Amyotrophic Lateral Sclerosis/parkinsonism-dementia Complex Research Articles

Examining the interaction of apo E and neurotoxicity on a murine model of ALS-PDC

Epidemiological studies have shown a positive relationship between cycad flour consumption and the development of the neurodegenerative disorder, amyotrophic lateral sclerosis - parkinsonism - dementia complex (ALS-PDC). Apolipoprotein E (apo E) allele variations have been associated with genetic susceptibility in neurodegenerative diseases, including ALS-PDC. We have studied cycad toxicity in a mouse model of ALS-PDC with a particular interest in its impact on the central nervous system (CNS) in both apo E knock-out (KO) mice and their wild-type (WT) counterparts. Behavioral motor tests, motor neuron counts, and immunohistochemical staining in brain and spinal cord, as well as routine histological examinations on internal organs, were performed to evaluate cycad toxicity. Plasma cholesterol levels were also measured before and during the study. Cycad treatment was associated with higher levels of plasma cholesterol only in apo E KO mice; increased levels of plasma cholesterol did not result in increased athero genesis. Cycad-fed wild-type mice developed progressive behavioral deficits including ALS-PDC-like pathological outcomes, while cycad-fed apo E KO mice were not significantly affected. Cycad-fed wild-type mice had shorter gait length measurements along with higher active caspase-3 levels in the striatum, substantia nigra, primary motor cortex, and spinal cord as compared with corresponding controls. These changes were associated with decreased labeling for glutamate transporter 1B and tyrosine hydroxylase activity levels. No evidence of cycad toxicity was observed in internal organs of either wild-type or apo E KO mice. Our data demonstrate that apo E KO mice are less susceptible to cycad toxicity, suggesting a role for apo E as a possible genetic susceptibility factor for some forms of toxin-induced neurodegeneration.

OPL046 Variation in clinical and neuropathological phenotypes of a family of amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the Kii peninsula of Japan

OPL046 Variation in clinical and neuropathological phenotypes of a family of amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of the Kii peninsula of Japan

Quantitative measurement of neurodegeneration in an ALS–PDC model using MR microscopy

Exposure to cycad ( Cycas micronesica K.D. Hill) toxins via diet has been shown to induce neurodegeneration in vivo that mimics the progressive neurological disease, amyotrophic lateral sclerosis–parkinsonism dementia complex (ALS–PDC). In previous studies, specific cortical and subcortical cell loss was measured with conventional stained sections. In the present study, magnetic resonance (MR) microscopy was used to examine neurodegeneration in three dimensions (3D) in isolated intact brains and spinal cords. Mice were fed washed cycad for 2 months and showed progressive motor deficits resembling human ALS–PDC. CNS tissue was imaged at 17.6 T. T 2* scans were acquired on both spinal cord and brain samples with an isotropic resolution of 41 μm. Through MR volumetrics, cycad-fed mice showed significantly decreased volumes in lumbar spinal cord gray matter, substantia nigra, striatum, basal nucleus/internal capsule, and olfactory bulb. Cortical measurements of conventionally stained sections revealed that cycad-fed mice also showed decreased cortical thickness. These results show that MR microscopy (MRM) is sensitive enough to measure degeneration in this early stage model of a progressive neurological disease with strong correlations to behavioral deficits and histological results and may be applicable in vivo to the same model. Similar analysis may be used in the future as a diagnostic aid in tracking the early progression of neurological disorders in preclinical human subjects.

Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam.

