LUHMES Cells as Human In Vitro Model to Study Mechanisms of Parkinsonian Neurodegeneration

Abstract

Background: Parkinson’s disease (PD) is characterized by a progressive loss of dopaminergic neurons in the substantia nigra. As degeneration processes run very slowly and single cells are hard to track in vivo, the demand for in vitro neurodegeneration models for mechanistic studies and the discovery of new pharmacological targets is growing.Aims: To study both genetic and toxic factors of parkinsonianlike neurodegeneration, a human in vitro model system (LUHMES) resembling classical features of in vivo dopaminergic neurons was established. We aim to clarify PD-like neurodegeneration mechanisms in these cells.Methods: For characterization of LUHMES and toxicological experiments state-of-the-art methods were used. Degeneration was induced by the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP1).Results: LUHMES cells develop a pronounced dopaminergic phenotype following differentiation for 6 days: Dopamine transporter (DAT), dopamine receptor 2 (DRD2), tyrosine hydroxylase (TH), vesicular monoamine transporter (VMAT-2) as well as the neuronal form of ß-III-tubulin and alpha-synuclein (ASYN) are endogenously expressed and a complex neurite network is formed. Hereby, dopamine levels increase, and differentiated LUHMES, in contrast to undifferentiated cells, become susceptible to low micromolar concentrations of the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP1), while they do not react to the parental compound 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To investigate cell death pathways involved in MPP1 toxicity, LUHMES were treated with various inhibitors acting on different levels of the involved signalling pathways prior to MPP1 stimulation. Inhibition on kinase levels with CEP1347, inhibition on the level of mitochondrial contribution as well as the antioxidant ascorbic acid effectively protect from MPP1 toxicity. Also, high concentrations of pan-caspase inhibitors completely protected from cell death. Furthermore, genetic components, as e.g. the contribution of ASYN to dopamine-dependent MPP1 toxicity can be studied in LUHMES cells, as siRNA-based knockdown of ASYN leads to attenuated toxicity of MPP1.Discussion/Conclusion: Since the LUHMES/MPP1 in vitro model displays characteristic features observed in PD brain, this system will be used to study both genetic and toxic factors of parkinsonian neurodegeneration and seems to be suited for larger screen situations for pharmacological inhibitors of dopaminergic neurodegeneration.