Biology subjects

Lein, P. J.

Publications and source records attributed to Lein, P. J..

14 recordsLinked to original sources

bioRxiv2026

PCB-Exposed Gut Microbiota Recapitulate Neurobehavioral Deficits and Reprogram Hippocampal Transcriptomic Signatures

Polychlorinated biphenyls (PCBs) are persistent environmental pollutants associated with developmental neurotoxicity and autism spectrum disorder (ASD). However, whether PCB-induced alterations in gut microbiota contribute to neurodevelopmental toxicity independently of direct chemical exposure remains unclear. Here, we investigated whether transplantation of PCB-associated gut microbiota produces early neurobehavioral alterations and disrupts brain molecular pathways in offspring. Gut microbiota from conventional dams exposed to the Fox River PCB mixture (FRM; 6.0 mg/kg/day) or vehicle were transplanted into germ-free recipient females without direct PCB administration. At postnatal day 7 (P7), offspring underwent ultrasonic vocalization (USV) testing, whole-brain RNA sequencing (n = 4/sex/group), and untargeted serum metabolomics. Untargeted serum metabolomics were also performed in donor dams. PCB-associated fecal microbiota transplantation (FMT) significantly reduced offspring USVs, reproducing effects observed following direct maternal PCB exposure. Brain transcriptomics in offspring identified substantially more differentially expressed genes in males (9,339) than females (1,082), including 512 shared genes. Concordantly regulated genes revealed downregulation of neurotransmitter signaling and ion channel pathways alongside upregulation of RNA processing pathways. Male-associated responses involved altered protein synthesis and neuronal signaling, whereas female-associated responses involved DNA repair and extracellular matrix remodeling. Despite these molecular differences, behavioral responses showed no significant sex-dependent effect. Maternal metabolomics revealed increased bile acids and decreased indole-3-propionic acid, highlighting potential AhR-NF{kappa}B and bile acid-associated signaling mechanisms. Offspring metabolomics further identified alterations in lipid-associated metabolites and metabolic profiles. Collectively, these findings show that transferring PCB-exposed microbiota reproduces early neurobehavioral alterations and induces shared and sex-biased molecular responses, identifying the maternal gut microbiome as a candidate mediator linking PCB exposure to developmental neurotoxicity.

pharmacology and toxicology↗

bioRxiv2026

Chronic NLRP3 inflammasome activation drives neutrophil brain entry and interactions with microglia

NOD-like receptor family pyrin domain-containing 3 (NLRP3) is a cytosolic regulator of an inflammasome-mediated innate immune response. In the central nervous system (CNS), NLRP3 inflammasome activation has been implicated in multiple neurodegenerative diseases, yet the mechanisms by which it contributes to disease remain unclear. Here, we investigated the CNS effects of chronic NLRP3 activation using a humanized NLRP3 gain-of-function mouse model (hNLRP3D305N). Bulk brain analyses confirmed constitutive inflammasome activation, widespread cytokine induction, and the increased presence of blood-associated proteins suggestive of dysfunction at CNS border sites and the blood-brain barrier (BBB). Furthermore, cerebrospinal fluid (CSF) neurofilament light chain levels were elevated, indicating neuronal damage. Single-cell RNA-sequencing of CD45+ immune cells in the brain demonstrated that microglia adopt distinct reactive states and that peripheral immune cells infiltrate the CNS, with neutrophils emerging as the predominant infiltrating immune cell type. This finding was confirmed by untargeted bulk brain and CSF proteomics that also suggest neutrophil reactivity. Immunohistochemistry further revealed regional neutrophil entry into the brain parenchyma, concurrent with reactive microglia and engulfment of neutrophils, suggesting functional microglia-neutrophil interactions. Collectively, these findings establish a direct pathogenic role for the NLRP3 inflammasome in the CNS independent of other neurodegeneration-related disease pathologies.

neuroscience↗

bioRxiv2025

Spatial and spectral mapping of traffic-related air pollution (TRAP) nanoparticles in relation to plaques and inflammatory markers in an Alzheimer disease model

