ß-N-methylamino-L-alanine production, photosynthesis and transcriptional expression in a possible mutation strain and a wild strain of Thalassiosira minima.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) produced by marine diatoms has been implicated as an important environmental trigger of neurodegenerative diseases in humans. However, the biosynthesis mechanism of BMAA in marine diatoms is still unknown. In the present study, the strain of diatom Thalassiosira minima almost lost the biosynthesis ability for BMAA after a long-term subculture in our laboratory. The production of BMAA-containing proteins in the mutant strain of T. minima reduced to 18.2 % of that in the wild strain, meanwhile the cell size decreased but pigment content increased in the mutant strain. Take consideration of our previous transcriptional data on the mixed diatom and cyanobacterium cultures, the current transcriptome analysis showed four identical and highly correlated KEGG pathways associated with the accumulation of misfolded proteins in diatom, including ribosome, proteasome, SNARE interactions in vesicle transport, and protein processing in the endoplasmic reticulum. Analysis of amino acids and transcriptional information suggested that amino acid synthesis and degradation are associated with the biosynthesis of BMAA-containing proteins. In addition, a reduction in the precision of ubiquitination-mediated protein hydrolysis and vesicular transport by the COPII system will exacerbate the accumulation of BMAA-containing proteins in diatoms.

Microbial Diversity Impacts Non-Protein Amino Acid Production in Cyanobacterial Bloom Cultures Collected from Lake Winnipeg.

Lake Winnipeg in Manitoba, Canada is heavily impacted by harmful algal blooms that contain non-protein amino acids (NPAAs) produced by cyanobacteria: N-(2-aminoethyl)glycine (AEG), ß-aminomethyl-L-alanine (BAMA), ß-N-methylamino-L-alanine (BMAA), and 2,4-diaminobutyric acid (DAB). Our objective was to investigate the impact of microbial diversity on NPAA production by cyanobacteria using semi-purified crude cyanobacterial cultures established from field samples collected by the Lake Winnipeg Research Consortium between 2016 and 2021. NPAAs were detected and quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using validated analytical methods, while Shannon and Simpson alpha diversity scores were determined from 16S rRNA metagenomic sequences. Alpha diversity in isolate cultures was significantly decreased compared to crude cyanobacterial cultures (p < 0.001), indicating successful semi-purification. BMAA and AEG concentrations were higher in crude compared to isolate cultures (p < 0.0001), and AEG concentrations were correlated to the alpha diversity in cultures (r = 0.554; p < 0.0001). BAMA concentrations were increased in isolate cultures (p < 0.05), while DAB concentrations were similar in crude and isolate cultures. These results demonstrate that microbial community complexity impacts NPAA production by cyanobacteria and related organisms.

How does the neurotoxin ß-N-methylamino-L-alanine exist in biological matrices and cause toxicity?

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been deemed as a risk factor for some neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). This possible link has been proved in some primate models and cell cultures with the appearance that BMAA exposure can cause excitotoxicity, formation of protein aggregates, and/or oxidative stress. The neurotoxin BMAA extensively exists in the environment and can be transferred through the food web to human beings. In this review, the occurrence, toxicological mechanisms, and characteristics of BMAA were comprehensively summarized, and proteins and peptides were speculated as its possible binding substances in biological matrices. It is difficult to compare the published data from previous studies due to the inconsistent analytical methods and components of BMAA. The binding characteristics of BMAA should be focused on to improve our understanding of its health risk to human health in the future.

Cell cycle of microalga Isochrysis galbana arrested by neurotoxin ß-N-methylamino-l-alanine and corresponding molecular mechanisms.

The phycotoxin ß-N-methylamino-l-alanine (BMAA) has attracted attention due to its risks to marine organisms and human health. In this study, approximately 85 % of synchronized cells of the marine microalga Isochrysis galbana were arrested at the cell cycle G1 phase by BMAA at 6.5 µM for a 24-h exposure. The concentration of chlorophyll a (Chl a) gradually decreased, while the maximum quantum yield of PSII (Fv/Fm), the maximum relative electron transport rate (rETRmax), light utilization efficiency (α) and half-saturated light irradiance (Ik) reduced early and recovered gradually in I. galbana exposed to BMAA in 96-h batch cultures. Transcriptional expression of I. galbana analyzed at 10, 12, and 16 h disclosed multiple mechanisms of BMAA to suppress the microalgal growth. Production of ammonia and glutamate was limited by the down-regulation of nitrate transporters, glutamate synthase, glutamine synthetase, cyanate hydrolase, and formamidase. Diverse extrinsic proteins related to PSII, PSI, cytochrome b6f complex, and ATPase were influenced by BMAA at transcriptional level. Suppression of the DNA replication and mismatch repair pathways increased the accumulation of misfolded proteins, which was reflected by the up-regulated expression of proteasome to accelerate proteolysis. This study improves our understanding of the chemical ecology impacts of BMAA in marine ecosystems.

