Exercise Ameliorates Dopaminergic Neurodegeneration in Parkinson's Disease Mice by Suppressing Microglia-Regulated Neuroinflammation Through Irisin/AMPK/Sirt1 Pathway

Abstract

Although exercise is known to exert anti-inflammatory effects in neurodegenerative diseases, its specific impact and underlying mechanisms in Parkinson's disease (PD) remain poorly understood. This study explores the effects of exercise on microglia-mediated neuroinflammation and apoptosis in a PD model, focusing on the role of irisin signaling in mediating these effects. Using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model, we found that a 10-week treadmill exercise regimen significantly enhanced motor function, reduced dopaminergic neuron loss, attenuated neuronal apoptosis, and alleviated neuroinflammation. Exercise also shifted microglia from a pro-inflammatory to an anti-inflammatory phenotype. Notably, levels of irisin, phosphorylated AMP-activated protein kinase (p-AMPK), and sirtuin 1 (Sirt1), which were decreased in the PD brain, were significantly increased following exercise. These beneficial effects were abolished by blocking the irisin receptor with cyclic arginine-glycine-aspartic acid-tyrosine-lysine (cycloRGDyk). Our results indicate that exercise promotes neuroprotection in PD by modulating microglial activation and the AMPK/Sirt1 pathway through irisin signaling, offering new insights into exercise-based therapeutic approaches for PD.

Keywords: Parkinson’s Disease; apoptosis; exercise; irisin; microglia; neuroinflammation.

Figure 1

(A) Schematic overview of animal experimental procedures. (B,C) Representative images of TH expression in the substantia nigra (Scale bars, 200 μm) and striatum (Scale bars, 500 μm). (D) The amount of time the mice stayed on the pole in the rotarod test after MPTP injection. (E,F) Quantitative analysis of TH staining in the substantia nigra and striatum. (G) The amount of time the mice stayed on the pole in the rotarod test after all interventions. (H,I) Representative images of TH expression in the substantia nigra (Scale bars, 200 μm) and striatum (Scale bars, 500 μm), and relative quantitative analysis. ** and *** represent p < 0.01 and p < 0.001, respectively, when comparing with the CON group; ^†, ^††, and ^††† indicate p < 0.05, p < 0.01, and p < 0.001, respectively, in comparison with the PD group. CON, control; PD, Parkinson’s disease; PE, PD plus exercise.

Figure 2

(A) Representative images and enlargements of fluorescent double staining of TH (red), caspase-3 (green), and DAPI (blue) (scale bars, 200 μm). (B) Quantitative analysis of caspase-3 staining. (C) Number of co-localized TH⁺ caspase-3⁺ cells (per field). (D) Western blot bands of Bax and Bcl-2 protein in mice substantia nigra. (E,F) Quantitative analysis of Bax and Bcl-2 proteins. All immunofluorescence images were processed with uniform brightness/contrast adjustments using ImageJ software without altering the original data interpretation. *** represent p < 0.001 when comparing with the CON group; ^†† and ^††† indicate p < 0.01 and p < 0.001, respectively, in comparison with the PD group. CON, control; PD, Parkinson’s disease; PE, PD plus exercise.

Figure 3

(A,B) Representative images of Iba-1 (red), pro-inflammatory cytokine IL-1β and TNF-α (green), and DAPI (blue) immunofluorescence staining (scale bars, 100 μm). (C) Quantitative analysis of Iba-1 staining. (D) Number of co-localized Iba-1⁺ IL-1β ⁺ cells (mm²). (E) Number of co-localized Iba-1⁺ TNF-α⁺ cells (mm²). (F–I) Western blot bands of Iba-1, IL-1β, and TNF-α protein in mice substantia nigra and quantitative analysis. All immunofluorescence images were processed with uniform brightness/contrast adjustments using ImageJ software without altering the original data interpretation. ** and *** represent p < 0.01 and p < 0.001, respectively, when comparing with the CON group; ^†, ^††, and ^††† indicate p < 0.05, p < 0.01, and p < 0.001, respectively, in comparison with the PD group. CON, control; PD, Parkinson’s disease; PE, PD plus exercise.

Figure 4

(A) Images of co-staining of Iba-1 (green), iNOS (red), and DAPI (blue) (scale bars, 100 μm), and enlargements (scale bars, 50 μm). The white line in the enlarged microphotographs denotes a 260-pixel cross-section for quantitative analysis of fluorescence intensity (green for microglia, red for inflammatory factors), the arrow indicates the region of interest for the zoomed-in view. (B) Fluorescence density analysis of Iba1⁺iNOS⁺ co-localized cells images. (C) The percentage of co-localized areas of Iba1⁺iNOS⁺ cells. (D) Images of co-staining of Iba-1 (green), CD206 (red), and DAPI (blue) (scale bars, 100 μm; enlarged images, 50 μm). (E) Fluorescence density analysis of Iba1⁺CD206⁺ co-localized cell images. (F) The percentage of co-localized areas of Iba1⁺CD206⁺ cells. (G–I) mRNA levels, including iNOS, CD16, and CD11b. (J–L) mRNA levels, including CD206, YM-1, and Arg-1. All immunofluorescence images were processed with uniform brightness/contrast adjustments using ImageJ software without altering the original data interpretation. *** represent p < 0.001 when comparing with the CON group; ^†† and ^††† indicate p < 0.01 and p < 0.001, respectively, in comparison with the PD group. CON, control; PD, Parkinson’s disease; PE, PD plus exercise.

Figure 5

(A) Western blot image of irisin, AMPK, p-AMPK, and Sirt1 protein in mice substantia nigra. (B) Quantitative analysis of irisin protein. (C) Serum levels of irisin. (D) Quantitative analysis of p-AMPK/AMPK ratio. (E) Quantitative analysis of Sirt1 protein. *** represent p < 0.001 when comparing with the CON group; ^†† and ^††† indicate p < 0.01 and p < 0.001, respectively, in comparison with the PD group; ^§, ^§§, and ^§§§ denote p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the EX group. CON, control; PD, Parkinson’s disease; PE, PD plus exercise; PERG, PD plus exercise and cycloRGDyk.

Figure 6

(A) Representative images and enlargements of fluorescent double staining of TH (red) and caspase-3 (green) (scale bars, 200 μm). (B) Quantitative analysis of caspase-3 staining and number of co-localized TH⁺ caspase-3⁺ cells in the substantia nigra (per field). (C,D) Western blot bands of Bax and Bcl-2 protein and quantitative analysis. (E,F) Western blot bands of Iba-1, IL-1β, and TNF-α protein and quantitative analysis. (G–I) Co-staining of Iba-1 (green), iNOS/CD206 (red), and DAPI (blue) (scale bars, 100 μm), and enlarged images (scale bars, 50 μm), as well as relative quantitative analysis. The white line in the enlarged microphotographs denotes a 260-pixel cross-section for quantitative analysis of fluorescence intensity (green for microglia, red for inflammatory factors), the arrow indicates the region of interest for the zoomed-in view. (J) mRNA levels, including iNOS, CD16, CD11b, CD206, YM-1, and Arg-1. All immunofluorescence images were processed with uniform brightness/contrast adjustments using ImageJ software without altering the original data interpretation. ^§, ^§§, and ^§§§ denote p < 0.05, p < 0.01, and p < 0.001, respectively, compared with the EX group. CON, control; PD, Parkinson’s disease; PE, PD plus exercise; PERG, PD plus exercise and cycloRGDyk.