Background
Autism spectrum disorders (ASDs) and Attention Deficit Hyperactivity Disorder (ADHD) exhibit partially overlapping neurobehavioural symptoms, frequent comorbidity, altered monoaminergic function, and shared genetic aetiology [
1]. Both types of disorder are significantly more frequently diagnosed in males than in females, suggesting a potential role for sex-linked genetic risk variants [
2], and both can have long-term adverse consequences for example, increased risk for alcohol dependence in ADHD [
3] or lack of independence, close social ties and employment in ASDs [
4].
Both cytogenetic deletions at Xp22.32 encompassing the X-linked
STS gene (encoding the enzyme steroid sulphatase) and its immediate neighbours, and inactivating point mutations within
STS, appear to predispose to ADHD (particularly the inattentive subtype) [
5]; larger cytogenetic deletions encompassing
STS and more distant contiguous genes (notably
NLGN4X) seem to predispose to autism and related disorders [
5]. Polymorphisms within
STS are associated with ADHD risk [
6,
7] and cognitive function in individuals with ADHD [
8], whilst the gene is expressed in regions of the developing brain whose structure is known to be altered in ADHD cases [
8]. Steroid sulphatase cleaves sulphate groups from a variety of steroid hormones (for example, dehydroepiandrosterone sulphate, DHEAS) thereby altering their activity and/or specificity, and subsequent developmental and physiological effects [
9]. As sulphated and non-sulphated steroid hormones can act as modulators at key neurotransmitter receptors, including N-methyl-D-aspartic acid (NMDA) and γ-aminobutyric acid type A (GABA
A) receptors [
9], lack of STS developmentally could potentially elicit important effects on neuronal organisation processes mediated by these neurotransmitters [
10].
Inactivating mutations within the
ASMT gene, located within the pseudoautosomal region of the human X chromosome and encoding the enzyme acetylserotonin O-methyltransferase that catalyses the final step in melatonin biosynthesis, have been suggested as being potentially pathogenic in a variety of psychiatric and developmental conditions, including ASDs [
11‐
17]. Such mutations may act to reduce systemic melatonin levels, a reported feature of individuals with ASDs [
15]. Alternatively, or additionally, they could affect upstream substrate levels in the brain or blood platelets, for example, of the growth factor serotonin (5-hydroxytryptamine, 5-HT) or blood cell function [
15]; elevated platelet serotonin levels are a consistent finding in ASD cases [
18].
The 39,X
Y*O mouse lacks both the pseudoautosomal
Sts and
Asmt genes (and hence their expression in all tissues) as a consequence of an end-to-end fusion of the X and Y chromosomes [
19]; as such, it has some degree of construct validity as a genetic mouse model for neurodevelopmental disorders. On an MF1 outbred albino strain background, this mouse also exhibits considerable face validity for such disorders: it is inattentive [
20], hyperactive, emotionally hyper-reactive (showing increased indices of stress in novel or arousing environments), occasionally aggressive [
21], and perseverative (showing persistent responding in the absence of reinforcement) [
19,
22] and exhibits reduced systemic DHEA levels [
21]. Whilst melatonin levels in wildtype and 39,X
Y*O MF1 male mice remain to be determined, other outbred albino strains are known to produce significant quantities of the hormone [
23].
Currently, the neurobiology of the 39,X
Y*O mouse is poorly defined, although we have previously shown that it exhibits altered monoaminergic chemistry, notably elevated hippocampal and striatal serotonin levels and reduced 5-HT turnover in these regions [
19,
22]. Interestingly, however, the 39,X
Y*O mouse, in contrast to individuals with ADHD, exhibits enhanced behavioural inhibition relative to 40,XY male controls as indexed by performance on murine versions of the 5-choice Serial Reaction Time Task and the Stop Signal Reaction Time Task [20, S.T., O.A.O. and W.D., unpublished observations]. We have previously shown that acute administration of one dose of the specific steroid sulphatase inhibitor COUMATE to wildtype male mice also results in inattention [
20] and enhanced behavioural inhibition [S.T., O.A.O. and W.D., unpublished observations], suggesting that these phenotypes in the 39,X
Y*O mouse are due to the ongoing activity of the enzyme. Other phenotypes in the 39,X
Y*O mouse (for example, hyperactivity and anxiety) cannot be recapitulated by acute inhibition of steroid sulphatase [
21], suggesting that they may arise from the developmental effects of deficiency for the enzyme, or alternatively from neuroendocrinological abnormalities as a consequence of ASMT deficiency.
