BMAA Research Articles

Computational Investigation of BMAA and Its Carbamate Adducts as Potential GluR2 Modulators.

Beta-N-methylamino-l-alanine (BMAA) is a potential neurotoxic nonprotein amino acid, which can reach the human body through the food chain. When BMAA interacts with bicarbonate in the human body, carbamate adducts are produced, which share a high structural similarity with the neurotransmitter glutamate. It is believed that BMAA and its l-carbamate adducts bind in the glutamate binding site of ionotropic glutamate receptor 2 (GluR2). Chronic exposure to BMAA and its adducts could cause neurological illness such as neurodegenerative diseases. However, the mechanism of BMAA action and its carbamate adducts bound to GluR2 has not yet been elucidated. Here, we investigate the binding modes and the affinity of BMAA and its carbamate adducts to GluR2 in comparison to the natural agonist, glutamate, to understand whether these can act as GluR2 modulators. Initially, we perform molecular dynamics simulations of BMAA and its carbamate adducts bound to GluR2 to examine the stability of the ligands in the S1/S2 ligand-binding core of the receptor. In addition, we utilize alchemical free energy calculations to compute the difference in the free energy of binding of the beta-carbamate adduct of BMAA to GluR2 compared to that of glutamate. Our findings indicate that carbamate adducts of BMAA and glutamate remain stable in the binding site of the GluR2 compared to BMAA. Additionally, alchemical free energy results reveal that glutamate and the beta-carbamate adduct of BMAA have comparable binding affinity to the GluR2. These results provide a rationale that BMAA carbamate adducts may be, in fact, the modulators of GluR2 and not BMAA itself.

Brentuximab in Children, Adolescent and Young Adults with Relapsed/Refractory Anaplastic Large Cell Lymphoma

Background: Anaplastic Large Cell Lymphoma (ALCL) is an aggressive malignancy characterized by strong expression of CD30. Multi-agent chemotherapy cures 65-90% of newly diagnosed patients, the rate of relapse approaching 30%. Targeted agents eventually followed by allogeneic stem cell transplantation (SCT) may represent a valuable salvage therapy in patients with relapsed/refractory ALCL. Brentuximab vedotin is a CD30-directed monoclonal antibody-drug conjugate (ADC) that comprises a monoclonal human/murine antibody linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable linker. Design and methods: We retrospectively analyzed monocentric data of 20 patients with ALCL treated with Brentuximab from 2.10.2014 to 22.05.2022. Treatment was characterized by the intravenous infusion of 1.8 mg/kg Brentuximab in monotherapy or as part of a polychemotherapy regimen, in children with either relapsed disease or not fully responding to first-line treatment. More in detail, chemotherapy regimens administered in combination with the immunotherapy were: bendamustine, AM and BM blocks (ALCL99 Trial), or AA, BB and CC blocks (LNH97 Trial). Objective response rate (ORR) was assessed through computed tomography (CT)/FDG-PET scans. Results: Eight patients received Brentuximab for incomplete response to first-line treatment (2 of them had haemophagocytic lymphohistiocytosis at diagnosis, and underwent genetic testing that revealed the heterozygous variant c.1336C>T (p.Arg446Trp) in IKBKB gene and the hemizygous variant c.622T>C (p.Ala91Val) in IL2RG gene, respectively). In these patients, Brentuximab was mostly used in combination with the ALCL99 regimen. Three of these 8 patients experienced a subsequent relapse and were re-exposed to the drug after disease recurrence. Fifteen patients received Brentuximab at relapse. Median time between diagnosis and relapse was 10.3 months (range 3.4-34 months). The median number of Brentuximab infusions was 7 (range 2-25). The combination of Brentuximab 1.8 mg/kg/dose on Day 1 and Bendamustine 120 mg/m2/dose on Day 2 and 3, was the principal reinduction regimen in the relapsed cohort, having been infused in 9 patients with a median of 4 cycles (range 2 to 5). This drug combination was very well tolerated and showed a 100% complete response (CR) assessed by CT and FDG-PET scans, allowing to perform allogeneic SCT in all of them. Allogeneic SCT was performed in 15 patients (one of them was the child with haemophagocytic lymphohistiocytosis at diagnosis given Brentuximab during the first-line treatment because of incomplete response), while the remaining child, who had relapsed 34 months after the diagnosis, was treated with Brentuximab monotherapy and remains in CR. Nine patients received HLA-haploidentical SCT, while the remaining 6 were transplanted using either a matched sibling donor (3 patient) or an unrelated volunteer (3 patients) respectively. Sixteen out of the 20 patients (80%) are alive and disease free with a median follow-up of 28.6 months (range 3-78.7). Three patients died due to transplant related causes and 1 patient due to disease progression. Conclusions: Despite the limitations of a limited number of patients analyzed and the retrospective nature of the study, our data suggest that Brentuximab administered either in monotherapy or in combination with chemotherapy is a valuable salvage option and may represent a suitable bridge to allogeneic SCT.

