Elevated parkinsonism pathological markers in dopaminergic neurons with developmental exposure to atrazine.

Atrazine (ATZ) is one of the most used herbicides in the US and a known endocrine disruptor. ATZ is frequently detected in drinking water, especially in Midwestern regions of the United States, exceeding the EPA regulation of maximum contamination level (MCL) of 3 ppb. Epidemiology studies have suggested an association between ATZ exposure and neurodegeneration. Less, however, is known about the neurotoxic mechanism of ATZ, particularly for exposures at a developmental stage. Here, we exposed floor plate progenitors (FPPs) derived from human induced pluripotent stem cells (hiPSCs) to low concentrations of ATZ at 0.3 and 3 ppb for two days followed by differentiation into dopaminergic (DA) neurons in ATZ-free medium. We then examined the morphology, activity, pathological protein aggregation, and transcriptomic changes of differentiated DA neurons. We observed significant decrease in the complexity of neurite network, increase of neuronal activity, and elevated tau- and α-synuclein (aSyn) pathologies after ATZ exposure. The ATZ-induced neuronal changes observed here align with pathological characteristics in Parkinson's disease (PD). Transcriptomic analysis further corroborates our findings; and collectively provides a strong evidence base that low-concentration ATZ exposure during development can elicit increased risk of neurodegeneration.

Developmental Pb exposure increases AD risk via altered intracellular Ca2+ homeostasis in hiPSC-derived cortical neurons.

Exposure to environmental chemicals such as lead (Pb) during vulnerable developmental periods can result in adverse health outcomes later in life. Human cohort studies have demonstrated associations between developmental Pb exposure and Alzheimer's disease (AD) onset in later life which were further corroborated by findings from animal studies. The molecular pathway linking developmental Pb exposure and increased AD risk, however, remains elusive. In this work, we used human iPSC-derived cortical neurons as a model system to study the effects of Pb exposure on AD-like pathogenesis in human cortical neurons. We exposed neural progenitor cells derived from human iPSC to 0, 15, and 50 ppb Pb for 48 h, removed Pb-containing medium, and further differentiated them into cortical neurons. Immunofluorescence, Western blotting, RNA-sequencing, ELISA, and FRET reporter cell lines were used to determine changes in AD-like pathogenesis in differentiated cortical neurons. Exposing neural progenitor cells to low-dose Pb, mimicking a developmental exposure, can result in altered neurite morphology. Differentiated neurons exhibit altered calcium homeostasis, synaptic plasticity, and epigenetic landscape along with elevated AD-like pathogenesis markers, including phosphorylated tau, tau aggregates, and Aß42/40. Collectively, our findings provide an evidence base for Ca dysregulation caused by developmental Pb exposure as a plausible molecular mechanism accounting for increased AD risk in populations with developmental Pb exposure.

Electroacupuncture Induces Bilateral S1 and ACC Epigenetic Regulation of Genes in a Mouse Model of Neuropathic Pain.

Clinical and animal studies have shown that acupuncture may benefit controlling neuropathic pain. However, the underlying molecular mechanisms are poorly understood. In a well-established mouse unilateral tibial nerve injury (TNI) model, we confirmed the efficacy of electroacupuncture (EA) in reducing mechanical allodynia and measured methylation and hydroxy-methylation levels in the primary somatosensory cortex (S1) and anterior cingulate cortex (ACC), two cortical regions critically involved in pain processing. TNI resulted in increased DNA methylation of both the contra- and ipsilateral S1, while EA only reduced contralateral S1 methylation. RNA sequencing of the S1 and ACC identified differentially expressed genes related to energy metabolism, inflammation, synapse function, and neural plasticity and repair. One week of daily EA decreased or increased the majority of up- or downregulated genes, respectively, in both cortical regions. Validations of two greatly regulated genes with immunofluorescent staining revealed an increased expression of gephyrin in the ipsilateral S1 after TNI was decreased by EA; while TNI-induced increases in Tomm20, a biomarker of mitochondria, in the contralateral ACC were further enhanced after EA. We concluded that neuropathic pain is associated with differential epigenetic regulations of gene expression in the ACC and S1 and that the analgesic effect of EA may involve regulating cortical gene expression.

