Background
Autism spectrum disorders (ASD) are a heterogeneous set of neurodevelopmental disorders including autism, Asperger’s syndrome, childhood disintegrative disorder, and pervasive developmental disorder not otherwise specified (PDD-NOS). Autism is currently diagnosed by extensive behavioral and psychological testing and diagnosis is declared based on DSM-V characteristics. These characteristics include communication deficits, excessive dependence on routine, and obsessive tendencies showing up in early childhood. ASDs are more prevalent in boys than in girls, with ratios of 3:1 for classic autism and 10:1 for Asperger syndrome, suggesting the possible role of sex hormones in the pathophysiology of this disorder [
1]. It has been suggested that high levels of testosterone during early development may be a risk factor for ASDs [
2,
3]. The above hypothesis has been supported by a number of studies reporting an association between fetal testosterone levels and autistic features [
2].
Estradiol, the most potent estrogen, is formed from testosterone by the enzyme aromatase, also known as cytochrome P450, family 19 (CYP19A1). Estrogen is neuroprotective and plays an important role in emotional responses [
4] and in frontal cortical activity during cognitive task performance in humans [
5]. Estrogen acts through the binding to its receptor called estrogen receptor (ER). ER exists in two main forms, ERα and ERβ, which have distinct tissue expression patterns [
6]. ERα and ERβ are encoded by separate genes,
ESR1 and
ESR2, respectively, found at different chromosomal locations, and numerous mRNA splice variants exist for both receptors in both diseased and normal tissue [
7]. In the “classical” pathway of estrogen action, estrogen binds to ER, a ligand-activated transcription factor that regulates transcription of target genes in the nucleus by binding to estrogen response element regulatory sequences in target genes and recruiting co-regulatory proteins such as co-activators or co-repressors. The major co-regulators involved in estrogen signaling are steroid receptor co-activator 1 (SRC-1), transcriptional mediators/intermediary factor 2 (TIF-2), nuclear receptor co-repressor 1 (nCoR), CREB-binding protein (CBP), p300/CREB-binding protein-associated protein (P/CAF), amplified in breast 1 (AIB-1), and silencing mediator of retinoid and thyroid hormone receptors (SMRT) [
8]. Among these co-factors, SRC-1, TIF-2, CBP, AIB-1, and P/CAF are transcriptional co-activators, whereas SMRT and nCoR are transcriptional repressors. On the other hand, in the rapid or “non-genomic” pathway, activation of the membrane ER by estrogen leads to a rapid change in various intracellular signaling molecules including kinases, which in turn regulate gene transcription [
9].
ERα is widely distributed in the various brain regions including amygdala-hippocampal area, periamygdaloid cortex, and posterior cortical nucleus of the brain [
10]. Moreover, ERα influences various neurotransmitter systems, such as dopamine, serotonin (5-HT), and norepinephrine, indicating its role in neuropsychiatric disorders [
11,
12]. ERβ is the principal estrogen receptor expressed in brain areas such as cerebral cortex, hippocampus, and cerebellum [
13]. An earlier study has found a significant association of the ERβ gene with scores on the Autism Spectrum Quotient and the Empathy Quotient in ASD subjects [
14]. Moreover, ERβ mediates some of the effects of estrogens on anxiety, locomotor activity, fear responses, and learning behavior [
15]. Although the above studies are interesting, it is still unclear whether the expression of ER is impaired in the brain of ASD subjects. In the present study, we first examined the gene expression of ERα, ERβ, CYP19A1, and the co-regulators SRC-1, TIF-2, CBP, P/CAF, SMRT, AIB-1, and nCoR in postmortem middle frontal gyrus of ASD and control subjects. The middle frontal gyrus region was selected because a number of studies from neurocognitive as well as neuroimaging studies have implicated middle frontal gyrus in the pathophysiology of ASD [
16‐
18]. Moreover, a sexual dimorphic nature has been reported in the middle frontal gyrus [
19]. Based on our mRNA data, we then used western blotting to examine whether the changes found at the gene level of ERβ and CYP19A1 are significant at the protein level. We hypothesized that the expression of ERs are impaired in ASD and these abnormalities involve key co-regulators involved in ER regulation.
Discussion
We have found, for the first time, alterations in ERβ transcriptional regulation in the brain of ASD subjects. We also report a significant decrease in CYP19A1 expression in ASD subjects. The above changes were associated with alterations in ER co-activators in the same study subjects. We did not find a significant difference in ERα expression between ASD and controls. Together, these findings suggest alterations in ER signaling in ASD.
