Introducing the Role of Genotoxicity in Neurodegenerative Diseases and Neuropsychiatric Disorders.

Decades of research have identified genetic and environmental factors involved in age-related neurodegenerative diseases and, to a lesser extent, neuropsychiatric disorders. Genomic instability, i.e., the loss of genome integrity, is a common feature among both neurodegenerative (mayo-trophic lateral sclerosis, Parkinson's disease, Alzheimer's disease) and psychiatric (schizophrenia, autism, bipolar depression) disorders. Genomic instability is associated with the accumulation of persistent DNA damage and the activation of DNA damage response (DDR) pathways, as well as pathologic neuronal cell loss or senescence. Typically, DDR signaling ensures that genomic and proteomic homeostasis are maintained in both dividing cells, including neural progenitors, and post-mitotic neurons. However, dysregulation of these protective responses, in part due to aging or environmental insults, contributes to the progressive development of neurodegenerative and/or psychiatric disorders. In this Special Issue, we introduce and highlight the overlap between neurodegenerative diseases and neuropsychiatric disorders, as well as the emerging clinical, genomic, and molecular evidence for the contributions of DNA damage and aberrant DNA repair. Our goal is to illuminate the importance of this subject to uncover possible treatment and prevention strategies for relevant devastating brain diseases.

Novel Techniques for Mapping DNA Damage and Repair in the Brain.

DNA damage in the brain is influenced by endogenous processes and metabolism along with exogenous exposures. Accumulation of DNA damage in the brain can contribute to various neurological disorders, including neurodegenerative diseases and neuropsychiatric disorders. Traditional methods for assessing DNA damage in the brain, such as immunohistochemistry and mass spectrometry, have provided valuable insights but are limited by their inability to map specific DNA adducts and regional distributions within the brain or genome. Recent advancements in DNA damage detection methods offer new opportunities to address these limitations and further our understanding of DNA damage and repair in the brain. Here, we review emerging techniques offering more precise and sensitive ways to detect and quantify DNA lesions in the brain or neural cells. We highlight the advancements and applications of these techniques and discuss their potential for determining the role of DNA damage in neurological disease.

MGMT, a risk factor for both genetic and environmental forms of dementia.

MGMT, the gene coding for the DNA-repair protein O6 -methylguanine methyltransferase, which has been recently shown to be a risk factor for inherited forms of Alzheimer's disease (AD), notably among women, might also be linked to Western Pacific amyotrophic lateral sclerosis and Parkinsonism-dementia complex (ALS/PDC), one phenotype of which is an AD-like dementia. Guam ALS/PDC is strongly considered to be an environmental disorder caused by oral exposure to natural toxins (i.e., genotoxic/epigenotoxic chemicals), notably methylazoxymethanol (MAM) that alkylates guanine to form O6 -methylguanine, found in the seed of cycad plants traditionally used for food. Thus, the DNA-repair protein MGMT might participate in both AD and in the AD-related disorder ALS/PDC.

Role of Hydrazine-Related Chemicals in Cancer and Neurodegenerative Disease.

Hydrazine-related chemicals (HRCs) with carcinogenic and neurotoxic potential are found in certain mushrooms and plants used for food and in products employed in various industries, including aerospace. Their propensity to induce DNA damage (mostly O6-, N7- and 8-oxo-guanine lesions) resulting in multiple downstream effects is linked with both cancer and neurological disease. For cycling cells, unrepaired DNA damage leads to mutation and uncontrolled mitosis. By contrast, postmitotic neurons attempt to re-enter the cell cycle but undergo apoptosis or nonapoptotic cell death. Biomarkers of exposure to HRCs can be used to explore whether these substances are risk factors for sporadic amyotrophic laterals sclerosis and other noninherited neurodegenerative diseases, which is the focus of this paper.

Animal models of brain maldevelopment induced by cycad plant genotoxins.

