Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways associated with autism spectrum disorders

Autism spectrum disorder (ASD) defines a complex group of neurodevelopmental disabilities characterized by a wide range of impairments in social and communicative skills, stereotyped behaviors, and restricted mental flexibility [1]. The neurodevelopmental and neuroanatomical bases of ASD have been confirmed by a number of clinical, neuroimaging and neuropathological studies [1‐3]. The most evident abnormality in ASD consists in an early (perinatal) brain overgrowth followed by an arrest of growth during the first year of age [4]. Neuropathological studies on post-mortem samples from ASD patients also showed a number of cellular and cytoarchitectural abnormalities at the level of the cerebral cortex, cerebellum, amygdala and forebrain limbic structures. Anomalies in the cerebellum are the most reproducible neuropathological alterations in ASD patients [3, 5].

A large series of evidence clearly indicates that neuropathological features and behavioral deficits of ASD have a primarily genetic origin. However, the etiology of ASD remains essentially unknown [6, 7]. Transcriptome analysis has also been used to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways dysregulated in ASD [6]. Several studies have analyzed genome-wide expression profiles of ASD patients using lymphoblastoid cell lines and blood samples, supporting upregulation of immune genes and downregulation of neurodevelopmental genes as key players in the pathogenesis of ASD (see [8] for a review). Recently, gene co-expression network analysis of autistic brain areas revealed defects in cortical patterning and an enrichment of differentially expressed genes associated with ASD [9].

The homeobox-containing transcription factor engrailed-2 (En2) is crucially involved in the regionalization, patterning and neuronal differentiation of the midbrain and hindbrain [10‐15]. Human studies indicated association of two intronic single-nucleotide polymorphisms (SNPs) in the human engrailed-2 (EN2) gene with ASD [16, 17]. Furthermore, the ASD associated A-C haplotype markedly affected EN2 promoter activity when tested with a luciferase reporter assay in rat, mouse and human cell lines [18]. A recent study of the epigenetic evaluation of EN2 in post-mortem cerebellar samples from autistic patients indicated a persistent upregulation of this homeobox gene induced by epigenetic abnormalities in histone methylation patterns that may contribute to Purkinje cell loss in some individuals with autism [19].

Mice lacking the homeobox domain of En2 (En2 ^hd/hd mice; [20], referred to as En2 ^-/- ) have been proposed as a model for ASD, due to their complex neuroanatomical and behavioral phenotype. En2 ^-/- mice display cerebellar hypoplasia, including a reduced number of Purkinje cells, and a defect in the antero-posterior pattern of cerebellar foliation [20‐23]. The behavior of En2 ^-/- mice is also reminiscent of some features of ASD individuals. Deficits in social behaviors were detected in En2 ^-/- mice, including decreased play and reduced social interactions; locomotor impairment, as well as defective spatial learning and memory, was also reported in these mice [24‐26]. Furthermore, we reported dysfunctions in GABAergic interneurons in adult En2 ^-/- mice and demonstrated engrailed protein expression in specific subpopulations of adult hippocampal and cortical interneurons [27].

To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2 ^-/- mouse model and performed genome-wide expression profiling on cerebellar and hippocampal adult tissues. Our transcriptome analysis of the cerebellum and hippocampus of En2 ^-/- mice suggests convergent pathological pathways with ASD, including abnormal synaptic transmission and increased immune response. Furthermore, we provide evidence for a significant enrichment of differentially expressed genes associated to ASD in this mouse model of the disease.

To date, more than 500 autism-associated genes have been identified (SFARI Gene; gene.sfari.org; updated mar/2013); yet the etiology of ASD remains essentially unknown [6, 7]. The significance of animal models in ASD research has been widely recognized as important for unraveling the molecular, cellular, anatomical, electrophysiological and behavioral consequences of gene dysfunction in ASD. Here, we present a transcriptome analysis in a mouse model of ASD of two brain areas, the cerebellum and the hippocampus, areas that are profoundly affected in ASD patients. Despite the small number of samples used for the microarray analysis and the sample gender heterogeneity, the low genetic variance among individuals allowed a reasonable statistical power for our bioinformatic analysis. Our study revealed that the molecular signature of these two brain regions shares convergent pathological pathways with ASD, including abnormal synaptic transmission and increased immune response. Furthermore, when directly compared to the repository of the SFARI database (gene.sfari.org), our differentially expressed genes in the hippocampus show an enrichment of ASD-associated genes significantly higher than previously reported [41].