Chronic exposure to traffic-related air pollution (TRAP) is linked to increased risk of neurodegenerative diseases, including Alzheimer disease (AD). Ultrafine particulate matter (UFPM) is a suspected driver of TRAP neurotoxicity, but its spatial interactions with AD pathology remain poorly defined. We investigated the distribution, composition, and pathological context of TRAP-derived UFPM in the hippocampus of TgF344-AD rats chronically exposed to TRAP or filtered air (FA) for 14 months. Using a multimodal imaging workflow that combines enhanced darkfield hyperspectral imaging (EDF-HSI) with confocal immunofluorescence for microglia (CD68/Iba1) and amyloid beta (A{beta}) plaques (Thioflavin S), we mapped the localization and spectral properties of UFPM in situ. UFPM accumulation was elevated in TRAP-exposed females, suggesting sex-specific vulnerability in blood-brain barrier (BBB) permeability or particle retention. Particles near plaques showed red-shifted spectral signatures, consistent with biochemical transformation. Dimension reduction revealed clustering of particle spectra by TRAP exposure and plaque proximity. However, UFPM was rarely found within plaques or microglia, implying indirect neuroimmune modulation. These findings highlight a novel spatial and spectral imaging approach for characterizing environmental nanoparticle interactions in the brain and suggest that chronic TRAP exposure may influence AD-related inflammation and pathology in a sex-and region-dependent manner. SynopsisThis study shows that chronic traffic-related air pollution alters ultrafine particulate matter deposition and neuroinflammation in a rodent Alzheimer model, revealing region- and sex-specific vulnerability in the brains response to environmental exposure.

pharmacology and toxicology↗

bioRxiv2025

Single-cell transcriptomics showed that maternal PCB exposure dysregulated ER stress-mediated cell type-specific responses in the liver of female offspring

Polychlorinated biphenyls (PCBs) are persistent environmental toxicants that bioaccumulate in the food chain and readily cross the placenta, raising concerns for developmental toxicity. While PCB exposure has been associated with metabolic and neurodevelopmental disorders, its cell type-specific effects on liver development remain poorly understood. This study aimed to investigate how maternal exposure to an environmentally relevant Fox River PCB mixture affects liver development in female offspring at single-cell resolution. We hypothesized that early-life PCB exposure disrupts hepatic metabolic and immune function in a cell type-specific manner. Using single-cell RNA sequencing (scRNA-seq) on liver tissue from postnatal day 28 female mice perinatally exposed to PCBs, we identified major hepatic and immune cell populations and assessed cell-specific transcriptional responses. PCB exposure significantly altered the proportions of endothelial cells and Kupffer cells and reduced neutrophil abundance. Transcriptomic analysis revealed that PCBs dysregulated key pathways in hepatocytes and non-parenchymal cells, including ER stress responses, drug metabolism, and glucose/insulin signaling. Notably, hepatocytes exhibited upregulation of phase-I drug-metabolizing enzymes and uptake transporters, but downregulation of phase-II enzymes and efflux transporters. Kupffer cells and endothelial cells had altered immune and metabolic gene expression, and intercellular communication analysis predicted disrupted fibronectin, collagen, and chemokine signaling due to PCB exposure. RT-qPCR validation confirmed increased hepatic ER stress marker expression. Together these findings demonstrate that perinatal PCB exposure induces persistent, cell type-specific transcriptomic reprogramming in the liver, impairing metabolic and immune functions. This study highlights the utility of single-cell transcriptomics for revealing toxicant effects with cellular precision during critical windows of development.

systems biology↗

bioRxiv2025

Female cortical cellular mosaicism underlies shared MeCP2 and PCB impacted gene pathways