The decrease in ERβ mRNA and protein expression in the middle frontal gyrus of ASD subjects found in this study is consistent with the increasing evidence for the role of estrogen signaling in the etiology of ASD. A significant association of the ERβ gene with autism traits as measured by the Autism Spectrum Quotient and the Empathy Quotient has been reported in ASD subjects [
14]. ERβ is abundantly expressed in the cortex [
22]. Moreover, ERβ plays an important role in neurodevelopment, and ERβ knockout, but not ERα knockout mice show defects of neuronal migration [
23]. It is known that ERα and ERβ have distinct tissue expression profiles, and have different cellular functions [
22‐
24]. ERα is involved in mediating estrogen action on reproductive organs and reproductive behavior, whereas ERβ is known to mediate some of the effects of estrogens on behaviors that are not specifically associated with reproduction, such as locomotor activity, fear responses, anxiety, and learning [
15]. ERβ knockdown has been shown to abolish E2-induced reductions in depressive behavior in mice [
25‐
27]. Moreover, administration of ERβ agonist or selective ligand has been shown to reduce anxiety-type behavior [
27] and depressive behavior [
28] in rats. Activation of ERβ with the specific agonist WAY-200070 in cortical neurons results in increased spine density and PSD-95 (postsynaptic density-95) accumulation in membrane [
29]. Together, these results suggest that ERβ-mediated mechanism(s) are important for E2-induced neuronal plasticity.
The decrease in ERβ expression found in the ASD subjects might be the result of transcriptional regulation, either through methylation or by the regulation of genes of specific transcription factors binding to the ERβ promoter. Hypermethylation of the ERβ promoter is associated with a marked decrease in ERβ mRNA expression [
30]. It is known that estrogen binds to ER leading to a conformational change in ER. The estrogen-ER complex can bind directly to DNA via an estrogen responsive element or become attached to a transcription factor [
24]. It recruits a variety of co-regulators that result in the activation or repression of target genes by modifying chromatin structure. The p160/SRC (steroid receptor co-activator) family is one of the most studied classes of co-activators [
31]. Among the co-activators, SRC-1 and CBP exhibit autonomous histone acetyltransferase activity that promotes efficient transcription. In contrast, in the absence of ligands, ER associates with co-repressors nCoR or SMRT to mediate transcriptional repression of target genes through the histone deacetylase activity of the co-repressors [
32‐
34]. The present study revealed a novel finding that ERβ expression levels correlated with ER co-activators, SRC-1, P/CAF, and CBP. Moreover, we observed that the above co-factors interact with ERβ in human middle frontal gyrus. It is known that ERβ can antagonize ERα-dependent transcription in cells [
35]. In addition, ERβ and its variant, ERβ2, have been shown to increase the proteolytic degradation of ERα [
36]. Thus, it is possible that the changes in the expression levels of ERs and co-factors observed in our study might influence the estrogen receptor signaling machinery and might play important roles in the pathophysiology of ASD.
Our findings demonstrate that CYP19A1 expression is significantly lower in the brain of ASD subjects. Earlier studies have reported the expression of CYP19A1, the key enzyme required for estrogen production, in the cortex [
37]. Furthermore, CYP19A1 is enriched at synapses and localizes to presynaptic structures in cortical neurons [
29], suggesting that brain-synthesized estrogen plays an important role in neuronal function [
38]. The decrease in CYP19A1 could lead to reduced conversion of testosterone to estradiol resulting in increased levels of testosterone as observed in ASD subjects [
39]. Our data is in agreement with a previous finding on reduced aromatase protein levels in the frontal cortex of ASD subjects [
40]. An earlier genetic study has reported association between androgen receptor and ASD, suggesting an important role of androgen signaling in ASD [
41]. Further studies should examine the mRNA and protein levels of androgen receptors in the brain samples from ASD and control subjects, and such information would be helpful to better understand the relationship between estrogen-related and testosterone-related signaling pathways in ASD.
Conclusions
We have identified dysregulation of ERβ, CYP19A1, and co-activators associated with ER signaling in the middle frontal gyrus of ASD subjects with a significant association between these molecules. Our data suggest that a coordinated regulation of ER signaling molecules plays an important role in ER signaling in the brain, and that this network may be impaired in subjects with ASD. Although we found a large, significant association between the co-factor mRNA transcripts (SRC-1, CBP, and nCoR) and ADI-R scores in ASD subjects, its implication is unclear. Moreover, the robust reduction found in CBP mRNA expression in ASD subjects needs further investigation. CBP is known to be associated with other steroid receptors, including progesterone receptor, thyroid hormone receptors, and retinoid receptors [
32]. It is important to examine whether the complex formation of ERβ with CBP is indispensible for ER-dependent neuronal plasticity and ASD-like behavior. Moreover, the present data was collected in a relatively smaller number of study subjects, which needs further investigation using large samples before a conclusion can be drawn. Since brain tissue from individuals with ASD is quite scarce, lymphoblastoid cell lines that are banked for ASD cohorts (though there are several limitations including difference in tissue type and difficulties in transformation procedure) could provide a large sample of biological material to understand the pathophysiology of ASD. Future studies will investigate the mechanism of regulation of ERβ in ASD, which might lead to a better understanding of the pathophysiology and provide new avenues of treatment of this disorder.
Availability of supporting data
The data sets supporting the results of this article are included within the article and its additional files.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AC and RT carried out the gene expression studies. AC performed the protein analysis. AOA carried out the statistical analysis and helped to draft the manuscript. AP designed the study and wrote the manuscript. All authors read and approved the final manuscript.