Experimental evidence challenges the presumed defensive function of a \u201cslow toxin\u201d in cycads

beta-methylamino-L-alanine (BMAA) is a neurotoxic non-protein amino acid found in the tissues of cycad plants. The demonstrated toxicity of BMAA to diverse organisms, including humans, is widely assumed to imply a defensive function of BMAA against herbivores; however, this hypothesis has not previously been tested in an ecologically relevant system. We investigated the effects of dietary BMAA, across a range of dosages matching and exceeding levels typically present in cycad leaves, on the feeding preferences and performance of a generalist lepidopteran herbivore (Spodoptera littoralis).We observed no effects of dietary BMAA on the survival or development of S. littoralis larvae, nor any larval preference between BMAA-laced and control diets. These findings suggest that BMAA in cycad tissues does not deter feeding by insect herbivores, raising questions about other potential physiological or ecological functions of this compound.

Use of a Mixed Cationic-Reverse Phase Column for Analyzing Small Highly Polar Metabolic Markers in Biological Fluids for Multiclass LC-HRMS Method

The determination of small highly polar metabolites at low concentrations is challenging when reverse-phase (RP) chromatography is used for multiclass analysis. A mixed cationic-RP column coupled to high-resolution tandem MS (HR-MS/MS) was tested for highly polar compounds in biological fluids, i.e., trimethylamine N-oxide (TMAO) and the isobaric molecules beta-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB). The efficient retention and separation of the above compounds were obtained with common and MS-friendly RP conditions, reaching high selectivity and sensitivity. The method was firstly assessed in plasma and urine, showing good linearity in the range 50–1000 µg/L and 500–10,000 µg/L for TMAO and both BMAA and DAB, respectively. Excellent precision (RDS < 3%) and good accuracies (71–85%) were observed except for BMAA in plasma, whose experimental conditions should be specifically optimized. Preliminary tests performed on compounds with biological relevance and a wider range of polarities proved the effectiveness of this chromatographic solution, allowing the simultaneous analysis of a larger panel of metabolites, from very small and polar compounds, like trimethylamine, to quite lipophilic molecules, such as corticosterone. The proposed LC-HRMS protocol is an excellent alternative to hydrophilic interaction liquid chromatography and ion-pairing RP chromatography, thus providing another friendly analytical tool for metabolomics.

The First Proteomics Study of Nostoc sp. PCC 7120 Exposed to Cyanotoxin BMAA under Nitrogen Starvation.

The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid.

Prenatal Exposure of Mice to Beta‐methyl‐amino‐L‐alanine (BMAA) as a Model for Amyotrophic Lateral Sclerosis‐Parkinson’s Dementia Complex (ALS‐PDC).

