecules and cells from blood to brain. 

His more recent research focuses on the biology of retroviruses and emerged from his postdoctoral position at the Cochin Institute in Paris in the late 1990s, where he investigated how retroviruses such as HIV and HTLV enter the brain by crossing the blood-brain barrier and cause neurological conditions. His work is carried out in vitro using culture systems designed to mimic the complex cellular environment of the human brain. Since joining the OU in 2000, he has taught courses in biological psychology and cell and molecular biology.