Giovanni Provenzano

Mutant mouse models of autism spectrum disorders.

Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental diseases characterized by a triad of specific behavioral traits: abnormal social interactions, communication deficits and stereotyped or repetitive behaviors. Several recent studies showed that ASDs have a strong genetic basis, contributing to the discovery of a number of ASD-associated genes. Due to the genetic complexity of these disorders, mouse strains with targeted deletion of ASD genes have become an essential tool to investigate the molecular and neurodevelopmental mechanisms underlying ASD. Here we will review the most relevant genetic mouse models developed by targeted inactivation of ASD-associated genes, and discuss their importance for the development of novel pharmacological therapies of these disorders.

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

BackgroundTranscriptome analysis has been used in autism spectrum disorder (ASD) to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways. To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2-/- mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues.MethodsCerebellar and hippocampal tissue samples from three En2-/- and wild type (WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categories overrepresented in the differentially expressed genes, we used integrated gene-network analysis, gene ontology enrichment and mouse phenotype ontology analysis. Furthermore, we performed direct enrichment analysis of ASD-associated genes from the SFARI repository in our differentially expressed genes.ResultsGiven the limited number of animals used in the study, we used permissive criteria and identified 842 differentially expressed genes in En2-/- cerebellum and 862 in the En2-/- hippocampus. Our functional analysis revealed that the molecular signature of En2-/- cerebellum and hippocampus shares convergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to the repository of the SFARI database, our differentially expressed genes in the hippocampus showed enrichment of ASD-associated genes significantly higher than previously reported. qPCR was performed for representative genes to confirm relative transcript levels compared to those detected in microarrays.ConclusionsDespite the limited number of animals used in the study, our bioinformatic analysis indicates the En2-/- mouse is a valuable tool for investigating molecular alterations related to ASD.

Neurobiological bases of autism–epilepsy comorbidity: a focus on excitation/inhibition imbalance

Autism spectrum disorders (ASD) and epilepsy are common neurological diseases of childhood, with an estimated incidence of approximately 0.5–1% of the worldwide population. Several genetic, neuroimaging and neuropathological studies clearly showed that both ASD and epilepsy have developmental origins and a substantial degree of heritability. Most importantly, ASD and epilepsy frequently coexist in the same individual, suggesting a common neurodevelopmental basis for these disorders. Genome‐wide association studies recently allowed for the identification of a substantial number of genes involved in ASD and epilepsy, some of which are mutated in syndromes presenting both ASD and epilepsy clinical features. At the cellular level, both preclinical and clinical studies indicate that the different genetic causes of ASD and epilepsy may converge to perturb the excitation/inhibition (E/I) balance, due to the dysfunction of excitatory and inhibitory circuits in various brain regions. Metabolic and immune dysfunctions, as well as environmental causes also contribute to ASD pathogenesis. Thus, an E/I imbalance resulting from neurodevelopmental deficits of multiple origins might represent a common pathogenic mechanism for both diseases. Here, we will review the most significant studies supporting these hypotheses. A deeper understanding of the molecular and cellular determinants of autism–epilepsy comorbidity will pave the way to the development of novel therapeutic strategies.

Hippocampal Dysregulation of Neurofibromin-Dependent Pathways Is Associated with Impaired Spatial Learning in Engrailed 2 Knock-Out Mice

Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly, En2 knock-out (En2−/−) mice show anatomical and behavioral “ASD-like” features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits in En2−/− mice are not known. Deficits in signaling pathways involving neurofibromin and extracellular-regulated kinase (ERK) have been associated with impaired learning. Here we investigated the neurofibromin-ERK cascade in the hippocampus of wild-type (WT) and En2−/− mice before and after spatial learning testing. When compared with WT littermates, En2−/− mice showed impaired performance in the Morris water maze (MWM), which was accompanied by lower expression of the activity-dependent gene Arc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treated En2−/− mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus of En2−/− mice after MWM. Treatment of En2−/− mice with lovastatin, an indirect inhibitor of ERK phosphorylation, markedly reduced ERK phosphorylation in the dentate gyrus, but was unable to rescue learning deficits in MWM-trained mutant mice. Further investigation is needed to unravel the complex molecular mechanisms linking dysregulation of neurofibromin-dependent pathways to spatial learning deficits in the En2 mouse model of ASD.

