GDNF and EphA3 ectodomain regulate retinal ganglion cell axon guidance.

SPELZINI, GONZALO; MEDORI, MARA; RONDON, JONATHAN; ROBALDO, MANUEL; TERUEL, LUISA R; SANCHEZ, VIVIANA; FIORE, LUCIANO; SCICOLONE, GABRIEL

Congreso; Neuroscience 2021. 50th Annual Meeting; 2021

Society for Neuroscience

GDNF and EphA3 ectodomain regulate retinal ganglion cell axon guidance.Gonzalo Spelzini1,2, Mara Medori1,2, Jonathan Rondón Martínez1,2, Manuel Robaldo1,2, Luisa R Teruel1,2, Viviana Sanchez1,2, Luciano Fiore1,2, Gabriel Scicolone1,2.1. CONICET ? Universidad de Buenos Aires, Instituto de Biología Celular y Neurociencias ?Prof. E. De Robertis? (IBCN). Ciudad de Buenos Aires, Argentina.2. Universidad de Buenos Aires, Facultad de Medicina, Departamento de Biología Celular, Histología, Embriología y Genética. Ciudad de Buenos Aires, Argentina.Previously we showed that tectal EphA3 stimulates axon growth of nasal retinal ganglion cells (RGC) toward the caudal tectum preventing them from branching in the rostral tectum. GDNF participates in the formation of topographic connections of motor neurons of the spinal cord with the muscles of the lower limbs, stimulating axon growth interacting with Eph/ephrin system. However its effect on RGC axon growth and guidance is not clear and there is no evidence about the combinatorial effects of EphA3 and GDNF. We demonstrated that GDNF and EphA3 stimulate RGC axon growth.Our purpose was to study the individual and combinatorial effects of EphA3 and GDNF on RGC axon guidance.We cultured chicken embryo nasal retinal explants and dissociated RGCs on poly-L-lysine/laminin and exposed them to control conditions, to EphA3 ectodomain (aggregated EphA3-Fc), to GDNF or to EphA3-Fc plus GDNF to evaluate their effects on axon guidance.To confirm that EphA3 has a positive effect on the axon guidance of nasal RGCs, we developed the technique of Stripe assay using retinal explants. Nasal RGCs axons preferentially grow on a substrate with EphA3 instead of a permissive substrate of laminin in the stripe assay. This axonal preference is not modified with the addition of GDNF.In order to evaluate the participation of soluble gradients of GDNF and EphA3 on the axonal guidance of nasal RGCs, we developed a chemotaxis test using the Dunn´s chamber. This consists of two concentric rings in one glass slide face. An annular bridge lower than the rest of the slide separates both wells. The external well contains a chemoattractant while the internal well contains culture medium. A linear gradient of chemoattractant is formed by diffusion across the bridge between two wells.The results showed that RGC axons change their directions of growth toward gradients of EphA3 and GDNF. Besides, EphA3-Fc and GDNF together present the highest effects in comparison to the effects produced by any of them alone.This demonstrates that GDNF and EphA3 not only stimulate RGC axon growth but their gradients also have chemoattractant effects on nasal RGC axons, suggesting that both of them present synergistic effects.Supported by UBACYT 0769.