miR-100a-5p-enriched exosomes derived from mesenchymal stem cells enhance the anti-oxidant effect in a Parkinson's disease model via regulation of Nox4/ROS/Nrf2 signaling

Abstract

Background: The pathogenesis of Parkinson's disease (PD) has not been fully elucidated, and there are no effective disease-modifying drugs for the treatment of PD. Mesenchymal stem cells have been used to treat several diseases, but are not readily available.

Methods: Here, we used phenotypically uniform trophoblast stage-derived mesenchymal stem cells (T-MSCs) from embryonic stem cells, which are capable of stable production, and their exosomes (T-MSCs-Exo) to explore the molecular mechanisms involved in dopaminergic (DA) neuron protection in PD models using experimental assays (e.g., western blotting, immunofluorescence and immunohistochemistry staining).

Results: We assessed the levels of DA neuron injury and oxidative stress in MPTP-induced PD mice and MPP⁺-induced MN9D cells after treating them with T-MSCs or T-MSCs-Exo. Furthermore, T-MSCs-Exo miRNA sequencing analysis revealed that miR-100-5p-enriched T-MSCs-Exo directly targeted the 3' UTR of NOX4, which could protect against the loss of DA neurons, maintain nigro-striatal system function, ameliorate motor deficits, and reduce oxidative stress via the Nox4-ROS-Nrf2 axis in PD models.

Conclusions: The study suggests that miR-100-5p-enriched T-MSCs-Exo may be a promising biological agent for the treatment of PD. Schematic summary of the mechanism underlying the neuroprotective actions of T-MSCs-Exo in PD. T-MSCs Exo may inhibit the expression level of the target gene NOX4 by delivering miR-100-5p, thereby reducing ROS production and alleviating oxidative stress via the Nox4-ROS-Nrf2 axis, thus improving DA neuron damage in PD.

Keywords: Dopaminergic neuron; Exosomes; Mesenchymal stem cell; NOX4; Parkinson's disease; miR-100-5p.

Fig. 1

Neuroprotective effects of T-MSCs in MPTP-induced PD mice. a Experimental protocol schematic in vivo. b–d Representative images and quantification of IHC of TH-positive neurons in the striatum and SNpc of the control, T-MSCs, MPTP, and MPTP + T-MSCs groups (n = 3 per group). Scale bars, 1000 µm for images in striatum; 500, 200, and 100 µm for the series of images in SNpc. e, f Western blotting analysis showed the TH expression levels in the SN of the control, T-MSCs, MPTP, and MPTP + T-MSCs groups (n = 3 per group). The results are shown as mean ± SD. One-way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01

Fig. 2

T-MSCs-Exo could be taken up by MN9D cells via endocytosis and enhanced cell viability in MPP⁺-induced MN9D cells. a T-MSCs-Exo were labelled using the Exosome Tracer Kit and co-cultured with MN9D cells. During this period, five time points (1, 3, 6, 12, and 24 h) were set. Cells from each time point were stained with DAPI and the uptake of T-MSCs-Exo by MN9D cells was observed by confocal microscopy. Scale bars, upper, 20 µm; lower, 5 µm. b CCK-8 was used to evaluate the MPP⁺-induced MN9D cell viability after treatment with 10, 50, 100, 200, and 300 μg/mL T-MSCs-Exo for 24 h. c, d Immunofluorescence staining and quantification of TH expression in MPP⁺-induced MN9D cells after treatment with different concentrations of T-MSCs-Exo for 24 h. e, f Western blotting analysis showed the TH expression on MPP⁺-induced MN9D cells after treatment with T-MSCs-Exo for 24 h. Each experiment was independently repeated three times, and the results are shown as mean ± SD. One-way ANOVA was used to analyze the data. **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

T-MSCs-Exo can cross the BBB to reach the SN and alleviate the degeneration of DA neurons in PD mice. a We injected PKH26-labeled T-MSCs-Exo into mice via the tail vein, and subjected them to cardiac perfusion for brain extraction at 0, 2, 6, 8, 10, 12 and 48 h. Next, we prepared frozen sections and detected the uptake of PKH26-labeled T-MSCs-Exo in the DA neurons of the midbrain at each time point using immunofluorescence. The number of mice at each time point was three. Scale bars, upper, 20 µm; lower, 5 µm. b Schematic of in vivo experimental protocol. c Open field experiment. d–f Representative images and quantification of IHC of TH-positive neurons in the striatum and SNpc of the control, T-MSCs-Exo, MPTP, and MPTP + T-MSCs-Exo groups (n = 3 per group). Scale bars, 2.5 mm for images in striatum; 625, 200, and 100 µm for the series of images in SNpc. g, h Immunofluorescence staining and quantification of TH expression in MPTP-induced PD mice after treatment with T-MSCs-Exo (n = 3 per group). Scale bars, upper, 100 µm; lower, 50 µm. i, j Western blotting analysis showed the TH expression levels in the SN of the control, T-MSCs-Exo, MPTP, and MPTP + T-MSCs-Exo groups (n = 3 per group). The results are shown as mean ± SD. One-way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01

