FIGURE 4. In vitro validation of a single-nucleus RNA-seq–discovered pathway with iPSC-derived neurons^a.

^a Panel A shows results of the enrichment of genome-wide association studies (GWASs) of posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) summary statistics in cell-type-specific markers for induced pluripotent stem cells (iPSCs), peripheral blood mononuclear cells (PBMCs), and cortical neurons. Each point represents the negative logarithm of the MAGMA-based enrichment p value (y-axis) for each literature-reported marker gene set (x-axis) (see marker gene sets in Table S14 in the online supplement). The shapes denote the two different input GWASs’ summary statistics (square=PTSD; triangle=MDD), and error bars indicate standard error of the mean. Opaque dots denote cell-type-specific markers that were false discovery rate (FDR) significant. Panel B illustrates iPSCs being differentiated into cortical neurons. The top left panel is a bright-field image of an iPSC colony displaying nominal cellular morphology; the bottom left panel is an immunofluorescence image of iPSCs expressing the canonical nuclear pluripotency marker NANOG (green). The top right panel is a bright-field image of iPSC-derived cortical cultures after ~60 days of differentiation displaying expansive neurite arborization and connectivity; the bottom right panel is an immunofluorescence image of neurons expressing the mature neuron markers for cytoskeleton and nucleus, MAP2 (green), and NeuN (red). DAPI is used as a nuclear counterstain. Scale bars are 100 mm. Panel C shows gene expression in control iPSC-derived neurons of literature-reported cell-type-specific markers for iPSCs, PBMCs, and neurons (see Table S14 in the online supplement). Blue denotes the iPSCs’ marker set, orange the PBMCs’ marker set, and red the neurons’ marker set. Data are represented as box plots of normalized expression (log₂ scale), with data points labeled with their corresponding marker gene. The between-gene-set differences in expression were estimated by two-tailed Wilcoxon signed-rank test. Panel D shows representative micrographs of glucocorticoid receptor (GR) in iPSC-derived neurons. Cells were stained with the neuronal nuclear marker NeuN (red), the GR protein (green), and the nuclear DNA marker DAPI (blue). The left panel is of iPSC-derived neurons in vehicle conditions, with minimal GR signal localized in the nucleus. The right panel is of iPSC-derived neurons exposed to 100 nM dexamethasone (DEX) for 1 hour, showing that GR signal has been translocated into the nuclei. White arrows indicate colocalization of GR with NeuN. Panel E shows nuclear GR levels, with quantification of the average ratio of GR signal to nucleus area for individual neuronal nuclei across control (0 nM) and 100 nM DEX conditions, with DEX-treated neurons having a higher ratio of GR signal in their nuclei (control group, N=122 nuclei; DEX group, N=157 nuclei). Data are represented as violin plots using a fold change (from the mean of the control group). The p value of the group difference was estimated by a two-tailed Wilcoxon signed-rank test. Panel F shows DEX-induced increased levels of phosphorylated GR at Ser211 (pGR-Ser211), indicative of an activated and nucleus-localized GR in iPSC-derived neurons (control group, 0 nM DEX; DEX group, 100 nM DEX). Data are represented as mean fold change (from the control group) ± standard error of the mean. The p value of the group difference was estimated by a two-tailed unpaired t test. Panel G shows DEX-induced increase in FKBP5 protein levels (in fold change), indicative of direct GR-binding to the FKBP5 gene leading to upregulation. FKBP5 protein was measured by capillary-based immunoblotting (control group, 0 nM DEX; DEX group, 100 nM DEX). Data are represented as mean fold change (from the control group) ± standard error of the mean. The p value of the group difference was estimated by a two-tailed unpaired t test. Panel H is a volcano plot illustrating the relationship of p value with fold changes of differential gene expression (DGE) results based on 100 nM versus 0 nM DEX in iPSC-derived neurons. Green denotes downregulation, and purple denotes upregulation. Dots with high-intensity color indicate differentially expressed genes with FDR-adjusted p<0.05. Low-intensity color denotes p<0.05. Gray dots indicate that the gene was not significant. The top genes with p<1.0–27 have been named. Panels I and J are rank-rank hypergeometric overlap (RRHO) plots between DEX-induced DGE in iPSC-derived neurons and PTSD and MDD neuronal DGE, respectively (excitatory neurons [EX] at left and inhibitory neurons[IN] at right). For this analysis, genes in each DGE list were ranked according to the product of the sign of log₂(fold change) with −log₁₀(p). The plotted RRHO heat map represents the extent of overlap with the colors based on −log₁₀(p) of the hypergeometric test measuring the significance of overlap of gene lists. Warm colors in the bottom left and top right quadrants reflect overlap in genes with upregulation or downregulation, respectively, in both data sets. Warm colors in the top left and bottom right quadrants reflect overlaps in genes with opposite direction of effects in the two data sets. The coefficient (ρ) of Spearman correlations based on the ranking metrics are given as reference of effect size of the relationship. Panels Kand L present scatterplots of z statistics of the PTSD effect and MDD effect, respectively, in EX neurons and IN neurons with DEX effect in iPSC-derived neurons. Nominally significant genes in each pair of analyses were selected, and genes with z score (of signed p value) >3 in the PTSD and/or the MDD analysis, respectively, are named. Each red circle corresponds to a gene and denotes the PTSD effect or the MDD effect in EX neurons paired with the DEX effect in iPSC-derived neurons, and each blue triangle corresponds to a gene and denotes the PTSD effect or the MDD effect in IN neurons paired with the DEX effect in iPSC-derived neurons. For each panel, the regression line was plotted along with the confidence interval, and the Pearson correlation coefficient, the p value, and the size of the gene set are listed. Inserts show also the respective normalized enrichment score (NES) of PTSD and MDD DGE signatures for DEX-regulated genes (up or down) in iPSC-derived neurons’ DGE. An asterisk indicates significant enrichment.