THE UPA-PAI1-UPAR COMPLEX PROMOTES NEURONAL MIGRATION AND NEURITOGENESIS IN VITRO.

BARBER MARIANA; LINO NOELIA; RAPACIOLI MELINA; TERUEL LUISA; DI NAPOLI JENNIFER; RODRIGUEZ CELIN ALEJANDRA; DUARTE SANTIAGO; SANCHEZ VIVIANA

Buenos Aires. Argentina

Congreso; 4th International Meeting of the Latin Ametican Society of developmental Biology; 2008

Latin American Society of Developmental Biology

The upa-pai1-upar complex promotes neuronal migration and neuritogenesis in vitro. Barber Mariana1, Lino Noelia1, Rapacioli Melina2, Teruel Luisa1, Di Napoli Jennifer1, Rodriguez Celin Alejandra2, Duarte Santiago1, Sanchez Viviana1. 1Insitute of Cell Biology and Neuroscience Medicine School, Buenos Aires University 2Interdisciplinary Group in Theoretical Biology. Favaloro University. vsanchez@fmed.uba.ar 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 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%CO2 at 37 ºC for 20 hours. Afterwards, explants were treated with a) uPA, b) PAI1 or c) 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. Inmunohistochemistry: After the treatment explants were fixed in 4% paraformaldehyde in 0.1M phosphate buffer, blocked with 0.5% normal goat serum and incubated with: anti-PAI-1 (Calbiochem®, final concentration 2,5 µg/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 4ºC. 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 to detect uPAR, PAI1or 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% +/-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 also 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 with 0.37nM or 1.5 nM of uPA-PAI complex. There were not significant differences between explants treated with 3.0 nM of uPA-PAI complex and the controls. The immunohistochemistry showed that the addition of uPA-PAI1 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-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 stress 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