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

Acta Pharmacol Sin. 2014 Jul;35(7):945-56. doi: 10.1038/aps.2014.36.


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 α-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 μmol/L. MPP(+) (80 μmol/L) significantly increased caspase 3/7 activity in the cells. Both the CDK inhibitor GW8510 and the GSK3β inhibitor SB216763 effectively rescued MPP(+)-induced reduction of ATP levels with EC50 values of 12 and 205 nmol/L, respectively. Overexpression of α-synuclein also significantly decreased intracellular ATP levels and increased caspase 3/7 activity in the cells. GW8510 and SB216763 effectively rescued α-synuclein overexpression-induced reduction of ATP levels, whereas GW8510, but not SB216763, ameliorated α-synuclein overexpression-induced increase of caspase 3/7 activity.

Conclusion: MPP(+)- and α-synuclein overexpression-induced cytotoxicity of differentiated LUHMES cells may serve as good alternative systems for identifying neuroprotective compounds for PD.

MeSH terms

  • 1-Methyl-4-phenylpyridinium
  • Cell Death / drug effects
  • Cell Differentiation
  • Cell Line
  • Cyclin-Dependent Kinase 2 / antagonists & inhibitors
  • Drug Evaluation, Preclinical / methods*
  • Glycogen Synthase Kinase 3 / antagonists & inhibitors
  • Humans
  • Indoles / pharmacology*
  • Maleimides / pharmacology*
  • Mesencephalon / cytology*
  • Neurons / cytology
  • Neurons / drug effects*
  • Neurons / metabolism
  • Neuroprotective Agents / pharmacology*
  • Parkinson Disease / drug therapy*
  • Parkinson Disease / genetics
  • Parkinson Disease, Secondary / chemically induced
  • alpha-Synuclein / genetics


  • GW8510
  • Indoles
  • Maleimides
  • Neuroprotective Agents
  • SB 216763
  • alpha-Synuclein
  • Cyclin-Dependent Kinase 2
  • Glycogen Synthase Kinase 3
  • 1-Methyl-4-phenylpyridinium