A Direct Analysis of ß-N-methylamino-l-alanine Enantiomers and Isomers and Its Application to Cyanobacteria and Marine Mollusks.

Of the wide variety of toxic compounds produced by cyanobacteria, the neurotoxic amino acid ß-N-methylamino-l-alanine (BMAA) has attracted attention as a result of its association with chronic human neurodegenerative diseases such as ALS and Alzheimer's. Consequently, specific detection methods are required to assess the presence of BMAA and its isomers in environmental and clinical materials, including cyanobacteria and mollusks. Although the separation of isomers such as ß-amino-N-methylalanine (BAMA), N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) from BMAA has been demonstrated during routine analysis, a further compounding factor is the potential presence of enantiomers for some of these isomers. Current analytical methods for BMAA mostly do not discriminate between enantiomers, and the chiral configuration of BMAA in cyanobacteria is still largely unexplored. To understand the potential for the occurrence of D-BMAA in cyanobacteria, a chiral UPLC-MS/MS method was developed to separate BMAA enantiomers and isomers and to determine the enantiomeric configuration of endogenous free BMAA in a marine Lyngbya mat and two mussel reference materials. After extraction, purification and derivatization with N-(4-nitrophenoxycarbonyl)-l-phenylalanine 2-methoxyethyl ester ((S)-NIFE), both L- and D-BMAA were identified as free amino acids in cyanobacterial materials, whereas only L-BMAA was identified in mussel tissues. The finding of D-BMAA in biological environmental materials raises questions concerning the source and role of BMAA enantiomers in neurological disease.

Genomic insights into the biosynthesis and physiology of the cyanobacterial neurotoxin 3-N-methyl-2,3-diaminopropanoic acid (BMAA).

Cyanobacteria are an ancient clade of photosynthetic prokaryotes, present in many habitats throughout the world, including water resources. They can present health hazards to humans and animals due to the production of a wide range of toxins (cyanotoxins), including the diaminoacid neurotoxin, 3-N-methyl-2,3-diaminopropanoic acid (ß-N-methylaminoalanine, BMAA). Knowledge of the biosynthetic pathway for BMAA, and its role in cyanobacteria, is lacking. Present evidence suggests that BMAA is derived by 3-N methylation of 2,3-diaminopropanoic acid (2,3-DAP) and, although the latter has never been reported in cyanobacteria, there are multiple pathways to its biosynthesis known in other bacteria and in plants. Here, we used bioinformatics analyses to investigate hypotheses concerning 2,3-DAP and BMAA biosynthesis in cyanobacteria. We assessed the potential presence or absence of each enzyme in candidate biosynthetic routes known in Albizia julibrissin, Lathyrus sativus seedlings, Streptomyces, Clostridium, Staphylococcus aureus, Pantoea agglomerans, and Paenibacillus larvae, in 130 cyanobacterial genomes using sequence alignment, profile hidden Markov models, substrate specificity/active site identification and the reconstruction of gene phylogenies. Most enzymes involved in pathways leading to 2,3-DAP in other species were not found in the cyanobacteria analysed. Nevertheless, two species appear to have the genes sbnA and sbnB, responsible for forming the 2,3-DAP constituent in staphyloferrin B, a siderophore from Staphylococcus aureus. It is currently undetermined whether these species are also capable of biosynthesising BMAA. It is possible that, in some cyanobacteria, the formation of 2,3-DAP and/or BMAA is associated with environmental iron-scavenging. The pam gene cluster, responsible for the biosynthesis of the BMAA-containing peptide, paenilamicin, so far appears to be restricted to Paenibacillus larvae. It was not detected in any of the cyanobacterial genomes analysed, nor was it found in 93 other Paenibacillus genomes or in the genomes of two BMAA-producing diatom species. We hypothesise that the presence, in some cyanobacterial species, of the enzymes 2,3-diaminopropionate ammonia-lyase (DAPAL) and reactive intermediate deaminase A (RidA) may explain the failure to detect 2,3-DAP in analytical studies. Overall, the taxonomic distribution of 2,3-DAP and BMAA in cyanobacteria is unclear; there may be multiple and additional routes, and roles, for the biosynthesis of 2,3-DAP and BMAA in these organisms.

Genomic insights into the biosynthesis and physiology of the cyanobacterial neurotoxin 2,4-diaminobutanoic acid (2,4-DAB).

