All Title Author
Keywords Abstract

PLOS ONE  2012 

Slit2 Inactivates GSK3β to Signal Neurite Outgrowth Inhibition

DOI: 10.1371/journal.pone.0051895

Full-Text   Cite this paper   Add to My Lib


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.


[1]  Dickson BJ (2002) Molecular mechanisms of axon guidance. Science 298: 1959–1964.
[2]  Huber AB, Kolodkin AL, Ginty DD, Cloutier JF (2003) Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu Rev Neurosci 26: 509–563.
[3]  Tessier-Lavigne M, Goodman CS (1996) The molecular biology of axon guidance. Science 274: 1123–1133.
[4]  Brose K, Bland KS, Wang KH, Arnott D, Henzel W, et al. (1999) Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96: 795–806.
[5]  Nguyen Ba-Charvet KT, Brose K, Ma L, Wang KH, Marillat V, et al. (2001) Diversity and specificity of actions of Slit2 proteolytic fragments in axon guidance. J Neurosci 21: 4281–4289.
[6]  Kidd T, Bland KS, Goodman CS (1999) Slit is the midline repellent for the robo receptor in Drosophila. Cell 96: 785–794.
[7]  Kidd T, Brose K, Mitchell KJ, Fetter RD, Tessier-Lavigne M, et al. (1998) Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors. Cell 92: 205–215.
[8]  Li HS, Chen JH, Wu W, Fagaly T, Zhou L, et al. (1999) Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons. Cell 96: 807–818.
[9]  Chedotal A (2007) Slits and their receptors. Adv Exp Med Biol 621: 65–80.
[10]  Ypsilanti AR, Zagar Y, Chedotal A (2010) Moving away from the midline: new developments for Slit and Robo. Development 137: 1939–1952.
[11]  Jaworski A, Tessier-Lavigne M (2012) Autocrine/juxtaparacrine regulation of axon fasciculation by Slit-Robo signaling. Nat Neurosci 15: 367–369.
[12]  Wang KH, Brose K, Arnott D, Kidd T, Goodman CS, et al. (1999) Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell 96: 771–784.
[13]  Ma L, Tessier-Lavigne M (2007) Dual branch-promoting and branch-repelling actions of Slit/Robo signaling on peripheral and central branches of developing sensory axons. J Neurosci 27: 6843–6851.
[14]  Yuasa-Kawada J, Kinoshita-Kawada M, Rao Y, Wu JY (2009) Deubiquitinating enzyme USP33/VDU1 is required for Slit signaling in inhibiting breast cancer cell migration. Proc Natl Acad Sci U S A 106: 14530–14535.
[15]  Narayan G, Goparaju C, Arias-Pulido H, Kaufmann AM, Schneider A, et al. (2006) Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression. Mol Cancer 5: 16.
[16]  Bedell VM, Yeo SY, Park KW, Chung J, Seth P, et al. (2005) roundabout4 is essential for angiogenesis in vivo. Proc Natl Acad Sci U S A 102: 6373–6378.
[17]  Wang B, Xiao Y, Ding BB, Zhang N, Yuan X, et al. (2003) Induction of tumor angiogenesis by Slit-Robo signaling and inhibition of cancer growth by blocking Robo activity. Cancer Cell 4: 19–29.
[18]  Jones CA, London NR, Chen H, Park KW, Sauvaget D, et al. (2008) Robo4 stabilizes the vascular network by inhibiting pathologic angiogenesis and endothelial hyperpermeability. Nat Med 14: 448–453.
[19]  Qi Y, Wang JK, McMillian M, Chikaraishi DM (1997) Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation. J Neurosci 17: 1217–1225.
[20]  Suri C, Fung BP, Tischler AS, Chikaraishi DM (1993) Catecholaminergic cell lines from the brain and adrenal glands of tyrosine hydroxylase-SV40 T antigen transgenic mice. J Neurosci 13: 1280–1291.
[21]  Hur EM, Zhou FQ (2010) GSK3 signalling in neural development. Nat Rev Neurosci 11: 539–551.
[22]  Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A, et al. (2005) GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell 120: 137–149.
[23]  Mao Y, Ge X, Frank CL, Madison JM, Koehler AN, et al. (2009) Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell 136: 1017–1031.