Amyotrophic Lateral Sclerosis‐Parkinson’s Dementia Complex (ALS‐PDC) is a severe neurological disorder, caused by degeneration of upper motor glutamine (UMG), nigrostriatal dopamine (NSDA) and basal forebrain acetylcholine (BFbA) neurons, and locally called Lytico‐bodig in Guam. The incidence of ALS‐PDC has decline, but the cause is still unknown. Several studies indicate that beta‐methylamino‐L‐alanine (BMAA), a non‐protein amino acid, consumed in food prepared from the cycad plant, is the cause of ALS‐PDC. The consumption of BMAA has been reduced, as well as the incidence of ALS‐PDC, and these implicate BMAA as a likely cause for ALS‐PDC, Studies, however, had produced no good model of ALS‐PDC. Since UMG, NSDA and MFbA neurons are impaired in ALS‐PDC, we targeted these neuronal groups in the fetus, by exposing C57BL/6J pregnant mice to BMAA during gestation day 8–18, the birth or neurogenesis of these neurons in the fetus. The goal was to produce impaired neurons that will succumb/die early during aging. The outcomes showed post‐natal changes in weight, behaviors, muscle functions as well as specific neurotransmitters and other neurochemicals. BMAA administered at 10, 50 and 100 mg/kg to the pregnant dams caused dose‐dependent reduction in the weight of two weeks pups by 12.5, 20.0 and 27.5% of the control. Body weight, however, recovered at 4–5 weeks post‐weaning. Prenatal BMAA also reduced open field motor activity: e.g. the total distance travelled was reduced by 8.8, 11.6 and 19% by the respective doses of BMAA. On the Rota‐Rod, the latency to fall was dramatically reduced to 71.4, 17.4 and 8.7% by the 10, 50 and 100 mg/kg of prenatal BMAA, as compared to control. Likewise, the no of revolution per min that the mice endured were reduced from the control value of 100%, to 67.8%, 25.4% and 16.9% for the 10, 50 and 100 mg/kg of BMAA. The data show that BMAA has effects on motor performance of offspring exposed prenatally. The continuation of the study will reveal underlying neurochemical changes that may be related to ALS‐PDC. The indication is that ALSPDC may have a fetal basis in which early subtle neuronal impairments occurred and symptoms precipitated later in life when UMN, NSDA and MFbA are impaired during aging.Support or Funding InformationNIH

A Locomotor Activity Study of the Effects of L‐Serine to Neurodegeneration Impacts of Beta‐Methylamino‐L‐alanine in Drosophila melanogaster

Amyotrophic Lateral Sclerosis‐Parkinsonism Dementia Complex (ALS‐PDC) is a neurodegenerative disease that described by loss of motor function, tremors, and dementia due to neurodegeneration. The disease is thought to be caused by Beta‐Methylamino‐L‐Alanine (BMAA), a neurotoxin from blue‐green algae. Since it has a structure that is similar to glutamate, BMAA act as an agonist to the glutamate receptor N‐methyl‐D‐aspartate which lead to excitotoxicity and degenerate neurons. L‐serine is an essential amino acid that has shown to interfere with the effect of BMAA in vivo studies. The purpose of this research is to further test the novel impact of L‐serine to BMAA‐induced fly in long‐term study. The interaction of BMAA and L‐serine was analyzed through circadian rhythm activity and viability of wild‐type Drosophila melanogaster in ALS‐PDC model. We used two Drosophila Activity Monitor (DAM) systems to measure activity of 64 fruit flies (Gender and Age‐Match). The experimental groups were fed with L‐serine, BMAA, BMAA+L‐serine and the control group was fed with standard fly agar. Fly activity was recorded by the number of fly pass the IR beam in the monitor for 10‐day (12L/12D at 22°C). After 10 days, circadian rhythm data showed BMAA flies increased activity over the first few days, and decreased gradually to the end of the period. BMAA‐induced flies (n = 12) does not show pattern of regular activity in L/D as compared to Control (n = 15) and L‐serine (n = 16). The co‐fed BMAA+L‐serine (n = 7) showed a similar pattern as the Control. For daytime vs nighttime activity (mean ± s.e.m), Control (n = 15) shown regular activity (day: 137 ± 13%, night: 21 ± 5%) as compared to BMAA (n = 12) that shown high activity in dark cycle (day: 162 ± 10%, night: 155 ± 21%). Moreover, the cofed L‐serine + BMAA (n = 7) exhibited normal daytime activity but not nighttime activity (day: 138 ± 14%, night: 167 ± 31%). The data from DAM shows the abnormal activity of flies in long term when inducing with BMAA such as abrupt sleep due to high activity during dark phase. L‐serine shows to reduce down the high activity when cofed with BMAA. This supports our hypothesis of L‐serine ameliorates the effects of BMAA in fly. This preliminary data is a starting point for further research in drug discovery for ALS‐PDC.Support or Funding InformationAcknowledgementsThis work was funded by the Fresno State Undergraduate Research Grant Award (2019–2020), and Howard P. Kubo, M.D. Award for Outstanding Achievement in Biomedical Science.

