Sex-specific impacts of prenatal bisphenol A exposure on genes associated with cortical development, social behaviors, and autism in the offspring's prefrontal cortex.

BACKGROUND: Recent studies have shown that prenatal BPA exposure altered the transcriptome profiles of autism-related genes in the offspring's hippocampus, disrupting hippocampal neuritogenesis and causing male-specific deficits in learning. However, the sex differences in the effects of prenatal BPA exposure on the developing prefrontal cortex, which is another brain region highly implicated in autism spectrum disorder (ASD), have not been investigated. METHODS: We obtained transcriptome data from RNA sequencing analysis of the prefrontal cortex of male and female rat pups prenatally exposed to BPA or control and reanalyzed. BPA-responsive genes associated with cortical development and social behaviors were selected for confirmation by qRT-PCR analysis. Neuritogenesis of primary cells from the prefrontal cortex of pups prenatally exposed to BPA or control was examined. The social behaviors of the pups were assessed using the two-trial and three-chamber tests. The male-specific impact of the downregulation of a selected BPA-responsive gene (i.e., Sema5a) on cortical development in vivo was interrogated using siRNA-mediated knockdown by an in utero electroporation technique. RESULTS: Genes disrupted by prenatal BPA exposure were associated with ASD and showed sex-specific dysregulation. Sema5a and Slc9a9, which were involved in neuritogenesis and social behaviors, were downregulated only in males, while Anxa2 and Junb, which were also linked to neuritogenesis and social behaviors, were suppressed only in females. Neuritogenesis was increased in males and showed a strong inverse correlation with Sema5a and Slc9a9 expression levels, whereas, in the females, neuritogenesis was decreased and correlated with Anxa2 and Junb levels. The siRNA-mediated knockdown of Sema5a in males also impaired cortical development in utero. Consistent with Anxa2 and Junb downregulations, deficits in social novelty were observed only in female offspring but not in males. CONCLUSION: This is the first study to show that prenatal BPA exposure dysregulated the expression of ASD-related genes and functions, including cortical neuritogenesis and development and social behaviors, in a sex-dependent manner. Our findings suggest that, besides the hippocampus, BPA could also exert its adverse effects through sex-specific molecular mechanisms in the offspring's prefrontal cortex, which in turn would lead to sex differences in ASD-related neuropathology and clinical manifestations, which deserves further investigation.

Investigating the impact of paternal aging on murine sperm miRNA profiles and their potential link to autism spectrum disorder.

Paternal aging has consistently been linked to an increased risk of neurodevelopmental disorders, including autism spectrum disorder (ASD), in offspring. Recent evidence has highlighted the involvement of epigenetic factors. In this study, we aimed to investigate age-related alterations in microRNA (miRNA) profiles of mouse sperm and analyze target genes regulated by differentially expressed miRNAs (DEmiRNAs). Microarray analyses were conducted on sperm samples from mice at different ages: 3 months (3 M), over 12 M, and beyond 20 M. We identified 26 miRNAs with differential expression between the 3 and 20 M mice, 34 miRNAs between the 12 and 20 M mice, and 2 miRNAs between the 3 and 12 M mice. The target genes regulated by these miRNAs were significantly associated with apoptosis/ferroptosis pathways and the nervous system. We revealed alterations in sperm miRNA profiles due to aging and suggest that the target genes regulated by these DEmiRNAs are associated with apoptosis and the nervous system, implying a potential link between paternal aging and an increased risk of neurodevelopmental disorders such as ASD. The observed age-related changes in sperm miRNA profiles have the potential to impact sperm quality and subsequently affect offspring development.

Fatty acid binding protein type 7 deficiency preserves auditory function in noise-exposed mice.