Cyanobacteria are an ancient clade of photosynthetic prokaryotes, whose worldwide occurrence, especially in water, presents health hazards to humans and animals due to the production of a range of toxins (cyanotoxins). These include the sometimes co-occurring, non-encoded diaminoacid neurotoxins 2,4-diaminobutanoic acid (2,4-DAB) and its structural analogue ß-N-methylaminoalanine (BMAA). Knowledge of the biosynthetic pathway for 2,4-DAB, and its role in cyanobacteria, is lacking. The aspartate 4-phosphate pathway is a known route of 2,4-DAB biosynthesis in other bacteria and in some plant species. Another pathway to 2,4-DAB has been described in Lathyrus species. Here, we use bioinformatics analyses to investigate hypotheses concerning 2,4-DAB biosynthesis in cyanobacteria. We assessed the presence or absence of each enzyme in candidate biosynthesis routes, the aspartate 4-phosphate pathway and a pathway to 2,4-DAB derived from S-adenosyl-L-methionine (SAM), in 130 cyanobacterial genomes using sequence alignment, profile hidden Markov models, substrate specificity/active site identification and the reconstruction of gene phylogenies. In the aspartate 4-phosphate pathway, for the 18 species encoding diaminobutanoate-2-oxo-glutarate transaminase, the co-localisation of genes encoding the transaminase with the downstream decarboxylase or ectoine synthase - often within hybrid non-ribosomal peptide synthetase (NRPS)-polyketide synthases (PKS) clusters, NRPS-independent siderophore (NIS) clusters and incomplete ectoine clusters - is compatible with the hypothesis that some cyanobacteria use the aspartate 4-phosphate pathway for 2,4-DAB production. Through this route, in cyanobacteria, 2,4-DAB may be functionally associated with environmental iron-scavenging, via the production of siderophores of the schizokinen/synechobactin type and of some polyamines. In the pathway to 2,4-DAB derived from SAM, eight cyanobacterial species encode homologs of SAM-dependent 3-amino-3-carboxypropyl transferases. Other enzymes in this pathway have not yet been purified or sequenced. Ultimately, the biosynthesis of 2,4-DAB appears to be either restricted to some cyanobacterial species, or there may be multiple and additional routes, and roles, for the synthesis of this neurotoxin.

Environmental distribution of the neurotoxin l-BMAA in Paenibacillus species.

The environmental distribution of the neurotoxic amino acid, 3-N-methyl-2,3-diaminopropanoic acid (BMAA), first isolated in 1967, was initially believed to be limited to tropical and subtropical plants of the genus Cycas. The seeds of one such species, which had been used historically on the Pacific island of Guam as a foodstuff, had a reputation for neurotoxicity. Some 40 years later the amino acid was detected in terrestrial and aquatic cyanobacteria and in other aquatic organisms. Overlooked was the discovery of BMAA in peptides of bizarre structure that had been isolated in 1975 from Paenibacillus pulvifaciens during a search for antibiotics. More recently (2014), peptides of similar structure were isolated from Paenibacillus larvae; this organism is causative of American Foulbrood, a lethal disease of honeybee colonies. These are interesting chemical and environmental observations, but knowledge of the bacterial distribution of BMAA is limited to just these two species of Paenibacillus, while more than 200 Paenibacillus spp. are known. Paenibacillus spp. are ever present naturally in the environment and are used agriculturally; recent research reports that some species infect human foods - including cow's milk - and have been isolated from human body fluids. We wish to stimulate interest in the environmental distribution of the neurotoxic BMAA in Paenibacillus spp. by drawing together previously isolated streams of research and by proposing experimental approaches by which this matter might be resolved.

50 years of research on α-amino-ß-methylaminopropionic acid (ß-methylaminoalanine).

The isolation of α-amino-ß-methylaminopropionic acid from seeds of Cycas circinalis (now C. micronesica Hill) resulted from a purposeful attempt to establish the cause of the profound neurological disease, amyotrophic lateral sclerosis/parkinsonism/dementia, that existed in high frequency amongst the inhabitants of the western Pacific island of Guam (Guam ALS/PD). In the 50 years since its discovery the amino acid has been a stimulus, and sometimes a subject of mockery, for generations of scientists in a remarkably diverse range of subject areas. The number of citations of the original paper has risen in the five decades from a few to 120 within the decade 2007-2016 and continues at a high rate into the next decade. The reasons for this remarkable outcome are discussed and examples from the literature are used to illustrate the wide range of scientific interest that the original paper generated.

Metabolic solutions to the biosynthesis of some diaminomonocarboxylic acids in nature: Formation in cyanobacteria of the neurotoxins 3-N-methyl-2,3-diaminopropanoic acid (BMAA) and 2,4-diaminobutanoic acid (2,4-DAB).

