Environmental carcinogens disproportionally mutate genes implicated in neurodevelopmental disorders.

Introduction: De novo mutations contribute to a large proportion of sporadic psychiatric and developmental disorders, yet the potential role of environmental carcinogens as drivers of causal de novo mutations in neurodevelopmental disorders is poorly studied. Methods: To explore environmental mutation vulnerability of disease-associated gene sets, we analyzed publicly available whole genome sequencing datasets of mutations in human induced pluripotent stem cell clonal lines exposed to 12 classes of environmental carcinogens, and human lung cancers from individuals living in highly polluted regions. We compared observed rates of exposure-induced mutations in disease-related gene sets with the expected rates of mutations based on control genes randomly sampled from the genome using exact binomial tests. To explore the role of sequence characteristics in mutation vulnerability, we modeled the effects of sequence length, gene expression, and percent GC content on mutation rates of entire genes and gene coding sequences using multivariate Quasi-Poisson regressions. Results: We demonstrate that several mutagens, including radiation and polycyclic aromatic hydrocarbons, disproportionately mutate genes related to neurodevelopmental disorders including autism spectrum disorders, schizophrenia, and attention deficit hyperactivity disorder. Other disease genes including amyotrophic lateral sclerosis, Alzheimer's disease, congenital heart disease, orofacial clefts, and coronary artery disease were generally not mutated more than expected. Longer sequence length was more strongly associated with elevated mutations in entire genes compared with mutations in coding sequences. Increased expression was associated with decreased coding sequence mutation rate, but not with the mutability of entire genes. Increased GC content was associated with increased coding sequence mutation rates but decreased mutation rates in entire genes. Discussion: Our findings support the possibility that neurodevelopmental disorder genetic etiology is partially driven by a contribution of environment-induced germ line and somatic mutations.

Gene-Environment Interactions in Repeat Expansion Diseases: Mechanisms of Environmentally Induced Repeat Instability.

Short tandem repeats (STRs) are units of 1-6 base pairs that occur in tandem repetition to form a repeat tract. STRs exhibit repeat instability, which generates expansions or contractions of the repeat tract. Over 50 diseases, primarily affecting the central nervous system and muscles, are characterized by repeat instability. Longer repeat tracts are typically associated with earlier age of onset and increased disease severity. Environmental exposures are suspected to play a role in the pathogenesis of repeat expansion diseases. Here, we review the current knowledge of mechanisms of environmentally induced repeat instability in repeat expansion diseases. The current evidence demonstrates that environmental factors modulate repeat instability via DNA damage and induction of DNA repair pathways, with distinct mechanisms for repeat expansion and contraction. Of particular note, oxidative stress is a key mediator of environmentally induced repeat instability. The preliminary evidence suggests epigenetic modifications as potential mediators of environmentally induced repeat instability. Future research incorporating an array of environmental exposures, new human cohorts, and improved model systems, with a continued focus on cell-types, tissues, and critical windows, will aid in identifying mechanisms of environmentally induced repeat instability. Identifying environmental modulators of repeat instability and their mechanisms of action will inform preventions, therapies, and public health measures.

Sex-specific neurobehavioral and prefrontal cortex gene expression alterations following developmental acetaminophen exposure in mice.

