SESSION 8C BEYOND GUAM: NEW ASPECTS OF THE CYNOBACTERIA/BMAA HYPOTHESIS

C65 BMAA AS A POSSIBLE TRIGGER FOR SPORADIC ALS/MND: INSIGHTS FROM THE CHAMORRO

COX P, BANACK S, JOHNSON H, BAKHSHI F, TOWN A

Institute for Ethnomedicine, Jackson, Wyoming, United States

E-mail address for correspondence: [email protected]

Keywords: BMAA, Guam, Neurotoxic Diet

Background: The hypothesis that the neurotoxic amino acid BMAA can trigger sporadic neurodegenerative illness in vulnerable individuals depends on four pillars: 1) BMAA is produced by cyanobacteria, which are ubiquitous throughout the world; 2) in the known ALS/PDC foci, exposure to high levels of BMAA can result from biomagnification and dietary ingestion from multiple sources, while in other areas, low-level exposure occurs is occurs through cyanobacterially-contaminated water supplies [1]; 3) vulnerable individuals accumulate BMAA in their neuroproteins [2], and 4) accumulation of BMAA in neuroproteins can trigger neurodegenerative illness.

Objectives: To determine if dietary exposure to BMAA among the Chamorro is associated with increased risk of ALS/PDC.

Methods: We interviewed 23 Chamorro villagers of varying ages from Umatac and Merizo villages after they signed an informed consent form. A code number was attached to a small locket of hair that each villager supplied and then the sample was blinded for BMAA analysis. BMAA was detected by protein hydrolysis and AQC-derivatization in a reverse phase HPLC-FD system.

Results: We found a significant relationship between disease and level of flying fox consumption (χ²=3.86, p < 0.05) with a highly significant relationship (χ²=26; p < 0.001) between disease status and moderate to high flying fox consumption. However, we found no significant relationship (χ²=0.91, NS) between flying fox consumption and the presence of BMAA in hair, since many villagers who had not consumed flying foxes still had detectable BMAA. Only small children who had not consumed possible BMAA sources (flying foxes, pigs, deer, land crabs, or cycad flour) had no detectable BMAA in their hair.

Discussion: These results support previous findings that protein-bound BMAA in cycad flour and feral animals that feed on cycad seeds result in significant BMAA inputs into the Chamorro diet [3]. The results of Borenstein et al. [4] also support the suggestion that protein-bound BMAA from multiple sources may contribute to neurological disease. Although they did not find a specific correlation linking disease state with flying fox consumption, they did find widespread consumption of flying foxes among Chamorros aged 65 and older and statistically significant correlations with disease state and cycad consumption. This suggests a broad exposure of the Chamorro population to dietary BMAA, which may serve as a trigger for neurodegenerative illness.

C66 CYANOBACTERIA, NEUROTOXICITY AND WATER RESOURCES

METCALF J¹, BANACK S², COX P², CODD G¹

¹Dundee University, Dundee, United Kingdom, ²Institute for Ethnomedicine, Jackson, WY, United States

E-mail address for correspondence: [email protected]

Keywords: Cyanobacteria, BMAA, neurotoxicity

Background: Cyanobacteria are ancient, photosynthetic bacteria that occur throughout the world in a wide range of environments. They are most commonly known for their ability to form mass occurrences in waterbodies used for recreation and the preparation of human drinking water. In addition to their effects on the aesthetic quality of waterbodies, cyanobacteria are capable of producing a wide range of bioactive compounds, including potent hepato- and neurotoxins. Although the majority of the neurotoxins are acutely toxic, little is known concerning their long-term health effects. Included in these is β-N-methylamino-L-alanine (BMAA) which has been associated with an Amyotrophic Lateral Sclerosis/Parkinsonism Dementia Complex (ALS-PDC) in Guam, thought to accumulate through the diet.

Objectives: The purpose of the study was to investigate the occurrence of BMAA in environmental cyanobacterial bloom material from waterbodies in the UK. The waterbodies selected all have a history of either being used for the preparation of human and/or animal drinking water, for recreation or as fisheries and are sites of known animal poisoning incidents as a result of cyanobacterial blooms.

