Paul Alan Cox

Co-occurrence of β-N-methylamino-l-alanine, a neurotoxic amino acid with other cyanobacterial toxins in British waterbodies, 1990–2004

The neurotoxic amino acid, beta-N-methylamino-L-alanine, was found to be present in all of 12 analysed samples of cyanobacterial blooms, scums and mats, which had been collected in seven years between 1990 and 2004 inclusive and stored at -20 degrees C. BMAA identification was by high performance liquid chromatography with fluorescence detection and by triple quadrapole mass spectrometry after derivatization. The samples originated from 11 freshwater lakes and 1 brackish waterbody, used either for drinking water, recreation, or both. BMAA was present at between 8 and 287 microg g(-1) cyanobacterial dry weight and was present as both the free amino acid and associated with precipitated proteins. Ten of the samples contained additional cyanotoxins (including microcystins, anatoxin-a, nodularin and saxitoxin) at the time of sample collection. Five of the samples were associated with animal deaths, attributable at the time of sample collection, to microcystins, nodularin or anatoxin-a. The data demonstrate the presence of BMAA by high performance liquid chromatography and mass spectrometry in a diverse range of cyanobacterial bloom samples from high resource waterbodies. Furthermore, samples collected over several years shows that BMAA can co-occur with other known cyanotoxins in such waterbodies. Health risk assessment of cyanobacterial BMAA in waterbodies is suggested.

BMAA selectively injures motor neurons via AMPA/kainate receptor activation

The toxin beta-methylamino-l-alanine (BMAA) has been proposed to contribute to amyotrophic lateral sclerosis-Parkinsonism Dementia Complex of Guam (ALS/PDC) based on its ability to induce a similar disease phenotype in primates and its presence in cycad seeds, which constituted a dietary item in afflicted populations. Concerns about the apparent low potency of this toxin in relation to estimated levels of human ingestion led to a slowing of BMAA research. However, recent reports identifying potential new routes of exposure compel a re-examination of the BMAA/cycad hypothesis. BMAA was found to induce selective motor neuron (MN) loss in dissociated mixed spinal cord cultures at concentrations ( approximately 30 muM) significantly lower than those previously found to induce widespread neuronal degeneration. The glutamate receptor antagonist NBQX prevented BMAA-induced death, implicating excitotoxic activation of AMPA/kainate receptors. Using microfluorimetric techniques, we further found that BMAA induced preferential [Ca(2+)](i) rises and selective reactive oxygen species (ROS) generation in MNs with minimal effect on other spinal neurons. Cycad seed extracts also triggered preferential AMPA/kainate-receptor-dependent MN injury, consistent with the idea that BMAA is a crucial toxic component in this plant. Present findings support the hypothesis that BMAA may contribute to the selective MN loss in ALS/PDC.

The Non-Protein Amino Acid BMAA Is Misincorporated into Human Proteins in Place of l-Serine Causing Protein Misfolding and Aggregation

Mechanisms of protein misfolding are of increasing interest in the aetiology of neurodegenerative diseases characterized by protein aggregation and tangles including Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy Body Dementia (LBD), and Progressive Supranuclear Palsy (PSP). Some forms of neurodegenerative illness are associated with mutations in genes which control assembly of disease related proteins. For example, the mouse sticky mutation sti, which results in undetected mischarging of tRNAAla with serine resulting in the substitution of serine for alanine in proteins causes cerebellar Purkinje cell loss and ataxia in laboratory animals. Replacement of serine 422 with glutamic acid in tau increases the propensity of tau aggregation associated with neurodegeneration. However, the possibility that environmental factors can trigger abnormal folding in proteins remains relatively unexplored. We here report that a non-protein amino acid, β-N-methylamino-L-alanine (BMAA), can be misincorporated in place of l-serine into human proteins. We also report that this misincorporation can be inhibited by l-serine. Misincorporation of BMAA into human neuroproteins may shed light on putative associations between human exposure to BMAA produced by cyanobacteria and an increased incidence of ALS.

