C35 GUAM ALS/PDC: A DOORWAY TO UNDERSTANDING SPORADIC ALS?
COX P
Institute for Ethnomedicine, Jackson Hole, Wyoming, USA
Email address for correspondence: [email protected]
Keywords: Guam, cyanobacteria, BMAA
BMAA in Guam: Studies of ALS/PDC foci in Guam, Japan's Kii Peninsula, and West Papua, have identified a variety of potential genetic and environmental risk factors. There is renewed interest in β-N-methylamino-l-alanine (BMAA), a neurotoxic amino acid originally isolated from Guam cycad seeds, as a possible trigger for sporadic ALS in gene/environment interactions (1).
BMAA occurs both as a free amino acid and as a protein-bound amino acid in multiple components of the traditional Chamorro diet, including tortillas, dumplings, and stews made from cycad seeds and the flesh of wild or feral animals that feed on cycad seeds (2,3). Protein-bound BMAA occurs in higher concentrations than the free amino acid in cycad gametophytes and other tissues. Washing cycad chips with water fails to remove bound BMAA, causing previous investigators to underestimate BMAA concentrations in cycad flour (4).
BMAA in other ecosystems: BMAA is produced by endosymbiotic cyanobacteria of the genus Nostoc in specialized, positively geotropic roots of cycads (5). BMAA is also produced by other cyanobacterial taxa, suggesting that exposure to BMAA may occur far from Guam (6). Double-blinded analyses of post mortem brain tissues detected BMAA in North American Alzheimer's patients but not healthy controls (2). This result was replicated by researchers at the Miami Brain Bank who also found BMAA in ALS patients but generally not in Huntington's patients or in healthy controls (7). Investigators in Sweden and Florida found BMAA accumulating in ascending trophic levels in marine ecosystems (8,9). Investigators in New Hampshire and France suggest that cyanobacterially-contaminated water and shellfish may be linked to clusters of sporadic ALS (10, W. Camu in press).
Neurotoxicity to motor neurons: BMAA has effects on all of the main types of glutamate receptors: NMDA, AMPA/kainate, and metabotropic receptors, particularly in the presence of bicarbonate (11,12). Selective toxicity of BMAA at concentrations of 30 μM to sub-populations of motor neurons distinguished by the presence of NADPH-diaphorase is mediated by AMPA/kainate receptors. BMAA also acts on the cystine/glutamate antiporter (system xc-), where it induces oxidative stress and glutamate release. At concentrations of 10 μM, BMAA potentiates neuronal injury induced by exposure to other neurotoxins, including amyloid-β or 1-methyl-4-phenylpyridinium ion (MPP +), which are used in models of Alzheimer's and Parkinson's disease respectively (11).
Protein misincorporation: New findings to be reported at this meeting by Australian researchers show that BMAA is misincorporated into human proteins through mischarged tRNA, causing changes in protein confirmation and collapse. Swedish and French investigators report that BMAA binds to melanin in animal models (13,14).
New approaches: Studies of BMAA suggest new approaches to ALS prevention and therapy, including a drug currently in Phase II human clinical trials (15).
References
- Cox PA, Bradley WG, (eds). ALS 2009;10(S2):1–126.
- Murch SJ, Cox PA, Banack SA. PNAS 2004:101: 12228–31.
- Banack SA, Murch SJ, Cox PA. J Ethnopharm. 2006;106:97–104.
- Cheng R, Banack SA. ALS 2009;10(S2):41–3.
- Cox PA, Banack SA, Murch SJ. PNAS 2003;100:13380–3.
- Cox PA, Banack SA, Murch SJ, et al. PNAS 2005;102:5074–8.
- Pablo J, Banack SA, Cox PA, et al. Acta Neuro Scan 2009; 120:216–25.
- Jonasson S, Eriksson J, Berntzon L. PNAS 2010; 107(20):9252–7.
- Brand LE. ALS 2009;10(S2):85–95.
- Caller TA, Doolin JW, Haney JF, et al. ALS 2009; 10(S2):101–8.
- Lobner D. ALS 2009;10(S2):56–60.
- Vyas KJ, Weiss JH. ALS 2009;10(S2):50–5.