Acetaminophen (N-acetyl-p-aminophenol (APAP), also known as paracetamol) is one of the most common medications used by the general population, including pregnant people. Although many human observational and animal model studies have shown associations between prenatal and early postnatal APAP exposure and attention deficit hyperactivity disorder, autism spectrum disorders, and altered neurodevelopment, the existing literature is limited. In particular, no mouse studies of prenatal APAP exposure have investigated offspring attention deficits in behavioral tasks specifically designed to measure attention, and no prior rodent studies have utilized 'omics' technologies, such as transcriptomics, for an untargeted exploration of potential mechanisms. We randomly assigned pregnant mice (starting embryonic day 4-10) to receive APAP (150 mg/kg/day) or vehicle control through postnatal day 14. We evaluated 111 mouse offspring in a battery of behavioral tests, including pup ultrasonic vocalizations, elevated plus-maze, open field test, CatWalk (gait), pre-pulse inhibition, and the automated 5-choice serial reaction time task. Prefrontal cortex was collected at birth from 24 pups for RNA sequencing. Developmental APAP treatment resulted in increased and hastened separation-induced pup vocalizations between postnatal days 2 and 11, as well as decreased ambulation and vertical rearings in the open field in male but not female adult offspring. APAP treatment was also associated with altered sex-specific prefrontal cortex gene expression relating to glutathione and cytochrome p450 metabolism, DNA damage, and the endocrine and immune systems. This study provides additional evidence for the neurodevelopmental harm of prenatal APAP exposure and generates hypotheses for underlying molecular pathways via RNA sequencing.

Deep phenotyping and lifetime trajectories reveal limited effects of longevity regulators on the aging process in C57BL/6J mice.

Current concepts regarding the biology of aging are primarily based on studies aimed at identifying factors regulating lifespan. However, lifespan as a sole proxy measure for aging can be of limited value because it may be restricted by specific pathologies. Here, we employ large-scale phenotyping to analyze hundreds of markers in aging male C57BL/6J mice. For each phenotype, we establish lifetime profiles to determine when age-dependent change is first detectable relative to the young adult baseline. We examine key lifespan regulators (putative anti-aging interventions; PAAIs) for a possible countering of aging. Importantly, unlike most previous studies, we include in our study design young treated groups of animals, subjected to PAAIs prior to the onset of detectable age-dependent phenotypic change. Many PAAI effects influence phenotypes long before the onset of detectable age-dependent change, but, importantly, do not alter the rate of phenotypic change. Hence, these PAAIs have limited effects on aging.

Convergent neural correlates of prenatal exposure to air pollution and behavioral phenotypes of risk for internalizing and externalizing problems: Potential biological and cognitive pathways.

Humans are ubiquitously exposed to neurotoxicants in air pollution, causing increased risk for psychiatric outcomes. Effects of prenatal exposure to air pollution on early emerging behavioral phenotypes that increase risk of psychopathology remain understudied. We review animal models that represent analogues of human behavioral phenotypes that are risk markers for internalizing and externalizing problems (behavioral inhibition, behavioral exuberance, irritability), and identify commonalities among the neural mechanisms underlying these behavioral phenotypes and the neural targets of three types of air pollutants (polycyclic aromatic hydrocarbons, traffic-related air pollutants, fine particulate matter < 2.5 µm). We conclude that prenatal exposure to air pollutants increases risk for behavioral inhibition and irritability through distinct mechanisms, including altered dopaminergic signaling and hippocampal morphology, neuroinflammation, and decreased brain-derived neurotrophic factor expression. Future studies should investigate these effects in human longitudinal studies incorporating complex exposure measurement methods, neuroimaging, and behavioral characterization of temperament phenotypes and neurocognitive processing to facilitate efforts aimed at improving long-lasting developmental benefits for children, particularly those living in areas with high levels of exposure.

Ten-eleven translocation methylcytosine dioxygenase 3-loaded microspheres penetrate neurons in vitro causing active demethylation and neurite outgrowth.

Epigenetic processes, such as DNA methylation and other chromatin modifications, are believed to be largely responsible for establishing a reduced capacity for growth in the mature nervous system. Ten-eleven translocation methylcytosine dioxygenase 3 (Tet3)-, a member of the Tet gene family, plays a crucial role in promoting injury-induced DNA demethylation and expression of regeneration-associated genes in the peripheral nervous system. Here, we encapsulate Tet3 protein within a clinically tolerated poly(lactide-co-glycolide) microsphere system. Next, we show that Tet3-loaded microspheres are internalized into mHippoE-18 embryonic hippocampal cells. We compare the outgrowth potential of Tet3 microspheres with that of commonly used nerve growth factor (NGF)-loaded microspheres in an in vitro injury model. Tet3-containing microspheres increased levels of nuclear 5-hydroxymethylcytosine indicating active demethylation and outperformed NGF-containing microspheres in measures of neurite outgrowth. Our results suggest that encapsulated demethylases may represent a novel avenue to treat nerve injuries.