Transcriptome analysis has been employed to unravel common pathways based on the assumption that the core phenotypes of ASD may be caused by convergent molecular mechanisms [6]. Several studies have analyzed genome-wide expression profiles of lymphoblastoid cell lines and blood samples from ASD patients, pointing to an upregulation of immune genes as key mechanisms in the pathogenesis of ASD [8]. Despite the limited source of brain tissue samples from ASD cases and the technical restrictions, studies of ASD brain transcriptome are emerging as strategic for uncovering functionally relevant alterations in gene expression. A previous microarray study found alterations of glutamatergic neurotransmission in ASD cerebellum [39], and expression profiles from ASD patient temporal cortices showed upregulation of genes involved in innate immune response and downregulation of several neurodevelopmental genes [40]. Moreover, the transcriptome profiles from three different brain regions (frontal cortex, temporal cortex and cerebellum) of nineteen autism cases and seventeen controls were investigated recently using classical differential expression analysis and a network-based approach [9]. These analyses showed upregulation of genes involved in immune response and downregulation of genes involved in synaptic function and vesicular transport [9]. Our results are in accordance with these findings. Using gene ontology enrichment, integrated gene-network analysis and mouse phenotypes analysis, we report significantly enriched functions and pathways that were previously associated to ASD [42]. In detail, we found increased immune response and major histocompatibility complex-related immunity in the En2 ^-/- cerebellum; decreased and abnormal neurotransmission and increased seizures in the En2 ^-/- hippocampus [see Additional file 5 for details]. Moreover, by direct comparison with the SFARI repository of ASD-related genes, we show that the gene expression changes observed in the En2 ^-/- hippocampus were significantly enriched in ASD-related genes. Furthermore, the proportion of ASD-associated genes enrichment in En2 ^-/- hippocampus was significantly higher than previous studies (Table 2) when compared with Voineagu et al.[9], likely the most comprehensive transcriptome study of ASD post-mortem brain to date. In the case of the cerebellum, in contrast to Voineagu et al.[9] we did not find significant enrichment of ASD-associated genes in En2 ^-/- mice. Such difference could be the result of the complex structural and cytoarchitectural abnormalities in En2 ^-/- cerebellum [20, 21] and the consequent phenotypical variability, or could simply reflect differences between mouse and human phenotypes, as the incidence of cerebellar hypoplasia was not reported in the diagnostic criteria used in the study [9]. Remarkably, EN2 was among the differentially expressed genes found in Voineagu et al.[9], confirming our evidence about the role of En2 in the neuropathology of ASD, and in anterior brain structures [27].

Among the differentially expressed genes, Grm5, Nrxn3 and Scn1a are of particular interest for ASD. Grm5 encodes mGluR5, a G-protein coupled receptor for the neurotransmitter glutamate [43]. In a recent study, mGluR5 has been shown to participate in the pathogenesis of fragile X syndrome (FXS) while genetic downregulation of Grm5 was able to compensate for some of the symptoms in a mouse model of FXS [44]. Furthermore, Grm5 was shown to be downregulated in hippocampal neurons lacking Shank3, another ASD-associated gene [45]. These data support a central role for Grm5 in neurobiological pathways related to ASD pathogenesis. Our results show an increased expression of Grm5 in the cerebellum of En2 ^-/- mice, suggesting a role of Grm5 in the cerebellar phenotype of these mice. The contribution of Grm5 and its interaction with Fmr1 in the En2 ^-/- hippocampus remains to be established and could open new perspective of pharmacological and genetic rescue of the ASD-related phenotype of these mice.

Nrxn3 encodes neuronal adhesion proteins of the Neurexin (NRXN) family. NRXNs are presynaptic cell adhesion proteins that form trans-synaptic complexes with their postsynaptic counterpart neuroligins (NLGNs) and have important roles in synapse development and function [46]. Recently, a report of hemizygous and de novo deletions involving NRXN3 in ASD families provided strong support for a causative link between the loss of NRXN3 and the development of ASD [47]. Our results of an increased expression of Nrxn3 in the cerebellum suggest alterations in Purkinje cell synaptic formation, where NRXNs have been shown to participate to the formation of glutamatergic synapses through interaction with Cerebellin 1 precursor protein (also downregulated in the En2 ^-/- cerebellum) and GluR∂2 [48, 49].

