DJ-1 isoforms in whole blood as potential biomarkers of Parkinson disease

Abstract

DJ-1 is a multifunctional protein that plays an important role in oxidative stress, cell death, and synucleinopathies, including Parkinson disease. Previous studies have demonstrated that total DJ-1 levels decrease in the cerebrospinal fluid, but do not change significantly in human plasma from patients with Parkinson disease when compared with controls. In this study, we measured total DJ-1 and its isoforms in whole blood of patients with Parkinson disease at various stages, Alzheimer disease, and healthy controls to identify potential peripheral biomarkers of PD. In an initial discovery study of 119 subjects, 7 DJ-1 isoforms were reliably detected, and blood levels of those with 4-hydroxy-2-nonenal modifications were discovered to be altered in late-stage Parkinson disease. This result was further confirmed in a validation study of another 114 participants, suggesting that, unlike total DJ-1 levels, post-translationally modified isoforms of DJ-1 from whole blood are candidate biomarkers of late-stage Parkinson disease.

Figure 1. Total DJ-1 levels in whole blood were not significantly different among patients with PD at various stages, AD, and controls.

Pooled whole blood samples from controls, patients with Alzheimer disease (AD) and patients with Parkinson disease (PD) at various stages (UPDRS motor score <15, 15–30, and >30) were analyzed by 1-DE western blotting. The samples were pooled according to Table 1 (five comparison groups with each pooled into three sub-groups). (A) Coomassie R-350 staining of the membrane indicated equal loading among the five samples. This is a representative result from one sub-group of three independent experiments. (B) Quantitative western blotting against DJ-1 of the 5 groups indicated total DJ-1 does not significantly change with disease status. The adjusted DJ-1 band volumes were normalized to the control values in each independent experiment/gel. A representative gel is shown in (C).

Figure 2. DJ-1 isoforms in whole blood were confirmed by MS/MS and western blotting.

(A) 2-DE western blotting against DJ-1 identified 7 spots as DJ-1 isoforms (Inset). LTQ-Obitrap MS/MS confirmed the 7 spots to be DJ-1. Shown is a MS2 spectrum of MH+ 1675.8035 indicating a DJ-1 peptide with the sequence GAEEMETVIPVDVMR. (B) MS2 spectrum indicating MH+ 1259.8259 is a DJ-1 peptide with the sequence DVVICPDASLEDAKK.

Figure 3. 4-HNE and phosphorylation modifications of DJ-1 in whole blood were identified by MS/MS.

(A) Western blotting confirmed HNE modification of DJ-1 isoforms. The right-hand inset shows HNE western blotting before stripping of the DJ-1 membrane. Left, LTQ-Obitrap MS/MS spectrum indicated MH+ 1212.7881 is a DJ-1 HNE-modified peptide in spots 4 and 6 at Lys-32. (B) MS2 spectrum indicated MH+ 687.8409 is a DJ-1 HNE-modified peptide in spot 6 at Lys-62. (C) MS2 spectrum indicated MH+ 1771.7635 is a DJ-1 phosphorylation-modified peptide in isoforms 4, 5 and 6 at Thr-19.

Figure 4. The expression of DJ-1 isoforms was significantly different among control and disease whole blood samples.

(A) 2-DE western blotting of DJ-1 isoforms in different pooled whole blood samples (Table 1); this is a representative result from one of the three sub-groups of three independent experiments. (B) Quantification of 2-DE western blotting against DJ-1; the adjusted DJ-1 isoform volumes over total DJ-1 levels were normalized to the control values in each independent experiment. Means ± SEM are shown. (C) Quantification of DJ-1 isoform 2 (DJ-1 isoform 2/total DJ-1) among control and disease whole blood samples; (D) Quantification of DJ-1 isoform 6 (DJ-1 isoform 6/total DJ-1) among control and disease whole blood samples. Asterisks denote data points representing an experimental group significantly different statistically (*p<0.05, **p<0.01, ***p<0.001) from the control group.

Figure 5. The expression of HNE-altered DJ-1 isoforms was significantly different in whole blood samples obtained from AD patients, PD patients at various stages, and controls in discovery and validation cohorts.

(A) 2-DE western blotting of HNE-modified DJ-1 isoforms in pooled whole blood samples. Left, blotting using an anti-HNE antibody; right, the same membrane was stripped and re-probed with an anti-DJ-1 antibody. (B)–(E) Quantification of 2-DE western blotting of DJ-1 isoforms 4 and 6 in discovery (solid bars) and validation set (open bars): HNE-modified isoform 4 as a relative fraction (mean ± SEM) of total isoform 4 (HNE-isoform 4/DJ-1 isoform 4 total) (B) and of total HNE-modified DJ-1 (HNE-isoform 4/[HNE-isoform 4 + 6]) (C), HNE-modified isoform 6 as a relative fraction (mean ± SEM) of total isoform 6 (D) and of total HNE-modified DJ-1 (E); the values in each independent experiment were normalized to the values of the control group. (* indicates p<0.05; ** indicates p<0.01).