Fatty acid-binding protein 7 (FABP7) is vital for uptake and trafficking of fatty acids in the nervous system. To investigate the involvement of FABP7 in noise-induced hearing loss (NIHL) pathogenesis, we used Fabp7 knockout (KO) mice generated via CRISPR/Cas9 in the C57BL/6 background. Initial auditory brainstem response (ABR) measurements were conducted at 9 weeks, followed by noise exposure at 10 weeks. Subsequent ABRs were performed 24 h later, with final measurements at 12 weeks. Inner ears were harvested 24 h after noise exposure for RNA sequencing and metabolic analyses. We found no significant differences in initial ABR measurements, but Fabp7 KO mice showed significantly lower thresholds in the final ABR measurements. Hair cell survival was also enhanced in Fabp7 KO mice. RNA sequencing revealed that genes associated with the electron transport chain were upregulated or less impaired in Fabp7 KO mice. Metabolomic analysis revealed various alterations, including decreased glutamate and aspartate in Fabp7 KO mice. In conclusion, FABP7 deficiency mitigates cochlear damage following noise exposure. This protective effect was supported by the changes in gene expression of the electron transport chain, and in several metabolites, including excitotoxic neurotransmitters. Our study highlights the potential therapeutic significance of targeting FABP7 in NIHL.

ADP Ribosylation Factor 4 (Arf4) Regulates Radial Migration through N-Cadherin Trafficking during Cerebral Cortical Development.

During the development of the cerebral cortex, N-cadherin plays a crucial role in facilitating radial migration by enabling cell-to-cell adhesion between migrating neurons and radial glial fibers or Cajar-Reztius cells. ADP ribosylation factor 4 (Arf4) and Arf5, which belong to the Class II Arf small GTPase subfamily, control membrane trafficking in the endocytic and secretory pathways. However, their specific contribution to cerebral cortex development remains unclear. In this study, we sought to investigate the functional involvement of Class II Arfs in radial migration during the layer formation of the cerebral cortex using mouse embryos and pups. Our findings indicate that knock-down of Arf4, but not Arf5, resulted in the stalling of transfected neurons with disorientation of the Golgi in the upper intermediate zone (IZ) and reduction in the migration speed in both the IZ and cortical plate (CP). Migrating neurons with Arf4 knock-down exhibited cytoplasmic accumulation of N-cadherin, along with disturbed organelle morphology and distribution. Furthermore, supplementation of exogenous N-cadherin partially rescued the migration defect caused by Arf4 knock-down. In conclusion, our results suggest that Arf4 plays a crucial role in regulating radial migration via N-cadherin trafficking during cerebral cortical development.

Whole-brain mapping of neuronal activity evoked by maternal separation in neonatal mice: An association with ultrasound vocalization.

Neonatal mice emit ultrasonic vocalizations (USVs) when separated from their mothers. Since the USVs attract their mothers' attention and trigger maternal retrieval, they are considered to serve as social signals for communication. We have modeled paternal aging effects on the vocal communication of offspring in mice. However, little is known about the neural basis underlying neonatal USV production. To identify responsible brain regions driving the vocal behavior, we comprehensively mapped the neuronal activity associated with USV production in the entire brain of mice at postnatal day 6 (P6). Using an expression of immediate-early gene c-Fos as a neuronal activity marker, correlations between the numbers of USVs and c-Fos positive neurons were analyzed. We identified 23 candidate brain regions associated with USV production in the mice at P6. Our study would be a first step toward comprehensively understanding the neuronal mechanisms that regulate and develop vocal behaviors in neonatal mice.

A novel feature of the ancient organ: A possible involvement of the subcommissural organ in neurogenic/gliogenic potential in the adult brain.

The subcommissural organ (SCO) is a circumventricular organ highly conserved in vertebrates from Cyclostomata such as lamprey to mammals including human. The SCO locates in the boundary between the third ventricle and the entrance of the aqueduct of Sylvius. The SCO functions as a secretory organ producing a variety of proteins such as SCO-spondin, transthyretin, and basic fibroblast growth factor (FGF) into the cerebrospinal fluid (CSF). A significant contribution of the SCO has been thought to maintain the homeostasis of CSF dynamics. However, evidence has shown a possible role of SCO on neurogenesis in the adult brain. This review highlights specific features of the SCO related to adult neurogenesis, suggested by the progress of understanding SCO functions. We begin with a brief history of the SCO discovery and continue to structural features, gene expression, and a possible role in adult neurogenesis suggested by the SCO transplant experiment.

