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. 2022 Apr 1;14(7):3070-3083.
doi: 10.18632/aging.203989. Epub 2022 Apr 1.

Hypoxic pretreatment of adipose-derived stem cell exosomes improved cognition by delivery of circ-Epc1 and shifting microglial M1/M2 polarization in an Alzheimer's disease mice model

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Hypoxic pretreatment of adipose-derived stem cell exosomes improved cognition by delivery of circ-Epc1 and shifting microglial M1/M2 polarization in an Alzheimer's disease mice model

Haining Liu et al. Aging (Albany NY). .

Abstract

Alzheimer's disease (AD) is the most common dementia in the world. Increasing evidence has shown that exosomes from hypoxic pretreated adipose-derived stem cells (ADSCs) could be an effective cognitive function therapeutic in AD-associated pathophysiology. However, their role and regulatory mechanism remain largely unknown. High-throughput sequencing was used to identify differentially expressed circRNAs from ADSCs or hypoxia pretreated ADSC exosomes. Luciferase reporter assays and RT-qPCR were used to investigate the relationships between circ-Epc1, miR-770-3p, and TREM2. APP/PS1 double transgenic AD model mice were then used to study therapeutic effects regarding circ-Epc1 in ADSC exosomes. BV2 cells were used to show the regulatory relationships between circ-Epc1, miR-770-3p, and TREM2 and to show how these interactions modulated phenotypic transformations and inflammatory cytokine expressions in microglia. The results showed that exosomes from hypoxia pretreated ADSCs had a good therapeutic effect on improving cognitive functions by decreasing neuronal damage in the hippocampus. High-throughput sequencing showed that circ-Epc1 played an important role in hypoxia-pretreated ADSC exosomes regarding their ability to improve cognitive functions. Luciferase reporter assays showed that TREM2 and miR-770-3p were downstream targets of circ-Epc1. Overexpressing miR-770-3p or downregulating TREM2 reversed the effects of circ-Epc1 on M2 microglia during lipopolysaccharide treatment. In vivo experiments showed that circ-Epc1-containing ADSC exosomes increased the therapeutic effect of exosomes by improving cognitive function, decreasing neuronal damage, and shifting hippocampal microglia from the M1 polarization to the M2 polarization stages. Taken together, the data show that hypoxic pretreatment of ADSC exosomes improved cognition by delivery of circ-Epc1 and by shifting microglial M1/M2 polarization in an AD mouse model.