Here, we further investigated the neurobiology of the 39,XY*O mouse using two methods, with a view to identifying biological correlates of the behaviours mentioned above. First, we compared gene expression in adult 40,XY and 39,XY*O whole brain tissue by microarray to identify non-obvious genetic changes between the two groups, that is changes that could not be predicted a priori on the basis of known biology; we assayed whole brain tissue given that Sts is widely expressed throughout the mouse brain, and because disruptions to multiple brain regions were likely to underpin the 39,XY*O behavioural phenotypes. We also tested to see whether any of the significant changes were seen in the 39,XY*O hippocampus or striatum (given the known changes in 5-HT levels), or in COUMATE-treated male mouse whole brain (and hence whether they could explain the effects of acute STS deficiency on attention and behavioural inhibition). Second, we characterised brain steroids in the adult 40,XY mouse for the first time, and compared this profile to that of the 39,XY*O mouse. We anticipated that these combined approaches might reveal new pathophysiological mechanisms underlying phenotypes associated with STS/ASMT deficiency specifically and neurodevelopmental disorders more generally, and might highlight novel factors protecting against behavioural disinhibition. We report that 39,XY*O mice show a highly specific pattern of gene expression changes, with several being of relevance to developmental disorders, but surprisingly, no large changes in brain steroid levels.
Discussion
The 39,XY*O mouse model exhibits face and construct validity for neurodevelopmental disorders, showing behavioural endophenotypes associated with such conditions, and lacking two genes (Sts and Asmt) whose human orthologues have been implicated in ADHD and autism pathogenesis respectively. In this study, we investigated the neurobiology of this model by two methods with a view to identifying mechanisms by which loss of function of these genes might contribute towards behavioural pathology; this work is important given the current lack of availability of single gene knockout models for Sts and Asmt.
Microarray and quantitative PCR analyses comparing 40,XY and 39,X
Y*O brain tissue identified a surprisingly small number of robust gene expression differences between the groups. Our inability to verify differential microarray expression calls at
P < 0.05 for multiple transcripts of relevance to autism and ADHD suggests that we are likely to have successfully identified all the genes that truly differ in their expression across the whole brain in 40,XY and 39,X
Y*O mice. However, it is possible that there are further group differences in gene expression within specific brain regions; indeed, our microarray analysis did not indicate altered expression of
Htr2c (encoding the serotonin 2c receptor) which we have previously shown to be upregulated in the 39,X
Y*O hippocampus [
22].
Erdr1 gene expression was significantly reduced in 39,X
Y*O whole brain, and in hippocampal and striatal dissections; this gene is retained in the 39,X
Y*O mouse and is located adjacent to the fusion point of the X and Y chromosomes [
19]. Our current results suggest that in addition to deleting of the
Sts and
Asmt genes, the lesion in 39,X
Y*O mice disrupts a genetic element that enhances
Erdr1 expression. Theoretically, in concert with loss of
Sts and
Asmt, reduced expression of the widely-expressed, but poorly-characterised, erythroid differentiation regulator 1 protein encoded by
Erdr1 could contribute towards downstream gene expression changes, abnormal striatal and hippocampal monoamine neurochemistry, and behavioural phenotypes in the 39,X
Y*O model; brain and behavioural investigations in mice in which the function of this gene alone is disrupted will help clarify the extent and specificity of this contribution.
