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

Abstract

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 IC 50 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 EC 50 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.

Major changes of cell function and toxicant sensitivity in cultured cells undergoing mild, quasi-natural genetic drift

“…LUHMES cells are used as highly reproducible test system for neurotoxicity investigations or to model aspects of neurodegenerative disease (Delp et al 2017, 2018; Harris et al 2017; Krug et al 2014; Poltl et al 2012; Sarkar et al 2017; Schildknecht et al 2013; Scholz et al 2011; Stiegler et al 2011; Szego et al 2017; Xiang et al 2013; Zhang et al 2014). Since the MPP + model for Parkinson’s disease is frequently used by us and others (Schildknecht et al 2017; Terron et al 2018), the finding that the induction of cell death in LUHMES purchased from ATCC required 16 times higher concentration of the neurotoxicant MPP + (Zhang et al 2014), than found for the original cells (“UKN”) (Krug et al 2014; Poltl et al 2012; Schildknecht et al 2009), raised concerns about the definitions of cell line identity.…”

“…Since the MPP + model for Parkinson’s disease is frequently used by us and others (Schildknecht et al 2017; Terron et al 2018), the finding that the induction of cell death in LUHMES purchased from ATCC required 16 times higher concentration of the neurotoxicant MPP + (Zhang et al 2014), than found for the original cells (“UKN”) (Krug et al 2014; Poltl et al 2012; Schildknecht et al 2009), raised concerns about the definitions of cell line identity. Even though we found here SP of LUHMES from different sources to be highly similar on the genomic level (same STR-profile; PI_HAT score ≈ 1), we identified clear phenotypic and toxicological differences using a targeted identity profiling.…”

Neuronal protection against oxidative insult by polyanhydride nanoparticle-based mitochondria-targeted antioxidant therapy

“…For these studies, we used human mesencephalon-derived neuronal LUHMES cells, which are used to model human dopaminergic degeneration (53). With the addition of cAMP and GDNF, LUHMES cells differentiate by forming long axons with relatively dense interconnections typically seen in the mesencephalic region in the brain (54).…”

SH‐SY5Y and LUHMES cells display differential sensitivity to MPP+, tunicamycin, and epoxomicin in 2D and 3D cell culture

“…Despite increased sensitivity to the tested neurotoxicants, the MPP+ concentrations used in this study are quite high (100–1,000 uM) compared to the 5‐μM concentrations previously reported to induce significant (<50% intracellular ATP) LUHMES cell death . While previous studies used a 72‐h MPP+ exposure, 48‐h MPP+ exposure has been reported to have an LD50 of approximately 100 μM . Experiments also produced variable results with our neurotoxicant screen producing LC50 values similar to previous literature in one trial (Figure S3) while subsequent trials resulted in considerably higher LC50s.…”