Synaptic pruning through glial synapse engulfment upon motor learning.

Synaptic pruning is a fundamental process of neuronal circuit refinement in learning and memory. Accumulating evidence suggests that glia participates in sculpting the neuronal circuits through synapse engulfment. However, whether glial involvement in synaptic pruning has a role in memory formation remains elusive. Using newly developed phagocytosis reporter mice and three-dimensional ultrastructural characterization, we found that synaptic engulfment by cerebellar Bergmann glia (BG) frequently occurred upon cerebellum-dependent motor learning in mice. We observed increases in pre- and postsynaptic nibbling by BG along with a reduction in spine volume after learning. Pharmacological blockade of engulfment with Annexin V inhibited both the spine volume reduction and overnight improvement of motor adaptation. These results indicate that BG contribute to the refinement of the mature cerebellar cortical circuit through synaptic engulfment during motor learning.

Deficiency of CHAMP1, a gene related to intellectual disability, causes impaired neuronal development and a mild behavioural phenotype.

CHAMP1 is a gene associated with intellectual disability, which was originally identified as being involved in the maintenance of kinetochore-microtubule attachment. To explore the neuronal defects caused by CHAMP1 deficiency, we established mice that lack CHAMP1. Mice that are homozygous knockout for CHAMP1 were slightly smaller than wild-type mice and died soon after birth on pure C57BL/6J background. Although gross anatomical defects were not found in CHAMP1 -/- mouse brains, mitotic cells were increased in the cerebral cortex. Neuronal differentiation was delayed in CHAMP1 -/- neural stem cells in vitro, which was also suggested in vivo by CHAMP1 knockdown. In a behavioural test battery, adult CHAMP1 heterozygous knockout mice showed mild memory defects, altered social interaction, and depression-like behaviours. In transcriptomic analysis, genes related to neurotransmitter transport and neurodevelopmental disorder were downregulated in embryonic CHAMP1 -/- brains. These results suggest that CHAMP1 plays a role in neuronal development, and CHAMP1-deficient mice resemble some aspects of individuals with CHAMP1 mutations.

Advanced paternal age diversifies individual trajectories of vocalization patterns in neonatal mice.

Infant crying is a communicative behavior impaired in neurodevelopmental disorders (NDDs). Because advanced paternal age is a risk factor for NDDs, we performed computational approaches to evaluate how paternal age affected vocal communication and body weight development in C57BL/6 mouse offspring from young and aged fathers. Analyses of ultrasonic vocalization (USV) consisting of syllables showed that advanced paternal age reduced the number and duration of syllables, altered the syllable composition, and caused lower body weight gain in pups. Pups born to young fathers had convergent vocal characteristics with a rich repertoire, whereas those born to aged fathers exhibited more divergent vocal patterns with limited repertoire. Additional analyses revealed that some pups from aged fathers displayed atypical USV trajectories. Thus, our study indicates that advanced paternal age has a significant effect on offspring's vocal development. Our computational analyses are effective in characterizing altered individual diversity.

Regulation of male germline transmission patterns by the Trp53-Cdkn1a pathway.

A small number of offspring are born from the numerous sperm generated from spermatogonial stem cells (SSCs). However, little is known regarding the rules and molecular mechanisms that govern germline transmission patterns. Here we report that the Trp53 tumor suppressor gene limits germline genetic diversity via Cdkn1a. Trp53-deficient SSCs outcompeted wild-type (WT) SSCs and produced significantly more progeny after co-transplantation into infertile mice. Lentivirus-mediated transgenerational lineage analysis showed that offspring bearing the same virus integration were repeatedly born in a non-random pattern from WT SSCs. However, SSCs lacking Trp53 or Cdkn1a sired transgenic offspring in random patterns with increased genetic diversity. Apoptosis of KIT+ differentiating germ cells was reduced in Trp53- or Cdkn1a-deficient mice. Reduced CDKN1A expression in Trp53-deficient spermatogonia suggested that Cdkn1a limits genetic diversity by supporting apoptosis of syncytial spermatogonial clones. Therefore, the TRP53-CDKN1A pathway regulates tumorigenesis and the germline transmission pattern.