We here report biomagnification (the increasing accumulation of bioactive, often deleterious molecules through higher trophic levels of a food chain) of the neurotoxic nonprotein amino acid beta-methylamino-l-alanine (BMAA) in the Guam ecosystem. Free-living cyanobacteria produce 0.3 microg/g BMAA, but produce 2-37 microg/g as symbionts in the coralloid roots of cycad trees. BMAA is concentrated in the developing reproductive tissues of the cycad Cycas micronesica, averaging 9 microg/g in the fleshy seed sarcotesta and a mean of 1,161 microg/g BMAA in the outermost seed layer. Flying foxes (Pteropus mariannus), which forage on the seeds, accumulate a mean of 3,556 microg/g BMAA. Flying foxes are a prized food item of the indigenous Chamorro people who boil them in coconut cream and eat them whole. Chamorros who die of amyotrophic lateral sclerosis/parkinsonism-dementia complex (AL-SPDC), a neurodegenerative disease with symptoms similar to amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease, have an average of 6.6 microg/g BMAA in their brain tissues. The biomagnification of BMAA through the Guam ecosystem fits a classic triangle of increasing concentrations of toxic compounds up the food chain. This may explain why the incidence of ALS-PDC among the Chamorro was 50-100 times the incidence of amyotrophic lateral sclerosis elsewhere. Biomagnification of cyanobacterial BMAA may not be unique to Guam; our discovery of BMAA in the brain tissue from Alzheimer's patients from Canada suggests alternative ecological pathways for the bioaccumulation of BMAA in aquatic or terrestrial ecosystems.

Conservation Implications of Chamorro Consumption of Flying Foxes as a Possible Cause of Amyotrophic Lateral Sclerosis–Parkinsonism Dementia Complex in Guam

Abstract: In the Pacific island of Guam, flying fox ( Pteropus mariannus and P. tokudae ) populations historically persisted for centuries despite subsistence foraging by the indigenous Chamorro people. However, the militarization of Guam in the twentieth century, with an increased emphasis on a cash rather than a subsistence economy, led to two features that had deleterious effects on the flying fox populations: easy access to firearms greatly increased hunter yields and disposable income facilitated commercial traffic in flying foxes. As a result, consumption of flying foxes dramatically increased, leading to the extinction of one indigenous species and near extinction of another. The demise of the flying fox populations was mirrored by a subsequent decrease in the health of the Chamorro people. Amyotrophic lateral sclerosis–Parkinsonism dementia complex ( ALS‐PDC ), a neurological disease with aspects of ALS, Alzheimer's, and Parkinson's disease, became a major cause of adult death in some villages. It has been recently hypothesized that consumption of flying foxes by the Chamorro may have generated sufficiently high cumulative doses of neurotoxins to result in the observed high levels of ALS‐PDC among the Chamorros in Guam because flying foxes forage on cycad seeds that are rich in neurotoxins. Commercialization of traffic in wild flying fox populations may have therefore produced grave health consequences for the Chamorro people.

Tau and α-Synuclein Pathology in Amygdala of Parkinsonism-Dementia Complex Patients of Guam

Amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is a progressive neurodegenerative disorder of Chamorro residents of Guam and the Mariana Islands, characterized by abundant neuron loss and tau neurofibrillary pathology similar to that observed in Alzheimer's disease (AD). A variety of neurodegenerative diseases with tau pathology including ALS/PDC also have alpha-synuclein positive pathology, primarily in the amygdala. We further characterized the tau and alpha-synuclein pathology in the amygdala of a large series of 30 Chamorros using immunohistochemical and biochemical techniques. Tau pathology was readily detected in both affected and unaffected Chamorros. In contrast, alpha-synuclein pathology was detected in 37% of patients with PDC but not detected in Chamorros without PDC or AD. The alpha-synuclein aggregates often co-localized within neurons harboring neurofibrillary tangles suggesting a possible interaction between the two proteins. Tau and alpha-synuclein pathology within the amygdala is biochemically similar to that observed in AD and synucleinopathies, respectively. Thus, the amygdala may be selectively vulnerable to developing both tau and alpha-synuclein pathology or tau pathology may predispose it to synuclein aggregation. Furthermore, in PDC, tau and alpha-synuclein pathology occurs independent of beta-amyloid deposition in amygdala thereby implicating the aggregation of these molecules in the severe neurodegeneration frequently observed in this location.

Behavioral and neurological correlates of ALS-parkinsonism dementia complex in adult mice fed washed cycad flour.

