expHtt: aggregate formation and striatal neuron death [10]. On this in vitro model system, DA exerted a dual role on striatal death, via two independent pathways. One was due to reactive oxygen species and activation of the pro-apoptotic cJun-N terminal Kinase (JNK) pathway, the other one was specifically linked to D2 receptor stimulation and aggregate formation. Importantly, D2 antagonist treatments in vitro and in vivo protected from aggregate formation and striatal dysfunctions induced by ExpHtt [10,11]. Aggregate formation is known to be involved in the destabiliza- tion of microtubules and disorganization of the dendritic arbors, which are early events in the pathogenic mechanisms involved in HD [12,13,14,15,16]. One critical intracellular signalling pathway involved in actin cytoskeleton rearrangements and neurites elongation is the Rho/ROCK pathway [17,18,19]. This pathway has recently been shown to be involved in aggregate formation and cell death induced by ExpHtt, in drosophila or cell line models [20,21,22]. In the present study, we show a coupling of D2 receptor to this signalling pathway. The inhibition of ROCK activity using selective inhibitors, of knock-down of ROCK-II expression reversed D2 agonist-mediated aggregate formation, neuritic retraction and neuronal death induced by ExpHtt. By contrast, these treatments failed to affect neuronal dysfunctions induced by ExpHtt itself. We thus conclude that striatal neurons vulnerability in HD may be at least in part, mediated by the Rho/ROCK signalling pathway. The inclusion of Rho/ROCK inhibitors could be an interesting therapeutic option aimed at forestalling the onset of the disease. Results DA-Mediated Potentiation of Aggregates Formation and Neuronal Death Induced by ExpHtt in Striatal Neurons Involved D2R but Not D1R Stimulation Using primary cultures of striatal neurons expressing exon 1 of Huntingtin with a polyglutamine stretch (103Q: ExpHtt) fused to Dopamine/D2 Receptor and HD EGFP, we recently showed that DA, via D2 receptor stimulation, potentiates aggregate formation and neurodegenerescence in- duced by ExpHtt. These results were based on pharmacological studies made on primary striatal neurons from WT or knock-out mice for D2 receptors (D2R) [10]. Other in vitro studies documented that D1 receptors (D1R) play a role in mutant huntingtin-mediated aggregates formation and apoptosis [23,24,25]. In order to clarify the specific contribution of D1R and D2R on DA-mediated potentiation of aggregates formation and neuronal death in our model system, we used molecular tools to abrogate D1R and D2R expression. We first showed expression of both D1R and D2R in primary cultures from E14 striatal neurons after 7 days in vitro (DIV 7) (Fig. 1A–D). Of interest, both the full-length and spliced version of D2 receptors (D2L and D2S) were present in these striatal neurons (Fig. 1B). Specific invalidation of D1R and D2R mRNA and protein expression was achieved following siRNA transfection in striatal neurons (Fig. 1). Then, using this siRNA molecular approach, we focused on the role of both D1and D2 receptors in DA-mediated potentiation of aggregates formation and neuronal death induced by ExpHtt. We took advantages of GFP-tagged cDNAsto visualize expression and localization of Htt and ExpHtt in transfected neurons (Fig. 2A). As a consequence, aggregates of ExpHtt was visualized owing to GFP labelling. Knockdown of D2R (both D2L and D2S) but not D1R, using specific siRNAs resulted in a total inhibition of DA-induced aggregates formation (Fig. 2B), as previously demonstrated from D2R-knock out mice [10]. As expected, D2R siRNA only partially inhibited DA-mediated neuronal death in ExpHtt expressing neurons (Fig. 2C). Further- more, specific knockdown of D1R did not affect neuronal death in ExpHtt expressing neurons (Fig. 2C). Thus, having shown the specific involvement of D2R but not D1R stimulation in aggregate formation and neuronal death, we used for the following experiments a specific D2R agonist, quinpirole (Quin), to study Figure 1. Validation of D1R and D2R specific siRNA in primary striatal neurons. A–D: primary striatal neurons were transfected or not (Control) with Cont siRNA, D1R siRNA and D2R siRNA. After 72 hours, mRNAs were extracted followed by RT-PCR (A and B) and protein expression levels detected by immunoblot (C and D). A: RT-PCR of D1R gene and, B: RT-PCR D2R. Note the presence of both D2R long and short isoforms. For A and B: ARP gene was used as an housekeeping gene. B and D: immunoblot using specific anti-D1R (C), and specific D2R (D) antibodies. b-tubulin was used for control loading. doi:10.1371/journal.pone.0008287.g001 PLoS ONE | www.plosone.org 2 December 2009 | Volume 4 | Issue 12 | e8287