Male autism spectrum disorder is linked to brain aromatase disruption by prenatal BPA in multimodal investigations and 10HDA ameliorates the related mouse phenotype
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Abstract
Male sex, early life chemical exposure and the brain aromatase enzyme have been implicated in autism spectrum disorder (ASD). In the Barwon Infant Study birth cohort (n = 1074), higher prenatal maternal bisphenol A (BPA) levels are associated with higher ASD symptoms at age 2 and diagnosis at age 9 only in males with low aromatase genetic pathway activity scores. Higher prenatal BPA levels are predictive of higher cord blood methylation across the CYP19A1 brain promoter I.f region (P = 0.009) and aromatase gene methylation mediates (P = 0.01) the link between higher prenatal BPA and brain-derived neurotrophic factor methylation, with independent cohort replication. BPA suppressed aromatase expression in vitro and in vivo. Male mice exposed to mid-gestation BPA or with aromatase knockout have ASD-like behaviors with structural and functional brain changes. 10-hydroxy-2-decenoic acid (10HDA), an estrogenic fatty acid alleviated these features and reversed detrimental neurodevelopmental gene expression. Here we demonstrate that prenatal BPA exposure is associated with impaired brain aromatase function and ASD-related behaviors and brain abnormalities in males that may be reversible through postnatal 10HDA intervention.
Introduction
Autism spectrum disorder (ASD or autism) is a clinically diagnosed neurodevelopmental condition in which an individual has impaired social communication and interaction, as well as restricted, repetitive behavior patterns1. The estimated prevalence of ASD is approximately 1–2% in Western countries2, with evidence that the incidence of ASD is increasing over time3. While increased incidence is partly attributable to greater awareness of ASD4, other factors including early life environment, genes and their interplay are important5. Strikingly, up to 80% of individuals diagnosed with ASD are male, suggesting sex-specific neurodevelopment underlies this condition5.
Brain aromatase, encoded by CYP19A1 and regulated via brain promoter I.f6,7,8 converts neural androgens to neural estrogens9. During fetal development, aromatase expression within the brain is high in males10 in the amygdala11,12. Notably, androgen disruption is implicated in the extreme male brain theory for ASD13, and postmortem analysis of male ASD adults show markedly reduced aromatase activity compared to age-matched controls. Furthermore, CYP19A1 aromatase expression was reduced by 38% in the postmortem male ASD prefrontal cortex14, as well as by 52% in neuronal cell lines derived from males with ASD15. Environmental factors, including exposure to endocrine-disrupting chemicals such as bisphenols, can disrupt brain aromatase function16,17,18.
Early life exposure to endocrine-disrupting chemicals, including bisphenols, has separately been proposed to contribute to the temporal increase in ASD prevalence19. Exposure to these manufactured chemicals is now widespread through their presence in plastics and epoxy linings in food and drink containers and other packaging products20. Although bisphenol A (BPA) has since been replaced by other bisphenols such as bisphenol S in BPA-free plastics, all bisphenols are endocrine-disrupting chemicals that can alter steroid signaling and metabolism21. Elevated maternal prenatal BPA levels are associated with child neurobehavioral issues20 including ASD-related symptoms22,23, with many of these studies reporting sex-specific effects20,22,23,24. Furthermore, studies in rodents have found that prenatal BPA exposure is associated with gene dysregulation in the male hippocampus accompanied by neuronal and cognitive abnormalities in male but not female animals20,23,24. One potential explanation is that epigenetic programming by bisphenols increases aromatase gene methylation, leading to its reduced cellular expression16 and a deficiency in aromatase-dependent estrogen signaling. If such is the case, it is possible that estrogen supplementation, such as with 10-hydroxy-2-decenoic acid (10HDA), a major lipid component of the royal jelly of honeybees, may be relevant as a nutritional intervention for ASD. Indeed, 10HDA is known to influence homeostasis through its intracellular effects on estrogen responsive elements that regulate downstream gene expression25,26, as well as its capacity to influence neurogenesis in vitro27.
