Research Article Alternatively Spliced Methionine Synthase in SH-SY5Y Neuroblastoma Cells: Cobalamin and GSH Dependence and Inhibitory Effects of Neurotoxic Metals and Thimerosal Mostafa Waly, 1 Verna-Ann Power-Charnitsky, 2 Nathaniel Hodgson, 3 Alok Sharma, 4 Tapan Audhya, 5 Yiting Zhang, 6 and Richard Deth 7 1 Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, 123 Al-Khoud, Oman 2 Natural Sciences Department, Regis College, Weston, MA 02493, USA 3 Department of Neurology, Boston Children’s Hospital, Boston, MA 02215, USA 4 Department of Pharmaceutical Sciences, MCPHS University, Manchester, NH 03101, USA 5 Department of Medicine, New York University Medical School, New York, NY 10016, USA 6 Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, USA 7 Department of Pharmaceutical Sciences, Nova Southeastern University, Fort Lauderdale, FL 33328, USA Correspondence should be addressed to Richard Deth; rdeth@nova.edu Received 6 September 2015; Revised 28 December 2015; Accepted 10 January 2016 Academic Editor: Antonio Ayala Copyright © 2016 Mostafa Waly et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Te folate and cobalamin (Cbl-) dependent enzyme methionine synthase (MS) is highly sensitive to oxidation and its activity afects all methylation reactions. Recent studies have revealed alternative splicing of MS mRNA in human brain and patient- derived fbroblasts. Here we show that MS mRNA in SH-SY5Y human neuroblastoma cells is alternatively spliced, resulting in three primary protein species, thus providing a useful model to examine cofactor dependence of these variant enzymes. MS activity was dependent upon methylcobalamin (MeCbl) or the combination of hydroxocobalamin (OHCbl) and S-adenosylmethionine (SAM). OHCbl-based activity was eliminated by depletion of the antioxidant glutathione (GSH) but could be rescued by provision of either glutathionylcobalamin (GSCbl) or MeCbl. Pretreatment of cells with lead, arsenic, aluminum, mercury, or the ethylmercury- containing preservative thimerosal lowered GSH levels and inhibited MS activity in association with decreased uptake of cysteine, which is rate-limiting for GSH synthesis. Timerosal treatment decreased cellular levels of GSCbl and MeCbl. Tese fndings indicate that the alternatively spliced form of MS expressed in SH-SY5Y human neuronal cells is sensitive to inhibition by thimerosal and neurotoxic metals, and lower GSH levels contribute to their inhibitory action. 1. Introduction MS is a multidomain enzyme which transfers a folate-derived methyl group to homocysteine (HCY), thereby creating methionine. Te cobalamin (Cbl) cofactor of MS, its Cbl[I] form, directly participates in the transfer reaction by abstract- ing a folate-derived methyl group, temporarily creating methylcobalamin (MeCbl), and then transferring the methyl group to HCY [1]. However, if Cbl[I] oxidizes prior to MeCbl formation, enzyme activity is temporarily halted, increasing HCY diversion to the transsulfuration pathway and augment- ing formation of cysteine, the rate-limiting metabolite for synthesis of the antioxidant GSH [2, 3]. In this manner Cbl serves as a redox sensor whose oxidation leads to increased antioxidant synthesis in proportion to cellular demand. MS inactivation is accompanied by decreased methylation activity, caused by lower levels of the methyl donor SAM and higher levels of the methylation inhibitor S-adenosylhomo- cysteine (SAH) [4]. Tus MS and Cbl link redox status to methylation status, including methylation of DNA and Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2016, Article ID 6143753, 11 pages http://dx.doi.org/10.1155/2016/6143753