Thirty Years' History since the Discovery of Pax6: From Central Nervous System Development to Neurodevelopmental Disorders.

Pax6 is a sequence-specific DNA binding transcription factor that positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system (CNS). As indicated by the morphological and functional abnormalities in spontaneous Pax6 mutant rodents, Pax6 plays pivotal roles in various biological processes in the CNS. At the initial stage of CNS development, Pax6 is responsible for brain patterning along the anteroposterior and dorsoventral axes of the telencephalon. Regarding the anteroposterior axis, Pax6 is expressed inversely to Emx2 and Coup-TF1, and Pax6 mutant mice exhibit a rostral shift, resulting in an alteration of the size of certain cortical areas. Pax6 and its downstream genes play important roles in balancing the proliferation and differentiation of neural stem cells. The Pax6 gene was originally identified in mice and humans 30 years ago via genetic analyses of the eye phenotypes. The human PAX6 gene was discovered in patients who suffer from WAGR syndrome (i.e., Wilms tumor, aniridia, genital ridge defects, mental retardation). Mutations of the human PAX6 gene have also been reported to be associated with autism spectrum disorder (ASD) and intellectual disability. Rodents that lack the Pax6 gene exhibit diverse neural phenotypes, which might lead to a better understanding of human pathology and neurodevelopmental disorders. This review describes the expression and function of Pax6 during brain development, and their implications for neuropathology.

The subcommissural organ maintains features of neuroepithelial cells in the adult mouse.

The subcommissural organ (SCO) is a part of the circumventricular organs located in the dorsocaudal region of the third ventricle at the entrance of the aqueduct of Sylvius. The SCO comprises epithelial cells and produces high molecular weight glycoproteins, which are secreted into the third ventricle and become part of Reissner's fibre in the cerebrospinal fluid. Abnormal development of the SCO has been linked with congenital hydrocephalus, a condition characterized by excessive accumulation of cerebrospinal fluid in the brain. In the present study, we characterized the SCO cells in the adult mouse brain to gain insights into the possible role of this brain region. Immunohistochemical analyses revealed that expression of Pax6, a transcription factor essential for SCO differentiation during embryogenesis, is maintained in the SCO at postnatal stages from P0 to P84. SCO cells in the adult brain expressed known neural stem/progenitor cell (NSPC) markers, Sox2 and vimentin. The adult SCO cells also expressed proliferating marker PCNA, although expression of another proliferation marker Ki67, indicating a G2 /M phase, was not detected. The SCO cells did not incorporate BrdU, a marker for DNA synthesis in the S phase. Therefore, the SCO cells have a potential for proliferation but are quiescent for cell division in the adult. The SCO cells also expressed GFAP, a marker for astrocytes or NSPCs, but not NeuN (for neurons). A few cells positive for Iba1 (microglia), Olig2 (for oligodendrocytes) and PDGFRα (oligodendrocyte progenitors) existed within or on the periphery of the SCO. These findings revealed that the SCO cells have a unique feature as secretory yet immature neuroepithelial cells in the adult mouse brain.

Detection of REST expression in the testis using epitope-tag knock-in mice generated by genome editing.

BACKGROUND: Repressor element 1-silencing transcription factor (REST) is a master regulator that is highly expressed in multipotent stem cells to repress gene networks involving a wide range of biological processes. A recent study has suggested that REST might be involved in a misregulation of its target genes in the embryonic brain of offspring derived from aged fathers. However, detailed analyses of the REST function in spermatogenesis are lacking due to difficulty in the detection of REST protein in specific cell types. RESULTS: To determine localization of REST, we generated an epitope tag knock-in (KI) mouse line with the C-terminus insertion of a podoplanin (PA)-tag at an endogenous Rest locus by the CRISPR/Cas9 system. Localization of the PA-tag was confirmed in neural stem cells marked with Pax6 in the embryonic brain. Moreover, PA-tagged REST was detected in undifferentiated and differentiating spermatogonia as well as Sertoli cells in both neonatal and adult testes. CONCLUSIONS: We demonstrate that REST is expressed at the early step of spermatogenesis and suggest a possibility that REST may modulate the epigenetic state of male germline cells. Our KI mice may be useful for studying REST-associated molecular mechanisms of neurodevelopmental and age-related disorders.

