A Single-Cell Transcriptomic Analysis of the Mouse Hippocampus After Voluntary Exercise

Abstract

Exercise has been recognized as a beneficial factor for cognitive health, particularly in relation to the hippocampus, a vital brain region responsible for learning and memory. Previous research has demonstrated that exercise-mediated improvement of learning and memory in humans and rodents correlates with increased adult neurogenesis and processes related to enhanced synaptic plasticity. Nevertheless, the underlying molecular mechanisms are not fully understood. With the aim to further elucidate these mechanisms, we provide a comprehensive dataset of the mouse hippocampal transcriptome at the single-cell level after 4 weeks of voluntary wheel-running. Our analysis provides a number of interesting observations. For example, the results suggest that exercise affects adult neurogenesis by accelerating the maturation of a subpopulation of Prdm16-expressing neurons. Moreover, we uncover the existence of an intricate crosstalk among multiple vital signaling pathways such as NF-κB, Wnt/β-catenin, Notch, and retinoic acid (RA) pathways altered upon exercise in a specific cluster of excitatory neurons within the Cornu Ammonis (CA) region of the hippocampus. In conclusion, our study provides an important resource dataset and sheds further light on the molecular changes induced by exercise in the hippocampus. These findings have implications for developing targeted interventions aimed at optimizing cognitive health and preventing age-related cognitive decline.

Keywords: Aerobic exercise; Cognitive decline; Dementia; Environmental enrichment; Gene-expression; Hippocampus; Learning and memory; Single-cell RNAseq.

Fig. 1

Single-nucleus RNA sequencing analysis of the whole hippocampus in exercising vs. sedentary mice reveals changes in the abundance of specific cell-types. A Experimental design: The cohort of WT mice belonging to the exercising experimental group, or “runners” (n = 4), had free access to running wheels in their cages for a duration of 4 weeks. Mice belonging to the control or “sedentary” group (n = 4) were similarly housed but the running wheels were blocked. The 4-week-long voluntary exercise paradigm was followed by the isolation of hippocampal nuclei for single-nucleus RNA sequencing. B UMAP plots showing clusters of nuclei or “cells,” colored by experimental group (left panel) and cell-type (right panel). (ExN1-13, excitatory neurons; InN1-4, inhibitory neurons; ODC, oligodendrocytes; OPC, oligodendrocyte precursor cells; AST, astrocytes; MGL, microglia) C Violin plots showing average module score (expression) of marker genes specific to the different cell types/hippocampal regions, after cell-type specific annotation of clusters (ExN, excitatory neurons; InN, inhibitory neurons; ODC, oligodendrocytes; OPC, oligodendrocyte precursor cells; AST, astrocytes; MGL, microglia; CA, Cornu Ammonis; DG, Dentate Gyrus). D Bar graph indicating the proportions of broad cell-types observed in the dataset (ExN, excitatory neurons; InN, inhibitory neurons; ODC, oligodendrocytes; OPC, oligodendrocyte precursor cells; AST, astrocytes; MGL, microglia). E UMAP plot with cell clusters colored by average module score (expression) of marker genes specific to the dentate gyrus (DG), highlighting the two DG excitatory neuron clusters. F Analysis of differences in cell-type proportions between cells from exercise and control samples, using permutation testing. The x-axis denotes the fold difference in cell-type proportions (log2 scale). Points marked in red indicate clusters with significantly different proportions of cells between the two groups. Horizontal lines around the points indicate the confidence interval for the magnitude of difference for a specific cluster, calculated via bootstrapping. G UMAP plot with cells colored by Augur cell-type prioritization upon perturbation resulting from exercise, measured using the area under the curve (AUC) scores

Fig. 2

Selective loss of InN2 inhibitory neuron cluster upon exercise indicates a role of the transcription factor Prdm16. A UMAP plot with cells colored by the experimental group, showing the loss of InN2 inhibitory neurons upon exercise. B Heatmap plots of functional annotation for specific genes among the top 50 markers of the InN2 cluster that had significantly enriched GO (biological process, molecular function, and cellular component) terms. C Cell-type specific regulons for InN2 cluster identified using the SCENIC workflow. The y-axis denotes the regulon specificity score (RSS) (with high RSS values indicating high cell-type/cluster specificity, and vice versa). The x-axis denotes the rank of each regulon within the selected cluster, based on the RSS. The top 5 ranked regulons for InN2 are labeled on the plot, with the number of genes comprising each regulon indicated within parentheses. (Regulons ending with “extended” also include motifs linked to the transcription factor by lower confidence annotations). D Dot plot showing significant GO biological process terms enriched among the collective list of genes and transcription factors (TFs) making up the top 5 regulons, as indicated in the RSS-Rank plot in (C). E Violin plots depicting the normalized expression of Prdm16 (top panel), and the average module score (expression) for the genes included in the Prdm16 regulon (bottom panel) in all clusters. F Violin plot showing the expression of Prdm16 in the InN2 cluster, split between exercise and control cells. G Network plot showing the 17 genes comprising the Prdm16 regulon. H Dot plot showing significant GO biological process terms enriched among the list of 17 Prdm16 regulon genes