Cycads are long-lived tropical and subtropical plants that contain azoxyglycosides (e.g., cycasin, macrozamin) and neurotoxic amino acids (notably ß-N-methylamino-l-alanine l-BMAA), toxins that have been implicated in the etiology of a disappearing neurodegenerative disease, amyotrophic lateral sclerosis and parkinsonism-dementia complex that has been present in high incidence among three genetically distinct populations in the western Pacific. The neuropathology of amyotrophic lateral sclerosis/parkinsonism-dementia complex includes features suggestive of brain maldevelopment, an experimentally proven property of cycasin attributable to the genotoxic action of its aglycone methylazoxymethanol (MAM). This property of MAM has been exploited by neurobiologists as a tool to study perturbations of brain development. Depending on the neurodevelopmental stage, MAM can induce features in laboratory animals that model certain characteristics of epilepsy, schizophrenia, or ataxia. Studies in DNA repair-deficient mice show that MAM perturbs brain development through a DNA damage-mediated mechanism. The brain DNA lesions produced by systemic MAM appear to modulate the expression of genes that regulate neurodevelopment and contribute to neurodegeneration. Epigenetic changes (histone lysine methylation) have also been detected in the underdeveloped brain after MAM administration. The DNA damage and epigenetic changes produced by MAM and, perhaps by chemically related substances (e.g., nitrosamines, nitrosoureas, hydrazines), might be an important mechanism by which early-life exposure to genotoxicants can induce long-term brain dysfunction.

Early-onset, conjugal, twin-discordant, and clusters of sporadic ALS: Pathway to discovery of etiology via lifetime exposome research.

The identity and role of environmental factors in the etiology of sporadic amyotrophic lateral sclerosis (sALS) is poorly understood outside of three former high-incidence foci of Western Pacific ALS and a hotspot of sALS in the French Alps. In both instances, there is a strong association with exposure to DNA-damaging (genotoxic) chemicals years or decades prior to clinical onset of motor neuron disease. In light of this recent understanding, we discuss published geographic clusters of ALS, conjugal cases, single-affected twins, and young-onset cases in relation to their demographic, geographic and environmental associations but also whether, in theory, there was the possibility of exposure to genotoxic chemicals of natural or synthetic origin. Special opportunities to test for such exposures in sALS exist in southeast France, northwest Italy, Finland, the U.S. East North Central States, and in the U.S. Air Force and Space Force. Given the degree and timing of exposure to an environmental trigger of ALS may be related to the age at which the disease is expressed, research should focus on the lifetime exposome (from conception to clinical onset) of young sALS cases. Multidisciplinary research of this type may lead to the identification of ALS causation, mechanism, and primary prevention, as well as to early detection of impending ALS and pre-clinical treatment to slow development of this fatal neurological disease.

Effects of sample collection and storage conditions on DNA damage in buccal cells from agricultural workers.

Buccal cells are becoming a widely used tissue source for monitoring human exposure to occupational and environmental genotoxicants. A variety of methods exist for collecting buccal cells from the oral cavity, including rinsing with saline, mouthwash, or scraping the oral cavity. Buccal cells are also routinely cryopreserved with dimethyl sulfoxide (DMSO), then examined later for DNA damage by the comet assay. The effects of these different sampling procedures on the integrity of buccal cells for measuring DNA damage are unknown. This study examined the influence of the collection and cryopreservation of buccal cells on cell survival and DNA integrity. In individuals who rinsed with Hank's balanced salt solution (HBSS), the viability of leukocytes (90%) was significantly (p<0.01) greater than that of epithelial cells (12%). Similar survival rates were found for leukocytes (88%) and epithelial cells (10%) after rinsing with Listerine(®) mouthwash. However, the viability of leukocytes after cryopreservation varied significantly (p<0.01) with DMSO concentration. Cell survival was greatest at 5% DMSO. Cryopreservation also influenced the integrity of DNA in the comet assay. Although tail length and tail moment were comparable in fresh or cryopreserved samples, the average head intensity for cryopreserved samples was ∼6 units lower (95% CI: 0.8-12 units lower) than for fresh samples (t(25)=-2.36, p=0.026). These studies suggest that the collection and storage of buccal samples are critical factors for the assessment of DNA damage. Moreover, leukocytes appear to be a more reliable source of human tissue for assessing DNA damage and possibly other biochemical changes.

Genotoxic Damage During Brain Development Presages Prototypical Neurodegenerative Disease.

Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex (ALS/PDC) is a disappearing prototypical neurodegenerative disorder (tau-dominated polyproteinopathy) linked with prior exposure to phytogenotoxins in cycad seed used for medicine and/or food. The principal cycad genotoxin, methylazoxymethanol (MAM), forms reactive carbon-centered ions that alkylate nucleic acids in fetal rodent brain and, depending on the timing of systemic administration, induces persistent developmental abnormalities of the cortex, hippocampus, cerebellum, and retina. Whereas administration of MAM prenatally or postnatally can produce animal models of epilepsy, schizophrenia or ataxia, administration to adult animals produces little effect on brain structure or function. The neurotoxic effects of MAM administered to rats during cortical brain development (specifically, gestation day 17) are used to model the histological, neurophysiological and behavioral deficits of human schizophrenia, a condition that may precede or follow clinical onset of motor neuron disease in subjects with sporadic ALS and ALS/PDC. While studies of migrants to and from communities impacted by ALS/PDC indicate the degenerative brain disorder may be acquired in juvenile and adult life, a proportion of indigenous cases shows neurodevelopmental aberrations in the cerebellum and retina consistent with MAM exposure in utero. MAM induces specific patterns of DNA damage and repair that associate with increased tau expression in primary rat neuronal cultures and with brain transcriptional changes that parallel those associated with human ALS and Alzheimer's disease. We examine MAM in relation to neurodevelopment, epigenetic modification, DNA damage/replicative stress, genomic instability, somatic mutation, cell-cycle reentry and cellular senescence. Since the majority of neurodegenerative disease lacks a solely inherited genetic basis, research is needed to explore the hypothesis that early-life exposure to genotoxic agents may trigger or promote molecular events that culminate in neurodegeneration.

Protocol for High-Throughput Screening of Neural Cell or Brain Tissue Protein Using a Dot-Blot Technique with Near-Infrared Imaging.

Dot blotting allows for the rapid screening of a larger number of samples and/or targets than more traditional methods, such as a western blot or in-tissue-based methods. We have developed a dot-blot assay specifically for use with a LiCor Odyssey CLx imager, which allows for sensitive detection of proteins in the infrared range. Here, we provide a detailed protocol for the preparation of brain tissue and neural cell culture lysates for analysis of protein targets by dot blotting.

Western Pacific ALS-PDC: Evidence implicating cycad genotoxins.

Amyotrophic Lateral Sclerosis and Parkinsonism-Dementia Complex (ALS-PDC) is a disappearing neurodegenerative disorder of apparent environmental origin formerly hyperendemic among Chamorros of Guam-USA, Japanese residents of the Kii Peninsula, Honshu Island, Japan and Auyu-Jakai linguistic groups of Papua-Indonesia on the island of New Guinea. The most plausible etiology is exposure to genotoxins in seed of neurotoxic cycad plants formerly used for food and/or medicine. Primary suspicion falls on methylazoxymethanol (MAM), the aglycone of cycasin and on the non-protein amino acid ß-N-methylamino-L-alanine, both of which are metabolized to formaldehyde. Human and animal studies suggest: (a) exposures occurred early in life and sometimes during late fetal brain development, (b) clinical expression of neurodegenerative disease appeared years or decades later, and (c) pathological changes in various tissues indicate the disease was not confined to the CNS. Experimental evidence points to toxic molecular mechanisms involving DNA damage, epigenetic changes, transcriptional mutagenesis, neuronal cell-cycle reactivation and perturbation of the ubiquitin-proteasome system that led to polyproteinopathy and culminated in neuronal degeneration. Lessons learned from research on ALS-PDC include: (a) familial disease may reflect common toxic exposures across generations, (b) primary disease prevention follows cessation of exposure to culpable environmental triggers; and (c) disease latency provides a prolonged period during which to intervene therapeutically. Exposure to genotoxic chemicals ("slow toxins") in the early stages of life should be considered in the search for the etiology of ALS-PDC-related neurodegenerative disorders, including sporadic forms of ALS, progressive supranuclear palsy and Alzheimer's disease.

Cycad ß-N-methylamino-L-alanine (BMAA), methylazoxymethanol, genotoxicity, and neurodegeneration.

Cycad-associated neurodegenerative disease is more strongly correlated with the gymnosperm's major neurotoxin cycasin (methylazoxymethanol glucoside) than with the minor neurotoxin ß-N-methylamino-L-alanine (L-BMAA).

Genotoxicants target distinct molecular networks in neonatal neurons.

