Neurology -- Correspondence for Steele and McGeer, 70 (21) 1984-1990

Correspondence published:

The ALS/PDC syndrome of Guam and the cycad hypothesis: Amy R. Borenstein, James A. Mortimer, Gerard D. Schellenberg ( Philadelphia, PA) , Douglas Galasko ( San Diego, CA.) (25 July 2008)

The ALS/PDC syndrome of Guam and the cycad hypothesis: Walter G. Bradley, Sandra Anne Banack, Paul Alan Cox (25 July 2008)

The ALS/PDC syndrome of Guam and the cycad hypothesis: Reyniel Cruz-Aguado, Christopher A. Shaw (25 July 2008)

The ALS/PDC syndrome of Guam and the cycad hypothesis: Peter S. Spencer, Valerie Palmer and Glen Kisby (25 July 2008)

Reply from the authors: Patrick L. McGeer, John C. Steele (25 July 2008)

The ALS/PDC syndrome of Guam and the cycad hypothesis 25 July 2008

Amy R. Borenstein,
University of South Florida
COPH, Epi and Biostat, 13201 Bruce B Downs Blvd., Tampa, FL 33612,
James A. Mortimer, Gerard D. Schellenberg ( Philadelphia, PA) , Douglas Galasko ( San Diego, CA.)
Send Correspondence to journal:
Re: The ALS/PDC syndrome of Guam and the cycad hypothesis

aborenst{at}health.usf.edu Amy R. Borenstein, et al.

In their review of the ALS/PDC syndrome of Guam, Steele and McGeer argue against the cycad hypothesis of causation, noting the persistence of this disorder after 1950 when the regular consumption of cycad flour (fadang) among the native Chamorros ceased. [1]
The rapid decline in incidence of both ALS and PDC beginning in the 1950s and persisting for more than 50 years is well established. [2] The occasional observation of new cases by Dr. Steele is consistent with a disappearing syndrome resulting from a point exposure in the 1940s. Analysis of the 29 cases of PDC identified in our 2003 island-wide prevalence study reveals that all subjects were born before 1940 and would have had ample opportunity to be exposed to an environmental toxin in the 1940s. [3]
Steele and McGeer state that our findings of an association of cycad exposure with PDC, MCI and Guam dementia (GD) �are inconsistent with any reasonable theory regarding fadang toxicity,� because of a protective effect of exposure in childhood and lack of association in adulthood. [1] We pointed out that the protective effect in childhood is likely due to recall bias related to study design. [4]
Our findings of significantly elevated odds ratios for picking, processing and eating fadang in early adulthood for all three outcomes (GD, MCI and PDC) are consistent with a point-source exposure. These findings are also consistent with epidemiologic evidence showing a preponderance of affected men as well as reflecting a commonality in causation of all three disorders. The fact that adult exposures were not associated is likely due to the rapid decline in exposure rates of Chamorros to cycad during the 1960s and later when subjects reached adulthood, reducing the statistical power to detect an effect. [4]
Long-term cycad toxicity is plausible, based on recent studies that implicate toxins other than BMAA. For example, phytosterol glucosides that result in the excitotoxic release of glutamate lead to motor neuron and Parkinsonian phenotypes and pathology in cycad extract-fed mice. [5]
While alternative hypotheses are welcome, there are negligible data to support them. Steele and McGeer suggest that an infection that causes retinopathy may predispose to ALS/PDC. [1] The infectious agent has never been identified, and there is no evidence that infectious retinal diseases trigger tangle formation in widespread regions of brain and spinal cord. Finally, the rapid decline in incidence is inconsistent with either a genetic or persisting environmental exposure.
References
1. Steele JC, McGeer PL. The ALS/PDC syndrome of Guam and the cycad hypothesis. Neurology 2008;70:1984-1990.
2. Plato CC, Garruto RM, Galasko D, et al. Amyotrophic lateral sclerosis and Parkinsonism-dementia complex of Guam: changing incidence rates during the past 60 years. American Journal of Epidemiology 2003;157:149-157.
3. Galasko D, Salmon D, Gamst A et al. Prevalence of dementia in Chamorros on Guam: relationship to age, gender, education and APOE. Neurology 2007;68:1772-1781.
4. Borenstein AR, Mortimer E, Schofield E et al. Cycad exposure and risk of dementia, MCI and PDC in the Chamorro population of Guam. Neurology 2007;68:1764-71.
5. Wilson J, Shaw CA. Commentary on: Return of the cycad hypothesis � does the amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC) of Guam have new implications for global health? Neuropathology and Applied Neurobiology 2006;32:341-343.
Disclosures: The authors have no disclosures

