Mechanisms and modes of β-N-methylamino-lalanine neurotoxicity: the basis for designing therapies

Since the discovery of the non-canonical amino acid β-N-methylamino-L-alanine (BMAA) and the demonstration of its acute neurotoxicity in chicks and rats, it has been postulated that BMAA might contribute to the development of neurodegenerative diseases worldwide due to its presence in numerous aquatic and terrestrial food webs. This hypothesized link was widely criticized because of the inability to reproduce symptoms in a BMAA-exposed animal model that resembled the symptoms observed in humans, and for the inability to achieve significant levels of toxicity in in vitro models via the postulated mechanisms of toxicity. The most widely described mechanism of BMAA toxicity was excitotoxicity by over-excitation of ionotropic and/or metabotropic glutamate receptors following activation by BMAA. However, the excitotoxic potency of BMAA is much lower than those of other known excitotoxins and it was not known whether BMAA could accumulate in significant concentrations in synapses to cause the said excitotoxicity. Therefore, uptake of BMAA into synaptic vesicles from where it can be released into synapses in high concentrations, was investigated and it was found that, unlike the uptake that was observed for glutamate, BMAA was not taken up into synaptic vesicles. This discovery suggests that BMAA is not released into synapses via synaptic vesicles and that excitotoxicity is an unlikely mechanism of BMAA toxicity in mammalian systems. Misincorporation of BMAA into proteins in the place of L-serine was suggested to be an important mechanism of BMAA toxicity that could lead to protein misfolding and the subsequent protein aggregates that are typically found in the central nervous system (CNS) of neurodegenerative disease patients. However, previous studies in prokaryotes and in a rat pheochromocytoma PC12 cell line showed that misincorporation of BMAA does not occur to any significant extent. However, these studies were criticized for not using human-derived model systems to show that misincorporation does not occur, and it was argued that due to differences in mitochondrial protein synthesis mechanisms, misincorporation of BMAA into human proteins could not be ruled out as a possible mechanism of toxicity. Therefore, misincorporation of BMAA was investigated in a number of human-derived non-neuronal cell lines and directly compared to the misincorporation of other known amino acid analogues. No evidence of misincorporation of BMAA into these cell lines was obtained and therefore it was concluded that misincorporation of BMAA into proteins does not occur in human-derived cell models. Although misincorporation of BMAA into proteins was refuted as a mechanism of toxicity, the strong interactions between BMAA and proteins that require extensive purification procedures to remove the associated BMAA, could not be discounted as a possible contributor to the toxicity of BMAA. Cell-free interactions between BMAA and enzymes, which resulted in reduced activity, were described previously but the nature of these interactions was never determined. Therefore, the direct interactions between BMAA and a range of commercial proteins and melanin (that is known to also have a strong affinity for BMAA) were investigated in an attempt to describe the nature of these interactions. It was discovered that BMAA has a high affinity for hydroxyl groups, and that if these hydroxyl groups in the form of hydroxyl containing amino acid residues occurred in important regulatory or active sites of proteins, BMAA reduced the enzyme activity. Catalase was subsequently selected as an important enzyme required for the maintenance of the delicate reactive oxygen species (ROS) balance in the CNS, to test the effect of BMAA on the activity of the enzyme. BMAA inhibited a human commercial extract of catalase in a cell free system, and this inhibition appeared to be non-competitive in nature. Subsequently, catalase in an extract from a human cell line was also shown to be inhibited by BMAA and it was concluded that this BMAA induced inhibition of catalase could be an important contributor to the toxicity of BMAA in in vivo systems. The affinity of BMAA for hydroxyl groups, especially the reactive L-tyrosine side chain hydroxyl, was recognized as a possible mechanism that can be utilized to protect against the toxicity of BMAA. It was subsequently shown that excess concentrations of L-serine and L-tyrosine could protect against the BMAA-induced enzyme inhibition and improper folding of proteins in a cell-free system. By administering an equimolar concentration of either L-phenylalanine (the soluble precursor of L-tyrosine) or L-serine an hour before administration of BMAA in a rat model, the BMAA-induced neurotoxicity was greatly reduced, especially by treatment with L-phenylalanine, which resulted in a decrease of between 60-70% in the observed neuropathologies. It was recognized that the protection offered by L-phenylalanine was greater than would be expected if protection was by virtue of direct hydroxyl binding alone and it was subsequently hypothesized that the conversion of L-phenylalanine to dopamine could have contributed to the observed protection. Subsequently, the possible protection offered by dopamine, administered as L-DOPA, against BMAA neurotoxicity was investigated in the same neonatal rat model and compared to the protection offered by L-tyrosine. It was discovered that dopamine protected against the BMAAinduced neuronal cell losses in the hippocampus, striatum and spinal cord but it was not as efficient as L-tyrosine in protection against the BMAA-induced proteinopathies, suggesting two distinct mechanisms of BMAA toxicity, one of which is a depletion of dopamine, which had not been previously described. Finally, the nature of the BMAA-induced dopamine depletion was investigated by administering BMAA in combination with other dopaminergic modifiers viz. apomorphine (a D1/D2 receptor agonist), a dopamine transporter inhibitor (GBR12783) and reserpine (a vesicular monoamine transporter -VMAT2- inhibitor) to the neonatal rat model in an attempt to describe how BMAA functions as a dopaminergic toxin. Based on these results it was concluded that BMAA inhibits uptake of dopamine into synaptic vesicles by inhibiting VMAT2-mediated uptake of dopamine, which causes neuronal loss in the hippocampus, striatum and substantia nigra pars compacta, and that the BMAA-induced inhibition of catalase contributes significantly to the toxicity of BMAA by causing an accumulation of hydrogen peroxide in the hippocampus, striatum and spinal cord, which results in extensive neuronal damage in these areas. This work was the first to thoroughly investigate the mechanisms that explain the observed pathologies caused by BMAA in an in vivo model, and was the first to suggest that BMAA can reduce the dopamine in the CNS by inhibiting VMAT2-mediated uptake of dopamine into synaptic vesicles, and increase damage by reactive oxygen species by inhibiting catalase. BMAA is therefore a multimechanistic and multimodal

