The team of Mol Psychiatry—Pang Zhiping/Chen Chao/Nobel laureate Thomas Südhof reveals a new mechanism of synapses acquired by autism risk mutations

Synaptic dysfunction is thought to be one of the key physiological pathogenic mechanisms of
neurodevelopmental and psychiatric disorders such as autism spectrum disorders (ASDs).
Neuroligin 3 is an essential cell adhesion molecule for synaptic formation in neuronal cells, and it was first found to encode a rare mutation in the gene NLGN3, R451C and ASD, in family genetic studies Closely related [1].

The Nlgn3^R451C gene knock-in mouse model, pioneered by Thomas C.
Südhof's research group, is also the earliest genetic model of autistic mice [2].

。 After the study of the Nlgn3 R451C mouse model system, it was found that rare mutations of ^R451C can cause social disorders and abnormal neurosynaptic transmission in mice Highly correlated phenotypes [2-3].

Still, the effects of the mutation on human neurons have not been studied
.

October 24, 2022, Professor Chen Chao of Central South University, Rutgers University, USA Professor Zhiping P.
Pang and Stanford University Nobel Prize winner Thomas C.
Südhof The professors' teamwork was published online in Molecular Psychiatry as a joint online title "Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism" (this article was selected as the cover article).
Gene knock into NLGN3-R451C in human embryonic stem cells (ESCs) was found to be rare mutations and induced neurons iNs)[3] significantly reduces the protein expression level of NLGN3, but enhances glutamatergic synaptic transmission
.
This study further uses human neuronal models to confirm that synaptic dysfunction is a key cellular phenotype leading to ASD, which provides an important theoretical basis
for finding possible ASD treatment strategies.
(Read more: Previous work by Nobel laureate Thomas C.
Südhof's team is detailed in the Logical Neuroscience report.
) Nature – Frontier! Mechanisms by which GluDs transduce different presynaptic signals to different postsynaptic receptor responses).

The human NLGNs family consists of NLGN1 (3q26), NLGN2 (17p13), and NLGN3 (Xq13), NLGN4X (Xp22.
3), and NLGN4Y ( YQ11.
2) five gene compositions
.
NLGNs, as synaptic adhesion molecules of nerve cells, play an extremely important role in the formation and function of nerve synapses [4].

Previous research on the NLGN3 gene has focused on genetically modified mouse models, and there have been few
studies on the function of the gene in human neurons.
To study the effects of rare genetic mutations in NLGN3-R451C in the context of human genetics, the authors used CRISPR-Cas9 to treat the ESC X chromosome NLGN3 The coding region is homologously specific knocked into the R451C mutation and uses the previously established iNs model [5], that is, the transcription factor Ngn2 is conditionally overexpressed in stem cells (Neurogenin 2) induces glutamatergic neurons (Ngn2 neurons) [6], through conditional overexpression of the transcription factor Ascl1 (Achaete-Scute Family BHLH Transcription Factor 1) and Dlx2 (Distal-Less Homeobox 2) induction GABAergic neurons (AD neurons) [7], respectively, functionally analyzed
the synaptic transmission of two neurons.
The results showed that NLGN3-R451C mutations significantly enhanced the frequency of mEPSCs in glutamatergic neurons, while gaba ghosts in mIPSCs There was no significant change
in frequency and amplitude.
In addition, gene knockout NLGN3 in human Ngn2 neurons significantly reduced mEPSC frequency
.
This suggests that R451C is a functionally acquired mutation in a human-derived neuronal model that enhances AMPA-mediated glutamatergic synaptic transmission (Figure 1).

。

Fig.
1 NLGN3 R451C enhances excitatory synaptic transmission

(Source: Wang, L.
et al.
, Mol Psychiatry, 2022).

Due to the lack of in vivo physiological environment of iNs models cultured in vitro, long-term in vitro culture often causes cell stress
.
Human nerve cells transplanted in rodent models have been used to study human neuronal development and function [8-9].

Therefore, the authors employed a strategy of labeling dual color genotypes by labeling control and R451C Ngn2 neurons (mCherry and mVenus, respectively).
Co-transplanted into the forebrain region of
neonatal immunodeficient mice.
Ngn2 neurons of both genotypes develop entirely in the same in vivo environment
.
In adult mice, human Ngn2 neurons were found to be morphologically and functionally integrated
with mouse endogenous neural circuits.
Furthermore, brain slice electrophysiology was used to record neurons of different genotypes transplanted in the brain of the same mouse, so as to reduce the bias
caused by the transplantation process and individual animals.
The results showed a substantial enhancement
of the mEPSC frequency of R451C genotype neurons.
This further confirms that the R451C mutation affects glutamatergic synaptic transmission through acquired function, thereby altering the excitability/inhibition balance in the brain and acting on the pathogenesis of autism (Figure 2).
）
。

Figure 2 Transplantation of WT and NLGN3R451C neurons with dual fluorescent genotypes

(Source: Wang, L.
et al.
, Mol Psychiatry, 2022).

To understand the association between molecular-level changes induced by NLGN3-R451C in human-derived neurons and synapse-associated phenotypes, the authors performed single-cell transcriptome analysis
on in vitro co-cultured neuronal models.
Differential expression analysis and functional annotations [10] showed that Differential expressed genes (DEGs) were significantly upregulated in different types of neurons and enriched in synaptic function-related gene pathways
.
To further understand the DEGs and ASD caused by R451C, schizophrenia (SCZ).
), Bipolar disorder (BD) and major depressive disorder (MDD).
whether there is a potential association
between neurodevelopmental and neuropsychiatric disorders.
The authors looked at DEGs for different nerve cell types versus collected ASD, SCZ, BD, and MDD Enrichment analysis
of disease-related gene sets such as genetic risk genes, postmortem human brain differentially expressed genes, and postmortem human brain gene co-expression module genes were enriched.
The results showed that DEGs of the major nerve cell types showed different degrees of significant enrichment than the risk gene sets of ASD, SCZ and BD types
.
MDD and the body mass index as a control did not have any degree of enrichment in the analysis
.
This suggests that alterations in gene expression networks caused by NLGN3-R451C mutations may play an equally important role in SCZ and BD, as in MDD The molecular mechanism may differ from the other three types of neuropsychiatric and neurodevelopmental diseases (Figure 3).

Figure 3 Human-induced single-cell sequencing of neurons

(Source: Wang, L.
et al.
, Mol Psychiatry, 2022).

In summary, the study combined gene editing, human stem cell-induced neuron model, dual genotype cell transplantation model, electrophysiological patch clamp, and single-cell sequencing revealed the acquired function of rare mutations in autism NLGN3R451C, and enhanced AMPA receptor-mediated glutamatergic neurosynaptic transmission
。 At the same time, the study provides new evidence
for the neurosynaptic mechanism of autism.
In addition, human stem cell-induced neuron models and bigenotypic cell transplantation models provide new research models
for studying neurodevelopmental and neuropsychiatric diseases.
On the downside, the molecular mechanism underlying this rare mutation is not well understood
.
In addition, NLGN3 is also widely expressed in glial cells as a postsynaptic membrane protein, and whether this mutation also mediates the interaction between nerves and glial cells needs to be further clarified
in future studies.