Incident dementia and long-term exposure to constituents of fine particle air pollution: A national cohort study in the United States.

Growing evidence suggests that fine particulate matter (PM2.5) likely increases the risks of dementia, yet little is known about the relative contributions of different constituents. Here, we conducted a nationwide population-based cohort study (2000 to 2017) by integrating the Medicare Chronic Conditions Warehouse database and two independently sourced datasets of high-resolution PM2.5 major chemical composition, including black carbon (BC), organic matter (OM), nitrate (NO3-), sulfate (SO42-), ammonium (NH4+), and soil dust (DUST). To investigate the impact of long-term exposure to PM2.5 constituents on incident all-cause dementia and Alzheimer's disease (AD), hazard ratios for dementia and AD were estimated using Cox proportional hazards models, and penalized splines were used to evaluate potential nonlinear concentration-response (C-R) relationships. Results using two exposure datasets consistently indicated higher rates of incident dementia and AD for an increased exposure to PM2.5 and its major constituents. An interquartile range increase in PM2.5 mass was associated with a 6 to 7% increase in dementia incidence and a 9% increase in AD incidence. For different PM2.5 constituents, associations remained significant for BC, OM, SO42-, and NH4+ for both end points (even after adjustments of other constituents), among which BC and SO42- showed the strongest associations. All constituents had largely linear C-R relationships in the low exposure range, but most tailed off at higher exposure concentrations. Our findings suggest that long-term exposure to PM2.5 is significantly associated with higher rates of incident dementia and AD and that SO42-, BC, and OM related to traffic and fossil fuel combustion might drive the observed associations.

Joint effects of prenatal exposure to indoor air pollution and psychosocial factors on early life inflammation.

It is hypothesized that air pollution and stress impact the central nervous system through neuroinflammatory pathways Despite this, the association between prenatal exposure to indoor air pollution and psychosocial factors on inflammatory markers in infancy has been underexplored in epidemiology studies. This study investigates the individual and joint effects of prenatal exposure to indoor air pollution and psychosocial factors on early life inflammation (interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)). We analyzed data from the South African Drakenstein Child Health Study (N = 225). Indoor air pollution and psychosocial factor measurements were taken in the 2nd trimester of pregnancy. Circulating inflammatory markers (IL-1ß, Il-6, and TNF-α) were measured in serum in the infants at 6 weeks postnatal. Linear regression models were used to investigate associations between individual exposures and inflammatory markers. To investigate joint effects of environmental and psychosocial factors, Self-Organizing Maps (SOM) were used to create exposure profile clusters. These clusters were added to linear regression models to investigate the associations between exposure profiles and inflammatory markers. All models were adjusted for maternal age, maternal HIV status, and ancestry to control for confounding. Most indoor air pollutants were positively associated with inflammatory markers, particularly benzene and TNF-α in single pollutant models. No consistent patterns were found for psychosocial factors in single-exposure linear regression models. In joint effects analyses, the SOM profile with high indoor air pollution, low SES, and high maternal depressive symptoms were associated with higher inflammation. Indoor air pollutants were consistently associated with increased inflammation in both individual and joint effects models, particularly in combination with low SES and maternal depressive symptoms. The trend for individual psychosocial factors was not as clear, with mainly null associations. As we have observed pro- and anti-inflammatory effects, future research should investigate joint effects of these exposures on inflammation and their health effects.

Associations between prenatal organophosphate pesticide exposure and placental gene networks.

