Salivary total α-synuclein, oligomeric α-synuclein and SNCA variants in Parkinson's disease patients

Abstract

The present study was to evaluate the diagnostic value of salivary total and oligomeric α-synuclein levels in PD. Furthermore, we sought to explore the relationship between salivary total α-synuclein and α-synuclein SNP variants levels. 201 PD patients and 67 controls were recruited, of which there also had the genetic information of two positive α-synuclein (SNCA) loci. Salivary total α-synuclein was assayed using a highly sensitive Luminex assay and oligomeric α-synuclein was quantified by the combination of Gel filtration chromatography and Western blot, respectively. From our analysis,No difference in salivary total α-synuclein levels was found between PD patients and healthy controls, it decreased with age in PD patients, and was closely associated with genotypic distribution of rs11931074 and rs894278 in PD, respectively. After controlled for age and genders, G allele of rs11931074 was correlated with lower salivary total α-synuclein levels, while G allele of rs894278 was also correlated with the higher levels. Simultaneously, the further study was shown that salivary oligomeric α-synuclein in PD patients significantly increased comparing to healthy controls. In conclusions,our study firstly demonstrated that salivary total α-synuclein levels could be manipulated by different α-synuclein SNPs and salivary oligomeric α-synuclein could be a potential diagnostic indicator of PD.

Figure 1. The characteristics of total salivary α-synuclein in PD group.

(A) Salivary α-synuclein levels were measured in individual normal controls (NC) and PD samples by Luminex. Quantitative Luminex analysis of salivary α-synuclein levels in patients with PD and NC, before and after controlling for potential contributions secondary to outliers (e.g. age, gender) by the nonparametric Mann-Whitney U test (for pair wise comparisons) and the covariance (ANCOVA) model analysis. There was no significant difference in mean salivary α-synuclein levels between cases and controls (p = 0.97), also no significant differences were found when normalized (p = 0.49). (B) Using linear regression analysis with spearman’s correlation, levels of α-synuclein in saliva tended to decrease with age in PD group (r = −0.16 p = 0.02, r = −0.21, p = 0.003 when normalized) but not in controls (p > 0.05; p > 0.05 when normalized). Error bars represent mean ± SEM.

Figure 2. Association between total salivary α-synuclein and two SNCA variants in PD.

(A) After controlling for potential contributions secondary to outliers (e.g. age, gender), α-synuclein levels in saliva were associated with genotype distribution of rs11931074. The levels in the GG group were significantly lower than those in the GT (p = 0.033, p = 0.022 when normalized), TT (p = 0.038, p = 0.027 when normalized). (B) The levels in the rs894278 GG group were higher than those in the GT (p = 0.023, p = 0.089 when normalized), TT (p = 0.052, p = 0.11 when normalized). Error bars represent mean ± SEM.

Figure 3. The diagnostic value of salivary oligomeric α-synuclein in PD.

Salivary proteins were analyzed by high-performance liquid chromatography on a Superdex75 (GE Health Care, USA) column that had been equilibrated with a solution containing 50 mM NH₄HCO₃ (pH = 7.4). Proteins were eluted from the column with the equilibration solution and the resulting fractions were subjected to immunoblotting analysis with antibodies to α-synuclein. All the gels have been run under the same experimental conditions and the representative cropped gels were shown in the figure.The positions of monomers, oligomers of α-synuclein are shown (Fig. 3A). From the analysis by Image J, the ratio of oligomer/total α-synuclein decreased significantly in H&Y stage I (p = 0.001) and increased in H&Y II-IV stages compared to normal controls (p = 0.037, p = 0.002, p = 0.000, respectively, Fig. 3B). The results also revealed significant differences at each H&Y stage (74.7%, 84.2%, 87.2%, 94.8%, respectively, p = 0.000, Fig. 3C). An analysis of covariance (ANCOVA) model was used.Error bars represent mean ± SD from 3 independent experiments.