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Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism

Molecular Psychiatry (2022)Cite this article

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Abstract

Mutations in many synaptic genes are associated with autism spectrum disorders (ASD), 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 NLGN3 R451C mutation on human neurons has not been investigated. Here, we generated human knockin neurons with the NLGN3 R451C and NLGN3 null mutations. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses; meanwhile NLGN3 knockout neurons showed reduction in excitatory synaptic strengths. Moreover, overexpression of NLGN3 R451C recapitulated the synaptic enhancement in human neurons. Notably, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons corresponding to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASD.

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Fig. 1: NLGN3 R451C mixed culture of induced neuronal (iN) cells.
Fig. 2: NLGN3 R451C increases excitatory neurotransmission in vitro.
Fig. 3: No major ER stress found in human neurons carrying NLGN3 R451C mutation.
Fig. 4: Dual-color genotype transplantation of ESC H1 and NLGN3 R451C human iN cells in vivo.
Fig. 5: Single-cell RNA seq of E/I mixed culture R451C human iNs.

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Data availability

Single-cell-RNAseq data were deposited and available on the NCBI Gene Expression Omnibus under accession number GSE180751.

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Acknowledgements

We thank the members of the Pang Lab for their insightful comments on the manuscript. We also thank Xueying Wang from Central South University for collecting the psychiatric risk summary gene list.

Funding

This study was supported by grants from the Robert Wood Johnson Foundation to the Child Health Institute of New Jersey (RWJF grant #74260), the Governor’s Council for Medical Research and Treatment of Autism (CAUT14APL028 to ZPP; CAUT16APL020 to DC), and the NIMH (MH092931 to TCS; MH122519 to CP; MH126420 to ZPP), and by a predoctoral fellowship from the NIMH (F30MH108321 to VRM).

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Author notes

  1. These authors contributed equally: Le Wang, Vincent R. Mirabella.

Authors and Affiliations

  1. Child Health Institute of New Jersey and Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, 08901, USA

    Le Wang, Vincent R. Mirabella, Xiao Su, Matteo Bernabucci, Ishnoor Singh, Davide Comoletti & Zhiping P. Pang

  2. Center for Medical Genetics & Hunan Key Laboratory of Medical Genetics, School of Life Sciences, and Department of Psychiatry, The Second Xiangya Hospital, Central South University, 410008, Changsha, China

    Le Wang, Yu Chen, Jianghua Tian, Chunyu Liu & Chao Chen

  3. Department of Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, CA, 94305, USA

    Vincent R. Mirabella & Thomas C. Südhof

  4. Department of Psychiatry, SUNY Upstate Medical University, Syracuse, NY, 13210, USA

    Rujia Dai & Chunyu Liu

  5. Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, 08854, USA

    Ranjie Xu, Azadeh Jadali, Peng Jiang, Kevin Y. Kwan & Ronald P. Hart

  6. Department of Biochemistry & Molecular Biology, University of Massachusetts, Amherst, MA, 01003, USA

    ChangHui Pak

  7. School of Psychology, Shaanxi Normal University, 710000, Xi’an, Shaanxi, China

    Chunyu Liu

  8. School of Biological Sciences, Victoria University of Wellington, Wellington, 6012, New Zealand

    Davide Comoletti

  9. National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, 410008, Changsha, Hunan, China

    Chao Chen

  10. Hunan Key Laboratory of Animal Models for Human Diseases, Central South University, 410008, Changsha, Hunan, China

    Chao Chen

  11. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, 94305, USA

    Thomas C. Südhof

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  1. Le Wang
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Contributions

LW conducted the experiments including cell culture, electrophysiology, morphological, cell transplantation and genomics analysis. VRM conducted the CRISPR/Cas9 gene targeting, related analyses, cloned the overexpression constructs and provided conceptual input on experimental design. RD, XS, RX, MB, IS, YC, and JT, conducted part of the analysis. RH conducted the single-cell analysis. KYK, PJ, DC, CP, TCS, CL, CC planned the experiments and analyzed the data. VRM, LW, DC, ZPP, and TCS conceived the project. TCS and ZPP wrote the paper with input from all authors.

Corresponding authors

Correspondence to Chao Chen, Thomas C. Südhof or Zhiping P. Pang.

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Wang, L., Mirabella, V.R., Dai, R. et al. Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism. Mol Psychiatry (2022). https://doi.org/10.1038/s41380-022-01834-x

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  • DOI: https://doi.org/10.1038/s41380-022-01834-x

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