The ALS/PDC syndrome of Guam and the cycad hypothesis 25 July 2008

Walter G. Bradley,
Department of Neurology, Miller School of Medicine, University of Miami
CRB 1315, 1120 NW 14th Street, Miami, FL 33136,
Sandra Anne Banack, Paul Alan Cox
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Re: The ALS/PDC syndrome of Guam and the cycad hypothesis

wbradley{at}med.miami.edu Walter G. Bradley, et al.

We read with interest the review by Steele and McGeer but disagree with their contention that cycads do not deserve serious consideration as possible causes of ALS/PDC in Guam. [1]
We read with interest the review by Steele and McGeer [1], but disagree with their contention that cycads do not deserve serious consideration as possible causes of ALS/PDC in Guam. In what might be better labeled �the cyanobacteria/BMAA hypothesis,� cyanobacteria resident in specialized cycad roots produce the amino acid BMAA [6]. This potent excitotoxin, produced by diverse species of cyanobacteria, is found in cycad seed flour and in animals which forage on cycad seeds, as well as in water supplies contaminated with cyanobacteria [7]. BMAA can biomagnify in the food chain, and occurs in a protein-bound form at concentrations 50-100 greater than as a free amino acid [6, 8] which may explain previous negative reports.
BMAA was found in blinded brain tissues of ALS/PDC patients from Guam and in brain tissues of Alzheimer Disease (AD) patients from North America, but not in healthy controls. [8] Researchers recently independently replicated the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-tag method of BMAA analysis with high performance liquid chromatography with fluorescence detection (HPLC-FD), and found protein-bound BMAA in brain tissues of North American AD and ALS patients. [9]
ALS cases (n=13) had 134 � 13 microg/g mean BMAA concentration, Alzheimer cases (n=12) had 111 � 15 microg/g mean BMAA concentration, while BMAA was undetected in 22/24 samples from 12 control patients (limit of detection = 2 ng on column), but was detected at 45 and 36 microg/g in 2/24 samples. [9]
Similar to how patients with phenylketonuria accumulate rather than metabolize the amino acid phenylalanine, vulnerable individuals may accumulate BMAA, which can then trigger latent neurodegeneration. It might seem surprising that two different clinicopathological diseases�ALS and AD�may arise from the action of one environmental neurotoxin. However, there are several instances where a single gene mutation causes different diseases defined clinicopathologically (e.g. ALS, frontotemporal dementia, corticobasal degeneration, AD, Parkinson disease from mutations of progranulin and MAPT genes, limb girdle muscular dystrophy and several different distal myopathies from mutations of dysferlin gene).
Chamorros are exposed to BMAA through consumption of cycad flour, flying foxes, and other animals which forage on cycad seeds. Elsewhere in the world, BMAA exposure comes primarily from water contaminated with cyanobacteria. The cyanobacteria hypothesis suggests that a worldwide environmental neurotoxin precipitates sporadic ALS and AD, generates testable predictions, and offers new approaches to prevention and therapy.
References
6. Cox PA, Banack SA, Murch SJ. Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam. Proc Natl Acad Sci USA 2003;100:13380-13383.
7. Metcalf JS, Banack SA, Lindsay J, Morrison LF, Cox PA, Codd GA. Co -occurrence of beta-N-methylamino-L-alanine, a neurotoxic amino acid, with other cyanobacterial toxins in British waterbodies, 1990�2004. Environ Microbiol 2008;10:702-708.
8. Murch SJ, Cox PA, Banack SA. A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam. Proc Natl Acad Sci USA 2004;101:12228-12231.
9. Mash D, Pablo J, Banack SA, et al. Neurotoxic non-protein amino acid BMAA in brain from patients dying with ALS and Alzheimer�s disease. American Academy of Neurology annual meeting. P06.127. Neurology 2008; 70(Suppl 1):A329.
Disclosures: The authors have no disclosures.