Our microarray and qPCR analyses also showed that the expression of
C1qc (encoding the protein complement component 1, q subcomponent, c chain) was upregulated in the brains of young, behaviourally-naïve 39,X
Y*O mice, but downregulated in the striata and hippocampi of older, behaviourally-trained mutant animals. These data suggest a potential basis for the neurochemical and behavioural abnormalities seen in the 39,X
Y*O mouse that may be sensitive to spatiotemporal or environmental regulation, and further suggest the possibility that the neurobehavioural pathology in individuals lacking functional STS and/or ASMT proteins may be due, in part, to altered C1QC levels. Previous animal and clinical studies have implicated aberrant expression of C1q family members in developmental and behavioural phenotypes. In rodents, C1q deletion results in altered synaptic elimination [
33,
34],
C1qc expression levels are altered in a model of developmental hippocampal pathology [
35], and
C1qc expression is associated with behavioural phenotypes (notably the consumption of ethanol relative to water) [
36]. In man, individuals with autism can exhibit elevated C1QC serum levels [
37] and altered gastrointestinal C1q deposition [
38,
39]. Whether the 39,X
Y*O mouse exhibits alterations in synaptic structure/function or hippocampal structure, or heightened alcohol preference remains to be investigated. It also remains to be seen whether individuals with elevated C1QC levels and autism possess genetic mutations in either
STS or
ASMT, and whether individuals lacking functional
STS and/or
ASMT genes are at increased risk of alcohol dependence.
By the same logic, the present findings further indicate that disrupted expression of
Metap1d and/or
Sfi1 could play a role in 39,X
Y*O phenotypes, and in developmental phenotypes associated with Xp22.3 mutations, but probably not through influencing striatal or hippocampal physiology. There is some evidence for an association between a linkage block at 2q31.1 containing
METAP1D and autism [
40], whilst copy number variants encompassing
SFI1 have previously been identified in autism and related developmental disorders [
41‐
45].
We also showed that the genetic mutation in 39,X
Y*O mice resulted in reduced hippocampal expression of the
Dhcr7 gene. DHCR7 is a known modulator of serotonergic system development in mammals [
32]; therefore, its reduced expression represents a strong candidate mechanism for abnormal serotonin levels in the 39,X
Y*O hippocampus and associated behavioural phenotypes [
19,
22]. In man, defects in the DHCR7 enzyme underlie Smith-Lemli-Opitz syndrome (SLOS). Individuals with SLOS exhibit a range of behavioural symptoms with some overlap with autism, including: hyperactivity, aggression, insomnia, self-injurious behaviour, sensory hypersensitivity and repetitive behaviours [
32]; interestingly, several of these behavioural abnormalities are also observed in the 39,X
Y*O mouse [
19,
21,
22], indicating that hippocampal loss of DHCR7 function may underlie key SLOS phenotypes, and suggesting the 39,X
Y*O mouse as a potential novel model for aspects of the syndrome.
Genetic and functional work in mice has indicated a link between steroid sulphatase and aggressive behaviour [
46,
47]. Consistent with this, 39,X
Y*O mice [
21], and mice co-administered COUMATE and DHEAS [
48], exhibit elevated levels of aggression. Previous rodent studies have demonstrated that major urinary proteins may elicit aggressive behaviour through their actions at sensory neurons expressing Vmn2r putative pheromone receptors [
49]. Of the 124 genes within the
Vmn2r family, the expression of just one,
Vmn2r86, was significantly altered in 39,X
Y*O brain; this increased expression thus represents an excellent candidate mechanism underlying aggression in these mutant mice. The fact that there is no human orthologue of
Vmn2r86 may explain why individuals with Xp22.3 deletions encompassing
STS and/or
ASMT do not consistently show obvious aggressive tendencies.
