Molecular Architecture of Contactin-associated Protein-like 2 (CNTNAP2) and Its Interaction with Contactin 2 (CNTN2).
ABSTRACT: Contactin-associated protein-like 2 (CNTNAP2) is a large multidomain neuronal adhesion molecule implicated in a number of neurological disorders, including epilepsy, schizophrenia, autism spectrum disorder, intellectual disability, and language delay. We reveal here by electron microscopy that the architecture of CNTNAP2 is composed of a large, medium, and small lobe that flex with respect to each other. Using epitope labeling and fragments, we assign the F58C, L1, and L2 domains to the large lobe, the FBG and L3 domains to the middle lobe, and the L4 domain to the small lobe of the CNTNAP2 molecular envelope. Our data reveal that CNTNAP2 has a very different architecture compared with neurexin 1α, a fellow member of the neurexin superfamily and a prototype, suggesting that CNTNAP2 uses a different strategy to integrate into the synaptic protein network. We show that the ectodomains of CNTNAP2 and contactin 2 (CNTN2) bind directly and specifically, with low nanomolar affinity. We show further that mutations in CNTNAP2 implicated in autism spectrum disorder are not segregated but are distributed over the whole ectodomain. The molecular shape and dimensions of CNTNAP2 place constraints on how CNTNAP2 integrates in the cleft of axo-glial and neuronal contact sites and how it functions as an organizing and adhesive molecule.
Project description:Genetic studies are rapidly identifying variants that shape risk for disorders of human cognition, but the question of how such variants predispose to neuropsychiatric disease remains. Noninvasive human brain imaging allows assessment of the brain in vivo, and the combination of genetics and imaging phenotypes remains one of the only ways to explore functional genotype-phenotype associations in human brain. Common variants in contactin-associated protein-like 2 (CNTNAP2), a neurexin superfamily member, have been associated with several allied neurodevelopmental disorders, including autism and specific language impairment, and CNTNAP2 is highly expressed in frontal lobe circuits in the developing human brain. Using functional neuroimaging, we have demonstrated a relationship between frontal lobar connectivity and common genetic variants in CNTNAP2. These data provide a mechanistic link between specific genetic risk for neurodevelopmental disorders and empirical data implicating dysfunction of long-range connections within the frontal lobe in autism. The convergence between genetic findings and cognitive-behavioral models of autism provides evidence that genetic variation at CNTNAP2 predisposes to diseases such as autism in part through modulation of frontal lobe connectivity.
Project description:Intragenic deletions of the contactin-associated protein-like 2 gene (CNTNAP2) have been found in patients with Gilles de la Tourette syndrome, intellectual disability (ID), obsessive compulsive disorder, cortical dysplasia-focal epilepsy syndrome, autism, schizophrenia, Pitt-Hopkins syndrome, stuttering, and attention deficit hyperactivity disorder. A variety of molecular mechanisms, such as loss of transcription factor binding sites and perturbation of penetrance and expressivity, have been proposed to account for the phenotypic variability resulting from CNTNAP2 mutations. Deletions of both CNTNAP2 alleles produced truncated proteins lacking the transmembrane or some of the extracellular domains, or no protein at all. This observation can be extended to heterozygous intragenic deletions by assuming that such deletion-containing alleles lead to expression of a Caspr2 protein lacking one or several extracellular domains. Such altered forms of Capr2 proteins will lack the ability to bridge the intercellular space between neurons by binding to partners, such as CNTN1, CNTN2, DLG1, and DLG4. This presumed effect of intragenic deletions of CNTNAP2, and possibly other genes involved in connecting neuronal cells, represents a molecular basis for the postulated neuronal hypoconnectivity in autism and probably other neurodevelopmental disorders, including epilepsy, ID, language impairments and schizophrenia. Thus, CNTNAP2 may represent a paradigmatic case of a gene functioning as a node in a genetic and cellular network governing brain development and acquisition of higher cognitive functions.
Project description:Autism is a genetically complex neurodevelopmental syndrome in which language deficits are a core feature. We describe results from two complimentary approaches used to identify risk variants on chromosome 7 that likely contribute to the etiology of autism. A two-stage association study tested 2758 SNPs across a 10 Mb 7q35 language-related autism QTL in AGRE (Autism Genetic Resource Exchange) trios and found significant association with Contactin Associated Protein-Like 2 (CNTNAP2), a strong a priori candidate. Male-only containing families were identified as primarily responsible for this association signal, consistent with the strong male affection bias in ASD and other language-based disorders. Gene-expression analyses in developing human brain further identified CNTNAP2 as enriched in circuits important for language development. Together, these results provide convergent evidence for involvement of CNTNAP2, a Neurexin family member, in autism, and demonstrate a connection between genetic risk for autism and specific brain structures.