Impact factors on the production of β-methylamino-L-alanine (BMAA) by cyanobacteria.

Cyanobacteria produce a series of secondary metabolites, one of which is beta-N-methylamino-l-alanine (BMAA). BMAA is considered to be the cause of human neurodegeneration. Compared with other cyanotoxins, the role of BMAA in cyanobacteria remains unclear. To investigate this question, six strains of cyanobacteria were cultured and tested in this experiment with an optimized and validated BMAA determination method. The results show that four strains can produce BMAA. The effects of nutrient levels on the production of BMAA by Anabaena sp. FACHB-418 were studied by changing the initial concentrations of nitrate (NaNO3) and phosphate (K2HPO4) in mediums. Bound BMAA was detected in all samples and the concentrations were within 50-100 ng/g. Free BMAA was presence when the concentration of nitrogen was lower than 1.7 mg/L (121.43 μM). Free BMAA was released from the dead and ruptured cells during the cell decline period, so dissolved BMAA cannot be detectable in the adaptation and logarithmic periods, but could be abundant in the decline periods. Statistical analyses show that free BMAA concentrations were negatively correlated with nitrogen strongly (p = 2.334 × 10-10 and r = -0.842), but positively correlated with phosphorus weakly (p = 0.016 and r = 0.405). Moreover, the results of culture experiments indicated that exogenous BMAA could inhibit the growth of cyanobacteria that cannot produce BMAA, and the effect was enhanced as the concentration of exogenous BMAA increased. This phenomenon implies that the production of BMAA may be the stress response by some cyanobacteria to low nitrogen conditions to kill other cyanobacteria, i.e., their competitors.

Assessment of the cytotoxic impact of cyanotoxin beta-N-methylamino-L-alanine on a fish immune cell line

Beta-N-methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid produced by several cyanobacteria species. It is considered to be a potent neurotoxin. Although its neurotoxic effects are well studied, other negative effects of BMAA have not yet been completely elucidated. In the present study, we studied the cytotoxic effects of a wide range of concentrations of BMAA (0.25–2.0 mM) on a stable fish immune cell line (CLC) obtained from carp monocytes. The cells exposed to higher concentrations of BMAA exhibited an altered morphology, changed ATP levels, and reduced proliferation. On the basis of toxic effects of BMAA on lysosomes, mitochondrial dehydrogenases activity, and cell membrane integrity, we determined its cytotoxic concentrations. We also investigated effects of the toxin at non-cytotoxic concentrations on the basic functions of CLC cells. BMAA did not affect the production and release of IL-1β or phagocytic activity of the cells. However, higher non-toxic BMAA concentrations altered the levels of extracellular and intracellular total proteins compared to those in control cells.

Cyanobacterial Neurotoxin Beta-Methyl-Amino-l-Alanine Affects Dopaminergic Neurons in Optic Ganglia and Brain of Daphnia magna.

