Paternal fenvalerate exposure causes autism-like behavior partly by altering epigenetic reprogramming of the imprinted gene IGF2 in fetal brain and paternal sperm.

Maternal infection during pregnancy is a well-established risk factor for neurodevelopmental disorders (NDDs), yet the underlying molecular mechanisms remain poorly understood. Lipocalin-2 (Lcn2), an innate immune protein, is highly upregulated during infection, known to affect neuronal and glial function. This study investigates the role of Lcn2 in shaping brain development, particularly after maternal immune activation (MIA). To mimic maternal infection, pregnant mice received intraperitoneal injections of either lipopolysaccharide (LPS) or saline on embryonic days 16 to 18 to model infection during the second trimester of pregnancy in humans. We first showed that Lcn2 mRNA was expressed in the fetal brain and that MIA significantly upregulated Lcn2 mRNA in the hippocampus and neocortex of both sexes. To assess functional relevance, we employed Lcn2 heterozygous females to generate wild-type and Lcn2 KO offspring from the MIA and control groups. Both female and male offspring underwent a battery of behavioral assays. Strikingly, both Lcn2 deletion and MIA independently led to social deficits and increased repetitive behaviors, hallmark features of NDDs, but their combination did not produce additive effects, suggesting a shared or converging mechanism. These alterations were specific to social and repetitive behavior, as no deficits were observed in the learning and memory task. To investigate potential shared molecular mechanisms, we quantified pro-inflammatory cytokine levels in the placenta and fetal forebrain at 4 h after the final LPS injection. No genotype-dependent differences were observed in IL-6, TNF-α, or IL-1β expression. However, RNA sequencing analysis revealed an overlapping group of differentially expressed genes in the Lcn2 KO and MIA groups, indicating convergence on similar transcriptional pathways that may underlie the observed behavioral phenotypes. Together, these findings reveal a previously unrecognized role for Lcn2 in brain development and suggest that while Lcn2 may not directly mediate the damaging effects of maternal immune challenge, it intersects with critical developmental pathways that shape social behavior.

Organophosphate Flame Retardants Disrupt Autism-Relevant Gene Networks Across Development: A Cross-Species Multi-Omics Study.

Pyrethroid insecticides represent a broad class of chemicals used widely in agriculture and household applications. Human studies show mixed effects of maternal pyrethroid exposure on fetal growth and neurodevelopment. Assessment of shared pyrethroid metabolites as a biomarker for exposure obscures effects of specific chemicals within this broader class. To better characterize pyrethroid effects on fetal development, we investigated maternal exposure to permethrin, a type I pyrethroid, and α-cypermethrin, a type II pyrethroid, on fetal development in mice. Pregnant CD1 mice were exposed to permethrin (1.5, 15, or 50 mg/kg), α-cypermethrin (0.3, 3, or 10 mg/kg), or corn oil vehicle via oral gavage on gestational days (GDs) 6 to 16. Effects on fetal growth, placental toxicity, and neurodevelopment were evaluated at GD 16. Cypermethrin, but not permethrin, significantly reduced fetal growth and altered placental layer morphology. Placental RNAseq analysis revealed downregulation of genes involved in extracellular matrix remodeling in response to α-cypermethrin. Both pyrethroids induced shifts in fetal dorsal forebrain microglia morphology from ramified to ameboid states; however, the effects of α-cypermethrin were more pronounced. The α-cypermethrin transcriptome of fetal dorsal forebrain implicated altered glutamate receptor signaling, synaptogenesis, and c-AMP signaling. Coregulated gene modules in individual placenta and fetal dorsal forebrain pairs were correlated and overlapped in biological processes characterizing synapses, mitotic cell cycle, and chromatin organization, suggesting placenta–fetal brain shared mechanisms with α-cypermethrin exposure. In summary, maternal exposure to the type II pyrethroid α-cypermethrin, but not type I pyrethroid permethrin, significantly affected placental development, fetal growth, and neurodevelopment, and these effects were linked.

