Evaluation of altered cell-cell communication between glia and neurons in the hippocampus of 3xTg-AD mice at two time points

Abstract

Alzheimer's disease (AD) is the most common form of dementia and is characterized by progressive memory loss and cognitive decline, affecting behavior, speech, and motor abilities. The neuropathology of AD includes the formation of extracellular amyloid-β plaque and intracellular neurofibrillary tangles of phosphorylated tau, along with neuronal loss. While neuronal loss is an AD hallmark, cell-cell communication between neuronal and non-neuronal cell populations maintains neuronal health and brain homeostasis. To study changes in cellcell communication during disease progression, we performed snRNA-sequencing of the hippocampus from female 3xTg-AD and wild-type littermates at 6 and 12 months. We inferred differential cell-cell communication between 3xTg-AD and wild-type mice across time points and between senders (astrocytes, microglia, oligodendrocytes, and OPCs) and receivers (excitatory and inhibitory neurons) of interest. We also assessed the downstream effects of altered glia-neuron communication using pseudobulk differential gene expression, functional enrichment, and gene regulatory analyses. We found that glia-neuron communication is increasingly dysregulated in 12-month 3xTg-AD mice. We also identified 23 AD-associated ligand-receptor pairs that are upregulated in the 12-month-old 3xTg-AD hippocampus. Our results suggest increased AD association of interactions originating from microglia. Signaling mediators were not significantly differentially expressed but showed altered gene regulation and TF activity. Our findings indicate that altered glia-neuron communication is increasingly dysregulated and affects the gene regulatory mechanisms in neurons of 12-month-old 3xTg-AD mice.

Keywords: Alzheimer’s disease; cell-cell communication; microglia; networks; neurodegeneration; signaling; snRNA-seq.

Figure 1.. Schematic overview of our study design.

(A) We sequenced the hippocampus of female 3xTg-AD and WT littermates at 6 and 12 months. (B) We inferred ligands and receptors between senders (astrocytes, microglia, oligodendrocytes, OPCs) and receivers (excitatory and inhibitory neurons) using differential gene expression (log2FC > 0.5) and known ligand-receptor information. We also predicted target genes potentially regulated by the predicted ligand-receptor pairs. (C) We generated cell-type-specific count matrices with condition and time point information for downstream analyses. (D) Using the cell-type-specific count matrices, we identified differentially expressed genes between 3xTg-AD and WT hippocampus for every cell type and at each time point (absolute log2FC > 0.2, Wald test padj < 0.05). (E) To determine changes in gene regulation of signaling mediators, we built cell-type- and time-point-specific gene regulatory networks (GRNs) and calculated the differential gene targeting score of signaling mediators between 12-month 3xTg-AD and WT GRNs. (F) Using differential expression information, we inferred the differential TF activity of signaling mediators that are TFs between conditions at 12 months.

Figure 2.. Overview of our 3xTgAD snRNA-seq data from the hippocampus across time points

(A) Uniform Manifold Approximation and Projection (UMAP) of 184,858 nuclei after quality control colored by cell type. (B) Stacked violin plot of the expression of representative marker genes used for cell type annotation. (C) Integrated UMAP split by condition (AD, WT). (D) Stacked barplot of the proportion of cell types annotated by condition. (E) UMAP split by time point (6 and 12 months) in both conditions. (F) Stacked barplot of cell type proportions by time point in both conditions.

Figure 3.. CCC dysregulation is increased at 12 months in the 3xTg-AD hippocampus.

(A) Bar plot of the number of interactions up-regulated (log2FC > 0.5) in each time point and condition grouped by senders. Color and pattern indicate condition and time point, respectively. (B) UpSet plot of the overlap of interactions between individual groups (group = time point and condition pairs). The top bar plot denotes intersection size, circles represent which comparisons have overlap, and the set size reflects the total number of interactions for individuals groups. The intersection size bar plot is colored by the receiver. (C) Stacked bar plot of the interactions shared between time points and conditions colored by condition (3xTg-AD, WT). (D) Stacked bar plot of the interactions specific to the 6-month time point colored by group (6mAD, 6mWT). (E) Stacked bar plot of the interactions specific to the 12-month time point colored by group (12mAD, 12mWT).

Figure 4.. Increased target gene expression differences in the 3xTg-AD hippocampus at 12 months.

Volcano plot of differential target gene expression in excitatory and inhibitory neurons between 3xTg-AD and WT mouse hippocampus at (A) 6 months and (B) 12 months. Color refers to differential gene expression direction and significance, and shape indicates receiver cell type. NS/DE = not significant or differentially expressed. Significant genes had an absolute log2FC > 0.2 with a Wald test padj < 0.05. (C) Bubble plot of functional enrichment terms of target genes at 6 months and (D) at 12 months. Condition denotes the context in which the target-associated interaction (i.e., the ligand-receptor pair) was upregulated. Plotted terms had Bonferroni adjusted p-values < 0.05.

Figure 5.. Significant AD-risk gene-associated interactions are specific to the 12-month 3xTg-AD hippocampus.

(A) Alluvial plot of ligand-receptor pairs with an AD risk gene as a target. Color refers to the sender, and grey indicates the involvement of more than one sender. Asterisks indicate ligands and receptors that are an AD-risk gene. (B) Split heatmap of log2FC of target genes that are AD-risk genes (top) and their predicted signaling mediators (bottom) in 12-month receiver cell types. Asterisks indicate genes with an absolute log2FC > 0.2 and Wald test padj < 0.05 in the 3xTg-AD hippocampus.

Figure 6.. Signaling mediators were differentially regulated and had altered TF activity at 12 months.

(A) Heatmap of differential gene targeting scores of signaling mediators in 12-month receivers. Differential gene targeting scores are in reference to AD. Red and blue represent increased (i.e., top quartile) and decreased (i.e., bottom quartile) gene targeting in the 3xTg-AD hippocampus, respectively. NE = not enriched in 3xTg-AD hippocampus (i.e., not among the highest and lowest quartiles/targeting scores). (B) Heatmap of differential TF activity scores of signaling mediators between 3xTg-AD and WT hippocampus, annotated by receiver, at 12 months. Green and purple represent TF activity as increased (i.e., active) or decreased (i.e., inactive), respectively.