Genetic Implication of Prenatal GABAergic and Cholinergic Neuron Development in Susceptibility to Schizophrenia

Abstract

Background: The ganglionic eminences (GE) are fetal-specific structures that give rise to gamma-aminobutyric acid (GABA)- and acetylcholine-releasing neurons of the forebrain. Given the evidence for GABAergic, cholinergic, and neurodevelopmental disturbances in schizophrenia, we tested the potential involvement of GE neuron development in mediating genetic risk for the condition.

Study design: We combined data from a recent large-scale genome-wide association study of schizophrenia with single-cell RNA sequencing data from the human GE to test the enrichment of schizophrenia risk variation in genes with high expression specificity for developing GE cell populations. We additionally performed the single nuclei Assay for Transposase-Accessible Chromatin with Sequencing (snATAC-Seq) to map potential regulatory genomic regions operating in individual cell populations of the human GE, using these to test for enrichment of schizophrenia common genetic variant liability and to functionally annotate non-coding variants-associated with the disorder.

Study results: Schizophrenia common variant liability was enriched in genes with high expression specificity for developing neuron populations that are predicted to form dopamine D1 and D2 receptor-expressing GABAergic medium spiny neurons of the striatum, cortical somatostatin-positive GABAergic interneurons, calretinin-positive GABAergic neurons, and cholinergic neurons. Consistent with these findings, schizophrenia genetic risk was concentrated in predicted regulatory genomic sequence mapped in developing neuronal populations of the GE.

Conclusions: Our study implicates prenatal development of specific populations of GABAergic and cholinergic neurons in later susceptibility to schizophrenia, and provides a map of predicted regulatory genomic elements operating in cells of the GE.

Keywords: GABA; GWAS; acetylcholine; gene expression; genetic; genomic; neurodevelopment; schizophrenia.

Fig. 1.

Initial analyses of single-cell RNA sequencing data from the ganglionic eminences. Cells were clustered according to gene expression profile using Seurat 4.3.0 and visualized in two-dimensional space using UMAP. Cells that were labeled by Shi et al, as deriving from tissue outside of the GE were excluded prior to clustering. (A) Broad “Level 1” cell types: developing neurons from the MGE (MGE-N), developing neurons from the CGE (CGE-N), developing neurons from the LGE (LGE-N), intermediate progenitor cells (IPC), progenitor cells and microglia. (B) Violin plots showing cell marker gene expression across level 1 cell types.

Fig. 2.

Mean of the MAGMA and SLDSR –log₁₀P values for enrichment of schizophrenia common variant liability in genes in the top expression specificity decile of 6 broad level 1 cell types of the ganglionic eminences. The dotted vertical line indicates nominal (P < .05) significance and the dashed vertical line indicates the Bonferroni-corrected P-value threshold for 6 tested cell populations (P < .0083). CGE-N, developing neurons from the CGE; IPC, intermediate progenitor cells; LGE-N, developing neurons from the LGE; MGE-N, developing neurons from the MGE.

Fig. 3.

Mean of the MAGMA and SLDSR –log₁₀P values for enrichment of schizophrenia common variant liability in genes in the top expression specificity decile of 36 “level 2” cellular subpopulations of the ganglionic eminences. The dotted vertical line indicates the nominal (P < .05) significance level and the dashed vertical line indicates the Bonferroni-corrected P-value threshold for 36 tested cell populations (P < .0014). CGE-N, developing neurons from the CGE; LGE-N, developing neurons from the LGE; IPC, intermediate progenitor cells; MGE-N, developing neurons from the MGE.

Fig. 4.

Single nuclei ATAC-Seq of the human ganglionic eminences. (A) snATAC-Seq clusters after integration with scRNA-Seq data from the study of Shi et al. Nuclei were clustered according to open chromatin profile using ArchR and visualized in 2D space using Uniform Manifold Approximation and Projection (UMAP). CGE-N, developing neurons from the CGE; LGE-N, developing neurons from the LGE; MGE-N, developing neurons from the MGE. (B) Clustered nuclei were classed as deriving from major cell types based on gene scores (inferred gene expression based on chromatin accessibility at the gene locus) for known marker genes. (C) Number of open chromatin regions identified in each cell population according to genomic annotation (promoter sequence is arbitrarily defined as within 1000 bp upstream and 100 bp downstream of a transcription start site). (D) Overlap of OCRs identified in developing neurons of the CGE, LGE, and MGE. (E) Overlap between OCRs identified in developing neurons of the MGE, LGE, and CGE and those identified through ATAC-Seq of bulk tissue from those regions by Markenscoff-Papadimitriou et al.

Fig. 5.

−Log₁₀P values for enrichment of schizophrenia SNP heritability in open chromatin regions mapped within individual cell populations of the ganglionic eminences. Analyses were performed using SLDSR; enrichment P values were derived from Z scores accounting for 53 (v1.2) baseline genomic annotations and OCRs identified in all other cell populations of the GE. The dotted vertical line indicates nominal (P < .05) significance and the dashed vertical line indicates the Bonferroni-corrected P-value threshold for 4 tested cell populations (P < .0125). CGE-N, developing neurons of the CGE; LGE-N, developing neurons of the LGE; MGE-N, developing neurons of the MGE.