Amelioration of pathologic α-synuclein-induced Parkinson's disease by irisin

Abstract

Physical activity provides clinical benefit in Parkinson's disease (PD). Irisin is an exercise-induced polypeptide secreted by skeletal muscle that crosses the blood-brain barrier and mediates certain effects of exercise. Here, we show that irisin prevents pathologic α-synuclein (α-syn)-induced neurodegeneration in the α-syn preformed fibril (PFF) mouse model of sporadic PD. Intravenous delivery of irisin via viral vectors following the stereotaxic intrastriatal injection of α-syn PFF cause a reduction in the formation of pathologic α-syn and prevented the loss of dopamine neurons and lowering of striatal dopamine. Irisin also substantially reduced the α-syn PFF-induced motor deficits as assessed behaviorally by the pole and grip strength test. Recombinant sustained irisin treatment of primary cortical neurons attenuated α-syn PFF toxicity by reducing the formation of phosphorylated serine 129 of α-syn and neuronal cell death. Tandem mass spectrometry and biochemical analysis revealed that irisin reduced pathologic α-syn by enhancing endolysosomal degradation of pathologic α-syn. Our findings highlight the potential for therapeutic disease modification of irisin in PD.

Keywords: Parkinson’s disease; irisin; neurodegeneration; synuclein.

Fig. 1.

Irisin protects neurons against α-syn PFF neurotoxicity. (A) Representative images of pS129-α-syn (green) in primary cortical neurons preincubated for 1 h followed by sustained treatment with indicated concentration of irisin, and further incubated with α-syn PFF (1 μg/mL) for 7 d. DAPI (blue) was used for nuclei staining. (Scale bar, 20 μm.) (B) Quantification of p-α-syn signals in (A) normalized with DAPI. Bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test (n = 3). (C) Representative immunoblots of pS129-α-syn and α-syn in the Triton X-100-soluble and insoluble fraction from primary cortical neurons preincubated for 1 h followed by sustained treatment with indicated concentration of irisin followed by incubation with α-syn PFF for 7 d. (D) Quantification of levels of pS129-α-syn and α-syn in the Triton X-100-insoluble fraction normalized to β-actin shown in (C). Bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test (n = 4). (E) Cell death assay quantified from Hoechst and propidium iodide (PI) staining in primary cortical neurons treated for 1 h followed by sustained treatment with indicated concentration of irisin and further incubated with α-syn PFF (5 μg/mL) for 14 d. Bars represent mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 4). (F) Cell death assay quantified from Hoechst and propidium iodide (PI) staining in primary cortical neurons preincubated 1 h followed by sustained treatment with irisin (50 ng/mL) and further incubated with α-syn PFF (5 μg/mL) for 14 d as well as delayed treatment (1 d, 2 d, 4 d, and 7 d) after α-syn PFF treatment. Bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test (n = 4). *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig. 2.

Irisin protects α-syn PFF-induced pathology in vivo. (A) Diagram of in vivo experiments. Two-month-old WT mice were injected with PBS or α-syn PFF (5 μg/mouse) into the striatum. Two weeks after α-syn PFF injection, the mice were injected with AAV8-GFP or AAV8-Irisin-FLAG (1E10 G.C./mouse) via the tail vein. The mice were subjected to behavioral test (pole test and grip strength test), stereology and biochemical analysis 6 mo after α-syn PFF injection. (B) Representative TH and Nissl staining of SNpc DA neurons of PBS or α-syn PFF injected mice treated with AAV-GFP or AAV-Irisin. (Scale bars, 400 μm.) (C, D) Stereological counts of (C) TH⁺ and (D) TH⁺/Nissl⁺ cells. Data are mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 5 mice per group). i = ipsilateral, c = contralateral. (E) Representative immunoblots and quantification of TH and DAT in the ipsilateral striatum of injected mice. Bars represent the mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 4). (F) DA concentrations in the striatum of PBS or α-syn PFF injected mice treated with AAV-GFP or AAV-Irisin determined by HPLC. Bars represent mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test. (n = 7 mice per group). (G) Representative immunoblots of pS129-α-syn and α-syn in the detergent-soluble and insoluble fraction from the SNpc of injected mice. (H) Quantification of pS129-α-syn and α-syn levels in the detergent-insoluble fraction normalized to β-actin. Bars represent mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 3). (I, J) Pole test (I) and grip strength (J) test were performed 6 mo after PBS or α-syn PFF injection. Data are the mean ± SEM *P < 0.05, ***P < 0.0005, two-way ANOVA followed by Tukey’s post hoc test (n = 12–13 mice per group). *P < 0.05, **P < 0.005, ***P < 0.0005.

