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Autism Spectrum Disorder (ASD) is characterized by substantial, yet highly heterogeneous abnormalities in functional brain connectivity. However, the origin and significance of this phenomenon remain unclear. To unravel ASD connectopathy and relate it to underlying etiological heterogeneity, we carried out a bi-center cross-etiological investigation of fMRI-based connectivity in the mouse, in which specific ASD-relevant mutations can be isolated and modeled minimizing environmental contributions. By performing brain-wide connectivity mapping across 16 mouse mutants, we show that different ASD-associated etiologies cause a broad spectrum of connectional abnormalities in which diverse, often diverging, connectivity signatures are recognizable. Despite this heterogeneity, the identified connectivity alterations could be classified into four subtypes characterized by discrete signatures of network dysfunction. Our findings show that etiological variability is a key determinant of connectivity heterogeneity in ASD, hence reconciling conflicting findings in clinical populations. The identification of etiologically-relevant connectivity subtypes could improve diagnostic label accuracy in the non-syndromic ASD population and paves the way for personalized treatment approaches., Molecular Psychiatry, 26 (12), ISSN:1359-4184, ISSN:1476-5578

Abnormal tactile response is an integral feature of Autism Spectrum Disorders (ASDs), and hypo-responsiveness to tactile stimuli is often associated with the severity of ASDs core symptoms. Patients with Phelan-McDermid syndrome (PMS), caused by mutations in the SHANK3 gene, show ASD-like symptoms associated with aberrant tactile responses. The neural underpinnings of these abnormalities are still poorly understood. Here we investigated, in Shank3b−/− adult mice, the neural substrates of whisker-guided behaviors, a key component of rodents’ interaction with the surrounding environment. We assessed whisker-dependent behaviors in Shank3b−/− adult mice and age-matched controls, using the textured novel object recognition (tNORT) and whisker nuisance (WN) test. Shank3b−/− mice showed deficits in whisker-dependent texture discrimination in tNORT and behavioral hypo-responsiveness to repetitive whisker stimulation in WN. Sensory hypo-responsiveness was accompanied by a significantly reduced activation of the primary somatosensory cortex (S1) and hippocampus, as measured by c-fos mRNA induction, a proxy of neuronal activity following whisker stimulation. Moreover, resting-state fMRI showed a significantly reduced S1-hippocampal connectivity in Shank3b mutants, in the absence of altered connectivity between S1 and other somatosensory areas. Impaired crosstalk between hippocampus and S1 might underlie Shank3b−/− hypo-reactivity to whisker-dependent cues, highlighting a potentially generalizable somatosensory dysfunction in ASD.

Abnormal tactile response is considered an integral feature of Autism Spectrum Disorders (ASDs), and hypo-responsiveness to tactile stimuli is often associated with the severity of ASDs core symptoms. Patients with Phelan-McDermid syndrome (PMS), caused by mutations in the SHANK3 gene, show ASD-like symptoms associated with aberrant tactile responses. However, the neural underpinnings of these somatosensory abnormalities are still poorly understood. Here we investigated, in Shank3b−/− adult mice, the neural substrates of whisker-guided behaviors, a key component of rodents’ interaction with the surrounding environment. To this aim, we assessed whisker-dependent behaviors in Shank3b−/− adult mice and age-matched controls, using the textured novel object recognition (tNORT) and whisker nuisance (WN) test. Shank3b−/− mice showed deficits in whisker-dependent texture discrimination in tNORT and behavioral hypo-responsiveness to repetitive whisker stimulation in WN. Notably, sensory hypo-responsiveness was accompanied by a significantly reduced activation of the primary somatosensory cortex (S1) and hippocampus, as measured by c-fos mRNA in situ hybridization, a proxy of neuronal activity following whisker stimulation. Moreover, resting-state fMRI showed a significantly reduced S1-hippocampal connectivity in Shank3b mutant mice. Together, these findings suggest that impaired crosstalk between hippocampus and S1 might underlie Shank3b−/− hypo-reactivity to whisker-dependent cues, highlighting a potentially generalizable form of dysfunctional somatosensory processing in ASD.Significance StatementPatients with Phelan-McDermid syndrome, a syndromic form of ASD caused by mutation of the SHANK3 gene, often show aberrant responses to touch. However, the neural basis of atypical sensory responses in ASD remains undetermined. Here we used Shank3 deficient mice to investigate the neural substrates of behavioral responses to repetitive stimulation of the whiskers, a highly developed sensory organ in mice. We found that mice lacking the Shank3 gene are hypo-responsive to repetitive whisker stimulation. This trait was associated with reduced engagement and connectivity between the primary somatosensory cortex and hippocampus. These results suggest that dysfunctional cortico-hippocampal coupling may underlie somatosensory processing deficits in SHANK3 mutation carriers and related syndromic forms of ASD.

