Prevalence of ß-methylamino-L-alanine (BMAA) and its isomers in freshwater cyanobacteria isolated from eastern Australia.

Environmental exposure to the amino acid ß-methylamino-L-alanine (BMAA) was linked to the high incidence of neurodegenerative disease first reported on the island of Guam in the 1940s and has more recently been implicated in an increased incidence of amyotrophic lateral sclerosis (ALS) in parts of the USA. BMAA has been shown to be produced by a range of cyanobacteria and some marine diatoms and dinoflagellates in different parts of the world. BMAA is commonly found with two of its constitutional isomers: 2,4- diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl) glycine (AEG). These isomers are thought to be co-produced by the same organisms that produce BMAA and MS/MS analysis following LC separation can add an additional level of specificity over LC-FL. Although the presence of BMAA and 2,4-DAB in surface scum samples from several sites in Australia has been reported, which Australian cyanobacterial species are capable of BMAA, 2,4-DAB and AEG production remains unknown. The aims of the present studies were to identify some of the cyanobacterial genera or species that can produce BMAA, 2,4-DAB and AEG in freshwater cyanobacteria blooms in eastern Australia. Eleven freshwater sites were sampled and from these, 19 single-species cyanobacterial cultures were established. Amino acids were extracted from cyanobacterial cultures and analysed using liquid chromatography-tandem mass spectrometry. BMAA was detected in 17 of the 19 isolates, 2,4-DAB was detected in all isolates, and AEG was detected in 18 of the 19 isolates, showing the prevalence of these amino acids in Australian freshwater cyanobacteria. Concentrations of all three isomers in Australian cyanobacteria were generally higher than the concentrations reported elsewhere. This study confirmed the presence of BMAA and its isomers in cyanobacteria isolated from eastern Australian freshwater systems, and determined which Australian cyanobacterial genera or species were capable of producing them when cultured under laboratory conditions.

Detection of the suspected neurotoxin ß-methylamino-l-alanine (BMAA) in cyanobacterial blooms from multiple water bodies in Eastern Australia.

The emerging toxin ß-methylamino-l-alanine (BMAA) has been linked to the development of a number of neurodegenerative diseases in humans including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and Parkinson's disease. BMAA has been found to be produced by a range of cyanobacteria, diatoms, and dinoflagellates worldwide, and is present in freshwater, saltwater, and terrestrial ecosystems. Surface scum samples were collected from waterways in rural and urban New South Wales, Australia and algal species identified. Reverse phase liquid chromatography-tandem mass spectrometry was used to analyse sixteen cyanobacterial scum for the presence of BMAA as well as its toxic structural isomer 2,4-diaminobutyric acid (2,4-DAB). BMAA was detected in ten of the samples analysed, and 2,4-DAB in all sixteen. The presence of these toxins in water used for agriculture raises concerns for public health and food security in Australia.

Investigation of the interaction of ß-methylamino-L-alanine with eukaryotic and prokaryotic proteins.

There is a strong body of evidence linking the non-protein amino acid (NPAA) ß-methylamino-L-alanine (BMAA) to the development of a number of neurodegenerative diseases. BMAA has been found globally, is produced by a number of organisms including cyanobacteria, diatoms, and dinoflagellates; and has been shown to biomagnify through trophic levels. The role of BMAA in neurodegenerative disease is highlighted by its presence in the brains of a number of neurodegenerative disease patients, where it was found in a protein-bound form. We have previously shown that BMAA is bound to cell proteins, and results in the upregulation of the unfolded protein response, an endoplasmic reticulum stress response activated by the presence of misfolded proteins within the cell. Structurally aberrant proteins are features of a number of neurodegenerative diseases, and further investigation of how BMAA interacts with proteins is crucial to our understanding of its toxicity. Here we use radiolabelled BMAA to investigate the interaction and binding of BMAA to eukaryotic and prokaryotic proteins. We found differences in the presence and distribution of protein-bound BMAA between E. coli and neuroblastoma cells, with an increase in binding over time only seen in the eukaryotic cells. We also found that BMAA was unable to bind to pure proteins, or cell lysate in native or denaturing conditions, indicating that biological processing is required for BMAA to bind to proteins.

Assessing the Combined Toxicity of BMAA and Its Isomers 2,4-DAB and AEG In Vitro Using Human Neuroblastoma Cells.