Pre-differentiation GenX exposure induced neurotoxicity in human dopaminergic-like neurons.

GenX, also known as hexafluoropropylene oxide dimer acid (HFPO-DA) was introduced as a safer alternative to perfluorooctanoic acid (PFOA) in 2009. After nearly two decades of applications there are increasing safety concerns about GenX due to its association with various organ damages. Few studies, however, have systematically assessed the molecular neurotoxicity of low-dose GenX exposure. Here, we evaluated the effects of pre-differentiation exposure of GenX on dopaminergic (DA) -like neurons using SH-SY5Y cell line; and assessed changes in epigenome, mitochondrion, and neuronal characteristics. Low dose GenX exposure at 0.4 and 4 µg/L prior to differentiation induced persistent changes in nuclear morphology and chromatin arrangements, manifested specifically in the facultative repressive marker H3K27me3. We also observed impaired neuronal network, increased calcium activity along with alterations in Tyrosine hydroxylase (TH) and α-Synuclein (αSyn) after prior exposure to GenX. Collectively, our results identified neurotoxicity of low-dose GenX exposure in human DA-like neurons following a developmental exposure scheme. The observed changes in neuronal characteristics suggest GenX as a potential neurotoxin and risk factor for Parkinson's disease.

Acute Transcriptomic and Epigenetic Alterations at T12 After Rat T10 Spinal Cord Contusive Injury.

Spinal cord injury is a severely debilitating condition affecting a significant population in the USA. Spinal cord injury patients often have increased risk of developing persistent neuropathic pain and other neurodegenerative conditions beyond the primary lesion center later in their life. The molecular mechanism conferring to the "latent" damages at distal tissues, however, remains elusive. Here, we studied molecular changes conferring abnormal functionality at distal spinal cord (T12) beyond the lesion center (T10) by combining next-generation sequencing (RNA- and bisulfite sequencing), super-resolution microscopy, and immunofluorescence staining at 7 days post injury. We observed significant transcriptomic changes primarily enriched in neuroinflammation and synaptogenesis associated pathways. Transcription factors (TFs) that regulate neurogenesis and neuron plasticity, including Egr1, Klf4, and Myc, are significantly upregulated. Along with global changes in chromatin arrangements and DNA methylation, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), bisulfite sequencing further reveals the involvement of DNA methylation changes in regulating cytokine, growth factor, and ion channel expression. Collectively, our results pave the way towards understanding transcriptomic and epigenomic mechanism in conferring long-term disease risks at distal tissues away from the primary lesion center and shed light on potential molecular targets that govern the regulatory mechanism at distal spinal cord tissues.

Pre-differentiation exposure of PFOA induced persistent changes in DNA methylation and mitochondrial morphology in human dopaminergic-like neurons.

Perfluorooctanoic acid (PFOA) is abundant in environment due to its historical uses in consumer products and industrial applications. Exposure to low doses of PFOA has been associated with various disease risks, including neurological disorders. The underlying mechanism, however, remains poorly understood. In this study, we examined the effects of low dose PFOA exposure at 0.4 and 4 µg/L on the morphology, epigenome, mitochondrion, and neuronal markers of dopaminergic (DA)-like SH-SY5Y cells. We observed persistent decreases in H3K4me3, H3K27me3 and 5 mC markers in nucleus along with alterations in nuclear size and chromatin compaction percentage in DA-like neurons differentiated from SH-SY5Y cells exposed to 0.4 and 4 µg/L PFOA. Among the selected epigenetic features, DNA methylation pattern can be used to distinguish between PFOA-exposed and naïve populations, suggesting the involvement of epigenetic regulation. Moreover, DA-like neurons with pre-differentiation PFOA exposure exhibit altered network connectivity, mitochondrial volume, and TH expression, implying impairment in DA neuron functionality. Collectively, our results revealed the prolonged effects of developmental PFOA exposure on the fitness of DA-like neurons and identified epigenome and mitochondrion as potential targets for bearing long-lasting changes contributing to increased risks of neurological diseases later in life.