The non-proteinogenic amino acid beta-methyl-amino-l-alanine (BMAA) is a neurotoxin produced by cyanobacteria. BMAA accumulation in the brain of animals via biomagnification along the food web can contribute to the development of neurodegenerative diseases such as Amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC), the latter being associated with a loss of dopaminergic neurons. Daphnia magna is an important microcrustacean zooplankton species that plays a key role in aquatic food webs, and BMAA-producing cyanobacteria often form part of their diet. Here, we tested the effects of BMAA on putative neurodegeneration of newly identified specific dopaminergic neurons in the optic ganglia/brain complex of D. magna using quantitative tyrosine-hydroxylase immunohistochemistry and fluorescence cytometry. The dopaminergic system was analysed in fed and starved isogenic D. magna adults incubated under different BMAA concentrations over 4 days. Increased BMAA concentration showed significant decrease in the stainability of dopaminergic neurons of D. magna, with fed animals showing a more extreme loss. Furthermore, higher BMAA concentrations tended to increase offspring mortality during incubation. These results are indicative of ingested BMAA causing neurodegeneration of dopaminergic neurons in D. magna and adversely affecting reproduction. This may imply similar effects of BMAA on known human neurodegenerative diseases involving dopaminergic neurons.

Oxidation of cyanobacterial neurotoxin beta-N-methylamino-L-alanine (BMAA) with chlorine, permanganate, ozone, hydrogen peroxide and hydroxyl radical

Beta-N-methylamino-L-alanine (BMAA), a new cyanobacterial neurotoxin produced by more than 20 genera of cyanobacteria, has been associated with amyotrophic lateral sclerosis/parkinsonism–dementia complex (ALS/PDC) or Alzheimer's disease. Although BMAA has been shown to be removed in drinking water treatment plants (DWTPs), studies regarding the reactions between BMAA and the commonly used oxidants in DWTPs are limited to chlorine under specific conditions. In this study, the reaction kinetics between BMAA and five oxidants commonly used in DWTPs, including chlorine, potassium permanganate, ozone, hydrogen peroxide and hydroxyl radical were investigated. The oxidation of BMAA by chlorine, ozone or OH radical followed the second order reaction rate law, and the reaction rate was in the order of OH radicals > ozone >> chlorine. The rate constants increased by 20 times from 2 × 103 M−1s−1 at pH 5.8 to 4.93 × 104 M−1s−1 at pH 7, and kept in a relatively stable level at pH 7–9.5; rate constants of OH radicals were 1.11 × 108 M−1s−1 at pH 6.5 and 5.51 × 109- 1.35 × 1010 M−1s−1 at pH > 6.5. For both permanganate and H2O2 only, the removal of BMAA was negligible. The pH dependency of chlorine and the OH radical may be attributed to the neutral form of BMAA with free lone pair electrons readily to be attacked by oxidants. However, for ozonation of BMAA, the rate constants were 1.88 × 106–3.72 × 1010 M−1s−1, with a linear dependency on pH, implying that the hydroxide concentration governs the reaction. In addition, the rate of BMAA degradation was found to be slower in natural water if compared with that in deionized water.

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.

Exposure to BMAA mirrors molecular processes linked to neurodegenerative disease.

The goal of this study is to investigate the molecular pathways perturbed by in vitro exposure of beta-methylamino-L-alanine (BMAA) to NSC-34 cells via contemporary proteomics. Our analysis of differentially regulated proteins reveals significant enrichment (p<0.01) of pathways related to ER stress, protein ubiquitination, the unfolded protein response, and mitochondrial dysfunction. Upstream regulator analysis indicates that exposure to BMAA induces activation of transcription factors (X-box binding protein 1; nuclear factor 2 erythroid like 2; promyelocytic leukemia) involved in regulation of the UPR, oxidative stress, and cellular senescence. Furthermore, the authors examine the hypothesis that BMAA causes protein damage via misincorporation in place of L-Serine. The authors are unable to detect misincorporation of BMAA into protein via analysis of cellular protein, secreted protein, targeted detection of BMAA after protein hydrolysis, or through the use of in vitro protein translation kits.

