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[Preprint]. 2024 May 5:2024.05.02.592289.
doi: 10.1101/2024.05.02.592289.

Multi-omics delineate growth factor network underlying exercise effects in an Alzheimer's mouse model

Xin Li  1 , Chaozhong Liu  2 , Wenbo Li  1 , Yanwan Dai  2 , Chaohao Gu  2 , Wenjun Zhou  1 , Veronica C Ciliberto  1 , Jing Liang  1   3 , Kumar S Udhaya  1 , Dongyin Guan  1 , Zhaoyong Hu  4 , Hui Zheng  5 , Hu Chen  2 , Zhandong Liu  2 , Ying-Wooi Wan  2 , Zheng Sun  1   6
Affiliations

Multi-omics delineate growth factor network underlying exercise effects in an Alzheimer's mouse model

Xin Li et al. bioRxiv. .

Abstract

Physical exercise represents a primary defense against age-related cognitive decline and neurodegenerative disorders like Alzheimer's disease (AD). To impartially investigate the underlying mechanisms, we conducted single-nucleus transcriptomic and chromatin accessibility analyses (snRNA-seq and ATAC-seq) on the hippocampus of mice carrying AD-linked NL-G-F mutations in the amyloid precursor protein gene (APPNL-G-F) following prolonged voluntary wheel-running exercise. Our study reveals that exercise mitigates amyloid-induced changes in both transcriptomic expression and chromatin accessibility through cell type-specific transcriptional regulatory networks. These networks converge on the activation of growth factor signaling pathways, particularly the epidermal growth factor receptor (EGFR) and insulin signaling, correlating with an increased proportion of immature dentate granule cells and oligodendrocytes. Notably, the beneficial effects of exercise on neurocognitive functions can be blocked by pharmacological inhibition of EGFR and the downstream phosphoinositide 3-kinases (PI3K). Furthermore, exercise leads to elevated levels of heparin-binding EGF (HB-EGF) in the blood, and intranasal administration of HB-EGF enhances memory function in sedentary APPNL-G-F mice. These findings offer a panoramic delineation of cell type-specific hippocampal transcriptional networks activated by exercise and suggest EGF-related growth factor signaling as a druggable contributor to exercise-induced memory enhancement, thereby suggesting therapeutic avenues for combatting AD-related cognitive decline.

Keywords: Alzheimer’s disease; Physical exercise; cognition; growth factor; hippocampus.

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Conflict of interest statement

CONFLICT OF INTEREST The authors declare no financial conflict of interest.