Etiologies of neurodevelopmental disorders involve genes and environment however their interactions are understudied. Rett Syndrome (RTT) is an X-linked, dominant neurodevelopmental disorder caused by mutations in MECP2, encoding the epigenetic regulator methyl CpG binding protein. Epigenetic features of MECP2 expression due to X-linked cellular mosaicism and the variability in severity and timing of progression in RTT suggest interaction with environmental neurotoxicants such as lipophilic polychlorinated biphenyls (PCBs). To understand shared mechanisms, we exposed WT and Mecp2e1-/+ female mice to a human-relevant PCB mixture and dose, then performed single-nucleus 5 RNA-seq from cortex. We identified significant overlap in dysregulated genes and 71 shared pathways between the effects of PCB exposure and MeCP2 mutation, and co-mitigation of their transcriptional impacts. PCBs influenced the non-cell- autonomous transcriptional effects of MeCP2 mutations in wild-type-expressing neurons within the mosaic mutant female cortex in both mouse and human, suggesting that the interactions predominantly involve homeostatic gene networks.

systems biology↗

bioRxiv2024

Placental-brain axis in females detected within broadly impacted metabolic gene networks protects against prenatal PCB exposure

BackgroundNeurodevelopmental disorders have a strong male bias that is poorly understood. Placenta is a rich source of molecular information about environmental interactions with genetics (including biological sex), that affect the developing brain. We investigated placental-brain transcriptional responses in an established mouse model of prenatal exposure to a human-relevant mixture of polychlorinated biphenyls (PCBs). ResultsTo understand sex, tissue, and dosage effects in embryonic (E18) brain and placenta by RNAseq, we used weighted gene correlation network analysis (WGCNA) to create correlated gene networks that could be compared across sex or tissue. WGCNA revealed that expression within most correlated gene networks was significantly and strongly associated with PCB exposures, but frequently in opposite directions between male-female and placenta-brain comparisons. In both WGCNA and differentially expressed gene analyses, male brain showed more PCB-induced transcriptional changes than male placenta, but the reverse pattern was seen in females. Furthermore, non-monotonic dose responses to PCBs were observed in most gene networks but were most prominent in male brain. The transcriptomic effects of low dose PCB exposure were significantly reversed by dietary folic acid supplementation across both sexes, but these effects were strongest in female placenta. PCB-dysregulated and folic acid-reversed gene networks were commonly enriched in functions in metabolic pathways involved in energy usage and translation, with female-specific protective effects enriched in PPAR, thermogenesis, glycerolipids, and O-glycan biosynthesis, as opposed to toxicant responses in male brain. ConclusionsThe female protective effect in prenatal PCB exposures appears to be mediated by dose-dependent sex differences in transcriptional modulation of metabolism in placenta. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/603326v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@14d4936org.highwire.dtl.DTLVardef@101b87eorg.highwire.dtl.DTLVardef@1f787e6org.highwire.dtl.DTLVardef@1fb1b62_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Rat primary cortical cell tri-culture to study effects of amyloid-beta on microglia function

IntroductionThe etiology and progression of sporadic Alzheimers Disease (AD) have been studied for decades. One proposed mechanism is that amyloid-beta (A{beta}) proteins induce neuroinflammation, synapse loss, and neuronal cell death. Microglia play an especially important role in A{beta} clearance, and alterations in microglial function due to aging or disease may result in A{beta} accumulation and deleterious effects on neuronal function. However, studying these complex factors in vivo, where numerous confounding processes exist, is challenging, and until recently, in vitro models have not allowed sustained culture of microglia, astrocytes and neurons in the same culture. Here, we employ a tri-culture model of rat primary neurons, astrocytes, and microglia and compare it to co-culture (neurons and astrocytes) and mono-culture enriched for microglia to study microglial function (i.e., motility and A{beta} clearance) and proteomic response to exogenous A{beta}. MethodsWe established cortical co-culture (neurons and astrocytes), tri-culture (neurons, astrocytes, and microglia), and mono-culture (microglia) from perinatal rat pups. On days in vitro (DIV) 7 - 14, the cultures were exposed to fluorescently-labeled A{beta} (FITC-A{beta}) particles for varying durations. Images were analyzed to determine the number of FITC-A{beta} particles after specific lengths of exposure. A group of cells were stained for {beta}III-tubulin, GFAP, and Iba1 for morphological analysis via quantitative fluorescence microscopy. Cytokine profiles from conditioned media were obtained. Live-cell imaging with images acquired every 5 minutes for 4 hours was employed to extract microglia motility parameters (e.g., Euclidean distance, migration speed, directionality ratio). Results and discussionFITC-A{beta} particles were more effectively cleared in the tri-culture compared to the co-culture. This was attributed to microglia engulfing FITC-A{beta} particles, as confirmed via epifluorescence and confocal microscopy. Adding FITC-A{beta} significantly increased the size of microglia, but had no significant effect on neuronal surface coverage or astrocyte size. Analysis of the cytokine profile upon FITC-A{beta} addition revealed a significant increase in proinflammatory cytokines (TNF-, IL-1, IL-1{beta}, IL-6) in tri-culture, but not co-culture. In addition, A{beta} addition altered microglia motility marked by swarming-like motion with decreased Euclidean distance yet unaltered speed. These results highlight the importance of cell-cell communication in microglia function (e.g., motility and A{beta} clearance) and the utility of the tri-culture model to further investigate microglia dysfunction in AD.

