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

Figures & data

Table 1. Clinical information of donated human brain tissues.

	Ctrl			PD
	1	2	3	1	2	3
	BB19-5	BB19-11	BB18-9	BB18-10	BB17-10	BB17-16
Sex	M	M	M	F	M	M
Age	61	87	55	74	69	79
Postmortem interval (h)	12.5	9	9	19	6	4
Clinical Diagnosis	Stomach-carcinoma	Cholangio-carcinoma	Hypoparynx cancer, diabetes mellitus	PD	PD	PD
Braak Stage	I/VI^a	I/VI^a	0/VI^b	I/II	I	I
NIA-AA ABC Score	NA	NA	NA	LOW	LOW	LOW

^a Only mild pTau/p62 positive neuropil threads in entorhinal cortex; ^bOnly mild pTau positive neuropil threads in entorhinal cortex and amygdala; NA: not applicable.

Table 2. Summary of human urine samples.

	Age	Sex	Duration (years)	L-DOPA dose (mg)
non-PD	72.75 (±9.16)	F = 6, M = 2 (n = 8)	NA	NA
PD	72.85 (±6.48)	F = 7, M = 6 (n = 13)	7.77 (±2.17)	130.77 (±138.13)

NA: not applicable.

Table 3. Summary of used antibodies for western blot.

Antibody name	Dilution
Recombinant Anti-LRRK2 (phospho S1292) rabbit monoclonal antibody [MJFR-19-7-8] (abcam, sb203181)	1:500
Anti-c-Myc mouse monoclonal antibody [9E10] (Santa Cruz, sc-40)	1:2000
Anti-LRRK2/Dardarin mouse monoclonal antibody [N241A/34] (Neuromab, 75-253)	1:1000
Anti-β-actin mouse monoclonal antibody [C4] (Santa Cruz. Sc-47778)	1:4000
Anti-α-tubulin mouse monoclonal antibody [B-5-1-2] (Sigma-aldrich, T5168)	1:10000
Peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (#115-035-003; Jackson Immunoresearch Laboratories Inc.)	1:5000
Peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (#111-035-144; Jackson Immunoresearch Laboratories Inc.).	1:5000

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).

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.