The uPA-PAI1-uPAr complex promotes neuronal migration and neuritogenesis in vitro

BARBER, MARIANA; LINO NOELIA; RAPACIOLI MELINA; TERUEL LUISA; VIVIANA SANCHEZ.

Buenos Aires

Congreso; 4th International meeting of the Latin American Society of developmental Biology; 2008

Latin American Society of developmental Biology

Objectives. The aim of this work is to investigate the rol of the urokinase-type plasminogen activator, plasminogen activator inhibitor-1 (uPA-PAI1) complex and the uPA receptor in cell migration and neuritogenesis using the avian optic tectum (OT) as experimental model. Methods: Explants cultures: Ots obteined from 6 days-old-chick embryos were dissected. Explants of cephalic portions of Ots were culture in F12/DMEM with methylcellulose (0.4%), glutamine and N2, 5% CO2 at 37ºC for 20 hours. Afterwards, explantas were treated with a) uPA, b) PAI1 or c) both al different concentrations ranging from 0.37 to 3 nM. After 4 hours, the explants were observed by phase-contrast microscopy and photographed. The images were assembled and four parameters were recorded: a) number of migrasting cells and distance from the explant and b) axonal length and density. Inmunohistochemistry: After treatment explants were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PBS), blocked with 5% normal serum goat in PBS and incubated with: anti-PAI-1 (Calbiochem®, final concentration of 25 µg/ml) or with anti-urikinase-type plasminogen activator receptor (uPAR, Santa Cruz®, final concentration of 0.6 µg/ml) or anti-neurofilaments (final concentration of 1.6 µg/ml) overnight at 4ºC. Coinmunolocalization was performed with explants representative of each experimental conditions. For this purpose, explantas were incubated two of the primary antibodies. After incubation, the primary antibodies were removed and explants were incubated with the appropiate secondary antibodies Alexa Fluor (Molecular Probes®) in order to detec uPAR, PAI1 or neurofilaments. Some of them were incubated with phalloidin-rhodamine (Sigma, final concentration 3 mg/ml) for detection of actin filaments. Results: The Results showed a dose-dependent response. The addition of 0.74 nM of uPA-PAI1 complex produced a significant increase (61.16%+/-1.6%) in the number og migrating cells as well as in the distance of these cells from explans border (19% +/-1.8%). Besides, there was an increase (33.3%+/- 10.9%) in the number and also in the length of axons. The percentages represent the increase respect to the control explantas. The increase in the above mentioned parameters was lower with 0.37 nM or 1.5 nM of uPA-PAI complex. There were no significant differences between explants treatment with 3.0 nM of uPA-PAI complex and the controls.The inmunohistochesmistry showed that the addition of uPA-PAI1 complex produce an increase inthenumber of cells displaying stress fibers and that these cells were inmunolabeled with both the anti+uPAR and the anti-PAI1 antibodies. Conclusion: these results suggest that the uPA-PAI1 complex could be involved in promoting cell migration and neuritogenesis during the central nervous system development. The presence of dense networks of strss fibers in uPAR positive neurons suggests that a reorganization of the actin cytoskeleton may be mediated by a uPAR-dependent intracellular signaling pathway. Work supported by grants of UBACyT and CONICET.