Figures

Figure 1.
Figure 1.. Exercise improves memory without altering the overall hippocampal cellular composition.
Discrimination index and exploration time of the standard object-in-place memory test (a-c) and the Y-maze test (d-f) in male WT and APPNL-G-F (APP) mice at 10 months old after 6 months wheel-running exercise (EX) or rest (RT) (n = 12 for WT_RT; 8 for WT_EX; 10 for APP_RT; and 9 for APP_EX). Asterisks indicate significant differences with the two-way ANOVA and Fisher’s LSD multiple comparisons test. (g-i) Discrimination index and exploration time of a modified 5-object in-place test in 4-month-old male WT mice after 2 months wheel-running (n = 12 for RT, 13 for EX). Bar graphs show the mean with S.E.M. Asterisks indicate significant differences by 2-sided t-test. (j) Violin plot of cell type-specific marker gene expression levels. (k) UMAP of major cell types of the hippocampus based on snRNA-seq and snATAC-seq datasets. Excitatory neurons (EN), inhibitory neurons (IN), microglia (MG), astrocytes (AST), oligodendrocytes (OLG), and oligodendrocyte progenitor cells (OPC). APP/Exercise (APP_EX), APP/Rest (APP_RT), WT/Exercise (WT_EX), WT/Rest (WT_RT).
Figure 2.
Figure 2.. Exercise counteracts amyloid-dependent transcriptomic changes.
(a) Scatter plot of gene expression showing a negative correlation between exercise effects (APP_EX vs. APP_RT) and amyloid effects (APP_RT vs. WT_RT) across different cell types. (b) Heat map of 833 differentially expressed genes (DEGs) with the reversed pattern in at least one cell type. (c) Number of overlapping reversed DEGs across different cell types. (d) Overlapping pathways enriched in reversed DEGs across different cell types. (e-f) GSEA analysis of the insulin signaling pathway, a top common enriched pathway in reversed genes in different cell types.
Figure 3.
Figure 3.. Exercise activates the EGFR/insulin signaling.
(a) Heat map of reversed DEGs within the insulin signaling in different cell clusters. (b-c) RNAscope analysis of Stxbp1, a gene of the insulin/EGFR pathway with known function in neurotransmission and a robust reversed expression pattern in EN. Scale bar, 200 μm. n = 3 mice. Bar graphs show the mean with S.E.M. Asterisks indicate significant differences by t-test. ns, non-significant. (d) Some of the DEGs in the EGFR pathway. (e) Some of the DEGs in the insulin pathway. Images were generated from the KEGG pathway database.
Figure 4.
Figure 4.. Exercise stimulates cell type-specific transcriptional regulatory networks.
(a) Heat map of the relative levels of the reversed differentially accessible regions (DARs) in each cluster. DARs upregulated by APPNL-G-F (APP_RT vs. WT_RT) and downregulated by exercise (APP_EX vs. APP_RT) were referred to as “U>D”, while those DARs downregulated by APPNL-G-F and upregulated by exercise were referred to as “D>U”. (b) Top enriched motifs in the DARs in each cell cluster. (c-h) Top network showing direction-specific enrichment in EN, IN, and OLG populations.
Figure 5.
Figure 5.. Exercise impacts excitatory neurons and oligodendrocytes.
(a-b) UMAP of snRNA-seq data in EN sub-clusters: Prox1+ granule cells and Ociad2+ pyramidal cells. Expression is based on library-size-normalized log values. (c) UMAP of hippocampal EN sub-clusters in each individual group. (d) The proportion of granule and pyramidal cells within the EN cluster in each group. (e) Proportions of granule cells and INs in the DG. (f) Box plot of predicted GC immature score (imGC). 514 of 534 genes in the model were found in our data, and missing genes had only small weights in the model. The log-transformed and max-normalized counts matrix were taken as the input to predict the final imGC score from the logistic regression model. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. Asterisks indicate significant differences. ns, non-significant. (g) UMAP of the OLG for trajectory analysis. (h) Module score along the pseudotime trajectory of the OLG. (i) Distribution of each group on the OLG trajectory.
Figure 6.
Figure 6.. EGFR and PI3K signaling are required for the cognitive-improving effects of exercise.
(a) Average actogram profiles of wheel-running activity of 8-month-old male mice treated with EGFR inhibitor Gefitinib and PI3K inhibitor Wortmannin through oral gavage once every other day from 4 to 8 months old. Mice were simultaneously subjected to wheel-running from 4 to 8 months old. (b) Average daily wheel-running activity (n = 7 cages per group with 2 mice per cage). (c) Body weight. (d-o) Object-in-place, Y-maze, 3-chamber sociability and social memory, Morris water maze, elevated plus maze, open field arena, and light-dark tests (n = 15 mice for RT, 15 mice for EX, and 16 mice for EX + inhibitor). 2 RT, 2 EX and 1 EX+inhibitor mice were excluding due to discrimination index were greater or smaller than ± 0.7 (p-q) Immunostaining of β-amyloid in mice treated with inhibitors. Scale bar: 600 μm. n = 6 mice per group. All bar graphs show the mean with S.E.M. Asterisks indicate significant differences by one-way ANOVA with Fisher’s LSD multiple comparisons except Morris water maze was analyzed with repeated-measure 2-way ANOVA with Fisher’s LSD multiple comparisons where asterisks indicate differences between RT vs. EX and EX + inhibitor vs. EX.
Figure 7.
Figure 7.. Intranasal HB-EGF mimics exercise-induced cognitive improvement.
(a) Replot of gene expression levels of EGF family members in human skeletal muscles after long-term exercise training from a published transcriptomic dataset. (b) Replot of protein levels of EGF family members in the human blood after long-term exercise training from a published proteomics dataset. (c) Serum HB-EGF levels in APPNL-G-F male mice after chronic wheel-running exercise for 6 months (n = 11 mice for RT, and 11 mice for EX). (d) Body weight gain during intranasal HB-EGF administration in female APPNL-G-F mice. Administration started at 4 months old, with once every other day (n = 13 mice for the vehicle; 13 mice for HB-EGF). (e-o) Object-in-place, Y-maze, 3-chamber sociability and social memory, Morris water maze test, open field arena test, and light-dark test in female mice at 7-8-months old after chronic HB-EGF administration (n = 13 mice for the vehicle; 13 mice for HB-EGF; one mouse from the HB-EGF group was excluding from MWM due to low mobility). (p-q) Immunostaining of β-amyloid in 8-month-old female mice treated with intranasal HB-EGF for 4 months. Scale bar: 600 μm. n = 6 mice per group. All bar graphs show the mean with S.E.M. Asterisks indicate significant differences by one-way ANOVA with Fisher’s LSD multiple comparisons or repeated-measure ANOVA with Fisher’s LSD multiple comparisons.
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