Chemotactic effects of Urokinase-type Plasminogen Activator (uPA) in postmitotic neurons and growth cones in the development of the central nervous system

CROMBERG, LUCAS; LINO, NOELIA; TERUEL, LUISA; RAPACIOLI, MELINA; DUARTE, SANTIAGO; FLORES, VLADIMIR; SANCHEZ, VIVIANA

Córdoba. Argentina

Congreso; First Joint Meeting of the Argentine Society for Neuroscience (SAN) and the Argentine Workshop in Neurosciences (TAN); 2009

Sociedad Argentina de Neurociencias

Several signalling molecules have been implicated in controlling neuronal migration and a number of these same molecules play important roles in axonal branching and guidance. These molecules modify the cell motility promoting either inhibition or promotion of cell motility. The growth cones at the tip of the neurites senses the concentration gradient of these same molecules and translates them into changes in the rate of outgrowth and promotes the axonal branching. The aim of this study was to investigate the possible chemotactic effect of uPA on the neuronal migration and the neuritogenis Optic tectum explants from chicken embryos of 6 days, were grown in DMEM/F12, incubated at 37ºC for 20 hours. After that, the explants were exposed to a gradient of uPA using acrylic beads embedded in uPA at a final concentration of 20 nM. After 15 or 30 minutes, the explants were photographed. The images were assembled and the explants were divided in two halves, in order to compare the side next to the beads with the opposing side. Three different variables were measured: cell migration pattern, number of filopodia and growth cones size. After the treatment, the explants were fixed in paraformaldehyde 4% in 0.1M phosphate buffer, and incubated with primary antibody anti-b3tubulin. After that the explants were incubated with secondary antibody Alexa Fluor 488 and phalloidin-rhodamine to detect microtubules and actin cytoskeleton. The region exposed to the gradient showed an significant increase in the number of neurons that left the explants and in the number of filopodia in comparison with the control condition. The submitted to the uPA gradient halves showed large growth cones with microtubule loops whose area were bigger than the control conditions. The results indicate that uPA acts as a chemotactic molecule promoting the directional neuronal migration and the formation of large growth cones regulating the axon outgrowth.