Ascorbate Prevents Cell Death From Prolonged Exposure to Glutamate in an In Vitro Model of Human Dopaminergic Neurons Santiago Ballaz, 1,2 * Ingrid Morales, 2,3 Manuel Rodr  ıguez, 2,3 and Jos e A. Obeso 1,2 1 Laboratory of Movement Disorders, Department of Neuroscience, Centre for Applied Medicine Research (CIMA), University of Navarra, Pamplona, Spain 2 Centro de Investigaci on Biom edica en Red sobre Enfermedades Neurodegenerativas, CIBERNED, Madrid, Spain 3 Laboratory of Neurobiology and Experimental Neurology, Department of Physiology, Medical School, University of La Laguna, La Laguna, Tenerife, Canary Islands, Spain Ascorbate (vitamin C) is a nonenzymatic antioxidant highly concentrated in the brain. In addition to mediating redox balance, ascorbate is linked to glutamate neurotransmis- sion in the striatum, where it renders neuroprotection against excessive glutamate stimulation. Oxidative stress and glutamatergic overactivity are key biochemical features accompanying the loss of dopaminergic neurons in the substantia nigra that characterizes Parkinson’s disease (PD). At present, it is not clear whether antiglutamate agents and ascorbate might be neuroprotective agents for PD. Thus, we tested whether ascorbate can prevent cell death from prolonged exposure to glutamate using dopa- minergic neurons of human origin. To this purpose, dopamine-like neurons were obtained by differentiation of SH-SY5Y cells and then cultured for 4 days without antioxi- dant (antiaging) protection to evaluate glutamate toxicity and ascorbate protection as a model system of potential factors contributing to dopaminergic neuron death in PD. Glutamate dose dependently induced toxicity in dopa- minergic cells largely by the stimulation of AMPA and metabotropic receptors and to a lesser extent by N-methyl- D-aspartate and kainate receptors. At relatively physiologi- cal levels of extracellular concentration, ascorbate protected cells against glutamate excitotoxicity. This neuroprotection apparently relies on the inhibition of oxidative stress, because ascorbate prevented the pro-oxidant action of the scavenging molecule quercetin, which occurred over the course of prolonged exposure, as is also seen with gluta- mate. Our findings show the relevance of ascorbate as a neuroprotective agent and emphasize an often underappre- ciated role of oxidative stress in glutamate excitotoxicity. Occurrence of a glutamate–ascorbate link in dopaminergic neurons may explain previous contradictions regarding their putative role in PD. V C 2013 Wiley Periodicals, Inc. Key words: Parkinson’s disease; SH-SY5Y; AMPA receptor; glutamate metabotropic receptors; kainate receptor; NMDA receptor Parkinson’s disease (PD) is a neurodegenerative dis- order characterized mainly by the progressive loss of mid- brain dopaminergic neurons in the substantia nigra pars compacta. It is generally accepted that dopaminergic degeneration has a multifactorial origin (Przedborski, 2005; Obeso et al., 2010), including a-synuclein aggrega- tion (the hallmark of PD), mitochondrial dysfunction (Schapira, 2008), presence of enhanced oxidative stress (Jenner and Olanow, 1992), free radical production asso- ciated with both monoamine metabolism and glutamate (GLU) toxicity (Maher and Davis, 1996), and increased GLU activity (Blandini et al., 1996; Rodriguez et al., 1998). GLU could modulate dopaminergic activity and release by stimulating various GLU receptor subtypes expressed by dopaminergic neurons in the substantia nigra (Counihan et al., 1998; Tang et al., 2003; Mueller et al., 2004; Schiemann et al., 2012). Even if preclinical findings suggest a role for excitotoxicity in PD (Turski et al., 1991; Battaglia et al., 2003), neither antiglutamate agent (Jankovic and Hunter, 2002; Uitti et al., 1996) nor anti- oxidant (Weber and Ernst, 2006) has yet proved effica- cious to halt or modify disease progression. Ascorbate [the water-soluble form of ascorbic acid (AA), or vitamin C] is a nonenzymatic scavenging mole- cule that is highly concentrated in the brain to fulfill phys- iological roles that are only beginning to be elucidated (Rice, 2000; Harrison and May, 2009). In the striatum, the activation of extracellular GLU uptake generally involves the release of AA to the extracellular medium, Contract grant sponsor: R&D National Plan of the Science and Technol- ogy Ministry, Spanish Government. *Correspondence to: Santiago Ballaz, PhD, Department of Neuroscien- ces, CIMA, Pio XII 55, E-31008 Pamplona, Spain. E-mail: sballazg@ gmail.com Received 19 April 2013; Revised 15 June 2013; Accepted 17 June 2013 Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23276 V C 2013 Wiley Periodicals, Inc. Journal of Neuroscience Research 00:00–00 (2013)