Epigenetic Gene-Regulatory Loci in Alu Elements Associated with Autism Susceptibility in the Prefrontal Cortex of ASD.

Alu elements are transposable elements that can influence gene regulation through several mechanisms; nevertheless, it remains unclear whether dysregulation of Alu elements contributes to the neuropathology of autism spectrum disorder (ASD). In this study, we characterized transposable element expression profiles and their sequence characteristics in the prefrontal cortex tissues of ASD and unaffected individuals using RNA-sequencing data. Our results showed that most of the differentially expressed transposable elements belong to the Alu family, with 659 loci of Alu elements corresponding to 456 differentially expressed genes in the prefrontal cortex of ASD individuals. We predicted cis- and trans-regulation of Alu elements to host/distant genes by conducting correlation analyses. The expression level of Alu elements correlated significantly with 133 host genes (cis-regulation, adjusted p < 0.05) associated with ASD as well as the cell survival and cell death of neuronal cells. Transcription factor binding sites in the promoter regions of differentially expressed Alu elements are conserved and associated with autism candidate genes, including RORA. COBRA analyses of postmortem brain tissues showed significant hypomethylation in global methylation analyses of Alu elements in ASD subphenotypes as well as DNA methylation of Alu elements located near the RNF-135 gene (p < 0.05). In addition, we found that neuronal cell density, which was significantly increased (p = 0.042), correlated with the expression of genes associated with Alu elements in the prefrontal cortex of ASD. Finally, we determined a relationship between these findings and the ASD severity (i.e., ADI-R scores) of individuals with ASD. Our findings provide a better understanding of the impact of Alu elements on gene regulation and molecular neuropathology in the brain tissues of ASD individuals, which deserves further investigation.

Alteration of Extracellular Matrix Components in the Anterior Pituitary Gland of Neonatal Rats Induced by a Maternal Bisphenol A Diet during Pregnancy.

The extracellular matrix (ECM) plays crucial roles in the anterior pituitary gland via the mechanism of cell-ECM interaction. Since bisphenol A (BPA), a well-known endocrine disruptor, can cross through the placenta from mother to fetus and bind with estrogen receptors, cell populations in the neonatal anterior pituitary gland could be the target cells affected by this chemical. The present study treated maternal rats with 5000 µg/kg body weight of BPA daily throughout the pregnancy period and then investigated the changes in ECM-producing cells, i.e., pericytes and folliculostellate (FS) cells, including their ECM production in the neonatal anterior pituitary at Day 1. We found that pericytes and their collagen synthesis reduced, consistent with the increase in the number of FS cells that expressed several ECM regulators-matrix metalloproteinase (MMP) 9 and the tissue inhibitors of metalloproteinase (TIMP) family. The relative MMP9/TIMP1 ratio was extremely high, indicating that the control of ECM homeostasis was unbalanced. Moreover, transmission electron microscopy showed the unorganized cell cluster in the BPA-treated group. This study revealed that although the mother received BPA at the "no observed adverse effect" level, alterations in ECM-producing cells as well as collagen and the related ECM balancing genes occurred in the neonatal anterior pituitary gland.

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.

Are endocrine disrupting compounds environmental risk factors for autism spectrum disorder?

Recent research on the etiology of autism spectrum disorder (ASD) has shifted in part from a singular focus on genetic causes to the involvement of environmental factors and their gene interactions. This shift in focus is a result of the rapidly increasing prevalence of ASD coupled with the incomplete penetrance of this disorder in monozygotic twins. One such area of environmentally focused research is the association of exposures to endocrine disrupting compounds (EDCs) with elevated risk for ASD. EDCs are exogenous chemicals that can alter endogenous hormone activity and homeostasis, thus potentially disrupting the action of sex and other natural hormones at all stages of human development. Inasmuch as sex hormones play a fundamental role in brain development and sexual differentiation, exposure to EDCs in utero during critical stages of development can have lasting neurological and other physiological influences on the developing fetus and, ultimately, the child as well as adult. This review will focus on the possible contributions of EDCs to autism risk and pathogenesis by first discussing the influence of endogenous sex hormones on the autistic phenotype, followed by a review of documented human exposures to EDCs and associations with behaviors relevant to ASD. Mechanistic links between EDC exposures and aberrant neurodevelopment and behaviors are then considered, with emphasis on EDC-induced transcriptional profiles derived from animal and cellular studies. Finally, this review will discuss possible mechanisms through which EDC exposure can lead to persistent changes in gene expression and phenotype, which may in turn contribute to transgenerational inheritance of ASD.