Project description:Contactins and Contactin-Associated Proteins, and Contactin-Associated Protein-Like 2 (CNTNAP2) in particular, have been widely cited as autism risk genes based on findings from homozygosity mapping, molecular cytogenetics, copy number variation analyses, and both common and rare single nucleotide association studies. However, data specifically with regard to the contribution of heterozygous single nucleotide variants (SNVs) have been inconsistent. In an effort to clarify the role of rare point mutations in CNTNAP2 and related gene families, we have conducted targeted next-generation sequencing and evaluated existing sequence data in cohorts totaling 2704 cases and 2747 controls. We find no evidence for statistically significant association of rare heterozygous mutations in any of the CNTN or CNTNAP genes, including CNTNAP2, placing marked limits on the scale of their plausible contribution to risk.
Project description:Autism is a childhood neuropsychiatric disorder that, despite exhibiting high heritability, has largely eluded efforts to identify specific genetic variants underlying its etiology. We performed a two-stage genetic study in which genome-wide linkage and family-based association mapping was followed up by association and replication studies in an independent sample. We identified a common polymorphism in contactin-associated protein-like 2 (CNTNAP2), a member of the neurexin superfamily, that is significantly associated with autism susceptibility. Importantly, the genetic variant displays a parent-of-origin and gender effect recapitulating the inheritance of autism.
Project description:The contactin-associated protein-like 2 (CNTNAP2) gene is highly expressed in the frontal lobe circuits in the developing human brain. Mutations in this gene have been associated with several neurodevelopmental disorders such as autism and specific language impairment. Here we describe a 450?kb deletion within the CNTNAP2 gene that is maternally inherited in two male siblings, but with a variable clinical phenotype. This variability is described in the context of a limited number of other cases reported in the literature. The in-frame intragenic deletion removes a critical domain of the CNTNAP2 protein, and this case also highlights the challenges of correlating genotype and phenotype.
Project description:GABAergic interneurons are emerging as prominent substrates in the pathophysiology of multiple neurodevelopmental disorders, including autism spectrum disorders, schizophrenia, intellectual disability, and epilepsy. Interneuron excitatory activity is influenced by 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid receptors (AMPARs), which in turn affects excitatory transmission in the central nervous system. Yet how dysregulation of interneuronal AMPARs distinctly contributes to the molecular underpinning of neurobiological disease is drastically underexplored. Contactin-associated protein-like 2 (CNTNAP2) is a neurexin-related adhesion molecule shown to mediate AMPAR subcellular distribution while calcium/calmodulin-dependent serine protein kinase (CASK) is a multi-functional scaffold involved with glutamate receptor trafficking. Mutations in both genes have overlapping disease associations, including autism spectrum disorders, intellectual disability, and epilepsy, thus suggesting converging perturbations of excitatory/inhibitory balance. Our lab has previously shown that CNTNAP2 stabilizes interneuron dendritic arbors through CASK and that CNTNAP2 regulates AMPAR subunit GluA1 trafficking in excitatory neurons. The interaction between these three proteins, however, has not been studied in interneurons. Using biochemical techniques, structured illumination microscopy (SIM) and shRNA technology, we first confirm that these three proteins interact in mouse brain, and then examined relationship between CNTNAP2, CASK and GluA1 in mature interneurons. Using SIM, we ascertain that a large fraction of endogenous CNTNAP2, CASK, and GluA1 molecules collectively colocalize together in a tripartite manner. Finally, individual knockdown of either CNTNAP2 or CASK similarly alter GluA1 levels and localization. These findings offer insight to molecular mechanisms underlying GluA1 regulation in interneurons.