The non-encoded diaminomonocarboxylic acids, 3-N-methyl-2,3-diaminopropanoic acid (syn: α-amino-ß-methylaminopropionic acid, MeDAP; ß-N-methylaminoalanine, BMAA) and 2,4-diaminobutanoic acid (2,4-DAB), are distributed widely in cyanobacterial species in free and bound forms. Both amino acids are neurotoxic in whole animal and cell-based bioassays. The biosynthetic pathway to 2,4-DAB is well documented in bacteria and in one higher plant species, but has not been confirmed in cyanobacteria. The biosynthetic pathway to BMAA is unknown. This review considers possible metabolic routes, by analogy with reactions used in other species, by which these amino acids might be biosynthesised by cyanobacteria, which are a widespread potential environmental source of these neurotoxins. Where possible, the gene expression that might be implicated in these biosyntheses is discussed.

Analysis of BMAA enantiomers in cycads, cyanobacteria, and mammals: in vivo formation and toxicity of D-BMAA.

Chronic dietary exposure to the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA) triggers neuropathology in non-human primates, providing support for the theory that BMAA causes a fatal neurodegenerative illness among the indigenous Chamorro people of Guam. However, since there are two stereoisomers of BMAA, it is important to know if both can occur in nature, and if so, what role they might play in disease causation. As a first step, we analysed both BMAA enantiomers in cyanobacteria, cycads, and in mammals orally dosed with L-BMAA, to determine if enantiomeric changes could occur in vivo. BMAA in cyanobacteria and cycads was found only as the L-enantiomer. However, while the L-enantiomer in mammals was little changed after digestion, we detected a small pool of D-BMAA in the liver (12.5%) of mice and in the blood plasma of vervets (3.6%). Chiral analysis of cerebrospinal fluid of vervets and hindbrain of mice showed that the free BMAA in the central nervous system was the D-enantiomer. In vitro toxicity investigations with D-BMAA showed toxicity, mediated through AMPA rather than NMDA receptors. These findings raise important considerations concerning the neurotoxicity of BMAA and its relationship to neurodegenerative disease.

The Regulation and Function of Lactate Dehydrogenase A: Therapeutic Potential in Brain Tumor.

There are over 120 types of brain tumor and approximately 45% of primary brain tumors are gliomas, of which glioblastoma multiforme (GBM) is the most common and aggressive with a median survival rate of 14 months. Despite progress in our knowledge, current therapies are unable to effectively combat primary brain tumors and patient survival remains poor. Tumor metabolism is important to consider in therapeutic approaches and is the focus of numerous research investigations. Lactate dehydrogenase A (LDHA) is a cytosolic enzyme, predominantly involved in anaerobic and aerobic glycolysis (the Warburg effect); however, it has multiple additional functions in non-neoplastic and neoplastic tissues, which are not commonly known or discussed. This review summarizes what is currently known about the function of LDHA and identifies areas that would benefit from further exploration. The current knowledge of the role of LDHA in the brain and its potential as a therapeutic target for brain tumors will also be highlighted. The Warburg effect appears to be universal in tumors, including primary brain tumors, and LDHA (because of its involvement with this process) has been identified as a potential therapeutic target. Currently, there are, however, no suitable LDHA inhibitors available for tumor therapies in the clinic.

Brain glutathione as a target for aetiological factors in neurolathyrism and konzo.

Both neurolathyrism and konzo are associated with the nutritional dependence of human populations on a single plant food. These diseases express themselves as chronic disorders of upper motor neurones, leading to signs and symptoms that characterise amyotrophic lateral sclerosis (motor neurone disease). The plant food associated with neurolathyrism is grass pea, which contains the neurotoxic ß-N-oxalyl-α,ß-diaminopropionic acid (ß-ODAP). The plant food associated with konzo is cassava, which may contain significant concentrations of cyanogenic glycosides and their degradation products. A monotonous diet of grass pea is likely to generate nutritional deficiencies; it is proposed that one of these, plasma methionine deficiency, may predispose neurones to the neurotoxic effects of ß-ODAP. Subjects suffering from konzo also have low concentrations of plasma methionine as a result of a dietary deficiency of this amino acid. However, the plasma cystine concentration is also compromised because cyanide released from cyanogenic glycosides in cassava probably reacts with plasma cystine non-enzymatically. The product of this reaction is 2-imino-4-thiazolidine carboxylic acid. Since both plasma methionine and cystine are used for glutathione synthesis it seems likely that one common feature that leads to motor neurone death in neurolathyrism and konzo is the depletion of glutathione in the central nervous system.