Characteristics of pericytes in diethylstilbestrol (DES)-induced pituitary prolactinoma in rats.

Prolactinomas are the most common tumor of the human pituitary. They result in excessive prolactin secretion and important changes in the vasculature. Pericytes are perivascular cells associated with capillaries and have crucial roles in physiological and pathological neovascularization. We previously reported that pericytes produce type I and III collagens in the anterior pituitary of adult rats. In addition, pituitary pericytes contained well-developed cell organelles and actively synthesized collagens during early postnatal development. However, the characteristics of pericytes in pituitary tumors are unclear. In this study, we used diethylstilbestrol (DES)-treated rats as an animal model of prolactinoma. Using five common pericyte markers, more pericytes were observed in rats treated with DES for 3 months (prolactinoma) compared to the control. Transmission electron microscopy revealed that attached and semidetached pericytes exhibited active cell organelles. Moreover, we identified pericyte migration between capillaries. Although the fine structure of pituitary pericytes was active in prolactinoma, expressions of type I and III collagen mRNAs were greatly diminished. In sum, the characteristics and functions of pericytes were altered in pituitary tumors. This study is the first to clarify fine structural changes of pericytes in rat prolactinomas and improves our understanding of the function of pericytes under pathological conditions.

miR-570 interacts with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets.

Loss of platelet quality during ex vivo storage is a major concern in the transfusion medicine field and it has been known that platelet mitochondrial dysfunction is associated with storage time. In the last decade, small noncoding RNAs also known as microRNAs (miRNAs) have been reported to regulate key cellular processes through their target sequence interactions with selected mRNAs. In this study, we focused on understanding the mechanisms of platelet mitochondrial dysfunction during storage through miRNA regulation of mRNAs. RNA was isolated from day 0, day 5, and day 9 of stored human leukocyte-depleted platelets and subjected to differential miRNA and mRNA profiling. The miRNA profiling identified several miRNAs at low levels including a set of 12 different miR-548 family members (miR-548a-3p, miR-548aa, miR-548x, miR-548ac, miR-548c-3p, miR-603, miR-548aj, miR-548ae, miR-548z, miR-548u, miR-548al, and miR-570-3p). The mRNA profiling identified, among many, the mitochondrial ATP synthase subunit g (ATP5L) mRNA at high levels during storage. Target Scan algorithm for potential targets of miR-570-3p also identified ATP5L as one of its targets. We further identified two target sites for miR-570-3p in the 3' untranslated region (3'UTR) of ATP5L mRNA. While ATP5L is a subunit of F0ATPase complex, its function is not established yet. Overexpression of miR-570-3p in platelets resulted in reduced levels of ATP5L mRNA and concomitant ATP loss. These experimental results provide first-time insights into the miRNA-mRNA interactions underlying mitochondrial dysfunction in ex vivo stored platelets and warrants further investigation.

Evaluation of small noncoding RNAs in ex vivo stored human mature red blood cells: changes in noncoding RNA levels correlate with storage lesion events.