Multiple Functions of the Dmrt Genes in the Development of the Central Nervous System.

The Dmrt genes encode the transcription factor containing the DM (doublesex and mab-3) domain, an intertwined zinc finger-like DNA binding module. While Dmrt genes are mainly involved in the sexual development of various species, recent studies have revealed that Dmrt genes, which belong to the DmrtA subfamily, are differentially expressed in the embryonic brain and spinal cord and are essential for the development of the central nervous system. Herein, we summarize recent studies that reveal the multiple functions of the Dmrt genes in various aspects of vertebrate neural development, including brain patterning, neurogenesis, and the specification of neurons.

Autism-Related Transcription Factors Underlying the Sex-Specific Effects of Prenatal Bisphenol A Exposure on Transcriptome-Interactome Profiles in the Offspring Prefrontal Cortex.

Bisphenol A (BPA) is an environmental risk factor for autism spectrum disorder (ASD). BPA exposure dysregulates ASD-related genes in the hippocampus and neurological functions of offspring. However, whether prenatal BPA exposure has an impact on genes in the prefrontal cortex, another brain region highly implicated in ASD, and through what mechanisms have not been investigated. Here, we demonstrated that prenatal BPA exposure disrupts the transcriptome-interactome profiles of the prefrontal cortex of neonatal rats. Interestingly, the list of BPA-responsive genes was significantly enriched with known ASD candidate genes, as well as genes that were dysregulated in the postmortem brain tissues of ASD cases from multiple independent studies. Moreover, several differentially expressed genes in the offspring's prefrontal cortex were the targets of ASD-related transcription factors, including AR, ESR1, and RORA. The hypergeometric distribution analysis revealed that BPA may regulate the expression of such genes through these transcription factors in a sex-dependent manner. The molecular docking analysis of BPA and ASD-related transcription factors revealed novel potential targets of BPA, including RORA, SOX5, TCF4, and YY1. Our findings indicated that prenatal BPA exposure disrupts ASD-related genes in the offspring's prefrontal cortex and may increase the risk of ASD through sex-dependent molecular mechanisms, which should be investigated further.

Transgenerational epigenetic information through the sperm: Sperm cells not just merely supply half of the genome for new life; they also seem to transmit additional information via epigenetic modifications.

The biological cause for the increase in Autism Spectrum Disorder may be fathers' older age and the epigenetic marks it leaves on sperm cells.

Low-intensity pulsed ultrasound therapy promotes recovery from stroke by enhancing angio-neurogenesis in mice in vivo.

Since the treatment window of thrombolytic therapy for stroke is limited, new therapy remains to be developed. We have recently developed low-intensity pulsed ultrasound (LIPUS) therapy to improve cognitive dysfunction in mouse models of vascular dementia and Alzheimer's disease. Here, we further aimed to examine whether our LIPUS therapy improves neurological recovery from ischemic stroke, and if so, to elucidate the mechanisms involved. In a mouse model of middle cerebral artery occlusion (MCAO), we applied LIPUS (32 cycles, 193 mW/cm2) to the whole brain 3 times in the first week (days 1, 3, and 5) after MCAO. We evaluated neurological functions using behavioral tests and performed histological analyses. Furthermore, to elucidate how LIPUS works within the injured brain, we also tested the effects of LIPUS in endothelial nitric oxide synthase (eNOS)-deficient (eNOS-/-) mice. In wild-type mice, the LIPUS therapy markedly improved neurological functions in the tightrope and rotarod tests at 28 days after MCAO. Histological analyses showed that the LIPUS therapy significantly increased the numbers of CD31-positive blood vessels in the perifocal lesion and doublecortin (DCX)-positive neurons in the ischemic striatum, indicating the angio-neurogenesis effects of the therapy. Importantly, these beneficial effects of the LIPUS therapy were totally absent in eNOS-/- mice. No adverse effects of the LIPUS therapy were noted. These results indicate that the LIPUS therapy improves neurological functions after stroke through enhanced neuro-angiogenesis in mice in vivo in an eNOS-dependent manner, suggesting that it could a novel and non-invasive therapeutic option for stroke.