Fig. 4

Sequencing analysis of T-MSCs-Exo miRNAs, and miR-100-5p directly targets the 3' UTR NOX4. a Size distribution of miRNA reads was between 18 and 26 nt with a peak of 23 nt. b Summary of known and predicted miRNAs. c KEGG pathway analysis. d, e qRT-PCR assay of miR-100-5p and NOX4 expression after T-MSCs-Exo treatment of control or MPTP-induced PD mice. f, g qRT-PCR assay of miR-100-5p and NOX4 expression in control or MPP⁺-induced MN9D cells after miR-100-5p inhibitor/mimic transfection. h Wild-type and mutated-type binding sites between miR-100-5p and NOX4. i Dual luciferase reporter assay of MN9D cells in the presence of indicated treatments. Each experiment was independently repeated three times. The results are shown as mean ± SD. One-way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns: no significant

Fig. 5

T-MSCs alleviate oxidative stress injury in MPP⁺-induced MN9D cells through the Keap1-Nrf2-SOD pathway. a Representative graphs of ROS generation after T-MSCs treatment of control or MPP⁺-induced MN9D cells. Scale bars, 20 µm. b, d–h Western blotting analysis of Keap1, Nrf2, HO-1, SOD-1, and SOD-2 expression levels after T-MSCs treatment of control or MPP⁺-induced MN9D cells. c Quantification of the relative fluorescence intensity of ROS levels. i–l Representative blots and quantification showed the levels of cytoplasmic Nrf2 and Nuclear Nrf2 in MN9D cells. m, n Immunofluorescence staining and quantification of Nrf2 in the control, T-MSCs, MPP⁺, and MPP⁺ + T-MSCs groups. Scale bars, upper, 10 µm; lower, 5 µm. Each experiment was independently repeated three times. The results are shown as mean ± SD. One-way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

T-MSCs resist oxidative stress injury in PD mice through Keap1-Nrf2-SOD. a, b The content of GSSG and relative GSH/GSSG ratio after T-MSCs treatment of control or MPTP-induced PD mouse model (n = 3 per group). c–h Western blotting analysis of Keap1, Nrf2, HO-1, SOD-1, and SOD-2 expression levels after T-MSCs treatment of control or MPTP-induced PD mouse model (n = 3 per group). The results are shown as mean ± SD. One-way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01

Fig. 7

T-MSCs-Exo alleviate oxidative stress injury in PD models through Keap1-Nrf2-SOD. a Representative graphs of ROS generation after T-MSCs-Exo treatment of control or MPP⁺-induced MN9D cells. Scale bars, 20 µm. b, d–h Western blotting analysis of Keap1, Nrf2, HO-1, SOD-1, and SOD-2 expression levels after T-MSCs-Exo treatment of control or MPP⁺-induced MN9D cells. c Quantification of the relative fluorescence intensity of ROS levels. i, j Representative blots and quantification showed the levels of nuclear Nrf2 in MN9D cells. k, l Immunofluorescence staining and quantification of Nrf2 in the control, T-MSCs-Exo, MPP⁺, and MPP⁺ + T-MSCs-Exo groups. m-r Western blotting analysis of Keap1, Nrf2, HO-1, SOD-1, and SOD-2 expression levels after T-MSCs-Exo treatment of control or MPTP-induced PD mouse model (n = 3 per group). Scale bars, upper, 10 µm; lower, 5 µm. Each experiment was independently repeated three times. The results are shown as mean ± SD. One-way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01

Fig. 8

miR-100-5p relieves oxidative stress injury in MPP⁺-induced MN9D cells via the Nox4-ROS-Nrf2 axis. a–c Representative blots and quantification showed the levels of Nox4 and TH in control and MPP⁺-induced MN9D cells after miR-100-5p inhibitor/mimics transfection. d–j Western blotting analysis of PI3K, Keap1, Nrf2, HO-1, SOD-1, and SOD-2 in control or MPP⁺-induced MN9D cells after miR-100-5p inhibitor/mimic transfection. Each experiment was independently repeated three times. The results are shown as mean ± SD. One-way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01, ns no significant

Fig. 9

miR-100-5p relieves oxidative stress injury in MPTP-induced PD mice via the Nox4-ROS-Nrf2 axis. a Design and timeline of the animal experimental procedure. b Representational diagram of AAV injection site in mice and the co-labeling of TH with miR-100-5p showed that miR-100-5p was mainly expressed within the SNpc DA neurons. Scale bars, 100 µm; upper right, 10 µm. c–e Representative images and quantification of IHC of TH-positive neurons in the striatum and SNpc of the control, AAV-miR-NC, AAV-miR-100-5p, MPTP, MPTP + AAV-miR-NC, and MPTP + AAV-miR-100-5p groups (n = 3 per group). Scale bars, 1.000 mm for images in striatum; 500, 200, and 100 µm for the series of images in SNpc. f–g Immunofluorescence staining and quantification of TH expression in MPTP-stimulated PD mice after treatment with AAV-miR-NC or AAV-miR-100-5p (n = 3 per group). Scale bars, upper, 200 µm; lower, 100 µm. h–j Western blotting analysis showed the Nox4 and TH expression levels in the SN of the six groups (n = 3 per group). k–q Western blotting analysis showed the expression levels of PI3K, Keap1, Nrf2, HO-1, SOD-1, and SOD-2 in the SN of the six groups (n = 3 per group). The results are shown as mean ± SD. One-way ANOVA was used to analyze the data. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001