Background: Preeclampsia (PE) is a disorder associated with prenatal brain damage.This study investigated the effect of cinnamic acid (CIN) on the parameters of oxidative stress, oxidative DNA damage and cholinesterase (AChE) enzyme activity in the forebrain and the cell structure of the fetal medial frontal cortex (mFC) of l-NAME-induced PE rat model.Materials and methods: 25 pregnant rats were randomly divided into 5 groups: control group (no treatment), PE+NS group (intraperitoneal administration of 250 mg l-NAME from embryonic day (ED) 15 to ED20 and gavage normal saline), PE+CIN25, PE+CIN50 and PE+CIN100 groups (gavage of cinnamic acid with doses of 25, 50 and 100 mg/kg, after l-NAME injection).On the ED21, with cesarean section of live fetuses, the fetal brain was dissected.AChE, catalase (CAT), superoxide dismutase (SOD), 8hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA) levels in the forebrain and cell density in the mFC were measured.Results: A significant decrease in CAT and SOD, and a significant increase in MDA, 6-OHdG and AChE in the forebrain along with a decrease in neuronal density in the mFC of the PE+NS compared to the control were seen (p<0.05).While in the CIN-treated groups, they showed a significant increase in CAT and SOD, a significant decrease in MDA, 6-OHdG, and AChE in the forebrain and an increase in mFC neuronal density compared to the PE+NS (p<0.05).Conclusion: It seems that CIN with antioxidant effects in the fetal forebrain improves brain function and reduces cell damage in the middle frontal cortex of PE model fetuses.

Pyrethroid insecticides represent a broad class of chemicals used widely in agriculture and household applications. Human studies show mixed effects of maternal pyrethroid exposure on fetal growth and neurodevelopment. Assessment of shared pyrethroid metabolites as a biomarker for exposure obscures effects of specific chemicals within this broader class. To better characterize pyrethroid effects on fetal development, we investigated maternal exposure to permethrin, a type I pyrethroid, and α-cypermethrin, a type II pyrethroid, on fetal development in mice. Pregnant CD1 mice were exposed to permethrin (1.5, 15, or 50 mg/kg), α-cypermethrin (0.3, 3, or 10 mg/kg), or corn oil vehicle via oral gavage on gestational days (GD) 6–16. Effects on fetal growth, placental toxicity, and neurodevelopment were evaluated at GD 16. Cypermethrin, but not permethrin, significantly reduced fetal growth and altered placental layer morphology. Placental RNAseq analysis revealed downregulation of genes involved in extracellular matrix remodeling in response to α-cypermethrin. Both pyrethroids induced shifts in fetal dorsal forebrain microglia morphology from ramified to ameboid states; however, effects of α-cypermethrin were more pronounced. The α-cypermethrin transcriptome of fetal dorsal forebrain implicated altered glutamate receptor signaling, synaptogenesis, and c-AMP signaling. Coregulated gene modules in individual placenta and fetal dorsal forebrain pairs were correlated and overlapped in biological processes characterizing synapses, mitotic cell cycle, and chromatin organization, suggesting placenta-fetal brain shared mechanisms with α-cypermethrin exposure. In summary, maternal type II pyrethroid α-cypermethrin exposure but not type I pyrethroid permethrin significantly affected placental development, fetal growth, and neurodevelopment, and these effects were linked.

Background: Pre-eclampsia (PE) can cause brain damage before birth. However, its mechanism is not clear. The present study evaluated the effect of cinnamic acid (CIN) on the expression of inflammatory cytokines of the forebrain and neuronal damage in the hippocampus of PE model fetuses induced with l-NAME. Materials and methods: 25 pregnant female rats were randomly divided into 5 groups: control group (no treatment), PE+NS group (daily injection of 250 mg l-NAME from embryonic day (ED) 15 to 20 to induce PE and then one hour later normal saline gavage), PE+CIN25, PE+CIN50 and PE+CIN100 groups (CIN gavage with doses of 25, 50 and 100 mg, respectively, one hour after l-NAME injection). On the ED21, after cesarean section, the fetal brain was dissected. The content of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin-1 beta (IL-1β) in the forebrain and cell density in the CA1 and CA3 regions of the fetal hippocampus were measured. Results: A significant increase in TNF-α, IL-6 and IL-1β in the forebrain along with a decrease in neuronal density in the CA1/CA3 regions was seen in the PE+NS group compared to the control group (p<0.05). While in the groups receiving CIN, they showed a significant decrease in TNF-α, IL-6 and IL-1β in the forebrain and an increase in CA1/CA3 neuronal density compared to the PE+NS group (p<0.05). Conclusion: CIN improved the inflammation and reduced cell damage in the hippocampus of PE model fetuses through modulating the level of anti-inflammatory cytokines in the fetal forebrain.