Scn1a encodes the voltage-gated sodium channel alpha subunit. De novo null mutations in SCN1A result in severe myoclonic epilepsy of infancy [50]. SCN1A mutations have been associated to a number of neurological disorders, including generalized epilepsy with febrile seizures plus, Dravet syndrome, borderline myoclonic epilepsy in infancy, intractable childhood epilepsy with generalized tonic-clonic seizures, familial hemiplegic migraine, and a number of cryptogenic focal and generalized epilepsies. Recently, de novo mutations in SCN1A have been associated with ASD [51], and a report of a recognized mutation in SCN1A suggests a wide phenotypic variation of the gene mutations causing a variety of neurologic disorders, including ASD [52]. In mice, heterozygous loss-of-function mutation in Scn1a (Scn1a ^+/- ), reproduces several of the symptoms associated to the human mutation, such as thermally induced and spontaneous seizures, premature death, ataxia and sleep disorder [53, 54]. Scn1a ^+/- mice show both cognitive deficits and autistic traits that are caused by impaired GABAergic neurotransmission and can be rescued by drug treatment. Scn1a down-regulation in the En2 ^-/- hippocampus could contribute to the abnormal excitability and altered GABAergic neurotransmission shown in these mice by our previous studies [27, 30]. Pharmacological rescue of the hippocampal phenotype in the En2 ^-/- with GABAergic drugs is currently under investigation.

Anomalies in the cerebellum are the most reproducible neuropathological alterations in ASD patients. Several cerebellar abnormalities have been observed in mouse models of both En2 gain- and loss-of-function. Ectopic overexpression of En2 in Purkinje cells during late embryonic and postnatal cerebellar development results in reduced cerebellar volume and loss of Purkinje cells and other cerebellar neurons [55, 56]. Interestingly, En2 knock-out causes defective cerebellar patterning, reduced Purkinje cell number and abnormal dendritic foliation [10, 57], indicating that alterations in En2 expression levels during development cause similar phenotypes. Furthermore, deficits in social behaviors as well as defective spatial learning and memory were also reported in En2 ^-/- mice [24‐26]. A recent epigenetic analysis of EN2 promoter methylation in the cerebellum of ASD individuals indicated hypermethylation of the promoter region and persistent upregulation of the gene. The authors report that promoter hypermethylation is normally associated with a decrease in gene expression and suggest the possibility of a developmental mechanism intended to support downregulation of EN2 during perinatal development [19]. Taken together, this evidence suggests that an overall imbalance in EN2 expression may be relevant for ASD pathogenesis, as it could produce alterations in critical brain functions. Comparable evidence of a similar dosage effect has been reported in the case of mutations of other genes critically involved in gene expression regulation and maintenance of synaptic and neuronal homeostasis, such as MECP2 and ARX[58, 59]. It remains to be established whether En2 overexpressing mice display abnormal behaviors relevant to autism. Microarray data have been produced for En2 overexpressing Purkinje cells on a different platform; however, the results overlap only marginally with the herein reported study [60].

Using transcriptome analysis, we identified over 800 genes differentially expressed in the cerebellum and hippocampus of En2 ^-/- mice. Despite the small number of samples used and the relatively small statistical power, our study is the first to analyze molecular changes occurring in two brain structures with neuroanatomical alterations relevant to ASD in a mouse model of this disease. Our bioinformatic analysis of the molecular signature of En2 ^-/- cerebellum and hippocampus shows a significant convergence of neurobiological pathways previously linked to ASD pathology in brain samples from ASD patients. Overall, the present study points to a strong impact of transcriptome analysis on mouse models for identifying neurobiological pathways commonly altered when ASD genes are disrupted in a human patient and in a mouse model alike. Furthermore, together with the frequent association of cerebellar neuroanatomical alterations to the neuropathology of ASD, our molecular analysis suggests a contribution also for the hippocampus, where molecular changes relevant to ASD may occur also in human patients. This notion is supported by the consistent enrichment of ASD-related genes in the En2 ^-/- hippocampus compared to the cerebellum and to other similar studies performed on ASD patient tissue samples [41]. Finally, our results confirm the En2 ^-/- mouse model of ASD as a valuable tool for investigating neuroanatomical, behavioral, as well as molecular alterations related to ASD.

The data sets supporting the results of this article are available in the GEO repository, GSE51612.