The evolution of vivipary in flowering plants

Vivipary in flowering plants is defined as the precocious and continuous growth of the offspring when still attached to the maternal parent. Two main types, true vivipary (involving sexually produced offspring) and pseudovivipary (asexual offspring), may be identified. Vivipary has been described from slightly less than a hundred different species of flowering plants, of which we classify approx. 50% as having true vivipary, with the remaining species being pseudoviviparous. Species with true vivipary tend to inhabit shallow marine habitats while those with pseudovivipary are mostly found in arctic, alpine, or arid habitats. We suggest that all of these habitats are characterized by extraordinarily coarse-grained environments for seedling establishment, albeit with major differences in patch size. In all of these situations the probability of an offspring being dispersed in time or space to a patch better than the parental patch is very low, hence taxa with seed dormancy or seed dispersal mechanisms enjoy no particular advantage. We suggest that future research focus on species that are facultatively pseudoviviparous, as well as on comparisons of ecological and biomechanical aspects of viviparous and non-viviparous mangroves and seagrasses.

Cyanobacteria (Nostoc commune) used as a dietary item in the Peruvian highlands produce the neurotoxic amino acid BMAA

In the mountains of Peru, globular colonies of Nostoc commune (Nostocales) are collected in the highland lakes by the indigenous people, who call them llullucha. They are consumed locally, traded for maize, or sold, eventually entering the folk markets of Cusco and other neighboring cities. Throughout highland Peru, Nostoc commune is highly salient as a seasonal dietary item, being eaten alone, or in picante -- a local stew -- and is said to be highly nutritious. Nostoc commune has been known to produce unusual amino acids, including those of the mycosporine group, which possibly function to prevent UV damage. We analyzed 21 different Nostoc commune spherical colonies from 7 different market collections in the Cusco area for the presence of beta-N-methylamino-L-alanine (BMAA), a neurotoxic amino acid produced by diverse taxa of cyanobacteria, using four different analytical techniques (HPLC-FD, UPLC-UV, UPLC/MS, LC/MS/MS). We found using all four techniques that BMAA was present in the samples purchased in the Peruvian markets. Since BMAA has been putatively linked to neurodegenerative illness, it would be of interest to know if the occurrence of ALS, Alzheimers, or Parkinsons Disease is greater among individuals who consume llullucha in Peru.

Dietary exposure to an environmental toxin triggers neurofibrillary tangles and amyloid deposits in the brain

Neurofibrillary tangles (NFT) and β-amyloid plaques are the neurological hallmarks of both Alzheimers disease and an unusual paralytic illness suffered by Chamorro villagers on the Pacific island of Guam. Many Chamorros with the disease suffer dementia, and in some villages one-quarter of the adults perished from the disease. Like Alzheimers, the causal factors of Guamanian amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC) are poorly understood. In replicated experiments, we found that chronic dietary exposure to a cyanobacterial toxin present in the traditional Chamorro diet, β-N-methylamino-l-alanine (BMAA), triggers the formation of both NFT and β-amyloid deposits similar in structure and density to those found in brain tissues of Chamorros who died with ALS/PDC. Vervets (Chlorocebus sabaeus) fed for 140 days with BMAA-dosed fruit developed NFT and sparse β-amyloid deposits in the brain. Co-administration of the dietary amino acid l-serine with l-BMAA significantly reduced the density of NFT. These findings indicate that while chronic exposure to the environmental toxin BMAA can trigger neurodegeneration in vulnerable individuals, increasing the amount of l-serine in the diet can reduce the risk.

Dietary BMAA Exposure in an Amyotrophic Lateral Sclerosis Cluster from Southern France

Background Dietary exposure to the cyanotoxin BMAA is suspected to be the cause of amyotrophic lateral sclerosis in the Western Pacific Islands. In Europe and North America, this toxin has been identified in the marine environment of amyotrophic lateral sclerosis clusters but, to date, only few dietary exposures have been described. Objectives We aimed at identifying cluster(s) of amyotrophic lateral sclerosis in the Hérault district, a coastal district from Southern France, and to search, in the identified area(s), for the existence of a potential dietary source of BMAA. Methods A spatio-temporal cluster analysis was performed in the district, considering all incident amyotrophic lateral sclerosis cases identified from 1994 to 2009 by our expert center. We investigated the cluster area with serial collections of oysters and mussels that were subsequently analyzed blind for BMAA concentrations. Results We found one significant amyotrophic lateral sclerosis cluster (p = 0.0024), surrounding the Thau lagoon, the most important area of shellfish production and consumption along the French Mediterranean coast. BMAA was identified in mussels (1.8 µg/g to 6.0 µg/g) and oysters (0.6 µg/g to 1.6 µg/g). The highest concentrations of BMAA were measured during summer when the highest picocyanobacteria abundances were recorded. Conclusions While it is not possible to ascertain a direct link between shellfish consumption and the existence of this ALS cluster, these results add new data to the potential association of BMAA with sporadic amyotrophic lateral sclerosis, one of the most severe neurodegenerative disorder.

