Figure 2. DA-mediated potentiation of aggregates formation and neuronal death induced by ExpHtt in striatal neurons involves D2R but not D1R stimulation. A: Immunocytochemical detection of Htt was performed on striatal neurons transfected with either GFP alone, GFP-tagged Htt or GFP-tagged ExpHtt. Nuclei were labelled using Hoechst (blue). Note that ExpHtt and GFP perfectly colocalize in aggregates. B and C: Seventy two hours after RNA interference procedure, primary neurons were transfected with Htt or ExpHtt and treated with DA (100 mM) for 10 (B) or 24 (C) hours. B: quantification of transfected neurons with aggregates of ExpHtt. C: quantification of surviving transfected neurons based on Hoechst labeling. Data were analyzed from at least 3 independent experiments (100 transfected neurons per condition and per experiment) and expressed as mean6SEM. (***P,0.001, **P,0.005, *P,0.05, ns: non significant). doi:10.1371/journal.pone.0008287.g002 Dopamine/D2 Receptor and HD D2R signalling pathway involved in the potentiation of ExpHtt- induced cellular dysfunctions. D2 Receptor Stimulation Potentiates Neuritic Retraction and Growth Cone Collapse Induced by ExpHtt in Striatal Neurons Once aggregates of ExpHtt are formed, they destabilize microtubules and disrupt the cellular architecture [15]. We first investigated whether ExpHtt had any effect on the dynamic organization of the cytoskeleton and neuritic outgrowth in our model system. Using MAP2 (Microtubule-Associated-protein) immunostaining we analyzed the mean neuritic length of transfected neurons (Fig. 3A, C). Statistical analyses revealed a significant decrease of neuritic length (225%) in ExpHtt expressing neurons when compared to Htt-transfected cells. We then analyzed a possible potentiation of DA and/or D2-Receptor stimulation on ExpHtt-mediated neuritic retraction. All experi- ments were performed in the presence of anti-oxidants, in order to avoid indirect effect of DA auto-oxidation. DA and quinpirole treatments potentiated significantly the reduction of neuritic length induced by ExpHtt alone (Fig. 3A, C). Importantly, DA and quinpirole treatments also induced a significant decrease of neuritic length in non-transfected striatal neurons (230%; data not shown) as well as in Htt-expressing neurons (Fig. 3A, C). In both Htt- and ExpHtt-expressing neurons, DA effects on neuritic retraction were reversed by the selective D2 receptor antagonist, raclopride (Fig. 3C). This indicates that the major effects exerted by DA on neuritic retraction are mediated by D2 receptor stimulation. Neuritic retraction is accompanied by a disorganization of actin filaments that can be visualized by rhodamine-phalloidin labelling. The filaments of actin are concentrated in filopodes and lamellipodes of growth cones (Fig. 3B). Actin filaments are mainly observed at the tips of the neurites and present very well structured and large growth cones, in most neurons expressing Htt. About 20% of collapse was found in Htt-expressing neurons (Fig. 3B, D). A significant increase of growth cone collapse was observed in ExpHtt-expressing neurons (42% of collapse) (Fig. 3B, D). This collapse was even higher upon quinpirole treatment, in both Htt and ExpHtt transfected neurons (30% and 65%, respectively, Fig. 3D), indicating that D2 receptor stimulation promotes a disorganization of actin filaments. D2-R Mediated Neuritic Retraction and Growth Cone Collapse Is Associated with ROCK-II Activation in Striatal Neurons Various signalling pathways participate in the dynamic reorganization of the cytoskeleton and neuritic elongation, among which the Rho/ROCK signalling pathway is thought to play a key role [17,18,19]. We thus investigated a possible involvement of this pathway in neuritic retraction induced by ExpHtt in the presence or not of the D2 agonist treatment. The ROCK pharmacological inhibitors Y27632 and Hydroxyfasudil were used as a first approach. These inhibitors, applied 30 minutes prior to, and during quinpirole treatment, reversed D2-mediated effects on neuritic retraction in Htt- and ExpHtt- expressing neurons (Fig. 4A). By contrast, they failed to affect neuritic retraction induced by ExpHtt alone (Fig. 4A). We then wished to confirm these observations using siRNAs to abolish ROCK protein expression. Two different genes encode ROCK, giving rise to ROCK-I and ROCK-II. Both proteins are implicated in the formation of stress fibers and suppression of neurite outgrowth and are ubiquitiously expressed, with an enrichment of ROCK-II in PLoS ONE | www.plosone.org 3 December 2009 | Volume 4 | Issue 12 | e8287