title: Differential involvement of Ras-GRF1 and Ras-GRF2 in L-DOPA-induced dyskinesia.
abstract: OBJECTIVE
  Recent findings have shown that pharmacogenetic manipulations of the Ras-ERK pathway provide a therapeutic means to tackle l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID). First, we investigated whether a prolonged l-DOPA treatment differentially affected ERK signaling in medium spiny neurons of the direct pathway (dMSNs) and in cholinergic aspiny interneurons (ChIs) and assessed the role of Ras-GRF1 in both subpopulations. Second, using viral-assisted technology, we probed Ras-GRF1 and Ras-GRF2 as potential targets in this pathway. We investigated how selective blockade of striatal Ras-GRF1 or Ras-GRF2 expression impacted on LID (induction, maintenance, and reversion) and its neurochemical correlates.
  
  
  METHODS
  We used both Ras-GRF1 knockout mice and lentiviral vectors (LVs) delivering short-hairpin RNA sequences (shRNAs) to obtain striatum-specific gene knockdown of Ras-GRF1 and Ras-GRF2. The consequences of these genetic manipulations were evaluated in the 6-hydroxydopamine mouse model of Parkinson's disease. Escalating doses of l-DOPA were administered and then behavioral analysis with immunohistochemical assays and in vivo microdialysis were performed.
  
  
  RESULTS
  Ras-GRF1 was found essential in controlling ERK signaling in dMSNs, but its ablation did not prevent ERK activation in ChIs. Moreover, striatal injection of LV-shRNA/Ras-GRF1 attenuated dyskinesia development and ERK-dependent signaling, whereas LV-shRNA/Ras-GRF2 was without effect, ruling out the involvement of Ras-GRF2 in LID expression. Accordingly, Ras-GRF1 but not Ras-GRF2 striatal gene-knockdown reduced l-DOPA-induced GABA and glutamate release in the substantia nigra pars reticulata, a neurochemical correlate of dyskinesia. Finally, inactivation of Ras-GRF1 provided a prolonged anti-dyskinetic effect for up to 7 weeks and significantly attenuated symptoms in animals with established LID.
  
  
  CONCLUSIONS
  Our results suggest that Ras-GRF1 is a promising target for LID therapy based on Ras-ERK signaling inhibition in the striatum.
authors.affiliation: Section of Pharmacology, Department of Medical Sciences, University of Ferrara Ferrara, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Bido
authors.affiliation: Division of Neuroscience, Institute of Experimental Neurology, IRCSS San Raffaele Scientific Institute Milan, Italy ; Istituto di Ricerche Farmacologiche "Mario Negri" Milan, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Solari
authors.affiliation: Division of Neuroscience, Institute of Experimental Neurology, IRCSS San Raffaele Scientific Institute Milan, Italy ; Istituto di Ricerche Farmacologiche "Mario Negri" Milan, Italy.
authors.forename: Marzia
authors.initials: M
authors.lastname: Indrigo
authors.affiliation: Division of Neuroscience, Institute of Experimental Neurology, IRCSS San Raffaele Scientific Institute Milan, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: D'Antoni
authors.affiliation: Division of Neuroscience, Institute of Experimental Neurology, IRCSS San Raffaele Scientific Institute Milan, Italy ; Division of Neuroscience, School of Biosciences, Neuroscience and Mental Health Research Institute, Cardiff University Cardiff, United Kingdom.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Brambilla
authors.affiliation: Section of Pharmacology, Department of Medical Sciences, University of Ferrara Ferrara, Italy ; Neuroscience Centre and National Institute of Neuroscience Ferrara, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Morari
authors.affiliation: Division of Neuroscience, Institute of Experimental Neurology, IRCSS San Raffaele Scientific Institute Milan, Italy ; Division of Neuroscience, School of Biosciences, Neuroscience and Mental Health Research Institute, Cardiff University Cardiff, United Kingdom.
authors.forename: Stefania
authors.initials: S
authors.lastname: Fasano