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Seventy years of research on Western Pacific amyotrophic lateral sclerosis and Parkinsonism-dementia Complex (ALS/PDC) have provided invaluable data on the etiology, molecular pathogenesis and latency of this disappearing, largely environmental neurodegenerative disease. ALS/PDC is linked to genotoxic chemicals (notably methylazoxymethanol, MAM) derived from seed of the cycad plant (Cycas spp.) that were used as a traditional food and/or medicine in all three disease-affected Western Pacific populations. MAM, nitrosamines and hydrazines generate methyl free radicals that damage DNA (in the form of O6-methylguanine lesions) that can induce mutations in cycling cells and degenerative changes in post-mitotic cells, notably neurons. This paper explores exposures to naturally occurring and manmade sources of nitrosamines and hydrazines in association with sporadic forms of ALS (with or without frontotemporal degeneration), progressive supranuclear palsy, and Alzheimer disease. Research approaches are suggested to examine whether these associations might have etiological significance. Lay Summary Unknown environmental exposures are thought to be risk factors for non-inherited forms of certain progressive brain diseases, such as sporadic forms of amyotrophic lateral sclerosis (sALS), progressive supranuclear palsy (sPSP), and Alzheimer's disease (sAD). Related progressive and fatal brain disorders coalesce in a single neurodegenerative disease of largely environmental origin (ALS-Parkinsonism-dementia Complex) that has affected three genetically distinct populations residing in islands of the Western Pacific region. Prolonged study of this prototypical neurodegenerative disease has provided invaluable information on the probable environmental cause (specific chemical genotoxins) and molecular mechanisms (unrepaired nerve cell DNA-damage) by which brain degeneration begins, evolves and, years or decades later, clinical signs appear, and progress. This information is used as a foundation to explore whether chemically related genotoxins (nitrosamines, hydrazines) are possible risk factors for sALS, sPSP, and sAD. Methods to test this hypothesis in the field and laboratory are proposed.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that has proved refractory to drug treatment. Given evidence of neuroprotection in animal models of ischemic stroke, we assessed the prenylflavonoid xanthohumol from the Common Hop (Humulus lupulus L.) for therapeutic potential in murine neuroblastoma N2a cells stably expressing human Swedish mutant amyloid precursor protein (N2a/APP), a well-characterized cellular model of AD. The ELISA and Western-blot analysis revealed that xanthohumol (Xn) inhibited Aβ accumulation and APP processing, and that Xn ameliorated tau hyperphosphorylation via PP2A, GSK3β pathways in N2a/APP cells. The amelioration of tau hyperphosphorylation by Xn was also validated on HEK293/Tau cells, another cell line with tau hyperphosphorylation. Proteomic analysis (2D-DIGE-coupled MS) revealed a total of 30 differentially expressed lysate proteins in N2a/APP vs. wild-type (WT) N2a cells (N2a/WT), and a total of 21 differentially expressed proteins in lysates of N2a/APP cells in the presence or absence of Xn. Generally, these 51 differential proteins could be classified into seven main categories according to their functions, including: endoplasmic reticulum (ER) stress-associated proteins; oxidative stress-associated proteins; proteasome-associated proteins; ATPase and metabolism-associated proteins; cytoskeleton-associated proteins; molecular chaperones-associated proteins, and others. We used Western-blot analysis to validate Xn-associated changes of some key proteins in several biological/pathogenic processes. Taken together, we show that Xn reduces AD-related changes in stably transfected N2a/APP cells. The underlying mechanisms involve modulation of multiple pathogenic pathways, including those involved in ER stress, oxidative stress, proteasome molecular systems, and the neuronal cytoskeleton. These results suggest Xn may have potential for the treatment of AD and/or neuropathologically related neurodegenerative diseases.

