Cell type characterization of spatiotemporal gene co-expression modules in Down syndrome brain

Abstract

Down syndrome (DS) is the most common genetic cause of intellectual disability and increases the risk of other brain-related dysfunctions, like seizures, early-onset Alzheimer's disease, and autism. To reveal the molecular profiles of DS-associated brain phenotypes, we performed a meta-data analysis of the developmental DS brain transcriptome at cell type and co-expression module levels. In the DS brain, astrocyte-, microglia-, and endothelial cell-associated genes show upregulated patterns, whereas neuron- and oligodendrocyte-associated genes show downregulated patterns. Weighted gene co-expression network analysis identified cell type-enriched co-expressed gene modules. We present eight representative cell-type modules for neurons, astrocytes, oligodendrocytes, and microglia. We classified the neuron modules into glutamatergic and GABAergic neurons and associated them with detailed subtypes. Cell type modules were interpreted by analyzing spatiotemporal expression patterns, functional annotations, and co-expression networks of the modules. This study provides insight into the mechanisms underlying brain abnormalities in DS and related disorders.

Keywords: Developmental neuroscience; Transcriptomics.

Graphical abstract

Figure 1

Cell type-enriched genes were differentially expressed in DS Differential expression patterns of each cell type-related gene between DS and control are displayed on a log2 scale across brain regions and developmental stages. A sliding window approach was used to investigate the temporal expression pattern. Human brain developmental periods were based on the criteria presented in Kang et al. Genes associated with astrocytes, microglia, and endothelial cells showed upregulated patterns in DS, whereas oligodendrocyte- and neuron-enriched genes showed downregulated patterns in DS. Expression differences were mostly increased in older brains. Differences in glutamatergic neuron- and GABAergic neuron-related genes appeared in fetal and infancy stages. There were no significant expression differences in glutamatergic neuron-enriched genes; however, GABAergic neuron-enriched genes showed significant expression differences in fetal and infancy stages. ∗, p< 0.05 (paired t-test).

Figure 2

Cell type-enriched WGCNA modules were identified (A and B) Cell-type enrichment analysis with cell type-enriched genes of brain cell types (A) and neuronal subtypes (B) was conducted on 43 co-expressed gene modules constructed by WGCNA. The-log10 (p-value) value is shown (BHA p< 0.05, Fisher’s exact test). Cell type-enriched modules (BHA p< 1 × 10⁻²) for each cell type hardly overlapped, even in glutamatergic and GABAergic neurons. Oligodendrocyte- and microglia-enriched genes were exclusively enriched in one module for each cell type, but many modules were enriched for astrocytes and neurons.

Figure 3

Gene expression patterns of astrocyte modules (A–D) Gene expression patterns of astrocyte modules (M5 and M29) are visualized in a line graph of module eigengenes (A and B) and a gene expression heatmap (C and D). Genes in astrocyte modules were relatively less expressed during prenatal stages and were upregulated in DS. Gene expressions of M5 genes were similar between the DFC and CBC, whereas M29 genes were less expressed in the CBC than in the DFC.

Figure 4

Gene expression patterns of oligodendrocyte and microglia modules (A–D) Gene expression patterns of oligodendrocyte and microglia modules (M14 and M41, respectively) are visualized in a line graph of module eigengenes (A and B) and a gene-expression heatmap (C and D). M14 genes became gradually more expressed with age and were downregulated in DS, especially in the DFC. M41 genes were highly expressed in the older DFC of DS.

Figure 5

Gene expression patterns of glutamatergic neuron modules (A–D) Gene expression patterns of glutamatergic neuron modules (M24 and M26) are visualized in a line graph of module eigengenes (A and B) and a gene expression heatmap (C and D). M24 and M26 genes showed high expression levels during fetal stages, unlike other modules. In addition, they were more expressed in the DFC than in the CBC and were downregulated in DS. M24 was more constrained by developmental stages and disease status than M26, which was more constrained by brain region.

Figure 6

Gene expression patterns of GABAergic neuron modules (A–D) Gene expression patterns of GABAergic neuron modules (M32 and M42) are visualized by a line graph of module eigengenes (A and B) and a gene expression heatmap (C and D). M32 and M42 genes were repressed during fetal stages and were downregulated in DS. Genes in M32 showed similar expression patterns between the DFC and CBC, but genes in M42 were substantially repressed in the CBC.

