Adipose stem cell-derived extracellular vesicles ameliorates corticosterone-induced apoptosis in the cortical neurons via inhibition of ER stress

Abstract

Background: Corticosterone (CORT) can induce neuronal damage in various brain regions, including the cerebral cortex, the region implicated in depression. However, the underlying mechanisms of these CORT-induced effects remain poorly understood. Recently, many studies have suggested that adipose stem cell-derived extracellular vesicles (A-EVs) protect neurons in the brain.

Methods: To investigated neuroprotection effects of A-EVs in the CORT-induced cortical neurons, we cultured cortical neurons from E15 mice for 7 days, and the cultured cortical neurons were pretreated with different numbers (5 × 10⁵-10⁷ per mL) of A-EVs (A-EVs⁵, A-EVs⁶, A-EVs⁷) for 30 min followed by administration of 200 μM CORT for 24 h.

Results: Here, we show that A-EVs exert antiapoptotic effects by inhibiting endoplasmic reticulum (ER) stress in CORT-induced cortical neurons. We found that A-EVs prevented neuronal cell death induced by CORT in cultured cortical neurons. More importantly, we found that CORT exposure in cortical neurons resulted in increased levels of apoptosis-related proteins such as cleaved caspase-3. However, pretreatment with A-EVs rescued the levels of caspase-3. Intriguingly, CORT-induced apoptosis involved upstream activation of ER stress proteins such as GRP78, CHOP and ATF4. However, pretreatment with A-EVs inhibited ER stress-related protein expression.

Conclusion: Our findings reveal that A-EVs exert antiapoptotic effects via inhibition of ER stress in CORT-induced cell death.

Keywords: Adipose stem cell-derived extracellular vesicles (A-EVs); Apoptosis; Cortical neurons; Corticosterone; ER stress.

Fig. 1

Corticosterone-induced apoptosis in cortical neurons. A Cell viability was measured after treatment with 50–500 μM corticosterone for 24 h in primary cultured cortical neurons. n = 6. B Expression of total or cleaved Caspase-3 expression was detected by immunoblot in corticosterone-treated cells. C Quantitative analysis was showed on cleaved Caspase 3 per total caspase 3 protein expression. n = 4. D Fluorescence imaging of TUNEL staining after corticosterone treated-cortical neurons. Scale bar = 50 μm E Quantitative analysis was performed for TUNEL positive cells. n = 4 Statistical significance was determined by ANOVA with Bonferroni correction test. Data are shown as relative changes versus controls. *p < 0.05; **p < 0.01 and ***p < 0.001

Fig. 2

Corticosterone-induced apoptosis of cortical neurons is caused by ER stress. A ER stress or apoptosis-related proteins was measured by immunoblot in corticosterone or ISRIB-treated cells. B Quantification of CHOP protein levels shown in A. The relative expression of protein was normalized to β-actin. n = 4 C Quantification of cleaved caspase3/total caspase-3 protein levels shown in A. n = 4 D Quantification of Bcl2 protein levels shown in A. The relative expression of protein was normalized to β-actin. n = 4 E Quantification of Bax protein levels shown in A. The relative expression of protein was normalized to β-actin. n = 4 Statistical significance was determined by ANOVA with Bonferroni correction test. Data are shown as relative changes versus controls. *p < 0.05; **p < 0.01 and ***p < 0.001

Fig. 3

The characterization of A-EVs. A Particle size distribution of A-EVs measured by NTA. B–F Flow cytometry analysis of EV surface markers (CD9, CD63, CD81) and internal protein markers (GM130 and Calnexin). G TEM images of A-EVs. White bar represents 100 μm

Fig. 4

The protective effect of A-EVs against corticosterone-induced apoptosis in cortical neurons. A Representative fluorescence images of TUNEL staining in corticosterone with or without A-EVs-treated primary cultured cortical neurons. Scale bar = 50 μm B TUNEL-positive cells/nuclei by image were quantified. n = 4 C Cell viability was measured after treatment in corticosterone with or without A-EVs-treated for 24 h. n = 6 D Apoptosis-related proteins was measured by immunoblot in corticosterone or A-EVs-treated cells. E Expression of cleaved caspase3/total caspase-3 protein level was quantified. n = 4 F Quantification of Bcl2 protein levels. n = 4 G Quantification of Bax protein levels. n = 4. The expression of protein was normalized to β-actin. Statistical significance was determined by ANOVA with Bonferroni correction test. Data are shown as relative changes versus controls. *p < 0.05; **p < 0.01 and ***p < 0.001

Fig. 5

Effect of A-EVs on corticosterone-induced ER stress in cortical neurons. A A-EVs restores CORT-induced ER-stress related GPR78, ATF4, and CHOP mRNA levels. ER stress-related mRNAs, GRP78, ATF4, and CHOP, were measured by RT-PCR. B Quantification of GRP78 mRNA level shown. The fold change of GRP78 was normalized to GAPDH. n = 4. C Quantification of ATF4 mRNA level shown. The fold change of ATF4 was normalized to GAPDH. n = 4. D Quantification of CHOP mRNA was analyzed. The fold change of CHOP was normalized to GAPDH. n = 4 E EV ameliorated CORT-induced ER stress in cortical neurons. ER stress-related proteins, GPR78, ATF4 and CHOP, were measured by immunoblot. F Expression of GRP78 was quantified and normalized to β-actin. n = 4 G Expression of ATF4 level is analyzed. The expression of ATF4 was normalized to β-actin. n = 4 H Quantification of CHOP protein level was analyzed. The expression of protein was normalized to β-actin. n = 4 Statistical significance was determined by ANOVA with Bonferroni correction test. Data are shown as relative changes versus controls. *p < 0.05; **p < 0.01 and ***p < 0.001

Fig. 6

A schematic model illustrating an effect of A-EVs on CORT-induced apoptosis in the cortical neurons via inhibition of ER stress