Mitochondrial dysfunction is implicated in the pathogenesis of Alzheimer’s disease (AD). However, the precise mitochondrial molecular deficits in AD remain poorly understood. Mitochondrial and nuclear proteomic analysis in mature male triple transgenic AD mice (PS1M146V/APPSwe/TauP301L) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF-MS/MS, bio-informatics analysis and immunofluorescent staining were performed in this study. In addition to impaired spatial memory impairment and intracellular accumulation of amyloid 1–42 (Aβ1–42) in the 3xTg-AD mice, a well-accepted mouse model of the human disease, we also found significantly increased DNA oxidative damage in entorhinal cortex, hippocampal CA1, CA3 and dental gyrus (DG), as evidenced by the positive staining of 8-hydroxyguanosine, a biomarker of mild cognitive impairment early in AD. We identified significant differences in 27 hippocampal mitochondrial proteins (11 increased and 16 decreased), and 37 hippocampal nuclear proteins (12 increased and 25 decreased) in 3xTg-AD mice compared with the wild-type (WT) mice. Differentially expressed mitochondrial and nuclear proteins were mainly involved in energy metabolism (>55%), synapses, DNA damage, apoptosis and oxidative stress. Two proteins were differentially expressed in both hippocampal mitochondria and nuclei, namely electron transport chain (ETC)-related protein ATP synthase subunit d (ATP5H) was significantly decreased, and apoptosis-related dynamin-1 (DYN1), a pre-synaptic and mitochondrial division-regulated protein that was significantly increased. In sum, perturbations of hippocampus mitochondrial energy metabolism-related proteins responsible for ATP generation via oxidation phosphorylation (OXPHOS), especially nuclear-encoded OXPHOS proteins, correlated with the amyloid-associated cognitive deficits of this murine AD model. The molecular changes in respiratory chain-related proteins and DYN1 may represent novel biomarkers of AD.

A commentary on Unraveling 50-year-old clues linking neurodegeneration and cancer to cycad toxins: are microRNAs common mediators? by Spencer, P., Fry, R. C., and Kisby, G. E. (2012). Front. Gene. 3:192. doi: 10.3389/fgene.2012.00192 The Western Pacific amyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS-PDC) has been described as a Rosetta Stone that bears the essential clue to understanding the etiopathogenesis of related neurodegenerative diseases. The three clinical forms (ALS, atypical parkinsonism with dementia, and dementia alone) have a single pathology (polyproteinopathy, notably tauopathy), just as the Rosetta Stone is inscribed with three distinct scripts bearing a common message. As recently discussed (Kisby and Spencer, 2011), studies of ALS-PDC in the three geographically separate and genetically distinct island populations (Chamorros on Guam; Japanese in Honshu Island's Kii Peninsula; and Papuan New Guineans in Irian Jaya, Indonesia) show: ALS-PDC is primarily if not exclusively an environmental disease: no gene mutations identified in related neurodegenerative disorders are found in Guam and Kii-Japan cases, and disease rates have steadily declined in the three affected populations. Emigrants from Guam may develop ALS-PDC years or decades later, but disease risk is absent in their offspring who were born and live abroad. Conversely, Filipino and other immigrants who adopt the Chamorro lifestyle on Guam may acquire the disease. As ALS-PDC declined during the twentieth century, the disease changed its clinical face from ALS in the first third of life, to PD in the second, and D in the third, a pattern consistent with a dose-related response to an environmental exposure that waned with modernization. With this hypothesis, those with the highest dose of the putative environmental factor develop fatal ALS (with sub-clinical nigrostriatal damage) relatively shortly after exposure; those with intermediate doses survive with amyotrophy long enough to develop atypical parkinsonism; those with low doses reach old age and display dementia, while others with the lowest exposure have subclinical neurofibrillary disease reminiscent of early aging. Other features of ALS-PDC variably include loss of olfaction, retinal pigment epitheliopathy, and atypical skin cytology. The most plausible but unproven trigger decades before the disease surfaces in clinical form is exposure to certain plant-derived neurotoxins in food or medicine, or both. The raw seed of the neurotoxic cycad plant (Cycas spp.) was used to heal skin lesions (Guam, Irian Jaya) and as a tonic (Kii). On