Assessment of non-derivatized β-N-methylamino-l-alanine (BMAA) neurotoxin in free form in urine of patients with nonspecific neurological symptoms

The beta-N-methylamino-l-alanine (BMAA) is a non-proteinogenic amino acid discussed to be produced by cyanobacteria forming harmful blooms. Since BMAA is suspected etiological agent in neurodegenerative diseases, there is a need to study and validate whether and in what concentrations can BMAA be present in human tissues. The aim of the present study was to validate analytical and extraction procedures for quantification of non-derivatized BMAA in the urine using liquid chromatography and commercial ELISA Kit. The study was focused on BMAA in different forms - dissolved, protein associated and total. The validated protocol included SPE followed by HILIC MS/MS for analyses of non-derivatized free form of BMAA with a limit of quantification 20 ng/mL. The methods for other BMAA forms (i.e. protein-associated and total) were also assessed but high matrix interferences did not allow their implementation. The method was used for analyses of free BMAA in 23 urine samples from healthy volunteers and psychiatric patients suffering from nonspecific neurological symptoms. Traces of BMAA were suspectedly detected in a single urine sample but they were not unequivocally proved according to all conservative analytical criteria. BMAA was also not confirmed in a repeatedly collected sample from the same person. The evaluated commercial BMAA ELISA Kit (Abraxis) was not suitable for determination of BMAA in extracted urine samples because of systematically highly false positive results. In agreement with recent findings, analyses of BMAA appear to methodologically challenging, and further research on BMAA in human tissues (or its precursors with potency to form BMAA under natural conditions or - eventually - during sample processing) is needed to clarify its potential ethiological role in neurodegenerative diseases.

Embryonic Exposure to the Environmental Neurotoxin BMAA Negatively Impacts Early Neuronal Development and Progression of Neurodegeneration in the Sod1-G93R Zebrafish Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to progressive paralysis and death within 2-5 years after diagnosis. Sporadic cases (SALS) comprise approximately 90% of cases with the remaining 10% familial (FALS) caused by mutations in approximately 27 genes. The vast heterogeneity seen in age and location of disease onset, rate of progression, and duration of disease has been linked with genetic and environmental influences in both SALS and FALS cases. Increased ALS incidence clusters in Guam, southern France, and Maryland have been linked with exposure to Beta-methylamino-L-alanine (BMAA), a nonproteinogenic amino acid produced by cyanobacteria, dinoflaggelates, and diatoms. We embryonically exposed zebrafish, Danio rerio, (transgenically overexpressing a FALS-causing SOD1-G93R mutation) to BMAA to investigate early motor neuron outgrowth in larvae and endurance and fatigability in 5-month adults. SOD1-G93R zebrafish showed decreased embryonic nerve length with increased BMAA dose, a phenotypic change mirrored in 5-month performance measures of weaker swimming and increased fatigability. In contrast, transgenic fish overexpressing wild-type SOD1 were resistant to phenotypic changes, indicating a potential neuroprotective function of healthy SOD1. We show that the etiology of genetic ALS animal models can be influenced by environmental exposures, and that embryonic toxin exposures can result in changes to both early and adult measures. We demonstrate that zebrafish can be a robust model for investigating causes of ALS heterogeneity. Establishing these links between developmental and adult ALS-like symptoms in the zebrafish increases the power of this model for toxicological and drug screens.

Vervets and macaques: Similarities and differences in their responses to l-BMAA

Vervets and macaques: Similarities and differences in their responses to l-BMAA

9. Beta-N-methylamino-L-alanine (BMAA): Optimization of identification and quantification in biological samples

9. Beta-N-methylamino-L-alanine (BMAA): Optimization of identification and quantification in biological samples

Analysis of β-N-methylamino-L-alanine (BMAA) in spirulina-containing supplements by liquid chromatography-tandem mass spectrometry.

Over the last decade the amino acid beta-N-methylamino-L-alanine (BMAA) has come under intense scrutiny. International laboratory and epidemiological research continues to support the hypothesis that environmental exposure to BMAA (e.g., through dietary practices, water supply) can promote the risk of various neurodegenerative diseases. A wide variety of cyanobacteria spp. have previously been reported to produce BMAA, with production levels dependent upon species, strain and environmental conditions. Since spirulina (Arthrospira spp.) is a member of the cyanobacteria phylum frequently consumed via dietary supplements, the presence of BMAA in such products may have public health implications. In the current work, we have analyzed ten spirulina-containing samples for the presence of BMAA; six pure spirulina samples from two separate raw materials suppliers, and four commercially-available multi-ingredient products containing 1.45 g of spirulina per 8.5 g serving. Because of controversy surrounding the measurement of BMAA, we have used two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: one based on reversed phase LC (RPLC) with derivatization and the other based on hydrophilic interaction LC (HILIC). Potential matrix effects were corrected for by internal standardization using a stable isotope labeled BMAA standard. BMAA was not detected at low limits of detection (80 ng/g dry weight) in any of these product samples. Although these results are reassuring, BMAA analyses should be conducted on a wider sample selection and, perhaps, as part of ongoing spirulina production quality control testing and specifications.