The ALS/PDC syndrome of Guam and the cycad hypothesis 25 July 2008

Reyniel Cruz-Aguado,
University of British Columbia
386-828 W.10th Ave., Vancouver, BC, V5Z1L8, Canada,
Christopher A. Shaw
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Re: The ALS/PDC syndrome of Guam and the cycad hypothesis

reynielc{at}yahoo.com Reyniel Cruz-Aguado, et al.

A recent article on ALS-PDC of Guam reviews some of the recent epidemiological, genetic, and pathological findings on this intriguing disorder and rightfully underscores its relevance as an important geographical isolate that might shed light on similar disorders elsewhere. [1]
In their examination of the etiological factors, Steele and McGeer also revisit some of the environmental hypothesis and refute the cycad hypothesis. The authors state that the cycad hypothesis lacks scientific merit due to lack of supportive evidence. As an example of these failures, a brief discussion of the widely criticized the cyanobacteria �-methylamino L-alanine (BMAA) biomagnification hypothesis is presented.
However, to further refute the cycad hypothesis, the authors omit scientific literature. A basic PubMed search reveals at least 25% of the published articles on this subject in the last five years including a feature review. [10] Whatever the flaws in the recent Borenstein et al. article [4], this study recapitulates the original conclusions of Whiting and others.
The authors of the current review present their objections based on age of cycad exposure and use the apparent discrepancies to dismiss the entire hypothesis. However, age-dependent impacts of neurotoxins are well known.
We have published over a dozen original articles clearly demonstrating an ALS-PDC phenotype in vivo and plausible putative neurotoxins in vivo and in vitro. The in vivo studies have shown that chronic dietary exposure to cycad flour leads to neurodegeneration in mice after prolonged exposure. [11] These data confirm those of Dastur et al. who fed macaque monkeys washed and unwashed cycad flour.
In addition, we have identified water-insoluble molecules that could be responsible for cycad neurotoxicity. [12] We welcome critiques of the hypothesis that cycad toxins are not causal to ALS-PDC.
References
10. Miller G. Guam's Deadly Stalker: On the Loose Worldwide? Science 313: 428-431
11. Wilson JM, Khabazian I, Wong MC, et al. Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour. Neuromolecular Med. 2002: 207-221.
12. Tabata RC, Wilson JM, Ly P, et al. Chronic Exposure to Dietary Sterol Glucosides is Neurotoxic to Motor Neurons and Induces an ALS-PDC Phenotype. Neuromolecular Med. 2008: 24-39.
Disclosures: The authors have no disclosures.

The ALS/PDC syndrome of Guam and the cycad hypothesis 25 July 2008

Peter S. Spencer,
CROET/Oregon Health & Science University (OHSU)
3181 SW Sam Jackson Park Rd,, Portland, OR 97239,
Valerie Palmer and Glen Kisby
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Re: The ALS/PDC syndrome of Guam and the cycad hypothesis

spencer{at}ohsu.edu Peter S. Spencer, et al.