Fig. 3

Increased abundance of neurons in the ExN11 cluster suggests increased neurogenesis upon exercise. A (left panel) UMAP plots highlighting the ExN11 cluster, split by the experimental conditions (exercise and control). B Dotplot showing significant GO biological process terms enriched among the top 50 genes differentially expressed between ExN11 and ExN1 clusters, and among the top 50 genes differentially expressed between the ExN11 cluster and the ODC and OPC clusters. C Partition-based graph abstraction (PAGA) graph for InN2, ExN11, and ExN1 clusters, with velocity-directed edges constructed from RNA velocity measurements. Edges denote either connectivities (dashed) or transitions (solid/arrows). D Box-plots showing a significant decrease in the proportion of developing cells (ExN11/InN2) and an increase in the proportion of mature granule cells (ExN1) upon exercise. Each dot indicates one sample (n = 4/4 for exercise and control samples). E Violin plots showing normalized expression of selected marker genes for radial glia/immature neurons/exercise-mediated neurogenesis. F Cell-type specific regulons for ExN11 cluster identified using the SCENIC workflow. The y-axis denotes the regulon specificity score (RSS) (with high RSS values indicating high cell-type/cluster specificity, and vice versa). The x-axis denotes the rank of each regulon within the selected cluster, based on the RSS. The top 5 ranked regulons for ExN11 are labeled on the plot, with the number of genes comprising each regulon indicated within parentheses. G Dot plot showing significant GO biological process terms enriched among the collective list of genes and transcription factors (TFs) making up the top 5 regulons, as indicated in the RSS-Rank plot above. H Combined gene-regulatory network plot with TFs specific to InN2 and ExN11 clusters, and their respective regulon genes (larger nodes represent genes/TFs that connect two or more regulon networks)

Fig. 4

Differentially expressed genes in excitatory neurons suggest regulation of synaptic plasticity and neuron differentiation to promote increased neurogenesis upon exercise. A (top panel) and B (top panel) UpSet plots depicting A commonly upregulated and B commonly downregulated genes upon exercise, between different clusters. Each row corresponds to a cluster, and bar charts on the right show the size of the set of genes up/downregulated upon exercise in that cluster. Each column corresponds to a possible intersection (commonly up/downregulated gene(s)): the filled-in cells show which cluster set is part of an intersection. Gene names are labeled for intersections with < 2 common genes among clusters. A (bottom panel) and B (bottom panel) Dot plots showing GO biological process terms enriched among A genes significantly upregulated and B genes significantly downregulated upon exercise, in clusters with significantly enriched terms. C Heatmap depicting the average regulon activity scores (RAS) for regulons with significant differential activity between cells from exercise and control conditions, in specific clusters. The number of genes comprising each regulon is indicated within parentheses. Regulons within red boxes indicate those upregulated upon exercise, and the ones within blue boxes indicate the ones downregulated upon exercise. D Cell-type specific regulons for ExN6 cluster identified using the SCENIC workflow. The y-axis denotes the regulon specificity score (RSS) (with high RSS values indicating high cell-type/cluster specificity, and vice versa). The x-axis denotes the rank of each regulon within the selected cluster, based on the RSS. The top 5 ranked regulons for ExN6 are labeled on the plot, with the number of genes comprising each regulon indicated within parentheses. E Violin plots showing the average module score (expression) of the 3 regulons specific to ExN6 that also show upregulated activity upon exercise (Hes1, Klf8, Nr2f2). F Dot plot showing significant GO biological process terms enriched among the collective list of genes and transcription factors (TFs) making up the top 5 regulons, as indicated in the RSS-Rank plot in (C). G Network plot with TFs and their respective regulon genes, specific to or showing differential activity in ExN6 upon exercise, indicating gene regulatory interactions in ExN6 cluster (genes/TFs that are significantly deregulated upon exercise or are specifically overexpressed in ExN6 are represented bigger in size, while selected genes belonging to these regulons which are deregulated in the same direction as the regulon TF are colored in red or blue)