Peripheral and central compensatory mechanisms for impaired vagus nerve function during peripheral immune activation.

BACKGROUND: Determining the etiology and possible treatment strategies for numerous diseases requires a comprehensive understanding of compensatory mechanisms in physiological systems. The vagus nerve acts as a key interface between the brain and the peripheral internal organs. We set out to identify mechanisms compensating for a lack of neuronal communication between the immune and the central nervous system (CNS) during infection. METHODS: We assessed biochemical and central neurotransmitter changes resulting from subdiaphragmatic vagotomy and whether they are modulated by intraperitoneal infection. We performed a series of subdiaphragmatic vagotomy or sham operations on male Wistar rats. Next, after full, 30-day recovery period, they were randomly assigned to receive an injection of Escherichia coli lipopolysaccharide or saline. Two hours later, animal were euthanized and we measured the plasma concentration of prostaglandin E2 (with HPLC-MS), interleukin-6 (ELISA), and corticosterone (RIA). We also had measured the concentration of monoaminergic neurotransmitters and their metabolites in the amygdala, brainstem, hippocampus, hypothalamus, motor cortex, periaqueductal gray, and prefrontal medial cortex using RP-HPLC-ED. A subset of the animals was evaluated in the elevated plus maze test immediately before euthanization. RESULTS: The lack of immunosensory signaling of the vagus nerve stimulated increased activity of discrete inflammatory marker signals, which we confirmed by quantifying biochemical changes in blood plasma. Behavioral results, although preliminary, support the observed biochemical alterations. Many of the neurotransmitter changes observed after vagotomy indicated that the vagus nerve influences the activity of many brain areas involved in control of immune response and sickness behavior. Our studies show that these changes are largely eliminated during experimental infection. CONCLUSIONS: Our results suggest that in vagotomized animals with blocked CNS, communication may transmit via a pathway independent of the vagus nerve to permit restoration of CNS activity for peripheral inflammation control.

Limited efficacy of somatic cell lysis buffer to purify laboratory mouse sperm.

AIM: Epigenetic analyses of sperm require pure samples devoid of diploid cell contamination. We sought to determine the efficacy of somatic cell lysis buffer (SCLB) treatment to purify mouse epididymis sperm samples. MATERIALS & METHODS: Sperm cell concentration, sperm purity, small RNA contents and sperm and somatic marker gene expression was compared in SCLB-treated sperm samples and two different control conditions. RESULTS: The SCLB condition as well as the control condition mimicking the additional pelleting and re-suspension steps resulted in substantial cell loss without evidence of enhanced purification of sperm cells as compared with epididymis-derived sperm samples that were not manipulated further. CONCLUSION: Molecular analyses focused on sperm cells require high levels of purity in order to avoid high-confounding RNA and cytosolic contributions of nonsperm cells. Our findings advocate gradient or cell sorting-based purification approaches where pure samples are required for sensitive molecular assays.

Epigenetic alterations in longevity regulators, reduced life span, and exacerbated aging-related pathology in old father offspring mice.

Advanced age is not only a major risk factor for a range of disorders within an aging individual but may also enhance susceptibility for disease in the next generation. In humans, advanced paternal age has been associated with increased risk for a number of diseases. Experiments in rodent models have provided initial evidence that paternal age can influence behavioral traits in offspring animals, but the overall scope and extent of paternal age effects on health and disease across the life span remain underexplored. Here, we report that old father offspring mice showed a reduced life span and an exacerbated development of aging traits compared with young father offspring mice. Genome-wide epigenetic analyses of sperm from aging males and old father offspring tissue identified differentially methylated promoters, enriched for genes involved in the regulation of evolutionarily conserved longevity pathways. Gene expression analyses, biochemical experiments, and functional studies revealed evidence for an overactive mTORC1 signaling pathway in old father offspring mice. Pharmacological mTOR inhibition during the course of normal aging ameliorated many of the aging traits that were exacerbated in old father offspring mice. These findings raise the possibility that inherited alterations in longevity pathways contribute to intergenerational effects of aging in old father offspring mice.

