Unfolded Protein Response activated by an autism-associated mutation in Neuroligin3

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders with a strong genetic background. Several mutations identified in association with ASDs are found in genes encoding synaptic cell adhesion molecules (sCAMs), such as Neuroligins (NLGNs). The best characterized among the ASD-linked mutations is the R451C substitution in NLGN3. In vitro studies indicate that R451C substitution causes a local misfolding in the extracellular domain of NLGN3, which induces the retention of the protein in the Endoplasmic Reticulum (ER). Accumulation of misfolded proteins in the ER activates the Unfolded Protein Response (UPR), a program finalized to restore normal ER functions and recently correlated to many neurological diseases. We have used HEK-293 and PC12 Tet-On cells expressing wild-type or mutant NLGN3 in order to investigate whether the mutant protein leads to UPR activation. Our results show that NLGN3 R451C activates distinct UPR signaling pathways as shown by the induction of a reporter gene containing ER stress responsive elements, by the splicing of XBP1 mRNA and the eIF2α phosphorylation. In addition, we have developed a cellular system to quantify protein trafficking in HEK-293, cloning a soluble NLGN3 form fused to a fluorescent protein, that is secreted in the culture media, whose fluorescence can be quantified. This system will be used to screen libraries of compounds, in order to rescue the trafficking alteration caused by the mutation.