Enhancement of dopaminergic properties and protection mediated by neuronal activation of Ras in mouse ventral mesencephalic neurones Koushik Chakrabarty, Tsvetan Serchov,* Stefan A. Mann,* Irmgard D. Dietzel and Rolf Heumann Department of Molecular Neurobiochemistry, NC7 ⁄ 174, Ruhr University, 44780 Bochum, Germany Keywords: dopaminergic neurones, neuroprotection, neurospheres, Nurr1, Parkinson’s disease, synRas Abstract The poor differentiation and survival of dopaminergic neurones are practical constraints in their therapeutic applications. Here we explored the role of neuronally activated Ras in ventral mesencephalon-derived neurospheres generated from synRas mouse embryos. The expression of Val12 Ha-Ras transgene and enhanced Ras activity was evident after differentiation of the neurospheres with a corresponding activating phosphorylation of mitogen-activated protein kinase. Phosphorylation of Akt ⁄ PKB, the target kinase of phosphoinositide 3-kinase, along with phosphorylation of Bad and CREB were enhanced in synRas-derived differentiated neurosphere cultures. Furthermore, increased Nurr1 expression was associated with elevated numbers of dopaminergic neurones in synRas-derived cultures compared with the wild-type. Correspondingly, tyrosine hydroxylase promoter assays revealed enhanced transcriptional activation of the promoter in synRas-derived cultures. synRas-derived dopaminergic neurones were greatly resistant to degeneration induced by various noxious stimuli. Consistently, the transgenic expression of activated Ras attenuated the adverse 6-hydroxydopamine effects on dopaminergic neurones. Dopaminergic neurones derived from both wild-type and synRas cultures expressed voltage-gated potassium and sodium currents, fired action potentials and exhibited electrical network activity. Thus, expression of the transgene promotes survival and enhances differentiation towards a dopaminergic cell fate without altering their basic electrical properties. Our results suggest that intracellular cell therapy mimicking trophic signalling may offer potential benefit in models of human disease associated with dopamine neurone dysfunction. Introduction Cell replacement strategies using neural stem cells have potential therapeutic applications. The generation of dopamine (DA) neurones from central nervous system precursors is of special interest given the promising results of fetal cell transplantation in patients with Parkinson’s disease (Olanow et al., 2001). The main practical constraints of neural grafting are the paucity of dopaminergic neurones and their poor survival (Schierle et al., 1999). Research in the area of enhancing the differentiation and survival of dopaminergic neurones generated from stem cells has employed and demonstrated the efficacy of various neurotrophic factors and cytokines. A variety of these molecules involved in cellular growth, differentiation and dynamic morphoregulation converges on Ras, a regulated GTPase switch (Marshall, 1996). In order to test Ras functions in adult brain neurones we used transgenic mice expressing constitutively activated Val12-Ha-Ras under the control of the promoter for the synapsin I gene (synRas mice). Using this synRas mouse model, we have previously demon- strated that neuronal activation of Ras enhances dendritic differenti- ation and protects against lesion-induced degeneration (Heumann et al., 2000; Arendt et al., 2004; Hansen et al., 2004). The major pathways induced by Ras are Raf-1 ⁄ B-raf ⁄ mitogen- activated protein kinase (MAPK) and phosphoinositide 3-kinase ⁄ Akt-1 [phosphoinositide-dependent protein kinase (PKB)] (Katz & McCor- mick, 1997). The effects of Ras are thought to be mediated in large part by its ability to regulate neuronal gene expression (Xia et al., 1996), at least partly through its contribution to the activation of the CREB transcription factor (Lonze & Ginty, 2002). With respect to DA neurones, the Nurr1 gene, which has been shown to play a prominent role in both DA differentiation and survival, is selectively activated by phosphorylated CREB, which binds as a dimer to the conserved cAMP response element to the cAMP response element site present in the Nurr1 promoter (Lee & Nikodem, 2004). Ras protein transduces cellular responses and the physiological downstream effects of its activity are strictly cell type specific (Mulcahy et al., 1985; Borasio et al., 1989). Here we tested the role of enhanced neuronal activated Ras in its ability to promote differentiation and enhance survival of dopaminergic neurones derived from ventral mesencephalon (VM) precursor cells and to unravel the molecular mechanisms involved in this phenomenon. We show an increase in the number of dopaminergic neurones along with strong neuroprotective features of Ras. Furthermore, we report enhanced phosphorylation of Ras effector proteins and a stronger activation of the tyrosine hydroxylase (TH) promoter in synRas-derived differentiated VM neurospheres compared with wild-type (WT) littermate-derived cul- tures. Evidence of enhanced Nurr1 expression in synRas cultures suggests a specific enhancement of the differentiation towards a dopaminergic cell fate. Characterization involving electrophysiological Correspondence: Dr Rolf Heumann, as above. E-mail: rolf.heumann@rub.de *T.S. and S.A.M. contributed equally to this work. Received 20 September 2006, revised 20 December 2006, accepted 6 February 2007 European Journal of Neuroscience, Vol. 25, pp. 1971–1981, 2007 doi:10.1111/j.1460-9568.2007.05457.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience