R451C autism linked substitution in Neuroligin3 activates the unfolded protein response in a neuronal inducible system

Several forms of monogenic autism spectrum disorders (ASDs) are associated to mutations in the Neuroligin (NLGNs) genes. The autism-linked substitution of arginine 451 by a cysteine (R451C) in NLGN3 induces local misfolding of the extracellular domain, causing partial retention in the endoplasmic reticulum (ER). ER stress due to the accumulation of misfolded proteins can result in the unfolded protein response (UPR). We have generated a PC12 Tet-On cell lines with inducible expression WT or R451C NLGN3 to investigate if there is activation of the unfolded protein response (UPR) as a result of misfolded protein retention. Our data show that overexpression of R451C mutant protein leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6, IRE1 and PERK. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that upregulation of BiP and CHOP was induced by both mutant proteins but not by wild type neuroligin3, both in proliferative cells and cells differentiated to a neuronal–like phenotype. Downstream effects of UPR have been found in PC12 NLGN3 Tet-On cells differentiated to a neuronal phenotype. This supports the relevance of the UPR elicited by the ER retention of misfolded R451C NLGN3, which might in turn play a role in neuronal behaviour. In fact, although the UPR is classically linked to protein folding stress under pathological conditions, it is becoming clear that UPR signalling also regulates various processes, including synaptic functions. At the molecular levels, subtype-selective modulation of cell surface receptors by CHOP has been reported and the phosphorylation of eIF2α has been associated to synaptic plasticity, learning and memory. Moreover, in vivo administration of GSK2606414 has been shown to affect memory consolidation, supporting the role of UPR and its mediators in mediating synaptic functions. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in PC12 cells, suggesting that this response may lead to neuronal circuit alterations and consequently have a role in the autistic phenotype.