Comparative Gene Expression Analysis of Two Mouse Models of Autism: Transcriptome Profiling of the BTBR and En2−/− Hippocampus

Autism spectrum disorders (ASD) are characterized by a high degree of genetic heterogeneity. Genomic studies identified common pathological processes underlying the heterogeneous clinical manifestations of ASD, and transcriptome analyses revealed that gene networks involved in synapse development, neuronal activity, and immune function are deregulated in ASD. Mouse models provide unique tools to investigate the neurobiological basis of ASD; however, a comprehensive approach to identify transcriptional abnormalities in different ASD models has never been performed. Here we used two well-recognized ASD mouse models, BTBR T+ Itpr3tf/J (BTBR) and Engrailed-2 knockout (En2−/−), to identify conserved ASD-related molecular signatures. En2−/− mice bear a mutation within the EN2 transcription factor homeobox, while BTBR is an inbred strain with unknown genetic defects. Hippocampal RNA samples from BTBR, En2−/− and respective control (C57Bl/6J and En2+/+) adult mice were assessed for differential gene expression using microarrays. A total of 153 genes were similarly deregulated in the BTBR and En2−/− hippocampus. Mouse phenotype and gene ontology enrichment analyses were performed on BTBR and En2−/− hippocampal differentially expressed genes (DEGs). Pathways represented in both BTBR and En2−/− hippocampal DEGs included abnormal behavioral response and chemokine/MAP kinase signaling. Genes involved in abnormal function of the immune system and abnormal synaptic transmission/seizures were significantly represented among BTBR and En2−/− DEGs, respectively. Interestingly, both BTBR and En2−/− hippocampal DEGs showed a significant enrichment of ASD and schizophrenia (SCZ)-associated genes. Specific gene sets were enriched in the two models: microglial genes were significantly enriched among BTBR DEGs, whereas GABAergic/glutamatergic postsynaptic genes, FMRP-interacting genes and epilepsy-related genes were significantly enriched among En2−/− DEGs. Weighted correlation network analysis (WGCNA) performed on BTBR and En2−/− hippocampal transcriptomes together identified six modules significantly enriched in ASD-related genes. Each of these modules showed a specific enrichment profile in neuronal and glial genes, as well as in genes associated to ASD comorbidities such as epilepsy and SCZ. Our data reveal significant transcriptional similarities and differences between the BTBR and En2−/− hippocampus, indicating that transcriptome analysis of ASD mouse models may contribute to identify novel molecular targets for pharmacological studies.

Reduced phosphorylation of synapsin I in the hippocampus of Engrailed-2 knockout mice, a model for autism spectrum disorders

Mice lacking the homeodomain transcription factor Engrailed-2 (En2(-/-) mice) are a well-characterized model for autism spectrum disorders (ASD). En2(-/-) mice present molecular, neuropathological and behavioral deficits related to ASD, including down-regulation of ASD-associated genes, cerebellar hypoplasia, interneuron loss, enhanced seizure susceptibility, decreased sociability and impaired cognition. Specifically, impaired spatial learning in the Morris water maze (MWM) is associated with reduced expression of neurofibromin and increased phosphorylation of extracellular-regulated kinase (ERK) in the hippocampus of En2(-/-) adult mice. In the attempt to better understand the molecular cascades underlying neurofibromin-dependent cognitive deficits in En2 mutant mice, we investigated the expression and phosphorylation of synapsin I (SynI; a major target of neurofibromin-dependent signaling) in the hippocampus of wild-type (WT) and En2(-/-) mice before and after MWM. Here we show that SynI mRNA and protein levels are down-regulated in the hippocampus of naïve and MWM-treated En2(-/-) mice, as compared to WT controls. This down-regulation is paralleled by reduced levels of SynI phosphorylation at Ser549 and Ser553 residues in the hilus of mutant mice, before and after MWM. These data indicate that in En2(-/-) hippocampus, neurofibromin-dependent pathways converging on SynI phosphorylation might underlie hippocampal-dependent learning deficits observed in En2(-/-) mice.

Hippocampal dysregulation of FMRP/mGluR5 signaling in engrailed-2 knockout mice: a model of autism spectrum disorders.

Many evidences indicate that mice lacking the homeobox transcription factor engrailed-2 (En2−/− mice) represent a reliable model to investigate neurodevelopmental basis and gene expression changes relevant to autism spectrum disorders. Dysfunctions in fragile X mental retardation protein (FMRP), metabotropic glutamate receptor 5 (mGluR5), and GABAergic signaling pathways have been proposed as a possible pathogenic mechanism of autism spectrum disorders. Here, we exploited En2−/− mice to investigate hippocampal expression of FMRP, mGluR5, and GABAA receptor &bgr;3 subunit (GABRB3). Quantitative reverse-transcription PCR showed that all these mRNAs were significantly downregulated in En2−/− mice compared with wild-type littermates. Western blot and immunohistochemistry confirmed the downregulation of FMRP and GABRB3 proteins, while showing a significant increase of mGluR5 protein in the En2−/− hippocampus. Our results suggest that the dysregulation of FMRP–mGluR5 signaling pathway, accompanied with a downregulation of GABRB3 expression, may contribute to the ‘autistic-like’ features observed in En2−/− mice, providing possible molecular targets for future pharmacological studies.

