EFFECT OF LRRK2 PATHOLOGICAL MUTATION ON DOPAMINE METABOLISM IN PC12 CELLS

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease. Li et al.1 have very recently shown that a LRRK2 transgenic mouse model recapitulates cardinal features of the disease; moreover, they showed that in vivo spontaneous dopamine (DA) release from striatal dopaminergic endings was significantly decreased. Since PC12 cells are a reliable model for studying in vitro dopamine (DA) metabolism, we deemed of interest the study of DA metabolism in PC12 cells with LRRK2 pathological mutation. Methods-Experiments were performed on PC12 cells during their exponential phase of growth. At the start of each experiment, 140x103 cells/cm2 were plated; after 24 h or 48 h DA and metabolites (DOPAC, HVA and 3-MT) were assayed both in medium and cell lysates by HPLC-EC, as previously described1. Four different PC12 lines (WT controls and 3 with LRRK2 pathological mutation) were used. Results – After 24 h of PC12 cell culture at confluence, DA content in lysates from the WT control was 3.78±0.38 µg/mg protein. In all PC12 lines with LRRK2 pathological mutation, DA content in cell lysates had increased (maximum increase by about 44%). Conversely, DA concentrations in the medium of the 3 lines with LRRK2 pathological mutation had drastically decreased from 2.66±0.61 µM of WT control to a maximum of 172±0.14 nM. Concentrations of 3-MT showed a similar decrease. After 48 h of of PC12 cell culture at confluence, DA content in cell lysates from the WT control was 3.98±0.38 µg/mg protein. In all PC12 lines with LRRK2 pathological mutation, DA content in cell lysates had significantly increased (maximum increase by about 82%). Conversely, DA concentrations in the medium of the 3 lines with LRRK2 pathological mutation had drastically decreased from 3.57±0.2 µM of WT control to a maximum of 311±0.27 nM. Concentrations of 3-MT showed a similar decrease. DOPAC and HVA content were always unaffected. Discussion – The results of the present study clearly show that LRRK2 pathological mutation greatly affects DA secretion from PC12 cells. Conversely, DA oxidative metabolism was unaffected. These data are in agreement with the in vivo results of Li et al.