CRC 629 B02 - Pertussis toxin-induced dynamic permeabilisation of cerebral endothelial barriers: retrograde transport, subcellular organization and cell migration

Basic data for this project

Description

Two complex cellular barriers maintain the homeostasis of the brain: the blood-brain-barrier (BBB) and the blood-liquor-barrier (BLB). The integrity of these barriers can be compromised by infectious and inflammatory processes, which may result in a permanent reduction of brain function. The exotoxin of Bordetella pertussis pertussis toxin (PT) exclusively disrupts the integrity of the BBB and might be instrumental for encephalopathies associated with pertussis disease (whooping cough). We could show that retrograde transport to the Golgi apparatus and the ER is essential for the intoxication of human brain microvascular endothelial cells (HBMEC) that leads to a transient parallel reduction of barrier function, the transient disintegration of Golgi organization and a transient increase in the diapedesis of human monocytes in a tissue culture model system mimicking the BBB. Barrier disintegration is associated with the PT-mediated ADP-ribosylation of Gai-2 in Golgi structures and with a removal of Claudin-1 from the tight junctions (TJ). In this project we want to understand the molecular mechanisms and signalling pathways underlying these effects with a particular emphasis on the alterations in TJ. In parallel, we will investigate whether the PT–induced transient disruption of barrier integrity enhances the translocation of pathogenic bacteria either directly and/or alternatively following a ’Trojan horse’ mechanism by employing cells of the immune system. For this we will use molecular biology (e. g. siRNA, DNA-microarrays) and cell biology methods and techniques (immunofluorescence, electron microscopy techniques, live imaging - LSM). Transendothelial resistance (TER) of HBMEC monolayers upon incubation with PT. HBMEC grown on Transwell filters were incubated with or without PT (200 ng/ml) and the TER was determined after different time points. After the TER decreased for 3-12 h it slowly approached the initial value after about 24 h. Specific objectives: What are the molecular mechanisms and signaling processes induced by PT resulting in transient barrier disruption in endothelia ? How is the inhibition of Gai-2 linked to the signalling to the TJ complexes ? Are in addition to claudin-1 also other TJ-associated proteins affected ? Which factors are involved in retrograde transport and the transient changes in the cellular organization of cerebral endothelial cells ? How does the transient disruption of barrier function enhance the translocation of pathogenic bacteria such as e.g. Neisseria, Haemophilus, Pneumococcus ? Does the ’Trojan horse’ mechanism represent a feasible alternative ? What are the consequences of the BBB-disrupting activities of PT in vivo ? Does PT also enhance the translocation in vivo (immune cells, pathogens, small molecules) ?