Studio del meccanismo di sintesi e rilascio della dopamina in cellule PC 12 TET-ON contenenti la dardarina (LRRK2) WT e mutata

Mutations in the gene coding for dardarin (LRRK2) cause autosomal-dominant Parkinson’s disease. Using PC12 as a model for studying in vitro dopamine (DA) metabolism,we studied the effect of LRRK2 on DA secretion and their metabolites in PC12 cells with human LRRK2, in order to distinguish between the effects of transgene expression, activated in presence of doxycycline (DOX),and the potential effects of nicotine. At the start of experiment,50x103cells/cm2were plated and treated 24h later (time 0) with DOX (0.2-1.0 µg/ml); after 48h,through microdialysis procedure, was treated with nicotine 5 mM and DA and metabolites (DOPAC, HVA and 3-MT) were assayed by HPLC-EC. Four PC12-ON cells lines were used for experiments and in 3 PC12-ON cell lines,expressing human LRRK2, pathological mutation was induced as follow: PC12-ON, PC12-ON LRRK2 WT,PC12-ON LRRK2G2019Sand PC12-ON LRRK2 R1441C. Changes in baseline levels of DA secretion were observed, in PC12-ON expressing either mutant LRRK2 or WT,48h after treatment with 0.2 µg/ml DOX.DA was higher in mutant LRRK2G2019S,and lower in WT-LRRK2, when compared with the control. A reduction of DA secretion was detected,48h after treatment with 1 µg/ml DOX,only in mutant LRRK2G2019S. In all the experiments with PC12 cell with mutant or WT-LRRK2,the baseline of DA metabolites was lower than the control. The results on DA secretion and their metabolites were confirmed when the cells were treated with different concentrations of DOX and infused with nicotine.