In vivo and In vitro investigations to elucidate the associations of B-N-methylamino-L-alanine with proteins

The cyanobacterially synthesized non-canonical amino acid β-N-methylamino-ʟ-alanine (BMAA) has been proposed to be a causative agent in the development of sporadic neurodegenerative diseases. This neurotoxin bioaccumulates and biomagnifies with increasing trophic levels in ecosystems by associating with proteins. It has been suggested that these associations with host proteins also serve as a reservoir from where BMAA is slowly released with normal protein catabolism, resulting in a continuous low level exposure. However, the nature of these associations remains poorly defined. The widely accepted hypothesis regarding the nature of these associations is that BMAA associates with proteins through primary incorporation into proteins with specific replacement of serine. In addition to excitotoxicity, BMAA misincorporation has been proposed as a potential mechanism of toxicity because of its link to protein tangle diseases. Interactions between BMAA and proteins that are not the result of misincorporation, have also been observed. However, the nature of these non-primary associations has not been investigated. This study focussed on establishing whether BMAA is misincorporated into host proteins with consequent toxicity, and on elucidating the nature of the BMAA-protein associations not linked to primary incorporation. In comparative studies between BMAA and canavanine, an arginine analogue known to misincorporate, exposure to BMAA did not result in any toxicity in prokaryotes or in an undifferentiated eukaryotic mammalian cell line, in contrast to what was observed upon canavanine exposure. Differentiation of the cell line with nerve growth factor to express glutamate receptors resulted in marked toxicity upon BMAA exposure, highlighting excitoxicity as the main mechanism of BMAA toxicity. Furthermore, it was demonstrated that BMAA interacts with free amino acids and proteins in the absence of de novo protein synthesis, causing enzyme inhibition and protein misfolding. It was concluded that BMAA does not interact with proteins through primary incorporation and that the observed associations are the result of an interaction between BMAA and amino acid side chains to form covalent bonds

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

Misincorporation of β-N-methylamino-l-alanine (BMAA) into proteins has been proposed to be a mechanism of toxicity to explain the role of BMAA in neurodegenerative disease development. However, studies have shown that all detectable BMAA can be removed from proteins by SDS-PAGE purification and that the toxicity of l-canavanine cannot be reproduced in prokaryotes or in a rat pheochromocytoma cell line, strongly indicating that the misincorporation hypothesis of BMAA should be re-investigated. The aim of this study was therefore to determine if BMAA misincorporates into proteins in cells of human origin with subsequent misincorporation-type toxicity. Almost complete loss of viability in response to exposure to l-4-fluorophenylalanine and l-m-tyrosine was observed in all of the cell lines, corresponding to a concentration-dependent increase of the analogues in protein extracts from exposed cells. In contrast, BMAA exposure resulted in slight toxicity in one of the cell lines but the observed toxicity was not the result of misincorporation of BMAA into proteins, as no BMAA was detected in any of the SDS-PAGE purified protein extracts that were obtained from the cells following BMAA exposure. The results show that BMAA is not misincorporated into human proteins and that misincorporation is not a valid mechanism of toxicity