Putative biosynthesis mechanism of the neurotoxin ß-N-methylamino-L-alanine in marine diatoms based on a transcriptomics approach.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been presumed as an environmental cause of human neurodegenerative disorders, such as Alzheimer's disease. Marine diatoms Thalassiosira minima are demonstrated here to produce BMAA-containing proteins in axenic culture while the isomer diaminobutyric acid was bacterially produced. In the co-culture with Cyanobacterium aponinum, diatom growth was inhibited but the biosynthesis of BMAA-containing proteins was stimulated up to seven times higher than that of the control group by cell-cell interactions. The stimulation effect was not caused by the cyanobacterial filtrate. Nitrogen deprivation also doubled the BMAA content of T. minima cells. Transcriptome analysis of the diatom in mixed culture revealed that pathways involved in T. minima metabolism and cellular functions were mainly influenced, including KEGG pathways valine and leucine/isoleucine degradation, endocytosis, pantothenate and CoA biosynthesis, and SNARE interactions in vesicular transport. Based on the expression changes of genes related to protein biosynthesis, it was hypothesized that ubiquitination and autophagy suppression, and limited COPII vesicles transport accuracy and efficiency were responsible for biosynthesis of BMAA-containing proteins in T. minima. This study represents a first application of transcriptomics to investigate the biological processes associated with BMAA biosynthesis in diatoms.

Degradation of ß-N-methylamino-l-alanine (BMAA) by UV/peracetic acid system: Influencing factors, degradation mechanism and DBP formation.

ß-N-methylamino-l-alanine (BMAA) is a cyanobacterial neurotoxin associated with human neurodegenerative diseases, and its removal in drinking water is receiving increasing attention. In this study, the degradation of BMAA in UV/peracetic acid (UV/PAA) system was investigated. BMAA degradation followed the pseudo-first-order kinetic model. The synergistic effect of UV and PAA exhibited a great potential for BMAA degradation, which was attributed to the generation of a large number of reactive radicals, of which R-C• was the most dominant contributor. We also explored the effects of different factors on BMAA degradation. The results showed that there was a positive correlation between BMAA degradation and PAA dosage, and the optimal effect was achieved at pH 7. Notably, the existence of water matrices such as bicarbonate (HCO3－), chloride ion (Cl－), humic acid (HA) and algal intracellular organic matter (IOM) all inhibited the degradation of BMAA. Based on the identified intermediates, this study suggested that reactive radicals degraded BMAA mainly by attacking the carbon-nitrogen bonds on BMAA. Besides, comparing the effect of Cl－ on disinfection byproduct (DBP) formation in UV/PAA-post-PAA oxidation and UV/chlorine-post-chlorination systems, it was found that the former was more sensitive to the presence of Cl－.

Neuropathological Mechanisms of ß-N-Methylamino-L-Alanine (BMAA) with a Focus on Iron Overload and Ferroptosis.

The incidence of neurodegenerative diseases and cyanobacterial blooms is concomitantly increasing worldwide. The cyanotoxin ß-N-methylamino-L-alanine (BMAA) is produced by most of the Cyanobacteria spp. This cyanotoxin is described as a potential environmental etiology factor for some sporadic neurodegenerative diseases. Climate change and eutrophication significantly increase the frequency and intensity of cyanobacterial bloom in water bodies. This review evaluates different neuropathological mechanisms of BMAA at molecular and cellular levels and compares the related studies to provide some useful recommendations. Additionally, the structure and properties of BMAA as well as its microbial origin, especially by gut bacteria, are also briefly covered. Unlike previous reviews, we hypothesize the possible neurotoxic mechanism of BMAA through iron overload. We also discuss the involvement of BMAA in excitotoxicity, TAR DNA-binding protein 43 (TDP-43) translocation and accumulation, tauopathy, and other protein misincorporation and misfolding.