Atrazine exposure in zebrafish induces aberrant genome-wide methylation.

Atrazine (ATZ) is the second most common agricultural herbicide used in the United States and is an endocrine disrupting chemical (EDC). Developmental exposure to ATZ can lead to significant behavioral and morphological alterations in exposed animals and their progeny suggesting the involvement of an epigenetic mechanism. Specific epigenetic mechanisms responsible for these alterations, however, are yet to be elucidated. In this study, we exposed zebrafish embryos to 0, 0.3, 3, or 30 ppb (µg/L) of ATZ from 1 to 72 h post fertilization (hpf). Chemical exposure was ceased and zebrafish maintained until 9 months post fertilization (mpf), when whole-genome bisulfite sequencing (WGBS) was performed to assess the effects of embryonic ATZ exposure on DNA methylation in female fish brains. The number of differentially methylated genes (DMGs) increased with increasing treatment concentration. DMGs were enriched in neurological pathways with extensive methylation changes consistently observed in neuroendocrine pathways. Specifically, DMGs with methylation changes in promoter regions showed hypomethylation in estrogen receptor signaling and hypermethylation in androgen signaling. DMGs with methylation changes in genebody were primarily enriched for mitochondrion-related pathways associated with healthy aging. Integrated analysis with transcriptomic data at 9 mpf exhibited a similar trend identifying CABLES1 and NDUFA4 as shared targets at all concentrations. We then compared the predicted upstream regulators of transcriptomic changes with DMGs and identified CALML3 as a common upstream regulator at both 0.3 and 30 ppb that exhibit significant methylation changes. Collectively, our study identified long-lasting DNA methylation changes in genome after embryonic ATZ exposure and elucidated potential gene targets whose aberrant methylation features may drive alterations in gene transcription in long-term.

Single-Cell Image-Based Analysis Reveals Chromatin Changes during the Acquisition of Tamoxifen Drug Resistance.

Cancer drug resistance is the leading cause of cancer related deaths. The development of drug resistance can be partially contributed to tumor heterogeneity and epigenetic plasticity. However, the detailed molecular mechanism underlying epigenetic modulated drug resistance remains elusive. In this work, we systematically analyzed epigenetic changes in tamoxifen (Tam) responsive and resistant breast cancer cell line MCF7, and adopted a data-driven approach to identify key epigenetic features distinguishing between these two cell types. Significantly, we revealed that DNA methylation and H3K9me3 marks that constitute the heterochromatin are distinctively different between Tam-resistant and -responsive cells. We then performed time-lapse imaging of 5mC and H3K9me3 features using engineered probes. After Tam treatment, we observed a slow transition of MCF7 cells from a drug-responsive to -resistant population based on DNA methylation features. A similar trend was not observed using H3K9me3 probes. Collectively, our results suggest that DNA methylation changes partake in the establishment of Tam-resistant breast cancer cell lines. Instead of global changes in the DNA methylation level, the distribution of DNA methylation features inside the nucleus can be one of the drivers that facilitates the establishment of a drug resistant phenotype in MCF7.

Lead exposure induces dysregulation of constitutive heterochromatin hallmarks in live cells.

Lead (Pb) is a heavy metal contaminant commonly found in air, soil, and drinking water due to legacy uses. Excretion of ingested Pb can result in extensive kidney damages due to elevated oxidative stress. Epigenetic alterations induced by exposure to Pb have also been implied but remain poorly understood. In this work, we assessed changes in repressive epigenetic marks, namely DNA methylation (meCpG) and histone 3 lysine 9 tri-methylation (H3K9me3) after exposure to Pb. Live cell epigenetic probes coupled to bimolecular fluorescence complementation (BiFC) were used to monitor changes in the selected epigenetic marks. Exposure to Pb significantly lowered meCpG and H3K9me3 levels in HEK293T cells suggesting global changes in constitutive heterochromatin. A heterodimeric pair of probes that tags chromatin regions enriched in both meCpG and H3K9me3 further confirmed our findings. The observed epigenetic changes can be partially attributed to aberrant transcriptional changes induced by Pb, such as overexpression of TET1 after Pb exposure. Lastly, we monitored changes in selected heterochromatin marks after removal of Pb and found that changes in these markers do not immediately recover to their original level suggesting potential long-term damages to chromatin structure.

