DJ-1 and alpha-synuclein in human cerebrospinal fluid as biomarkers of Parkinson's disease

Abstract

Biomarkers are urgently needed for the diagnosis and monitoring of disease progression in Parkinson's disease. Both DJ-1 and alpha-synuclein, two proteins critically involved in Parkinson's disease pathogenesis, have been tested as disease biomarkers in several recent studies with inconsistent results. These have been largely due to variation in the protein species detected by different antibodies, limited numbers of patients in some studies, or inadequate control of several important variables. In this study, the nature of DJ-1 and alpha-synuclein in human cerebrospinal fluid was studied by a combination of western blotting, gel filtration and mass spectrometry. Sensitive and quantitative Luminex assays detecting most, if not all, species of DJ-1 and alpha-synuclein in human cerebrospinal fluid were established. Cerebrospinal fluid concentrations of DJ-1 and alpha-synuclein from 117 patients with Parkinson's disease, 132 healthy individuals and 50 patients with Alzheimer's disease were analysed using newly developed, highly sensitive Luminex technology while controlling for several major confounders. A total of 299 individuals and 389 samples were analysed. The results showed that cerebrospinal fluid DJ-1 and alpha-synuclein levels were dependent on age and influenced by the extent of blood contamination in cerebrospinal fluid. Both DJ-1 and alpha-synuclein levels were decreased in Parkinson's patients versus controls or Alzheimer's patients when blood contamination was controlled for. In the population aged > or = 65 years, when cut-off values of 40 and 0.5 ng/ml were chosen for DJ-1 and alpha-synuclein, respectively, the sensitivity and specificity for patients with Parkinson's disease versus controls were 90 and 70% for DJ-1, and 92 and 58% for alpha-synuclein. A combination of the two markers did not enhance the test performance. There was no association between DJ-1 or alpha-synuclein and the severity of Parkinson's disease. Taken together, this represents the largest scale study for DJ-1 or alpha-synuclein in human cerebrospinal fluid so far, while using newly established sensitive Luminex assays, with controls for multiple variables. We have demonstrated that total DJ-1 and alpha-synuclein in human cerebrospinal fluid are helpful diagnostic markers for Parkinson's disease, if variables such as blood contamination and age are taken into consideration.

Figure 1

The effects of blood contamination on DJ-1 or α-synuclein levels in CSF. CSF DJ-1 (A) and α-synuclein (SNCA) (B) levels were measured in individual control (CTL) and disease (Alzheimer’s disease, AD; Parkinson’s disease, PD) samples by Luminex, while the haemoglobin levels were measured using an ELISA kit. The haemoglobin variable was log transformed (log10) for the correlation study due to its positively skewed distribution. A fit line is shown for all the samples tested. A positive correlation between haemoglobin and DJ-1 (C) or α-synuclein (D) levels was also observed in whole blood serially diluted in the Luminex assay diluent (0.1% bovine serum albumin/phosphate buffered saline).

Figure 2

Rostrocaudal gradient of CSF and DJ-1 or α-synuclein levels in CSF. The early CSF fractions (<10th ml, Early F), middle fractions (10–15th ml, Middle F) and late fractions (15–20th ml, Late F) in the control group were assessed. Samples included young (mean 29.1, range 21–40, n = 15), middle age (mean 59.8, range 55–64, n = 15) and old (mean 78.8, range 75–83, n = 15) controls. No significant difference was observed in different fractions when the average DJ-1 (A) or α-synuclein (SNCA) (C) levels were compared, with or without eliminating the samples with >200 ng/ml haemoglobin levels. The levels of DJ-1 (B) or α-synuclein (D) in different CSF fractions in individual samples were also compared after cases with high haemoglobin levels were omitted. Each symbol represented one participant and the three fractions from the same participant were linked. No significant difference among fractions was observed in individual samples.

Figure 3

Age dependence of DJ-1 or α-synuclein levels in CSF. CSF DJ-1 (A, B) and α-synuclein (SNCA) (C, D) levels were measured in individual control (CTL) and disease (Alzheimer’s disease, AD; Parkinson’s disease, PD) samples by Luminex. Data shown are before (A, C) or after (B, D) elimination of cases with high haemoglobin levels. The correlation coefficient (R) and P-value of linear regression for each group were as the following: DJ-1 in CTL, R = 0.50 (P < 0.0001) and R = 0.51 (P < 0.0001), for before and after elimination of contaminated cases, respectively; DJ-1 in Parkinson’s disease, R = 0.23 (P = 0.01) and R = 0.26 (P = 0.02), for before and after elimination of contaminated cases, respectively; DJ-1 in Alzheimer’s disease, R = 0.05 (P = 0.72) and R =−0.02 (P = 0.89), for before and after elimination of contaminated cases, respectively. α-synuclein in the control, R = 0.16 (P = 0.07) and R = 0.40 (P < 0.0001), for before and after elimination of contaminated cases, respectively; α-synuclein in Parkinson’s disease, R = 0.07 (P = 0.47) and R = 0.16 (P = 0.14), for before and after elimination of contaminated cases, respectively; α-synuclein in Alzheimer’s disease, R = 0.05 (P = 0.71) and R = 0.00 (P = 0.98) for before and after elimination of contaminated cases, respectively.

Figure 4

Cross-sectional examination of CSF DJ-1 and α-synuclein by Luminex. (A, C) Quantitative Luminex analysis of CSF DJ-1 or α-synuclein (SNCA) levels in patients with Parkinson’s disease, Alzheimer’s disease and healthy controls (CTL) before and after elimination of contaminated cases (200 ng/ml haemoglobin was used as a cut-off). Only age matched samples were included. *P < 0.05 versus control group; ^#P < 0.05 versus Parkinson’s disease group. (B, D) Statistical analysis of CSF DJ-1 and α-synuclein levels in Hoehn and Yahr's stages of Parkinson’s disease. Data shown are mean ± SEM; cases studies in each group can be found in Table 1.

Figure 5

ROC curves to evaluate the CSF DJ-1 and/or α-synuclein as Parkinson’s disease biomarkers. (A, B) Combined ROC curves for the DJ-1 (dotted line) and α-synuclein (solid line) of control versus Parkinson’s disease with donor’s age <65 (A) or ≥65 years (B). (C, D) Combined ROC curves for the DJ-1 (dotted line) and α-synuclein (solid line) of Parkinson’s disease versus Alzheimer’s disease with donor’s age <65 (C) or ≥65 years (D).

Figure 6

Relationship between DJ-1 and α-synuclein in CSF. Data shown are before (A) or after (B) eliminating cases containing high haemoglobin levels (200 ng/ml was used as a cut-off). The correlation coefficient and P-value of linear regression for controls are shown in each panel. CTL = control; PD = Parkinson's disease; AD = Alzheimer's disease.