Consumption of cycad seed products (Cycas circinalis) is one of the strongest epidemiological links to the Guamian neurological disorder amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), however, the putative toxin which causes neurodegeneration has never been identified definitively. To reexamine this issue, 6-7-mo-old, male CD-1 mice were assessed for motor and cognitive behaviours during and following feeding with pellets made from washed cycad flour. Cycad-fed animals showed early evidence of progressive motor and cognitive dysfunctions. Neurodegeneration measured using TUNEL and caspase-3 labeling was found in neocortex, various hippocampal fields, substantia nigra, olfactory bulb, and spinal cord. In vitro studies using rat neocortex have identified toxic compounds in washed cycad flour that induce depolarizing field potentials and lead to release of lactate dehydrogenase (LDH), both blocked by AP5. High-performance liquid chromatography (HPLC)/mass spectrometry of cycad flour samples failed to show appreciable amounts of other known cycad toxins, cycasin, MAM, or BMAA; only trace amounts of BOAA were present. Isolation procedures employing these techniques identified the most toxic component as beta-sitosterol beta-D-glucoside (BSSG). The present data suggest that a neurotoxin, or a toxic metabolite, not previously identified in cycad, is able to gain access to central nervous system (CNS) resulting in neurodegeneration of specific neural populations and in motor and cognitive dysfunctions. These data are consistent with a number of major features of ALS-PDC in humans.

Age-Dependent Emergence and Progression of a Tauopathy in Transgenic Mice Overexpressing the Shortest Human Tau Isoform

Filamentous tau aggregates are hallmarks of tauopathies, e.g., frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and amyotrophic lateral sclerosis/parkinsonism–dementia complex (ALS/PDC). Since FTDP-17 tau gene mutations alter levels/functions of tau, we overexpressed the smallest human tau isoform in the CNS of transgenic (Tg) mice to model tauopathies. These mice acquired age-dependent CNS pathology similar to FTDP-17 and ALS/PDC, including insoluble, hyperphosphorylated tau and argyrophilic intraneuronal inclusions formed by tau-immunoreactive filaments. Inclusions were present in cortical and brainstem neurons but were most abundant in spinal cord neurons, where they were associated with axon degeneration, diminished microtubules (MTs), and reduced axonal transport in ventral roots, as well as spinal cord gliosis and motor weakness. These Tg mice recapitulate key features of tauopathies and provide models for elucidating mechanisms underlying diverse tauopathies, including Alzheimer's disease (AD).

Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration.

Neuronal inclusions with bundles of abnormal filaments made of tau polymers are found in numerous diseases with neurofibrillary degeneration. Tau proteins are the basic components of paired helical filaments (PHF) in Alzheimer's disease (AD), and are abnormally phosphorylated. A disease-specific phosphorylation site at serine422 was demonstrated on PHF, but not on tau proteins from biopsy-derived brain samples. In the present study, we report the characterization of a polyclonal antibody (988) against the serine422 phosphorylation site. By using biochemical and immunohistochemical methods, we confirmed that it is not found on tau proteins from biopsy- or autopsy-derived control samples, and we investigated the presence of this epitope on tau proteins in several neurodegenerative disorders, including AD, Down syndrome (DS), Guamanian amyotrophic lateral sclerosis/Parkinsonism-dementia complex (ALS/PDC), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), postencephalitic parkinsonism (PEP) and Pick's disease (PiD). By Western blotting, antibody 988 labeled the characteristic tau triplet (tau 55, 64, 69) in AD, DS, Guamanian ALS/PDC and PEP. PSP and CBD exhibited their typical tau doublet (tau 64, 69), whereas the doublet tau 55 and 64 was detected in PiD. In all of these neurodegenerative disorders, antibody 988 clearly labeled NFT and dystrophic neurites, as well as Pick bodies in PiD cases, whereas no staining was observed in control cases. These data indicate that phosphorylation of serine422 on tau proteins is a common feature among neurodegenerative disorders and is therefore not specific of AD. Moreover, phosphorylation of this epitope permits the distinction between normal tau proteins and pathological tau proteins.

Amyloid plaques in Guam amyotrophic lateral sclerosis/parkinsonism-dementia complex contain species of A beta similar to those found in the amyloid plaques of Alzheimer's disease and pathological aging.