Hyperexcitability and Pharmacological Responsiveness of Cortical Neurons Derived from Human iPSCs Carrying Epilepsy-Associated Sodium Channel Nav1.2-L1342P Genetic Variant.

With the wide adoption of genomic sequencing in children having seizures, an increasing number of SCN2A genetic variants have been revealed as genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is predominantly expressed in the pyramidal excitatory neurons and supports action potential (AP) firing. One recurrent SCN2A genetic variant is L1342P, which was identified in multiple patients with epileptic encephalopathy and intractable seizures. However, the mechanism underlying L1342P-mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human-induced pluripotent stem cell (hiPSC) line from a male donor, in which L1342P was introduced by CRISPR/Cas9-mediated genome editing. Using patch-clamping and microelectrode array (MEA) recordings, we revealed that cortical neurons derived from hiPSCs carrying heterozygous L1342P variant have significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, suggesting hyperexcitability phenotypes. Interestingly, L1342P neuronal culture displayed a degree of resistance to the anticonvulsant medication phenytoin, which recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, can potently alleviate spontaneous and chemically-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.SIGNIFICANCE STATEMENT A mounting number of SCN2A genetic variants have been identified from patients with epilepsy, but how SCN2A variants affect the function of human neurons contributing to seizures is still elusive. This study investigated the functional consequences of a recurring SCN2A variant (L1342P) using human iPSC-derived neurons and revealed both intrinsic and network hyperexcitability of neurons carrying a mutant Nav1.2 channel. Importantly, this study recapitulated elements of clinical observations of drug-resistant features of the L1342P variant, and provided a platform for in vitro drug testing. Our study sheds light on cellular mechanism of seizures resulting from a recurring Nav1.2 variant, and helps to advance personalized drug discovery to treat patients carrying pathogenic SCN2A variant.

Live-Cell Probe for In Situ Single-Cell Monitoring of Mitochondrial DNA Methylation.

Mitochondria, as the center of energy production, play an important role in cell homeostasis by regulating the cellular metabolism and mediating the cellular response to stress. Epigenetic changes such as DNA and histone methylation have been increasingly recognized to play a significant role in homeostasis and stress response. The cross-talking between the metabolome and the epigenome has attracted significant attention in recent years but with a major focus on how metabolism contributes to epigenomic changes. Few studies have focused on how epigenetic modifications may alter the mitochondrial composition and activity. In this work, we designed a novel probe targeting methylated CpGs of mitochondrial DNA (mtDNA). We demonstrated the capability of our probe to reveal the spatial distribution of methylated mtDNA and capture the mtDNA methylation changes at a single-cell level. We were also able to track single-cell mtDNA and nDNA methylation simultaneously and discovered the unsynchronized dynamics of the nucleus and mitochondria. Our tool offers a unique opportunity to understand the epigenetic regulation of mtDNA and its dynamic response to the microenvironment and cellular changes.

Identifying distinct heterochromatin regions using combinatorial epigenetic probes in live cells.

The 3D spatial organization of the genome controls gene expression and cell functionality. Heterochromatin (HC), which is the densely compacted and largely silenced part of the chromatin, is the driver for the formation and maintenance of nuclear organization in the mammalian nucleus. It is functionally divided into highly compact constitutive heterochromatin (cHC) and transcriptionally poised facultative heterochromatin (fHC). Long regarded as a static structure, the highly dynamic nature of the heterochromatin is being slowly understood and studied. These changes in HC occur on various temporal scales during the cell cycle and differentiation processes. Most methods that capture information about the heterochromatin are static techniques that cannot provide a readout of how the HC organization evolves with time. The delineation of specific areas such as fHC are also rendered difficult due to its diffusive nature and lack of specific features. Another degree of complexity in characterizing changes in heterochromatin occurs due to the heterogeneity in the HC organization of individual cells, necessitating single cell studies. Overall, there is a need for live cell compatible tools that can stably track the heterochromatin as it undergoes re-organization. In this work, we present an approach to track cHC and fHC based on the epigenetic hallmarks associated with them. Unlike conventional immunostaining approaches, we use small recombinant protein probes that allow us to dynamically monitor the HC by binding to modifications specific to the cHC and fHC, such as H3K9me3, DNA methylation and H3K27me3. We demonstrate the use of the probes to follow the changes in HC induced by drug perturbations at the single cell level. We also use the probe sets combinatorically to simultaneously track chromatin regions enriched in two selected epigenetic modifications using a FRET based approach that enabled us tracking distinctive chromatin features in situ.

