The Role Of P190 Protein In Maintaining Blood-Brain Barrier Function

Background and PurposeP190 is a neuronal transmembrane protein, which was initially found at the paranodes of the myelinated fibers, and mainly expressed in vertebrate brain tissues. Ovary, pancreas, intestines, lung, heart and testis also have low expression for P190 protein. P190 can contact with NF155 and together form paranodal junction at the paranodes of myelinated fibers. Paranodal junction is similar with septate junction, which is widely distributed at blood-nerve barrier in invertebrate such as Drosophila. Present studies indicate that the main functions of septate like junction in vertebrate are maintaining rapid and efficient propagation of action potential, messages transmition between axon and glia, specific axon domain formation, and barrier formation between axon-glia gap and extracellular matrix. The abnormal expression of P190 can cause serious nervous system disorder. P190-/-type mouse have low nerve impulse conduction speed and ill or death with neural degeneration after delivery. Other studies indicate that the expression of P190 is obviously low in multiple sclerosis patients. Low expression of P190 protein can cause axon demyelination and nerve degeneration. The diabetic neuropathies rats also have low P190 expression. Those indicate the important function of P190 protein, but its specific functions and mechanisms still need studies.The blood-brain barrier (BBB) is a highly specialized neurovascular system which is composed of brain microvascular endothelial cells, astrocytes, basilar membrane and pericytes. The brain microvascular endothelial cells are the most important components for BBB, and they are connected together by tight junctions and adhesion junctions. Many central nervous diseases have dispersed endothelial cell-cell junctions, including Alzheimer's disease and multiple sclerosis. Now people already have certain understanding on the function mechanism of tight junctions and adhesion junctions, but the mechanism is still not so clear. Our laboratory found that besides nerve axons, HBMECs also express P190 highly, and the P190 potentially assembled on the membrane at cell-cell junctions. Based on this phenomenon, we construct different P190 mutants and test the function of different mutants and normal HBMEC. We also exploit yeast two hybrid method to screen proteins interacting with P190 intracellular region form human fetal brain library, and wish to find out the P190 protein function mechanism.Methods1,Overlap PCR method to obtain P190 band4.1 binding domain deletion gene sequences. Then eukaryotic expression plasmid pcDNA 3.1/myc-His B-P190 was constructed by molecular cloning technology, and then was transfected into P26 HBMECs.2,In vitro tube formation assay detect the tube formation ability of different mutated endothelial cells or control cells.3,Hanging-drop aggregation assay test the cell-cell adhesion of different mutated cells or control cells.4,Test the permeability of cell monolayers formed by different mutated cells or control cells to HRP.5,Western blot investigate the expression of tight junction protein ZO-1 and adhesion junction protein VE-cadherin in different mutated cells or in control cells.6,Immunocytochemical method investigate the distribution of tight junction protein ZO-1 and adhesion junction protein VE-cadherin in different mutated cells and in control cells.7,Screening proteins interact with P190 intracellular domain by yeast two hybrid method and confirm the interaction by GST-pull down. Results1,Successfully obtained band4.1 binding domain deletion gene sequences, and stable transfected to P26 HBMECs.2,In vitro tube formation assay suggested that there is no significant difference between P190 mutated cells and the control cells3,Hanging-drop aggregation assay indicated that the intercellular adhesion was obviously weak in P190 intracellular domain deleted cells than in the control cells or other kinds of mutated cells.4,P190 intracellular domain deleted cells monolayer show a high permeability to HRP.5,The expression of tight junction protein ZO-1and adhesion junction protein VE-cadherin has no difference in P190 different mutated cells or control cells.6,Immunofluorescence show a line continuous distribution of junctional protein ZO-1 and VE-cadhetin at cell-cell junctions in control cells and SH3 or band4.1 domain deleted cells; While there is an intermittent distribution of ZO-1 and VE-cadhetin and interrupted cell-cell junction in P190 intracellular domain deleted cells.7,Obtained 14 interacted gene sequence but need further certification.ConclusionDeletion of the intracellular domain (1305-1384aa) of P190 can decrease the adhesion between human brain microvascular endothelial cells and increase the permeability of the endothelial cells monolayer. But whether the P190 protein participates in or regulates the junctions between HBMECs is still need further study.