Every-other-day feeding extends lifespan but fails to delay many symptoms of aging in mice.

Dietary restriction regimes extend lifespan in various animal models. Here we show that longevity in male C57BL/6J mice subjected to every-other-day feeding is associated with a delayed onset of neoplastic disease that naturally limits lifespan in these animals. We compare more than 200 phenotypes in over 20 tissues in aged animals fed with a lifelong every-other-day feeding or ad libitum access to food diet to determine whether molecular, cellular, physiological and histopathological aging features develop more slowly in every-other-day feeding mice than in controls. We also analyze the effects of every-other-day feeding on young mice on shorter-term every-other-day feeding or ad libitum to account for possible aging-independent restriction effects. Our large-scale analysis reveals overall only limited evidence for a retardation of the aging rate in every-other-day feeding mice. The data indicate that every-other-day feeding-induced longevity is sufficiently explained by delays in life-limiting neoplastic disorders and is not associated with a more general slowing of the aging process in mice.Dietary restriction can extend the life of various model organisms. Here, Xie et al. show that intermittent periods of fasting achieved through every-other-day feeding protect mice against neoplastic disease but do not broadly delay organismal aging in animals.

Environmental Chemicals and Aging.

PURPOSE OF REVIEW: Innovations in agriculture and medicine as well as industrial and domestic technologies are essential for the growing and aging global population. These advances generally require the use of novel natural or synthetic chemical agents with the potential to affect human health. Here, we attempt to highlight environmental chemicals and select drugs with the potential to exacerbate aging by directly affecting molecular aging cascades focusing particular attention on the brain. Finally, we call attention to some potential fruitful areas of research, particularly with advanced molecular profiling that could aid in prevention or mitigation of environmental chemical toxic influences in the periphery and the brain. RECENT FINDINGS: We briefly summarize new research and highlight a recent study designed to prospectively identify agrochemicals with the potential to induce neurological diseases and place these discoveries into the already rich neurodegeneration and aging literature. Collectively, the research reviewed briefly here highlight chemicals with the true potential to accelerate aging, particularly in the brain, by eliciting elevated free radical stress and mitochondrial dysfunction. We make general recommendations about improved methodological approaches toward identification and regulation of chemicals that are gerontogenic to the brain.

Curiosity as an approach to ethoexperimental analysis: Behavioral neuroscience as seen by students and colleagues of Bob Blanchard.

This review is a synopsis of an International Behavioral Neuroscience Society (IBNS) symposium which focused on the elements of Behavioral Neuroscience for which Robert J. Blanchard was a Pioneer, Leading Expert, Advocate, Mentor, and Sage. Bob Blanchard's work demonstrably changed our broad understanding of animal behavior, and led the way to experimental design and analysis for studies of animal behavior that helped to clarify the deep complexity and subtleties of behavior. Bob's impact on the field of Behavioral Neuroscience includes the behavior, neurocircuitry, neurochemistry, and pharmacology related to social interactions, aggressive behavior, defensive behaviors, flight, freezing, threat, attack, risk assessment, anxiety disorders, animal models, models of social behavior, and autism. The methods and designs developed by Bob Blanchard over a lifetime have been adopted by scientists around the world, and form a standard of excellence in the field. The article addresses these topics in a way that presents developments in the field, describes the newest research data, and pays tribute to a great scientist and founder of this field of work, Bob Blanchard.

Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration.