neuroscience↗

bioRxiv2024

The alpha4 nicotinic acetylcholine receptor is necessary for the initiation of organophosphate-induced neuronal hyperexcitability

Acute intoxication with organophosphorus (OP) cholinesterase inhibitors can produce seizures that rapidly progress to life-threatening status epilepticus. Significant research effort has been invested investigating the involvement of muscarinic acetylcholine receptors (mAChRs) in OP-induced seizure activity. In contrast, there has been far less effort focused on nicotinic AChRs (nAChRs) in this context. Here, we address this data gap using a combination of in vitro and in vivo models. Pharmacological antagonism and genetic deletion of 4, but not 7, nAChR subunits prevented or significantly attenuated OP-induced electrical spike activity in acute hippocampal slices and seizure activity in mice, indicating that 4 nAChR activation is necessary for neuronal hyperexcitability triggered by acute OP exposures. These findings not only suggest that therapeutic strategies for inhibiting the 4 nAChR subunit warrant further investigation as prophylactic and acute treatments for acute OP-induced seizures, but also provide mechanistic insight into the role of the nicotinic cholinergic system in seizure generation.

pharmacology and toxicology↗

bioRxiv2023

Activation of the aryl hydrocarbon receptor inhibits neuropilin-1 upregulation on IL-2 responding CD4+ T cells

Neuropilin-1 (Nrp1), a transmembrane protein expressed on CD4+ T cells, is mostly studied in the context of regulatory T cell (Treg) function. More recently, there is increasing evidence that Nrp1 is also highly expressed on activated effector T cells and that increases in these Nrp1-expressing CD4+ T cells correspond with immunopathology across several T cell-dependent disease models. Thus, Nrp1 may be implicated in the identification and function of immunopathologic T cells. Nrp1 downregulation in CD4+ T cells is one of the strongest transcriptional changes in response to immunoregulatory compounds that act though the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. To better understand the link between AhR and Nrp1 expression on CD4+ T cells, Nrp1 expression was assessed in vivo and in vitro following AhR ligand treatment. In the current study, we identified that the percentage of Nrp1 expressing CD4+ T cells increases over the course of activation and proliferation in vivo. The actively dividing Nrp1+Foxp3- cells express the classic effector phenotype of CD44hiCD45RBlo, and the increase in Nrp1+Foxp3- cells is prevented by AhR activation. In contrast, Nrp1 expression is not modulated by AhR activation in non-proliferating CD4+ T cells. The downregulation of Nrp1 on CD4+ T cells was recapitulated in vitro in cells isolated from C57BL/6 and NOD (non-obese diabetic) mice. CD4+Foxp3- cells expressing CD25, stimulated with IL-2, or differentiated into Th1 cells, were particularly sensitive to AhR-mediated inhibition of Nrp1 upregulation. IL-2 was necessary for AhR-dependent downregulation of Nrp1 expression both in vitro and in vivo. Collectively, the data demonstrate that Nrp1 is a CD4+ T cell activation marker and that regulation of Nrp1 could be a previously undescribed mechanism by which AhR ligands modulate effector CD4+ T cell responses.

