Human Neuropsychiatric Disease Modeling using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1

Abstract

Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ES cells with different heterozygous conditional NRXN1 mutations, and analyzed two different types of isogenic control and NRXN1-mutant neurons derived from these ES cells. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, NRXN1-mutant human neurons exhibited increased levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations.

Carbonic anhydrase-related protein CA10 is an evolutionarily conserved pan-neurexin ligand

“…(C) Nrxn1 levels in human-induced neurons infected with lentivirus to express control (empty vector) or FLAG-tagged CA10. The iN cells are genome-edited to carry an HA tag in the NRXN1 locus (28). Samples were immunoblotted (Left) using antibodies against HA (to detect tagged NRXN1), CA10 (to detect overexpressed CA10; endogenous levels are low in these cultures), and actin (as loading control).…”

“…S7D). Furthermore, we used human embryonic stem cells that had been genome-edited to carry an HA-tagged NRXN1 allele (28). We differentiated those cells into neuronal cells (iN cells) by a direct lineage-conversion protocol (29) and found that CA10 had a similar effect on neurexin protein levels and isoform distribution in human neurons (Fig.…”

Analysis of conditional heterozygous STXBP1 mutations in human neurons

“…Two independent heterozygous and homozygous ES cell clones were selected for analyses (Figure 1B and Supplemental Figure 1, A and C; supplemental material available online with this article; doi:10.1172/JCI78612DS1). This cKO approach was designed to allow analysis of the effects of hetero-and homozygous mutations in human cells on a controlled genetic background, thereby eliminating potentially confounding effects induced by genetic background changes or selection of cell clones (28).…”

Synaptic Neurexin Complexes: A Molecular Code for the Logic of Neural Circuits

“…In human neurons carrying conditional heterozygous NRXN1 loss-of-function mutations, heterozygous NRXN1 inactivation produced a significant synaptic impairment and caused an increase in the levels of CASK, which binds to the cytoplasmic sequences of Nrxn1 (Pak et al, 2015). The nature of he synaptic impairment suggested a dysregulation of neurotransmitter release, not a global loss of synaptic strength.…”

“…CASK phosphorylates neurexins, and heterozygous NRXN1 deletions in human neurons cause an increase in CASK levels (Pak et al, 2015), suggesting that CASK physiologically interacts with neurexins. Hundreds of mutations in the X-linked CASK gene have been reported, with a range of symptoms whose severity appears to depend on how severely the mutation disrupts CASK function (Hackett et al, 2010; Moog et al, 2015).…”