Impaired function of GABAergic interneurons, and the subsequent alteration of excitation/inhibition balance, is thought to contribute to autism spectrum disorders (ASD). Altered numbers of GABAergic interneurons and reduced expression of GABA receptors has been detected in the brain of ASD subjects and mouse models of ASD. We previously showed a reduced expression of GABAergic interneuron markers parvalbumin (PV) and somatostatin (SST) in the forebrain of adult mice lacking the Engrailed2 gene (En2-/- mice). Here, we extended this analysis to postnatal day (P) 30 by using in situ hybridization, immunohistochemistry, and quantitative RT-PCR to study the expression of GABAergic interneuron markers in the hippocampus and somatosensory cortex of En2-/- and wild type (WT) mice. In addition, GABA receptor subunit mRNA expression was investigated by quantitative RT-PCR in the same brain regions of P30 and adult En2-/- and WT mice. As observed in adult animals, PV and SST expression was decreased in En2-/- forebrain of P30 mice. The expression of GABA receptor subunits (including the ASD-relevant Gabrb3) was also altered in young and adult En2-/- forebrain. Our results suggest that GABAergic neurotransmission deficits are already evident at P30, confirming that neurodevelopmental defects of GABAergic interneurons occur in the En2 mouse model of ASD.

Foxg1 is an ancient transcription factor gene orchestrating a number of neurodevelopmental processes taking place in the rostral brain. In this study, we investigated its impact on neocortical activity. We found that mice overexpressing Foxg1 in neocortical pyramidal cells displayed an electroencephalography (EEG) with increased spike frequency and were more prone to kainic acid (KA)-induced seizures. Consistently, primary cultures of neocortical neurons gain-of-function for Foxg1 were hyperactive and hypersynchronized. That reflected an unbalanced expression of key genes encoding for ion channels, gamma aminobutyric acid and glutamate receptors, and was likely exacerbated by a pronounced interneuron depletion. We also detected a transient Foxg1 upregulation ignited in turn by neuronal activity and mediated by immediate early genes. Based on this, we propose that even small changes of Foxg1 levels may result in a profound impact on pyramidal cell activity, an issue relevant to neuronal physiology and neurological aberrancies associated to FOXG1 copy number variations.

The Engrailed-2 (En2) gene codes for a homeobox-containing transcription factor, involved in midbrain-hindbrain embryonic development. In postnatal brain, En2 is expressed in the ventral mesencephalon, cerebellum, hippocampus and neocortex. Two single-nucleotide polymorphisms (SNPs) that are associated to autism spectrum disorders (ASD) have been identified in the human EN2 gene. Accordingly, mice lacking the En2 homeodomain (En2hd/hd, referred to as En2-/-) show molecular, anatomical and behavioral "ASD-like" features. Among these, we previously showed a partial loss of GABAergic interneurons in the En2-/- postnatal hippocampus and neocortex, accompanied by a marked decrease of brain-derived neurotrophic factor (BDNF) signaling, a crucial determinant of GABAergic differentiation. In order to better investigate the role of En2 in GABAergic interneuron differentiation, we generated and subsequently differentiated neural stem cells (NSCs) from basal ganglia and neocortex of En2+/+ and En2-/- mouse embryos. Wild-type NSCs from both basal ganglia and neocortex express En2, while mutant ones do not, as expected. As compared to En2+/+ NSCs, En2-/- NSCs derived from basal ganglia show impaired GABAergic differentiation accompanied by a reduced expression of the BDNF receptor trkB. Conversely, En2-/- NSCs derived from the neocortex expressed high levels of trkB and readily differentiated into neurons, as En2+/+ NSCs. Our results suggest that En2 contributes to GABAergic neuron differentiation from basal ganglia NSCs through a trkB-dependent BDNF signaling, thus providing a possible explanation for the reduced number of GABAergic interneurons detected in the En2-/- postnatal forebrain.