BACKGROUND: Exposure to organophosphate (OP) pesticides during pregnancy has been linked to deficiencies of neurobehavioral development in childhood; however, the molecular mechanisms underlying this association remain elusive. The placenta plays a crucial role in protecting the fetus from environmental insults and safeguarding proper fetal development including neurodevelopment. The aim of our study is to evaluate changes in the placental transcriptome associated with prenatal OP exposure. METHODS: Pregnant farm workers from two agricultural districts in northern Thailand were recruited for the Study of Asian Women and Offspring's Development and Environmental Exposures (SAWASDEE) from 2017 to 2019. For 254 participants, we measured maternal urinary concentrations of six nonspecific dialkyl phosphates (DAP) metabolites in early, middle, and late pregnancy. In parallel, we profiled the term placental transcriptome from the same participants using RNA-Sequencing and performed Weighted Gene co-expression Network Analysis (WGCNA). Generalized linear regression modeling was used to examine associations of urinary OP metabolites and placental co-expression module eigenvalues. RESULTS: We identified 21 gene co-expression modules in the placenta. From the six DAP metabolites assayed, diethylphosphate (DEP) and diethylthiophosphate (DETP) were detected in more than 70% of the urine samples. Significant associations between DEP at multiple time points and two specific placental gene modules were observed. The 'black' module, enriched in genes involved in epithelial-to-mesenchymal transition (EMT) and hypoxia, was negatively associated with DEP in early (p = 0.034), and late pregnancies (p = 0.016). The 'lightgreen' module, enriched in genes involved in myogenesis and EMT, was negatively associated with DEP in late pregnancy (p = 0.010). We observed 2 hub genes (CELSR1 and PYCR1) of the 'black' module to be negatively associated with DEP in early and late pregnancies. CONCLUSIONS: Our results suggest that prenatal OP exposure may disrupt placental gene networks in a time-dependent manner. Such transcriptomic effects may lead to down-stream changes in placental function that ultimately affect the developing fetus.

The association between organophosphate insecticides and blood pressure dysregulation: NHANES 2013-2014.

BACKGROUND: Organophosphate (OP) insecticides represent one of the largest classes of sprayed insecticides in the U.S., and their use has been associated with various adverse health outcomes, including disorders of blood pressure regulation such as hypertension (HTN). METHODS: In a study of 935 adults from the NHANES 2013-2014 cycle, we examined the relationship between systolic and diastolic blood pressure changes and urinary concentrations of three OP insecticides metabolites, including 3,5,6-trichloro-2-pyridinol (TCPy), oxypyrimidine, and para-nitrophenol. These metabolites correspond to the parent compounds chlorpyrifos, diazinon, and methyl parathion, respectively. Weighted, multivariable linear regression analysis while adjusting for potential confounders were used to model the relationship between OP metabolites and blood pressure. Weighted, multivariable logistic regression analysis was used to model the odds of HTN for quartile of metabolites. RESULTS: We observed significant, inverse association between TCPy on systolic blood pressure (ß-estimate = -0.16, p < 0.001) and diastolic blood pressure (ß-estimate = -0.15, p < 0.001). Analysis with para-nitrophenol revealed a significant, positive association with systolic blood pressure (ß-estimate = 0.03, p = 0.02), and an inverse association with diastolic blood pressure (ß-estimate = -0.09, p < 0.001). For oxypyrimidine, we observed significant, positive associations between systolic blood pressure (ß-estimate = 0.58, p = 0.03) and diastolic blood pressure (ß-estimate = 0.31, p < 0.001). Furthermore, we observed significant interactions between TCPy and ethnicity on systolic blood pressure (ß-estimate = 1.46, p = 0.0036). Significant interaction terms were observed between oxypyrimidine and ethnicity (ß-estimate = -1.73, p < 0.001), as well as oxypyrimidine and BMI (ß-estimate = 1.51 p < 0.001) on systolic blood pressure, and between oxypyrimidine and age (ß-estimate = 1.96, p = 0.02), race (ß-estimate = -3.81 p = 0.004), and BMI on diastolic blood pressure (ß-estimate = 0.72, p = 0.02). A significant interaction was observed between para-nitrophenol and BMI for systolic blood pressure (ß-estimate = 0.43, p = 0.01), and between para-nitrophenol and ethnicity on diastolic blood pressure (ß-estimate = 2.19, p = 0.006). Lastly, we observed a significant association between the odds of HTN and TCPy quartiles (OR = 0.65, 95% CI [0.43,0.99]). CONCLUSION: Our findings support previous studies suggesting a role for organophosphate insecticides in the etiology of blood pressure dysregulation and HTN. Future studies are warranted to corroborate these findings, evaluate dose-response relationships between organophosphate insecticides and blood pressure, determine clinical significance, and elucidate biological mechanisms underlying this association.

Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease.

Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction.

