Cell-based assays for Parkinson's disease using differentiated human LUHMES cells

Aim: Lund human mesencephalic (LUHMES) cells can be differentiated to post-mitotic cells with biochemical, morphological and functional features of dopaminergic (DAergic) neurons. Given the limited scale of primary DAergic neuron culture, we developed differentiated LUHMES cell-based cytotoxicity assays for identifying neuroprotective agents for Parkinson's disease (PD). Methods: LUHMES cells were incubated in a differentiation medium containing cAMP and GDNF for 6 d, and then differentiated cells were treated with MPP+ or infected with baculovirus containing alpha-synuclein. Cytotoxicity was determined by measuring intracellular ATP levels and caspase 3/7 activity in the cells. DAergic neuron-specific marker protein and mRNA levels in the cells were analyzed using Western blotting and RT-PCR, respectively. Results: LUHMES cells grew extensive neurites and became post-mitotic neuron-like cells during differentiation period, and three DAergic neuron markers TH, DAT and Nurr1 exhibited different expression profiles. MPP+ dose-dependently reduced ATP levels in the cells with an IC50 value of 65 mu mol/L. MPP+ (80 mu mol/L) significantly increased caspase 3/7 activity in the cells. Both the CDK inhibitor GW8510 and the GSK3 beta inhibitor SB216763 effectively rescued MPP+-induced reduction of ATP levels with EC50 values of 12 and 205 nmol/L, respectively. Overexpression of alpha-synuclein also significantly decreased intracellular ATP levels and increased caspase 3/7 activity in the cells. GW8510 and SB216763 effectively rescued alpha-synuclein overexpression-induced reduction of ATP levels, whereas GW8510, but not SB216763, ameliorated alpha-synuclein overexpression-induced increase of caspase 3/7 activity. Conclusion: MPP+- and alpha-synuclein overexpression-induced cytotoxicity of differentiated LUHMES cells may serve as good alternative systems for identifying neuroprotective compounds for PD.