Unfolded protein response activated by Neuroligin autism-linked mutation causing retention in the endoplasmic reticulum

Rare mutations in genes encoding synaptic proteins, specifically neuroligins 3 and 4 contribute to 1% of Autism Spectrum Disorders diagnoses. Most of the mutations characterized so far, map to the extracellular domain and affect protein folding. In particular the R451C substitution in Neuroligin3 causes a local misfolding in the extracellular protein domain, which induces the partial retention of the protein in the Endoplasmic Reticulum (ER). Accumulation of misfolded proteins in the ER might activate an ER stress response characterized by a signaling cascade named the Unfolded Protein Response (UPR), a program finalized to restore normal ER functions and recently correlated to many neurological diseases. In order to investigate whether the misfolded protein is leading to the activation of the unfolded protein response, we have generated PC12 clones where either wild-­‐type or R451C mutant Neuroligin3 can be induced by doxycycline. Our results show that the R451C mutation in Neuroligin3 activates single UPR branches downstream of the ER stress sensors: IRE1, ATF6 and PERK. Also we show increased expression levels of the main UPR target genes in undifferentiated and neuron-­‐like differentiated PC12 cells. Our results show activation of a cellular response due to the intracellular accumulation of the protein that could underlie possible functional alterations. The R451C Neuroligin3 Knock-­‐In mice represent a valid model system for investigating the involvment of ER stress in vivo and its possible correlation to a “gain of function” effect caused by the mutation.