- Karlsson O, Berg C, Brittebo EB. Pigm Cell Melan Res. 2009;22:120–30.
- Santucci S, Zsürger N, Chabry J. J Neurochem 2009; 109(3):819–25.
- Bradley WG. ALS 2009;10(S2):114–23.
C36 AN ENVIRONMENTAL NEUROTOXIN, BMAA, IN GUAM
BANACK S1, METCALF J1, NUNN P2
1Institute for Ethnomedicine, Jackson, WY, USA, 2School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, UK
Email address for correspondence: [email protected]
Keywords: BMAA, neurotoxin, environment
Background: ALS/PDC in Guam has been examined from many different research perspectives and the idea of an environmental link to the disease is long-standing. The data demonstrating that the Chamorro diet is rich in the neurotoxin β-methylamino-L-alanine (BMAA) suggest a mechanism for chronic dietary exposure in traditional villages. Cyanobacterial symbionts in specialized cycad roots produce BMAA. Free-living cyanobacteria from habitats worldwide also produce BMAA. The hypothesis that cyanobacterially produced BMAA can trigger sporadic ALS in vulnerable individuals is testable and has the potential to lead to target-based therapy.
Objectives: We established analytical techniques to separate BMAA from related compounds and to quantify BMAA concentrations in a variety of complex physiological matrices including cyanobacteria, plant, animal, and human tissues.
Methods: Using AQC precolumn derivatization with HPLC-FD, UPLC-UV, LC/MS, and LC/MS/MS detection, we analytically separated BMAA from other diamino acids and amides particularly those that might occur in complex physiological matrices, such as 2,6-diaminopimelic acid. An amino acid analyzer which separates underivatized BMAA using an ion-exchange method with post-column colorimetric detection employing ninhydrin was also used. Free amino acids are analyzed using a TCA extraction method and compared with water, methanol, and acetone extractions. Protein-bound amino acids were analyzed using standard hydrolysis techniques and with enzymatic degradation (glucosidases and Pronase). Verification across multiple instruments, by multiple investigators in different laboratories, using different techniques provides increased confidence in the determination and quantification of BMAA, particularly within complex physiological matrices.
Results: We found that standard methods of amino acid analysis clearly distinguish BMAA from other diamino acids and amides. Free and protein-bound BMAA was detected throughout the tissues of 9 Guamanian flying fox specimens, Pteropus mariannus mariannus, including internal organs, muscles, skin, hair and in the skin, hair, and in the liver of 3 Pteropus mariannus yapensis but not in the kidney or muscle. In 2 dried specimens of Pteropus tonganus, BMAA was not detected. Hydrolyzed protein samples released higher concentrations of BMAA than free-BMAA extractions in washed cycad flour samples. Enzyme degradation experiments demonstrated that BMAA is released with Pronase but not with glucosidases in cycad gametophyte tissues.
Conclusions: Current analytical methods to quantify BMAA using AQC derivatization reliably separate BMAA from other diamino acids and amides. BMAA is associated with both the free and protein fraction of animal and plant tissues. Enzyme degradations suggest that BMAA is covalently bound to the insoluble seed material from Cycas micronesica: that it is not bound to the carbohydrate component, but is bound to the protein component of the seed. The current data support the hypothesis that cyanobacterially produced BMAA may be a trigger for sporadic ALS in vulnerable individuals.
C37 DETECTION OF BMAA IN THE MARINE ENVIRONMENT OF A SPORADIC ALS CLUSTER IN SOUTHERN FRANCE
MASSERET E1, BANACK S2, BOUMEDIENE F3, ABADIE E4, BRIENT L5, VAQUER A1, MORALES R6, PAGEOT N6, METCALF J2, COX P2, CAMU W6
1Ecologie des Systèmes Marins Côtiers, UMR 5119 UM2-CNRS-IRD-IFREMER-UM1, UM2, Montpellier, France, 2Institute for Ethnomedicine, Jackson Hole ,WY, USA, 3Service GEOMATIQUE, GEOLAB UMR CNRS 6042, GERHICO-CERHILIM EA 4270, FLSH, Limoges, France, 4Laboratoire Environnement Ressouces, Sète, France, 5Université de Rennes I, UMR Eco-Bio, Rennes, France, 6ALS center, CHU and UM1, Montpellier, France
Email address for correspondence: [email protected]
Keywords: cluster, BMAA, standardized incidence rate
Background: The causes of sporadic ALS (SALS) remain unknown in the vast majority of the cases but several environmental studies have pointed to the role of exposure to BMAA as a potential risk factor for developing ALS. Recent research determined that BMAA, produced by cyanobacteria, is present in marine areas with cyanobacterial blooms and can bioaccumulate from invertebrates to animals of higher trophic levels, ending in human consumption.
