the central nervous system [17]. We checked ROCK-I and ROCK-II expression in striata from adult mice (Fig. 4B: striatal extracts), in striatal neurons in culture (Fig. 4B striatal culture), and in HEK cells extracts as a positive control by western blot. Both proteins were found at 160 KDa, their expected molecular weight, in HEK cells (Fig. 4B). Very low levels of ROCK-I were found when compared to ROCK-II in striatal cells both in vivo and in vitro (Fig. 4B). Further studies were thus focused on ROCK-II knock-down. Having established the efficiency of siRNAs on ROCK-II expression by western-blot (Fig. 4C), we transfected them (DIV 4) along with cDNAs encoding Htt or ExpHtt (DIV7). The ROCK-II knock-down effects were analyzed on neuritic retraction 14 hours later, in the presence of quinpirole (Fig. 4D). ROCK-II knock-down totally reversed potentiation by quinpirole of ExpHtt- induced neuritic retraction. Similar results were found using specific inhibitors of ROCK, Y27632 and Hydroxyfasudil. By contrast, neither molecular nor pharmacological inhibition of Rho/ROCK signalling reversed neuritic retraction induced by ExpHtt alone. A total reversal of D2-mediating effects was also found on growth cone collapse, without modification of ExpHtt- induced effects (Fig. 4E). Altogether our data provide a strong, albeit indirect, evidence of a coupling between D2 receptors and Rho/ROCK activation. The DA/D2 receptor subtype has been classically coupled to inhibition of adenylate cyclase [26], but recent evidence in the literature also supported a coupling between D2 receptors and Rho-GTP binding protein [27]. We thus wished to provide a direct evidence of Rho/ROCK activation by D2 receptor stimulation instriatal neurons. Rho activity was measured using the ratio of Rho-GTP levels by comparison to total Rho proteins (Fig. 5A,B). Both DA and quinpirole treatments significantly increased Rho activity after 15 min of treatment. Once activated by Rho, ROCK proteins can phosphorylate several substrates, including LIM kinase which phosphorylates Cofilin at Ser3 [28]. Cofilin critically controls actin filament dynamics and reorganiza- tion. Using immunocytochemical approaches, we investigated phosphorylation of cofilin after quinpirole treatments on striatal neurons and found an hyperphosphorylation after 20 minutes of treatment in about 35% of striatal neurons (Fig. 5C–E). This hyperphosphorylation was reversed by ROCK inhibitors (Fig. 5D) and ROCK-II siRNA treatments (Fig. 5E). Altogether these data provide the first evidence that D2 receptor stimulation induces phosphorylation of cofilin via the Rho/ROCK signalling pathway in striatal neurons. D2-R Stimulation Potentiates ExpHtt-Induced Aggregate Formation via ROCK-II Activation The pattern of ExpHtt expression into striatal neurons can be observed owing to the GFP tag at the C-terminal region of exon 1 ExpHtt. Thus, ten hours after transfection, ExpHtt shows a diffuse pattern in 65% of transfected neurons (Fig. 6A, upper panel), while aggregates of ExpHtt are observed in the remaining transfected neurons (35%) (Fig. 6A, lower panel). According to our previous observations [10], D2 agonist (quinpirole) treatment significantly increased the number of transfected neurons containing aggregates of ExpHtt (52% versus 35%) (Fig. 6B). To gain insights into the mechanisms that lead to D2 effects on aggregate formation, we investigated the role of ROCK inhibitors and ROCK siRNAs. D2-mediated aggregate formation was totally reversed by ROCK inhibitors, without any significant effects on aggregates formed by ExpHtt alone. These data were confirmed using ROCK-II siRNAs (Fig. 6B). Thus, our findings indicate that Dopamine/D2 Receptor and HD ROCK-II signalling is implicated, downstream D2 receptors, in the potentiation of ExpHtt aggregate formation. D2-R Stimulation Potentiates ExpHtt-Induced Neuronal Death via ROCK-II Activation We previously showed [10] that D2 agonist treatment potentiated striatal death induced by ExpHtt. Cell survival was analyzed 24 hours after transfection by appreciation of nucleus morphology (DNA fragmentation) by Hoechst staining (Fig. 7A). Quinpirole significantly potentiated ExpHtt-induced neuronal death without affecting cell survival in Htt-expressing neurons. The ROCK inhibitors (Y-27632 or Hydroxyfasudil) and ROCK- II siRNA totally rescued toxicity induced by the D2 agonist in ExpHtt-expressing striatal neurons with no effects against toxicity induced by ExpHtt itself (Fig. 7B). Altogether, these data indicate that the Rho/ROCK-II signalling pathway is a major pathway downstream D2 receptor for mediating toxicity in ExpHtt- expressing neurons. Discussion In the present work, we wished to analyze how DA via DA receptors stimulation might account for aggregate formation and potentiation of neuronal death in ExpHtt expressing striatal neurons. We first confirm our previous results [10,11] showing that DA potentiation of ExpHtt toxicity is mediated via D2R but not D1R stimulation, in our model system, i.e. in striatal neurons expressing a truncated version of ExpHtt. We show the first evidence for a coupling of D2 receptors to the Rho/ROCK-II signalling pathway. In cultured striatal neurons, inhibition of this pathway reversed DA/D2 receptor-mediated neuritic retraction, growth cone collapse, aggregate formation and neuronal death induced by ExpHtt in the presence of D2 agonist. By contrast with findings in other model systems of HD (Drosophila, and HEK cells), we failed to demonstrate a clear involvement of ROCK in ExpHtt induced cellular phenotypes in striatal neurons. Neurodegeneration in HD occurs most prominently in the basal ganglia and neocortex. Striatal neurons show a range of vulnerabilities in Huntington’s disease. Medium Spiny Neurons (MSNs) are the first affected striatal cells while interneurons are spared, even at a late stage of the disease. Among the MSNs, the GABAergic/enkephalinergic, which preferentially express D2 receptors and project to the external segment of globus pallidus (Gpe) tend to degenenerate before the substance P/dynorphiner- gic striato-nigral neurons expressing D1 receptors [29,30,31]. In HD patients, primates and mouse models of HD, aggregates appear first in the neuropil [9,14,16,32,33,34], where they are thought to be toxic by poisoning the axonal transport [11,16,35]. The reduction of these aggregates leads to an improvement of the phenotype in HD mice [36]. Interestingly, the Substancia Nigra (SN) and the GPe, which receive afferents from the D2R- expressing striatal neurons, are the first brain areas where neuropil aggregates appear, in mice and human [16,34,37,38]. Hyperdo- paminergic HD mice display an accelerated clinical and neuropathological phenotype with early formation of neuropil aggregates that predominate in the GPe [9], which express D2R but not D1R. In primary culture of striatal neurons expressing ExpHtt, D2 receptor stimulation increases the formation of neuropil aggregates, while striatal neurons from D2R knock-out mice fail to show any vulnerability to DA for aggregate formation (11). By contrast, D1R stimulation did not influence the total number of mutant Htt aggregates in SK-N-MC expressing ExpHtt (N-term-103Q) [24]. These observations were strengthened in the present work, where D2R- but not D1R- siRNAs prevented DA- PLoS ONE | www.plosone.org 6 December 2009 | Volume 4 | Issue 12 | e8287