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Series GSE168762 Query DataSets for GSE168762
Status Public on Mar 12, 2021
Title Cross-Platform Validation of Neurotransmitter Release Impairments in Schizophrenia Patient-Derived NRXN1-Mutant Neurons
Organisms Homo sapiens; Mus musculus
Experiment type Expression profiling by high throughput sequencing
Summary 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.
Overall design 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.
Contributor(s) Pak C, Wang J, Zhang X, Qu P, Danko T, Mittelpunkt A, Urban AE, Pang ZP, Jin K, Dexheimer P, Bardes E, Aronow BJ, Levinson DF, Wernig M, Südhof TC
Citation(s) 34035170
NIH grant(s)
Grant ID Grant title Affiliation Name
U19 MH104172 Multimodal analysis of high-risk psychosis mutations in induced neuronal cells STANFORD UNIVERSITY Thomas C. Sudhof
Submission date Mar 11, 2021
Last update date Jun 11, 2021
Contact name Bruce J Aronow
Organization name Cincinnati Children's Hospital Medical Center
Department Biomedical Informatics, Developmental Biology
Lab Genome Informatics
Street address 240 Albert Sabin Way
City Cincinnati
State/province OH
ZIP/Postal code 45229
Country USA
Platforms (2)
GPL18573 Illumina NextSeq 500 (Homo sapiens)
GPL19415 Illumina NextSeq 500 (Homo sapiens; Mus musculus)
Samples (30)
GSM5169059 CTRL_iPSC_3
GSM5169060 CTRL_iPSC_4
GSM5169061 CTRL_iPSC_5
BioProject PRJNA713833

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE168762_Human_iPSC+iN_expressionMatrix_log2TPM+1.xlsx 3.3 Mb (ftp)(http) XLSX
GSE168762_RAW.tar 39.3 Mb (http)(custom) TAR (of TSV)
Processed data provided as supplementary file
Processed data are available on Series record
Raw data not provided for this record

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