title: Cell-based assays for Parkinson's disease using differentiated human LUHMES cells.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  MPP(+)- and α-synuclein overexpression-induced cytotoxicity of differentiated LUHMES cells may serve as good alternative systems for identifying neuroprotective compounds for PD.
authors.forename: Xiao-Min
authors.initials: XM
authors.lastname: Zhang
authors.forename: Ming
authors.initials: M
authors.lastname: Yin
authors.forename: Min-Hua
authors.initials: MH
authors.lastname: Zhang
mesh_terms: D015655:1-Methyl-4-phenylpyridinium; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation; D002460:Cell Line; D051357:Cyclin-Dependent Kinase 2 / Q000037:antagonists & inhibitors; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology*; D008636:Mesencephalon / Q000166:cytology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D010300:Parkinson Disease / Q000188:drug therapy* / Q000235:genetics; D010302:Parkinson Disease, Secondary / Q000139:chemically induced; D051844:alpha-Synuclein / Q000235:genetics
chemical_list: C500810:GW8510; D007211:Indoles; D008301:Maleimides; D018696:Neuroprotective Agents; C417521:SB 216763; D051844:alpha-Synuclein; D051357:Cyclin-Dependent Kinase 2; D038362:Glycogen Synthase Kinase 3; D015655:1-Methyl-4-phenylpyridinium