Here, we have investigated whether higher prenatal BPA exposure leads to an elevated risk of ASD in males and explore aromatase as a potential underlying mechanism. We demonstrate in a preclinical (mouse) model that postnatal administration of 10HDA, an estrogenic fatty acid, can ameliorate ASD-like phenotypes in young mice prenatally exposed to BPA.
Human studies
We examined the interplay between prenatal BPA, aromatase function and sex in relation to human ASD symptoms and diagnosis in the Barwon Infant Study (BIS) birth cohort28. By the BIS cohort health review at 7-11 years (mean = 9.05, SD = 0.74; hereafter referred to as occurring at 9 years), 43 children had a pediatrician- or psychiatrist- confirmed diagnosis of ASD against the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria, as of the 30th of June 2023. ASD diagnosis was over-represented in boys with a 2.1:1 ratio at 9 years (29 boys and 14 girls; Supplementary Table 1). In BIS, the DSM-5 oriented autism spectrum problems (ASP) scale of the Child Behavior Checklist (CBCL) at age 2 years29 predicted diagnosed autism strongly at age 4 and moderately at age 9 in receiver operating characteristic (ROC) curve analyses; area under the curve (AuC) of 0.92 (95% CI 0.82, 1.00)30 and 0.70 (95% CI 0.60, 0.80), respectively. The median CBCL ASP score in ASD cases and non-cases at 9 years was 51 (IQR = 50, 58) and 50 (IQR = 50, 51), respectively. Only ASD cases with a pediatrician-confirmed diagnosis of ASD against the DSM-5, as verified by the 30th of June 2023, were included in this report. We thus examined both outcomes (ASP scale and ASD diagnosis) as indicators of ASD over the life course from ages 2 to 9 years (Supplementary Table 1). Quality control information for the measurement of BPA is presented in Supplementary Table 2.
BPA effects on ASD symptoms at age 2 years are most evident in boys genetically predisposed to low aromatase enzyme activity
Of the 676 infants with CBCL data in the cohort sample, 249 (36.8%) had an ASP score above the median based on CBCL normative data (Supplementary Table 1). From a whole genome SNP array (Supplementary Methods), a CYP19A1 genetic score for aromatase enzyme activity was developed based on five single nucleotide polymorphisms (SNPs; rs12148604, rs4441215, rs11632903, rs752760, rs2445768) associated with lower estrogen levels31. Among 595 children with prenatal BPA and CBCL data, those in the top quartile of the genetic predisposition score, that is, children with three or more variants associated with lower levels of estrogens were classified as ‘low aromatase activity’ with the remaining classified as ‘high aromatase activity’ (Fig. 1). Regression analyses stratified by this genetic score and child’s sex were performed and an association between high prenatal BPA exposure (top quartile (>2.18 μg/L) and greater ASP scores was only seen in males with low aromatase activity, with a matched OR of 3.56 (95% CI 1.13, 11.22); P = 0.03 (Supplementary Table 4). These findings were minimally altered following adjustment for additional potential confounders. Among males with low aromatase activity, the fraction with higher than median ASP scores attributable to high BPA exposure (the population attributable fraction) was 11.9% (95% CI 4.3%, 19.0%). These results indicate a link between low aromatase function and elevated ASP scores. A sensitivity analysis using an independent weighted CYP19A1 genetic score confirmed these findings. For the additional score, the Genotype-Tissue Expression (GTEx) portal was first used to identify the top five expression quantitative trait loci (eQTLs; rs7169770, rs1065778, rs28757202, rs12917091, rs3784307) for CYP19A1 in any tissue type that showed a consistent effect direction in brain tissue. A functional genetic score was then computed for each BIS participant by summing the number of aromatase-promoting alleles they carry across the five eQTLs, weighted by their normalized effect size (NES) in amygdala tissue. This score captures genetic contribution to cross-tissue aromatase activity with a weighting towards the amygdala, a focus in our animal studies. The score was then reversed so that higher values indicate lower aromatase activity and children in the top quartile were classified as ‘low aromatase activity’ with the remaining classified as ‘high aromatase activity’. Again, a positive association between prenatal BPA exposure and ASP scores was only seen in males with low aromatase activity, with a matched OR of 3.74 (95% CI 1.12, 12.50); P = 0.03. Additional adjustment for individual potential confounders provided matched ORs between 3.13 to 3.85 (Supplementary Table 5).
