BackgroundHuntington's disease (HD) is a polyglutamine-expanded related neurodegenerative disease. Despite the ubiquitous expression of expanded, polyQ-Huntingtin (ExpHtt) in the brain, striatal neurons present a higher susceptibility to the mutation. A commonly admitted hypothesis is that Dopaminergic inputs participate to this vulnerability. We previously showed that D2 receptor stimulation increased aggregate formation and neuronal death induced by ExpHtt in primary striatal neurons in culture, and chronic D2 antagonist treatment protects striatal dysfunctions induced by ExpHtt in a lentiviral-induced model system in vivo. The present work was designed to elucidate the signalling pathways involved, downstream D2 receptor (D2R) stimulation, in striatal vulnerability to ExpHtt.Methodology/Principal FindingsUsing primary striatal neurons in culture, transfected with a tagged-GFP version of human exon 1 ExpHtt, and siRNAs against D2R or D1R, we confirm that DA potentiates neuronal dysfunctions via D2R but not D1R stimulation. We demonstrate that D2 agonist treatment induces neuritic retraction and growth cone collapse in Htt- and ExpHtt expressing neurons. We then tested a possible involvement of the Rho/ROCK signalling pathway, which plays a key role in the dynamic of the cytoskeleton, in these processes. The pharmacological inhibitors of ROCK (Y27632 and Hydroxyfasudil), as well as siRNAs against ROCK-II, reversed D2-related effects on neuritic retraction and growth cone collapse. We show a coupling between D2 receptor stimulation and Rho activation, as well as hyperphosphorylation of Cofilin, a downstream effector of ROCK-II pathway. Importantly, D2 agonist-mediated potentiation of aggregate formation and neuronal death induced by ExpHtt, was totally reversed by Y27632 and Hydroxyfasudil and ROCK-II siRNAs.Conclusions/SignificanceOur data provide the first demonstration that D2R-induced vulnerability in HD is critically linked to the activation of 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.

Activation of the extracellular signal-regulated kinase (ERK) signaling pathway in the striatum is crucial for molecular adaptations and long-term behavioral alterations induced by cocaine. In response to cocaine, ERK controls the phosphorylation levels of both mitogen and stress-activated protein kinase 1 (MSK-1), a nuclear kinase involved in histone H3 (Ser10) and cAMP response element binding protein phosphorylation, and Elk-1, a transcription factor involved in serum response element (SRE)-driven gene regulations. We recently characterized the phenotype of msk-1 knock-out mice in response to cocaine. Herein, we wanted to address the role of Elk-1 phosphorylation in cocaine-induced molecular, morphological, and behavioral responses. We used a cell-penetrating peptide, named TAT-DEF-Elk-1 (TDE), which corresponds to the DEF docking domain of Elk-1 toward ERK and inhibits Elk-1 phosphorylation induced by ERKs without modifying ERK or MSK-1 in vitro. The peptide was injected in vivo before cocaine administration in mice. Immunocytochemical, molecular, morphological, and behavioral studies were performed. The TDE inhibited Elk-1 and H3 (Ser10) phosphorylation induced by cocaine, sparing ERK and MSK-1 activation. Consequently, TDE altered cocaine-induced regulation of genes bearing SRE site(s) in their promoters, including c-fos, zif268, ⌬FosB, and arc/arg3.1 (activity-regulated cytoskeleton-associated protein). In a chronic cocaine administration paradigm, TDE reversed cocaine-induced increase in dendritic spine density. Finally, the TDE delayed the establishment of cocaine-induced psychomotor sensitization and conditioned-place preference. We conclude that Elk-1 phosphorylation downstream from ERK is a key molecular event involved in long-term neuronal and behavioral adaptations to cocaine.

Ceramide-1-phosphate (C1P), the product of ceramide kinase, is a sphingophospholipid with recently recognized signaling properties. In particular, it was reported to be mitogenic and capable of direct stimulation of cytosolic phospholipase A 2a . Much of the present knowledge has relied on the use of C1P of various acyl chain lengths, together with diverse protocols to deliver it to cultured cells. A mixture of ethanol (or methanol) with dodecane, as the vehicle, has become popular. However, the contribution of this solvent to the observed effects of C1P has not been documented. Here, we show that addition of C1P in ethanoldodecane to culture medium leads to irreversible cytotoxic effects. These culminate in mitochondrial swelling, vacuole formation, and cell death. Not only the toxicity of C1P, but also its ability to trigger prostaglandin E 2 release, is fully dependent upon addition of a premade C1P-dodecane mixture. Furthermore, we show that these effects are not restricted to C1P. They result from the capacity of dodecane to interact with phospholipids; hence, they go undetected with a vehicle control. This study should raise awareness about the use of dodecane for phospholipid delivery and, in turn, help in unraveling C1P signaling, which is still poorly