Abstract
Various single nucleotide polymorphisms (SNPs) in the oxytocin receptor (OXTR) gene have been associated with behavioral traits, autism spectrum disorder (ASD) and other diseases. The non-synonymous SNP rs4686302 results in the OXTR variant A218T and has been linked to core characteristics of ASD, trait empathy and preterm birth. However, the molecular and intracellular mechanisms underlying those associations are still elusive. Here, we uncovered the molecular and intracellular consequences of this mutation that may affect the psychological or behavioral outcome of oxytocin (OXT)-treatment regimens in clinical studies, and provide a mechanistic explanation for an altered receptor function. We created two monoclonal HEK293 cell lines, stably expressing either the wild-type or A218T OXTR. We detected an increased OXTR protein stability, accompanied by a shift in Ca2+ dynamics and reduced MAPK pathway activation in the A218T cells. Combined whole-genome and RNA sequencing analyses in OXT-treated cells revealed 7823 differentially regulated genes in A218T compared to wild-type cells, including 429 genes being associated with ASD. Furthermore, computational modeling provided a molecular basis for the observed change in OXTR stability suggesting that the OXTR mutation affects downstream events by altering receptor activation and signaling, in agreement with our in vitro results. In summary, our study provides the cellular mechanism that links the OXTR rs4686302 SNP with genetic dysregulations associated with aspects of ASD.
Highlights
The neuropeptide oxytocin (OXT) regulates multiple social and emotional behaviors, such as social bonding, reciprocal trust, aggression, fear, and anxiety, both in animals and humans [1,2,3]
Transduction of HEK293 cells with OXT receptors (OXTR) gene variants For a stable integration of the OXTR variants in the genome, moloney murine leukemia virus (MMLV) vectors were designed (VectorBuilder, NeuIsenburg, Germany), and HEK293 cells were transduced according to the Molecular modeling Both monomeric and homodimeric forms of OXTR may be present at the OXTR expression levels in the HEK293 cellular lines studied here [43]
We focused on the transmembrane helix 5 (TM5)/TM5’ dimer, for which the A218T variant is expected to have a more significant effect
Summary
The neuropeptide oxytocin (OXT) regulates multiple social and emotional behaviors, such as social bonding, reciprocal trust, aggression, fear, and anxiety, both in animals and humans [1,2,3]. By coupling to different G-proteins [10], the OXTR is linked to multiple intraneuronal signaling cascades, including Ca2+, protein kinase C and mitogen-activated protein kinase (MAPK) kinase (MEK1/2) signaling [11, 12], the myocyte enhancer factor 2A (MEF2A) [13], as well as mitochondrial respiration [14]. Some of these pathways, e.g., MEF2A signaling and mitochondrial functioning, have been associated with autism spectrum disorder (ASD) [15,16,17]. Synthetic OXT, which can be applied intranasally and penetrates brain tissue [21, 22] has been shown to improve social deficits in autistic children [23, 24]
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