John C. Steele

Occurrence of beta-methylamino-l-alanine (BMAA) in ALS/PDC patients from Guam.

We tested the brain tissues of the Chamorro people of Guam who died of amyotrophic lateral sclerosis/Parkinsonism dimentia complex (ALS/PDC) for the neurotoxin β‐methylamino‐l‐alanine (BMAA). We used validated high‐pressure liquid chromatography and liquid chromatography‐mass spectrometry analyses to test well‐characterized archival tissues of the superior frontal gyrus from eight Chamorros from Guam and a comparison group of 15 Canadians. BMAA was found as a free amino acid in 83% of Chamorro ALS/PDC patients (3–10 μg/g) as a protein‐associated amino acid in 100% of the Chamorro individuals (149–1190 μg/g). Both forms of BMAA were also found at comparable levels in two Canadians who died of progressive neurodegenerative disease. BMAA, which is produced by cyanobacteria, may be associated with some cases of neurodegenerative disease.

2‐Amino‐3‐(methylamino)‐propanoic acid (BMAA) in cycad flour: An unlikely cause of amyotrophic lateral sclerosis and parkinsonism‐dementia of Guam

We conducted an investigation of the levels of the neurotoxin 2-amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour. Analysis of 30 flour samples processed from the endosperm of Cycas circinalis seeds collected on Guam indicated that more than 87% of the total BMAA content was removed during processing. Furthermore, in ½ the samples almost all (≥99%) of the total BMAA was removed. We found no significant regional differences in the BMAA content of flour prepared from cycad seeds collected from several villages on Guam. Testing of different samples prepared by the same Chamorro woman over 2 years suggests that the washing procedure probably varies in thoroughness from preparation to preparation but is routinely efficient in removing at least 85% of the total BMAA from all batches. Analysis of a flour sample that had undergone only 24 hours of soaking indicated that this single wash removed 90% of the total BMAA. We conclude that processed cycad flour as prepared by the Chamorros of Guam and Rota contains extremely low levels of BMAA, which are in the order of only 0.005% by weight (mean values for all samples). Thus, even when cycad flour is a dietary staple and eaten regularly, it seems unlikely that these low levels could cause the delayed and widespread neurofibrillary degeneration of nerve cells observed in amyotrophic lateral sclerosis and the parkinsonism-dementia complex of Guam (ALS-PD).

The ALS/PDC syndrome of Guam and the cycad hypothesis.

THE ALS/PDC SYNDROME OF GUAM AND THE CYCAD HYPOTHESIS To the Editor: In their review of the amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC) syndrome of Guam, Steele and McGeer1 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. 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 Steele and McGeer 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 McGeer1 suggest that an infection that causes retinopathy may predispose to ALS/PDC. 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.

The tau H2 haplotype is almost exclusively Caucasian in origin.

We have assessed the distribution of the tau H1/H2 haplotype in the publicly available reference series of samples with representatives of most racial groups. This analysis shows that the H2 haplotype is probably exclusively Caucasian in origin and its marginal occurrence in other racial groups is likely to reflect admixture. We discuss this observation in terms of the origin of the H2 haplotype and the epidemiology of the tauopathies.

Odor identification deficit of the parkinsonism‐dementia complex of Guam Equivalence to that of Alzheimer's and idiopathic Parkinson's disease

Olfactory dysfunction is among the first signs of Alzheimers disease (AD), idiopathic Parkinsons disease (PD), and the parkinsonism-dementia complex (PDC) of Guam. We have recently demonstrated that the odor identification and detection deficits of patients with PD are equivalent to those of patients with mild AD when subtle differences in cognitive function are statistically controlled for by analysis of covariance. In contrast, patients with progressive supranuclear palsy (PSP) and patients with l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism evidence olfactory function much more similar to that of normal controls. In the present study, we administered the University of Pennsylvania Smell Identification Test and the Picture Identification Test to 24 patients with early signs of the PDC of Guam and statistically compared their test scores to those of 24 early-stage AD and 24 early-stage PD patients of similar age and gender from the United States mainland. Although the PDC group evidenced slightly more difficulty in identifying pictures than did the other 2 groups, the odor identification deficit associated with this disorder was of the same magnitude as that observed in AD and PD, suggesting that olfactory testing cannot be used to distinguish among these 3 diseases and that the olfactory dysfunction of these disorders may reflect a common neurologic substrate.

Neurofibrillary tangles of Guam parkinson-dementia are associated with reactive microglia and complement proteins.

Guamanian parkinsonism-dementia, locally described as bodig, is characterized by the widespread appearance of neurofibrillary tangles in cortical and subcortical areas. These tangles have similar regional distribution and immunohistochemical profile to those found in Alzheimer disease (AD). We studied the immunohistochemical staining of these tangles, as well as those of AD, using antibodies to complement proteins and related molecules. In bodig, as in AD, extracellular tangles were intensely decorated with antibodies to C1q, C4d and C3d, but not fraction Bb of factor B, properidin or immunoglobulins. This is evidence that the classical, but not the alternative complement pathway is activated on extracellular tangles and that the activation is independent of antibodies. Immunohistochemical staining for amyloid P, an in vitro activator of complement, was remarkably similar to that for the C1q, C4d and C3d in both bodig and AD. This was not the case for beta-amyloid protein (BAP), another in vitro complement activator. Positive staining was observed in only a minority of extracellular tangles in bodig and was only rarely observed in those of AD. BAP would therefore not appear to be a candidate for activating complement on extracellular neurofibrillary tangles. Reactive microglia and reactive astrocytes were closely associated with complement positive extracellular neurofibrillary tangles, indicating an inflammatory response similar to that seen in AD.

