Analyses Of The Autism-associated Neuroligin-3 R451C Mutation In Human Neurons Reveal A Gain-of-Function Synaptic Mechanism

Background:Mutations in many synaptic genes are associated with autism spectrum disorders（ASDs）,suggesting that synaptic dysfunction is a key driver of ASD pathogenesis.Among these mutations,the R451C-substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs.In mice,the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities,but the impact of the NLGN3R451C-mutation on human neurons has not been investigated.Objective:We aim to study the impact of NLGN3^R451C on synaptic function impact and the molecular mechanism in human induced neuron model.Methods:1.We generated isogenic knock in（KI）human embryonic stem（ES）cell lines harboring NLGN3-R451C allele.2.To examine the impact of NLGN3-R451C on cellular function,we generated excitatory and inhibitory induced human neurons co-culture system.3.We co-grafted control and R451C human excitatory neurons into the forebrain of neonatal（postnatal day 0-3）RAG2^-/-immunocompromised mice.4.To gain insight into the underlying molecular mechanism of NLGN3-R451C in human neurons,we conducted single-cell gene expression profiling and examined the differential expressed genes（DEGs）.5.To determine whether NLGN3-R451C affects the connection between neurons,we performed cell-cell interaction analysis.6.To further determine whether NLGN3-R451C induced DEGs are associated with psychiatric disorders including ASDs,schizophrenia（SCZ）,bipolar disorder（BP）,and major depression disorder（MDD）,we performed enrichment tests for the risk genes of ASD,SCZ,BP,MDD,and body mass index（BMI）as a control.Results:1.Analysis of co-cultured excitatory and inhibitory induced neurons（i Ns）revealed enhanced excitatory synapse number and synaptic strength in neuronal culture harboring R451C mutation compared to isogenic control.2.No significant cell apoptosis and endoplasmic reticulum stress were found to be associated with R451C mutation.3.The augmentation in excitatory transmission was also confirmed in vivo mouse forebrain transplanted model.4.Using single-cell RNA seq on co-cultured excitatory and inhibitory i Ns,we identified differentially expressed genes（DEGs）in the subgroup of relatively mature human neurons and found NLGN3-R451C alters gene5.In ligand-receptor binding pair cell-cell communication analysis,the interaction number and strength of excitatory neurons are significantly increased.6.Risk genes enrichment analysis suggested DEGs identified in R451C human neuron subtypes associated strongly with ASD,SCZ,and BP but not with MDD and obesity.Conclusions:Our finding suggests that the NLGN3-R451C mutation could impact synaptic transmission,i.e.gain-of-function in excitatory synaptic transmission,which may cause the imbalance between excitatory and inhibitory synaptic transmission and represent the possible synaptic mechanism underlying pathophysiology of ASDs.