BACKGROUND: While biomarkers of storage lesions (SLs) for red blood cells (RBCs) abound, the physiologic consequences of SLs and associated important events are poorly understood. Previously we have identified differentially expressed regulatory small noncoding RNAs (ncRNAs) in stored RBCs, suggesting their role in the RBC SL process and their potential as quality biomarkers of stored RBCs. STUDY DESIGN AND METHODS: Comprehensive ncRNA expression analysis of RBCs stored for up to 56 days was performed on RNAs collected from enriched mature RBCs on Days 0, 7, 14, 28, 42, and 56. Three known RBC SL processes, that is, mature RBCs' suicidal death (eryptosis), ATP loss, and changes in RBC indices, were correlated with differentially expressed ncRNAs to gain knowledge on the SL molecular processes. RESULTS: The analysis identified four ncRNAs whose changes in the expression levels were correlated with the selected three SL processes. Differential expression on Days 14 and 28 of the four selected ncRNAs was confirmed by TaqMan quantitative reverse transcription-polymerase chain reaction analysis. Bioinformatics analysis identified potential targets and biologic functions of these ncRNAs. Overexpression of one such ncRNA, hsa-miR-196a, in a human erythroblast cell line confirmed its protective effects against the cell death and ATP loss. CONCLUSION: Overall, this study demonstrates that changes in the levels of small ncRNAs of stored RBCs correlate with some of the SL events and thus they have the potential to serve as the storage quality markers.

Investigation of chimeric transcripts derived from LINE-1 and Alu retrotransposons in cerebellar tissues of individuals with autism spectrum disorder (ASD).

LINE-1 and Alu retrotransposons are components of the human genome and have been implicated in many human diseases. These elements can influence human transcriptome plasticity in various mechanisms. Chimeric transcripts derived from LINE-1 and Alu can also impact the human transcriptome, such as exonization and post-transcriptional modification. However, its specific role in ASD neuropathology remains unclear, particularly in the cerebellum tissues. We performed RNA-sequencing of post-mortem cerebellum tissues from ASD and unaffected individuals for transposable elements profiling and chimeric transcript identification. The majority of free transcripts of transposable elements were not changed in the cerebellum tissues of ASD compared with unaffected individuals. Nevertheless, we observed that chimeric transcripts derived from LINE-1 and Alu were embedded in the transcripts of differentially expressed genes in the cerebellum of ASD, and these genes were related to developments and abnormalities of the cerebellum. In addition, the expression levels of these genes were correlated with the significantly decreased thickness of the molecular layer in the cerebellum of ASD. We also found that global methylation and expression of LINE-1 and Alu elements were not changed in ASD, but observed in the ASD sub-phenotypes. Our findings showed associations between transposable elements and cerebellar abnormalities in ASD, particularly in distinct phenotypic subgroups. Further investigations using appropriate models are warranted to elucidate the structural and functional implications of LINE-1 and Alu elements in ASD neuropathology.

Aquilaria crassna Extract Exerts Neuroprotective Effect against Benzo[a]pyrene-Induced Toxicity in Human SH-SY5Y Cells: An RNA-Seq-Based Transcriptome Analysis.

Benzo[a]pyrene (B[a]P) is known to inhibit neurodifferentiation and induce neurodegeneration. Agarwood or Aquilaria crassna (AC), a plant with health-promoting properties, may counteract the neurotoxic effects of B[a]P by promoting neuronal growth and survival. This study investigated the protective effect of AC leaf ethanolic extract (ACEE) on the B[a]P-induced impairment of neuronal differentiation. A transcriptomic analysis identified the canonical pathway, the biological network, and the differentially expressed genes (DEGs) that are changed in response to neuronal differentiation and neurogenesis. Several genes, including CXCR4, ENPP2, GAP43, GFRA2, NELL2, NFASC, NSG2, NGB, BASP1, and NEUROD1, in B[a]P-treated SH-SY5Y cells were up-regulated after treatment with ACEE. Notably, a Western blot analysis further confirmed that ACEE increased the protein levels of GAP43 and neuroglobin. B[a]P treatment led to decreased phosphorylation of Akt and increased phosphorylation of ERK in SH-SY5Y cells; however, ACEE was able to reverse these effects. Clionasterol and lupenone were identified in ACEE. Molecular docking showed that these two phytochemicals had significant interactions with CXCR4, GDNF family receptor alpha (GFRA), and retinoid X receptors (RXRs). In conclusion, ACEE may be a potential alternative medicine for the prevention of impaired neuronal differentiation and neurodegenerative diseases.

