Slit2 Inactivates GSK3β to Signal Neurite Outgrowth Inhibition

Justin Byun,Bo Taek Kim,Eun-Mi Hur,Yun Tai Kim,Zhongxian Jiao,Feng-Quan Zhou,Henning Ulrich

doi:10.1371/journal.pone.0051895

Abstract

Slit molecules comprise one of the four canonical families of axon guidance cues that steer the growth cone in the developing nervous system. Apart from their role in axon pathfinding, emerging lines of evidence suggest that a wide range of cellular processes are regulated by Slit, ranging from branch formation and fasciculation during neurite outgrowth to tumor progression and to angiogenesis. However, the molecular and cellular mechanisms downstream of Slit remain largely unknown, in part, because of a lack of a readily manipulatable system that produces easily identifiable traits in response to Slit. The present study demonstrates the feasibility of using the cell line CAD as an assay system to dissect the signaling pathways triggered by Slit. Here, we show that CAD cells express receptors for Slit (Robo1 and Robo2) and that CAD cells respond to nanomolar concentrations of Slit2 by markedly decelerating the rate of process extension. Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth. Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons. Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.

Highlights

The study of directed axon growth has led to the identification of four canonical families of axon guidance molecules, namely, ephrins, netrins, semaphorins, and Slits [1,2,3]
We show that Slit2 regulates GSK3 and axon growth in dorsal root gangion (DRG) neurons, validating Slit2 signaling in primary neurons
CAD cells Express Robos, the Receptors for Slit CAD cell line is a variant of Cath.a, a central nervous system (CNS) catecholaminergic cell line derived from a brain tumor that arose in a transgenic mouse [20]

Summary

Introduction

The study of directed axon growth has led to the identification of four canonical families of axon guidance molecules, namely, ephrins, netrins, semaphorins, and Slits [1,2,3]. The Slit molecules are secreted glycoproteins that are best known for their role in the regulation of axon guidance at the midline, an imaginary line that runs along the longitudinal axis of the central nervous system (CNS) [4,5]. Slits exert their functions by binding to single-pass transmembrane cell surface receptors, roundabout (Robo) [4,6,7,8,9]. Molecular analyses of the downstream events of Slit-Robo signaling have been limited, in part, because of the lack of a model system that produces a readily identifiable phenotype in response to Slit and that can be conveniently manipulated using standard molecular biological methods

Methods

Results

Conclusion