Genetic implication of prenatal GABAergic and cholinergic neuron development in susceptibility to schizophrenia

Abstract

Background: The ganglionic eminences are fetal-specific structures that give rise to gamma-aminobutyric acid (GABA)- and acetylcholine- releasing neurons of the forebrain. Given evidence for GABAergic and cholinergic disturbances in schizophrenia, as well as an early neurodevelopmental component to the disorder, we tested the potential involvement of developing cells of the ganglionic eminences 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 ganglionic eminences to test enrichment of schizophrenia risk variation in genes with high expression specificity for particular developing cell populations within these structures. 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 ganglionic eminences, using these to additionally 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 also concentrated in predicted regulatory genomic sequence mapped in developing neuronal populations of the ganglionic eminences.

Conclusions: Our study provides evidence for a role of prenatal GABAergic and cholinergic neuron development in later susceptibility to schizophrenia.

Figure 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 labelled 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.

Figure 2:. −Log₁₀ P-values for enrichment of schizophrenia (A) and height (B) common variant associations in genes in the top expression specificity decile of 6 broad level 1 cell types of the ganglionic eminences using MAGMA and SLDSR.

The dotted vertical line indicates nominal (P < 0.05) significance and the dashed vertical line indicates the Bonferroni-corrected P-value threshold for 6 tested cell populations (P < 0.0083). CGE-N = developing neurons from the CGE; LGE-N = developing neurons from the LGE; MGE-N = developing neurons from the MGE; IPC = intermediate progenitor cells.

Figure 3.. −Log₁₀ P-values for enrichment of schizophrenia and height common variant associations in genes in the top expression specificity decile of 36 ‘level 2’ ganglionic eminence subpopulations using MAGMA and SLDSR.

The dotted vertical line indicates the nominal (P < 0.05) significance level and the dashed vertical line indicates the Bonferroni-corrected P-value threshold for 36 tested cell populations (P < 0.0014). CGE-N = developing neurons from the CGE; LGE-N = developing neurons from the LGE; MGE-N = developing neurons from the MGE; IPC = intermediate progenitor cells.

Figure 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 open chromatin regions identified in developing neurons of the CGE, LGE and MGE. E) Overlap between open chromatin regions 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 .

Figure 5:. −Log₁₀ P-values for enrichment of SNP heritability for schizophrenia and height in OCRs 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 < 0.05) significance and the dashed vertical line indicates the Bonferroni-corrected P-value threshold for 4 tested cell populations (P < 0.0125). CGE-N = developing neurons of the CGE; LGE-N = developing neurons of the LGE; MGE-N = developing neurons of the MGE.