The non-protein amino acid (NPAA) ß-methylamino-L-alanine (BMAA) is produced by a diverse range of cyanobacteria, diatoms and dinoflagellates, and is present in both aquatic and terrestrial ecosystems globally. Exposure to BMAA has been implicated in the development of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD). BMAA is often found in nature along with its structural isomers 2,4-diaminobutyric acid (2,4-DAB) and aminoethylglycine (AEG); however, the toxicity of these NPAAs in combination has not been examined. We have previously demonstrated that BMAA induces endoplasmic reticulum (ER) stress and increases caspase and cathepsin activity in human neuroblastoma cells (SH-SY5Y), effects consistent with proteotoxic stress due to disturbances in protein synthesis, folding or turnover. The current study investigates whether 2,4-DAB and AEG share a similar mechanism of toxicity to BMAA, and if simultaneous exposure of cells to BMAA and its isomers results in increased toxicity in vitro. We show that a 48-h treatment with both 500 µM BMAA and 2,4-DAB decreases cell viability in vitro whereas AEG was not cytotoxic under the same conditions. Treatment of SH-SY5Y cells with 2,4-DAB did not increase expression of ER stress markers. Combined treatment of cells with BMAA and 2,4-DAB resulted in increased caspase activity and increased apoptosis above that of BMAA or 2,4-DAB on their own. These results suggest that 2,4-DAB does not share the same mechanism of toxicity as BMAA but the presence of 2,4-DAB increases the toxicity of BMAA to human cells in vitro.

Cyanobacterial Neurotoxins: Their Occurrence and Mechanisms of Toxicity.

Cyanobacteria are some of the oldest organisms on earth, and have evolved to produce a battery of toxic metabolites, including hepatotoxins, dermatoxins, and neurotoxins. In this review, we focus on the occurrence and mechanisms of toxicity of a number of neurotoxins synthesised by these ancient photosynthetic prokaryotes. We discuss the evidence linking ß-methylamino-L-alanine (BMAA), a non-protein amino acid, to an unusual neurological disease complex reported on the island of Guam in the 1950s, and how 60 years later, the role that BMAA plays in human disease is still unclear. There is now evidence that BMAA is also produced by some eukaryotes, and can bioaccumulate in food chains; this combined with higher frequency of cyanobacterial blooms globally, increases the potential for human exposure. Three BMAA isomers that often co-occur with BMAA have been identified, and the current knowledge on the toxicity of these molecules is presented. The acute alkaloid toxins; anatoxin-a, homoanatoxin-a and the saxitoxins, and the organophosphate neurotoxin anatoxin-a(S) are also discussed. In many cases, human exposure to a cocktail of cyanobacterial neurotoxins is likely; however, the implications of combined exposure to these toxins have not been fully explored. Increased understanding of the combined effects of cyanobacterial neurotoxins is required to fully understand how these molecules impact on human health.

The use of L-serine to prevent ß-methylamino-L-alanine (BMAA)-induced proteotoxic stress in vitro.

ß-methylamino-L-alanine (BMAA), a non-protein amino acid synthesised by cyanobacteria, has been linked to a complex neurological disorder on Guam and more recently to other cases of sporadic ALS (sALS), however the mechanisms of BMAA toxicity are not completely understood. We have previously demonstrated that BMAA is misincorporated into newly synthesised proteins by human neuroblastoma cells and fibroblasts, resulting in the formation of autofluorescent material and the induction of apoptotic cell death. In the present study we show that BMAA at low levels does not cause an acute toxicity in neuroblastoma cells but increases the expression of the ER stress marker, C/EBP homologous protein (CHOP) and increases the activity of the pro-apoptotic enzyme caspase-3. We also observed an increase in the activity of the lysosomal cysteine proteases cathepsin B and L, characteristic of the accumulation of proteins in the lysosomal system. We were able to prevent these proteotoxic effects in neuroblastoma cells through co-treatment with l-serine suggesting that they resulted from incorporation of BMAA into proteins. Misincorporation provides a possible mechanism whereby BMAA could initiate misfolding, and the accumulation of aggregate-prone proteins in neurons. This build-up of misfolded proteins could explain the long latency period of the disease previously reported on Guam.