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.

Transcriptomic analysis of glutamate-induced HT22 neurotoxicity as a model for screening anti-Alzheimer's drugs.

Glutamate-induced neurotoxicity in the HT22 mouse hippocampal neuronal cell line has been recognized as a valuable cell model for the study of neurotoxicity associated with neurodegenerative diseases including Alzheimer's disease (AD). However, the relevance of this cell model for AD pathogenesis and preclinical drug screening remains to be more elucidated. While there is increasing use of this cell model in a number of studies, relatively little is known about its underlying molecular signatures in relation to AD. Here, our RNA sequencing study provides the first transcriptomic and network analyses of HT22 cells following glutamate exposure. Several differentially expressed genes (DEGs) and their relationships specific to AD were identified. Additionally, the usefulness of this cell model as a drug screening system was assessed by determining the expression of those AD-associated DEGs in response to two medicinal plant extracts, Acanthus ebracteatus and Streblus asper, that have been previously shown to be protective in this cell model. In summary, the present study reports newly identified AD-specific molecular signatures in glutamate-injured HT22 cells, suggesting that this cell can be a valuable model system for the screening and evaluation of new anti-AD agents, particularly from natural products.

Investigation of autism-related transcription factors underlying sex differences in the effects of bisphenol A on transcriptome profiles and synaptogenesis in the offspring hippocampus.

BACKGROUND: Bisphenol A (BPA) has been linked to susceptibility to autism spectrum disorder (ASD). Our recent studies have shown that prenatal BPA exposure disrupted ASD-related gene expression in the hippocampus, neurological functions, and behaviors associated with ASD in a sex-specific pattern. However, the molecular mechanisms underlying the effects of BPA are still unclear. METHODS: Transcriptome data mining and molecular docking analyses were performed to identify ASD-related transcription factors (TFs) and their target genes underlying the sex-specific effects of prenatal BPA exposure. Gene ontology analysis was conducted to predict biological functions associated with these genes. The expression levels of ASD-related TFs and targets in the hippocampus of rat pups prenatally exposed to BPA were measured using qRT-PCR analysis. The role of the androgen receptor (AR) in BPA-mediated regulation of ASD candidate genes was investigated using a human neuronal cell line stably transfected with AR-expression or control plasmid. Synaptogenesis, which is a function associated with genes transcriptionally regulated by ASD-related TFs, was assessed using primary hippocampal neurons isolated from male and female rat pups prenatally exposed to BPA. RESULTS: We found that there was a sex difference in ASD-related TFs underlying the effects of prenatal BPA exposure on the transcriptome profiles of the offspring hippocampus. In addition to the known BPA targets AR and ESR1, BPA could directly interact with novel targets (i.e., KDM5B, SMAD4, and TCF7L2). The targets of these TFs were also associated with ASD. Prenatal BPA exposure disrupted the expression of ASD-related TFs and targets in the offspring hippocampus in a sex-dependent manner. Moreover, AR was involved in the BPA-mediated dysregulation of AUTS2, KMT2C, and SMARCC2. Prenatal BPA exposure altered synaptogenesis by increasing synaptic protein levels in males but not in females, but the number of excitatory synapses was increased in female primary neurons only. CONCLUSIONS: Our findings suggest that AR and other ASD-related TFs are involved in sex differences in the effects of prenatal BPA exposure on transcriptome profiles and synaptogenesis in the offspring hippocampus. These TFs may play an essential role in an increased ASD susceptibility associated with endocrine-disrupting chemicals, particularly BPA, and the male bias of ASD.