Mitochondria are unique organelles that have their own genome (mtDNA) and perform various pivotal functions within a cell. Recently, evidence has highlighted the role of mitochondria in the process of stem cell differentiation, including differentiation of neural stem cells (NSCs). Here we studied the importance of mtDNA function in the early differentiation process of NSCs in two cell culture models: the CGR8-NS cell line that was derived from embryonic stem cells by a lineage selection technique, and primary NSCs that were isolated from embryonic day 14 mouse fetal forebrain. We detected a dramatic increase in mtDNA content upon NSC differentiation to adapt their mtDNA levels to their differentiated state, which was not accompanied by changes in mitochondrial transcription factor A expression. As chemical mtDNA depletion by ethidium bromide failed to generate living ρ° cell lines from both NSC types, we used inhibition of mtDNA polymerase-γ by 2'-3'-dideoxycytidine to reduce mtDNA replication and subsequently cellular mtDNA content. Inhibition of mtDNA replication upon NSC differentiation reduced neurogenesis but not gliogenesis. The mtDNA depletion did not change energy production/consumption or cellular reactive oxygen species (ROS) content in the NSC model used. In conclusion, mtDNA replication is essential for neurogenesis but not gliogenesis in fetal NSCs through as yet unknown mechanisms, which, however, are largely independent of energy/ROS metabolism.

Keratan sulfate (KS) is a proteoglycan secreted in the fetal brain astrocytes and radial glia into extracellular parenchyma as granulofilamentous deposits. KS surrounds neurons except dendritic spines, repelling glutamatergic and facilitating GABAergic axons. The same genes are expressed in both neuroblast migration and axonal growth. This study examines timing of KS during morphogenesis of some normally developing human fetal forebrain structures. Twenty normal human fetal brains from 9-41 weeks gestational age were studied at autopsy. KS was examined by immunoreactivity in formalin-fixed paraffin sections, plus other markers including synaptophysin, S-100β protein, vimentin and nestin. Radial and tangential neuroblast migratory pathways from subventricular zone to cortical plate were marked by KS deposits as early as 9wk GA, shortly after neuroblast migration initiated. During later gestation this reactivity gradually diminished and disappeared by term. Long axonal fascicles of the internal capsule and short fascicles of intrinsic bundles of globus pallidus and corpus striatum also appeared as early as 9-12wk, as fascicular sleeves before axons even entered. Intense KS occurs in astrocytic cytoplasm and extracellular parenchyma at 9wk in globus pallidus, 15wk thalamus, 18wk corpus striatum, 22wk cortical plate, and hippocampus postnatally. Corpus callosum and anterior commissure do not exhibit KS at any age. Optic chiasm shows reactivity at the periphery but not around intrinsic subfasciculi. We postulate that KS forms a chemical template for many long and short axonal fascicles before axons enter and neuroblast migratory pathways at initiation of migration. Cross-immunoreactivity with aggrecan may render difficult molecular distinction.