Steele and McGeer acknowledge the possible role of environmental factors in the etiology of the �Guam syndrome� while refuting the cycad hypothesis (specifically the minor cycad component L-BMAA) as a plausible environmental etiology for this prototypical neurodegenerative disease complex. [1]
The authors present incomplete cycad exposure information for the Japanese residents of the disease-affected region of Kii Peninsula of Honshu Island and the absence of any consideration of the comparable neurodegenerative disease focus in Auyu and Jaqai linguistic groups of West Papua, Indonesia. The only common environmental factor in these three genetically distinct, ALS/PDC-affected populations is the use of raw, non-detoxified Cycas spp. seed for medicinal purposes. It is used as a poultice for open wounds in New Guinea and Guam, and as an oral tonic in the Kii Peninsula. [13,14] Food use of poorly detoxified cycad seed is an additional historical exposure factor on Guam yet fully detoxified cycad products (stem and/or seed) have been used for food in the Japanese Ryukyu Islands and Northern Australia where Guam-like neurodegenerative disease foci are absent. [14]
There is a strong epidemiological relationship for sampled Guam villages between the concentration of residual methylazoxymethanol (MAM, the aglycone of the principal cycad neurotoxin cycasin) in washed cycad flour and the 10-year age-adjusted incidence of ALS and PD. Cycad components and cycasin produce motor system disease in adult cattle. [14] MAM is an established developmental neurotoxin in rodents, where it induces formation of ectopic, multinucleated Purkinje-like cells comparable to those reported in the cerebellum of deceased Guam and Kii ALS patients. [14] It also damages rodent neuronal DNA and modulates tau expression in vitro [15] and induces the accumulation of synuclein in the brain of young mice. [16] The ability of MAM to disrupt the metabolism of both synuclein and tau (including its phosphorylation) is evidence that is consistent with the classification of ALS/PDC as a synucleinopathy and tauopathy.
Contrary to the view expressed by Steele and McGeer [1], these facts demonstrate the plausibility of a cycad hypothesis for western Pacific ALS/PDC that emphasizes the role of the plant�s principal neurotoxin, MAM-beta-glucoside, while not excluding a role for BMAA.
References
13. Spencer PS, Palmer VS, Ludolph AC. On the decline and etiology of high-icidence motor system disease in West Papua (southwest New Guinea). Movement Disorders 2005;20:S119-126.
14. Spencer, PS, Kisby GE, Palmer VS, Obendorf P. Cycasin, methylazoxymethanol and related compounds. In: Experimental and Clinical Neurotoxicology, 2nd edition (eds. Spencer, P.S. and Schaumburg, H.H.), Oxford University Press, New York, 2000, pp 436-446.
15. Esclaire F, Kisby G, Spencer P, Milne J, Lesort M, Hugon J. The Guam cycad toxin methylazoxymethanol damages neuronal DNA and modulates tau mRNA expression and excitotoxicity. Experimental Neurology 1999;155:11-21.
16. Kisby GE, Standley M, Park T, et al. Proteomic analysis of the genotoxicant methylazoxymethanol (MAM) induced changes in the developing cerebellum. J Proteome Res 2006;5:2656-2665.
Disclosures: The authors have no disclosures.

Reply from the authors 25 July 2008

Patrick L. McGeer,
University of British Columbia
Kinsmen Laboratory of Neurological Research, 2255 Wesbrook Mall, Vancouver, BC Canada V6T1Z3,
John C. Steele
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Re: Reply from the authors

mcgeerpl{at}interchange.ubc.ca Patrick L. McGeer, et al.

