Etude génétique et fonctionnelle de CNTNAP2 / Caspr2 dans les troubles du spectre autistique

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterised by deficits in social communication and repetitive behaviours. Alterations in brain connectivity are the most replicated findings in ASD patients. The CNTNAP2 gene, coding Caspr2, has been associated to ASD, with a large number of missense heterozygous variants identified in patients. However, some missense variants have also been found in the control population, questioning their pathogenicity. Here, using in vitro and in vivo approaches we identify new functions for Caspr2 in axonal development and provided proof of principle that some variants could impact these functions. We report that Caspr2 is involved in axon growth of cortical neurons in culture in a dose-dependent manner, with Cntnap2+/- neurons presenting an intermediate phenotype between wild type and Cntnap2-/- neurons. We also show that Caspr2 is required in vivo for the development of axons projecting into two major interhemispheric myelinated tracts, the corpus callosum and the anterior commissure. Performing morphometric and electron microscopy analyses we detect morphological modifications of these structures and alterations in axo-axonal contacts and axonal diameter in both Cntnap2+/- and Cntnap2-/- mice throughout development. Using in vitro assays and axon growth as read-out, we further show that some of the variants display either a dominant-negative effect or a loss-of-function in a Cntnap2+/- genetic background, suggesting that they could alter brain connectivity and thus contribute to the manifestations of ASD.Les troubles du spectre autistique (TSA) sont caractérisés par des déficits de communication sociale et des comportements répétitifs, qui résultent d’altérations de la connectivité cérébrale. Le gène CNTNAP2, codant Caspr2, a été associé aux TSA, avec de nombreux variants hétérozygotes faux-sens identifiés chez les patients. Cependant, des variants faux-sens sont également présents dans les sujets contrôles, questionnant leur pouvoir pathogène. Par des approches in vitro et in vivo, nous identifions ici de nouvelles fonctions de Caspr2 au cours du développement axonal et apportons la preuve que certains des variants pourraient impacter ces fonctions. Nous démontrons que Caspr2 régule de manière dose-dépendante la croissance axonale des neurones corticaux en culture, les neurones Cntnap2+/- présentant un phénotype intermédiaire par rapport aux neurones sauvages et Cntnap2-/-. Nous montrons également que Caspr2 participe in vivo au développement des axones de deux tractus interhémisphériques myélinisés majeurs, le corps calleux et la commissure antérieure. Des analyses morphométriques et de microscopie électronique révèlent des modifications morphologiques de ces structures et des altérations des contacts axo-axonaux et du diamètre axonal chez les souris Cntnap2+/- et Cntnap2-/- tout au long du développement. En utilisant des approches in vitro et la croissance axonale comme test biologique, nous montrons en outre que certains variants présentent un effet dominant-négatif ou une perte de fonction dans un contexte génétique Cntnap2+/-, suggérant qu’ils pourraient affecter la connectivité cérébrale et ainsi contribuer aux manifestations des TSA

CNTNAP2 Heterozygous Missense Variants: Risk Factors for Autism Spectrum Disorder and/or Other Pathologies?

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Genetic variants in autism-related CNTNAP2 impair axonal growth of cortical neurons

International audienceThe CNTNAP2 gene, coding for the cell adhesion glycoprotein Caspr2, is thought to be one of the major susceptibility genes for autism spectrum disorder (ASD). A large number of rare heterozygous missense CNTNAP2 variants have been identified in ASD patients. However, most of them are inherited from an unaffected parent, questioning their clinical significance. In the present study, we evaluate their impact on neurodevelopmental functions of Caspr2 in a heterozygous genetic background. Performing cortical neuron cultures from mouse embryos, we demonstrate that Caspr2 plays a dose-dependent role in axon growth in vitro. Loss of one Cntnap2 allele is sufficient to elicit axonal growth alteration, revealing a situation that may be relevant for CNTNAP2 heterozygosity in ASD patients. Then, we show that the two ASD variants I869T and G731S, which present impaired binding to Contactin2/TAG-1, do not rescue axonal growth deficits. We find that the variant R1119H leading to protein trafficking defects and retention in the endoplasmic reticulum has a dominant-negative effect on heterozygous Cntnap2 cortical neuron axon growth, through oligomerization with wild-type Caspr2. Finally, we identify an additional variant (N407S) with a dominant-negative effect on axon growth although it is well-localized at the membrane and properly binds to Contactin2. Thus, our data identify a new neurodevelopmental function for Caspr2, the dysregulation of which may contribute to clinical manifestations of ASD, and provide evidence that CNTNAP2 heterozygous missense variants may contribute to pathogenicity in ASD, through selective mechanisms

Image_2_Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse.TIF

Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2–/– and Cntnap2+/– mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2+/– mice, either specifically, more severely, or oppositely as compared to Cntnap2–/– mice. In addition, Cntnap2+/– mice, but not Cntnap2–/– mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.</p

Image_1_Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse.TIF

Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2–/– and Cntnap2+/– mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2+/– mice, either specifically, more severely, or oppositely as compared to Cntnap2–/– mice. In addition, Cntnap2+/– mice, but not Cntnap2–/– mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.</p

Data_Sheet_1_Differential impacts of Cntnap2 heterozygosity and Cntnap2 null homozygosity on axon and myelinated fiber development in mouse.PDF

Over the last decade, a large variety of alterations of the Contactin Associated Protein 2 (CNTNAP2) gene, encoding Caspr2, have been identified in several neuronal disorders, including neurodevelopmental disorders and peripheral neuropathies. Some of these alterations are homozygous but most are heterozygous, and one of the current challenges is to estimate to what extent they could affect the functions of Caspr2 and contribute to the development of these pathologies. Notably, it is not known whether the disruption of a single CNTNAP2 allele could be sufficient to perturb the functions of Caspr2. To get insights into this issue, we questioned whether Cntnap2 heterozygosity and Cntnap2 null homozygosity in mice could both impact, either similarly or differentially, some specific functions of Caspr2 during development and in adulthood. We focused on yet poorly explored functions of Caspr2 in axon development and myelination, and performed a morphological study from embryonic day E17.5 to adulthood of two major brain interhemispheric myelinated tracts, the anterior commissure (AC) and the corpus callosum (CC), comparing wild-type (WT), Cntnap2–/– and Cntnap2+/– mice. We also looked for myelinated fiber abnormalities in the sciatic nerves of mutant mice. Our work revealed that Caspr2 controls the morphology of the CC and AC throughout development, axon diameter at early developmental stages, cortical neuron intrinsic excitability at the onset of myelination, and axon diameter and myelin thickness at later developmental stages. Changes in axon diameter, myelin thickness and node of Ranvier morphology were also detected in the sciatic nerves of the mutant mice. Importantly, most of the parameters analyzed were affected in Cntnap2+/– mice, either specifically, more severely, or oppositely as compared to Cntnap2–/– mice. In addition, Cntnap2+/– mice, but not Cntnap2–/– mice, showed motor/coordination deficits in the grid-walking test. Thus, our observations show that both Cntnap2 heterozygosity and Cntnap2 null homozygosity impact axon and central and peripheral myelinated fiber development, but in a differential manner. This is a first step indicating that CNTNAP2 alterations could lead to a multiplicity of phenotypes in humans, and raising the need to evaluate the impact of Cntnap2 heterozygosity on the other neurodevelopmental functions of Caspr2.</p