Toxicity of non-protein amino acids to humans and domestic animals.

Non-protein amino acids are common in plants and are present in widely consumed animal feeds and human foods such as alfalfa (Medicago sativa), which contains canavanine, and lentil (Lens culinaris), which contains homoarginine. Some occur in wild species that are inadvertently harvested with crop species. Some non-protein amino acids and metabolites can be toxic to humans, e.g. Lathyrus species contain a neurotoxic oxalyl-amino acid. Some potential toxins may be passed along a food chain via animal intermediates. The increased interest in herbal medicines in the Western countries will increase exposure to such compounds.

Three phases of research on beta-N-methylamino-L-alanine (BMAA)--a neurotoxic amino acid.

This paper discusses various aspects of the research that lead from the discovery of beta-N-methylamino-L-alanine (BMAA) to consider a variety of mechanisms that might explain the acute and chronic toxicities of this non-protein amino acid. Such is the fashion of science that current work represents the third phase of research on this compound over a period of more than 40 years. BMAA is now known to exist not only in the plant genus Cycas, where it is synthesized by symbiotic cyanobacteria in the coralloid roots of the plants, but to be widely distributed in the many sites at which free living cyanobacteria abound.

Beta-N-methylaminoalanine (BMAA): metabolism and metabolic effects in model systems and in neural and other tissues of the rat in vitro.

The non-protein amino acid, beta-N-methylaminoalanine (BMAA), is neurotoxic and has been implicated in the amyotrophic lateral sclerosis-Parkinsonism-dementia (ALS-PD) complex of Guam. This concept remains controversial, in part because of the lack of a convincing animal model. The neuropharmacology of BMAA is well established, but little is known of its metabolism. This paper reports aspects of the metabolism, and metabolic effects, of BMAA in rat tissues. BMAA changed the distribution of taurine, glycine and serine between rat brain slices and their incubation medium; the glutamate/glutamine cycle between neurones and glia was also compromised. In model experiments BMAA reacted non-enzymatically with pyridoxal-5'-phosphate, releasing methylamine. Rat liver and kidney homogenates, but not brain homogenates, also formed methylamine and 2,3-diaminopropanoic acid when incubated with BMAA. These results provide evidence that several biochemical mechanisms are involved in the neurotoxicity of BMAA. The novel discovery that methylamine is formed from BMAA in rat liver and kidney preparations may be significant since chronic administration of methylamine to rats causes oxidative stress. The extent to which this reaction occurs in different animal species might be a decisive factor in selecting an animal model.

The Lathyrus excitotoxin beta-N-oxalyl-L-alpha,beta-diaminopropionic acid is a substrate of the L-cystine/L-glutamate exchanger system xc-.

Beta-N-oxalyl-L-alpha-beta-diaminopropionic acid (beta-L-ODAP) is an unusual amino acid present in seeds of plants from the Lathyrus genus that is generally accepted as the causative agent underlying the motor neuron degeneration and spastic paraparesis in human neurolathyrism. Much of the neuropathology produced by beta-L-ODAP appears to be a direct consequence of its structural similarities to the excitatory neurotransmitter L-glutamate and its ability to induce excitotoxicity as an agonist of non-NMDA receptors. Its actions within the CNS are, however, not limited to non-NMDA receptors, raising the likely possibility that the anatomical and cellular specificity of the neuronal damage observed in neurolathyrism may result from the cumulative activity of beta-L-ODAP at multiple sites. Accumulating evidence suggests that system xc-, a transporter that mediates the exchange of L-cystine and L-glutamate, is one such site. In the present work, two distinct approaches were used to define the interactions of beta-L-ODAP with system xc-: Traditional radiolabel-uptake assays were employed to quantify inhibitory activity, while fluorometrically coupled assays that follow the exchange-induced efflux of L-glutamate were used to assess substrate activity. In addition to confirming that beta-L-ODAP is an effective competitive inhibitor of system xc-, we report that the compound exhibits a substrate activity comparable to that of the endogenous substrate L-cystine. The ability of system xc- to transport and accumulate beta-L-ODAP identifies additional variables that could influence its toxicity within the CNS, including the ability to limit its access to EAA receptors by clearing the excitotoxin from the extracellular synaptic environment, as well as serving as a point of entry through which beta-L-ODAP could have increased access to intracellular targets.