Estradiol facilitates neurite maintenance by a Src/Ras/ERK signalling pathway Alfredo Miñano a,b , Xavier Xifró a, 1 , Virgili Pérez a,2 , Bruna Barneda-Zahonero a,b , Carlos A. Saura a,b , José Rodríguez-Alvarez a,b, ⁎ a Institut de Neurociències and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Spain b Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Spain abstract article info Article history: Received 20 September 2007 Revised 3 April 2008 Accepted 3 June 2008 Available online 14 June 2008 Keywords: Cerebellum Estradiol Cultures Kinases Different reports suggest the estrogens are involved in neuritic outgrowth, maintenance of dendritic morphology and spine formation in the CNS. However, the molecular mechanisms regulated by estrogens on neuronal integrity are not fully understood. We have addressed the relationship between 17β-estradiol- dependent ERK pathway stimulation and the maintenance of neuritic morphology in cerebellar granule cell cultures (CGC). We report that 17β-estradiol clearly activates ERK phosphorylation in CGC cultured in low potassium via ERα localized in the plasma membrane and without the activation of the insulin-like growth factor-I receptor. 17β-estradiol activates the ERK pathway through Ras-dependent Src kinase activity. A concomitant activation of the cAMP-response element-binding protein (CREB) is observed. Moreover, we demonstrate that 17β-estradiol-mediated ERK activation is involved in the maintenance of neuritic arborisation and neuronal morphology in proapoptotic conditions. © 2008 Elsevier Inc. All rights reserved. Introduction 17β-estradiol has been associated with a variety of CNS effects including cell survival, differentiation and morphology. Thus, 17β- estradiol regulates the density of spines and dendrites in the hippocampus (Gould et al., 1990; Woolley and McEwen 1994; Murphy and Segal 1997) and in the cerebellum (Sakamoto et al., 2003). It has been also described that 17β-estradiol induces neurite outgrowth and branching in cholinergic neurons (Dominguez et al., 2004). The morphological effects of 17β-estradiol in the CNS are observed in normal and damaged tissue. The effect of 17β-estradiol seems to be partly mediated by changes in the expression pattern of several cytoskeletal proteins such as MAP2 isoforms or tau (Reyna-Neyra et al., 2002; Shah et al., 2003). The molecular mechanisms regulated by 17β-estradiol that elicit the effects described above are not fully understood. The mitogen- activated protein kinase kinase (MEK)/extracellular signal-regulated kinases (ERK) pathway is considered a major contributor of neuronal plasticity by affecting activity-dependent formation of dendrites (Vaillant et al., 2002; Goldin and Segal 2003). Several studies have reported that 17β-estradiol activates the MEK/ERK pathway in various tumour cell lines, adipocytes and neurons (Singer et al.,1999), (Watters et al., 1997; Wong et al., 2003), likely by activating 17β-estradiol receptors (ER) located in the plasma membrane (Stevis et al., 1999; Toran-Allerand et al., 2002). Although still controversial, several studies supported that activation of ERα induces MEK/ERK pathway stimulation (Watters et al., 1997; Singh et al., 2000; Dos Santos et al., 2002). In some cases the transactivation of IGF-IR is needed for ERα- mediated activation of MEK/ERK pathway (Kahlert et al., 2000; Cardona-Gomez et al., 2002). Besides ERα, it has been recently described that 17β-estradiol-mediated MEK/ERK activation could be also mediated by GPR30, a seven-transmembrane G-protein-coupled receptor located in the endoplasmic reticulum (Revankar et al., 2005). A role of MEK/ERK pathway in the 17β-estradiol-mediated effect on structural plasticity in the CNS has been reported in the hippocampus (Bi et al., 2000) and in basal forebrain cholinergic neurons (Dominguez et al., 2004). However, the mechanisms upstream and downstream of ERK activation that mediates the effects of 17β-estradiol on neuronal morphology are largely unknown. Some reports suggest that activation of the cAMP-response element-binding protein (CREB) could be involved. CREB has been implicated in synaptic plasticity (Finkbeiner et al., 1997) and phosphorylation of CREB by 17β-estradiol is dependent on ERK activity in hippocampal cultures (Lee et al., 2004) and in the basal forebrain (Szego et al., 2006). During the last years it has become apparent that 17β-estradiol has an important role in the development of the cerebellum. It has been suggested that estrogens could be involved in the regulation of dendritic growth, spine formation and neuron number in the cerebellum (Wong et al., 2003)(Sakamoto et al., 2003). Interestingly, expression levels of ERβ correlate with the differentiation of Purkinje Molecular and Cellular Neuroscience 39 (2008) 143–151 ⁎ Corresponding author. Institut de Neurosciències, Edifici M, Campus de Bellaterra, Universitat Autonòma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain. Fax: +34 935 814 152. E-mail address: jose.rodriguez@uab.es (J. Rodríguez-Alvarez). 1 Present address: Departament de Biología Cellular, Universitat de Barcelona and IDIBAPS, Barcelona, Spain. 2 Present address: Laboratori de Neuropsicofarmacologia, Institut de Recerca de l'Hospital de la Santa Creu i de Sant Pau, Barcelona, Spain. 1044-7431/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2008.06.001 Contents lists available at ScienceDirect Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne