A gene therapy approach to knock-down Fyn in the striatum reduces levodopa induced dyskinesia in the 6-hydroxydopamine mouse model of Parkinson?s disease.

DAMIANICH A; GERSHANIK OS; BORDONE M; BERNARDI MA; FERRARIO JE; SANZ BLASCO S; AVALE, M. ELENA

San Diego

Congreso; Society for Neuroscience Congress 2018; 2018

International Society for Neurochemistry (ISN)

Levodopa (L-DOPA) induced dyskinesia (LID) is the main side effect that appears after long-termtreatment with that drug for Parkinson?s disease (PD). To reduce the development and severity of LID is asignificant challenge because to date there are no available pharmacological alternative therapies to PDwith full clinical benefit. We have recently proposed Fyn as a novel target to control LID. Fyn is a Srctyrosine 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 receptorstimulation. Here we propose to genetically manipulate Fyn expression aiming to downregulate LID in amouse 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 silencingefficacy and selected the one with the highest silencing effect. Next, we generated the PD mouse model inC57 female mice (3 months of age) by injecting 6-hydroxydopamine (6-OHDA) into the medial forebrainbundle, selected the animals with a remarked deficit of the contralateral forepaw in the cylinder test andchallenged them with L-DOPA to induce LID before (n=16) or after (n=19) the intra-striatal injection of miRFynor a control sequence (n=5 for each group). A group of non-injected animals with LV particles was runin 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 tyrosinehydroxylase 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. Wefound that the selected miR-Fyn reduces Fyn protein by ~50% in N2a neuronal cell line, and it significantlydownregulated LID in a pretreatment paradigm of dyskinesia by ~60% or ~40% compared to the noninjectedanimals or the group injected with the control sequence, respectively. Also, the miR-Fyn was ableto reduce the severity of already established dyskinesia (postreatment paradigm) by ~35% compared toboth control groups. Our results demonstrate that Fyn is a potential target to control LID and set the grounds for a potential translation to therapeutic use in PD.