α-synuclein promotes progression of Parkinson's disease by upregulating autophagy signaling pathway to activate NLRP3 inflammasome

Abstract

Mechanism by which α-synuclein affects the progression of Parkinson's disease through Pyrin Domain Containing Protein 3 (NLRP3) was explored. Peripheral blood plasma of 40 Parkinson's disease patients and 40 normal healthy people attending the department of neurology of the Third Affiliated Hospital of Qiqihar Medical University were collected from March 2018 to January 2019. The expression levels of oligomers, phosphorylated α-synuclein, interleukin-1β (IL-1β), interleukin-6 (IL-6) and transforming growth factor-α (TGF-α) in plasma were detected by ELISA. Astrocytes in mouse brain tissues were extracted by primary culture method, the cells were divided into drug group and the drug + inhibitor group. After adding 0, 5, 10 and 20 µg oligomerized α-synuclein or 5 mM autophagy inhibitor 3-Methyladenine (3-MA), the expression level of NLRP3, caspase-1, IL-1β and Atg5 proteins in the cells was detected. The expression level of IL-1β in peripheral blood of PD patients was significantly increased (0.604±0.136 µmol/l vs. 1.876±0.327 µmol/l, P=0.002), while there was no significant difference between IL-6 and TGF-α. Both oligomers (0.171±0.045 µmol/l vs. 0.676±0.084 µmol/l, P<0.0001) and phosphorylated α-synuclein (0.128±0.041 µmol/l vs. 0.849±0.108 µmol/l, P<0.0001) in peripheral blood of PD patients were significantly elevated. The expression levels of NLRP3, caspase-1 and IL-1β in mouse astrocytes all increased with the increase of the concentration of oligomerized α-synuclein, and Atg5 protein expression also increased gradually with the concentration, and reached the highest level when the concentration was 10 µg/ml. The expression levels of NLRP3, caspase-1 and IL-1β were inhibited after the addition of autophagy inhibitor 3-MA. α-synuclein mediates the activation of NLRP3 inflammasome in PD patients by upregulating Atg5 protein expression.

Keywords: NLRP3 inflammasome; Parkinson's disease; autophagy; α-synuclein.

Figure 1.

Plasma levels of inflammatory factors IL-1β, IL-6 and TGF-α in PD patients. (A) IL-1β expression level in peripheral blood; (B) IL-6 expression level in peripheral blood; (C) TGF-α expression level in peripheral blood. **P<0.01. IL-1β, interleukin-1β; IL-6, interleukin-6; TGF-α, transforming growth factor-α; PD, Parkinson's disease.

Figure 2.

Plasma oligomerized and phosphorylated α-synuclein expression levels in PD patients. (A) Expression level of oligomerized α-synuclein in peripheral blood; (B) Expression level of phosphorylated α-synuclein in peripheral blood. ***P<0.001. O-α-synuclein, oligomerized α-synuclein; Ps-α-synuclein, phosphorylated α-synuclein; PD, Parkinson's disease.

Figure 3.

NLRP3 and related molecules in astrocytes at different drug concentrations. (A) Western blot of expression and quantification of (B) NLRP3, (C) caspase-1 and (D) IL-1β protein in mouse astrocytes after adding 0, 5, 10 and 20 µg/ml oligomerized α-synuclein. IL-1β, interleukin-1β; NLRP3, Pyrin Domain Containing Protein 3. *P<0.05; ***P<0.001.

Figure 4.

Expression levels of Atg5 and NLRP3 related proteins in mouse astrocytes at different drug concentrations. (A) Western blot of expression of Atg5 protein in mouse astrocytes after adding 0, 5, 10, and 20 µg/ml oligomerized α-synuclein. (B) Expression and statistical representation of NLRP3, caspase-1 and IL-1β in cells after administration of 10 µg/ml oligomerized α-synuclein and/or 3-MA in mouse astrocytes. (C and D) The western blot of expression levels and quantification of NLRP3, caspase-1 and IL-1β expression were inhibited by 3-MA. NLRP3, Pyrin Domain Containing Protein 3, 3-MA, 3-methyladenine; IL-1β, interleukin-1β. *P<0.05; **P<0.01; ***P<0.001.