Sortilin deletion in the prefrontal cortex and hippocampus ameliorates depressive-like behaviors in mice via regulating ASM/ceramide signaling

Abstract

Major depressive disorder (MDD) is a common psychiatric disorder characterized by persistent mood despondency and loss of motivation. Although numerous hypotheses have been proposed, the possible pathogenesis of MDD remains unclear. Several recent studies show that a classic transporter protein, sortilin, is closely associated with depression. In the present study, we investigated the role of sortilin in MDD using a well-established rodent model of depression. Mice were subjected to chronic unpredictable mild stress (CUMS) for 6 weeks. We showed that the expression levels of sortilin were significantly increased in the prefrontal cortex and hippocampus of CUMS mice. The depressive-like behaviors induced by CUMS were alleviated by specific knockdown of sortilin in the prefrontal cortex and hippocampus. We revealed that sortilin facilitated acid sphingomyelinase (ASM)/ceramide signaling, which activated RhoA/ROCK2 signaling, ultimately causing the transformation of dendritic spine dynamics. Specific overexpression of sortilin in the prefrontal cortex and hippocampus induced depressive-like behaviors, which was mitigated by injection of ASM inhibitor SR33557 (4 µg/μL) into the prefrontal cortex and hippocampus. In conclusion, sortilin knockdown in the prefrontal cortex and hippocampus plays an important role in ameliorating depressive-like behavior induced by CUMS, which is mainly evidenced by decreasing the trafficking of ASM from the trans-Golgi network to the lysosome and reducing the ceramide levels. Our results provide a new insight into the pathology of depression, and demonstrate that sortilin may be a potential therapeutic target for MDD.

Keywords: ASM/ceramide; RhoA/ROCK2; SR33557; cofilin; dendritic spines; depression; hippocampus; prefrontal cortex; sortilin.

Fig. 1. Increased sortilin expression and ASM/ceramide levels in the prefrontal cortex and hippocampus in mice exposed to CUMS.

a, b Western blot analysis of sortilin protein expression in the prefrontal cortex (PFC), hippocampus (Hipp), nucleus accumbens (NAcc), amygdala (Amyg) of mice. n = 4–5 per group. ***P < 0.001 compared with control. c, d Representative images of immunostaining for sortilin in the PFC (c) and Hipp (d) of mice. Scale bars = 200 μm. e Quantitative of relative fluorescence intensity (RFI) in the PFC and Hipp of mice. n = 3–4 per group. ***P < 0.001 compared with control. f, g Levels of acid sphingomyelinase (ASM) activity (f) and quantitative ELISA analysis of total ceramide (g) in the PFC, Hipp, NAcc, Amyg of mice. n = 3–4 per group. **P < 0.01, ***P < 0.001 compared with control. One-way ANOVA, Holm–Sidak’s multiple comparisons test. All data are represented as mean ± SEM.

Fig. 2. Specific knockdown of sortilin in the prefrontal cortex and hippocampus of mice ameliorates CUMS-induced depressive-like behaviors.

a Body weight changes from the beginning to the end of the experiment in the mice. n = 8–16 per group. b Total distance, immobility time, time spent in central zone in the OFT. n = 8–16 per group. c Immobility time in the TST. n = 8–16 per group. d Immobility time in the FST. e Latency to feed time in the NSFT. n = 8–16 per group. f Percentage of sucrose preference in the SPT. n = 8–16 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with CUMS + Ctrl AAV. One-way ANOVA, Holm–Sidak’s multiple comparisons test. All data are represented as mean ± SEM.

Fig. 3. ASM distribution between the TGN and lysosome regulated by sortilin affects ASM/ceramide levels.

a, b Levels of ASM activity (a) and quantitative ELISA analysis of total ceramide (b) in the PFC and Hipp of mice. n = 5 per group. c, d Representative images of immunostaining for ASM, TGN38, Hoechst (c) and ASM, LAMP1, Hoechst (d) in the PFC of mice. e, f Representative images of immunostaining for ASM, TGN38, Hoechst (e) and ASM, LAMP1, Hoechst (f) in the Hipp of mice. Scale bars = 5 μm. g, h Quantitative of ASM/TGN38 (g) and ASM/LAMP1 (h) co-localization Pearson’s correlation coefficient in the PFC of mice. n = 3–4 per group. i, j Quantitative of ASM/TGN38 (i) and ASM/LAMP1 (j) co-localization Pearson’s correlation coefficient in the Hipp of mice. n = 3–4 per group. ^#P < 0.05, ^##P < 0.01 compared with Ctrl AAV; *P < 0.05, **P < 0.01, ***P < 0.001 compared with Ctrl AAV or CUMS + Ctrl AAV. One-way ANOVA, Holm–Sidak’s multiple comparisons test. All data are represented as mean ± SEM.