Acute administration of COUMATE, a specific steroid sulphatase inhibitor, given at a dose known to induce behavioural changes, did not recapitulate any of the whole brain gene expression changes seen in the 39,X
Y*O mouse. There are two obvious possibilities why this might be the case: i) the expression changes in the 39,X
Y*O mouse result from abnormal developmental expression of STS and/or ii) the gene expression changes in the 39,X
Y*O mouse are the result of loss of function of ASMT, or reduced expression of
Erdr1. A previous study found no effect of acute administration of COUMATE on the concentrations of endogenous DHEAS or DHEA in whole mouse brain, although the drug did reduce entry of systemic DHEAS into the brain [
50]. Thus, the molecular basis of COUMATE-induced behavioural changes remains obscure; it is plausible that the drug induces brain region-specific gene expression changes that we were unable to detect, and this possibility remains to be investigated.
Our current analyses provide, for the first time, a systematic profile of the steroid milieu in the mouse brain. There was substantial overlap between the free and sulphated steroids that were detectable in the adult male mouse brain (present data), and those that were most readily detectable in the adult male rat brain [
26] consistent with a degree of cross-species homology, although, interestingly, concentrations of most compounds tended to be higher in mouse brain. We found no significant differences in the concentrations of the detectable compounds between 40,XY and 39,X
Y*O brains, consistent with an absence of large between-group differences in steroid brain biochemistry. This finding, taken together with our previous data showing reduced serum DHEA levels in 39,X
Y*O mice [
19] suggests the possibility that where STS is absent developmentally, as in 39,X
Y*O mice, a compensatory mechanism is recruited to cleave sulphated steroid esters in brain, but not in peripheral tissues. Due to the difficulty of generating 39,X
Y*O mice and precisely genetically-matched controls, and the apparent variability in brain steroid levels in the mutant group, our study had limited power, with several steroids below the limit of detection. As such, we cannot completely exclude the possibility that there are subtle differences in levels of one or more steroids within 40,XY and 39,X
Y*O brain tissue.
Conclusions
Here, we have shown that the genetic mutation in 39,XY*O mice, in addition to deleting the Sts and Asmt genes, also results in significant downregulation of the adjacent Erdr1 gene. In the absence of Sts or Asmt single gene knockout mice, the present study gives the first clues as to possible downstream gene expression changes that might result from the loss of one (or both) of these genes; of the limited number of robust resultant gene expression changes, several may be pertinent to 39,XY*O neurochemical and behavioural phenotypes, and hence, to similar phenotypes in individuals with loss of function mutations within STS or ASMT. Importantly, it should be noted that just because gene expression differences across genotypes are large, they might not necessarily be biologically significant.
Future functional validation and pathway analysis studies in the 39,XY*O mouse throughout development, incorporating an examination of the spatiotemporal dynamics of protein changes indicated by our present study, should further elucidate the neurobiological pathways by which the 39,XY*O mutation gives rise to behavioural phenotypes analogous to those seen in neurodevelopmental disorders. Future work might examine whether the gene expression changes seen here are recapitulated in accessible tissues from individuals lacking STS and/or ASMT, and could test for abnormalities in complement pathway function, mitochondrial metabolism and cholesterol biosynthesis. Should this be the case, these physiological abnormalities could modulate either the risk of developing ADHD or autism, and/or to the clinical course of these disorders.
Our analyses indicated no large differences in brain steroid concentration between 40,XY and 39,XY*O adult mice, and hence, suggest that altered steroid biochemistry may not be a significant contributor to abnormal brain and behavioural phenotypes in this mouse model, nor to similar phenotypes in individuals with mutations in STS and/or ASMT. However, a developmental difference in brain steroid levels between Sts/Asmt-deficient and wildtype subjects may plausibly exist and contribute towards between-group adult behavioural phenotypes.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ST, JPF and WD conceived and designed the experiments. ST, JPF and OAO performed the experiments. ST, JPF and WD analysed the data. ST, JFP, OAO and WD wrote the paper. All authors read and approved the final manuscript.