BPA effects on ASD diagnosis at 9 years are most evident in boys genetically predisposed to low aromatase enzyme activity
In subgroup analyses where we stratified by child’s sex and unweighted CYP19A1 genetic score, the results were consistent with those found at 2 years. A positive association between high prenatal BPA exposure and ASD diagnosis was only seen in males with low aromatase activity, with a matched OR of 6.24 (95% CI 1.02, 38.26); P = 0.05 (Supplementary Table 4). In this subgroup, the fraction of ASD cases attributable to high BPA exposure (the population attributable fraction) was 12.6% (95% CI 5.8%, 19.0%). In a sensitivity analysis where the weighted CYP19A1 genetic score was used, a similar effect size was observed in this subgroup; matched OR = 6.06 (95% CI 0.93, 39.43), P = 0.06 (Supplementary Table 4).
Higher prenatal BPA exposure predicts higher methylation of the CYP19A1 brain promoter PI.f in human cord blood
We investigated the link between BPA and aromatase further by evaluating epigenetic regulation of the aromatase gene at birth in the same BIS cohort. CYP19A1 (in humans; Cyp19a1 in the mouse) has eleven tissue-specific untranslated first exons under the regulation of tissue-specific promoters. The brain-specific promoters are PI.f6,7,8 and PII17. For a window positioned directly over the primary brain promoter PI.f, higher BPA was positively associated with average methylation, mean increase = 0.05% (95% CI 0.01%, 0.09%); P = 0.009 (Fig. 2). Higher BPA levels predicted methylation across both PI.f and PII as a composite, mean increase per log 2 increase = 0.06% (95% CI 0.01%, 0.10%); P = 0.009. Methylation of a control window, comprising the remaining upstream region of the CYP19A1 promoter and excluding both PI.f and PII brain promoters, did not associate with BPA, P = 0.12. These findings persisted after adjustment for the CYP19A1 genetic score for aromatase enzyme activity. Thus, higher prenatal BPA exposure was associated with increased methylation of brain-specific promoters in CYP19A1. Sex-specific differences were not observed. While these effects were identified in cord blood, methylation of CYP19A1 shows striking concordance between blood and brain tissue (Spearman’s rank correlation across the whole gene: ρ = 0.74 (95% CI 0.59, 0.84); over promoter PI.f window: ρ = 0.94 (95% CI 0.54, 0.99)32. Thus, prenatal BPA exposure significantly associates with disruption of the CYP19A1 brain promoter and hence likely the level of its protein product, aromatase.
Replication of the association between higher BPA levels and hypermethylation of the CYP19A1 brain promoter
Previously, the Columbia Centre for Children’s Health Study-Mothers and Newborns (CCCEH-MN) cohort (Supplementary Table 3) found BPA increased methylation of the BDNF CREB-binding region of promoter IV both in rodent blood and brain tissue at P28 and in infant cord blood in the CCCEH-MN cohort33. In rodents, BDNF hypermethylation occurred concomitantly with reduced BDNF expression in the brain33. Re-examining the CCCEH-MN cohort, BPA level was also associated with hypermethylation of the aromatase brain promoter P1.f (adjusted mean increase 0.0040, P = 0.0089), replicating the BIS cohort finding.
Molecular mediation of higher BPA levels and hypermethylation of BDNF through higher methylation of CYP19A1
In BIS, we aimed to reproduce these BDNF findings and extend them to investigate aromatase methylation as a potential mediator of the BPA-BDNF relationship. A link between aromatase and methylation of the BDNF CREB-binding region is plausible given that estrogen (produced by aromatase) is known to elevate brain expression of CREB34,35. In BIS, male infants exposed to BPA (categorized as greater than 4 µg/L vs. rest, following the CCCEH-MN study) had greater methylation of the BDNF CREB-binding site (adjusted mean increase = 0.0027, P = 0.02). This was also evident overall (adjusted mean increase = 0.0023, P = 0.006), but not for females alone (adjusted mean increase = 0.0019, P = 0.13). We then assessed whether methylation of aromatase promoter P1.f mediates this association. In both cohorts, aromatase methylation was positively associated with BDNF CREB-binding-site methylation in males (BIS, adjusted mean increase = 0.07, P = 0.0008; CCCEH-MN, adjusted mean increase = 0.91, P = 0.0016). In the two overall cohorts, there was evidence that the effect of increased BPA on BDNF hypermethylation was mediated partly through higher aromatase methylation (BIS, indirect effect, P = 0.012; CCCEH-MN, indirect effect, P = 0.012).
