Abstract
Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations.
Highlights
Genetic studies of monogenic forms of autism spectrum disorder (ASD) have identified synaptic function as one of the molecular pathways underlying neurodevelopmental disorders [1]
We show that up-regulation of immunoglobulin heavy-chain-binding protein (BiP) and C/EBP-homologous protein (CHOP) [C/EBP (CCAAT/enhancer-binding protein)homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype
We show that all three endoplasmic reticulum (ER) stress sensors, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), are activated by the mutant R451C NLGN3 protein, eliciting the corresponding signalling cascades in a time-dependent manner upon NLGN3 synthesis
Summary
Genetic studies of monogenic forms of ASDs (autism spectrum disorders) have identified synaptic function as one of the molecular pathways underlying neurodevelopmental disorders [1]. Rare human autism-linked mutations in the NLGN genes have been shown to affect protein folding and trafficking to the cell surface. The R451C single point mutation in NLGN3, an X-linked gene, was found in association with highly penetrant autism in a Swedish family [3]. Whereas most of the mutant protein was degraded, a small fraction of the protein was correctly trafficked to the cell surface [8]. Consistent with these in vitro studies, in vivo work on knockin R451C NLGN3 mice has shown that this mutation caused a 90 % reduction in NLGN3 protein levels [9]
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