BACKGROUND: Exposure of the brain to environmental agents during critical periods of neuronal development is considered a key factor underlying many neurologic disorders. OBJECTIVES: In this study we examined the influence of genotoxicants on cerebellar function during early development by measuring global gene expression changes. METHODS: We measured global gene expression in immature cerebellar neurons (i.e., granule cells) after treatment with two distinct alkylating agents, methylazoxymethanol (MAM) and nitrogen mustard (HN2). Granule cell cultures were treated for 24 hr with MAM (10-1,000 microM) or HN2 (0.1-20 microM) and examined for cell viability, DNA damage, and markers of apoptosis. RESULTS: Neuronal viability was significantly reduced (p < 0.01) at concentrations > 500 microM for MAM and > 1.0 microM for HN2; this correlated with an increase in both DNA damage and markers of apoptosis. Neuronal cultures treated with sublethal concentrations of MAM (100 microM) or HN2 (1.0 microM) were then examined for gene expression using large-scale mouse cDNA microarrays (27,648). Gene expression results revealed that a) global gene expression was predominantly up-regulated by both genotoxicants; b) the number of down-regulated genes was approximately 3-fold greater for HN2 than for MAM; and c) distinct classes of molecules were influenced by MAM (i.e, neuronal differentiation, the stress and immune response, and signal transduction) and HN2 (i.e, protein synthesis and apoptosis). CONCLUSIONS: These studies demonstrate that individual genotoxicants induce distinct gene expression signatures. Further study of these molecular networks may explain the variable response of the developing brain to different types of environmental genotoxicants.

Seeking environmental causes of neurodegenerative disease and envisioning primary prevention.

Pathological changes of the aging brain are expressed in a range of neurodegenerative disorders that will impact increasing numbers of people across the globe. Research on the causes of these disorders has focused heavily on genetics, and strategies for prevention envision drug-induced slowing or arresting disease advance before its clinical appearance. We discuss a strategic shift that seeks to identify the environmental causes or contributions to neurodegeneration, and the vision of primary disease prevention by removing or controlling exposure to culpable agents. The plausibility of this approach is illustrated by the prototypical neurodegenerative disease amyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS-PDC). This often-familial long-latency disease, once thought to be an inherited genetic disorder but now known to have a predominant or exclusive environmental origin, is in the process of disappearing from the three heavily affected populations, namely Chamorros of Guam and Rota, Japanese residents of Kii Peninsula, Honshu, and Auyu and Jaqai linguistic groups on the island of New Guinea in West Papua, Indonesia. Exposure via traditional food and/or medicine (the only common exposure in all three geographic isolates) to one or more neurotoxins in seed of cycad plants is the most plausible if yet unproven etiology. Neurotoxin dosage and/or subject age at exposure might explain the stratified epidemic of neurodegenerative disease on Guam in which high-incidence ALS peaked and declined before that of PD, only to be replaced today by a dementing disorder comparable to Alzheimer's disease. Exposure to the Guam environment is also linked to the delayed development of ALS among a subset of Chamorro and non-Chamorro Gulf War/Era veterans, a summary of which is reported here for the first time. Lessons learned from this study and from 65 years of research on ALS-PDC include the exceptional value of initial, field-based informal investigation of disease-affected individuals and communities, the results of which can provide an invaluable guide to steer cogent epidemiological and laboratory-based research.

Mitochondria as a target of environmental toxicants.

Enormous strides have recently been made in our understanding of the biology and pathobiology of mitochondria. Many diseases have been identified as caused by mitochondrial dysfunction, and many pharmaceuticals have been identified as previously unrecognized mitochondrial toxicants. A much smaller but growing literature indicates that mitochondria are also targeted by environmental pollutants. We briefly review the importance of mitochondrial function and maintenance for health based on the genetics of mitochondrial diseases and the toxicities resulting from pharmaceutical exposure. We then discuss how the principles of mitochondrial vulnerability illustrated by those fields might apply to environmental contaminants, with particular attention to factors that may modulate vulnerability including genetic differences, epigenetic interactions, tissue characteristics, and developmental stage. Finally, we review the literature related to environmental mitochondrial toxicants, with a particular focus on those toxicants that target mitochondrial DNA. We conclude that the fields of environmental toxicology and environmental health should focus more strongly on mitochondria.