immunology↗

bioRxiv2022

Pro-resolving lipid mediators within brain esterified lipid pools are reduced in female rats chronically exposed to traffic-related air pollution or genetically susceptible to Alzheimer's Disease phenotype

Traffic-related air pollution (TRAP) is a risk factor for Alzheimers disease (AD) where neuroinflammation underlies disease progression and pathogenesis. Unresolved inflammation in AD is known to be exacerbated by brain deficits in unesterified pro-resolving lipid mediators enzymatically synthesized from polyunsaturated fatty acids. Recently, we reported that in the brain, unesterified pro-resolving lipid mediators which are bioactive, can also be supplied from less bioactive esterified lipid pools such as neutral lipids (NLs) and phospholipids (PLs). It is not known whether esterified pro-resolving lipid mediators are affected by AD pathology and exacerbated by TRAP exposure. In the present study we addressed this data gap using TgF344-AD male and female rats that express human AD risk genes and their wildtype littermates exposed to filtered air (FA) or TRAP from 1 to 15 months of age. Esterified lipid mediators within NLs and PLs were quantified by mass-spectrometry. We observed a significant reduction in pro-resolving lipid mediators in both NLs and PLs of female TgF344-AD rats compared to wildtype controls. TRAP exposure also reduced pro-resolving lipids in the female brain, mainly in PL pools, but did not exacerbate changes observed in TgF344-AD rats. Minimal changes were observed in males. Our findings indicate that AD genotype and chronic TRAP exposure result in sex-specific deficits in brain esterified pro-resolving lipid mediators, the pool that supplies free and bioactive lipid mediators. These data provide new information on lipid-mediated mechanisms regulating impaired inflammation resolution in AD, and show for the first time that chronic TRAP exposure targets the same lipid network implicated in AD.

neuroscience↗

bioRxiv2021

Diisopropylfluorophosphate (DFP) volatizes and cross-contaminates wells in a common 96-well plate zebrafish larvae exposure method

Diisopropylfluorophosphate (DFP) is an organophosphate (OP) that is commonly used to study the neurotoxic effects of acutely intoxicating OP exposure. In preliminary studies, we discovered abnormal deaths in DMSO-only exposed larvae housed in the same plate as DFP-exposed larvae, and hypothesized that DFP volatilizes and cross-contaminates wells when using a 96-well plate exposure method for exposing zebrafish larvae. Survivability and acetylcholinesterase activity assays confirmed DFP presence in the tissues of zebrafish ostensibly exposed to DMSO only. These findings indicate DFP cross-contamination, which raises concerns for the experimental design of studies evaluating the toxicity of volatile and semi-volatile substances.

pharmacology and toxicology↗

bioRxiv2021

Genome-Wide DNA Methylation Profiles of Neurodevelopmental Disorder Genes in Mouse Placenta and Fetal Brain Following Prenatal Exposure to Polychlorinated Biphenyls

BackgroundPolychlorinated biphenyls (PCBs) are developmental neurotoxicants implicated as environmental risk factors for neurodevelopmental disorders (NDD), including autism spectrum disorders (ASD). ObjectiveWe examined the effects of prenatal exposure to a human-relevant mixture of PCBs on the DNA methylome of fetal mouse brain and placenta to determine if there was a shared subset of differentially methylated regions (DMRs). MethodsA PCB mixture formulated to model the 12 most abundant congeners detected in the serum of pregnant women from a prospective high-risk ASD cohort was administered to female mice prior to and during pregnancy. Whole-genome bisulfite sequencing (WGBS) was performed to assess genome-wide DNA methylation profiles of placenta and brain on gestational day 18. ResultsWe found thousands of significant (empirical p < 0.05) DMRs distinguishing placentas and brains from PCB-exposed embryos from sex-matched vehicle controls. In both placenta and brain, PCB-associated DMRs were significantly (p < 0.005) enriched for functions related to neurodevelopment, cellular adhesion, and cellular signaling, and significantly (Odds Ratio > 2.4, q < 0.003) enriched for bivalent chromatin marks. The placenta and brain PCB DMRs overlapped significantly (Z-score = 4.5, p = 0.0001) by genomic coordinate and mapped to a shared subset of genes significantly (q < 0.05) enriched for Wnt signaling, Slit/Robo signaling, and genes differentially expressed in multiple NDD/ASD models. The placenta and brain DMRs also significantly (q < 0.05) overlapped by genomic coordinate with brain samples from humans with Rett syndrome and Dup15q syndrome. DiscussionThese results demonstrate that placenta can be used as a surrogate for embryonic brain DNA methylation changes over genes relevant to NDD/ASD in a mouse model of prenatal PCB exposure.