Fig. 4. Sortilin overexpression induces depressive-like behaviors by promoting ASM/ceramide levels.

a Body weight gain of the mice. n = 15–20 per group. b Total distance, immobility time, time spent in central zone in the OFT. n = 10–14 per group. c Immobility time in the TST. n = 15–20 per group. d Immobility time in the FST. n = 15–20 per group. e Latency to feed time in the NSFT. n = 15–20 per group. f Percentage of sucrose preference in the SPT. n = 15–20 per group. g, h Levels of ASM activity (g) and quantitative ELISA analysis of total ceramide (h) in the PFC and Hipp of mice. n = 4–5 per group. i, j Representative images of immunostaining for ASM, TGN38, Hoechst (i) and ASM, LAMP1, Hoechst (j) in the PFC of mice. k, l Representative images of immunostaining for ASM, TGN38, Hoechst (k) and ASM, LAMP1, Hoechst (l) in the Hipp of mice. Scale bars = 10 μm. m, n Quantitation of ASM/TGN38 (m) and ASM/LAMP1 (n) co-localization Pearson’s correlation coefficient in the PFC of mice. n = 5 per group. o, p Quantitative of ASM/TGN38 (o) and ASM/LAMP1 (p) co-localization Pearson’s correlation coefficient in the Hipp of mice. n = 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Ctrl AAV. Two-sided Student’s t-test. All data are represented as mean ± SEM.

Fig. 5. Sortilin regulates dendritic spine dynamics.

a, b Representative images of Golgi staining for dendritic spine and quantitative analysis of dendritic spine dynamics in the PFC (a) and Hipp (b) of mice. n = 4 per group. Scale bars = 2 μm. ***P < 0.001 compared with Ctrl AAV or CUMS + Ctrl AAV; ^#P < 0.05, ^###P < 0.001 compared with CUMS + Ctrl AAV. One-way ANOVA, Holm–Sidak’s multiple comparisons test. c, d Western blot analysis of SYP38 and PSD95 expression in the PFC (c) and Hipp (d) of Sort1 KD mice. n = 3–5 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Ctrl AAV or CUMS + Ctrl AAV. One-way ANOVA, Holm–Sidak’s multiple comparisons test. e, f Western blot analysis of SYP38 and PSD95 expression in the PFC (e) and Hipp (f) of Sort1 OE mice. n = 4–6 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Ctrl AAV. Two-sided Student’s t-test. All data are represented as mean ± SEM.

Fig. 6. Sortilin plays a regulatory role in RhoA/ROCK2 signaling.

a, b RhoA activity test in the PFC (a) and Hipp (b) of mice. n = 4 per group. c, d Western blot analysis of ROCK2 protein expression in the PFC (c) and Hipp (d) of mice. n = 4–5 per group. e, f Western blot analysis of phosphorylation levels of cofilin in the PFC (e) and Hipp (f) of mice. n = 3–4 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Ctrl AAV1; ^#P < 0.05, ^##P < 0.01 compared with CUMS + Ctrl AAV1; ^$P < 0.05, ^$$P < 0.01, ^$$$P < 0.001 compared with Ctrl AAV2. One-way ANOVA, Holm–Sidak’s multiple comparisons test. All data are represented as mean ± SEM.

Fig. 7. ASM inhibitor reverses depressive-like behaviors induced by sortilin overexpression.

a Body weight gain of the mice. n = 15–17 per group. b Total distance, immobility time, time spent in central zone in the OFT. n = 14–17 per group. c Immobility time in the TST. n = 15–17 per group. d Immobility time in the FST. n = 15–17 per group. e Latency to feed time in the NSFT. n = 15–17 per group. f Percentage of sucrose preference in the SPT. n = 15–17 per group. **P < 0.01, ***P < 0.001 compared with Ctrl AAV + SR33557; ^#P < 0.05, ^###P < 0.001 compared with Sort1 OE AAV + Solvent. One-way ANOVA, Holm–Sidak’s multiple comparisons test. All data are represented as mean ± SEM.

Fig. 8. Repression of ASM reverses loss of dendritic spines induced by sortilin overexpression.

a, b Representative images of Golgi staining for dendritic spines and quantitative analysis of dendritic spine dynamics in the PFC (a) and Hipp (b) of mice. n = 4–5 per group. Scale bars = 2 μm. c, d Western blot analysis of SYP38 and PSD95 expression in the PFC (c) and Hipp (d) of mice. n = 4 per group. e, f Western blot analysis of ROCK2 expression in the PFC (e) and Hipp (f) of mice. n = 4 per group. g, h Western blot analysis of phosphorylation levels of cofilin in the PFC (g) and Hipp (h) of mice. n = 4 per group. *P < 0.05, **P < 0.01, ***P < 0.001 compared with Ctrl AAV + SR33557; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with Sort1 OE AAV + Solvent. One-way ANOVA, Holm–Sidak’s multiple comparisons test. All data are represented as mean ± SEM.