The Guamanian amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) is characterized by abundant neurofibrillary pathology and neuron loss. In contrast to Alzheimer's disease (AD), where extensive neurofibrillary lesions always occur with deposits of A beta in numerous amyloid plaques, A beta-rich amyloid plaques are absent or rare in most ALS/PDC patients. To characterize the amyloid plaques in the latter patients, we probed plaque-rich sections of their brains by immunohistochemistry using well-characterized antibodies to specific epitopes in the N and C termini of A beta as well as to defined epitopes in hyperphosphorylated tau (PHFtau). The results indicate that the species of A beta in the amyloid plaques of ALS/PDC patients resemble those detected in the amyloid plaques of cognitively intact subjects with pathological aging as well as patients with AD. However, the paucity of PHFtau-positive neurites in the ALS/PDC plaques suggests that they reflect pathological aging rather than AD.

Familial nature and continuing morbidity of the amyotrophic lateral sclerosis-parkinsonism dementia complex of Guam.

Chamorros suffer from two neurologic syndromes known as ALS and the parkinsonism-dementia complex (PDC) of Guam. We report mortality figures for these syndromes during 1991 to 1995 and compare them with those at 5-year intervals dating back to 1951. In contrast to predictions of disease disappearance, both syndromes remain prevalent. However, age of onset and age at death have increased for both syndromes, suggesting that shifting environmental factors are causing disease postponement. We also report the clinical, familial, neuropathologic, and immunohistochemical findings on a consecutive autopsy series of Guamanian Chamorro cases. Twelve cases were diagnosed as PDC, known locally as "bodig," and three as ALS, known locally as "lytico." All but three of these fifteen patients had a pronounced family history of similar illness. Eight of twelve boding patients had siblings who were also affected with bodig; two of three lytico cases had siblings afflicted with lytico. The family histories are compatible with genetic transmission of each syndrome. The neuropathology of bodig is characterized by severe and widespread neurofibrillary tangle (NFT) development. NFTs are surrounded by reactive microglia and reactive astrocytes, and complement proteins and other molecules connected with inflammation are associated with them. Similar inflammatory responses also occur in Alzheimer's disease (AD) but have been largely attributed to the presence of senile plaques. These data indicate that tangles, as well as plaques, generate inflammatory reactions that such reactions may exacerbate the fundamental pathology in bodig as well as in AD.

Apolipoprotein E in Guamanian amyotrophic lateral sclerosis/ parkinsonism-dementia complex: genotype analysis and relationships to neuropathological changes

Apolipoprotein E (Apo E) has been recently identified within amyloid deposits and neurofibrillary tangles in the brains of Alzheimer's disease (AD) patients. A strong association of the Apo E epsilon 4 allele with higher risk of developing AD has also been reported. In the present study, the distribution of Apo E and the possible relationship between Apo E alleles and neuropathological alterations were analyzed in a series of Guamanian amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) cases, a neurodegenerative condition characterized neuropathologically by widespread, severe neurofibrillary tangle formation but rare amyloid deposits. ApoE immunoreactivity was consistently observed in both type of lesions in these cases. Compared to tau protein immunoreactivity, there were generally fewer Apo E-immunoreactive neurofibrillary tangles, particularly in the deep layers of the neocortex and in the hippocampus. Genotype analysis revealed that the epsilon 4 allele frequency was 5.9%, the epsilon 3 allele frequency 88.2%, and the epsilon 2 allele frequency 5.9% in this series. Recent data suggest that the Apo E4 variant may induce amyloidogenesis, while E2 could have a neuroprotective role. However, the rare Guamanian patients with amyloid deposits in cortical areas were not related to the epsilon 4 allele, since all cases with senile plaques were epsilon 3/epsilon 3. In addition, compared to unaffected Guamanian cases and other Asian-Pacific populations previously reported, the observed low frequency of the epsilon 2 allele in the present cases, which may be consistent with the notion that this allele, may represent a neuroprotective factor in several neurodegenerative disorders. The present data indicate that there is a strong interaction between Apo E deposition and neurofibrillary changes in Guamanian ALS-PDC.