Figure 7

Functional annotation of glial cell type-related modules (A–D) Functional enrichment analysis with genes related to GO terms and KEGG pathways on the astrocyte (A and B), oligodendrocyte (C), and microglia (D) modules. The five most significant terms (p< 0.05) are shown in each category. GO: gene ontology, BP: biological process, CC: cellular component, MF: molecular function, KEGG: Kyoto Encyclopedia of Genes and Genomes pathway. M5 genes were enriched for growth-related terms (“regulation of cell proliferation”, “heart growth”, “growth factor binding”, “fibroblast growth factor binding”, and “signaling pathways regulating pluripotency of stem cells”) and cell–cell interaction-related terms (“extracellular vesicle” and “cell junction”). M29 was enriched for biomolecule transport and metabolism-related terms (“nitrogen compound transport”, “organonitrogen compound catabolic process”, “carboxylic acid transport”, “carboxylic acid biosynthetic process”, and “dicarboxylic acid metabolic process”). M14 was enriched for oligodendrocyte-related terms (“axon ensheathment”, “myelin sheath”, and “compact myelin”), and M41 was enriched for immune-related terms (“immune response”, “leukocyte activation”, “inflammatory response”, and “myeloid leukocyte activation”).

Figure 8

Functional annotation of neuronal cell type-related modules (A–D) Functional enrichment analysis with genes related to GO terms and KEGG pathways on the glutamatergic (A and B) and GABAergic (C and D) neuron modules. The five most significant terms (p< 0.05) are shown in each category. GO: gene ontology, BP: biological process, CC: cellular component, MF: molecular function, KEGG: Kyoto Encyclopedia of Genes and Genomes pathway. M24 and M26 were enriched for neurodevelopment-related terms (“cell projection morphogenesis”, “muscle organ development”, and “growth cone” in M24, and “neuron development”, “neuron projection development”, “central nervous system neuron differentiation”, and “filopodium” in M26). M24 was enriched for a dendrite-related term (“dendritic shaft”), whereas M26 was enriched for axon-related terms (“axon”, “delayed rectifier potassium channel activity”, “ephrin receptor activity”, and “axon guidance”). M32 was enriched for synapse-related terms (“secretion by cell”, “regulation of neurotransmitter levels”, “presynapse”, “syntaxin-1 binding”, and “synaptic vesicle cycle”). M42 was enriched for calcium–calmodulin signaling-related terms (“cAMP metabolic process”, “calmodulin binding”, and “calcium signaling pathway”).

Figure 9

Co-expression networks and hub genes of glial cell type-related modules (A–D) Intramodular co-expression networks of the astrocyte (A and B), oligodendrocyte (C), and microglia (D) modules. Nodes indicate genes. The width and blueness of edges are proportional to the co-expression level between two genes. The 10 genes with the highest kME values were selected as hub genes and marked with large red labels. The nodes of genes that showed higher expression in the cell type of each module when compared with other cell types are colored yellow.

Figure 10

Co-expression networks and hub genes of neuronal cell type-related modules (A–D) Intramodular co-expression networks of glutamatergic neuron (A and B) and GABAergic neuron (C and D) modules. Nodes indicate genes, and the width and blueness of edges are proportional to the co-expression level between two genes. The 10 genes with the highest kME values were selected as hub genes and marked with large red labels. The nodes of genes that showed higher expression in the cell type of each module when compared with other cell types are colored yellow.

Figure 11

Characterization of the most DS-associated module (A–D) Gene expression patterns of the most DS-associated module (M40) are visualized in a line graph of module eigengenes (A) and a gene expression heatmap (B). Functional enrichment analysis with genes related to GO terms and KEGG pathways on M40 (C). The five most significant terms (p< 0.05) are shown in each category. GO: gene ontology, BP: biological process, CC: cellular component, MF: molecular function, KEGG: Kyoto Encyclopedia of Genes and Genomes pathway. Intramodular co-expression networks of M40 (D). Nodes indicate genes, and the width and darkness of edges are proportional to the co-expression level between two genes. The 10 genes with the highest kME values were selected as hub genes and are marked with large red labels. The nodes of genes that showed higher expression in a brain cell type are colored (astrocyte: blue, microglia: green, endothelial cell: grey, and oligodendrocyte: yellow), and the nodes of genes that are not related to any cell type are colored white. Genes of M40 were upregulated in DS and related to cell signaling, immune response, vasculature, and extracellular space.