Prenatal programming laboratory studies—BPA effects on cellular aromatase expression in vitro, neuronal development as well as behavioral phenotype in mice
BPA reduces aromatase expression in human neuroblastoma SH-SY5Y cell cultures
To validate the findings of our human observational studies on BPA and aromatase expression, we began by studying the effects of BPA exposure on aromatase expression in the human neuroblastoma cell line SH-SY5Y (Fig. 3A). Indeed, the aromatase protein levels more than halved in the presence of BPA 50 μg/L (P = 0.01; Fig. 3B) by Western Blot analysis.
The effects of prenatal BPA exposure on aromatase-expressing neurons within the amygdala of male mice
There is a prominent expression of aromatase within cells of the male medial amygdala (MeA)11. To visualize aromatase-expressing cells, we studied genetically modified, Cyp19-EGFP transgenic mice harboring a single copy of a bacterial artificial chromosome (BAC) encoding the coding sequence for enhanced green fluorescent protein (EGFP) inserted upstream of the ATG start codon for aromatase (Cyp19a1)11 (see Methods). As shown, EGFP (EGFP+) expression in male mice was detected as early as embryonic day (E) 11.5 (Supplementary Fig. 1), indicating that aromatase gene expression is detectable in early CNS development.
To study the effects of prenatal BPA exposure on brain development, pregnant dams were subject to BPA at a dose of 50 μg/kg/day via subcutaneous injection, or a vehicle injection during a mid-gestation window of E10.5 to E14.5, which coincides with amygdala development. This dose matches current USA recommendations36,37 as well as the Tolerable Daily Intake (TDI) set by the European Food Safety Authority (EFSA) at the time that the mothers in our human cohort were pregnant28,38. In these experiments, we observed that prenatal BPA exposure led to a 37% reduction (P = 0.004) in EGFP+ neurons in the MeA of male EGFP+ mice compared to control mice (Fig. 3C). These results are consistent with our findings in SH-SY5Y cells that indicate that BPA exposure leads to a marked reduction in the cellular expression of aromatase.
Prenatal BPA exposure at mid-gestation influences social approach behavior in male mice
Next, we evaluated post-weaning social approach behavior (postnatal (P) days P21-P24) using a modified three-chamber social interaction test39 (Fig. 4C). As shown, male mice with prenatal exposure to BPA were found to spend less time investigating sex- and age-matched stranger mice, when compared with vehicle-treated males (with a mean time ± SEM of 101.2 sec ± 11.47 vs. 177.3 s ± 26.97, P = 0.0004; Fig. 4A). Such differences were not observed for female mice prenatally exposed to BPA (Fig. 4A). As a control for these studies, we found that the presence of the EGFP BAC transgene is not relevant to behavioral effects in the test (Supplementary Fig. 2), and the proportions of EGFP transgenic mice were not significantly different across BPA-exposed and vehicle-exposed cohorts.
To determine if the effects of prenatal BPA exposure were developmentally restricted, we delivered subcutaneous injections (50 µg/kg/day) of BPA to pregnant dams at early (E0.5–E9.5), mid (E10.5–E14.5), and late (E15.5–E20.5) stages of gestation. From these experiments, we found that while male pups exposed to BPA in mid-gestation developed a social approach deficit, such behavioral impairments were not observed for early or late gestation BPA exposure (Supplementary Fig. 3). In addition, we performed experiments in which BPA was available to dams by voluntary, oral administration (50 µg/kg/day) during mid-gestation. As shown, a social approach deficit was again observed in male mice (Supplementary Fig. 4), consistent with results from prenatal (mid-gestation) BPA exposure by subcutaneous injections. Thus, we find that prenatal BPA exposure at mid-gestation (E10.5-E14.5) in mice leads to reduced social approach behavior in male, but not female offspring. Notably, the amygdala of embryonic mice undergoes significant development during mid-gestation40.