Sex differences in the effects of prenatal bisphenol A exposure on autism-related genes and their relationships with the hippocampus functions.

Our recent study has shown that prenatal exposure to bisphenol A (BPA) altered the expression of genes associated with autism spectrum disorder (ASD). In this study, we further investigated the effects of prenatal BPA exposure on ASD-related genes known to regulate neuronal viability, neuritogenesis, and learning/memory, and assessed these functions in the offspring of exposed pregnant rats. We found that prenatal BPA exposure increased neurite length, the number of primary neurites, and the number of neurite branches, but reduced the size of the hippocampal cell body in both sexes of the offspring. However, in utero exposure to BPA decreased the neuronal viability and the neuronal density in the hippocampus and impaired learning/memory only in the male offspring while the females were not affected. Interestingly, the expression of several ASD-related genes (e.g. Mief2, Eif3h, Cux1, and Atp8a1) in the hippocampus were dysregulated and showed a sex-specific correlation with neuronal viability, neuritogenesis, and/or learning/memory. The findings from this study suggest that prenatal BPA exposure disrupts ASD-related genes involved in neuronal viability, neuritogenesis, and learning/memory in a sex-dependent manner, and these genes may play an important role in the risk and the higher prevalence of ASD in males subjected to prenatal BPA exposure.

Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF.

Advanced paternal age can have deleterious effects on various traits in the next generation. Here, we establish a paternal-aging model in mice to understand the molecular mechanisms of transgenerational epigenetics. Whole-genome target DNA methylome analyses of sperm from aged mice reveal more hypo-methylated genomic regions enriched in REST/NRSF binding motifs. Gene set enrichment analyses also reveal the upregulation of REST/NRSF target genes in the forebrain of embryos from aged fathers. Offspring derived from young mice administrated with a DNA de-methylation drug phenocopy the abnormal vocal communication of pups derived from aged fathers. In conclusion, hypo-methylation of sperm DNA can be a key molecular feature modulating neurodevelopmental programs in offspring by causing fluctuations in the expression of REST/NRSF target genes.

Identification of FMRP target mRNAs in the developmental brain: FMRP might coordinate Ras/MAPK, Wnt/ß-catenin, and mTOR signaling during corticogenesis.

Corticogenesis is one of the most critical and complicated processes during embryonic brain development. Any slight impairment in corticogenesis could cause neurodevelopmental disorders such as Fragile X syndrome (FXS), of which symptoms contain intellectual disability (ID) and autism spectrum disorder (ASD). Fragile X mental retardation protein (FMRP), an RNA-binding protein responsible for FXS, shows strong expression in neural stem/precursor cells (NPCs) during corticogenesis, although its function during brain development remains largely unknown. In this study, we attempted to identify the FMRP target mRNAs in the cortical primordium using RNA immunoprecipitation sequencing analysis in the mouse embryonic brain. We identified 865 candidate genes as targets of FMRP involving 126 and 118 genes overlapped with ID and ASD-associated genes, respectively. These overlapped genes were enriched with those related to chromatin/chromosome organization and histone modifications, suggesting the involvement of FMRP in epigenetic regulation. We further identified a common set of 17 FMRP "core" target genes involved in neurogenesis/FXS/ID/ASD, containing factors associated with Ras/mitogen-activated protein kinase, Wnt/ß-catenin, and mammalian target of rapamycin (mTOR) pathways. We indeed showed overactivation of mTOR signaling via an increase in mTOR phosphorylation in the Fmr1 knockout (Fmr1 KO) neocortex. Our results provide further insight into the critical roles of FMRP in the developing brain, where dysfunction of FMRP may influence the regulation of its mRNA targets affecting signaling pathways and epigenetic modifications.