Sprouty2 down-regulation promotes axon growth by adult sensory neurons Barbara Hausott a , Natalie Vallant a , Maria Auer a , Lin Yang a,c , Fangping Dai b , Beate Brand-Saberi b,d , Lars Klimaschewski a, ⁎ a Division of Neuroanatomy, Innsbruck Medical University, Muellerstrasse 59, A-6020 Innsbruck, Austria b Institute of Anatomy and Cell Biology II, Freiburg University, Albertstrasse 17, D-79104 Freiburg, Germany c Division of Anatomy and Embryology, Capital Medical University, Beijing, 100069, P.R. China d Institute of Anatomy, Bochum University, Universitätsstrasse 150, D-44801 Bochum, Germany abstract article info Article history: Received 28 April 2009 Revised 21 July 2009 Accepted 4 August 2009 Available online xxxx Fibroblast growth factors (FGFs) play a prominent role in axonal growth during development and repair. Treatment with FGF-2 or overexpression of FGF receptors promotes peripheral axon regeneration mainly by activation of extracellular signal-regulated kinase (ERK). The Ras/Raf/ERK pathway is under the control of Sprouty proteins acting as negative feedback inhibitors. We investigated the expression of Sprouty isoforms in adult sensory neurons of dorsal root ganglia (DRG) as well as the effects of Sprouty inhibition on axon growth by small interfering RNAs (siRNAs). Sprouty2 revealed the highest expression level in DRG neurons. Down-regulation of Sprouty2 promoted elongative axon growth by adult sensory neurons accompanied by enhanced FGF-2-induced activation of ERK and Ras, whereas Sprouty2 overexpression inhibited axon growth. Sprouty2 was not regulated in vivo in response to a sciatic nerve lesion. Together, our results imply that Sprouty2 is highly expressed in adult peripheral neurons and its down-regulation strongly promotes elongative axon growth by activation of the Ras/Raf/ERK pathway. © 2009 Elsevier Inc. All rights reserved. Introduction Fibroblast growth factors (FGFs) belong to the neurotrophic factor family (Baird, 1994) and play a prominent role in axonal growth during development and after lesion in the adult nervous system (for reviews see Reuss and von Bohlen und Halbach, 2003; Grothe et al., 2006; Mason, 2007). FGF-2 is up-regulated in response to nerve injury and exogenously applied FGF-2 promotes neuronal survival and neurite outgrowth in vitro and in vivo. FGFs mediate their responses by activation of four types of high affinity tyrosine kinase receptors (FGFR1–4) with FGFR1 being the most abundant FGFR in the nervous system (Ford-Perriss et al., 2001). FGFR1, -2 and -4 are expressed in adult sensory neurons obtained from dorsal root ganglia with FGFR1 being expressed at much higher levels than FGFR2 or FGFR4. Over- expression of FGFR1 enhances axon growth which is further increased by lysosomal inhibition of receptor degradation (Hausott et al., 2008). Ligand binding in cooperation with the accessory heparin sulfate proteoglycans leads to FGFR dimerization and autophosphorylation of their cytoplasmatic domains, which recruits a number of signaling molecules relevant for axonal growth (Mason, 2007). The Ras/Raf/ERK (extracellular signal-regulated kinase) and the PI3K (phosphatidylino- sitol-3 kinase)/Akt pathway are required for regenerative axon growth mediated by neurotrophic factors (for review see Zhou and Snider (2006)). Activation of Raf is necessary for axon growth by DRG neurons during development (Zhong et al., 2007) and induces axonal elongation in sensory neurons whereas active Akt increases axon caliber and branching (Markus et al., 2002). In addition to its role in gene regulation, ERK signaling is also required for local axon assembly induced by neurotrophins (Atwal et al., 2000) and for retrograde transport of regeneration signaling in DRG neurons (Perlson et al., 2005). FGFR signaling is attenuated by negative feedback inhibitors such as Sprouty or Sef (for reviews see Dikic and Giordano (2003); Tsang and Dawid (2004); Mason et al. (2006)). Sprouty proteins were first discovered in 1998 as regulators of tracheal development in the fruitfly Drosophila (Hacohen et al. 1998). They represent a major class of negative feedback loop modulators that limit the intensity and duration of tyrosine kinase activation, thereby controlling growth and differenti- ation processes. Mammals exhibit four Sprouty isoforms (Sprouty1–4) which are functionally conserved in vertebrates and of which Sprouty1, -2 and -4 represent the major isoforms, whereas expression of Sprouty3 seems to be restricted (Minowada et al., 1999; Ozaki et al., 2005). Sprouty isoforms block tyrosine kinase signaling induced by FGF (Hacohen et al. 1998; Impagnatiello et al., 2001), BDNF (brain-derived neurotrophic factor, Gross et al., 2007), GDNF (glial cell line-derived neurotrophic factor, Ishida et al., 2007), PDGF (platelet-derived growth factor, Gross et al., 2001) and VEGF (vascular epithelial Molecular and Cellular Neuroscience xxx (2009) xxx–xxx Abbreviations: FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; NGF, nerve growth factor; TrkA, tropomyosin related kinase A; DRG, dorsal root ganglia; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol-3 kinase. ⁎ Corresponding author. Fax: +43 512 9003 73112. E-mail address: lars.klimaschewski@i-med.ac.at (L. Klimaschewski). YMCNE-02376; No. of pages: 13; 4C: 4 1044-7431/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2009.08.005 Contents lists available at ScienceDirect Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne ARTICLE IN PRESS Please cite this article as: Hausott, B., et al., Sprouty2 down-regulation promotes axon growth by adult sensory neurons, Mol. Cell. Neurosci. (2009), doi:10.1016/j.mcn.2009.08.005