Ergosterol isolated from cloud ear mushroom (Auricularia polytricha) attenuates bisphenol A-induced BV2 microglial cell inflammation.

Bisphenol A (BPA) has been reported to have neurotoxic properties that may increase the risk of neurodegenerative diseases by inducing neuroinflammation. Auricularia polytricha (AP) is an edible mushroom with several medicinal properties. Herein, the anti-neuroinflammatory effects of AP extracts against BPA-induced inflammation of BV2 microglial cells were investigated. Hexane (APH) and ethanol (APE) extracts of AP inhibited BPA-induced neuroinflammation in BV2 microglia by reducing microglial activation and the expression of pro-inflammatory cytokines. These anti-inflammatory effects were regulated by the NF-κB signaling pathway. In addition, APH and APE exhibited antioxidative effects by increasing the activity of the SOD-1 enzyme and restoring the accumulation of reactive oxygen species (ROS) in BPA-induced BV2 cells. Moreover, the conditioned medium prepared using BPA-induced BV2 cells demonstrated that the presence of APH or APE could attenuate ROS production in HT-22 cells. Further, ergosterol was isolated from APE and also showed anti-inflammatory and antioxidative activities. In conclusion, AP extracts and ergosterol attenuated neuroinflammation against BPA induction in BV2 microglial cells through the NF-κB signaling pathway.

LINE-1 and Alu methylation signatures in autism spectrum disorder and their associations with the expression of autism-related genes.

Long interspersed nucleotide element-1 (LINE-1) and Alu elements are retrotransposons whose abilities cause abnormal gene expression and genomic instability. Several studies have focused on DNA methylation profiling of gene regions, but the locus-specific methylation of LINE-1 and Alu elements has not been identified in autism spectrum disorder (ASD). Here we interrogated locus- and family-specific methylation profiles of LINE-1 and Alu elements in ASD whole blood using publicly-available Illumina Infinium 450 K methylation datasets from heterogeneous ASD and ASD variants (Chromodomain Helicase DNA-binding 8 (CHD8) and 16p11.2del). Total DNA methylation of repetitive elements were notably hypomethylated exclusively in ASD with CHD8 variants. Methylation alteration in a family-specific manner including L1P, L1H, HAL, AluJ, and AluS families were observed in the heterogeneous ASD and ASD with CHD8 variants. Moreover, LINE-1 and Alu methylation within target genes is inversely related to the expression level in each ASD variant. The DNA methylation signatures of the LINE-1 and Alu elements in ASD whole blood, as well as their associations with the expression of ASD-related genes, have been identified. If confirmed in future larger studies, these findings may contribute to the identification of epigenomic biomarkers of ASD.

Gold Nanoparticles Affect Pericyte Biology and Capillary Tube Formation.

Gold nanoparticles (AuNPs) are used for diagnostic and therapeutic purposes, especially antiangiogenesis, which are accomplished via inhibition of endothelial cell proliferation, migration, and tube formation. However, no research has been performed on the effects of AuNPs in pericytes, which play vital roles in endothelial cell functions and capillary tube formation during physiological and pathological processes. Therefore, the effects of AuNPs on the morphology and functions of pericytes need to be elucidated. This study treated human placental pericytes in monoculture with 20 nm AuNPs at a concentration of 30 ppm. Ki-67 and platelet-derived growth factor receptor-ß (PDGFR-ß) mRNA expression was measured using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell migration was assessed by Transwell migration assay. The fine structures of pericytes were observed by transmission electron microscopy. In addition, 30 ppm AuNP-treated pericytes and intact human umbilical vein endothelial cells were cocultured on Matrigel to form three-dimensional (3D) capillary tubes. The results demonstrated that AuNPs significantly inhibited proliferation, reduced PDGFR-ß mRNA expression, and decreased migration in pericytes. Ultrastructural analysis of pericytes revealed AuNPs in late endosomes, autolysosomes, and mitochondria. Remarkably, many mitochondria were swollen or damaged. Additionally, capillary tube formation was reduced. We found that numerous pericytes on 3D capillary tubes were round and did not extend their processes along the tubes, which resulted in more incomplete tube formation in the treatment group compared with the control group. In summary, AuNPs can affect pericyte proliferation, PDGFR-ß mRNA expression, migration, morphology, and capillary tube formation. The findings highlight the possible application of AuNPs in pericyte-targeted therapy for antiangiogenesis.

