Abstract
Autism spectrum disorder (ASD) is a neurodevelopmental disorder with an unknown molecular pathogenesis. A recent molecular focus has been the mutated neuroligin 3, neuroligin 3(R451C), in gain-of-function studies and for its role in induced impairment of synaptic function, but endoplasmic reticulum (ER) stress induced by mutated molecules also deserves investigation. We previously found two missense mutations, H246N and Y251S, in the gene-encoding synaptic cell adhesion molecule-1 (CADM1) in ASD patients, including cleavage of the mutated CADM1 and its intracellular accumulation. In this study, we found that the mutated CADM1 showed slightly reduced homophilic interactions in vitro but that most of its interactions persist. The mutated CADM1 also showed morphological abnormalities, including shorter dendrites, and impaired synaptogenesis in neurons. Wild-type CADM1 was partly localized to the ER of C2C5 cells, whereas mutated CADM1 mainly accumulated in the ER despite different sensitivities toward 4-phenyl butyric acid with chemical chaperone activity and rapamycin with promotion activity for degradation of the aggregated protein. Modeling analysis suggested a direct relationship between the mutations and the conformation alteration. Both mutated CADM1 and neuroligin 3(R451C) induced upregulation of C/EBP-homologous protein (CHOP), an ER stress marker, suggesting that in addition to the trafficking impairment, this CHOP upregulation may also be involved in ASD pathogenesis.
Highlights
NLGNs are postsynaptic cell adhesion proteins that interact with neurexins on the presynaptic membrane,[5] and they are required for synapse maturation.[6]
We show that the Autism spectrum disorder (ASD)-related cell adhesion molecule-1 (CADM1) mutations, H246N and Y251S, and the NLGN3 mutation, R451C, cause an unfolded protein response (UPR) response with upregulation of CHOP as a gain of function
We examined the trans-interaction between wild-type and mutated CADM1 molecules in vitro by pull-down and western blot analysis (Figure 1a)
Summary
NLGNs are postsynaptic cell adhesion proteins that interact with neurexins on the presynaptic membrane,[5] and they are required for synapse maturation.[6]. Its extracellular domain displays calcium-independent homophilic trans-cell adhesion activity,[10,11] and its intracellular domain associates with calmodulin-associated serine/threonine kinase via individual PDZ-binding domains (PDZ: post synaptic density protein/Drosophila disc large tumor suppressor/ zonula occludens-1 protein).[10] Recently, we identified two missense mutations, C739A (amino acid: H246N) and A755C(Y251S), in the CADM1 gene of male Caucasian patients with ASD and their family members.[3] Both mutations are located in the third Ig (Ig3) domain of CADM1, which is essential for trans-active interactions These ASD-related mutations stimulate the cleavage of CADM1 and induce defective trafficking to the cell surface.[3] These results suggest an association between impaired synaptogenesis and the pathogenesis of ASD. ER stress is a gain-of-function associated with the mutated protein
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