Second Laboratory Validation of ß-N-Methylamino-L-Alanine, N-(2aminoethyl)Glycine, and 2,4-Diaminobuytric Acid by Ultra-Performance Liquid Chromatography and Tandem Mass Spectrometry.

The analysis of ß-N-methylamino-L-alanine (BMAA) has been validated according to AOAC international standards by a single laboratory (Glover et al. 2015). Using the same validated method, we add a second laboratory validation optimizing for different equipment. Given publicized concerns about standardizing methods across laboratories and recent reviews indicating superior results using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization for the separation of BMAA and its isomers N-(2aminoethyl)glycine (AEG), and 2,4-diaminobuytric acid (DAB) (Bishop and Murch 2019), we add a second laboratory validation to this method demonstrating that the method is robust across laboratories using different equipment. Using the US Food and Drug Administration (FDA 2018) method for evaluating instrument parameters, we calculated a limit of detection (LOD) of 10 pg/ml for BMAA, AEG, and DAB and lower limits of quantification (LLOQ) of 37 pg/ml based on reagent blanks. In biological matrices, a higher LLOQ may be warranted for AEG and DAB. We demonstrate that the endogenous BMAA in mussel tissue can be lost by drying the hydrolyzed preparation and suggest sample preparation parameters be evaluated for robustness.

A Single Laboratory Validation for the Analysis of Underivatized ß-N-Methylamino-L-Alanine (BMAA).

ß-N-Methylamino-L-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria that can accumulate in ecosystems and food webs. Human exposure to cyanobacterial and algal blooms may be a risk factor for neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Analytical chemists have struggled to find reliable methods for BMAA analysis in complex sample matrices. Analysis of BMAA is complicated by at least 3 naturally occurring isomers: N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB), and ß-aminomethyl-L-alanine (BAMA). More than 350 publications have reported detection and quantification of BMAA and its isomers, but varying results have led to controversy in the literature. The objective of this study was to perform a single laboratory validation (SLV) of a frequently published method for BMAA analysis using a ZIC-HILIC column. We investigated the selectivity, linearity, accuracy, precision, and sensitivity of the method and our data show that this HILIC method fails many of the criteria for a validated method. The method fails the criterion for selectivity as the chromatography does not separate BMAA from its isomer BAMA. Sensitivity of the method greatly decreased over the experimental period and it demonstrated a higher limit of detection (LOD) (7.5 pg on column) and a higher lower limit of quantification (LLOQ) (30 pg on column) than other published validated methods. The method demonstrated poor precision of repeated injections of standards of BMAA with % relative standard deviation (%RSD) values that ranged from 37 to 107% while HorRat values for BMAA had a fail rate of 80% and BAMA had a fail rate of 73%. No HorRat values between 0.5 and 2 were found for repeated injections of standards of AEG and DAB. Recovery of 13C3,15N2-BMAA in a cyanobacterial matrix was < 10% in experiments and we were also unable to accurately detect other protein amino acids including methionine, cysteine, or alanine, indicating matrix effects. The results of this study demonstrate that the ZIC-HILIC column is not fit for purpose for the analysis of BMAA in cyanobacterial matrices and further provides explanations for the high level of negative results reported by researchers using this method.

Microbial BMAA and the Pathway for Parkinson's Disease Neurodegeneration.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) is a natural non-proteinogenic diamino acid produced by several species of both prokaryotic (cyanobacteria) and eukaryotic (diatoms and dinoflagellates) microorganisms. BMAA has been shown to biomagnify through the food chain in some ecosystems, accumulating for example in seafood such as shellfish and fish, common dietary sources of BMAA whose ingestion may have possible neuronal consequences. In addition to its excitotoxic potential, BMAA has been implicated in protein misfolding and aggregation, inhibition of specific enzymes and neuroinflammation, all hallmark features of neurodegenerative diseases. However, the exact molecular mechanisms of neurotoxicity remain to be elucidated in detail. Although BMAA is commonly detected in its free form, complex BMAA-containing molecules have also been identified such as the paenilamicins, produced by an insect gut bacterial pathogen. On the other hand, production of BMAA or BMAA-containing molecules by members of the human gut microbiota, for example by non-photosynthetic cyanobacteria, the Melainabacteria, remains only hypothetical. In any case, should BMAA reach the gut it may interact with cells of the mucosal immune system and neurons of the enteric nervous system (ENS) and possibly target the mitochondria. Here, we review the available evidence and hint on possible mechanisms by which chronic exposure to dietary sources of this microbial neurotoxin may drive protein misfolding and mitochondrial dysfunction with concomitant activation of innate immune responses, chronic low-grade gut inflammation, and ultimately the neurodegenerative features observed across the gut-brain axis in Parkinson's disease (PD).