Aromatase knockout (ArKO) male mice have reduced social behavior
Having demonstrated that prenatal BPA exposure reduces aromatase expression in SH-SY5Y cells and affects the postnatal behavior of mice, we next asked if the aromatase gene (Cyp19a1) is central to these phenotypes. To address this, we performed social approach behavioral testing (Supplementary Fig. 5) on aromatase knockout (ArKO) mice41 which have undetectable aromatase expression. The social preference towards the stranger interaction zone compared to the empty zone was only evident for the wildtype (P = 0.003 Fig. 4B) but not the ArKO (P = 0.45 Fig. 4B). This male-specific social interaction deficit is similar to the BPA exposed pups. Further, postnatal estrogen replacement could reverse the ArKO reduction in sociability seen in males (P = 0.03 Supplementary Fig. 5) resulting in a similar stranger-to-empty preference in the E2-treated ArKO as observed for wildtype. The female ArKO pups did not have a sociability deficit (Supplementary Fig. 5).
Further, we did not observe any behavioral differences between ArKO vs WT (or BPA exposed vs unexposed) mice of both sexes in Y-maze test. All groups were able to distinguish the novel arm from the familiar arm. All groups spent significantly more time in the novel arm compared to the familiar arm (Supplementary Fig. 6), excluding major short-term memory, motor and sensory intergroup difference contributions.
Prenatal exposure to BPA affects repetitive behavior in male mice
Using the water squirt test, we have previously reported that male ArKO, but not female ArKO mice displayed excessive grooming, a form of repetitive behavior, compared to WT mice42. Thus, we conducted the water squirt test on BPA-exposed mice to find that male but not female mice exhibited excessive grooming behavior (P = 0.048; Fig. 4D). Thus, male prenatal BPA-exposed mice and ArKO mice, but not females, exhibited such repetitive behaviors compared to control mice.
The development of the MeA is altered in male ArKO mice as well as in prenatal BPA-exposed male mice
The development and function of the amygdala are highly relevant to human brain development and ASD43,44. Notably, the medial amygdala (MeA) is central to emotional processing45, and this tissue is a significant source of aromatase-expressing neurons. Given that aromatase function in the amygdala is significant for human cognition46 and behavior12,47, and that aromatase is highly expressed in the mammalian MeA, as particularly observed in male mice11, we investigated changes to the structure and function of this brain region. We performed stereology analyses on cresyl violet (Nissl)-stained sections of male MeA, we observed a 13.5% reduction in neuron (defined by morphology, size, and presence of nucleolus) number. Compared to the vehicle-exposed males, BPA-exposed males had significantly reduced total neuron number (mean count of 91,017 ± SEM of 2728 neurons vs 78,750 ± SEM of 3322 neurons, P = 0.046; Supplementary Fig. 7).
We further examined the characteristics of cells within this amygdala structure in detail using Golgi staining (Fig. 5A, B). We found that the apical and basal dendrites in the MeA were significantly shorter in male BPA-exposed mice vs. vehicle-treated mice (apical: 29.6% reduction, P < 0.0001; basal, P < 0.0001). This phenotype was also observed for male ArKO vs. WT mouse brains (apical 35.0% reduction, P < 0.0001; basal 31.9% reduction, P < 0.0001; Fig. 5A). Dendritic spine densities of apical and basal dendrites of male ArKO mice, as well as male mice exposed to BPA, were also significantly reduced (KO vs WT apical, P = 0.01; KO vs WT basal, P < 0.0001; BPA treated vs vehicle treated apical P < 0.0001; BPA treated vs vehicle treated basal, P = 0.004; Fig. 5B). The dendritic lengths (Fig. 5A) and spine densities (Fig. 5B) for apical and basal neurites within the MeA of female ArKO mice or BPA-exposed mice were not significantly different compared to control. Thus, in the context of aromatase suppression by prenatal BPA-exposure, or in ArKO mice lacking aromatase, we find that the apical and basal dendrite features within the MeA are affected in a sexually dimorphic manner.