Developmental pesticide exposure reproduces features of attention deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is estimated to affect 8-12% of school-age children worldwide. ADHD is a complex disorder with significant genetic contributions. However, no single gene has been linked to a significant percentage of cases, suggesting that environmental factors may contribute to ADHD. Here, we used behavioral, molecular, and neurochemical techniques to characterize the effects of developmental exposure to the pyrethroid pesticide deltamethrin. We also used epidemiologic methods to determine whether there is an association between pyrethroid exposure and diagnosis of ADHD. Mice exposed to the pyrethroid pesticide deltamethrin during development exhibit several features reminiscent of ADHD, including elevated dopamine transporter (DAT) levels, hyperactivity, working memory and attention deficits, and impulsive-like behavior. Increased DAT and D1 dopamine receptor levels appear to be responsible for the behavioral deficits. Epidemiologic data reveal that children aged 6-15 with detectable levels of pyrethroid metabolites in their urine were more than twice as likely to be diagnosed with ADHD. Our epidemiologic finding, combined with the recapitulation of ADHD behavior in pesticide-treated mice, provides a mechanistic basis to suggest that developmental pyrethroid exposure is a risk factor for ADHD.

Developmental exposure to the organochlorine insecticide endosulfan alters expression of proteins associated with neurotransmission in the frontal cortex.

Exposure to environmental contaminants, such as organochlorine insecticides during critical periods of neurodevelopment has been shown to be a major contributor to several neuropsychological deficits seen in children, adolescence, and adults. Although the neurobehavioral outcomes resulting from exposure to these compounds are known the neurotransmitter circuitry and molecular targets that mediate these endpoints have not been identified. Given the importance of the frontal cortex in facilitating numerous neuropsychological processes, our current study sought to investigate the effects of developmental exposure to the organochlorine insecticide, endosulfan, on the expression of specific proteins associated with neurotransmission in the frontal cortex. Utilizing in vitro models we were able to show endosulfan reduces cell viability in IMR-32 neuroblastoma cells in addition to reducing synaptic puncta and neurite outgrowth in primary cultured neurons isolated from the frontal cortex of mice. Elaborating these findings to an in vivo model we found that developmental exposure of female mice to endosulfan during gestation and lactation elicited significant alterations to the GABAergic (GAT1, vGAT, GABAA receptor), glutamatergic (vGlut and GluN2B receptor), and dopaminergic (DAT, TH, VMAT2, and D2 receptor) neurotransmitter systems in the frontal cortex of male offspring. These findings identify damage to critical neurotransmitter circuits and proteins in the frontal cortex, which may underlie the neurobehavioral deficits observed following developmental exposure to endosulfan and other organochlorine insecticides.

Effects of prenatal pesticide exposure on the fetal brain and placenta transcriptomes in a rodent model.

Organophosphate and pyrethroid pesticides are among the most extensively used insecticides worldwide. Prenatal exposures to both classes of pesticides have been linked to a wide range of neurobehavioral deficits in the offspring. The placenta is a neuroendocrine organ and the crucial regulator of the intrauterine environment; early-life toxicant exposures could impact neurobehavior by disrupting placental processes. Female C57BL/6 J mice were exposed via oral gavage to an organophosphate, chlorpyrifos (CPF) at 5 mg/kg, a pyrethroid, deltamethrin (DM), at 3 mg/kg, or vehicle only control (CTL). Exposure began two weeks before breeding and continued every three days until euthanasia at gestational day 17. The transcriptomes of fetal brain (CTL n = 18, CPF n = 6, DM n = 8) and placenta (CTL n = 19, CPF n = 16, DM n = 12) were obtained through RNA sequencing, and resulting data was evaluated using weighted gene co-expression networks, differential expression, and pathway analyses. Fourteen brain gene co-expression modules were identified; CPF exposure disrupted the module related to ribosome and oxidative phosphorylation, whereas DM disrupted the modules related to extracellular matrix and calcium signaling. In the placenta, network analyses revealed 12 gene co-expression modules. While CPF exposure disrupted modules related to endocytosis, Notch and Mapk signaling, DM exposure dysregulated modules linked to spliceosome, lysosome and Mapk signaling pathways. Overall, in both tissues, CPF exposure impacted oxidative phosphorylation, while DM was linked to genes involved in spliceosome and cell cycle. The transcription factor Max involved in cell proliferation was overexpressed by both pesticides in both tissues. In summary, gestational exposure to two different classes of pesticide can induce similar pathway-level transcriptome changes in the placenta and the brain; further studies should investigate if these changes are linked to neurobehavioral impairments.

Long-term effects of PM2.5 components on incident dementia in the northeastern United States.

