title: Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system.
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.
authors.affiliation: Department of Biology and Biotechnology "Charles Darwin" and Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.
authors.forename: Lisa
authors.initials: L
authors.lastname: Ulbrich
authors.affiliation: Department of Biology and Biotechnology "Charles Darwin" and Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.
authors.forename: Flores Lietta
authors.initials: FL
authors.lastname: Favaloro
authors.affiliation: Department of Biology and Biotechnology "Charles Darwin" and Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Trobiani
authors.affiliation: Department of Internal Medicine, University of Rome Tor Vergata, Via Montpellier, 100133 Rome, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Marchetti
authors.affiliation: Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Vruti
authors.initials: V
authors.lastname: Patel
authors.affiliation: Department of Psychology, "Daniel Bovet" Neurobiology Research Center Sapienza University of Rome, Santa Lucia Foundation, European Center for Brain Research CERC, via dei Marsi 78, 00185 Rome, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Pascucci
authors.affiliation: Department of Neuroscience and Cell Biology and Department of Pediatrics, Child Health Institute of New Jersey, Rutgers, Robert Wood Johnson Medical School, 125 Paterson Street, New Brunswick, NJ 08901, U.S.A.
authors.forename: Davide
authors.initials: D
authors.lastname: Comoletti
authors.affiliation: Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: Marciniak
authors.affiliation: Department of Biology and Biotechnology "Charles Darwin" and Pasteur Institute - Cenci Bolognetti Foundation, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy antonella.dejaco@uniroma1.it.
authors.forename: Antonella
authors.initials: A
authors.lastname: De Jaco
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001321:Autistic Disorder / Q000235:genetics*; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016716:PC12 Cells; D010766:Phosphorylation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D056811:Unfolded Protein Response; D015854:Up-Regulation
chemical_list: D015816:Cell Adhesion Molecules, Neuronal; D015852:Eukaryotic Initiation Factor-2; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C098004:neuroligin 3
keywords: ER stress; autism; molecular chaperones; neuroligin; protein misfolding; unfolded protein response