genomics↗

bioRxiv2020

Assessment of autism zebrafish mutant models using a high-throughput larval phenotyping platform

In recent years zebrafish have become commonly used as a model for studying human traits and disorders. Their small size, high fecundity, and rapid development allow for more high-throughput experiments compared to other vertebrate models. Given that zebrafish share >70% gene homologs with humans and their genomes can be readily edited using highly efficient CRISPR methods, we are now able to rapidly generate mutations impacting practically any gene of interest. Unfortunately, our ability to phenotype mutant larvae has not kept pace. To address this challenge, we have developed a protocol that obtains multiple phenotypic measurements from individual zebrafish larvae in an automated and parallel fashion, including morphological features (i.e., body length, eye area, and head size) and movement/behavior. By assaying wild-type zebrafish in a variety of conditions, we determined optimal parameters that avoid significant developmental defects or physical damage; these include morphological imaging of larvae at two time points (3 days post fertilization (dpf) and 5 dpf) coupled with motion tracking of behavior at 5 dpf. As a proof-of-principle, we tested our approach on two novel CRISPR-generated mutant zebrafish lines carrying predicted null-alleles of syngap1b and slc7a5, orthologs to two human genes implicated in autism-spectrum disorder, intellectual disability, and epilepsy. Using our optimized high-throughput phenotyping protocol, we recapitulated previously published results from mouse and zebrafish models of these candidate genes. In summary, we describe a rapid parallel pipeline to characterize morphological and behavioral features of individual larvae in a robust and consistent fashion, thereby improving our ability to better identify genes important in human traits and disorders. AUTHOR SUMMARYZebrafish (Danio rerio) are a well-established model organism for the study of neurodevelopmental disorders. Due to their small size, fast reproduction, and genetic homology with humans, zebrafish have been widely used for characterizing and screening candidate genes for many disorders, including autism-spectrum disorder, intellectual disability, and epilepsy. Although several studies have described the use of high-throughput morphological and behavioral assays, few combine multiple assays in a single zebrafish larva. Here, we optimized a platform to characterize morphometric features at two developmental time points in addition to behavioral traits of zebrafish larvae. We then used this approach to characterize two autism candidate genes (SYNGAP1 and SLC7A5) in two CRISPR-generated zebrafish null mutant models we developed in house. These data recapitulate previously published results related to enhanced seizure activity, while identifying additional defects not previously reported. We propose that our phenotyping platform represents a feasible method for maximizing the use of single zebrafish larvae in the characterization of additional mutants relevant to neurodevelopmental disorders.

genetics↗

bioRxiv2020

A Primary Neural Cell Culture Model for Neuroinflammation

Interactions between neurons, astrocytes and microglia critically influence neuroinflammatory responses to insult in the central nervous system. Studying neuroinflammation in vitro has been difficult because most primary culture models do not include all three critical cell types. We describe an in vitro model of neuroinflammation comprised of neurons, astrocytes and microglia. Primary rat cortical cells were cultured in a serum-free medium used to co-culture neurons and astrocytes that is supplemented with three factors (IL-34, TGF-{beta} and cholesterol) used to support isolated microglia. This "tri-culture" can be maintained for at least 14 days in vitro while retaining a physiologically-relevant representation of all three cell types. Additionally, we demonstrate that the tri-culture system responds to lipopolysaccharide, mechanical trauma and excitotoxicity with both neurotoxic and neuroprotective aspects of the neuroinflammatory response observed in vivo. We expect the tri-culture model will enable mechanistic studies of neuroinflammation in vitro with enhanced physiological relevance.

neuroscience↗