Cross-Platform Validation of Neurotransmitter Release Impairments in Schizophrenia Patient-DerivedNRXN1-Mutant Neurons

Abstract

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation predisposing to schizophrenia. Previous studies showed that engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multi-center effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. We show that in neurons that were trans-differentiated from induced pluripotent stem cells derived from three NRXN1-deletion patients, the same impairment in neurotransmitter release was observed as in engineered NRXN1-deficient neurons. This impairment manifested as a decrease in spontaneous synaptic events and in evoked synaptic responses, and an alteration in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

Schizophrenia-associated somatic copy number variants from 12,834 cases reveal contribution to risk and recurrent, isoform-specific NRXN1 disruptions

“…Our analysis of Hi-C data using the same hiPSC neurons from Flaherty et al, suggests a potential formation of a cryptic promoter once the NRXN1 alpha-promoter is deleted, potentially forming an N-terminal truncated form of NRXN1, leading to a novel dominant negative mechanism by trapping NRXN1α in the cytoplasm. This mechanism is consistent with higher intracellular protein levels of a NRXN1-binding protein CASK in human iPSC lines from SCZ patients with 5' NRXN1 deletions (Pak et al, 2021). This potential cryptic promoter might have been missed in previous studies due to the difficulty of developing targeted transcript primers not anchored at the 5' end (Flaherty et al, 2019).…”

“…This truncated protein would lack the signal peptide required for shuttling to the cell surface, potentially causing abnormal trafficking. Similar germline NRXN1 deletions have been shown to cause accumulation of the 7 NRXN1 intracellular binding protein CASK in human induced pluripotent cells (iPSC) from SCZ patients (Pak et al, 2021).…”

“…The copyright holder for this preprint this version posted January 1, 2022. ; https://doi.org/10.1101/2021.12.24.21268385 doi: medRxiv preprint NRXN1 intracellular binding protein CASK in human induced pluripotent cells (iPSC) from SCZ patients (Pak et al, 2021).…”

Loss-of-function of OTUD7A in the schizophrenia-associated 15q13.3 deletion impairs synapse development and function in human neurons

“…Although our transcriptomic analyses suggested a possible mechanistic link between OTUD7A LoF and the impaired neuronal phenotypes, the profound transcriptomic effects of OTUD7A LoF in CRISPR/Cas9-engineered iNs may need to be interpreted cautiously. It has been a general challenge in the field that transcriptomic analysis of hiPSC-derived neurons, even for isogenic pairs of iPSC lines, can be confounded by line-to-line and/or clonal variations (Pak et al, 2021b). However, such limitations may be mitigated by our use of multiple lines/clones and the congruence between GO analysis of the DEGs and the neuronal phenotypic alterations between isogenic pairs of neurons.…”

Loss of Neurodevelopmental Gene CASK Disrupts Neural Connectivity in Human Cortical Excitatory Neurons

“…Using transient transfection at day 4, neurons were sparsely labeled with green fluorescent protein (GFP) expressed from Synapsin promoter (SYN-EGFP vector). At day 7, we fixed these cells and performed confocal imaging and neurite outgrowth analysis to measure various parameters as previously performed (Pak et al, 2015(Pak et al, , 2021. Compared to WT, CASK KO neurons showed an overall increase in total dendritic length and number of branch points without significant changes in soma size and the number of primary processes (Fig.…”