Amyotrophic lateral sclerosis/parkinsonism-dementia complex of Guam: quantitative neuropathology, immunohistochemical analysis of neuronal vulnerability, and comparison with related neurodegenerative disorders

Amyotrophic lateral sclerosis/parkinsonism-dementia complex (lytico-bodig) is a chronic neurodegenerative disorder with high prevalence among the native Chamorro population of Guam. Neuropathological, biochemical, and immunohistochemical analyses were performed on a relatively large series of Guamanian cases and compared to Alzheimers disease cases. Thioflavin S and antibodies to amyloid βA4 and tau proteins were used for analysis of pathological changes, and antibodies to the calcium-binding proteins parvalbumin and calretinin, and to a nonphosphorylated epitope on neurofilament protein to study select neuronal populations. A differential distribution of neurofibrillary tangles was observed in the neocortex of Guamanian cases compared to Alzheimers disease cases, with much higher lesion counts in supragranular than in infragranular layers. Also, Guamanian cases with predominant parkinsonism had generally higher neurofibrillary tangle densities than cases with predominant amyotrophic lateral sclerosis. In addition, there was a certain degree of heterogeneity, qualitatively and quantitatively, in the biochemical distribution of tau proteins among Guamanian and Alzheimers disease cases as revealed by Western blot analysis. Previous studies have suggested that the clinical symptomatology observed in patients suffering from Alzheimers disease is related to the dramatic loss of specific corticocortically projecting neurons in the neocortex. Interestingly, a subset of neurofilament-rich pyramidal neurons known to be dramatically affected in Alzheimers disease appears to be resistant in lytico-bodig. Finally, as in Alzheimers disease, calcium-binding protein-containing interneurons are not affected. These data suggest that the set of projection neurons affected in Guamanian cases may not correspond to those involved in Alzheimers disease, and that both disorders are characterized by specific patterns of neuronal vulnerability.

Neurodegenerative diseases of Guam: analysis of TAU

Article abstract Mutations in the tau gene have been described in families affected by frontotemporal dementia with parkinsonism linked to chromosome 17. The authors performed a genetic and biochemical analysis of this gene and its product in the parkinsonism dementia complex of Guam, a disorder characterized by the extensive formation of neurofibrillary tangles. The tau gene is not a primary cause of the parkinsonism dementia complex of Guam.

Parkinsonism–dementia complex of Guam

On Guam and in two other Pacific locales, indigenous residents and immigrants are prone to familial neurodegeneration that manifests as atypical parkinsonism, dementia, motor neuron disease, or a combination of these three phenotypes. This progressive and fatal disease of the Mariana islands, the Kii peninsula of Japan, and the coastal plain of West New Guinea is similar and the pathological features have close affiliation with universal tauopathies, including progressive supranuclear palsy, Alzheimers disease, and amyotrophic lateral sclerosis. The Chamorros of Guam call the disease lytico‐bodig, and neuroscientists refer to it as the amyotrophic lateral sclerosis/Parkinsonism–dementia complex. During recent decades, its prevalence has declined progressively, and the age at onset has steadily increased. In 2004, motor neuron disease, once 100 times more common than elsewhere is rare, atypical parkinsonism is declining, and only dementia remains unusually common in elderly females. The cause of this obscure malady remains uncertain, despite 60 years of international research, but its ending implicates environmental influences rather than genetic predisposition.

Amyotrophic lateral sclerosis and parkinsonism-dementia from Guam : differences in neurofibrillary tangle distribution and density in the hippocampal formation and neocortex

Amyotrophic lateral sclerosis/parkinsonism-dementia complex is a highly prevalent neurodegenerative disorder among the native Chamorro population of Guam, and is characterized by widespread formation of neurofibrillary tangles. In the present study, the distribution of neurofibrillary tangles was quantitatively assessed in the cerebral cortex of cases presenting with either predominant amyotrophic lateral sclerosis or parkinsonism-dementia symptomatology. Results show that although the regional and laminar lesion distribution is qualitatively similar in both groups, cases with predominant parkinsonism-dementia generally have higher lesion densities than cases with amyotrophic lateral sclerosis. Interestingly, layer II of the entorhinal cortex was affected to the same degree in both conditions. In both groups, the CA1 field of the hippocampus, subiculum, and entorhinal cortex were the most affected areas. In the neocortex, the perirhinal and inferior temporal cortex consistently had higher lesion densities than the frontal, parietal, and cingulate cortex, whereas the visual cortex was practically devoid of lesions. Also, most of the neurofibrillary tangles were located in the supragranular layers of the neocortex, with relatively low densities in the infragranular layers, in both brain groups. Interestingly, the primary motor cortex contained more neurofibrillary tangles in parkinsonism-dementia than in amyotrophic lateral sclerosis cases. It is possible that the differences in regional neurofibrillary tangle densities reflect the variable severity of the dementing process observed between the two groups of patients. Several studies on Alzheimers disease and related disorders indicate that the regional and laminar cortical localization of neurofibrillary tangles may parallel the degeneration of specific corticocortical projections. The present data suggest that the population of corticocortical projections involved in Guamanian cases differs substantially from that affected in Alzheimers disease. The differential distribution and densities of the lesions may contribute to the differences in symptomatology and severity of dementia among Alzheimers disease and Guamanian cases, although these neurodegenerative disorders as well as related illnesses may share certain etiopathogenetic mechanisms.