[24]  Hartigan JA, Xiong WC, Johnson GV (2001) Glycogen synthase kinase 3beta is tyrosine phosphorylated by PYK2. Biochem Biophys Res Commun 284: 485–489.
[25]  Cole A, Frame S, Cohen P (2004) Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event. Biochem J 377: 249–255.
[26]  Zhou FQ, Walzer M, Wu YH, Zhou J, Dedhar S, et al. (2006) Neurotrophins support regenerative axon assembly over CSPGs by an ECM-integrin-independent mechanism. J Cell Sci 119: 2787–2796.
[27]  Yeo SY, Miyashita T, Fricke C, Little MH, Yamada T, et al. (2004) Involvement of Islet-2 in the Slit signaling for axonal branching and defasciculation of the sensory neurons in embryonic zebrafish. Mech Dev 121: 315–324.
[28]  Ye BQ, Geng ZH, Ma L, Geng JG (2010) Slit2 regulates attractive eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 expression during lung inflammation. J Immunol 185: 6294–6305.
[29]  Kim WY, Zhou FQ, Zhou J, Yokota Y, Wang YM, et al. (2006) Essential roles for GSK-3s and GSK-3-primed substrates in neurotrophin-induced and hippocampal axon growth. Neuron 52: 981–996.
[30]  Jiang H, Guo W, Liang X, Rao Y (2005) Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3beta and its upstream regulators. Cell 120: 123–135.
[31]  Lundstrom A, Gallio M, Englund C, Steneberg P, Hemphala J, et al. (2004) Vilse, a conserved Rac/Cdc42 GAP mediating Robo repulsion in tracheal cells and axons. Genes Dev 18: 2161–2171.
[32]  Wong K, Ren XR, Huang YZ, Xie Y, Liu G, et al. (2001) Signal transduction in neuronal migration: roles of GTPase activating proteins and the small GTPase Cdc42 in the Slit-Robo pathway. Cell 107: 209–221.
[33]  Chivatakarn O, Kaneko S, He Z, Tessier-Lavigne M, Giger RJ (2007) The Nogo-66 receptor NgR1 is required only for the acute growth cone-collapsing but not the chronic growth-inhibitory actions of myelin inhibitors. J Neurosci 27: 7117–7124.
[34]  Hur EM, Yang IH, Kim DH, Byun J, Saijilafu, et al (2011) Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules. Proc Natl Acad Sci U S A 108: 5057–5062.
[35]  Andrews WD, Barber M, Parnavelas JG (2007) Slit-Robo interactions during cortical development. J Anat 211: 188–198.
[36]  McGrath LM, Smith SD, Pennington BF (2006) Breakthroughs in the search for dyslexia candidate genes. Trends Mol Med 12: 333–341.
[37]  Hannula-Jouppi K, Kaminen-Ahola N, Taipale M, Eklund R, Nopola-Hemmi J, et al. (2005) The axon guidance receptor gene ROBO1 is a candidate gene for developmental dyslexia. PLoS Genet 1: e50.
[38]  Nugent AA, Kolpak AL, Engle EC (2012) Human disorders of axon guidance. Curr Opin Neurobiol.
[39]  Jen JC, Chan WM, Bosley TM, Wan J, Carr JR, et al. (2004) Mutations in a human ROBO gene disrupt hindbrain axon pathway crossing and morphogenesis. Science 304: 1509–1513.
[40]  Tseng RC, Lee SH, Hsu HS, Chen BH, Tsai WC, et al. (2010) SLIT2 attenuation during lung cancer progression deregulates beta-catenin and E-cadherin and associates with poor prognosis. Cancer Res 70: 543–551.
[41]  Dallol A, Da Silva NF, Viacava P, Minna JD, Bieche I, et al. (2002) SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers. Cancer Res 62: 5874–5880.
[42]  Dallol A, Krex D, Hesson L, Eng C, Maher ER, et al. (2003) Frequent epigenetic inactivation of the SLIT2 gene in gliomas. Oncogene 22: 4611–4616.
[43]  Pujic Z, Giacomantonio CE, Unni D, Rosoff WJ, Goodhill GJ (2008) Analysis of the growth cone turning assay for studying axon guidance. J Neurosci Methods 170: 220–228.
[44]  Hur EM, Saijilafu, Lee BD, Kim SJ, Xu WL, et al. (2011) GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules. Genes Dev 25: 1968–1981.


comments powered by Disqus