GH Dysfunction in Engrailed-2 Knockout Mice, a Model for Autism Spectrum Disorders

Insulin-like growth factor 1 (IGF-1) signaling promotes brain development and plasticity. Altered IGF-1 expression has been associated to autism spectrum disorders (ASD). IGF-1 levels were found increased in the blood and decreased in the cerebrospinal fluid of ASD children. Accordingly, IGF-1 treatment can rescue behavioral deficits in mouse models of ASD, and IGF-1 trials have been proposed for ASD children. IGF-1 is mainly synthesized in the liver, and its synthesis is dependent on growth hormone (GH) produced in the pituitary gland. GH also modulates cognitive functions, and altered levels of GH have been detected in ASD patients. Here, we analyzed the expression of GH, IGF-1, their receptors, and regulatory hormones in the neuroendocrine system of adult male mice lacking the homeobox transcription factor Engrailed-2 (En2−/− mice). En2−/− mice display ASD-like behaviors (social interactions, defective spatial learning, increased seizure susceptibility) accompanied by relevant neuropathological changes (loss of cerebellar and forebrain inhibitory neurons). Recent studies showed that En2 modulates IGF-1 activity during postnatal cerebellar development. We found that GH mRNA expression was markedly deregulated throughout the neuroendocrine axis in En2−/− mice, as compared to wild-type controls. In mutant mice, GH mRNA levels were significantly increased in the pituitary gland, blood, and liver, whereas decreased levels were detected in the hippocampus. These changes were paralleled by decreased levels of GH protein in the hippocampus but not other tissues of En2−/− mice. IGF-1 mRNA was significantly up-regulated in the liver and down-regulated in the En2−/− hippocampus, but no differences were detected in the levels of IGF-1 protein between the two genotypes. Our data strengthen the notion that altered GH levels in the hippocampus may be involved in learning disabilities associated to ASD.

Genetic control of social behavior: Lessons from mutant mice

Graphical abstract Figure. No caption available. HighlightsAltered social behavior is a pathological trait of autism spectrum disorder (ASD).In mice, mutations in ASD genes result in altered social behavior.Mouse models are a crucial tool to develop novel treatments for ASD. Abstract Social behavior is evolutionary conserved, and is thought to be evolved since it increased reproductive and survival fitness of living species. In humans, disturbances of social behavior are a peculiar pathological trait of neurodevelopmental disorders, namely autism spectrum disorder (ASD). ASD is defined by deficits in two core domains (social interaction/communication and repetitive/restrictive behaviors), which emerge during early postnatal development. ASD has a strong genetic component: copy number variations, de novo and familial mutations, as well as epigenetic modifications have been reported in a huge number of genes. Recent studies in mice demonstrate that mutations in a wide variety of ASD‐associated genes can cause neurodevelopmental defects, which subsequently result in social behavior disturbances during early postnatal age and adulthood. From these studies, it clearly emerges that functionally interrelated cellular mechanisms underlie social behavior and its disturbances in ASD. Indeed, most of ASD‐associated genes control neuronal differentiation and migration, growth of neuronal connections and synaptic function. Here we will present the recent advances in understanding the genetic determinants of social behavior, as they emerge from the study of ASD mouse models, and discuss the importance of these studies for the development of novel therapeutic approaches to overcome social disturbances in ASD.

A New Splicing Isoform of Cacna2d4 Mimicking the Effects of c.2451insC Mutation in the Retina: Novel Molecular and Electrophysiological Insights.

PURPOSE Mutations in CACNA2D4 exon 25 cause photoreceptor dysfunction in humans (c.2406C→A mutation) and mice (c.2451insC mutation). We investigated the feasibility of an exon-skipping therapeutic approach by evaluating the splicing patterns and functional role of targeted exons. METHODS Splicing of the targeted α2δ4 (CACNA2D4) exons in presence and absence of the mutation was assessed by RT-PCR in vivo on mouse retinae and in vitro in HEK293T cells using splicing-reporter minigenes. Whole-cell patch-clamp recordings were performed to evaluate the impact of different Cacna2d4 variants on the biophysical properties of Cav1.4 L-type calcium channels (CACNA1F). RESULTS Splicing analysis revealed the presence of a previously unknown splicing isoform of α2δ4 in the retina that truncates the gene open reading frame (ORF) in a similar way as the c.2451insC mutation. This isoform originates from alternative splicing of exon 25 (E25) with a new exon (E25b). Moreover, the c.2451insC mutation has an effect on splicing and increases the proportion of transcripts including E25b. Our electrophysiological analyses showed that only full-length α2δ4 was able to increase Cav1.4/β3-mediated currents while all other α2δ4 variants did not mediate such effect. CONCLUSIONS The designed exon-skipping strategy is not applicable because the resulting skipped α2δ4 are nonfunctional. α2δ4 E25b splicing variant is normally present in mouse retina and mimics the effect of c.2451insC mutation. Since this variant does not promote significant Cav1.4-mediated calcium current, it could possibly mediate a different function, unrelated to modulation of calcium channel properties at the photoreceptor terminals.