Guam, processed cycad seed was a Chamorro staple, and the cycad seed-eating flying fox (Pteropus sp.) that bioaccumulates the cycad-derived neurotoxin β-N-methylamino-L-alanine (L-BMAA) was a delicacy. Among the many bioactive chemicals in cycad seed, two with neurotoxic properties are singled out as potential triggers of ALS-PDC: (a) methylazoxymethanol (MAM), a potent genotoxin, carcinogen and developmental neurotoxin that is stored in the plant as an inactive β-glucoside, the concentration of which in cycad flour correlates significantly with incidence rates for ALS and PD in males and females on Guam, unlike the concentration of (b) L-BMAA, a weak excitotoxic amino acid that is taken up by brain tissue and possibly undergoes proteogenesis, resulting in misfolded proteins; daily oral dosing of macaques with L-BMAA for up to 3 months induces a L-dopa-responsive, non-progressive motorsystem disorder with non-excitotoxic cortical and spinal motor neuron pathology. Both L-BMAA and MAM are metabolized to formaldehyde, an established genotoxic agent and human carcinogen. Whereas, genotoxin-induced DNA damage is rapidly repaired in non-nervous tissue (i.e., cycling cells), this can persist in the brain because some DNA-repair mechanisms are weakly expressed in post-mitotic cells. MAM-induces O6-methylguanine (O6-mG) DNA damage, promutagenic lesions that induce uncontrolled mitoses (tumorigenesis) in mouse epithelial tissues and widespread degeneration in the developing murine brain. O6-mG lesions are clearly responsible for the MAM-induced neuronal loss because the pathology is greater in mice that lack the DNA-repair enzyme O6-mG methyltransferase (Mgmt−/−) and reduced or absent in mice that overexpress MGMT (Kisby and Spencer, 2011). Motor deficits in these mouse mutants are consistent with the extent of DNA damage. In sum, these findings suggest DNA damage is an initial event that leads to brain pathology (Figure ​(Figure11). Figure 1 Proposed common pathway underlying cancer and neurodegenerative disease that is mediated by DNA damage and epigenetic changes (modified from Spencer et al., 2012). MGMT activity is very low in the young adult human brain and may be absent in mature nerve cells. Mgmt−/− mice, an animal model of the young adult human brain, develop persistent brain O6-mG DNA lesions following a single dose of MAM. In the days following MAM treatment, these DNA lesions modulate key brain cell-signaling pathways that are also perturbed in human neurological disease, notably Alzheimer's disease, Parkinson's disease, and inherited and sporadic forms of ALS. Pathway analysis of MAM-modulated genes that are anchored to DNA damage reveals links with human cancer, genetic disorders, and skin and hair development (Kisby et al., 2011a). Several of these cell-signaling pathways continued to be modulated in the brain of Mgmt−/− mice 6 months later, with de novo expression changes of numerous genes involving olfaction (Kisby et al., 2011b). These findings emphasize the relationship between acquired brain tissue DNA damage, modulation of cell-signaling pathways, and the induction of early and persistent molecular changes that lead to neuronal demise. They also reveal important relationships between seemingly disparate diseases—cancer and neurodegeneration—the phenotype of MAM-induced DNA damage being determined, respectively, by the presence or absence of the proliferative capacity of target tissues. The genotoxic properties of MAM and formaldehyde, a common metabolite of MAM and L-BMAA, also involve non-coding RNAs with functional roles in both neurodegeneration and cancer (Spencer et al., 2012) (Figure ​(Figure11). While proof is lacking that cycad toxin(s) trigger ALS-PDC, the results of recent biochemical and systems biology studies, coupled with the absence of known mutations in related neurodegenerative disorders, encourage further efforts to examine the molecular and cellular actions of MAM and L-BMAA. These studies not only highlight the response of the brain to unrepaired DNA damage-induced by a genotoxin (e.g., alkylating agent) as a potential initiator of a neurodegenerative process, they also provide a foundation for understanding whether such effects can lead to persistent changes at the protein level (e.g., tau and synuclein), including the erroneous incorporation of foreign amino acids, a subject of recent interest. The ability of MAM to perturb synuclein and several other classes of brain proteins (e.g., calcium homeostasis, mitochondrial and RNA processing) is consistent with this hypothesis (Kisby et al., 2006).