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 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.
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References
de Rijk MC, Launer LJ, Berger K, Breteler MM, Dartigues JF, Baldereschi M, et al. Prevalence of Parkinson's disease in Europe: A collaborative study of population-based cohorts. Neurologic Diseases in the Elderly Research Group. Neurology 2000; 54: S21–3.
Dorsey ER, Constantinescu R, Thompson JP, Biglan KM, Holloway RG, Kieburtz K, et al. Projected number of people with Parkinson disease in the most populous nations, 2005 through 2030. Neurology 2007; 68: 384–6.
Fearnley JM, Lees AJ . Ageing and Parkinson's disease: substantia nigra regional selectivity. Brain 1991; 114: 2283–301.
Lees AJ, Hardy J, Revesz T . Parkinson's disease. Lancet 2009; 373: 2055–66.
Fahn S . The spectrum of levodopa-induced dyskinesias. Ann Neurol 2000; 47: S29.
Obeso JA, Rodriguez-Oroz MC, Chana P, Lera G, Rodriguez M, Olanow CW . The evolution and origin of motor complications in Parkinson's disease. Neurology 2000; 55: S13–20.
Alberio T, Lopiano L, Fasano M . Cellular models to investigate biochemical pathways in Parkinson's disease. FEBS J 2012; 279: 1146–55.
Phlman S, Mamaeva S, Meyerson G, Mattsson ME, Bjelfman C, Ortoft E, et al. Human neuroblastoma cells in culture: a model for neuronal cell differentiation and function. Acta Physiol Scand Suppl 1990; 592: 25–37.
Grau CM, Greene LA . Use of PC12 cells and rat superior cervical ganglion sympathetic neurons as models for neuroprotective assays relevant to Parkinson's disease. Methods Mol Biol 2012; 846: 201–11.
Schule B, Pera RA, Langston JW . Can cellular models revolutionize drug discovery in Parkinson's disease? Biochim Biophys Acta 2009; 1792: 1043–51.
Son JH, Chun HS, Joh TH, Cho S, Conti B, Lee JW . Neuroprotection and neuronal differentiation studies using substantia nigra dopaminergic cells derived from transgenic mouse embryos. J Neurosci 1999; 19: 10–20.
Schmidt F, Champy P, Seon-Meniel B, Franck X, Raisman-Vozari R, Figadere B . Chemicals possessing a neurotrophin-like activity on dopaminergic neurons in primary culture. PLoS One 2009; 4: e6215.
Lotharius J, Falsig J, van Beek J, Payne S, Dringen R, Brundin P, et al. Progressive degeneration of human mesencephalic neuron-derived cells triggered by dopamine-dependent oxidative stress is dependent on the mixed-lineage kinase pathway. J Neurosci 2005; 25: 6329–42.
Scholz D, Pöltl D, Genewsky A, Weng M, Waldmann T, Schildknecht S, et al. Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line. J Neurochem 2011; 119: 957–71.
Langston JW, Langston EB, Irwin I . MPTP-induced parkinsonism in human and non-human primates--clinical and experimental aspects. Acta Neurol Scand Suppl 1984; 100: 49–54.
Singer TP, Ramsay RR, McKeown K, Trevor A, Castagnoli NE Jr . Mechanism of the neurotoxicity of 1-methyl-4-phenylpyridinium (MPP+), the toxic bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Toxicology 1988; 49: 17–23.
Lotharius J, O'Malley KL . The parkinsonism-inducing drug 1-methyl-4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity. J Biol Chem 2000; 275: 38581–8.
Polymeropoulos MH, Lavedan C, Leroy E, Ide SE, Dehejia A, Dutra A, et al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 1997; 276: 2045–7.
Baba M, Nakajo S, Tu PH, Tomita T, Nakaya K, Lee VM, et al. Aggregation of alpha-synuclein in Lewy bodies of sporadic Parkinson's disease and dementia with Lewy bodies. Am J Pathol 1998; 152: 879–84.
Chartier-Harlin MC, Kachergus J, Roumier C, Mouroux V, Douay X, Lincoln S, et al. Alpha-synuclein locus duplication as a cause of familial Parkinson's disease. Lancet 2004; 364: 1167–9.
Chandra S, Fornai F, Kwon HB, Yazdani U, Atasoy D, Liu X, et al. Double-knockout mice for alpha- and beta-synucleins: effect on synaptic functions. Proc Natl Acad Sci U S A 2004; 101: 14966–71.
Studer L . Culture of substantia nigra neurons. Curr Protoc Neurosci 2001; Chapter 3: Unit.
Hongo H, Kihara T, Kume T, Izumi Y, Niidome T, Sugimoto H, et al. Glycogen synthase kinase-3beta activation mediates rotenone-induced cytotoxicity with the involvement of microtubule destabilization. Biochem Biophys Res Commun 2012; 426: 94–9.
Diaz-Corrales FJ, Asanuma M, Miyazaki I, Miyoshi K, Hattori N, Ogawa N . Dopamine induces supernumerary centrosomes and subsequent cell death through Cdk2 up-regulation in dopaminergic neuronal cells. Neurotox Res 2008; 14: 295–305.
Smith PD, O'Hare MJ, Park DS . CDKs: taking on a role as mediators of dopaminergic loss in Parkinson's disease. Trends Mol Med 2004; 10: 445–51.
Alvira D, Tajes M, Verdaguer E, de Arriba SG, Allgaier C, Matute C, et al. Inhibition of cyclin-dependent kinases is neuroprotective in 1-methyl-4-phenylpyridinium-induced apoptosis in neurons. Neuroscience 2007; 146: 350–65.
Petit-Paitel A, Brau F, Cazareth J, Chabry J . Involvment of cytosolic and mitochondrial GSK-3beta in mitochondrial dysfunction and neuronal cell death of MPTP/MPP-treated neurons. PLoS One 2009; 4: e5491.
Spillantini MG, Schmidt ML, Lee VM, Trojanowski JQ, Jakes R, Goedert M . Alpha-synuclein in Lewy bodies. Nature 1997; 388: 839–40.
Braak H, Ghebremedhin E, Rub U, Bratzke H, Del TK . Stages in the development of Parkinson's disease-related pathology. Cell Tissue Res 2004; 318: 121–34.
Volpicelli-Daley LA, Luk KC, Patel TP, Tanik SA, Riddle DM, Stieber A, et al. Exogenous alpha-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 2011; 72: 57–71.
Luk KC, Song C, O'Brien P, Stieber A, Branch JR, Brunden KR, et al. Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc Natl Acad Sci U S A 2009; 106: 20051–6.
Schildknecht S, Karreman C, Pöltl D, Efrémova L, Kullmann C, Gutbier S, et al. Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases. ALTEX 2013; 30: 427–444.
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Zhang, Xm., Yin, M. & Zhang, Mh. Cell-based assays for Parkinson's disease using differentiated human LUHMES cells. Acta Pharmacol Sin 35, 945–956 (2014). https://doi.org/10.1038/aps.2014.36
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DOI: https://doi.org/10.1038/aps.2014.36
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