Overreactivity and defensive behaviors in response to tactile stimuli are common symptoms in autism spectrum disorder (ASD) patients. Similarly, somatosensory hypersensitivity has also been described in mice lacking ASD-associated genes such asFmr1(fragile X mental retardation protein 1).Fmr1knock-out mice also show reduced functional connectivity between sensory cortical areas, which may represent an endogenous biomarker for their hypersensitivity. Here, we measured whole-brain functional connectivity inEngrailed-2knock-out (En2−/−) adult mice, which show a lower expression ofFmr1and anatomical defects common toFmr1knock-outs. MRI-based resting-state functional connectivity in adultEn2−/−mice revealed significantly reduced synchronization in somatosensory-auditory/associative cortices and dorsal thalamus, suggesting the presence of aberrant somatosensory processing in these mutants. Accordingly, when tested in the whisker nuisance test,En2−/−but not WT mice of both sexes showed fear behavior in response to repeated whisker stimulation.En2−/−mice undergoing this test exhibited decreased c-Fos-positive neurons (a marker of neuronal activity) in layer IV of the primary somatosensory cortex and increased immunoreactive cells in the basolateral amygdala compared with WT littermates. Conversely, when tested in a sensory maze,En2−/−and WT mice spent a comparable time in whisker-guided exploration, indicating that whisker-mediated behaviors are otherwise preserved inEn2mutants. Therefore, fearful responses to somatosensory stimuli inEn2−/−mice are accompanied by reduced basal connectivity of sensory regions, reduced activation of somatosensory cortex, and increased activation of the basolateral amygdala, suggesting that impaired somatosensory processing is a common feature in mice lacking ASD-related genes.SIGNIFICANCE STATEMENTOverreactivity to tactile stimuli is a common symptom in autism spectrum disorder (ASD) patients. Recent studies performed in mice bearing ASD-related mutations confirmed these findings. Here, we evaluated the behavioral response to whisker stimulation in mice lacking the ASD-related geneEngrailed-2(En2−/−mice). Compared with WT controls,En2−/−mice showed reduced functional connectivity in the somatosensory cortex, which was paralleled by fear behavior, reduced activation of somatosensory cortex, and increased activation of the basolateral amygdala in response to repeated whisker stimulation. These results suggest that impaired somatosensory signal processing is a common feature in mice harboring ASD-related mutations.

Defective cortical processing of visual stimuli and altered retinal function have been described in autism spectrum disorder (ASD) patients. In keeping with these findings, anatomical and functional defects have been found in the visual cortex and retina of mice bearing mutations for ASD-associated genes. Here we sought to investigate the anatomy and function of the adult retina of Engrailed 2 knockout (En2-/-) mice, a model for ASD. Our results showed that En2 is expressed in all three nuclear layers of the adult retina. When compared to age-matched En2+/+ controls, En2-/- adult retinas showed a significant decrease in the number of calbindin+ horizontal cells, and a significant increase in calbindin+ amacrine/ganglion cells. The total number of ganglion cells was not altered in the adult En2-/- retina, as shown by Brn3a+ cell counts. In addition, En2-/- adult mice showed a significant reduction of photoreceptor (rhodopsin) and bipolar cell (Pcp2, PKCα) markers. Functional defects were also present in the retina of En2 mutants, as indicated by electroretinogram recordings showing a significant reduction in both a-wave and b-wave amplitude in En2-/- mice as compared to controls. These data show for the first time that anatomical and functional defects are present in the retina of the En2 ASD mouse model.

The homeobox-containing transcription factor Engrailed-2 (En2) is involved in patterning and neuronal differentiation of the midbrain/hindbrain region, where it is prominently expressed. En2 mRNA is also expressed in the adult mouse hippocampus and cerebral cortex, indicating that it might also function in these brain areas. Genome-wide association studies revealed that En2 is a candidate gene for autism spectrum disorders (ASD), and mice devoid of its expression (En2(-/-) mice) display anatomical, behavioral and clinical "autistic-like" features. Since reduced GABAergic inhibition has been proposed as a possible pathogenic mechanism of ASD, we hypothesized that the phenotype of En2(-/-) mice might include defective GABAergic innervation in the forebrain. Here we show that the Engrailed proteins are present in postnatal GABAergic neurons of the mouse hippocampus and cerebral cortex, and adult En2(-/-) mice show reduced expression of GABAergic marker mRNAs in these areas. In addition, reduction in parvalbumin (PV), somatostatin (SOM) and neuropeptide Y (NPY) expressing interneurons is detected in the hippocampus and cerebral cortex of adult En2(-/-) mice. Our results raise the possibility of a link between altered function of En2, anatomical deficits of GABAergic forebrain neurons and the pathogenesis of ASD.

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(+) Itpr3 (tf) /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.

