Increased stability of α-synuclein aggregates in blood plasma of patients with Parkinson's disease
10.3760/cma.j.cn115354-20211203-00786
- VernacularTitle:帕金森病患者血浆增强α-突触核蛋白聚集体稳定性的研究
- Author:
Caixia PENG
1
;
Hanjiang LUO
;
Fan YANG
;
Chengwei LIU
;
Min CHEN
Author Information
1. 桂林医学院附属医院神经科学实验室，广西神经系统疾病临床医学研究中心，广西脑与认知神经科学重点实验室，桂林 541001
- Keywords:
Parkinson's disease;
α-synuclein;
Aggregate;
Digestion;
Stability
- From:
Chinese Journal of Neuromedicine
2022;21(4):325-332
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the stability of α-synuclein (α-Syn) aggregates formed by incubation with blood plasma of patients with Parkinson's disease (PD).Methods:Peripheral blood samples were collected from 10 patients diagnosed as having PD in our hospital from June 2020 to December 2020 and 10 healthy control subjects (HC) at the same time period. The 1 mg recombinant human α-Syn was dissolved in 140 μL 0.01 mol/L phosphate buffer solution (PBS), and then, incubated with plasma from HC and PD patients and PBS at 37 ℃ for 7 d (HC group, PD group and PBS group). Preformed fiber (PFF) group was used for subsequent experiment with 10 μg PFF. After digestion with different concentrations of trypsin (concentration ratios of trypsin/α-Syn=1:80, 1:40, and 1:20) and protease K (PK, 1.0, 1.5, and 2.0 μg/mL), the differences of α-Syn levels before and after digestion were detected by Western blotting.Results:(1) Effect of trypsin on PFF digestion: PFF gradually decreased with the increase of trypsin doses; when trypsin/α-Syn ratio=1:20, α-Syn aggregates with molecular weight greater than 35 000 were almost completely digested, and its digestion was significantly different as compared with that in other concentration ratios ( P<0.05). (2) Effect of trypsin on digestion of α-Syn aggregates: at the relative molecular weight<25 000, when the concentration ratio of trypsin/α-Syn=1:20, as compared with HC and PD groups, the PBS group had significantly more obvious decrease ( P<0.05). At the relative molecular weight 35 000-40 000, the α-Syn levels in PBS, HC and PD groups were significantly decreased as compared with those before digestion at all concentration ratios; as compared with HC and PD groups, the PBS group had significantly more obvious decrease ( P<0.05). At molecular weight>40 000, α-Syn decreased significantly in PD group only when the concentration ratio of trypsin/α-Syn=1:20, and the degrees of digestion was PBS group>HC group>PD group, with significant differences among groups ( P<0.05). (3) Effect of PK on PFF digestion: when PK concentration was 1.0, 1.5 or 2.0 μg/mL, α-Syn was basically digested at relative molecular weight>35 000, and α-Syn monomer was reduced and small fragment appeared at relative molecular weight<25 000; as compared with negative controls (0 μg/mL PK), these changes in groups of PK concentration of 1.0, 1.5 or 2.0 μg/mL were significantly different ( P<0.05). (4) Effect of PK on digestion of α-Syn aggregates: at relative molecular weight<25 000, α-Syn in PBS group was digested into smaller fragments while α-Syn in HC and PD groups was not digested; significant differences was noted among groups at the same concentration ( P<0.05). At relative molecular weight of 35 000-40 000, with the increase of PK concentration, the amount of α-Syn dimer in PBS group decreased (increased digestibility), that in HC group increased, however, that in PD group did not change obviously; significant difference was noted at the same concentration among the three groups ( P<0.05). At relative molecular weight>40 000, with the increase of PK concentration, α-Syn in PBS, HC and PD groups decreased to a certain extent, and significant difference was noted among groups at the same concentration ( P<0.05). Conclusion:The stability of α-Syn aggregates formed by incubation with plasma from PD patients is higher than that formed by incubation with HC plasma and PBS.
