ININFA   02677
INSTITUTO DE INVESTIGACIONES FARMACOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
A gene therapy approach to knock-down Fyn in the striatum reduces levodopa induced dyskinesia in the 6-OHDA mice model of Parkinson?s disease.
Autor/es:
DAMIANICH, ANA; GERSHANIK, OSCAR S.; BERNARDI, M. ALEJANDRA; AVALE, M. ELENA; BORDONE, MELINA P.; SANZ-BLASCO, SARA; FERRARIO, JUAN E.
Lugar:
San Diego
Reunión:
Congreso; SFN Neuroscience 2018; 2018
Resumen:
Levodopa (L-DOPA) induced dyskinesia (LID) is the main side effect that appears after long-term treatment with that drug for Parkinson?s disease (PD). To reduce the development and severity of LID is a significant challenge because to date there are no available pharmacological alternative therapies to PD with full clinical benefit. We have recently proposed Fyn as a novel target to control LID. Fyn is a Src tyrosine kinase located at the postsynaptic density zone that regulates the N-methyl-D-aspartate (NMDA) receptor by phosphorylation of the NR2B subunit at Tyr-1472 in response to dopamine D1 receptor stimulation. Here we propose to genetically manipulate Fyn expression aiming to downregulate LID in a mouse model of PD, by intra-striatal injection of lentiviral (LV) particles carrying a micro-RNA against Fyn (miR-Fyn). Four miR-Fyn sequences were designed, cloned in LV vectors, tested in vitro for silencing efficacy and selected the one with the highest silencing effect. Next, we generated the PD mouse model in C57 female mice (3 months of age) by injecting 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, selected the animals with a remarked deficit of the contralateral forepaw in the cylinder test and challenged them with L-DOPA to induce LID before (n=16) or after (n=19) the intra-striatal injection of miRFyn or a control sequence (n=5 for each group). A group of non-injected animals with LV particles was run in parallel as a positive control for dyskinesia (n=9). LIDs were determined every 3 days for 2 weeks. Postmortem, we determined the degree of dopaminergic denervation by counting the number of tyrosine hydroxylase positive cells in the substantia nigra, to ensure an equal level of degeneration in all groups, and the level of striatal TH, Fyn silencing and the marker of LID, FosB-ΔFosB, all by Western blot. We found that the selected miR-Fyn reduces Fyn protein by ~50% in N2a neuronal cell line, and it significantly downregulated LID in a pretreatment paradigm of dyskinesia by ~60% or ~40% compared to the noninjected animals or the group injected with the control sequence, respectively. Also, the miR-Fyn was able to reduce the severity of already established dyskinesia (postreatment paradigm) by ~35% compared to both control groups. Our results demonstrate that Fyn is a potential target to control LID and set thegrounds for a potential translation to therapeutic use in PD.