Objectives: To identify potential SALS clusters from Languedoc-Roussillon and investigate the existence of BMAA in the surrounding area.
Methods: Potential SALS clusters were identified from our database from 1994 and 2010. Our cases were then studied with the standardized incidence ratio (SIR) method. Environmental investigations were done in the most important cluster with local collection of environmental samples. These samples were analysed for BMAA and its neurotoxic isomer 2,4-DAB.
Results: From the several potential clusters identified in our data base, 9 had a significant SIR. Two of them, Mèze and Balaruc, corresponded to cities surrounding the Thau Lagoon with an SIR of 3.31 and 3.47, respectively. In these areas, food intake as well as the economic structure were focused on the culture and collection of molluscs. Local investigations found that oysters from the lagoon were macroscopically positive for phycocyanin, a marker for the presence of cyanobacteria. LC/MS/MS analysis of mussels and oysters, collected at different periods, showed BMAA concentrations ranging from 1.83 to 6.04 μg/g in mussels and from 0.52 to 2.11 μg/g in oysters, while no difference in DAB concentrations could be noted between oysters and mussels. The highest concentrations of BMAA were noted during summertime when the highest picocyanobacterial abundances have been recorded. Otherwise, seasons did not apparently influence DAB levels.
Discussion: BMAA is found in invertebrates of the Thau Lagoon, an area in which their consumption by indigenous populations is particularly important all-year-long. If the relationship with the occurrence of SALS cannot be formally ascertained, it is nevertheless tempting to speculate that populations from the cluster are at high risk of BMAA intake as they still eat shellfish during summer, unlike other people. The highest cyanobacteria densities are regularly observed during summer in Thau lagoon. Thus, this period could potentially present risks because of the transfer of BMAA in the trophic chain via the microbial loop. We plan to analyze brain tissues from deceased patients to determine whether their content in BMAA is high as it has been demonstrated in patients from other areas such as Guam or Florida.
C38 THE CYANOBACTERIA-DERIVED NEUROTOXIN BMAA CAN BE INCORPORATED INTO CELL PROTEINS AND COULD THUS BE AN ENVIRONMENTAL TRIGGER FOR ALS AND OTHER NEUROLOGICAL DISEASES ASSOCIATED WITH PROTEIN MISFOLDING
RODGERS K1, DUNLOP R2
1University of Technology, Sydney, Australia, 2Heart Research Institute, Sydney, Australia
Email address for correspondence: [email protected]
Keywords: neurotoxin, protein aggregation, BMAA
Background: Cyanobacteria (blue-green algae) are ubiquitously distributed in fresh water and marine environments. β-methylamino-L-alanine (BMAA), a non-protein amino acid produced by cyanobacteria has been linked to neurodegenerative disease in the South Pacific and to ALS and Alzheimer's disease (AD) in Canada and the USA (1,2). BMAA is bioconcentrated in aquatic species raising the possibility of ‘silent’ exposure to BMAA (3,4). BMAA is most commonly found in a protein-associated form [5]; the nature of this association is not known but there is evidence linking it to the chronic neurotoxicity of BMAA (6).
Hypothesis: BMAA is incorporated into cell proteins by protein synthesis in place of a ‘protein’ amino acid (7). The non-native proteins generated can misfold, inducing protein aggregation and a decline in cell function.
Objectives: To determine how BMAA is associated with proteins in mammalian cells in vitro.
Methods: Human fibroblasts (MRC-5) and neuroblastoma cells (SH-SY5Y) were exposed to culture medium containing 3H-BMAA, and levels of intracellular free and protein-associated 3H-BMAA quantified under a range of culture conditions. Release of 3H-BMAA from the isolated cell proteins was then examined.