Methods: Cyanobacterial bloom material collected from 12 waterbodies was examined for the presence of cyanobacteria by microscopy. Material was lyophilised and extracted to analyse for anatoxin-a, saxitoxins and microcystins using mouse bioassay, high performance liquid chromatography (HPLC) and ELISA. BMAA was derivatised and determined by HPLC with fluorescence detection and LC-MS/MS as either a free amino acid or associated with a precipitable protein fraction.

Results: Bloom samples were found to contain cyanotoxins, and in the case of acute animal intoxication, cyanotoxins were considered to be the proximal cause. With respect to BMAA, all 12 samples contained BMAA as either a free amino acid and/or associated with precipitable protein at between 2 and 275µg g^-1 dry weight of material.

Discussion and Conclusions: BMAA was found to co-occur with known cyanotoxins in cyanobacterial blooms from UK waterbodies. Although animal intoxications occurred at these sites, cyanotoxins other than BMAA were considered to be the cause of the mortalities. However, the results suggest that BMAA may occur in the environment and in places where humans are potentially exposed to this amino acid. Due to its associations with human neurological disease, further assessment of BMAA in the environment is required.

C67 PRODUCTION OF THE NEUROTOXIN BMAA BY CYANOBACTERIA THROUGHOUT THE WORLD: IMPLICATIONS FOR HUMAN HEALTH

JOHNSON H, CHENG R, BANACK S

Institute for Ethnomedicine, Jackson, Wyoming, United States

E-mail address for correspondence: [email protected]

Keywords: Sporadic ALS, Cyanobacteria, Environmental Neurotoxins

Background: BMAA (?-N-methylamino-l-alanine) is putatively linked to ALS/PDC [1]. Early reports concluded that washed cycad flour contains extremely low levels of BMAA [2]. However, they failed to analyze the protein fraction of this flour that contains up to 169 µ g/g of BMAA [3]. BMAA is produced by symbiotic cyanobacteria present in the cycad roots. BMAA is now known to be produced by cyanobacteria throughout the world ([4], [5]).

Objectives: We sought to determine if free-living species of the genus Nostoc [6] from around the world produce BMAA and if preservation methods affect detection in paired frozen or fluid preserved human tissues.

Methods: We used 5 different analytical methods, HPLC-FD, Amino Acid Analyzer, UPLC-UV, UPLC/MS, and triple quadrupole LC/MS/MS to detect BMAA in Nostoc and frozen and fluid human tissues.

Results: All five methods detect BMAA in free-living Nostoc. Fluid fixation of human tissues results in an underestimate of total BMAA.

Conclusions: Detection of BMAA at low concentrations in a complex physiological matrix, is best accomplished with multiple methods of detection. Verification using LC/MS/MS is definitive. Since low concentrations of BMAA (10–30 µ M) kill motor neurons ([7], [8]), our demonstration that free-living Nostoc produces BMAA supports the suggestion that human exposure to low concentrations of BMAA can occur throughout the world [9]. To test whether such exposure is associated with sporadic ALS/MND will require further epidemiological studies.

C68 IN VITRO NEUROTOXICITY OF THE CYCAD TOXIN, BETA-METHYLAMINO-L-ALANINE (BMAA)

WEISS J, RAO S

University of California, Irvine, CA, United States

E-mail address for correspondence: [email protected]

Keywords: BMAA, motor neuron, neurotoxicity

Background: The cycad toxin, beta-methylamino-L-alanine (BMAA) was first proposed as a contributor to the amyotrophic lateral sclerosis-Parkinsonism dementia complex of Guam (ALS/PDC) based on the ability of large amounts of this compound to induce a similar disease phenotype in primates. However, concerns about the apparent low potency of this toxin in relation to estimated levels of human ingestion led to doubts about its disease relevance.

Objectives: We have examined the in vitro neurotoxicity of BMAA in order to gain insights into both its mechanisms of toxicity and potency as a neurotoxin.

Methods: Neurotoxicity and imaging studies were carried out on mixed cortical or spinal neuronal cultures.

