Astrocytes underlie a faster-onset antidepressant effect of hypidone hydrochloride (YL-0919)

Abstract

Introduction: Major depression disorder (MDD) is a common and potentially life-threatening mental illness; however, data on its pathogenesis and effective therapeutic measures are lacking. Pathological changes in astrocytes play a pivotal role in MDD. While hypidone hydrochloride (YL-0919), an independently developed antidepressant, has shown rapid action with low side effects, its underlying astrocyte-specific mechanisms remain unclear. Methods: In our study, mice were exposed to chronic restraint stress (CRS) for 14 days or concomitantly administered YL-0919/fluoxetine. Behavioral tests were applied to evaluate the depression model; immunofluorescence and immunohistochemistry staining were used to explore morphological changes in astrocytes; astrocyte-specific RNA sequencing (RNA-Seq) analysis was performed to capture transcriptome wide alterations; and ATP and oxygen consumption rate (OCR) levels of primary astrocytes were measured, followed by YL-0919 incubation to appraise the alteration of energy metabolism and mitochondrial oxidative phosphorylation (OXPHOS). Results: YL-0919 alleviated CRS-induced depressive-like behaviors faster than fluoxetine and attenuated the number and morphologic deficits in the astrocytes of depressed mice. The changes of gene expression profile in astrocytes after CRS were partially reversed by YL-0919. Moreover, YL-0919 improved astrocyte energy metabolism and mitochondrial OXPHOS in astrocytes. Conclusion: Our results provide evidence that YL-0919 exerted a faster-onset antidepressant effect on CRS-mice possibly via astrocyte structural remodeling and mitochondria functional restoration.

Keywords: RNA-seq; YL-0919; astrocytes; depression; mitochondria.

FIGURE 1

YL-0919 concomitant treatment alleviates depression-like behaviors induced by CRS. (A) Experimental procedure. Timeline of mice adaption, CRS exposure, drug administration strategy, and behavioral tests. (B) Representative trace recordings of the OFT. Quantification of the distance traveled in the central zone (C, left) (F (3, 36) = 8.569, p = 0.0002) and the total distance moved in the OFT (C, right) (F (3, 36) = 0.5748, p = 0.6353). (D) Percentage of 1% sucrose consumption in the SPT after CRS exposure and drug treatment (F (3, 27) = 3.14, p = 0.0416). (E,F) Quantification of the time spent immobile in the TST (F (3, 36) = 5.918, p = 0.0022) and FST (F (3, 36) = 9.143, p = 0.0001). Data are expressed as mean ± SEM, n = 10 in each group. For SPT, one-way ANOVA, followed by Fisher’s LSD, was used; otherwise, Bonferroni’s multiple comparisons test was applied. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 2

YL-0919 reverses astrocyte loss in mPFC and dHIP. (A) Schematic representation depicting coronal section areas of the mPFC and HIP. (B) Quantitative analysis of S100β⁺ cell numbers in the mPFC (F (3,36) = 2.184, p = 0.1069). (C) Typical images of S100β immunohistochemical staining in the PFC (upper panel, scale bar, 500 μm) and mPFC (lower panel, scale bar, 200 μm). Immunohistochemical staining of GFAP in the dorsal hippocampus subfields including dVA1 (D), dCA3 (F), and dDG (G) (scale bar, 200 μm and 100 μm). Quantitative analysis of GFAP⁺ cell numbers in dCA1 (E) (F (3, 32) = 5.835, p = 0.0027), dCA3 (F) (F (3, 32) = 7.919, p = 0.0004), and dDG (G) (F (3, 32) = 7.671, p = 0.0005). Data are expressed as mean ± SEM, n = 10 in each group. For S100β⁺ and GFAP⁺ numbers in the vCA1 analysis, one-way ANOVA, followed by Fisher’s LSD, was used; otherwise, Bonferroni’s multiple comparisons test was applied. *p < 0.05; **p < 0.01.

FIGURE 3

Effects of antidepressants on the morphology of astrocytes in the vHIP. (A) Immunofluorescent confocal images in the vHIP with astrocyte markers GFAP (green) and DAPI (blue) (scale bar: 200 µm). (B) Typical images of the vCA1 and (D) vCA3 shown at higher magnification (upper panel) and schematic representations of the Sholl analysis (lower panel, scale bar: 20 µm). Sum intersections of vCA1 (C) (F (3, 116) = 20.99, p < 0.0001) and vCA3 (E) astrocytes. Number of intersections in vCA1 (F) (F (3, 1624) = 69.96, p < 0.0001) and vCA3 (G) (F (3, 1624) = 35.19, p < 0.0001) astrocytes in each group. Data are mean ± SEM. n = 30 cells from five mice per group. For Sholl analysis, two-way ANOVA, followed by Bonferroni’s multiple comparisons test, was used. For sum intersection analysis, one-way ANOVA, followed by Tukey’s multiple comparisons test, was applied. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 4

Gene expression profiling of astrocytes in CRS and YL-0919-treated mice revealed by RNA-Seq analysis. (A) Schemes depicting the column-based magnetic cell selection. (B) Heatmap and (C) volcano plot visualization of the gene profiling expression between the Ctrl and CRS groups. The heatmap (D) and volcano (E) show DEGs between the YL and CRS groups. Red: significantly upregulated genes; gray: genes with no significant differential expression; blue: significantly downregulated genes. The top 20 genes are shown (p < 0.05 and |log2FC| > 1). (F) Summary of gene ontology (GO) analysis of DEGs from the Ctrl, CRS, and YL groups. (G) Comprehensive comparison of significantly enriched pathways (Q < 0.05) of all comparison groups. The bubble colors and sizes, respectively, indicate the Q value and the number of differential genes of the pathway in the enrichment analysis. Q value > 0.05, the bubble is missing.

FIGURE 5

Venn screening and validation of oppositely changed DEGs. (A) Venn diagrams depicting the extent of overlap between genes downregulated (upregulated) in CRS mice and upregulated (downregulated) in YL-0919 treated mice. (B) Functional enrichment analysis of the overlapping genes indicating significantly enriched GO terms in the biological processes (red), cellular component (yellow), and molecular function (blue) categories. (C) qRT-PCR validation of the RNA-Seq data set with selected genes. Data are represented as mean ± SEM. One-way ANOVA, followed by Fisher’s LSD, was used. n = 3 in each group. Ctrl/YL vs. CRS. *p < 0.05; ****p < 0.0001.

FIGURE 6

Impact of YL-0919 on mitochondria function in primary astrocytes. (A) Summary of GSEA performed on the FDR-ranked gene list. (B) Representative image of cultured primary astrocytes in vitro. GFAP (red) and DAPI (blue); scale bar, 200 μm. (C) Astrocyte cell viability was expressed as a percentage of the control, which was set to 100%. Cells were treated with different concentrations of YL-0919 for 24 h. Data are expressed as means ± SEM and analyzed by one-way ANOVA, F (5, 24) = 1.046, p = 0.4139, and n = 10. Intracellular (D) and extracellular (E) ATP levels of Veh and YL-0919-treated astrocytes. (F) OCR measurements of Veh and YL-0919-treated astrocytes. Statistics on basic OCR (G left) and maximum OCR (G right). Data are expressed as means ± SEM and analyzed by two-tailed Student’s t-test. n = 10 in ATP. n = 5 in OCR. *p < 0.05.