Insights Into the Role of Contactin 4, -5 and -6 in Autism Spectrum Disorders

Background: Autism spectrum disorders (ASD) are a heterogeneous group of severe developmental disorders. A strong genetic component contributes to the aetiologies of ASD, which may vary from monogenic forms of ASD with the presence of de novomutations to oligo – and polygenic forms with the interaction of multiple hits within the genome. We have previously shown the involvement of genes encoding proteins involved in the formation and maintenance of synapses such as the Neuroligin-Shank-Neurexin pathway, with rare variants having a strong effect found in a relatively limited number of families.

Objectives: We focused our study on contactins (CNTNs), a family of six cell adhesion proteins of the immunoglobulin superfamily which are involved in brain development, neuronal wiring, and promote neurite outgrowth in vitro. Our genetic studies have led to the identification of copy number variations as well as non-synonymous rare variations in CNTN4, CNTN5 and CNTN6 genes in patients with ASD, and also in control individuals. Most of these variants were inherited from healthy parents. For CNTN5 and CNTN6, the identified mutations are distributed among the immunoglobulin and fibronectin domains of CNTN molecules. For CNTN4, we observed a single stop mutation located in the first fibronectin-like domain, leading to a truncated protein. We investigated the effects of wild-type and mutated rat Cntn4, Cntn5 and Cntn6 on different aspects of neurite outgrowth. In a second set of experiments, we analyzed the molecular interactions of human PTPRG and human CNTN4, -5, and -6 by generating the respective 3D structures of each complex, respectively. We then tested the effects of mutations located in the first four Ig-domains of CNTN5 and CNTN6 on the 3D structures of these proteins. 

Methods: We set up a co-culture system using rat cortical neurons and HEK293 cells over-expressing and delivering the secreted forms of wild-type and mutated rat contactin4, -5 and -6 in the vicinity of neurons. The molecular interaction of CNTN with PTPRG was studied using homology modeling and protein docking calculations.

Results: Our results show different promoting effects of wild-type rat Cntn4, Cntn5 and Cntn6 on neurite outgrowth and branching. We also observed significant differences, at the crucial Ig2-Ig3 domains, in the structure of human CNTN5 and CNTN6 as compared to CNTN4, with no direct consequence on PTPRG binding. The functional screening of contactin mutations identified in patients with ASD revealed significant alterations in the neurite outgrowth properties as compared to wild-type contactin proteins. 

Conclusions: Our data suggest that the differential contactin effects on neurite outgrowth do not result from distinct interactions with PTPRG. Results from our mutational screening indicate that rare variations of the contactins might represent inherited risk factors for ASD.