CONICET | Buscador de Institutos y Recursos Humanos

We have previously demonstrated that EphA3 stimulates axons of retinal ganglion cells to grow toward caudal tectum. We suggested that ephrin-As act as receptors for EphA3 and it is known that ephrin-As are located in cholesterol-rich membrane microdomains (rafts). Our objective was to examine if membrane-cholesterol microenvironment is necessary for axon growth and guidance. We used cultures of chicken embryos retinal explants exposed to methyl-β-cyclodextrin (MCD) at different concentrations and/or aggregated EphA3Fc. We analyzed the axonal length and morphology, and studied the expression pattern of EphAs and ephrin-As by immunocytochemistry. Explants exposed to MCD presented an early significant increase in the number of axonal varicosities and of collapsed growth cones followed by a significant decrease in the axonal length. Ephrin-A2 was reduced in the growth cones and was increased in the axonal varicosities. Explants exposed to EphA3Fc presented a significant increase in the number of expanded growth cones. Exposure to EphA3Fc and MCD together produced intermediate results than those produced by them separately. Results obtained with MCD suggest that cholesterol depletion decreases the ephrin-As located in growth cones and the axonal growth by stimulating endocytosis. The substractive effects of EphA3 and MCD suggest: a) axonal growth mediated by EphA3 is reduced by MCD for resting membrane by endocytosis and/or reducing the ephrin-As in the growth cones, and b) the interaction between ephrin-As and EphA3 reduces the endocytosis mediated by MCD. These data suggest that cholesterol environment is necessary for regulating the axon guidance mediated by EphA/ephrin-A system. Funded by CONICET and UBA.