Hippocampal exosomes from stroke aggravate post-stroke depression by regulating the expression of proBDNF and p75NTR and altering spine density

Abstract

Post-stroke depression (PSD) affects millions of patients who suffer cerebral stroke. However, the molecular mechanisms and pathophysiology are poorly understood. Previous studies have shown that exosomes have been proven to be involved in neuropsychiatric disorders such as stroke and post-stroke depression in neurotransmitter release, neuronal remodeling, and neuron angiogenesis. Here we extracted and purified hippocampal exosomes from stroke mouse model to investigate mechanisms of hippocampal exocytosis in PSD assessed by using behavioral tests and biochemical methods. Aiming at the effect of hippocampal exosomes from stroke on the development of PSD, behavioral test was compared including sugar water preference experiment, open fields, forced swimming test, to explore it in depth. Further, the expression of depression-related protein (proBDNF and p75NTR) and synapse-associated proteins (Synaptotagmin and PSD95) was evaluated by Western blotting, RT-qPCR or immunofluorescence staining. Density of dendritic protrusions of neurons was assessed by Golgi staining to measure changes in spine density after the treatment of hippocampal exosomes from stroke. Our results revealed that injection of exosomes from stroke models significantly aggravated depressive-like behaviors, increase of depression-related protein (proBDNF and p75NTR) expression and deficiency of synapse-associated proteins (Synaptotagmin and PSD95) expression, and the decreased number of spin density. Our findings together suggest that hippocampal exosomes from stroke cause exacerbation of depressive-like behavior in mice, possibly resulting from the regulation of neurogenesis by its depression-associated proteins (proBDNF and p75NTR). Therefore, hippocampal exosomes from stroke are promising targets for the diagnosis and treatment of PSD.

Keywords: Hippocampal exosomes; PSD95; Post-stroke depression; Synaptotagmin; p75NTR; proBDNF.

Fig. 1

Effects of PSD on the PSD-related and synapse-associated proteins expression in PSD. (A-D) Open field test. Mice were exposed to an open field environment for 5 min. (A) Behavioral trajectories. The locomotion of different treatments was determined using the distances travelled (B), velocity (C), and activity (D) (n = 6 per group). Results are presented as mean ± SD or percentage (%) and analyzed with one-way ANOVA analysis. (E, F) Image of PSD-related (proBDNF and p75NTR) and synapse-associated proteins (PSD95 and Synaptotagmin) expression in PSD in the hippocampus. β-actin was used as an internal reference. (G) the expression level of proBDNF protein, (H) the expression level of p75NTR protein, (I) the expression level of PSD95 protein, and (J) the expression level of Synaptotagmin protein. **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with the control group. (K) Example images of neuronal dendritic branch segments (n = 4). Scale bars show 50 μm. Results are presented as mean ± SD and analyzed with one-way ANOVA analysis. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2

The Morphological changes for disease model mouse in subgroups. Mouse brain tissue sections were made into slides for immunofluorescence staining from CA1 region of the hippocampus as described in methods. (A-D) Nuclei were marked in blue from a DAPI staining. The immunofluorescent staining of proBDNF (red immunofluorescence), p75NTR (red immunofluorescence), and merged immunofluorescence in four subgroups. The number of proBDNF or p75NTR positive cells were counted (n = 3). Results are presented as mean ± SD and analyzed with one-way ANOVA analysis and t test. Scale bars show 100 μm. “*” on the top of each bar represents the P value compared to the control group. Error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3

Typical characteristics of hippocampal exosomes from stroke. (A) Morphology of exosomes was observed by transmission electron microscopy (scale: 100 nm). (B) The markers of exosomes (CD9, CD63, and Alix) were detected by Western blotting.

Fig. 4

Effects of hippocampal exosomes on the mRNA and protein levels of proBDNF, receptor p75NTR, and synapse-associated protein (Synaptotagmin and PSD95). 30 µg exosomes from PSD and stroke mouse models were added into per group vein prior to PSD. (A) Cell viability (%) were detected using CCK-8; (B, and C) the mRNA level of p75NTR and proBDNF were detected using RT-qPCR; (D) the concentration of proBDNF (pg/mL) were detected using ELISA; (E-J) the expression levels of proBDNF, p75NTR, Synaptotagmin and PSD95 were detected using Western blotting. OGD-Exo represents “hippocampal exosomes from stroke mice”. Results are presented as mean ± SD and analyzed with one-way ANOVA analysis and t test. “*” on the top of each bar represents the P value compared to the control group. Error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 5

AnnexinV-FITC flow cytometry to detect the rate of apoptosis in subgroups. The rate of apoptosis is marked in the right upper quadrant. Results are presented as mean ± SD and analyzed with one-way ANOVA analysis and t test.

Fig. 6

The morphological changes for primary neurons in subgroups. Cultured neurons were made into slides for immunofluorescence staining as described in methods. (A-D) Nuclei were marked in blue from a DAPI staining. The immunofluorescent staining of Synaptotagmin (red immunofluorescence), PSD95 (red immunofluorescence), and merged immunofluorescence in four subgroups. The number of Synaptotagmin or PSD95 positive cells were counted (n = 3). Results are presented as mean ± SD and analyzed with one-way ANOVA analysis and t test. Scale bars show 100 μm. “*” on the top of each bar represents the P value compared to the control group. Error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7

Effects of PSD-Exo on the depressive behavior, the mRNAs and proteins expression. (A-B) forced swimming test (FST): The locomotion of different treatments was determined using the swimming time and immobility time (n = 6). Results are presented as mean ± SD and analyzed with one-way ANOVA analysis. (C) sucrose preference test (SPT). (D-E) The mRNA expressions of proBDNF, p75NTR were tested with RT-qPCR as described in methods, n = 6 per group. (F-K) PSD-related (proBDNF and p75NTR) and synapse-associated proteins (Synaptotagmin and PSD95) expression in PSD. (L) Example images of neuronal dendritic branch segments (n = 4). Stroke-Exo represents “hippocampal exosomes from stroke mice”. Scale bars show 50 μm. Results are presented as mean ± SD and analyzed with one-way ANOVA analysis and t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8

Effects of Stroke-Exo on the morphological changes for disease model mouse in PSD. Mouse brain tissue sections were made into slides for immunofluorescence staining from CA1 region of the hippocampus as described in methods. (A-D) Nuclei were marked in blue from a DAPI staining. The number of Synaptotagmin or PSD95 positive cells were counted (n = 3). Results are presented as mean ± SD and analyzed with one-way ANOVA analysis and t test. Scale bars show 100 μm. “*” on the top of each bar represents the P value compared to the control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.