Ryk Regulates Axon Guidance Through Modulating Planar Cell Polarity Signaling

Objective:In the mammalian central nervous system, the corticospinal tract(CST) is the major motor pathway contributing to the control of voluntary movements of the body. Our previous studies reported a role for Ryk in mediating Wnt5 a repulsion of CST in mice. Recent evidence has shown that Ryk regulates planar cell polarity(PCP) signaling through interacting with Vangl2. The aim of this study is to investigate the molecular mechanism of cross-talk between Ryk and PCP signaling pathway, which leads to repulsion of CST axons in response to the Wnt5 a gradient.Methods:1. Immunofluorescence was used to detect the expression of PCP components in corticospinal neurons.2. shRNA lentivirus was used to knockdown the expression of PCP components and test whether PCP components mediate growth cone turning and CST axon guidance.3. Immunofluorescence and co-immunoprecipitation were used to detect the interaction of Ryk and Vangl2.4. Immunofluorescence and western blot were used to detect the expression of Ryk and Vangl2 in subcellular fractions under different concentrations of Wnt5 a, and test the role of endocytosis of Ryk and Ryk–Vangl2 interaction in endocytosis of Vangl2.5. Dunn chamber was used to expose Wnt5 a gradients and test whether the endocytosis of Ryk and Vangl2 mediates growth cone turning.6. Inhibitor or overexpression lentivirus was injected to block Ryk endocytosis or Ryk–Vangl2 interaction and test their importance in CST pathfinding.Results:1. Immunofluorescence results showed that expression of PCP components is upregulated in neonatal corticospinal neurons.2. Knockdown of PCP components by shRNA lentivirus could interrupt the Wnt5 a repulsion of growth cone turning and lead to CST axons presented random extension.3. Immunofluorescence and co-immunoprecipitation results showed that Ryk and Vangl2 interact in corticospnal neurons.4. Immunofluorescence and western blot results showed that endocytosis of Ryk and Vangl2 is activated under high concentration of Wnt5 a, endocytosis of Ryk and Ryk–Vangl2 interaction facilitate endocytosis of Vangl2.5. Ryk and Vangl2 undergo asymmetric endocytosis in growth cone under Wnt5 a gradient, which is essential for growth cone turning.6. Inhibitor or overexpression lentivirus injection experiments showed that blocking Ryk endocytosis or Ryk–Vangl2 interaction could lead to CST axons presented random extension.Conclusion:Ryk and PCP pathway components play important roles in CST anterior–posterior guidance. Ryk–Vangl2 interaction is crucial for PCP signaling to mediate Wnt5 a repulsion of CST axons. Endocytosis of Ryk and Vangl2 is activated under high concentration of Wnt5 a, leading to inhibition of PCP downstream signaling, growth cone turning towards the lower Wnt5 a concentration, which further leads to repulsive behavior of corticospinal axons in response to the Wnt5 a gradient.