Screening Small Molecules With Neuroprotective Activity By Establishing Cell-based Assays For Parkinson’s Disease

Aim: Human mesencephalic neuron-derived LUHMES cells can be differentiatedto post-mitotic cells with biochemical, morphological and functional features ofdopaminergic neurons. In an effort to identify neuroprotective agents for Parkinson’sdisease (PD), we developed cell-based cytotoxicity assays using differentiatedLUHMES cells to model dopaminergic neuronal degeneration.Methods: LUHMES cells were cultured and differentiated using cAMP andGDNF. To induce cell death relevant to dopaminergic neuronal degeneration, thedifferentiated cells were treated with MPP+or infected with baculovirus tooverexpress α-synuclein. The cytotoxicity after the treatment was measured asintracellular ATP levels and caspase3/7activity. The CDK inhibitor GW8510and theGSK3β inhibitor SB216763were used as the reference compounds to rescue MPP+orα-synuclein-induced toxicity.Results: During the differentiation, LUHMES cells gradually expressed manydopaminergic neuron-specific markers, grew extensive neurites and becamepost-mitotic neuron-like cells. MPP+dose dependently reduced ATP levels andincreased caspase3/7activity in the differentiated cells. Both CDK inhibitor GW8510and GSK3β inhibitor SB216763effectively rescued MPP+-induced toxicity with EC50 values of12nmol/L and205nmol/L, respectively. Overexpression of α-synucleinalso significantly decreased intracellular ATP levels and caspase3/7activity, whichcould be ameliorated by GW8510, but not SB216763..Conclusion: In this study, we established and validated two PD-relevantcell-based cytotoxicity assays. Given high technical demand and limited scale ofprimary dopaminergic neuron culture, the differentiated LUHMES cells may be agood alternative assay system to screen for neuroprotective compounds forParkinson’s disease.