Cycad toxin-induced damage of rodent and human pancreatic β-cells

Environmental toxins may be risk factors for some forms of diabetes mellitus and neurodegenerative diseases. The medicinal and food use of seed from the cycad plant ( Cycas spp.), which contains the genotoxin cycasin, is a proposed etiological factor for amyotrophic lateral sclerosis/ Parkinsonism-dementia complex (ALS/PDC), a prototypical neurodegenerative disease found in the western Pacific. Patients with ALS/PDC have a very high prevalence of glucose intolerance and diabetes mellitus (in the range of 50–80%). We investigated whether the cycad plant toxin cycasin (methylazoxymethanol (MAM) β- d-glucoside) or the aglycone MAM are toxic in vitro to mouse or human pancreatic islets of Langerhans. Mouse pancreatic islets treated for 6 days with cycasin impaired the β-cell insulin response to glucose, but this effect was reversible after a further 4 days in culture without the toxin. When mouse islets were exposed for 24 hr to MAM/MAM acetate (MAMOAc; 0.1–1.0 mM), there was a dose-dependent impairment in insulin release and glucose metabolism, and a significant decrease in islet insulin and DNA content. At higher MAM/MAMOAc concentrations (1.0 mM), widespread islet cell destruction was observed. Glucose-induced insulin release remained impaired even after removal of MAM and a further culturing for 4 days without the toxin. MAM damages islets by two possible mechanisms: (a) nitric oxide generation, as judged by increased medium nitrite accumulation; and (b) DNA alkylation, as judged by increased levels of O 6-methyldeoxyguanosine in cellular DNA. Incubation of mouse islets with hemin (10 or 100 μM), a nitric oxide scavenger, or nicotinamide (5–20 mM) protected β-cells from a decrease in glucose oxidation by MAM. In separate studies, a 24 hr treatment of human β-islet cells with MAMOAc (1.0 mM) produced a significant decrease in both insulin content and release in response to glucose. In conclusion, the present data indicate that cycasin and its aglycone MAM impair both rodent and human β-cell function which may lead to the death of pancreatic islet cells. These data suggest that a “slow toxin” may be a common aetiological factor for both diabetes mellitus and neurodegenerative disease.

Screening for non-protein amino acids in seeds of the Guam cycad, Cycas circinalis, by an improved GC-MS method.

The non-protein amino acid, beta-N-methyl-amino-L-alanine (L-BMAA) from Cycas circinalis seeds, has been implicated as a causative agent of amyotrophic lateral sclerosis-Parkinsonism dementia complex (ALS-PDC), a disease known from Guam and other areas in the western Pacific. We analyzed C. circinalis seeds for additional free non-protein amino acids by a recently developed GC-MS method. The samples were prepared by water extraction of seed flour. The amino acids present in the extract were derivatized by N(O,S)-isobutyloxycarbonylation of the amine functional groups and then tert-butyldimethylsilylation of the carboxyl functional groups. Peaks for a total of seventeen derivatives of non-protein amino acids were detected by GC-MS. In addition to L-BMAA, four other non-protein amino acids were identified as beta-alanine, gamma-amino-butyric acid, pyroglutamic acid, and alpha-aminoadipic acid.

Transport of cycasin by the intestinal Na +/glucose cotransporter

The medicinal and food use of seed from the cycad plant ( Cycas spp.), which contains the neurotoxin cycasin, is a proposed etiological factor for amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC), a prototypical neurodegenerative disease found in the western Pacific. Cycasin, the β- d-glucoside of methylazoxymethanol might enter neurons and other cells via a glucose transporter. Since the intestinal brush-border Na +/glucose cotransporter plays a major role in the absorption of monosaccharides, the following studies were conducted to determine if cycasin, the β- d-glucoside of methylazoxymethanol, is a substrate for the transporter. We measured the ability of cycasin to (i) inhibit Na +/glucose uptake into rabbit intestinal brush-border membrane vesicles, and (ii) to generate current by the cloned Na +/glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes. The results show that cycasin inhibits Na +-dependent sugar transport in the vesicles, and cycasin generates phlorizin-sensitive currents in oocytes. We conclude that cycasin is a substrate for the intestinal brush-border Na +/glucose cotransporter, albeit with a lower affinity than d-glucose. This suggests that cycasin may be absorbed from the gut lumen by the cotransporter, and as a result either cycasin or the aglycone is presented to the blood-brain barrier for uptake into the brain.