Results: Although BMAA had reported to induce excitotoxic neuronal injury, largely via activation of NMDA receptors, in our early studies we found that BMAA had no direct excitatory effects unless the exposure was carried out in the present of bicarbonate. We and others concluded that in the presence of bicarbonate, a carbamate adduct of the side chain amino group of BMAA results in a structure resembling glutamate and capable of activating glutamate receptors ([1],[2]). In further studies we found evidence that although BMAA appeared to be a fairly weak NMDA receptor agonist, low levels of BMAA could selectively damage vulnerable subpopulations of neurons via activation of AMPA/kainate type glutamate receptors [3], leading us to hypothesize that these receptors might be of particular importance to motor neuronal (MN) degeneration, an idea supported by a large number of intervening studies. More recently, we have used mixed spinal neuronal cultures to examine the specific vulnerability of MNs to BMAA. We found that BMAA induced selective MN loss at concentrations (∼30 µM) which were significantly lower than those causing widespread neuronal degeneration [4]. Furthermore, this MN injury was blocked by the selective AMPA/kainate receptor antagonist, NBQX. Using imaging 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.

Discussion and Conclusions: Recent studies have identified new and diverse environmental sources of BMAA and have found evidence that BMAA can be incorporated into proteins, possibly providing an endogenous reservoir from which it can be slowly released [5]. Thus, in light of our in vitro studies, we propose that protein bound BMAA in vivo might contribute to slow MN injury in part via release from proteins, and formation of carbamate adducts, causing activation of AMPA/kainate type glutamate receptors.

C69 NEW ASPECTS OF THE CYANOBACTERIA/BMAA HYPOTHESIS AND FUTURE DIRECTIONS

BRADLEY W

Department of Neurology, Miller School of Medicine, University of Miami, United States

E-mail address for correspondence: [email protected]

Keywords: Cyanobacteria, BMAA, sporadic ALS

Background:The cause of ALS in the 85% of cases with sporadic disease is unknown and presumably environmental. Even in the 15% of familial cases there must be an environmental factor that precipitates the onset. Evidence is compelling that a cyanobacterial product, probably BMAA, in cycads causes Guamanian ALS/PDC. BMAA is a non-natural neurotoxic amino acid that becomes incorporated into the proteins of higher organisms. Protein-bound BMAA has been found in millimolar concentrations in brains of Chamorros dying from ALS/PDC and North American ALS and Alzheimer's disease patients, but not in control brains. Cyanobacteria that produce BMAA and other neurotoxins are ubiquitous, particularly in water resources associated with animal deaths.

Objectives: To discuss: 1) How BMAA is incorporated into brain proteins; 2) How BMAA might cause neurodegeneration; 3) Testable predictions of the cyanobacteria/BMAA hypothesis; and 4) Therapeutic predictions derived from the hypothesis.

Discussion: C.A.Shaw and colleagues have argued that steryl glucosides produced by cyanobacteria and other organisms are the cause of Guamanian ALS/PDC. Their hypothesis would suggest that BMAA is simply a marker of exposure to cyanobacteria. However, several in-vitro and in-vivo studies have demonstrated that BMAA is neurotoxic at concentrations comparable to that found in human brains, though most were studies of acute toxicity. No true animal model of BMAA chronic neurodegeneration (developing after several months of feeding) has yet been reported.

BMAA is transported across the blood-brain barrier via the high affinity saturatable L1 system that carries large neutral essential amino acids (LNEAA-leucine, valine, methionine, histidine, iso-leucine, tryptophan, phenylalanine, threonine). These LNEAA are actively exported from the brain by several Na⁺-dependent amino acid transporters to maintain a CSF concentration 10% of that of plasma. It is not known whether BMAA is actively exported across the blood-brain barrier, or which tRNA is responsible for incorporating BMAA into protein. The metabolic pathways for BMAA are not known. More basic information about BMAA is required.

The cyanobacteria/BMAA hypothesis predicts that blocking access of BMAA to the brain would prevent neurodegeneration. At least five trials of branch-chain amino acids in ALS have been reported; one showed benefit, one worsening, and three no effect. The dose required to saturate the L1-system and inhibit entry of BMAA is approximately six-times that used in these trials. Studies of ultra-high-doses of LNEAAs in ALS may be indicated. However, such treatment would be ineffective in patients already suffering from ALS unless BMAA is a recycling neurotoxin, as Murch and colleagues hypothesized, and unless high-dose LNEAA promotes the efflux of BMAA.

Conclusions: The cyanobacteria/BMAA hypothesis is an important advance in identifying the cause of sporadic ALS and the precipitation of familial ALS. Research to understand more about the association of cyanobacteria with ALS, the metabolism of BMAA and the production of models of chronic neurodegeneration may lead to a breakthrough in our understanding of ALS.