Prenatal BPA exposure or loss of aromatase in ArKO male mice leads to amygdala hypoactivation and alters behavioral response to a novel social stimulus
The amygdala, a social processing brain region, is hyporesponsive in ASD (see review ref. 48). A post-mortem stereology study reported that adolescents and adults diagnosed with ASD feature an ~15% decrease in the numbers of neurons within the amygdala43. Also, functional MRI studies report amygdala hypoactivation in participants with ASD compared to controls49. Given that the amygdala is a significant source of aromatase-expressing neurons, we next conducted a series of studies to explore how aromatase deficiency influences the male mouse amygdala, using a combination of c-Fos immunohistochemistry, Golgi staining of brain sections, as well as electrophysiological analyses.
To investigate amygdala activation responses after interacting with a stranger mouse, we performed c-Fos immunohistochemistry (a marker for neuronal activation50; Supplementary Fig. 8). As shown, prenatal BPA-exposed mice featured 58% fewer c-Fos positive neurons than in the amygdala of vehicle-exposed mice brains (P < 0.0001; Fig. 5C). Similarly, we found that the MeA of ArKO mice had a marked deficit of 67% c-Fos-positive neurons when compared with WT (P = 0.0002) mice, which was ameliorated by early postnatal estradiol replacement (Fig. 5C). Therefore, prenatal BPA exposure or loss of aromatase expression in ArKO mice leads to amygdala hypoactivation.
Next, we measured the synaptic excitability (I/O curve) of the MeA using multiple electrode analysis, with excitatory postsynaptic potential (EPSP) output indicative of electrical firing by local neurons. As shown, compared to corresponding controls, we find that MeA excitability (I/O curve) is significantly reduced in male mice prenatally exposed to BPA as well as in male ArKO mice (Figs. 5D and 9D). As shown, at 4-volt input, BPA treatment resulted in a 22.8% lower (P = 0.02) excitatory EPSP output than the vehicle treatment, while a 21% reduction (P = 0.03) in signal was observed for male ArKO mice compared to male WT mice. Thus, prenatal BPA exposure leads to functional hypoactivation of the amygdala of male mice, and this pattern is also evident in male ArKO mice.
Prenatal BPA exposure or loss of aromatase in ArKO male mice leads to abnormalities in neuronal cortical layer V as well as brain function
It has been reported that individuals with ASD show distinct anatomical changes within the somatosensory cortex, including in neurons of cortical layer V51. We previously reported that layer V within the somatosensory cortex is disrupted in ArKO mice52. Thus, we performed Golgi staining to study the apical and basal dendrites of neurons within layer V of the somatosensory cortex following prenatal BPA exposure, as well as in ArKO mice. As shown, we found that apical and basal dendrite lengths of layer V cortical neurons were significantly decreased in male mice prenatally exposed to BPA, compared with vehicle-treated mice (apical P = 0.04; basal P < 0.0001, Fig. 6A). Such reductions in dendrites were also reported in male ArKO vs. WT mice (apical P < 0.0001; basal P = 0.02; Fig. 6A). Furthermore, we found that dendritic spine densities on apical dendrites were also reduced (BPA-exposed mice vs. vehicle, P = 0.04; ArKO vs. WT mice, P = 0.01 (Fig. 6B).
To explore the effects of reduced aromatase on cortical activity, we performed electrocorticography (ECoG) recordings from mice in both experimental models (Fig. 6C). As shown, spectral analysis revealed an increased power in the range of 4–6 Hz for ArKO vs. WT mice (4 Hz P = 0.0006, 5 Hz P < 0.0001, 6 Hz P < 0.0001; Fig. 6D) and at 8 Hz for BPA-exposed vs. vehicle mice (P = 0.01; Fig. 6C). These data indicate that BPA-exposure or loss of aromatase in ArKO mice affects cortical activity, a result which is reminiscent of cortical dysfunction evidenced by EEG recordings on human participants diagnosed with ASD53.
