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

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Acta Pharmacol Sin 2014 Jul; 35(7):945-56

Authors: Xiao-Min Zhang, Ming Yin, Min-Hua Zhang

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 ?-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 ?mol/L. MPP(+) (80 ?mol/L) significantly increased caspase 3/7 activity in the cells. Both the CDK inhibitor GW8510 and the GSK3? inhibitor SB216763 effectively rescued MPP(+)-induced reduction of ATP levels with EC50 values of 12 and 205 nmol/L, respectively. Overexpression of ?-synuclein also significantly decreased intracellular ATP levels and increased caspase 3/7 activity in the cells. GW8510 and SB216763 effectively rescued ?-synuclein overexpression-induced reduction of ATP levels, whereas GW8510, but not SB216763, ameliorated ?-synuclein overexpression-induced increase of caspase 3/7 activity.Conclusion:MPP(+)- and ?-synuclein overexpression-induced cytotoxicity of differentiated LUHMES cells may serve as good alternative systems for identifying neuroprotective compounds for PD.

PMID: 24989254 [PubMed - as supplied by publisher]

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