Growing evidence has linked long-term fine particulate matter (PM2.5) exposure to neurological disorders. Less is known about the individual effects of PM2.5 components. A population-based cohort study investigated the association between long-term (1-year average) exposure to PM2.5 components and dementia incidence among the elderly population (age, ≥65 years) in the United States. We used data from the Medicare Chronic Conditions Warehouse and a high-resolution PM2.5 components dataset of the northeastern United States (2000-2017). We identified dementia diagnoses from patients' hospital and medical insurance records and carried out Cox proportional hazards regression to investigate their association with PM2.5 components. Among ∼2 million participants, 15.1% developed dementia. From the single-pollutant models, hazard ratios per interquartile range increase were 1.10 (95% confidence interval [CI]: 1.09-1.11) for black carbon, 1.08 (95% CI: 1.07, 1.10) for inorganic nitrate, 1.03 (95% CI: 1.02, 1.04) for organic matter, 1.13 (95% CI: 1.11, 1.15) for sulfate, 1.07 (95% CI: 1.06, 1.07) for soil particles, and 1.04 (95% CI: 1.03, 1.05) for sea salt. Increase in exposure to black carbon and sulfate per interquartile range had the strongest associations with dementia incidence. Penalized spline models indicated that dementia incidence increased linearly with elevated black carbon concentrations, whereas the incidence of dementia was only elevated significantly following sulfate concentrations above ∼2 µg/m3. Our study suggests that long-term exposure to PM2.5 components is significantly associated with increased dementia incidence and that different components have different neurotoxicity. Reduction of PM2.5 emissions, especially for main sources of black carbon and sulfate, may reduce the burden of dementia in the aging United States population.

Experimental colitis promotes sustained, sex-dependent, T-cell-associated neuroinflammation and parkinsonian neuropathology.

BACKGROUND: The etiology of sporadic Parkinson's disease (PD) remains uncertain, but genetic, epidemiological, and physiological overlap between PD and inflammatory bowel disease suggests that gut inflammation could promote dysfunction of dopamine-producing neurons in the brain. Mechanisms behind this pathological gut-brain effect and their interactions with sex and with environmental factors are not well understood but may represent targets for therapeutic intervention. METHODS: We sought to identify active inflammatory mechanisms which could potentially contribute to neuroinflammation and neurological disease in colon biopsies and peripheral blood immune cells from PD patients. Then, in mouse models, we assessed whether dextran sodium sulfate-mediated colitis could exert lingering effects on dopaminergic pathways in the brain and whether colitis increased vulnerability to a subsequent exposure to the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We assessed the involvement of inflammatory mechanisms identified in the PD patients in colitis-related neurological dysfunction in male and female mice, utilizing mice lacking the Regulator of G-Protein Signaling 10 (RGS10)-an inhibitor of nuclear factor kappa B (NFκB)-to model enhanced NFκB activity, and mice in which CD8+ T-cells were depleted. RESULTS: High levels of inflammatory markers including CD8B and NFκB p65 were found in colon biopsies from PD patients, and reduced levels of RGS10 were found in immune cells in the blood. Male mice that experienced colitis exhibited sustained reductions in tyrosine hydroxylase but not in dopamine as well as sustained CD8+ T-cell infiltration and elevated Ifng expression in the brain. CD8+ T-cell depletion prevented colitis-associated reductions in dopaminergic markers in males. In both sexes, colitis potentiated the effects of MPTP. RGS10 deficiency increased baseline intestinal inflammation, colitis severity, and neuropathology. CONCLUSIONS: This study identifies peripheral inflammatory mechanisms in PD patients and explores their potential to impact central dopaminergic pathways in mice. Our findings implicate a sex-specific interaction between gastrointestinal inflammation and neurologic vulnerability that could contribute to PD pathogenesis, and they establish the importance of CD8+ T-cells in this process in male mice.