Borenstein et al. defend the inconsistencies in their epidemiological study as recall bias. We drew attention in our review to the difficulty of reaching firm conclusions based on remembering details of events that took place over 50 years previously.
In this case-control study, the subjects were elderly Chamorros who in some cases were demented. However, this is not the main reason for rejecting the notion that the consumption of cycad flour, or the picking of its seeds, could be responsible for the Guam disease. It is the gap, amounting to orders of magnitude, between the amounts of cycad that Chamorros must have had to consume and the amounts required to produce even weak toxicity in animals.
If exposure to cycad itself fails for this reason, then exposure to its minor constituents also fails. This applies to the BMAA hypothesis put forward by Bradley et al, the �-sitosterol glucoside (BSSG) hypothesis put forward by Cruz-Aguado et al., and the methylazoxymethanol (MAM) hypothesis put forward by Spencer et al. Bradley et al. have gone beyond the BMAA hypothesis as a cause of the Guam syndrome by proposing that BMAA, produced by cyanobacteria, is contaminating world wide water supplies causing AD and ALS. They describe BMAA as �this potent excitotoxin�. However, BMAA is not a potent excitotoxin.
For example, rats were not seriously harmed by a continuous infusion of 100 mg/kg/day of BMAA over a two-week period. [17] Humans would have to consume a dose of about 5 kilograms per day of cycas micronesica to be exposed even to these minimally effective levels of BMAA. Potent excitotoxins, such as kainic and ibotenic acids, have a dicarboxylic acid structure similar to glutamate so that they produce a neurotoxic action at glutamate receptors. [18]
BMAA, as a monocarboxylic acid, lacks the necessary structure. Bradley et al. quote astonishingly high BMAA levels in the brains of Guam ALS/PDC as well as AD and ALS cases. The levels are at least four times higher than those obtained in the high dose BMAA rat infusion experiments. Significantly, there was no BMAA retained in the brain in these experiments. [17] This argues against the protein binding theory proposed by Bradley et al. Such binding would not be expected in any event since BMAA lacks the prosthetic groups normally involved in hydrogen bonding to proteins.
In our review, we pointed out that Montine et al., using a well established analytical technique, failed to detect any BMAA in the brains of AD cases, controls, or Chamorros with and without PDC. This indicates serious discrepancies in the analytical methods used for detecting BMAA. Those discrepancies need to be resolved. Bradley et al. state they have put forward a testable hypothesis. Since AD and ALS tissue is widely available, investigators will be able to determine whether or not their results can be replicated.
Cruz-Aguado et al. hypothesize that chronic exposure to BSSG induces an ALS/PDC phenotype yet overlooked the extremely low concentrations of this material that have been found in cycad preparations. Originally, only 7.3 mg was obtained from 200 grams of washed flour. [19] The authors fed mice 1 mg/day of synthetic BSSG for 15 weeks. This would be equivalent to humans eating 2.5 grams per day of this constituent. If the original report of its concentration in cycad is anywhere near quantitative, this would require a human to consume 68,000 grams of cycad flour per day. The mice survived this regimen and the authors did not identify motor deficits in their mice in such measures as open field behavior and motor strength, although they did report reduced counts of spinal motor neurons on autopsy. We do not consider that these results constitute an ALS PDC phenotype. Like the BMAA hypothesis, we do not consider that this hypothesis merits serious scientific consideration.
Spencer and colleagues propose that the toxic constituent in cycad flour is methylazoxymethanol (MAM). They suggest that the MAM hypothesis explains not just the Guam disease, but also the Kii peninsula disease and possibly a similar but unproven disease in West Papua, New Guinea. Kisby et al. injected a dose of 43 mg/kg of MAM to newborn mice and then observed small developmental changes in the cerebellum. [12] That dose would be equivalent to more than 2 grams of MAM to a human. MAM comes from cycasin, which constitutes 2-4% of unwashed cycad flour and 0.004- 75.93 micrograms/g of washed cycad flour. [11]
Since Guamanians were known to eat only washed cycad flour, this would translate to a consumption of between 1,300 and 250,000,000,000 grams of washed flour. Studies of adult rats fed cycasin or raw cycad flour failed to demonstrate major pathological changes or deficits in behavior. [20] In the Kii peninsula, there are two small foci each of five villages with ALS/PDC separated by 200 kilometers with no neurological disorders in other coastal and inland villages between them.
Kuzuhara et al. concluded that the epidemiological, clinical, pathological and immunological features of ALS/PDC were the same on Guam and in the Kii peninsula. [21] In October 2007, Kuzahara wrote: �None of the patients had the habit of eating the cycad, flying fox, or any other odd materials�. [22] We do not agree that the data presented by Spencer et al. demonstrate the plausibility of a cycad hypothesis.
References
17. Duncan MW, Nelly E, Villacreses PG, et al. 2-Amino-3-(methylamino)- propanoic acid (BMAA) pharmacokinetics and blood-brain barrier permeability in the rat. J Pharm Exp Ther 1991;258:27-35.
18. Kainic acid as a tool in neurobiology, McGeer EG, Olney J, McGeer PL Eds. Raven Press, New York NY, 1978.
19. Khabazian I, Bains, JS, Williams, DE, et al. Isolation of various forms of sterol B-D-glucoside from the seed of cycas circinalis: neurotoxicity and implications for the ALS-parkinsonin dementia complex. J Neurochem 2002;82:516-528.
20. Campbell ME et al. Effects of strain, age and diet on the response of rats to the ingestion of Cycas circinalis. J Nutr 1966; 88:115-124.
21. Kuzuhara S, Kokubo Y, Sasaki R, et al. Familial Amyotrophic lateral sclerosis and Parkinsonism-dementia complex of the Kii Peninsula of Japan: Clinical and neuropathological study and tau analysis. Ann Neurol 2001;49:501-511.
22. Kuzuhara S. Revisit to Kii ALS: The innovated concept of ALS/Parkinsonism-dementia complex, clinicopathological features, epidemiology and etiology (article in Japanese with English abstract) Brain Nerve 2007;59:1065-1074.
Disclosures: The authors have no disclosures.