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.

Prenatal exposure to bisphenol A alters the transcriptome-interactome profiles of genes associated with Alzheimer's disease in the offspring hippocampus.

Our recent study revealed that prenatal exposure to bisphenol A (BPA) disrupted the transcriptome profiles of genes in the offspring hippocampus. In addition to genes linked to autism, several genes associated with Alzheimer's disease (AD) were found to be differentially expressed, although the association between BPA-responsive genes and AD-related genes has not been thoroughly investigated. Here, we demonstrated that in utero BPA exposure also disrupted the transcriptome profiles of genes associated with neuroinflammation and AD in the hippocampus. The level of NF-κB protein and its AD-related target gene Bace1 were significantly increased in the offspring hippocampus in a sex-dependent manner. Quantitative RT-PCR analysis also showed an increase in the expression of Tnf gene. Moreover, the reanalysis of transcriptome profiling data from several previously published BPA studies consistently showed that BPA-responsive genes were significantly associated with top AD candidate genes. The findings from this study suggest that maternal BPA exposure may increase AD risk in offspring by dysregulating genes associated with AD neuropathology and inflammation and reveal a possible relationship between AD and autism, which are linked to the same environmental factor. Sex-specific effects of prenatal BPA exposure on the susceptibility of AD deserve further investigation.

Sex Differences in the Effects of Prenatal Bisphenol A Exposure on Genes Associated with Autism Spectrum Disorder in the Hippocampus.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder inexplicably biased towards males. Although prenatal exposure to bisphenol A (BPA) has recently been associated with the ASD risk, whether BPA dysregulates ASD-related genes in the developing brain remains unclear. In this study, transcriptome profiling by RNA-seq analysis of hippocampi isolated from neonatal pups prenatally exposed to BPA was conducted and revealed a list of differentially expressed genes (DEGs) associated with ASD. Among the DEGs, several ASD candidate genes, including Auts2 and Foxp2, were dysregulated and showed sex differences in response to BPA exposure. The interactome and pathway analyses of DEGs using Ingenuity Pathway Analysis software revealed significant associations between the DEGs in males and neurological functions/disorders associated with ASD. Moreover, the reanalysis of transcriptome profiling data from previously published BPA studies consistently showed that BPA-responsive genes were significantly associated with ASD-related genes. The findings from this study indicate that prenatal BPA exposure alters the expression of ASD-linked genes in the hippocampus and suggest that maternal BPA exposure may increase ASD susceptibility by dysregulating genes associated with neurological functions known to be negatively impacted in ASD, which deserves further investigations.

Phenotypic subgrouping and multi-omics analyses reveal reduced diazepam-binding inhibitor (DBI) protein levels in autism spectrum disorder with severe language impairment.