Nonmotor symptoms of Parkinson's disease revealed in an animal model with reduced monoamine storage capacity.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, culminating in severe motor symptoms, including resting tremor, rigidity, bradykinesia, and postural instability. In addition to motor deficits, there are a variety of nonmotor symptoms associated with PD. These symptoms generally precede the onset of motor symptoms, sometimes by years, and include anosmia, problems with gastrointestinal motility, sleep disturbances, sympathetic denervation, anxiety, and depression. Previously, we have shown that mice with a 95% genetic reduction in vesicular monoamine transporter expression (VMAT2-deficient, VMAT2 LO) display progressive loss of striatal dopamine, L-DOPA-responsive motor deficits, alpha-synuclein accumulation, and nigral dopaminergic cell loss. We hypothesized that since these animals exhibit deficits in other monoamine systems (norepinephrine and serotonin), which are known to regulate some of these behaviors, the VMAT2-deficient mice may display some of the nonmotor symptoms associated with PD. Here we report that the VMAT2-deficient mice demonstrate progressive deficits in olfactory discrimination, delayed gastric emptying, altered sleep latency, anxiety-like behavior, and age-dependent depressive behavior. These results suggest that the VMAT2-deficient mice may be a useful model of the nonmotor symptoms of PD. Furthermore, monoamine dysfunction may contribute to many of the nonmotor symptoms of PD, and interventions aimed at restoring monoamine function may be beneficial in treating the disease.

Altered vesicular dopamine storage in Parkinson's disease: a premature demise.

Dopamine is a potentially toxic neurotransmitter that has long been speculated to contribute to the pathogenesis of Parkinson's disease (PD). Recent work has demonstrated the importance of proper storage of dopamine in vesicles to maintain dopamine homeostasis, thus protecting neurons from the detrimental effects of dopamine accumulation and breakdown in the cytosol. These studies suggest that factors which affect dopamine storage might increase the susceptibility of dopamine neurons to further environmental or genetic insults, exacerbating the neuronal degeneration that characterizes PD. This review seeks to revisit the pathogenicity of cytosolic dopamine and further address the critical role of neurotransmitter storage in dopamine-mediated neurotoxicity.

Macrophage antigen complex-1 mediates reactive microgliosis and progressive dopaminergic neurodegeneration in the MPTP model of Parkinson's disease.

Neuronal death is known to trigger reactive microgliosis. However, little is known regarding the manner by which microglia are activated by injured neurons and how microgliosis participates in neurodegeneration. In this study we delineate the critical role of macrophage Ag complex-1 (MAC1), a member of the beta(2) integrin family, in mediating reactive microgliosis and promoting dopaminergic (DAergic) neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. MAC1 deficiency greatly attenuated the DAergic neurodegeneration induced by MPTP or 1-methyl-4-phenyl-pyridium iodide (MPP(+)) exposure both in vivo and in vitro, respectively. Reconstituted experiments created by adding microglia from MAC1(-/-) or MAC1(+/+) mice back to MAC1(+/+) neuron-enriched cultures showed that microglia with functional MAC1 expression was mandatory for microglia-enhanced neurotoxicity. Both in vivo and in vitro morphological and Western blot studies demonstrated that MPTP/MPP(+) produced less microglia activation in MAC1(-/-) mice than MAC1(+/+) mice. Further mechanistic studies revealed that a MPP(+)-mediated increase in superoxide production was reduced in MAC1(-/-) neuron-glia cultures compared with MAC1(+/+) cultures. The stunted production of superoxide in MAC1(-/-) microglia is likely linked to the lack of translocation of the cytosolic NADPH oxidase (PHOX) subunit (p47(phox)) to the membrane. In addition, the production of PGE(2) markedly decreased in neuron plus MAC1(-/-) microglia cocultures vs neuron plus MAC1(+/+) microglia cocultures. Taken together, these results demonstrate that MAC1 plays a critical role in MPTP/MPP(+)-induced reactive microgliosis and further support the hypothesis that reactive microgliosis is an essential step in the self-perpetuating cycle leading to progressive DAergic neurodegeneration observed in Parkinson's disease.

Biomarker discovery in neurodegenerative diseases: a proteomic approach.

Biomarkers for neurodegenerative disorders are essential to facilitate disease diagnosis, ideally at early stages, monitor disease progression, and assess response to existing and future treatments. Application of proteomics to the human brain, cerebrospinal fluid and plasma has greatly hastened the unbiased and high-throughput searches for novel biomarkers. There are many steps critical to biomarker discovery, whether for neurodegenerative or other diseases, including sample preparation, protein/peptide separation and identification, as well as independent confirmation and validation. In this review we have summarized current proteomics technologies involved in discovery of biomarkers for neurodegenerative diseases, practical considerations and limitations of several major aspects, as well as the current status of candidate biomarkers revealed by proteomics for Alzheimer and Parkinson diseases.

A Neurodevelopmental Model of Combined Pyrethroid and Chronic Stress Exposure.