Inhibitory effects of neurotoxin ß-N-methylamino-L-alanine on fertilization and early development of the sea urchin Lytechinus pictus.

Neurotoxin ß-N-methylamino-L-alanine (BMAA) has been widely detected in diverse aquatic organisms and hypothesized as an environmental risk to neurodegenerative diseases in humans. However, the knowledge of its toxicity to marine organisms requires attention. In the present study, embryos and sperm of the sea urchin, Lytechinus pictus, were used to assess the toxicity of BMAA. Effects of BMAA on fertilization and development of sea urchin embryos were measured, and its impacts on efflux transport of sea urchin blastula were also assayed. Results demonstrated that the fertilization and development of embryos were significantly inhibited by high concentrations of BMAA above 300 µg L-1. The EC50 values indicated by active swimming larvae and total larvae numbers at 96 HPF (hours post fertilization) were 165 µg L-1 (1.4 µmol L-1) and 329 µg L-1 (2.8 µmol L-1), respectively. Additionally, sperm exposed to BMAA for 10 min significantly reduced the fertilization ratio of sea urchin eggs. However, the ABC transport activity on the cytomembrane of sea urchin blastula was not inhibited by the presence of BMAA at 50 µg L-1, even up to 500 µg L-1. Abnormal division and developmental malformations occurred at different developmental stages for sea urchin embryos exposed to BMAA at 500 µg L-1. The inhibitory effects of BMAA on sea urchin embryos were reported at the first time in this study, for which the toxicological mechanisms will be explored in future studies.

The mechanism of ß-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation.

ß-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.

Accumulation and distribution of neurotoxin BMAA in aquatic animals and effect on the behavior of zebrafish in a T-maze test.

Neurotoxin ß-N-methylamino-L-alanine (BMAA) has been widely detected in diverse aquatic organisms within different ecosystem trophic levels in recent years. It was the goal of this study to investigate the accumulation and tissue distribution of BMAA in marine opossum shrimp (Neomysis awatschensis) and freshwater zebrafish (Danio rerio) in exposure experiments. A T-maze test was tentatively adopted to assess the effects of BMAA on the learning and memory ability of zebrafish. Interestingly, N. awatschensis was testified to be capable of accumulating free soluble BMAA from bathing seawater through a biological filtration pathway (max. 110.6 µg g-1 wet weight). BMAA was transferred quickly from viscus to muscle and head tissues of zebrafish after intraperitoneal administration of 16.3 µg BMAA per individual twice in two weeks. BMAA accumulated mainly as the total soluble form in both experimental organisms. Results do not support the hypothesis that free BMAA molecules can be largely incorporated into protein in aquatic animals. Behavior of zebrafish in the T-maze test demonstrated that the short-term learning and memory ability was negatively impacted to some degree after three-days exposure to BMAA. Moreover, on Day 3, certain individual zebrafish exhibited freezing and loitering behavior. However, further investigation will be required to discern the long-term effects of BMAA on animals in order to evaluate the risk of BMAA exposure to human health.

Ubiquity of the neurotoxin ß-N-methylamino-L-alanine and its isomers confirmed by two different mass spectrometric methods in diverse marine mollusks.

In recent years, the neurotoxin ß-N-methylamino-L-alanine (BMAA) has been reported in some marine mollusk species. To further discover BMAA in marine animals, a total of 59 samples belonging to 3 phyla, 22 families, and 43 species, were collected from Dalian, Rongcheng, and Zhoushan cities, China, in April 2017. All samples were quantified by a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) analysis of underivatized extract, and ten samples were also analyzed by a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis using a precolumn AQC (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate)-derivatization method. Results indicated that 48 mollusk samples contained BMAA with concentrations above the limit of detection (0.31 µg g-1 wet weight), and the isomers of BMAA, ß-amino-N-methylalanine (BAMA) and 2,4-diaminobutyric acid (DAB) were universally present in most samples. However, N-(2-aminoethyl) glycine (AEG) was not found in any sample. Comparison of both analytical methods showed that BMAA and BAMA were not completely separated by the HILIC column although they still could be identified by specific transitions. In contrast the C18 column provided good separation for the AQC-derivatives of BMAA and all of its isomers. Development of analytical methods and stable isotope tracing of BMAA should be carried out in the future.