Dopamine D2 receptor (D2R) signalling has been shown to modulate seizure-induced hippocampal cell death. D2R knockout (D2R−/−) mice are more susceptible to kainic acid (KA)-induced excitotoxicity, displaying cell death in the CA3 subfield of the hippocampus at KA doses not damaging in wild-type (WT) animals. Absence of D2R signalling in the hippocampus leads to activation (dephosphorylation) of glycogen synthase kinase 3β (GSK-3β) after KA (20 mg/kg), which is not associated with a change in the phosphorylation of the GSK-3β regulator Akt at the canonical threonine 308 residue. In the present study, we investigated alternative pathways responsible for the activation of GSK-3β in the hippocampus of the D2R−/− mice 24 h following KA-induced seizures. Here, we show that phosphorylation of Akt occurs at serine 473 (Ser473) in the CA3 region of WT but not D2R−/− mice following KA. Moreover, the CA1 subregion, which does not undergo neurodegeneration in either WT or D2R−/− mice, displays a strong induction of Akt (Ser473) phosphorylation after KA. Additionally, the vulnerability in the CA3 is not associated with changes to p38MAPK and Dishevelled activation, and β-catenin does not appear to be a downstream target of the GSK-3β. Thus, we propose that GSK-3β phosphorylation-mediated hippocampal cell survival may depend on Akt (Ser473) phosphorylation; loss of D2R-mediated signalling in the CA3 region of D2R−/− mice leads to reduced Akt (Ser473) phosphorylation rendering neurons more vulnerable to apoptosis. Further investigation is required to fully elucidate the GSK-3β targets involved in D2R-dependent response to excitotoxicity.

The major component of Lewy Bodies (LB), the pathological hallmark of Parkinson's disease (PD) is α-synuclein, most prominently phosphorylated at serine 129. G-protein coupled receptor kinase 5 (GRK5) has been reported to phosphorylate α-synuclein in vitro, enhancing the α-synuclein toxicity to dopaminergic neurons in Drosophila model. Moreover, GRK5 was found in LBs from brain of PD patients. A genetic association study performed in the Japanese population revealed haplotypic association of the GRK5 gene with susceptibility to sporadic PD. We aimed at investigating whether four polymorphisms within the GRK5 gene (rs871196, rs2420616, rs7069375, rs4752293) could represent a risk factor for sporadic PD in Southern Italy. We genotyped 446 patients with PD and 450 controls for these markers and did not find any significant association with the disease at allelic, genotypic and haplotypic level. Our results indicate that the GRK5 gene does not confer risk to sporadic PD in our sample from Southern Italy.

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 GABA(A) receptor β3 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.

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.

Genome-wide association studies indicated the homeobox-containing transcription factor Engrailed-2 (En2) as a candidate gene for autism spectrum disorders (ASD). Accordingly,En2knock-out (En2−/−) mice show anatomical and behavioral “ASD-like” features, including decreased sociability and learning deficits. The molecular pathways underlying these deficits inEn2−/−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) andEn2−/−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 geneArc. Quantitative RT-PCR, immunoblotting, and immunohistochemistry experiments showed a marked downregulation of neurofibromin expression in the dentate gyrus of both naive and MWM-treatedEn2−/−mice. ERK phosphorylation, known to be induced in the presence of neurofibromin deficiency, was increased in the dentate gyrus ofEn2−/−mice after MWM. Treatment ofEn2−/−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 theEn2mouse model of ASD.

Iron overload may lead to neurodegenerative disorders such as Parkinson’s disease (PD) and alterations of iron-related genes might be involved in the pathogenesis of this disease. The gene of haemochromatosis (HFE) encodes the HFE protein which interacts with the transferrin receptor (TFR), lowering its affinity for iron-bound transferrin (TF). We examined four known polymorphisms, C282Y and H63D in the HFE gene, G258S in the TF gene and S82G in the TFR gene, in 181 sporadic PD patients and 180 controls from Southern Italy to investigate their possible role in susceptibility to PD. No significant differences were found in genotype and allele frequencies between PD and controls for all the polymorphisms studied, suggesting that these variants do not contribute significantly to the risk of PD.

Mutations in the gene DJ-1 have been shown to be a rare cause of early-onset Parkinson's disease (EOPD). Since DJ-1 mutations have been found in patients with Parkinson's disease (PD) from southern Italy, we aimed to investigate whether polymorphisms within the DJ-1 gene could represent a risk factor for sporadic PD. First, we genotyped 294 patients with PD and 298 controls coming from southern Italy to assess the distribution of the insertion/deletion (Ins/Del) polymorphism. In a second phase, we identified five single-nucleotide polymorphisms (SNPs) useful to delimit a region potentially involved and genotyped all patients and controls for these markers. All the markers analyzed were significantly associated with PD at both allelic and genotypic level. The most significant association with the disease was found at the Ins/Del polymorphism (p = 0.0001; adjusted odds ratio (OR ) = 2.05; confidence interval (CI ) = 1.36-3.08). When we considered a three-marker sliding window, we found a highly significant association between the disease and the haplotypes including markers rs17523802, Ins/Del, and rs3766606 (p = 0.0007) and markers Ins/Del, rs3766606 and rs7517357 (p = 0.0054). Our results indicate that polymorphisms located in a region spanning 3535 bp from the promoter to the intron 2 of the DJ-1 gene confer risk to sporadic PD in southern Italy.

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.

Transcriptome 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. Cerebellar 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. Given 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. Despite 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.