Loss of RhoA in microglia disables glycolytic adaptation and impairs spinal cord injury recovery through Arhgap25/HIF-1α pathway

Abstract

RhoA, a small GTPase, plays a pivotal role in various diseases, including spinal cord injury (SCI). Although RhoA inhibition has been traditionally viewed as beneficial for SCI repair, recent clinical trials of RhoA inhibitors in SCI have failed to show significant therapeutic efficacy, suggesting functional heterogeneity across different cell types. The role of RhoA in microglia, the key immune cells involve in SCI, remains poorly understood. Using microglial RhoA conditional knockout mice, this study demonstrated that RhoA deficiency in microglia attenuates the morphological and functional repair of the SCI mice, and impairs the microglial biofunctions of proliferation, phagocytosis, and migration. Single-cell RNA sequencing, bulk RNA sequencing, and metabolomics revealed that RhoA deficiency can attenuate the microglial glycolytic enzyme expression, ATP production, ECAR and OCR levels through the Arhgap25/HIF-1α pathway. Overall, this is the first study to demonstrate that microglial RhoA is essential for SCI repair, the Arhgap25/HIF-1α pathway mediated glucose metabolism might enlighten a novel insight to enrich the understanding on the complex roles of RhoA and microglia in SCI repair. Moreover, this study highlights the importance of considering cell-specific roles of RhoA in SCI repair and provides a foundation for developing targeted therapies aimed at microglial metabolic reprogramming. Schematic representation of the proposed mechanism by which microglial RhoA regulates glycolytic adaptation and spinal cord repair. (Created by Figdraw.com with permission of # wgq=r7c74c).

Schematic representation of the proposed mechanism by which microglial RhoA regulates glycolytic adaptation and spinal cord repair. (Created by Figdraw.com with permission of # wgq=r7c74c).

Fig. 1. RhoA expression in microglia was up-regulated by spinal cord injury.

A Changes of RhoA mRNA expression in injured spinal cord obtained from different GEO databases. B Temporal changes of RhoA protein level in spinal cord following SCI (n = 5). C Major cell type classification in the GSE196928 dataset. D Distribution map of RhoA expression. E RhoA expression levels across different cell types. F Immunostaining shows the RhoA expression in microglia (IBA-1⁺) at different stages after SCI (n = 3). Data are presented as mean ± SD. Statistical significance: ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001 (vs. sham group).

Fig. 2. Microglial RhoA deficiency delays the spinal cord injury recovery.

A Representative foot print images of mice at 28 dpi. B BMS scores at different stages after SCI. C Swimming test was used to evaluate locomotor function in mice at 28 dpi. D Gastrocnemius muscle weight at 28 dpi. E H&E staining of the gastrocnemius muscle at 28 dpi. F NF200 Mean fluorescence intensity and the number of NeuN-positive cells at various distances from the SCI lesion center of the spinal cords present 1000 μm proximal (n = 3). Data are presented as mean ± SD; each dot represents an individual mouse. Statistical significance: n.s. nonsignificant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 3. RhoA knockout inhibits the microglial proliferation.

A Immunostainings show the number of Microglia (Iba-1⁺) cells in spinal cord tissue at 28 dpi. B Immunostainings show Ki67⁺ (green) and IBA-1⁺ (red) microglial cells in the SCI at 7 dpi. C Western blotting shows the related protein levels of proliferating and cell cycle in the primary cultured microglia. Immunostainings show the Ki67⁺ cells (D), PCNA⁺ cells (E) and Edu⁺ cells (F) in primary cultured microglia. Data are presented as mean ± SD; each dot represents an individual mouse. Statistical significance: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 4. RhoA knockout attenuates the microglial phagocytosis and migration.

A Immunostainings show IBA-1⁺ and ORO⁺ on the cross sections of the 7 dpi spinal cord tissue. B, C Immunostaining and Western blotting show the CD68 expression in the cultured microglia (IBA-1⁺). D, E Immunostaining and Western blotting demonstrate microglia engulfed myelin debris (MBP⁺) in the myelin debris treated microglial cultures. F Representative images of Transwell assay. Data are presented as mean ± SD; each dot represents an individual mouse. Statistical significance: ∗p < 0.05; ∗∗p < 0.01.

Fig. 5. Multi-omics insights into RhoA-mediated regulation of glycolysis and oxidative phosphorylation in microglia.

A t-SNE and unsupervised clustering of all cells based on biomarkers. B GSEA analysis highlighting differences in microglial subsets compared to other cell types. C High-resolution reclustering of microglia into ten distinct clusters. D Expression of key markers identified in the reclustered microglia. E Pseudotime trajectory analysis of microglial cells. F Visualization of ten microglial subclusters, each represented by a distinct color. Microglial subsets with significant differences in oxidative phosphorylation pathways (G subcluster 0, 2, 3, 4, 7) and glycolysis pathways (H subcluster 2, 3, 4, 7) identified through GSEA analysis. I CellChat analysis showing interactions between glycolysis-differentiated microglia (subcluster 2, 3, 4, 7) and other cell types. J PCA of bulk mRNA transcriptomics in primary microglial cells. K Heatmap illustrating the results of mRNA sequencing analysis. GSEA analysis of glycolysis-related (L) and oxidative phosphorylation-related (M) differences between flox and cKO groups. N PCA of metabolomic profiling data. O, P Comparative analysis of glycolysis and TCA cycle metabolic changes between cKO and flox groups. RhoA deletion decreases ATP production (Q), accompanied by reduced ECAR (R), OCR (S), and glucose consumption in the culture medium (T). Data are presented as mean ± SD; each dot represents an individual mouse. Statistical significance: ***p < 0.001.

Fig. 6. RhoA regulates the microglial biofunctions by regulating the HIF-1 pathway through Arhgap25.

A Highlight Arhgap25 in volcano diagram of mRNA-sequence. B Arhgap25 mRNA level in primary microglia in mRNA-sequence. C KEGG enrichment analysis of significant changes gene in mRNA-sequence. D Western blots show the Arhgap25, HIF-1α, HIF1AN, HK2, PKM2 and LDHA protein expression levels in primary cultured microglia. E, F Immunostainings show Arhgap25 and HIF-1α expression in primary cultured microglia. Data are presented as mean ± SD; each dot represents an individual mouse. Statistical significance: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 7. Arhgap25 knockdown reverses the regulatory effect of RhoA knockout on the biological function of microglia.

ATP concentration (A), ECAR (B) and OCR (C) levels in primary cultured microglia after siArhgap25 transfection. D Western blotting shows the expression of Arhgap25, HIF-1α, HK2, PKM2 and LDHA after siArhgap25 transfection. E Western blotting shows the expression of PCNA and PH3 protein after siArhgap25 transfection. Immunostaining shows the PCNA⁺(F), Ki67⁺ (G) and Edu⁺ (H) in primary cultured microglia after transfection with siArhgap25. Data are presented as mean ± SD; each dot represents an individual mouse. Statistical significance: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Fig. 8. Arhgap25 knockdown reverses the effects of RhoA cKO on the phagocytosis and migration of microglia.

A Immunostaining shows the CD68 expression in the cultured microglia (IBA-1⁺). B Immunostaining shows the myelin debris (MBP⁺) engulfed in the cultured microglia after the microglia were co-cultured with debris for 1 h. C Western blotting show the CD68 level in microglia (culture without debris) and MBP level in microglia (co-cultured with debris for 1 h). D Representative images of Transwell assay. Data are presented as mean ± SD; each dot represents an individual mouse. Statistical significance: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.