Project description:Although genetic variations in several genes encoding for synaptic adhesion proteins have been found to be associated with autism spectrum disorders, one of the most consistently replicated genes has been CNTNAP2, encoding for contactin-associated protein-like 2 (CASPR2), a multidomain transmembrane protein of the neurexin superfamily. Using immunofluorescence confocal microscopy and complementary biochemical techniques, we compared wild-type CASPR2 to 12 point mutations identified in individuals with autism. In contrast to the wild-type protein, localized to the cell surface, some of the mutants show altered cellular disposition. In particular, CASPR2-D1129H is largely retained in the endoplasmic reticulum (ER) in HEK-293 cells and in hippocampal neurons. BiP/Grp78, Calnexin and ERp57, key ER chaperones, appear to be responsible for retention of this mutant and activation of one signaling pathway of the unfolded protein response (UPR). The presence of this mutation also lowers expression and activates proteosomal degradation. A frame-shift mutation that causes a form of syndromic epilepsy (CASPR2-1253*), results in a secreted protein with seemingly normal folding and oligomerization. Taken together, these data indicate that CASPR2-D1129H has severe trafficking abnormalities and CASPR2-1253* is a secreted soluble protein, suggesting that the structural or signaling functions of the membrane tethered form are lost. Our data support a complex genetic architecture in which multiple distinct risk factors interact with others to shape autism risk and presentation.
Project description:Recent genetic studies have implicated a number of candidate genes in the pathogenesis of Autism Spectrum Disorder (ASD). Polymorphisms of CNTNAP2 (contactin-associated like protein-2), a member of the neurexin family, have already been implicated as a susceptibility gene for autism by at least 3 separate studies. We investigated variation in white and grey matter morphology using structural MRI and diffusion tensor imaging. We compared volumetric differences in white and grey matter and fractional anisotropy values in control subjects characterised by genotype at rs7794745, a single nucleotide polymorphism in CNTNAP2. Homozygotes for the risk allele showed significant reductions in grey and white matter volume and fractional anisotropy in several regions that have already been implicated in ASD, including the cerebellum, fusiform gyrus, occipital and frontal cortices. Male homozygotes for the risk alleles showed greater reductions in grey matter in the right frontal pole and in FA in the right rostral fronto-occipital fasciculus compared to their female counterparts who showed greater reductions in FA of the anterior thalamic radiation. Thus a risk allele for autism results in significant cerebral morphological variation, despite the absence of overt symptoms or behavioural abnormalities. The results are consistent with accumulating evidence of CNTNAP2's function in neuronal development. The finding suggests the possibility that the heterogeneous manifestations of ASD can be aetiologically characterised into distinct subtypes through genetic-morphological analysis.
Project description:The contactin-associated protein-like 2 (CNTNAP2) gene is a member of the neurexin superfamily. CNTNAP2 was first implicated in the cortical dysplasia-focal epilepsy (CDFE) syndrome, a recessive disease characterized by intellectual disability, epilepsy, language impairments and autistic features. Associated SNPs and heterozygous deletions in CNTNAP2 were subsequently reported in autism, schizophrenia and other psychiatric or neurological disorders. We aimed to comprehensively examine evidence for the role of CNTNAP2 in susceptibility to psychiatric disorders, by the analysis of multiple classes of genetic variation in large genomic datasets. In this study we used: i) summary statistics from the Psychiatric Genomics Consortium (PGC) GWAS for seven psychiatric disorders; ii) examined all reported CNTNAP2 structural variants in patients and controls; iii) performed cross-disorder analysis of functional or previously associated SNPs; and iv) conducted burden tests for pathogenic rare variants using sequencing data (4,483 ASD and 6,135 schizophrenia cases, and 13,042 controls). The distribution of CNVs across CNTNAP2 in psychiatric cases from previous reports was no different from controls of the database of genomic variants. Gene-based association testing did not implicate common variants in autism, schizophrenia or other psychiatric phenotypes. The association of proposed functional SNPs rs7794745 and rs2710102, reported to influence brain connectivity, was not replicated; nor did predicted functional SNPs yield significant results in meta-analysis across psychiatric disorders at either SNP-level or gene-level. Disrupting CNTNAP2 rare variant burden was not higher in autism or schizophrenia compared to controls. Finally, in a CNV mircroarray study of an extended bipolar disorder family with 5 affected relatives we previously identified a 131kb deletion in CNTNAP2 intron 1, removing a FOXP2 transcription factor binding site. Quantitative-PCR validation and segregation analysis of this CNV revealed imperfect segregation with BD. This large comprehensive study indicates that CNTNAP2 may not be a robust risk gene for psychiatric phenotypes.