Cross-Platform Validation of Neurotransmitter Release Impairments in Schizophrenia Patient-Derived NRXN1-Mutant Neurons

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and predispose to multiple other neurodevelopmental disorders. 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. Using four-weeks old, relatively mature human neurons generated from the three pairs of patient-derived NRXN1del and control iPS cells, we performed bulk RNA-sequencing analysis of total RNA from three independent culture batches. We also performed bulk RNAseq analyses on triplicate cultures of isogenic pairs of human neurons without or with the heterozygous NRXN1 deletion that were trans-differentiated from a de novo engineered iPS cell line carrying a conditional NRXN1del allele and compared this to 3 unrelated wild-type iPS cell lines (in duplicate). In total, we performed differential gene expression analyses on 30 samples (9 controls vs. 9 schizophrenia-NRXN1 mutants, 3 controls vs. 3 engineered NRXN1 mutants, and 6 wild-type iPS cell samples) in triplicates or duplicates (replicates refer to independent cultures performed at different time points). As a result of the co-culturing scheme of human neurons with mouse glia, the RNAseq data on neurons were composed of a mixture of human neuronal and mouse glia transcriptomes. A key step in processing of the RNAseq data was to deconvolve the two transcriptomes and to normalize the relative abundance of each mRNA from each species to the total number of mRNAs from that species only. As described in the Methods, the Kallisto program was able to unambiguously assign each paired 150 base-pair sequence to mouse and human reference transcriptomes. The mouse:human mRNA ratios differed between cultures of neurons trans-differentiated from various iPS cell lines. To control for these differences, we adjusted each sample’s species-specific mRNA abundance based on Transcripts per Million (TPM) for each gene, summed these for each gene, and then carried out per-sample quantile normalization steps for each sample. This approach provided a reproducible abundance measure for each gene in each sample. The resulting values were used to form a log2(TPM+1) gene-by-sample matrix that was used for differential gene expression analyses using LIMMA.