β-N-Methylamino-L-Alanine Toxicity in PC12: Excitotoxicity vs. Misincorporation

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 identified is excitotoxicity, with a second possible mechanism, the misincorporation of BMAA into the primary protein structure and consequent cell damage, having been more recently reported. However, studies on excitotoxicity and misincorporation have been conducted independently and there are therefore no data available on the relative contribution 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 expression 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 mitochondrial 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 serine. A slight increase in apoptosis was observed in undifferentiated cells treated with either BMAA or glutamate, and ROS production increased in glutamate-treated cells. However, the excitotoxicity was less pronounced than reported in previous studies with neuronal cells. In contrast, apoptosis was greatly increased in both BMAA- and glutamate-treated cells after differentiation and resulting mGluR1 increase, indicating that excitotoxicity is the main, if not only, mechanism of toxicity in PC12.

Investigating β-N-Methylamino-l-alanine Misincorporation in Human Cell Cultures: A Comparative Study with Known Amino Acid Analogues

“…Although Dunlop et al [ 16 ] reported the misincorporation of BMAA in a human lung fibroblast cell line and in human umbilical vein endothelial cells, by virtue of detection of BMAA in the protein fraction, no toxicity in these cell lines was reported whereas toxicity was reported for the neuroblastoma cell line used in the same study, suggesting excitotoxicity as a mechanism. Van Onselen et al [ 69 ] showed that differentiation of a rat pheochromocytoma cell line (PC12) with nerve growth factor, with resulting expression of glutamate receptors, was required in order to achieve BMAA toxicity, presumably via an excitotoxic mechanism. Differentiation of the same cell line was not required to observe toxicity in response to l -canavanine exposure, indicating different mechanisms of toxicity of these two compounds.…”

“…Evidence for non-primary protein association in favor of misincorporation also exists [ 34 ]. Furthermore, despite reports that BMAA does not misincorporate into proteins in bacteria or in a rat pheochromocytoma cell line [ 34 , 69 ] it has been suggested that the lack of prokaryote misincorporation may not hold true in eukaryotes [ 75 ], although the authors cite Glover et al [ 32 ] in support of their hypothesis, despite their use of a prokaryotic expression system. In this study, human cell lines were therefore used to evaluate BMAA misincorporation using both toxicological markers and amino acid compositional analysis of purified proteins from exposed cell lines, with reference to known misincorporating amino acid analogues.…”

Cellular and Molecular Aspects of the β-N-Methylamino-l-alanine (BMAA) Mode of Action within the Neurodegenerative Pathway: Facts and Controversy

“…It is also suggested that BMAA incorporates neuronal proteins, and substitutes for ala or ser residues, potentially inducing protein misfolding, aggregation mechanisms and cellular apoptosis [ 45 ]. But this hypothesis is controversial and recent in vitro data propose that BMAA does not misincorporate into proteins, adding to the complexity of its precise mode of action [ 92 ]. However, if confirmed, the incorporation of the toxin in proteins might explain its toxic bioaccumulation in cells coupled with a progressive release of BMAA in the CNS over the years, as a function of the protein turnover.…”

From Superficial Damage to Invasion of the Nucleosome: Ranking of Morbidities by the Biosemiotic Depth Hypothesis

“…However, the relevant research, at least in the case of BMAA shows that wholesale misincorporation of non-protoeinogenic amino acids is not easily achieved in living cells [253], [254]. BMAA misincorporation is resisted even in bacteria [242], though misincorporation can be humanly engineered and achieved efficiently in vitro under special conditions [251], [255], [256].…”