Attention-deficit hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders of childhood and previous studies indicate the dopamine system plays a major role in ADHD pathogenesis. Two environmental exposures independently associated with dopaminergic dysfunction and ADHD risk include exposure to deltamethrin, a pyrethroid insecticide, and chronic stress. We hypothesized that combined neurodevelopmental exposure to both deltamethrin and corticosterone (CORT), the major stress hormone in rodents, would result in additive changes within the dopamine system. To study this, we developed a novel dual exposure paradigm and exposed pregnant C57BL/6 dams to 3 mg/kg deltamethrin through gestation and weaning, and their offspring to 25 µg/mL CORT dissolved in the drinking water through adulthood. Midbrain RNA expression as well as striatal and cortical protein expression of key dopaminergic components were investigated, in addition to ADHD-like behavioral tasks and electrochemical dopamine dynamics via fast-scan cyclic voltammetry. Given the well-described sexual dimorphism of ADHD, males and females were assessed separately. Males exposed to deltamethrin had significantly decreased midbrain Pitx3 expression, decreased cortical tyrosine hydroxylase (TH) expression, increased activity in the Y maze, and increased dopamine uptake rate in the dorsal striatum. These effects did not occur in males exposed to CORT only, or in males exposed to both deltamethrin and CORT, suggesting that CORT may attenuate these effects. Additionally, deltamethrin- and CORT-exposed females did not display these dopaminergic features, which indicates these changes are sex-specific. Our results show dopaminergic changes from the RNA through the functional level. Moreover, these data illustrate the importance of testing multiple environmental exposures together to better understand how combined exposures that occur in certain vulnerable populations could affect similar neurodevelopmental systems, as well as the importance of studying sex differences of these alterations.

Embracing Systems Toxicology at Single-Cell Resolution.

As systems biology expands its multi-omic spectrum to increasing resolutions, distinguishing cells based on single-cell profiles becomes feasible. Unlike traditional bulk assays that average cellular responses and blur the distinct identities of responsive cells, single-cell technologies enable sensitive detection of small cellular changes and precise identification of those cells perturbed by toxicants. Among the suite of omic technologies that continue to expand and become affordable, single-cell RNA sequencing (scRNA-seq) is at the cutting edge and leading the way to transform systems toxicology. Single-cell systems toxicology can provide a wealth of information to elucidate cell-specific alterations and response trajectories, detect points-of-departure, map and develop dynamical models of toxicity pathways.

The second generation mixed lineage kinase-3 (MLK3) inhibitor CLFB-1134 protects against neurotoxin-induced nigral dopaminergic neuron loss.

Dopaminergic neurons express mixed lineage kinases which regulate the expression of cell death genes. In Parkinson's disease, cell death via apoptosis is prevalent, and previous work testing mixed lineage kinase inhibitors in animal models suggested the inhibitors had some neuroprotective potential. CLFB-1134 is a new, brain-penetrant inhibitor specific for MLK3, tested here in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of dopaminergic depletion and nigral neuron death in mice. After ensuring that treatment with CLFB-1134 did not alter conversion of MPTP to MPP+, we demonstrated CLFB-1134's inhibition of MLK3 and neuroprotective efficacy. Specifically we evaluated the integrity of the nigrostriatal dopamine system following MPTP by assessing protein expression, high performance liquid chromatography, and immunohistology with stereology. We found that CLFB-1134 achieves protection of striatal dopaminergic terminals and nigral cell bodies when dosed simultaneously or following MPTP treatment. By preventing phosphorylation of JNK and other downstream targets of MLK3, CLFB-1134 protects against the neurotoxin MPTP. Inhibition of MLK3 may be a valid target for future work investigating treatment of Parkinson's disease.

Reduced vesicular storage of dopamine causes progressive nigrostriatal neurodegeneration.