Environmental factors, including pesticides, have been linked to autism and neurodegeneration risk using retrospective epidemiological studies. Here we sought to prospectively identify chemicals that share transcriptomic signatures with neurological disorders, by exposing mouse cortical neuron-enriched cultures to hundreds of chemicals commonly found in the environment and on food. We find that rotenone, a pesticide associated with Parkinson's disease risk, and certain fungicides, including pyraclostrobin, trifloxystrobin, famoxadone and fenamidone, produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism, advanced age and neurodegeneration (Alzheimer's disease and Huntington's disease). These chemicals stimulate free radical production and disrupt microtubules in neurons, effects that can be reduced by pretreating with a microtubule stabilizer, an antioxidant, or with sulforaphane. Our study provides an approach to prospectively identify environmental chemicals that transcriptionally mimic autism and other brain disorders.

Applying the ethoexperimental approach to neurodevelopmental syndrome research reveals exaggerated defensive behavior in Mecp2 mutant mice.

Rett syndrome is a Pervasive Developmental Disorder (PDD) associated with de novo mutations of the methyl CpG-binding protein 2 (MECP2) gene. Mecp2 functions as a transcription factor that regulates the expression of hundreds of genes. Identification of the role of Mecp2 in specific neurodevelopmental symptoms remains an important research aim. We previously demonstrated that male mice possessing a truncation mutation in Mecp2 are hyper-social. We predicted that reduced fear or anxiety might underlie this enhanced affiliation. In order to probe risk assessment and anxiety-like behavior, we compared Mecp2 truncation mutants to their wild-type littermates in the elevated plus maze and elevated zero maze. Additionally, subjects were administered the mouse defense test battery to evaluate unconditioned fear- and panic-like behavior to a graded set of threat scenarios and a predator stimulus. Mutant mice showed no significant changes in anxiety-like behavior. Yet, they displayed hyper-reactive escape and defensive behaviors to an animate predatory threat stimulus. Notably, mutant mice engaged in exaggerated active defense responding to threat stimuli at nearly all phases of the fear battery. These results reveal abnormalities in emotion regulation in Mecp2 mutants particularly in response to ecologically relevant threats. This hyper-responsivity suggests that transcriptional targets of Mecp2 are critical to emotion regulation. Moreover, we suggest that detailed analysis of defensive behavior and aggression with ethologically relevant tasks provides an avenue to interrogate gene-behavior mechanisms of neurodevelopmental and other psychiatric conditions.

Crowding increases salivary cortisol but not self-directed behavior in captive baboons.

Reduced space can lead to crowding in social animals. Crowding increases the risk of agonistic interactions that, in turn, may require additional physiological defensive coping mechanisms affecting health. To determine the stress induced from increased social density in a group of nineteen baboons living in an indoor/outdoor enclosure, saliva cortisol levels and rates of anxiety-related behavior were analyzed across two unique crowding episodes. Initially, mean salivary cortisol levels when animals were restricted to their indoor quarters were compared to those when they also had access to their larger outdoor enclosure. Then, mean cortisol levels were compared before, during, and after two distinct crowding periods of long and short duration. Crowding resulted in significantly elevated cortisol during crowding periods compared to non-crowded periods. Cortisol levels returned to baseline following two crowding episodes contrasting in their length and ambient climate conditions. These cortisol elevations indicate greater metabolic costs of maintaining homeostasis under social stress resulting from reduced space. Self-directed behavior, conversely, was not reliably elevated during crowding. Results suggest that the potential for negative social interactions, and/or the uncertainty associated with social threat can cause physiological stress responses detected by salivary cortisol. Self-directed behavioral measures of stress may constitute inadequate indicators of social stress in colony-housed monkeys or represent subjective emotional arousal unrelated to hypothalamic-pituitary adrenal axis activation.

Topoisomerases facilitate transcription of long genes linked to autism.

