SRT2104 enhances dendritic outgrowth and spine formation through Sirtuin 1-mediated mTORC1 signaling

Abstract

Impaired neuroplasticity is a one of the key pathological mechanism of depression. Sirtuin 1 plays a crucial role in neuroplasticity; however, its precise mechanisms remain unclear. This study examined whether sirtuin 1 regulates dendritic outgrowth and spine formation via mTORC1 signaling in rat primary cortical cells under dexamethasone-induced neurotoxic conditions. Cortical cells were treated with SRT2104 (0.1, 1, and 10 µM), a selective sirtuin 1 activator, in the presence of dexamethasone (500 µM). Protein levels of sirtuin 1, mTORC1 signaling components, and synaptic markers (PSD-95 and GluA1) were analyzed by Western blotting, while dendritic outgrowth and spine density were assessed via immunofluorescence. SRT2104 significantly increased sirtuin 1 expression and ERK1/2 (a downstream target of sirtuin 1) phosphorylation. SRT2104 led to a substantial augmentation in the phosphorylation levels of mTORC1, as well as 4E-BP1 and p70S6K, which are downstream targets of mTORC1. Furthermore, SRT2104 led to an increase in dendritic outgrowth and spine density. Conversely, sirtuin 1 knockdown by siRNA transfection markedly reduced ERK1/2 and mTORC1 phosphorylation, as well as dendritic complexity and spine formation. These results suggest that sirtuin 1 promotes neuroplasticity by activating mTORC1 signaling, providing potential therapeutic implications for depression treatment.

Keywords: Depression; Dexamethasone; Neuroplasticity; SRT2104; Sirtuin 1; mTORC1 signaling.

Fig. 1

Effects of SRT2104 on cell viability in primary cortical cells. Cortical cells were treated with SRT2104 (0.1, 1, 10, and 50 µM) or DMSO (non-drug treated control, final concentration 1%) for 4 days in the presence or absence of dexamethasone (500 µM). An MTT assay was performed (n = 8). The values represent the mean ± SEM expressed as a percentage of the control cells (DMSO-treated, non-dexamethasone-treated cells). ^***P < 0.001 vs. control cells; ^†††P < 0.001 vs. DMSO-treated, dexamethasone-treated cells.

Fig. 2

Effects of SRT2104 on the levels of sirtuin 1 expression and ERK1/2 phosphorylation in dexamethasone-treated cells. Cortical cells were treated with SRT2104 (0.1, 1, and 10 µM) or DMSO (non-drug treated control, final concentration 1%) for 4 days in the presence of dexamethasone (500 µM). The levels of sirtuin 1 (A) and phospho-Thr²⁰²/Tyr²⁰⁴-ERK1/2 (B) were determined by Western blotting (n = 8). Full-length uncropped gels for all Western blots are available in the Supplementary Information. The values represent the mean ± SEM expressed as a percentage of the control cells (DMSO-treated, non-dexamethasone-treated cells). ^***P < 0.001 vs. control cells; ^†P < 0.05 vs. DMSO-treated, dexamethasone-treated cells.

Fig. 3

Effects of SRT2104 on the levels of mTORC1, 4E-BP1 and p70S6K phosphorylation in dexamethasone-treated cells. Cortical cells were treated with SRT2104 (0.1 and 1 µM) or DMSO for 4 days in the presence of dexamethasone. The levels of phospho-Ser²⁴⁴⁸-mTORC1 (A), phospho-Thr^37/46–4E-BP1 (B) and phospho-Thr³⁸⁹-p70S6K (C) were determined by Western blotting (n = 6–8). Full-length uncropped gels for all Western blots are available in the Supplementary Information. The values represent the mean ± SEM expressed as a percentage of control cells (DMSO-treated, non-dexamethasone-treated cells). ^*P < 0.05 or ^***P < 0.001 vs. control cells; ^†P < 0.05, ^††P < 0.01 or ^†††P < 0.001 vs. DMSO-treated, dexamethasone-treated cells.

Fig. 4

Effects of SRT2104 on the levels of PSD-95 and GluA1 expression in dexamethasone-treated cells. Cortical cells were treated with SRT2104 (0.1 and 1 µM) or DMSO for 4 days in the presence of dexamethasone. The levels of PSD-95 (A) and GluA1 (B) were determined by Western blotting (n = 6). Full-length uncropped gels for all Western blots are available in the Supplementary Information. The values represent the mean ± SEM expressed as a percentage of control cells (DMSO-treated, non-dexamethasone-treated cells). ^**P < 0.01 vs. control cells; ^†P < 0.05 or ^††P < 0.01 vs. DMSO-treated, dexamethasone-treated cells.

Fig. 5

Effects of SRT2104 on dendritic outgrowth and spine density in dexamethasone-treated cells. Cortical cells were treated with SRT2104 (0.1 and 1 µM) or DMSO for 5 days in the presence of dexamethasone. In total, 600 cells from each group were analyzed using anti-MAP2 to assess neurite length (A-a), neurite number (A-b), branch points (A-c), and soma area (A-d). In total, 35 dendritic segments from each group were analyzed using phalloidin dye to assess spine formation (B). All data are expressed as the mean ± SEM. ^***P < 0.001 vs. control cells (DMSO-treated, non-dexamethasone-treated cells); ^†††P < 0.001 vs. DMSO-treated, dexamethasone-treated cells.

Fig. 6

Effects of sirtuin 1 knockdown on the levels of ERK1/2 and mTORC1 phosphorylation in primary cortical cells. Cortical cells were transfected with fluorescein-labeled control or sirtuin 1 siRNA (100 nM) for 24 h. The levels of sirtuin 1 (A), phospho-Thr²⁰²/Tyr²⁰⁴-ERK1/2 (B) and phospho-Ser²⁴⁴⁸-mTORC1 (C) were determined by Western blotting (n = 6). Full-length uncropped gels for all Western blots are available in the Supplementary Information. The values represent the mean ± SEM expressed as a percentage of control cells (control siRNA-transfected cells). ^***P < 0.001 vs. control cells.

Fig. 7

Effects of sirtuin 1 knockdown on dendritic outgrowth and spine density in primary cortical cells. Cortical cells were transfected with fluorescein-labeled control or sirtuin 1 siRNA (green) for 24 h. In total, 600 cells from each group were analyzed using anti-MAP2 (red) to assess neurite length (A-a), neurite number (A-b), branch points (A-c), and soma area (A-d). In total, 45 dendritic segments from each group were analyzed using phalloidin dye (red) for spine formation (B). All data are expressed as the mean ± SEM. All data are expressed as the mean ± SEM. ^*P < 0.05, ^**P < 0.01 or ^***P < 0.001 vs. control cells (control siRNA-dtransfected cells).