Unraveling 50-Year-Old Clues Linking Neurodegeneration and Cancer to Cycad Toxins: Are microRNAs Common Mediators?

Recognition of overlapping molecular signaling activated by a chemical trigger of cancer and neurodegeneration is new, but the path to this discovery has been long and potholed. Six conferences (1962-1972) examined the puzzling neurotoxic and carcinogenic properties of a then-novel toxin [cycasin: methylazoxymethanol (MAM)-ß-d-glucoside] in cycad plants used traditionally for food and medicine on Guam where a complex neurodegenerative disease plagued the indigenous population. Affected families showed combinations of amyotrophic lateral sclerosis (ALS), parkinsonism (P), and/or a dementia (D) akin to Alzheimer's disease (AD). Modernization saw declining disease rates on Guam and remarkable changes in clinical phenotype (ALS was replaced by P-D and then by D) and in two genetically distinct ALS-PDC-affected populations (Kii-Japan, West Papua-Indonesia) that used cycad seed medicinally. MAM forms DNA lesions - repaired by O(6)-methylguanine methyltransferase (MGMT) - that perturb mouse brain development and induce malignant tumors in peripheral organs. The brains of young adult MGMT-deficient mice given a single dose of MAM show DNA lesion-linked changes in cell-signaling pathways associated with miRNA-1, which is implicated in colon, liver, and prostate cancers, and in neurological disease, notably AD. MAM is metabolized to formaldehyde, a human carcinogen. Formaldehyde-responsive miRNAs predicted to modulate MAM-associated genes in the brains of MGMT-deficient mice include miR-17-5p and miR-18d, which regulate genes involved in tumor suppression, DNA repair, amyloid deposition, and neurotransmission. These findings marry cycad-associated ALS-PDC with colon, liver, and prostate cancer; they also add to evidence linking changes in microRNA status both to ALS, AD, and parkinsonism, and to cancer initiation and progression.

Is neurodegenerative disease a long-latency response to early-life genotoxin exposure?

Western Pacific amyotrophic lateral sclerosis and parkinsonism-dementia complex, a disappearing neurodegenerative disease linked to use of the neurotoxic cycad plant for food and/or medicine, is intensively studied because the neuropathology (tauopathy) is similar to that of Alzheimer's disease. Cycads contain neurotoxic and genotoxic principles, notably cycasin and methylazoxymethanol, the latter sharing chemical relations with nitrosamines, which are derived from nitrates and nitrites in preserved meats and fertilizers, and also used in the rubber and leather industries. This review includes new data that influence understanding of the neurobiological actions of cycad and related genotoxins and the putative mechanisms by which they might trigger neurodegenerative disease.

Cigarette smoke induces DNA damage and alters base-excision repair and tau levels in the brain of neonatal mice.

The prenatal and perinatal periods of brain development are especially vulnerable to insults by environmental agents. Early life exposure to cigarette smoke (CS), which contains both genotoxicants and oxidants, is considered an important risk factor for both neurodevelopmental and neurodegenerative disorders. Yet, little is known regarding the underlying pathogenetic mechanisms. In the present study, neonatal Swiss ICR (CD-1) albino mice were exposed to various concentrations of CS for 4 weeks and the brain examined for lipid peroxides, DNA damage, base-excision repair (BER) enzymes, apoptosis, and levels of the microtubule protein tau. CS induced a dose-dependent increase in both malondialdehyde and various types of DNA damage, including single-strand breaks, double-strand breaks, and DNA-protein cross-links. However, the CS-induced DNA damage in the brain returned to basal levels 1 week after smoking cessation. CS also modulated the activity and distribution of the BER enzymes 8-oxoguanine-DNA-glycosylase (OGG1) and apyrimidinic/apurinic endonuclease (APE1) in several brain regions. Normal tau (i.e., three-repeat tau, 3R tau) and various pathological forms of tau were also measured in the brain of CS-exposed neonatal mice, but only 3R tau and tau phosphorylated at serine 199 were significantly elevated. The oxidative stress, genomic dysregulation, and alterations in tau metabolism caused by CS during a critical period of brain development could explain why CS is an important risk factor for both neurodevelopmental and neurodegenerative disorders appearing in later life.