Intrathecal infusion of BMAA induces selective motor neuron damage and astrogliosis in the ventral horn of the spinal cord

The neurotoxin beta-N-methylamino-l-alanine (BMAA) was first identified as a “toxin of interest” in regard to the amyotrophic lateral sclerosis–Parkinsonism Dementia Complex of Guam (ALS/PDC); studies in recent years highlighting widespread environmental sources of BMAA exposure and providing new clues to toxic mechanisms have suggested possible relevance to sporadic ALS as well. However, despite clear evidence of uptake into tissues and a range of toxic effects in cells and animals, an animal model in which BMAA induces a neurodegenerative picture resembling ALS is lacking, possibly in part reflecting limited understanding of critical factors pertaining to its absorption, biodistribution and metabolism. To bypass some of these issues and ensure delivery to a key site of disease pathology, we examined effects of prolonged (30day) intrathecal infusion in wild type (WT) rats, and rats harboring the familial ALS associated G93A SOD1 mutation, over an age range (80±2 to 110±2days) during which the G93A rats are developing disease pathology yet remain asymptomatic. The BMAA exposures induced changes that in many ways resemble those seen in the G93A rats, with degenerative changes in ventral horn motor neurons (MNs) with relatively little dorsal horn pathology, marked ventral horn astrogliosis and increased 3-nitrotyrosine labeling in and surrounding MNs, a loss of labeling for the astrocytic glutamate transporter, GLT-1, surrounding MNs, and mild accumulation and aggregation of TDP-43 in the cytosol of some injured and degenerating MNs. Thus, prolonged intrathecal infusion of BMAA can reproduce a picture in spinal cord incorporating many of the pathological hallmarks of diverse forms of human ALS, including substantial restriction of overt pathological changes to the ventral horn, consistent with the possibility that environmental BMAA exposure could be a risk factor and/or contributor to some human disease.

The persistence of cyanobacterial toxins in desert soils

Desert regions and arid soils are stabilized by a crust of cyanobacteria that bind sand to extruded polysaccharides. In these environments, cyanobacteria lie dormant during most of the year, but photosynthesize immediately when seasonal rains fall to take advantage of limited water resources. Desert cyanobacteria in biological soil crusts are known to produce a variety of toxins including microcystins and beta-N-methylamino-l-alanine (BMAA). We tested the hypothesis that toxin production of biological soil crusts increases directly following rainfall in arid environments. In the deserts of Qatar, meter square quadrats were divided into four treatment regimes: 1) intact crust; 2) watered intact crust; 3) disturbed crust; 4) watered disturbed crusts. Five replicates of each quadrat were monitored and tested for chlorophyll a as well as for the presence of three cyanobacterially-produced toxic isomers BMAA, 2,4-diaminobutyric acid (DAB), and N-(2-aminoethyl)glycine (AEG). Median chlorophyll content of soil samples from sections of the quadrats that were mechanically disturbed was only 37% of the chlorophyll a content from undisturbed sections, but neither watering nor disturbance had a significant impact on toxin content. These results suggest that low molecular weight cyanotoxins produced by biological soil crusts are persistent in the environment, and can survive even the removal of the crusts themselves. Thus, over many years a potentially large reservoir of cyanobacterial toxins can accumulate in desert soils, with possible health consequences for human exposure to particulate matter in dust storms, and from disturbance of desert soils from construction activities and military activities that result in loss of soil structure. Mechanical disturbance of either surface or below surface on dry or wet soils may equally contribute to human exposure to cyanotoxins through inhalation.