BACKGROUND: The mechanisms underlying autism spectrum disorder (ASD) remain unclear, and clinical biomarkers are not yet available for ASD. Differences in dysregulated proteins in ASD have shown little reproducibility, which is partly due to ASD heterogeneity. Recent studies have demonstrated that subgrouping ASD cases based on clinical phenotypes is useful for identifying candidate genes that are dysregulated in ASD subgroups. However, this strategy has not been employed in proteome profiling analyses to identify ASD biomarker proteins for specific subgroups. METHODS: We therefore conducted a cluster analysis of the Autism Diagnostic Interview-Revised (ADI-R) scores from 85 individuals with ASD to predict subgroups and subsequently identified dysregulated genes by reanalyzing the transcriptome profiles of individuals with ASD and unaffected individuals. Proteome profiling of lymphoblastoid cell lines from these individuals was performed via 2D-gel electrophoresis, and then mass spectrometry. Disrupted proteins were identified and compared to the dysregulated transcripts and reported dysregulated proteins from previous proteome studies. Biological functions were predicted using the Ingenuity Pathway Analysis (IPA) program. Selected proteins were also analyzed by Western blotting. RESULTS: The cluster analysis of ADI-R data revealed four ASD subgroups, including ASD with severe language impairment, and transcriptome profiling identified dysregulated genes in each subgroup. Screening via proteome analysis revealed 82 altered proteins in the ASD subgroup with severe language impairment. Eighteen of these proteins were further identified by nano-LC-MS/MS. Among these proteins, fourteen were predicted by IPA to be associated with neurological functions and inflammation. Among these proteins, diazepam-binding inhibitor (DBI) protein was confirmed by Western blot analysis to be expressed at significantly decreased levels in the ASD subgroup with severe language impairment, and the DBI expression levels were correlated with the scores of several ADI-R items. CONCLUSIONS: By subgrouping individuals with ASD based on clinical phenotypes, and then performing an integrated transcriptome-proteome analysis, we identified DBI as a novel candidate protein for ASD with severe language impairment. The mechanisms of this protein and its potential use as an ASD biomarker warrant further study.

Integrated genome-wide Alu methylation and transcriptome profiling analyses reveal novel epigenetic regulatory networks associated with autism spectrum disorder.

Background: Alu elements are a group of repetitive elements that can influence gene expression through CpG residues and transcription factor binding. Altered gene expression and methylation profiles have been reported in various tissues and cell lines from individuals with autism spectrum disorder (ASD). However, the role of Alu elements in ASD remains unclear. We thus investigated whether Alu elements are associated with altered gene expression profiles in ASD. Methods: We obtained five blood-based gene expression profiles from the Gene Expression Omnibus database and human Alu-inserted gene lists from the TranspoGene database. Differentially expressed genes (DEGs) in ASD were identified from each study and overlapped with the human Alu-inserted genes. The biological functions and networks of Alu-inserted DEGs were then predicted by Ingenuity Pathway Analysis (IPA). A combined bisulfite restriction analysis of lymphoblastoid cell lines (LCLs) derived from 36 ASD and 20 sex- and age-matched unaffected individuals was performed to assess the global DNA methylation levels within Alu elements, and the Alu expression levels were determined by quantitative RT-PCR. Results: In ASD blood or blood-derived cells, 320 Alu-inserted genes were reproducibly differentially expressed. Biological function and pathway analysis showed that these genes were significantly associated with neurodevelopmental disorders and neurological functions involved in ASD etiology. Interestingly, estrogen receptor and androgen signaling pathways implicated in the sex bias of ASD, as well as IL-6 signaling and neuroinflammation signaling pathways, were also highlighted. Alu methylation was not significantly different between the ASD and sex- and age-matched control groups. However, significantly altered Alu methylation patterns were observed in ASD cases sub-grouped based on Autism Diagnostic Interview-Revised scores compared with matched controls. Quantitative RT-PCR analysis of Alu expression also showed significant differences between ASD subgroups. Interestingly, Alu expression was correlated with methylation status in one phenotypic ASD subgroup. Conclusion: Alu methylation and expression were altered in LCLs from ASD subgroups. Our findings highlight the association of Alu elements with gene dysregulation in ASD blood samples and warrant further investigation. Moreover, the classification of ASD individuals into subgroups based on phenotypes may be beneficial and could provide insights into the still unknown etiology and the underlying mechanisms of ASD.