Topoisomerases are expressed throughout the developing and adult brain and are mutated in some individuals with autism spectrum disorder (ASD). However, how topoisomerases are mechanistically connected to ASD is unknown. Here we find that topotecan, a topoisomerase 1 (TOP1) inhibitor, dose-dependently reduces the expression of extremely long genes in mouse and human neurons, including nearly all genes that are longer than 200 kilobases. Expression of long genes is also reduced after knockdown of Top1 or Top2b in neurons, highlighting that both enzymes are required for full expression of long genes. By mapping RNA polymerase II density genome-wide in neurons, we found that this length-dependent effect on gene expression was due to impaired transcription elongation. Interestingly, many high-confidence ASD candidate genes are exceptionally long and were reduced in expression after TOP1 inhibition. Our findings suggest that chemicals and genetic mutations that impair topoisomerases could commonly contribute to ASD and other neurodevelopmental disorders.

Heparan sulfate deficiency in autistic postmortem brain tissue from the subventricular zone of the lateral ventricles.

Abnormal cellular growth and organization have been characterized in postmortem tissue from brains of autistic individuals, suggestive of pathology in a critical neurogenic niche, the subventricular zone (SVZ) of the brain lateral ventricles (LV). We examined cellular organization, cell proliferation, and constituents of the extracellular matrix such as N-sulfated heparan sulfate (HS) and laminin (LAM) in postmortem brain tissue from the LV-SVZ of young to elderly individuals with autism (n=4) and age-matched typically developing (TD) individuals (n=4) using immunofluorescence techniques. Strong and systematic reductions in HS immunofluorescence were observed in the LV-SVZ of the TD individuals with increasing age. For young through mature, but not elderly, autistic pair members, HS was reduced compared to their matched TDs. Cellular proliferation (Ki67+) was higher in the autistic individual of the youngest age-matched pair. These preliminary data suggesting that HS may be reduced in young to mature autistic individuals are in agreement with previous findings from the BTBR T+tf/J mouse, an animal model of autism; from mice with genetic modifications reducing HS; and with genetic variants in HS-related genes in autism. They suggest that aberrant extracellular matrix glycosaminoglycan function localized to the subventricular zone of the lateral ventricles may be a biomarker for autism, and potentially involved in the etiology of the disorder.

The BTBR T+ tf/J mouse model for autism spectrum disorders-in search of biomarkers.

Autism spectrum disorders (ASD) form a common group of neurodevelopmental disorders appearing to be under polygenic control, but also strongly influenced by multiple environmental factors. The brain mechanisms responsible for ASD are not understood and animal models paralleling related emotional and cognitive impairments may prove helpful in unraveling them. BTBR T+ tf/J (BTBR) mice display behaviors consistent with the three diagnostic categories for ASD. They show impaired social interaction and communication as well as increased repetitive behaviors. This review covers much of the data available to date on BTBR behavior, neuroanatomy and physiology in search for candidate biomarkers, which could both serve as diagnostic tools and help to design effective treatments for the behavioral symptoms of ASD.

Absence of social conditioned place preference in BTBR T+tf/J mice: relevance for social motivation testing in rodent models of autism.

A major goal of translation research in autism is to characterize the physiological and psychological processes underlying behavioral abnormalities. Since autism reflects impairments in social motivation, we modified the mouse three-chamber social approach apparatus for use as a social conditioned place preference arena. We paired one of two unique contexts with social interactions in juvenile mice for five or ten conditioning sessions in BTBR T+tf/J mice and a control strain with normal approach behaviors (C57BL/6J) since the BTBR T+tf/J inbred mouse strain displays a variety of behavioral alterations analogous to symptoms of autism spectrum disorders. While C57BL/6J mice formed a conditioned place preference to the context associated with social interactions, particularly those receiving ten days of conditioning, BTBR T+tf/J mice did not. Neither absence of social proximity nor avoidance due to high rates of autogrooming appeared to underlie the impaired positive incentive value of the unconditioned social stimulus in the BTBR T+tf/J strain. These data contribute to a growing body of evidence suggesting that the BTBR T+tf/J strain shows impairments in all diagnostic domains of autism including social motivation. Additionally, social conditioning testing might provide an important social motivation measure in other rodent models of neuropsychiatric disorders characterized by social abnormalities.