Effect of low-dose thalidomide on dopaminergic neuronal differentiation of human neural progenitor cells: A combined study of metabolomics and morphological analysis Xian-Yang Qin a,b , Hiromi Akanuma a , Feifei Wei c , Reiko Nagano a , Qin Zeng a , Satoshi Imanishi d , Seiichiroh Ohsako d , Jun Yoshinaga b , Junzo Yonemoto a , Masaru Tanokura c , Hideko Sone a, * a Health Risk Research Section, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8606, Japan b Department of Environmental Studies, Graduate School of Frontier Science, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 270-8563, Japan c Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan d Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 1. Introduction A number of neurodegenerative diseases, including Parkinson’s disease (PD), are characterized by the progressive loss of dopaminergic neurons in the substantia nigra and their projections to the striatum, which leads to various motor deficits (Ebert et al., 2008; Jeon et al., 2010). Human neural progenitor cells (hNPCs), which are capable of dividing and differentiating into many cells types of the nervous system including neurons, astrocytes and oligodendrocytes, can respond to environmental demands by increasing their proliferation and differentiation (Ryu et al., 2009). Transplantation of genetically modified hNPCs to replace and protect dopaminergic neurons, as a potential therapeutic strategy for the treatment of PD, has become increasingly popular among researchers (Kitiyanant et al., 2011; Lunn et al., 2011; Ryu et al., 2009). Furthermore, hNPCs have recently been proposed as a powerful model system for developmental neurotoxicity testing, as they have the capacity to differentiate into any cell type in the nervous system. The use of hNPCs also affords better predictive power, as there is no need to extrapolate results obtained with non-human species (Breier et al., 2008, 2010). Thalidomide was used as a sedative drug to treat morning sickness in pregnant women in the 1950s, but was subsequently withdrawn from the market in 1961 because of severe teratoge- nicity and neurotoxicity (Franks et al., 2004; Lenz, 1988; Matthews and McCoy, 2003). Interestingly, subsequent studies on the mechanisms of thalidomide teratogenicity revealed that the compound was an effective anticancer and anti-inflammatory agent. The US Food and Drug Administration approved thalido- mide for the treatment of lepromatous leprosy and multiple myeloma in 1998 and 2006, respectively (Kim and Scialli, 2011; Teo et al., 2005; Uhl et al., 2006). However, major obstacles to the use of thalidomide are its diverse neurological side effects. It has been hypothesized that impairment of energy metabo- lism might contribute to nerve cell death in neurodegenerative diseases (Beal et al., 1993). Dysregulation of metabolites in the methionine (Met) transmethylation and transsulfuration path- ways has been implicated in several neurodevelopmental diseases, NeuroToxicology 33 (2012) 1375–1380 A R T I C L E I N F O Article history: Received 17 February 2012 Accepted 31 August 2012 Available online 7 September 2012 Keywords: Thalidomide Dopamine Metabolomics Neuronal differentiation CE-TOFMS A B S T R A C T Thalidomide is increasingly used in anticancer and anti-inflammation therapies. However, it is known for its teratogenicity and ability to induce peripheral neuropathy, although the mechanisms underlying its neurological effect in humans are unclear. In this study, we investigated the effect of thalidomide on the metabolism and neuronal differentiation of human neural progenitor cells. We found that levels of tyrosine, phenylalanine, methionine and glutathione, which are involved in dopamine and methionine metabolism, were decreased following thalidomide treatment. Morphological analysis revealed that treatment with 100 nM thalidomide, which is much lower than clinical doses, significantly decreased the number of dopaminergic (tyrosine hydroxylase-positive) neurons, compared with control cells. Our results suggest that these adverse neurological effects of thalidomide should be taken into consideration prior to its use for the treatment of neurodegenerative and other diseases. ß 2012 Elsevier Inc. All rights reserved. Abbreviations: CE-TOFMS, capillary electrophoresis time-of-flight mass spectrom- etry; DoD, day of differentiation; GSH, glutathione; HCA, hierarchical clustering analysis; hNPCs, human neural progenitor cells; MAP2, microtubule-associated protein 2; Met, methionine; PCA, principal component analysis; PD, Parkinson’s disease; Phe, phenylalanine; TH, tyrosine hydroxylase; Tyr, tyrosine. * Corresponding author. Tel.: +81 29 850 2464; fax: +81 29 850 2546. E-mail address: hsone@nies.go.jp (H. Sone). Contents lists available at SciVerse ScienceDirect NeuroToxicology 0161-813X/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2012.08.016