Keywords: Alzheimer's disease; adipose derived stem cells; circ-Epc1; exosomes; microglia.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Characterization of exosomes released by adipose-derived mesenchymal stem cells (ADSCs). (A) ADSCs showed a typical cobblestone-like morphology. Scale bar: 30 μm. (BG) Immunofluorescence staining of cell surface markers. The antibodies were labeled with fluorescein isothiocyanate (FITC, green). CD29, CD90, CD44, and CD105 were positive. The von Willebrand Factor was negative. FITC- and PE-labeled mouse IgG isotype controls are shown (magnification: 200×). Scale bar: 30 μm. (H, I) Differentiation potential of ADSCs assessed by Oil Red O (H) and alkaline phosphatase staining (I). Scale bar: 50 μm. (J) Western blots of CD63 and CD9 expressions in exosomes from hypoxia-pretreated or wild-type ADSCs. (K) Transmission electron micrographs showing ADSC-exosome morphology. Scale bar: 100 nm. (L) Particle size distribution and concentration of ADSC-exosomes measured by nanoparticle tracking analysis.
Figure 2
Figure 2
Exosomes from hypoxia-pretreated adipose-derived mesenchymal stem cells have a more therapeutic effect at improving cognitive function by decreasing neuronal damage in the hippocampus. (A) The 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-labeled exosomes are red and nuclei are counterstained with 4′,6-diamidino-2-phenylindole (blue). The injected exosomes were detected in the cortex and hippocampus. (BD) ELISA assays showing expression of the inflammatory factors, TNF-α, IL-6, and IL-1β. Data represent mean ± SD (n=10). *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control; #P < 0.05 vs. exosomes derived from ADSCs (Exo). (E, F) Hippocampal neuron apoptosis was detected using the TUNEL assay. Data represent the mean ± SD (n=6). **P < 0.01, ***P < 0.001 vs. the control; #P < 0.05 vs. Exo. (G) Alzheimer’s disease mice exhibited a longer escape latency than exosome-treated animals. Data represent the mean ± SD (n=10). *P < 0.05, **P < 0.01 vs. the control; #P < 0.05 vs. Exo. (H) The number of platform crossings was increased in the exosome-treated group. Data represent the mean ± SD (n=10). *P < 0.05, ***P < 0.001 vs. the control; ###P < 0.001 vs. Exo.
Figure 3
Figure 3
Exosomes from hypoxia-pretreated adipose-derived mesenchymal stem cells had a greater therapeutic effect at improving cognitive function when delivering circ-Epc1. (A) Heat map showing the differentially expressed circRNAs. (B) Volcano plot showing the upregulated and downregulated circRNAs from exosomes derived from ADSCs (Ex)o and hypoxia-pretreated ADSC exosomes (HExo). (C) RT-qPCR detection showing the expression of 10 upregulated circRNAs between Exo and HExo. Data are the means ± SD. *P < 0.05, ***P < 0.001 vs. Exo. (D) The chromosomal location of circ-Epc1.
Figure 4
Figure 4
TREM2 and miR-770-3p are downstream targets of circ-Epc1. (A) Dual-luciferase reporter assays showed that co-transfecting wild-type (WT) and mimic miR-770-3p decreased the luciferase activity in HEK293T cells. Data are the means ± SD. **P < 0.01 vs. WT+NC. (B) Predicted binding sites of miR-770-3p in circ-Epc1. The mutant version of circ-Epc1 is shown. (C) Relative luciferase activity determined 48 h after transfecting HEK293T cells with the miR-770-3p mimic/NC or circ-Epc1 WT/Mut. Data are presented as the means ± SD. **P < 0.01. (D) Predicted binding sites of miR-770-3p within the 3′-UTR of TREM2. The mutant version of the TREM2 3′-UTR is shown. (E) Relative luciferase activity determined 48 h after transfecting HEK293T cells with the miR-770-3p mimic/NC or 3′-UTR-TREM2 WT/Mut. Data are presented as the means ± SD. **P < 0.01. (FH) RT-qPCR and western blot detection showing the expressions of circ-Epc1, miR-770-3p, and TREM2 in BV2 cells after transfection with circ-Epc1 overexpression (circ-Epc1), miR-770-3p mimic (mimic), TREM2 silencing vector (si-TREM2) individually, or in combination. Data are presented as means ± SD. ***P < 0.001 vs. NC; ###P < 0.001 vs. circ-Epc1.
Figure 5
Figure 5
Overexpressing miR-770-3p or downregulating TREM2 reversed the effect of circ-Epc1 on M2 microglial shifting during treatment with lipopolysaccharide (1 μg/mL). (AD) Immunofluorescence detection of macrophage polarization using Iba-I+, CD11b+, and CD206+staining. Data are presented as the mean ± SEM. ***P < 0.001 vs. NC; ###P < 0.001 vs. circ-Epc1. (EG) ELISA detection showing expression of the inflammatory factors, TNF-α, IL-6, and IL-1β. Data are presented the mean ± SD (n=10). ***P < 0.001 vs. NC; #P < 0.05 vs. circ-Epc1.
Figure 6
Figure 6
Circ-Epc1-modified ADSC exosomes (circ-Epc1-Exo) showed increased therapeutic effects at improving cognitive function by decreasing neuronal damage and shifting hippocampal microglia from M1 to M2. (A, B) Hippocampal neuron apoptosis was detected using the TUNEL assay. Data represent the mean ± SD (n=6). **P < 0.01, ***P < 0.001 vs. the control; #P < 0.05 vs. Exo. (C) Alzheimer’s disease mice exhibited a longer escape latency than exosome-treated animals. Data represent the mean ± SD (n=10). *P < 0.05, **P < 0.01 vs. the control; #P < 0.05 vs. hypoxia-pretreated ADSC exosomes (HExo). (D) The number of platform crossings was increased in the exosome-treated group. Data represent the mean ± SD (n=10). *P < 0.05, ***P < 0.001 vs. the control; ###P < 0.001 vs. HExo. (EH) Immunofluorescence detection of macrophage polarization using F4/80+, CD11b+, and CD206+staining. Data are presented as the mean ± SEM. ***P < 0.001 vs. control; ###P < 0.001 vs. HExo. (IK) ELISA results showing expressions of the inflammatory factors, TNF-α, IL-6, and IL-1β. Data are presented as the mean ± SEM. ***P < 0.001 vs. the control; ###P < 0.001 vs. HExo. (L, M) RT-qPCR showing the expressions of miR-770-3p and TREM2 in hippocampal tissues. Data are presented as the mean ± SEM. ***P < 0.001 vs. the control; ###P < 0.001 vs. HExo.

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