The vesicular monoamine transporter 2 (VMAT2; SLC18A2) is responsible for packaging dopamine into vesicles for subsequent release and has been suggested to serve a neuroprotective role in the dopamine system. Here, we show that mice that express approximately 5% of normal VMAT2 (VMAT2 LO) display age-associated nigrostriatal dopamine dysfunction that ultimately results in neurodegeneration. Elevated cysteinyl adducts to L-DOPA and DOPAC are seen early and are followed by increased striatal protein carbonyl and 3-nitrotyrosine formation. These changes were associated with decreased striatal dopamine and decreased expression of the dopamine transporter and tyrosine hydroxylase. Furthermore, we observed an increase in alpha-synuclein immunoreactivity and accumulation and neurodegeneration in the substantia nigra pars compacta in aged VMAT2 LO mice. Thus, VMAT2 LO animals display nigrostriatal degeneration that begins in the terminal fields and progresses to eventual loss of the cell bodies, alpha-synuclein accumulation, and an L-DOPA responsive behavioral deficit, replicating many of the key aspects of Parkinson's disease. These data suggest that mishandling of dopamine via reduced VMAT2 expression is, in and of itself, sufficient to cause dopamine-mediated toxicity and neurodegeneration in the nigrostriatal dopamine system. In addition, the altered dopamine homeostasis resulting from reduced VMAT2 function may be conducive to pathogenic mechanisms induced by genetic or environmental factors thought to be involved in Parkinson's disease.

Developmental heptachlor exposure increases susceptibility of dopamine neurons to N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)in a gender-specific manner.

Parkinson's disease (PD) is primarily thought of as a disease of aging. However, recent evidence points to the potential for exposure to xenobiotics during development to increase risk of PD. Here, we report that developmental exposure to the organochlorine pesticide heptachlor alters the dopamine system and increases neurotoxicity in an animal model of PD. Exposure of pregnant mice to heptachlor led to increased levels of the dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2) levels at both the protein and mRNA level in their offspring. Increased DAT and VMAT2 levels were accompanied by alterations of mRNA levels of nuclear transcription factors that control dopamine neuron development and regulate DAT and VMAT2 levels in adulthood. At 12 weeks of age, control and heptachlor-exposed offspring were administered a moderate dose (2 x 10mg/kg) of the parkinsonism-inducing agent MPTP. Greater neurotoxicity as evidenced by a greater loss of striatal dopamine and potentiation of increased levels of glial fibrillary acidic protein and alpha-synuclein was observed in heptachlor-exposed offspring. The neurotoxicity observed was greater in the male offspring than the female offspring, suggesting that males are more susceptible to the long-term effects of developmental heptachlor exposure. These data suggest that developmental heptachlor exposure causes long-term alterations of the dopamine system thereby rendering it more susceptible to dopaminergic damage in adulthood.

Per- and polyfluoroalkyl substances impact human spermatogenesis in a stem-cell-derived model.

Per- and polyfluoroalkyl substances (PFASs) represent a highly ubiquitous group of synthetic chemicals used in products ranging from water and oil repellents and lubricants to firefighting foam. These substances can enter and accumulate in multiple tissue matrices in up to 100% of people assessed. Though animal models strongly identify these compounds as male reproductive toxicants, with exposed rodents experiencing declines in sperm count, alterations in hormones, and DNA damage in spermatids, among other adverse outcomes, human studies report conflicting conclusions as to the reproductive toxicity of these chemicals. Using an innovative, human stem-cell-based model of spermatogenesis, we assessed the effects of the PFASs perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and a mixture of PFOS, PFOA, and PFNA for their impacts on human spermatogenesis in vitro under conditions relevant to the general and occupationally exposed populations. Here, we show that PFOS, PFOA, PFNA, and a mixture of PFOS, PFOA, and PFNA do not decrease in vitro germ cell viability, consistent with reports from human studies. These compounds do not affect mitochondrial membrane potential or increase reactive oxygen species generation, and they do not decrease cell viability of spermatogonia, primary spermatocytes, secondary spermatocytes, or spermatids in vitro under the conditions examined. However, exposure to PFOS, PFOA, and PFNA reduces expression of markers for spermatogonia and primary spermatocytes. While not having direct effects on germ cell viability, these effects suggest the potential for long-term impacts on male fertility through the exhaustion of the spermatogonial stem cell pool and abnormalities in primary spermatocytes. ABBREVIATIONS: CDC: Centers for Disease Control; DMSO: dimethyl sulfoxide; GHR: growth hormone receptor; hESCs: human embryonic stem cells; PFASs: per- and polyfluoroalkyl substances; PFCs: perfluorinated compounds; PFNA: perfluorononanoic acid; PFOS: perfluorooctanesulfonic acid; PFOA: perfluorooctanoic acid; PLZF: promyelocytic leukemia zinc finger; ROS: reactive oxygen species; HILI: RNA-mediated gene silencing 2; SSC: spermatogonial stem cell.