An Unusual Retinal Pigment Epitheliopathy Endemic to the Island of Guam

An unusual retinal pigment epitheliopathy resembling ophthalmomyiasis interna posterior (OMI) is endemic to Guam. Sixteen percent of a sample of the indigenous population were found to have the retinopathy. Fifty-two percent of a group of individuals suffering from amyotrophic lateral sclerosis/Parkinsonism-dementia complex (ALS/PDC) were found to have the same retinopathy. Although the retinal appearance is similar to that caused by a subretinal parasite, a definite etiology has not been identified. Three case reports are presented as samples of the clinical presentation of the condition.

Compensatory Mechanisms in Degenerative Neurologic Diseases

In animal models of parkinsonism, the ability to lose a substantial proportion of dopaminergic neurons without behavioral deficits does not derive from other systems taking over function of the dopaminergic pathway. The surviving nigrostriatal projection increases both the rate of synthesis and the release of dopamine, as compensatory adjustments. This capacity allows at least a fivefold rise in dopamine delivery per neuron, and this enhancement is potentiated further by receptor up-regulation. Decreased reuptake, due to loss of nerve endings, may also lead to augmented occupancy of dopamine receptors, and so constitute yet another compensatory mechanism. In humans, positron emission tomography has revealed subclinical impairment of the dopaminergic nigrostriatal pathway in subjects at risk for parkinsonism caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and Lytico-Bodig (the amyotrophic lateral sclerosis-parkinsonism dementia complex of Guam). However, the separation of patients with clinically overt idiopathic parkinsonism from controls is less marked in vivo (by positron emission tomography) than in postmortem analysis (by neurochemical assay). This disparity may be attributable to the reduction in the number of nigrostriatal nerve endings, leading, in vivo, to a relative increase of extracellular dopamine because active reuptake into the nerve endings is an important mechanism for removing dopamine from the synaptic cleft. In contrast, in a postmortem setting, dopamine that is not sequestered in the storage vesicles of nerve endings is readily available for biochemical degradation during the interval between death and brain analysis. Finally, it is also possible that differences may derive, in part, from dissimilar kinetic systems for handling exogenous and endogenous levodopa.

Amyotrophic lateral sclerosis parkinsonism dementia complex (ALS-PDC) of Guam as a model of Alzheimer's disease: An overview : ∗D.P. Perl, J.C. Steele Neuropathology Division, Mount Sinai Medical Center, New York, NY 10029; Guam Memorial Hospital, Agana, Guam

Amyotrophic lateral sclerosis parkinsonism dementia complex (ALS-PDC) of Guam as a model of Alzheimer's disease: An overview : ∗D.P. Perl, J.C. Steele Neuropathology Division, Mount Sinai Medical Center, New York, NY 10029; Guam Memorial Hospital, Agana, Guam

A Retinopathy on Guam with High Prevalence in Lytico-Bodig

A pigmentary retinopathy resembling that sometimes seen in posterior ophthalmomyiasis interna was found in 26 (53.1 %) of 49 Chamorro patients from Guam who have Lytico-Bodig (also known as amyotrophic lateral sclerosisParkinson ism-dementia complex of Guam). In nine cases, the retinopathy was bilateral. Similar changes were found in 6 (16.2%) of 37 clinically normal individuals from southern villages on Guam that have a high incidence of LyticoBodig, and in 1 (3.1%) of 32 individuals from Saipan, an island north of Guam that has a low incidence of the disease. These results indicate the possibility of an association between Lytico-Bodig and a retinopathy on Guam that may be parasitic in origin. Occurrence of a similar association in other debilitating diseases on Guam and frequency of occurrence of retinopathy in Guamanians with Lytico-Bodig after leaving Guam have yet to be determined.