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. 2021 Mar 31;24(4):102384.
doi: 10.1016/j.isci.2021.102384. eCollection 2021 Apr 23.

TLR7 and IL-6 differentially regulate the effects of rotarod exercise on the transcriptomic profile and neurogenesis to influence anxiety and memory

Yun-Fen Hung  1 , Yi-Ping Hsueh  1
Affiliations

TLR7 and IL-6 differentially regulate the effects of rotarod exercise on the transcriptomic profile and neurogenesis to influence anxiety and memory

Yun-Fen Hung et al. iScience. .

Abstract

Voluntary exercise is well known to benefit brain performance. In contrast, forced exercise induces inflammation-related stress responses and may cause psychiatric disorders. Here, we unexpectedly found that rotarod testing, a frequently applied assay for evaluating rodent motor coordination, induces anxiety and alters spatial learning/memory performance of mice. Rotarod testing upregulated genes involved in the unfolded protein response and stress responses and downregulated genes associated with neurogenesis and neuronal differentiation. It impacts two downstream pathways. The first is the IL-6-dependent pathway, which mediates rotarod-induced anxiety. The second is the Toll-like receptor 7 (TLR7)-dependent pathway, which is involved in the effect of rotarod exercise on gene expression and its impact on contextual learning and memory of mice. Thus, although rotarod exercise does not induce systemic inflammation, it influences innate immunity-related responses in the brain, controls gene expression and, consequently, regulates anxiety and contextual learning and memory.