Results: Up to 10% of intracellular 3H-BMAA was associated with cell proteins. The association between BMAA and cell proteins in vitro was both time and concentration dependent. Blocking protein synthesis using cycloheximide or increasing the concentration of protein amino acids in the culture medium, prevented any association between 3H-BMAA and proteins. 3H-BMAA could not be released from the isolated cell proteins by SDS, DTT or heat but could be released by proteolysis or acid hydrolysis.
Discussion: Release of 3H-BMAA from cell proteins required cleavage of peptide bonds by proteolysis or hydrolysis, providing evidence that 3H-BMAA was incorporated into proteins. The inclusion of a protein synthesis inhibitor in the culture medium prevented any association between 3H-BMAA and proteins, confirming that incorporation was a protein synthesis dependent process. The extent of incorporation of 3H-BMAA into protein was dependent on the level of amino acids present in the culture medium confirming direct competition between protein amino acids and BMAA for incorporation into protein.
Conclusions: BMAA, due to its ability to be incorporated into proteins by mammalian cells, could be an environmental trigger which promotes protein misfolding in genetically susceptible individuals.
References
- Murch SJ, et al. Proc Natl Acad Sci U S A, 2004;101(33): 12228–31.
- Pablo J, et al. Acta Neurol Scand, 2009;120(4):216–25.
- Brand LE, et al. Harmful Algae, 2010;9(6):620–35.
- Spacil Z, et al. 2011;135(1):127–32.
- Cox PA, ALS, 2009;10 Suppl 2:124–6.
- Karamyan VT, Speth RC. Life Sci, 2008;82(5–6):233–46.
- Rodgers KJ, Shiozawa N. Int J Biochem Cell Biol, 2008; 40(8):1452–66.
C39 TRACKING BRAIN UPTAKE AND PROTEIN INCORPORATION OF CYANOBACTERIAL TOXIN BMAA
XIE X1, MONDO K1, BASILE MJ1, BRADLEY WG1, MASH DC1,2
1Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA, 2Department of Cellular and Molecular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, USA
Email address for correspondence: [email protected]
Keywords: beta-N-methylamino-L-alanine (BMAA), Parkinsonism-Dementia Complex (PDC), environmental toxins
Beta-N-methylamino-L-alanine (BMAA) is a neurotoxic nonprotein amino acid. We and others have measured BMAA as a free and protein-bound amino acid in brains of Alzheimer's Disease (AD), Amyotrophic Lateral Sclerosis (ALS), and Guamanian ALS/Parkinsonism Dementia Complex (ALS-PDC) patients. A reservoir of BMAA misincorporated into proteins may function as a slow toxin that with continuous environmental exposure accumulates in brain. We report here on the biodistribution and time course of protein incorporation of custom synthesized [14C]-L-BMAA in adult male C57 mice following single dose intravenous (i.v.) administration. The uptake of [14C]-L-BMAA in brain was determined over time and quantified in soluble and protein fractions. The observed accumulation of radiolabeled BMAA following i.v. administration demonstrated that BMAA is taken up into the brain most likely through amino acid transport across the blood brain barrier. Accumulation of BMAA in brain was time dependent with increasing percentages of the total radiotracer occurring in the protein fraction at later time points. We observed over 70% of the radiotracer dose of BMAA incorporated into proteins at 3 days, suggesting an efficient transfer of BMAA from free to protein bound fractions. The uptake of BMAA and shift from the free to the protein bound fraction appeared to track the known rates of cerebral protein biosynthesis. Ex vivo autoradiography showed a relatively homogeneous distribution in brain at early time points after injection with increased BMAA labeling seen in the ventricles and choroid plexus. The regional pattern of BMAA uptake to the cerebral cortex and subcortical areas demonstrated that grey matter was elevated compared to white matter brain structures. These results demonstrate that the non-protein amino acid, BMAA, can be misincorporated into brain proteins and is in support of the toxic reservoir hypothesis. Environmental exposure to BMAA from the aquatic food web may lead to increased brain uptake and protein incorporation over time. Misincorporation of BMAA into cerebral proteins may alter normal function, folding and proteosomal degradation, increasing the risk for neurodegenerative diseases that affect aging populations.