Cyanotoxins in desert environments may present a risk to human health.

There have been few studies concerning cyanotoxins in desert environments, compared with the multitude of studies of cyanotoxins in aquatic environments. However, cyanobacteria are important primary producers in desert environments, where after seasonal rains they can grow rapidly both stabilising and fertilising arid habitats. Samples of cyanobacteria from wadis - dry, ephemeral river beds - and sabkha - supertidal salt flats - in Qatar were analysed for the presence of microcystins, nodularin, anatoxin-a, cylindrospermopsin and anatoxin-a(S). Microcystins were detected by HPLC-PDA and ELISA at concentrations between 1.5 and 53.7ngg(-1) dry wt of crust. PCR products for the mycD gene for microcystin biosynthesis were detected after amplification of DNA from desert crust samples at two out of three sample sites. The presence of anatoxin-a(S) was also indicated by acetylcholine esterase inhibition assay. As a function of area of desert crust, microcystin concentrations were between 3 and 56μgm(-2). Based on the concentration of microcystins detected in crust, with reference to the published inhalation NOAEL and LOAEL values via nasal spray inhalation of purified microcystin-LR in aqueous solution, and the amount of dust potentially inhaled by a person from these dried crusts, the dose of microcystins could exceed a calculated TDI value of 1-2ngkg(-1)day(-1) for an average adult. The presence of microcystins, and potentially of anatoxin-a(S), in desert crusts has important implications for human health. Further studies are required to monitor desert dust storms for the presence of cyanotoxins. An understanding of the risks of inhaling particles containing cyanotoxins is also warranted.

Restricted pollination on Oceanic Islands : Pollination of Ceiba pentandra by flying foxes in Samoa

The very low diversity of animal pollinators on remote oceanic islands can act as a potent biotic filter to potential plant immigrants. To assess the relative difficulties faced by colonizing plant species, we studied the pollination ecology of kapok, Ceiba pentandra (Bombacaceae), a widespread aboriginal introduction to Pacific islands. The Samoan form of this tree is self-fertile, but requires pollination for fruit set. In continental areas C. pentandra attracts a diverse assemblage of pollinators, but in Samoa it is pollinated only by Pteropus tonganus (Megachiroptera: Pteropodidae)an example of a plant exclusively dependent on one bat species for pollination. The pollination system is dystrophic, however, since foraging flying foxes may destroy up to 50 percent of C. pentandra flowers and developing fruits. Though resource defense is regarded as rare in nectarivorous bats, P. tonganus vigorously defends small feeding territories on the rich, but readily depleted, C. pentandra nectar resource. This intraspecies conflict probably shapes patterns of pollen transfer within and among trees, and enhances cross-pollination. Our study highlights the importance of conserving pollinators in remote oceanic islands with depauperate pollinator faunas.

Nitrogen starvation of cyanobacteria results in the production of β-N-methylamino-L-alanine.

β-N-Methylamino-L-alanine, an unusual amino acid implicated in neurodegenerative disease, has been detected in cultures of nearly all genera of environmentally ubiquitous cyanobacteria tested. The compound is present within cyanobacterial cells in free and protein-associated forms, with large variations occurring in the concentration of these pools between species as well as within single strains. With a lack of knowledge and supporting data on the regulation of BMAA production and the role of this compound in cyanobacteria, the association between BMAA and cyanobacteria is still subject to debate. In this study we investigated the biosynthesis of BMAA in axenic non-diazotrophic cyanobacterial cultures using the stable isotope ¹⁵N. Nitrogen starvation of nutritionally replete cells resulted in an increase in free cellular ¹⁵N BMAA suggesting that BMAA may be the result of catabolism to provide nitrogen or that BMAA is synthesised to serve a functional role in the cell in response to nitrogen deprivation. The addition of NO₃⁻ and NH₄⁺ to the culture medium following starvation resulted in a decrease of free cellular BMAA without a corresponding increase in the protein-associated fraction. The use of ammonia as a nitrogen source resulted in a more rapid reduction of BMAA when compared to nitrate. This study provides the first data regarding the regulation of intracellular BMAA concentrations in cyanobacteria with results conclusively showing the production of ¹⁵N BMAA by an axenic cyanobacterial culture.