Figure 6. D2-R increases ExpHtt aggregates formation via ROCK-II activation in striatal neurons. A) ExpHtt aggregates detection based on GFP fluorescence in primary striatal neurons transfected with ExpHtt (tagged with GFP) for 10 hours and MAP2 immunostaining (red). Note, at this time point, that ExpHtt present either a diffuse expression pattern (top panel) as well as aggregates formation (bottom panel). B) Quantification of ExpHtt-transfected neurons with aggregates after quinpirole treatment (10 mM) for 10 hours alone or in the presence of pharmacological inhibition of ROCK (Y27632, 10 mM and Hydroxyfasudil, 10 mM) or ROCK-II siRNA (50 nM). Data are expressed as mean6SEM from 3 independent experiments (100 transfected neurons per condition and per experi- ment) (***P,0.001, ns: non significant). doi:10.1371/journal.pone.0008287.g006 mutant Htt, either D1R or D2R seem to be differentially involved in DA-mediated toxicity on striatal neurons. In our in vitro model system, D2 receptor stimulation increased neuritic retraction and growth cone collapse in mutant- but also normal Htt-expressing neurons. This observation corroborates in vivo data showing that D2 receptors stimulation results in pruning of the terminal arbor of substantia nigra pars compacta (SNpc) DA neurons [39,40]. Furthermore, the genetic invalidation of D2 receptor in mice resulted in a significant increase in terminal tree size of these neurons, as well as the density of varicosities in the caudate-putamen (42,43). We can thus hypothesise that the disorganization of the neuritic arbors described in HD might be, at least in part, due to local D2 receptor stimulation. D2 receptors Dopamine/D2 Receptor and HD are classically described as negative regulators of the cAMP/PKA pathway, via coupling to Gai/o [41]. They can also couple to phospholipase (PLCb) via Gq, mobilize intracellular calcium stores [42] and activate the MAPK/ERK signalling cascade [42,43]. Dopaminergic stimulation of D2 receptors can also activate or inhibit the Akt phosphorylation level (Thr308), depending on the time of stimulation, short or long, respectively [43,44]. In the present study, we present the first evidence for a coupling between D2 receptors and the Rho/ROCK signalling pathway. Such coupling was suggested previously, by a study showing that D2- mediated activation of Phospholipase D (PLD) was abolished by preincubation of the cells with C3 exoenzyme, a specific inhibitor of the Rho proteins [27]. Other G protein-coupled receptors have been described for their ability to activate Rho proteins. The classical activator of this pathway is the lysophosphatidic acid (LPA) receptor which acts through G12/G13 to activate RhoA and ROCK and lead to neuritic retraction and growth cone collapse [45,46,47] The details of the mechanism by which D2 receptor stimulates Rho proteins remain unclear but could involve the scaffolding proteins b-arrestin that have been associated with the activation of Rho downstream Angiotensin A2 receptors [48] and are known to be critical for D2 receptor internalization [49]. ROCK proteins are principal mediators of RhoA activity in the nervous system where they have been largely reported as regulators of neuritic length and growth cone collapse [50]. Screening for biologically active molecules that inhibit polygluta- mine aggregation, lead to the identification of a pharmacological ROCK inhibitor, Y-27632 [21,22] When Y-27632 was adminis- trated in a Drosophila model of HD, it reduced Htt-exon1 (93Q) induced neurodegeneration. In HEK cell line expressing Htt exon1 (72Q), it was necessary to knock-down both ROCK-I and Protein Kinase C-related protein (PRK2) proteins to abolish polyglutamine aggregation [21] In Neuro2a cells expressing Htt- exon1 (150Q), knock-down of ROCK-II and ROCK-I reduced aggregation [51]. In striatal neurons, in vitro and in vivo, we found a poor expression of ROCK-I, while ROCK-II was strongly expressed. Global inhibitors of ROCK or knock-down of ROCK-II expression, failed to reverse neuronal dysfunctions induced by ExpHtt by itself. However, the pharmacological inhibitor of ROCK, as well as ROCK-II-siRNAs, reversed D2- mediated effects on ExpHtt-induced phenotype. We thus propose that, in striatal neurons, the Rho/ROCK-II signalling pathway is critically involved in D2-mediated vulnerability in HD. Interest- ingly, ROCK proteins have been proposed as a promising therapeutic target in various neurological conditions [52]. In the CNS, inhibition of ROCK using Y27632 promotes axon growth after spinal cord injury in experimental animal models [53] and reduces the expression of amyloid-bpeptide in a mouse model of Alzheimer’s disease [54]. Because long term pharmacological blockade of D2 receptors may be poorly tolerated in patients [55,56], the inclusion of Rho/ROCK inhibitors could be an interesting therapeutic option to target the vulnerability of D2-R expressing striatal neurons in HD. Materials and Methods Mouse Husbandry Mice were housed in a temperature-controlled room main- tained on a light/dark cycle. Food and water were available ad libitum. Experiments were performed in accordance with standard ethical guidelines (U.S. National Institutes of Health publication nu 85-23, revised 1985, and the European Committee Guidelines on the Care and Use of Laboratory Animals directive 86/609/EEC). PLoS ONE | www.plosone.org 8 December 2009 | Volume 4 | Issue 12 | e8287