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

BARBER, MARIANA; LINO, NOELIA; RAPACIOLI, MELINA; TERUEL, L; DI NAPOLI, J; RODRÍGUEZ CELÍN ALEJANDRA; DUARTE, SANTIAGO; SANCHEZ, VIVIANA

Buenos Aires, Argentima

Congreso; 4th International Meeting of the Latin American Society of Developmental Biology; 2008

Objectives. The aim of this work is to investigate the role of the urokinase-type plasminogenactivator, plasminogen activator inhibitor-1 (uPA-PAI1) complex and the uPA receptor in cellmigration and neuritogenesis using the avian optic tectum (OT) as experimental model. Methods. Explants cultures: OTs ´obtained from 6 days-old chick embryos were dissected. Explants of cephalic portions of OTs were cultured in F12/DMEM with methylcellulose (0,4%), glutamine and N2, 5%C02 at 37°C for 20 hours. Afterwards, explants were treated with a) uPA, b) PAI1 or e) both at different concentrations ranging from 0.37 to 3.0 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 migrating cells and distance from the explant and b) axonal length and density. Immunohistochemistry: After the treatment explants were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer: blocked with 0.5% normal goat serum and incubated with: anti-PAI-1 (Calbiochem®, final concentration 2,5 ìg/ml) or with anti-urokinase-type plasminogen activator receptor (uPAR, Santa CruZ®, final concentration of 0.6 mg/ml) or anti neurofilaments (final concentration 1.6mg/ml) overnight at 4°C. Coimmunolocalization was performed with explants representative of each experimental conditions. For this purpose, explants were incubated with two of the primary antibodies. After incubation the primary antibodies were removed and explants were incubated with the appropriate secondary antibodies Alexa Fluor (Molecular Probes®) in order todetect 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-PAl1 complex produced a significant increase (61.16% +/-16%) in the number of migrating cells as well as in the distance of these cells from the explants border (19% +/-1.8%). Besides, there was an increase (33.3 %+/-10.9%) in the number and lso in the length of axons. The percentages represent the increase respect to the control explants. The increase in the above mentioned parameters was lower with 0.37nM or 1.5 nM of uPA-PAI1 complex. There were not significant differences between explants treated with 3.0 nM of uPA-PAI1 complex and the controls. The imunohistochemistry showed that the addition of uPA-PAl1 complex produces an increase in the number cells displaying stress fibers and that these cells were immunolabeled with both the anti-uPAR and the anti-PAl1 antibodies. Conclusion. These results suggest that the uPA-PAl1 complex could be involved in promoting cell migration and neuritogenesis during the central nervous system development. The presence of dense networks of stress fibers in uPAR positive neurons suggests that a reorganization of the actin cytoskeleton may be mediated by a uPAR-dependent intracellular signaling pathway.