Fig. 3.

Irisin reduces the α-syn levels. (A) Schematic diagram of proteomic analysis. (B, C) Volcano plot of protein alterations. The proteins quantified from primary cortical neurons with or without preincubation of irisin (50 ng/mL) and further incubated with α-syn PFF (1 μg/mL) for (B) one or (C) 4 d were analyzed for differentially expressed proteins in PFF- and irisin-treated cells. The cutoff used to select differentially expressed proteins was q-value < 0.05. (D, E) Relative protein levels of (D) ApoE and (E) Snca in primary cortical neurons 1 and 4 d after PBS, α-syn PFF, or α-syn PFF with irisin administration analyzed by mass spec. Bars represent mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 3). (F) Representative immunoblots of pS129-α-syn, α-syn and α-syn-biotin in the detergent- insoluble and soluble fraction from cortical neurons 1 and 4 d after treatment. (G) Quantification of α-syn-biotin and α-syn levels in the detergent-soluble fraction normalized to β-actin. Bars represent mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 3). *P < 0.05, **P < 0.005, ***P < 0.0005. ND, not determined; ns, not significant.

Fig. 4.

Irisin increases the degradation of pathologic α-syn. (A, B) Primary cortical neurons from WT embryos were pretreated with 50 ng/mL Irisin for 1 h and further incubated with biotin-conjugated α-syn PFF (1 μg/mL) for 24 h. The levels of α-syn-biotin and α-syn in the endolysosome-enriched fraction were determined by immunoblotting using anti-streptavidin and an anti-α-syn antibody, respectively. Rab7 is a marker for endosome, Lamp2 is a marker for lysosome, HSP60 is a marker for mitochondria, and α-tubulin is a marker for cytoplasm. Bars represent mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 4). (C) Irisin promotes intracellular degradation of propagated α-syn PFF. Primary cortical neurons were pretreated with 50 ng/mL Irisin for 1 h and further incubated with biotin-conjugated α-syn PFF (1 μg/mL) in the presence of 50 ng/mL irisin for 12 h followed by media replacement with 50 ng/mL irisin not containing α-syn PFF. Intracellular biotin-conjugated α-syn PFF levels were determined by immunoblotting using anti-streptavidin antibody 3, 6, and 12 h after changing to fresh medium containing 50 ng/mL irisin. Graph represents mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 3). (D, E) Propagated α-syn PFF is degraded by the lysosome. Primary cortical neurons were pretreated with 50 ng/mL Irisin for 1 h and further incubated with biotin-conjugated α-syn PFF (1 μg/mL) for 12 h, followed by the fresh medium or medium containing NH₄Cl was replaced for 3 h. The levels of α-syn-biotin and α-syn were determined by immunoblotting using anti-streptavidin and α-syn antibodies, respectively. Graph represents mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 3). (F) Representative microscopic images of pS129-α-syn (green) in primary cortical neurons treated with α-syn PFF (1 μg/mL) for 4 d. Two days after α-syn PFF treatment, irisin and NH₄Cl were incubated for 2 d. DAPI (blue) is used for nuclei staining. (Scale bar, 20 μm.) Quantification of p-α-syn signals was normalized with DAPI. Bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test (n = 3). (G) Representative immunoblots of pS129-α-syn and α-syn in the detergent-soluble and insoluble fraction from primary cortical neurons incubated with α-syn PFF for 4 d followed by posttreated with irisin and NH₄Cl for 2 d. (H) Quantification of pS129-α-syn and α-syn levels in the detergent-insoluble fraction normalized to β-actin. Bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test (n = 4). *P < 0.05, **P < 0.005, ***P < 0.0005. ND, not determined; ns, not significant.