Abstract BACKGROUND Embryonal brain tumors (EBTs) such as medulloblastoma, atypical teratoid rhabdoid tumor (AT/RT), and embryonal tumor with multilayered rosettes (ETMR) uniquely arise in the developing brain. Traditional 2D models often fall short in capturing the tumoral microenvironment (TME) and intratumoral heterogeneity, which may limit their translational potential. METHODS To address this, we co-aggregated EBT cells from AT/RT (Chla02 and SHH310) and ETMR (BT183 and murine GEMM cells) with forebrain organoids derived from human induced pluripotent stem cells and developed an automated, screening-ready in vitro model (EBT-FBO). We used immunohistochemistry, whole-mount immunostaining, and single-cell RNA sequencing to characterize our model and compared it with a developmental forebrain atlas. We further used our model for cell type-specific drug screening and validated the results in vivo. RESULTS Our EBT-FBO model mimicked the differentiation states of a mid-gestational fetal forebrain, providing a natural immature neuronal environment for supratentorial EBT cells. This environment allowed the EBT cells from both AT/RT and ETMR to mimic in vivo tumor cell states and avoided the cycling cell selection common in other models. We screened over 160 drugs in ETMR-FBO and identified antracyclines and Triptolide as having anti-tumor, yet non-neurotoxic effects. Our in-depth transcriptomic analysis identified new therapeutic targets, including PDGFR-signaling for ETMR. Building on this insight, we disrupted tumor-TME interactions and observed significant anti-tumor effects both in vitro and in vivo. CONCLUSIONS EBT-FBO provide embryonal brain tumors like ETMR or AT/RT with their intrinsic, immature neuronal microenvironment essential for recapitulating natural tumor heterogeneity. This marks a significant advancement towards targeted therapy and patient-specific models in precision medicine.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder, characterized by social communication disability and stereotypic behavior. This study aims to investigate the impact of prenatal exposure to 1‐nitropyrene (1‐NP), a key component of motor vehicle exhaust, on autism‐like behaviors in a mouse model. Three‐chamber test finds that prenatal 1‐NP exposure causes autism‐like behaviors during the weaning period. Patch clamp shows that inhibitory synaptic transmission is reduced in medial prefrontal cortex of 1‐NP‐exposed weaning pups. Immunofluorescence finds that prenatal 1‐NP exposure reduces the number of prefrontal glutamate decarboxylase 67 (GAD67) positive interneurons in fetuses and weaning pups. Moreover, prenatal 1‐NP exposure retards tangential migration of GAD67‐positive interneurons and downregulates interneuron migration‐related genes, such as Nrg1, Erbb4, and Sema3F, in fetal forebrain. Mechanistically, prenatal 1‐NP exposure reduces hydroxymethylation of interneuron migration‐related genes through inhibiting ten‐eleven translocation (TET) activity in fetal forebrain. Supplement with alpha‐ketoglutarate (α‐KG), a cofactor of TET enzyme, reverses 1‐NP‐induced hypohydroxymethylation at specific sites of interneuron migration‐related genes. Moreover, α‐KG supplement alleviates 1‐NP‐induced migration retardation of interneurons in fetal forebrain. Finally, maternal α‐KG supplement improves 1‐NP‐induced autism‐like behaviors in weaning offspring. In conclusion, prenatal 1‐NP exposure causes autism‐like behavior partially by altering DNA hydroxymethylation of interneuron migration‐related genes in developing brain.

The protein fasciculation and elongation zeta-1 (FEZ1) is involved in axon outgrowth but potentially interacts with various proteins with roles ranging from intracellular transport to transcription regulation. Gene association and other studies have identified FEZ1 as being directly, or indirectly, implicated in schizophrenia susceptibility. To explore potential roles in normal early human forebrain neurodevelopment, we mapped FEZ1 expression by region and cell type. All tissues were provided with maternal consent and ethical approval by the Human Developmental Biology Resource. RNAseq data were obtained from previously published sources. Thin paraffin sections from 8 to 21 post-conceptional weeks (PCW) samples were used for RNAScope in situ hybridization and immunohistochemistry against FEZ1 mRNA and protein, and other marker proteins. Tissue RNAseq revealed that FEZ1 is highly expressed in the human cerebral cortex between 7.5-17 PCW and single cell RNAseq at 17-18 PCW confirmed its expression in all neuroectoderm derived cells. The highest levels were found in more mature glutamatergic neurons, the lowest in GABAergic neurons and dividing progenitors. In the thalamus, single cell RNAseq similarly confirmed expression in multiple cell types. In cerebral cortex sections at 8-10 PCW, strong expression of mRNA and protein appeared confined to post-mitotic neurons, with low expression seen in progenitor zones. Protein expression was observed in some axon tracts by 16-19 PCW. However, in sub-cortical regions, FEZ1 was highly expressed in progenitor zones at early developmental stages, showing lower expression in post-mitotic cells. FEZ1 has different expression patterns and potentially diverse functions in discrete forebrain regions during prenatal human development.