Chemistry and Chemical Equilibrium Dynamics of BMAA and Its Carbamate Adducts.

Beta-N-methylamino-L-alanine (BMAA) has been demonstrated to contribute to the onset of the ALS/Parkinsonism-dementia complex (ALS/PDC) and is implicated in the progression of other neurodegenerative diseases. While the role of BMAA in these diseases is still debated, one of the suggested mechanisms involves the activation of excitatory glutamate receptors. In particular, the excitatory effects of BMAA are shown to be dependent on the presence of bicarbonate ions, which in turn forms carbamate adducts in physiological conditions. The formation of carbamate adducts from BMAA and bicarbonate is similar to the formation of carbamate adducts from non-proteinogenic amino acids. Structural, chemical, and biological information related to non-proteinogenic amino acids provide insight into the formation of and possible neurological action of BMAA. This article reviews the carbamate formation of BMAA in the presence of bicarbonate ions, with a particular focus on how the chemical equilibrium of BMAA carbamate adducts may affect the molecular mechanism of its function. Highlights of nuclear magnetic resonance (NMR)-based studies on the equilibrium process between free BMAA and its adducts are presented. The role of divalent metals on the equilibrium process is also explored. The formation and the equilibrium process of carbamate adducts of BMAA may answer questions on their neuroactive potency and provide strong motivation for further investigations into other toxic mechanisms.

Seafood sold in Sweden contains BMAA: A study of free and total concentrations with UHPLC-MS/MS and dansyl chloride derivatization.

ß-N-Methylamino-l-alanine (BMAA) is a potential neurotoxin associated with the aquatic environment. Validated analytical methods for the quantification of both free and total concentrations of BMAA were used in an investigation of seafood purchased from different grocery stores in Uppsala, Sweden. The analysis was performed using ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and detection of BMAA as a dansyl derivate. The determined concentrations of free BMAA (after a simple trichloroacetic acid extraction) in mussels and scallops were up to 0.46 µg g-1 wet homogenate. The total BMAA (after hydrochloric acid hydrolysis) levels were between 0.29 and 7.08 µg g-1 wet mussel homogenate. The highest concentration of total BMAA was found in imported cooked and canned mussels which contained about ten times the quantity of BMAA measured in domestic cooked and frozen mussels. In this study it was also concluded that BMAA could be detected in seafood origin from four different continents. The risks associated with human exposure to BMAA through food are unknown today. However, the results of this study show that imported seafood in Sweden contain BMAA, indicating that this area needs more investigation, including a risk assessment regarding the consumption of e.g., mussels, scallops and crab.

BMAA in shellfish from two Portuguese transitional water bodies suggests the marine dinoflagellate Gymnodinium catenatum as a potential BMAA source.

The neurotoxin ß-N-methylamino-l-alanine (BMAA) and its putative role in multiple neurodegenerative diseases have been intensely studied since 2005 when the toxin was discovered to be produced by worldwide-distributed cyanobacterial species inhabiting terrestrial, marine, brackish, and freshwater ecosystems. Recently, BMAA production was also associated with one eukaryotic group, namely, diatoms, raising questions about its production by other phytoplanktonic groups. To test for BMAA bioavailability in ecosystems where abundant phytoplanktonic blooms regularly occur, samples of filter-feeding shellfish were collected in two Portuguese transitional water bodies. BMAA content in cockles (Cerastoderma edule) collected weekly between September and November 2009 from Ria de Aveiro and at least once a month from May to November from Ria Formosa, fluctuated from 0.079±0.055 to 0.354±0.066µg/g DW and from below the limit of detection to 0.434±0.110µg/g DW, respectively. Simultaneously to BMAA occurrence in cockles, paralytic shellfish toxins were detected in shellfish as a result of Gymnodinium catenatum blooms indicating a possible link between this marine dinoflagellate and BMAA production. Moreover, considerable high BMAA levels, 0.457±0.186µg/g DW, were then determined in a laboratory grown culture of G. catenatum. This work reveals for the first time the presence of BMAA in shellfish from Atlantic transitional water bodies and consubstantiate evidences of G. catenatum as one of the main sources of BMAA in these ecosystems.