Analysis of α-synuclein levels related to LRRK2 kinase activity: from substantia nigra to urine of patients with Parkinson's disease

Abstract

Research on Parkinson's disease (PD) has been focused on the development of PD diagnostic tools as much as the development of PD therapeutics. Several genetic culprits of PD, including DJ-1, Leucine-rich repeat kinase 2 (LRRK2), and α-synuclein (α-syn), have been investigated as markers of PD in human biofluids. Unfortunately, the approaches to develop PD diagnostic tools are impractical, and there is a considerable demand for an appropriate marker of PD. The measurement of α-syn in biofluids has recently been made more accurate by examining monomers and aggregates separately using enzyme-linked immunosorbent assay (ELISA). Previously, we reported on the development of two types of sandwich ELISA for total α-syn and MJFR-14-6-4-2 antibody-specific α-syn fibrillar oligomers. The pathogenic LRRK2 G2019S mutation is related to increased α-syn secretion in the extracellular space. We tested our established ELISA using differentiated SH-SH5Y cells transfected with LRRK2 G2019S. The secretory levels of fibrillar oligomeric α-syn divided by total α-syn were significantly increased in LRRK2 G2019S-expressing cells. Additionally, substantia nigra lysates or concentrated urine from PD patients and non-PD subjects were analyzed. We observed ambiguous changes in the levels of total or fibrillar oligomeric α-syn and their ratio between PD and non-PD. Despite the insignificant increase in the relative levels of fibrillar oligomeric α-syn to total α-syn in PD, the duration of disease progression after diagnosis significantly corresponded to the relative levels of fibrillar oligomeric α-syn to total α-syn in the urine. These results might provide greater understanding for the next stage of development of α-syn ELISAs.

Keywords: ELISA; Parkinson’s disease; leucine-rich repeat kinase 2; α-synuclein.

Figure 1.

Increases in fibrillar α-syn oligomer by the ectopic expression of G2019S LRRK2. (A) Expression of myc-tagged G2019S LRRK2 (myc-tag) and its autophosphorylation on the S1292 site (pS1292-LRRK2) analyzed with western blot. The measured intensity of protein bands normalized with β-actin levels and presented. Two-way ANOVA with Tukey post hoc test applied (n = 5). The lysates of cells transfected with vector or G2019S analyzed using ELISAs for Total-αS (B) and Fila-αS (C), and Fila-αS levels normalized with Total-αS are presented as the ratio of Fila-αS/Total-αS (D). Concentrates of culture media subjected to ELISAs of Total-αS (E) and Fila-αS (F), and the ratio of Fila-αS levels divided by Total-αS (G) are estimated. Student’s t-test was used for statistical analysis (n = 5).

Figure 2.

Analyses of α-syn along with LRRK2 in human brain SN tissues from PD patients and non-PD subjects. The lysates of SN tissues analyzed using the ELISA of Total-αS (A) and Fila-αS (B), and the ratio of Fila-αS/Total-αS (C) is estimated. Student’s t-test used for statistical analysis (n = 3). (D) For western blot, 20 µl of ELISA samples used, and the levels of total LRRK2 and pS1292-LRRK2 shown. (E) The levels of pS1292-LRRK2 or total LRRK2 of PD patients compared with the Total-αS (square), Fila-αS (opened circle), and the ratio of Fila-αS/Total-αS (reversed triangle). X-axis represents the densitometry levels of PD patients’ pS1292-LRRK2 and total LRRK2 protein bands. The correlation curve was interpolated using a linear standard curve and presented without the 95% confidence bands of the best-fit line.

Figure 3.

Quantification of urinary total α-syn and fibrillar oligomer α-syn. Urine samples from non-PD and PD patients was subjected to Total-αS (A) or Fila-αS (C), and the ratio of Fila-αS/Total- αS was estimated (E). The ROC curves for Total-αS (AUC = 0.75) (B), Fila-αS (AUC = 0.509) (D), or the ratio of Fila-αS/Total-αS (AUC = 0.74) (F) was computed. Dotted line: random classifier.

Figure 4.

Correlations of urinary α-syn with various parameters of PD. The levels of Total-αS in the urine compared with the on-set duration (A), age (B), and L-DOPA dosage (C). The levels of Fila-αS in the urine compared with the on-set duration (D), age (E), and L-DOPA (F). The ratio of Fila-αS/Total-αS estimated, and its correlation with the on-set duration (G), age (H), and the administration dose of L-DOPA determined (I). All XY correlations was computed by Pearson correlation coefficient (PD = 13).