Gestational 1-nitropyrene exposure causes anxiety-like behavior partially by altering hippocampal epigenetic reprogramming of synaptic plasticity in male adult offspring

BackgroundAutism spectrum condition or ‘autism’ is associated with numerous genetic risk factors including the polygenic 16p11.2 microdeletion. The balance between excitatory and inhibitory neurons in the cerebral cortex is hypothesised to be critical for the aetiology of autism making improved understanding of how risk factors impact on the development of these cells an important area of research. In the current study we aim to combine bioinformatics analysis of human foetal cerebral cortex gene expression data with anatomical and electrophysiological analysis of a 16p11.2+/- rat model to investigate how genetic risk factors impact on inhibitory neuron development.MethodsWe performed bioinformatics analysis of single cell transcriptomes from gestational week (GW) 8–26 human foetal prefrontal cortex and anatomical and electrophysiological analysis of 16p11.2+/- rat cerebral cortex and hippocampus at post-natal day (P) 21.ResultsWe identified a subset of human interneurons (INs) first appearing at GW23 with enriched expression of a large fraction of risk factor transcripts including those expressed from the 16p11.2 locus. This suggests the hypothesis that these foetal INs are vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2+/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells.LimitationsThe human foetal gene expression data was acquired from cerebral cortex between gestational week (GW) 8 to 26. We cannot draw inferences about potential vulnerabilities to genetic autism risk factors for cells not present in the developing cerebral cortex at these stages. The analysis 16p11.2+/- rat phenotypes reported in the current study was restricted to 3-week old (P21) animals around the time of weaning and to a single interneuron cell-type while in human 16p11.2 microdeletion carriers symptoms likely involve multiple cell types and manifest in the first few years of life and on into adulthood.ConclusionsWe have identified developing interneurons in human foetal cerebral cortex as potentially vulnerable to monogenic autism risk factors and the 16p11.2 microdeletion and report interneuron phenotypes in post-natal 16p11.2+/- rats.

Epidemiological studies show that prenatal exposure to nitrogen dioxide (NO2) might cause behavioral abnormalities in childhood. However, toxicological mechanisms for such effects remain unclear, and it is still difficult to define adverse outcome pathways linking exposures to behavioral phenotypes. In this study, by exposing pregnant mice to NO2 (2.5 ppm, 5 h/day) throughout gestation, we provided the first experimental evidence that prenatal NO2 exposure did cause anxiety- and depression-like behaviors in weaning male offspring but not females. Specifically, the behavioral abnormalities were associated with abnormal myelination and the alterations attributed to the delayed oligodendrocyte (OL) development in the fetus and the early stage after birth. The expression of platelet-derived growth factor receptor α (Pdgfr-α) and Olig2 significantly decreased in the NO2 group at E13.5 and E15.5, and the expression of Olig2, adenomatous polyposis coli colon (Cc1), and myelin basic protein (Mbp) was reduced in offspring at PNDs 1, 7, and 21. We performed the targeted metabolomic analysis of neurotransmitters in the placenta and found that prenatal exposure to NO2 disturbed the metabolism of placental neurotransmitters. Serotonin (5-HT) was transferred from the placenta to the fetus at E10.5, and its accumulation in the fetal forebrain might affect oligodendrocyte progenitor cell (OPC) differentiation and OL maturation and eventually be involved in behavioral abnormalities. Our findings provide new insights into the association between prenatal NO2 exposure with anxiety- and depression-like behaviors in male offspring.

Oligodendrogenesis in the human central nervous system has been observed mainly at the second trimester of gestation, a much later developmental stage compared to oligodendrogenesis in mice. Here, we characterize the transcriptomic neural diversity in the human forebrain at post-conception weeks (PCW) 8-10. Using single-cell RNA sequencing, we find evidence of the emergence of a first wave of oligodendrocyte lineage cells as early as PCW 8, which we also confirm at the epigenomic level through the use of single-cell ATAC-seq. Using regulatory network inference, we predict key transcriptional events leading to the specification of oligodendrocyte precursor cells (OPCs). Moreover, by profiling the spatial expression of 50 key genes through the use of in situ sequencing (ISS), we identify regions in the human ventral fetal forebrain where oligodendrogenesis first occurs. Our results indicate evolutionary conservation of the first wave of oligodendrogenesis between mice and humans and describe regulatory mechanisms involved in human OPC specification.