Pre-differentiation exposure to low-dose of atrazine results in persistent phenotypic changes in human neuronal cell lines.

Exposures to organic pesticides, particularly during a developmental window, have been associated with various neurodegenerative diseases later in life. Atrazine (ATZ), one of the most used pesticides in the U.S., is suspected to be associated with increased neurodegeneration later in life but few studies assessed the neurotoxicity of developmental ATZ exposure using human neuronal cells. Here, we exposed human SH-SY5Y cells to 0.3, 3, and 30 ppb of ATZ prior to differentiating them into dopaminergic-like neurons in ATZ-free medium to mimic developmental exposure. The differentiated neurons exhibit altered neurite outgrowth and SNCA pathology depending on the ATZ treatment doses. Epigenome changes, such as decreases in 5mC (for 0.3 ppb only), H3K9me3, and H3K27me3 were observed immediately after exposure. These alterations persist in a compensatory manner in differentiated neurons. Specifically, we observed significant reductions in 5mC and H3K9me3, as well as, an increase in H3K27me3 in ATZ-exposed cells after differentiation, suggesting substantial chromatin rearrangements after developmental ATZ exposure. Transcriptional changes of relevant epigenetic enzymes were also quantified but found to only partially explain the observed epigenome alteration. Our results thus collectively suggest that exposure to low-dose of ATZ prior to differentiation can result in long-lasting changes in epigenome and increase risks of SNCA-related Parkinson's Disease.

Low dose lead exposure induces alterations on heterochromatin hallmarks persisting through SH-SY5Y cell differentiation.

Lead (Pb) is a commonly found heavy metal due to its historical applications. Recent studies have associated early-life Pb exposure with the onset of various neurodegenerative disease. The molecular mechanisms of Pb conferring long-term neurotoxicity, however, is yet to be elucidated. In this study, we explored the persistency of alteration in epigenetic marks that arise from exposure to low dose of Pb using a combination of image-based and gene expression analysis. Using SH-SY5Y as a model cell line, we observed significant alterations in global 5-methycytosine (5 mC) and histone 3 lysine 27 tri-methylation (H3K27me3) and histone 3 lysine 9 tri-methylation (H3K9me3) levels in a dose-dependent manner immediately after Pb exposure. The changes are partially associated with alterations in epigenetic enzyme expression levels. Long term culturing (14 days) after cease of exposure revealed persistent changes in 5 mC, partial recovery in H3K9me3 and overcompensation in H3K27me3 levels. The observed alterations in H3K9me3 and H3K27me3 are reversed after neuronal differentiation, while reduction in 5 mC levels are amplified with significant changes in patterns as identified via texture clustering analysis. Moreover, correlation analysis demonstrates a strong positive correlation between trends of 5 mC alteration after differentiation and neuronal morphology. Collectively, our results suggest that exposure to low dose of Pb prior to differentiation can result in persistent epigenome alterations that can potentially be responsible for the observed phenotypic changes. Our work reveals that Pb induced changes in epigenetic repressive marks can persist through neuron differentiation, which provides a plausible mechanism underlying long-term neurotoxicity associated with developmental Pb-exposure.

Review: In vitro Cell Platform for Understanding Developmental Toxicity.

