ORIGINAL ARTICLE β-N-Methylamino-L-Alanine Toxicity in PC12: Excitotoxicity vs. Misincorporation R. van Onselen 1 & L. Venables 1 & M. van de Venter 1 & T. G. Downing 1,2 Received: 27 January 2017 /Revised: 12 April 2017 /Accepted: 21 April 2017 # Springer Science+Business Media New York 2017 Abstract The implication of β-N-methylamino-L-alanine (BMAA) in the development of neurodegenerative diseases worldwide has led to several investigations of the mechanism, or mechanisms, of toxicity of this cyanobacterially produced amino acid. The primary mechanism of toxicity that was iden- tified is excitotoxicity, with a second possible mechanism, the misincorporation of BMAA into the primary protein structure and consequent cell damage, having been more recently re- ported. However, studies on excitotoxicity and misincorporation have been conducted independently and there are therefore no data available on the relative contribu- tion of each of these mechanisms to the total toxicity of BMAA. The rat pheochromocytoma cell line PC12 is an ideal model for a study of this type, as glutamate receptor expres- sion is modified by cell differentiation, which can be affected by exposure to nerve growth factor. In this study, the PC12 cell line was evaluated as a model to study BMAA toxicity via the two proposed mechanisms: excitotoxicity and protein misincorporation. BMAA and canavanine treatment of cultures of PC12 were evaluated for depolarization of the mi- tochondrial membrane. In canavanine-treated cultures, this was evident after 9 days of treatment and was attributed to the primary mechanism of canavanine toxicity, protein misincorporation. However, no membrane depolarization was observed for BMAA-treated cultures even after 21 days of continuous treatment at 500 μM. Short-term exposure to both BMAA and canavanine resulted in a slight increase in necrosis in undifferentiated cells that was prevented in canavanine-treated cultures by co-incubation with arginine, but not in BMAA-treated cultures by co-incubation with ser- ine. A slight increase in apoptosis was observed in undiffer- entiated cells treated with either BMAA or glutamate, and ROS production increased in glutamate-treated cells. However, the excitotoxicity was less pronounced than report- ed in previous studies with neuronal cells. In contrast, apopto- sis was greatly increased in both BMAA- and glutamate- treated cells after differentiation and resulting mGluR1 in- crease, indicating that excitotoxicity is the main, if not only, mechanism of toxicity in PC12. Keywords BMAA . Canavanine . Glutamate . Differentiated Introduction The isolation of β-N-methylamino-L-alanine (BMAA) from the seeds of the Guam cycad, Cycas circinalis (renamed C. micronesica), in 1967 by Vega and Bell, and their subse- quent studies in animal models (Vega and Bell 1967; Vega et al. 1968 ) suggested a role for this natural non- proteinogenic amino acid in the development of the Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS/PDC) among the Chamorro people of Guam (Arnold et al. 1953; Kurland and Mulder 1954). Subsequent * T. G. Downing Tim.Downing@nmmu.ac.za R. van Onselen Rianita.VanOnselen@nmmu.ac.za L. Venables s204004039@nmmu.ac.za M. van de Venter Maryna.VanDeVenter@nmmu.ac.za 1 Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O. Box 77000, Port Elizabeth 6031, South Africa 2 Institute for Ethnomedicine, PO Box 3464, Jackson, WY 83001, USA Neurotox Res DOI 10.1007/s12640-017-9743-8