Keywords: Immunology; Molecular Neuroscience; Transcriptomics.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Rotarod training at 5 or 10 weeks of age induces anxiety in adult mice (A) Outline of our experimental design. (B) Arrangement of behavioral paradigms for (C and D). A rotarod test (RR) was carried out on mice at 5 weeks (w) of age, followed by open field (OF) and elevated plus maze (EPM) as indicated. (C) The results of open field test on mice with rotarod training at 5 weeks of age. (D) The results of elevated plus maze test of mice with rotarod training at 5 weeks of age (n = 9 for WT–RR, n = 8 for WT + RR). (E) Arrangement of behavioral paradigms for (F)-(G). Mice were subjected to rotarod training at 10 weeks of age. (F) The results of open field test on mice with rotarod training at 10 weeks of age. (G) The results of elevated plus maze test on mice with rotarod training at 10 weeks of age (n = 8 for WT–RR mice, n = 9 for WT + RR mice). Data represent mean plus SEM. Two-tailed Mann-Whitney U test (B-C). ∗p < 0.05; ∗∗∗p < 0.001.
Figure 2
Figure 2
Rotarod training at 10 week of age induces anxiety through IL-6 (A) Rotarod training at 10 weeks of age differentially regulates cytokine expression in brain tissue (cortices and hippocampi) and spleens. Quantitative PCR was performed to measure relative cytokine expression levels in WT and Tlr7–/y mice (n = 5 for WT, n = 6 for Tlr7–/y). (B) Deletion of Il6 mitigates rotarod-induced anxiety in an open field (n = 8 for Il6–/––RR mice, n = 9 for Il6−/−+RR mice). (C) Rotarod training does not alter the population of glial cells in hippocampi (n = 5 for ± RR). Representative images of GFAP (red) and IBA1 (green) dual staining are shown. (D) Quantification of IBA1 and GFAP immunoreactivities. Signal coverage (area) percentage of the CA3 and hilus regions of hippocampi was determined. Data represent mean +/− SEM and the results of individual samples are shown. Two-tailed Mann-Whitney U test (B and D); two-way ANOVA with Bonferroni's multiple comparisons test (A). ∗, p < 0.05; ∗∗, p < 0.01. Scale bar: (C) 100 μm.
Figure 3
Figure 3
Tlr7 deletion alters rotarod-induced behavioral patterns and neurogenesis (A) Experimental arrangements for mice with rotarod training at 10 weeks of age. (1) for (B) and (C); (2) for (D); (3) and (4) for (E) and (F). (B) The effect of rotarod training (RR) on open field (OF) responses. Rotarod training reduced the time spent in the center of an open field for both WT and Tlr7–/y mice, but not the number of rearing events of Tlr7–/y mice (n = 12 for WT–RR, n = 10 for WT + RR, n = 9 for Tlr7–/y–RR, n = 10 for Tlr7–/y + RR). (C) The effect of RR on fear conditioning. Rotarod training solely enhanced contextual memory of Tlr7–/y mice (n = 10 for WT ± RR, n = 9 for Tlr7–/y ± RR). AS, right after stimulation (representing pain sensation). (D) In Barnes maze, RR differentially impacted escape latency in WT and Tlr7–/y mice but did not affect short- (D5) or long-term (D12) memory (n = 10 for WT–RR, n = 15 for WT + RR, n = 10 for Tlr7–/y–RR, n = 13 for Tlr7–/y + RR). (E) Representative images of BrdU labeling. (F) The results of BrdU labeling. For short-term 24-hr BrdU labeling, RR enhanced neurogenesis in both WT and Tlr7–/y mice (n = 6 for WT–RR, n = 11 for WT + RR, n = 6 for Tlr7–/y–RR, n = 11 for Tlr7–/y + RR). For long-term 4-week BrdU labeling, RR reduced neural differentiation only in WT mice but not in Tlr7–/y mice (n = 9 for WT–RR, n = 8 for WT + RR, n = 7 for Tlr7–/y–RR, n = 7 for Tlr7–/y + RR). Data represent mean +/− SEM and the results of individual samples are shown. two-way ANOVA with Bonferroni's multiple comparisons test (B–D and F). ∗, p < 0.05; ∗∗, p < 0.01. Scale bar: (E) 100 μm.
Figure 4
Figure 4
Transcriptomic profiles and networks are differentially altered by rotarod training in WT and Tlr7–/y mice (A) Principal component analysis of RNA-seq data from WT and Tlr7–/y mouse brains with/without rotarod training. (B) Venn diagram showing overlap of upregulated (left) and downregulated (right) genes between WT and Tlr7–/y mice following rotarod training. Gene lists are provided in Tables S1 and S2. FC, fold-change; FDR, false discovery rate; TPM, transcripts per million. (C–G) Protein interaction networks analyzed by STRING. Node color represents an association with the different networks indicated at the bottom. Nodes with multiple colors indicate associations with multiple networks.
Figure 5
Figure 5
Genes regulated by rotarod training are highly correlated with neurological functions and diseases (A) Ingenuity Pathway Analysis (IPA) of genes regulated by rotarod training in WT and Tlr7–/y mouse brains. Both upregulated (red) and downregulated (blue) genes were combined for IPA. The functions of each network are indicated. Black font depicts genes unaffected by rotarod training but that interact with identified differentially expressed genes (DEG). (B) Overlapping DEG are highly relevant to anxiety. Eighteen and eight genes out of a total of 29 upregulated and 10 downregulated genes, respectively, are associated with anxiety. The related manuscripts and their PMIDs are indicated. (C) Heatmap of DEG. Color intensity represents relative TPM levels (log2 values) normalized to the WT–RR group. Gene groupings are also indicated. The color-coded bar represents the Z score.
Figure 6
Figure 6
Quantitative RT-PCR verifies differential expression of DEGs identified from RNA-seq A total of ten DEGs were selected from the heatmap shown in Figure 5C for quantitative RT-PCR. Data represent mean ± SEM. The results of individual animals are also shown. The sequences of primers and the numbers of Universal probes for PCR are available in Table S6. two-way ANOVA with Bonferroni's multiple comparisons test showed that rotarod treatment altered expression of all ten examined genes compared with mock control. The results of post-testing on significant differences are indicated. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. Sample size N = 4.
Figure 7
Figure 7
Tlr7 deletion influences the expression of DEG regulated by rotarod training (A) Heatmaps of downregulated DEG. The genes listed here have expression ratios of Tlr7–/y + RR to WT + RR greater than 1 (Table S3). Relative expression levels (log2) were normalized to WT–RR. The color-coded bar represents the Z score. (B) STRING analysis of the protein networks of selected genes (the same gene set in A). Node color represents an association with the different networks indicated at the bottom. (C) Gene ontology analysis of selected genes assessed using Metascape.
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