Modeling Synaptic Mechanisms of Autism-associated Neuroligin-3 R451C Mutation Using Human Neurons

Synaptic dysfunction represents a key pathophysiology in neurodevelopmental disorders such as autism spectrum disorder (ASD). Rare mutation R451C in human Neuroligin 3 (NLGN3, encoded by X-linked gene NLGN3), a cell adhesion molecule essential for synapse formation, has been linked to ASD. Despite success in recapitulating the social interaction behavioral deficits and the underlying synaptic abnormalities in mouse model, the impact of NLGN3 R451C on the human neuronal system remains elusive. Here, we generated isogenic knock-in human pluripotent stem cell lines harboring NLGN3 R451C allele and examined its impact on synaptic transmission. Analysis of co-cultured excitatory and inhibitory induced neurons (iNs) with mutation revealed an augmentation in excitatory synaptic strength comparing to isogenic control, but not in inhibitory synaptic transmission. Consistently, the augmentation in excitatory transmission was confirmed in iNs transplanted into mouse forebrain. Using single-cell RNA seq on co-cultured excitatory and inhibitory iNs, we identified differential expression genes (DEGs) and found NLGN3 R451C alters gene networks associated with synaptic transmission. Gene ontology and enrichment analysis revealed convergent gene networks associated with ASD and other mental disorders. Our finding suggests that the NLGN3 R451C mutation could preferentially impact excitatory neurons, which causes overall network properties changes and excitation-inhibition imbalance related to mental disorders. Three samples of mixed excitatory and inhibitory human neurons cultured on primary mouse glia were harvested and run through the 10X Genomics Chromium device for scRNAseq. One sample was prepared from unedited H1 hESC and two were isolates of CRISPR/Cas9-edited H1 cells with NLGN3 R451C introduced. Libraries were prepared using the 10X Genomics 3' GEM Reagent Kit v3.1. After aligning reads with a mixed human and mouse reference index in cellranger, and merging samples in Seurat, mouse-specific clusters were removed, leaving only human cell data for analysis.