Developmental toxicity and its affiliation to long-term health, particularly neurodegenerative disease (ND) has attracted significant attentions in recent years. There is, however, a significant gap in current models to track longitudinal changes arising from developmental toxicity. The advent of induced pluripotent stem cell (iPSC) derived neuronal culture has allowed for more complex and functionally active in vitro neuronal models. Coupled with recent progress in the detection of ND biomarkers, we are equipped with promising new tools to understand neurotoxicity arising from developmental exposure. This review provides a brief overview of current progress in neuronal culture derived from iPSC and in ND markers.

Profiling epigenetic changes in human cell line induced by atrazine exposure.

How environmental chemicals can affect and exert their toxic effect at a molecular level has gained significant interest in recent years, not only for understanding their immediate health implications over exposed individuals, but also for their subsequent progeny. Atrazine (ATZ) is a commonly used herbicide in the U.S. and a long-suspected endocrine disrupting chemical. The molecular mechanism conferring long-term adverse health outcomes, however, remain elusive. Here, we explored changes in epigenetic marks that arise after exposure to ATZ at selected doses using image-based analysis coupled with data clustering. Significant decreases in methylated CpG (meCpG) and histone 3 lysine 9 tri-methylated (H3K9me3) were observed in the selected human cell line with a clear spatial preference. Treating cells with ATZ leads to the loss of a subpopulation of cells with high meCpG levels as identified in our clustering and histogram analysis. A similar trend was observed in H3K9me3 potentially attributing to the cross-talking between meCpG and H3K9me3. Changes in meCpG are likely to be associated with alterations in epigenetic enzyme expression levels regulating meCpG and persist after the removal of ATZ source which collectively provide a plausible mechanism for long-term ATZ-induced toxicity.

A quantum dynamical treatment of symmetry-induced kinetic isotope effects in the formation of He2+.

Kinetic isotope effects for He(2)(+) formation are calculated quantum dynamically using high-quality Born-Oppenheimer (BO) potentials for two electronic states of He(2)(+) and an accurate treatment of all nonadiabatic BO corrections. The two potentials are coupled only when the helium isotopes are different, and the calculations reveal that this coupling is sufficient to allow the two sets of distinguishable reactants, (4)He(+) + (3)He or (3)He(+) + (4)He, to yield He(2)(+) with comparable efficiency over a wide temperature range. Consequently, the potential coupling provides a significant formation rate enhancement for the low isotopic symmetry reactants, as compared to the symmetrical cases (e.g., (4)He(+) + (4)He or (3)He(+) + (3)He). The computed symmetry-induced kinetic isotope effects (SIKIEs) are in substantial agreement with the available experimental results and represent the first theoretical demonstration of this unusual kinetic phenomenon. Possible application of SIKIE to ozone formation and other chemical systems is discussed.

Accurate, two-state ab initio study of the ground and first-excited states of He2+, including exact treatment of all Born-Oppenheimer correction terms.

Born-Oppenheimer (BO) potentials for the ground and first-excited electronic states of He2+ are determined using high level ab initio techniques for internuclear separations R of 1.2-100 bohrs and accurately fit to analytical functions. In the present formulation, the BO potentials are nuclear mass independent, and the corresponding BO approximation is obtained by ignoring four terms of the full rovibronic Hamiltonian. These four Born-Oppenheimer correction (BOC) terms are as follows: (1) mass polarization, (2) electronic orbital angular momentum, (3) first derivative with respect to R, and (4) second derivative with respect to R. In order to enable an exact rovibronic calculation, each of the four BOC terms are computed as a function of R, for the two electronic states and for their coupling, without any approximation or use of empirical parameters. Each of the BOC terms is found to make a contribution to the total energy over at least some portion of the range of R investigated. Interestingly, the most significant coupling contribution arises from the electronic orbital angular momentum term, which is evidently computed for the first time in this work. Although several BOC curves exhibit a nontrivial dependence on R, all are accurately fit to analytical functions. The resulting functions, together with the BO potentials, are used to compute exact rovibronic energy levels for 3He 3He+,3He 4He+) and 4He 4He+. Comparison to available high quality experimental data indicates that the present BOC potentials provide the most accurate representation currently available of both the low- and high-lying levels of the ground electronic state and the bound levels of the excited state.