L-dopa-induced dyskinesia (LID) is a common debilitating complication of dopamine replacement therapy in Parkinson's disease. Recent evidence suggests that LID may be linked causally to a hyperactivation of the Ras-ERK signaling cascade in the basal ganglia. We set out to determine whether specific targeting of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a brain-specific activator of the Ras-ERK pathway, may provide a therapy for LID. On the rodent abnormal involuntary movements scale, Ras-GRF1-deficient mice were significantly resistant to the development of dyskinesia during chronic L-dopa treatment. Furthermore, in a nonhuman primate model of LID, lentiviral vectors expressing dominant negative forms of Ras-GRF1 caused a dramatic reversion of dyskinesia severity leaving intact the therapeutic effect of Ldopa. These data reveal the central role of Ras-GRF1 in governing striatal adaptations to dopamine replacement therapy and validate a viable treatment for LID based on intracellular signaling modulation. P arkinson's disease (PD) is a neurodegenerative disorder characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) causing dopamine depletion in the striatum, the main input nucleus of the basal ganglia. Dopamine replacement therapy with L-dopa remains the most effective treatment for PD, but its use is associated with motor fluctuations and abnormal involuntary movements (AIMs), termed "L-dopa-induced dyskinesia" (LID), which are doselimiting and potentially disabling (1-3).A key objective for the future treatment of PD is to avoid dyskinesia altogether, but doing so will require an understanding of the molecular mechanisms that are involved. LID is attributed to a sequence of events, largely occurring in the striatum, that include pulsatile stimulation of dopamine D1 receptors, downstream changes in proteins and genes, dendritic alterations, and functional abnormalities in nondopaminergic transmitter systems, all of which concur to modify neuronal firing patterns in the basal ganglia-thalamocortical networks (1-4). Once symptoms have appeared, LID can be triggered easily by a single dose of L-dopa even after several weeks of treatment washout (4). Regulation of striatal gene expression is the likely mechanism underlying neuronal plasticity in LID. The ERK signaling cascade is a key regulator of striatal plasticity and an interesting candidate for drug targeting (5-8). Stimulation of dopamine and glutamate receptors on striatal neurons can switch on the small GTPases of the Ras family, which in turn activate the Raf/Mek/ Erk protein kinase cascade (5-8). Sustained activation of these biochemical pathways leads to synaptic rearrangements requiring de novo gene expression and protein synthesis. Importantly, in neurotoxic models of PD, such as the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rodent and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primate (NHP), a supersensitivity of striatal D1 receptors leads to ERK hyperactivation in ...

Background-Ras-extracellular signal-regulated kinase (Ras-ERK) signaling is central to the molecular machinery underlying cognitive functions. In the striatum, ERK1/2 kinases are coactivated by glutamate and dopamine D1/5 receptors, but the mechanisms providing such signaling integration are still unknown. The Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a neuronal specific activator of Ras-ERK signaling, is a likely candidate for coupling these neurotransmitter signals to ERK kinases in the striatonigral medium spiny neurons (MSN) and for modulating behavioral responses to drug abuse such as cocaine.

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The Extracellular Regulated Kinase 1 and 2 transduce a variety of extracellular stimuli regulating processes as diverse as proliferation, differentiation and synaptic plasticity. Once activated in the cytoplasm, ERK1 and ERK2 translocate into the nucleus and interact with nuclear substrates to induce specific programs of gene expression. ERK1/2 share 85% of aminoacid identity and all known functional domains and thence they have been considered functionally equivalent until recent studies found that the ablation of either ERK1 or ERK2 causes dramatically different phenotypes. To search a molecular justification of this dichotomy we investigated whether the different functions of ERK1 and 2 might depend on the properties of their cytoplasmic-nuclear trafficking. Since in the nucleus ERK1/2 is predominantly inactivated, the maintenance of a constant level of nuclear activity requires continuous shuttling of activated protein from the cytoplasm. For this reason, different nuclear-cytoplasmic trafficking of ERK1 and 2 would cause a differential signalling capability. We have characterised the trafficking of fluorescently tagged ERK1 and ERK2 by means of time-lapse imaging in living cells. Surprisingly, we found that ERK1 shuttles between the nucleus and cytoplasm at a much slower rate than ERK2. This difference is caused by a domain of ERK1 located at its N-terminus since the progressive deletion of these residues converted the shuttling features of ERK1 into those of ERK2. Conversely, the fusion of this ERK1 sequence at the N-terminus of ERK2 slowed down its shuttling to a similar value found for ERK1. Finally, computational, biochemical and cellular studies indicated that the reduced nuclear shuttling of ERK1 causes a strong reduction of its nuclear phosphorylation compared to ERK2, leading to a reduced capability of ERK1 to carry proliferative signals to the nucleus. This mechanism significantly contributes to the differential ability of ERK1 and 2 to generate an overall signalling output.