SummaryDefects in primary cilia, cellular antennas that control multiple intracellular signaling pathways, underlie several neurodevelopmental disorders, but it remains unknown how cilia control essential steps in human brain formation. Here, we show that cilia are present on the apical surface of radial glial cells in human fetal forebrain. Interfering with cilia signaling in human organoids by mutating the INPP5E gene leads to the formation of ventral telencephalic cell types instead of cortical progenitors and neurons. INPP5E mutant organoids also show increased Sonic hedgehog (SHH) signaling, and cyclopamine treatment partially rescues this ventralization. In addition, ciliary expression of SMO, GLI2, GPR161, and several intraflagellar transport (IFT) proteins is increased. Overall, these findings establish the importance of primary cilia for dorsal and ventral patterning in human corticogenesis, indicate a tissue-specific role of INPP5E as a negative regulator of SHH signaling, and have implications for the emerging roles of cilia in the pathogenesis of neurodevelopmental disorders.

Mitochondrial dysfunction is an underlying cause of childhood neurological disease secondary to the crucial role of mitochondria in proper neurodevelopment. We hypothesized that chronic intrauterine hypoxia (HPX) induces mitochondrial deficits by altering mitochondrial biogenesis and dynamics in the fetal brain. Pregnant guinea pigs were exposed to either normoxia (NMX, 21%O2) or HPX (10.5%O2) starting at 28-day (early onset, EO-HPX) or 50-day (late onset, LO-HPX) gestation until term (65days). Near-term male and female fetuses were extracted from anesthetized sows, and mitochondria were isolated from excised fetal forebrains (n = 6/group). Expression of mitochondrial complex subunits I-V (CI-CV), fission (Drp-1), and fusion (Mfn-2) proteins was measured by Western blot. CI and CIV enzyme activities were measured by colorimetric assays. Chronic HPX reduced fetal body wts and increased (P < 0.05) brain/body wt ratios of both sexes. CV subunit levels were increased in EO-HPX males only and CII levels increased in LO-HPX females only compared to NMX. Both EO- and LO-HPX decreased CIV activity in both sexes but had no effect on CI activity. EO-HPX increased Drp1 and decreased Mfn2 levels in males, while LO-HPX had no effect on either protein levels. In females, both EO-HPX and LO-HPX increased Drp1 but had no effect on Mfn2 levels. Chronic HPX alters abundance and activity of select complex subunits and shifts mitochondrial dynamics toward fission in a sex-dependent manner in the fetal guinea pig brain. This may be an underlying mechanism of reduced respiratory efficiency leading to disrupted metabolism and increased vulnerability to a second neurological injury at the time of birth in HPX fetal brains.

BackgroundPrenatal alcohol exposure is recognized for altering DNA methylation profiles of brain cells during development, and to be part of the molecular basis underpinning Fetal Alcohol Spectrum Disorder (FASD) etiology. However, we have negligible information on the effects of alcohol exposure during pre-implantation, the early embryonic window marked with dynamic DNA methylation reprogramming, and on how this may rewire the brain developmental program.ResultsUsing a pre-clinical in vivo mouse model, we show that a binge-like alcohol exposure during pre-implantation at the 8-cell stage leads to surge in morphological brain defects and adverse developmental outcomes during fetal life. Genome-wide DNA methylation analyses of fetal forebrains uncovered sex-specific alterations, including partial loss of DNA methylation maintenance at imprinting control regions, and abnormal de novo DNA methylation profiles in various biological pathways (e.g., neural/brain development).ConclusionThese findings support that alcohol-induced DNA methylation programming deviations during pre-implantation could contribute to the manifestation of neurodevelopmental phenotypes associated with FASD.

1133 Chronic intrauterine hypoxia shifts the balance of mitochondrial dynamics in the fetal guinea pig forebrain