Oxidized DJ-1 Levels in Urine Samples as a Putative Biomarker for Parkinson's Disease

Parkinson's disease (PD) is the second most common neurodegenerative disease. Oxidative stress is the most critical risk factor for neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). Numerous reports have demonstrated that oxidative stress aggravates cytotoxicity in dopaminergic neurons and accelerates the formation of protein inclusions. In addition, oxidative stress, such as 4-hydroxynonenal (HNE), oxidized protein, and dopamine quinone, are related to PD progression. DJ-1 is a PD-causative gene, and it plays a pivotal role as a sensor and eliminator of oxidative stress. Several studies have shown that oxidized DJ-1 (OxiDJ-1) formation is induced by oxidative stress. Hence, previous studies suggest that oxidized DJ-1 could be a biomarker for PD. We previously reported higher DJ-1 levels in Korean male PD patient urine exosomes than male non-PD controls. We speculate that OxiDJ-1 levels in PD patient urine might be higher than that in non-PD controls. In this study, we established an ELISA for OxiDJ-1 using recombinant DJ-1 treated with H2O2. Using Western blot assay and ELISA, we confirmed an increase of OxiDJ-1 from HEK293T cells treated with H2O2. Using our ELISA, we observed significantly higher, 2-fold, OxiDJ-1 levels in the urine of Korean PD patients than in non-PD controls.


Introduction
Parkinson's disease is the second most common neurodegenerative disease, and its primary symptoms include tremor, bradykinesia, and rigidity. e progression of PD is associated with an increase in reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide, hydroxyl radicals, singlet oxygen, and lipid peroxyl radicals [1]. Previous studies have revealed that 4-hydroxynonenal (HNE), a by-product of lipid peroxidation, is one of the most bioactive aldehydes, and oxidative stress initiates its production [2]. It has been implicated that oxidative modification of proteins is accelerated by increase of ROS [3,4]. e synthesis of dopamine quinone (DAQ) is also mediated by the oxidation of dopamine, thereby initiating mitochondrial damage and generation of DAQ-conjugated cytosolic adducts [5,6]. ese features are closely related to PD pathology.
DJ-1 is known as a PD-causative gene, and its function is associated with oxidative stress in PD [7]. A number of reports have demonstrated that DJ-1 senses ROS in the cytosol and conjugates ROS oxygen groups on its cysteine-106 residue [8,9]. Loss of ROS scavenger function in DJ-1 showed enhanced cytotoxicity and increase of α-synuclein (α-syn) aggregates, which are known as a culprit of PD-pathogenesis [10][11][12]. Interestingly, a previous report proposed that oxidized DJ-1 (OxiDJ-1) would be an efficient biomarker for PD diagnosis [13]. Levels of DJ-1 in biofluids, such as cerebrospinal fluid (CSF), whole blood, plasma, serum, saliva, and urine, were investigated as a biomarker for PD (Table 1). ere were higher levels of OxiDJ-1 in the erythrocytes of PD patients than in those of healthy subjects or medicated PD patients [13]. Intriguingly, MPTP, a drug that induces PD symptoms, increased OxiDJ-1 levels in mouse erythrocytes [14]. ese evidences suggest that screening for oxidized DJ-1 levels in urine could be a convenient and efficient tool for diagnosing PD. As such, we established an enzyme-linked immunosorbent assay (ELISA) for examining levels of OxiDJ-1 in patient urine. Using this ELISA, we found significantly higher levels of OxiDJ-1 in PD patient urine compared to controls. Center. e PD assessments, the unified Parkinson's disease rating (UPDR), and Hoehn and Yahr (HY) scales, were not used in this study. Instead, all PD patients were diagnosed by a certified neurologist, based on the UK Brain Bank criteria. Before starting our analysis, we excluded urine samples with proteinuria, hematuria, or a pH lower than 5.5. Two milliliters of patient urine samples were thawed slowly overnight at 4°C and then vortexed. Samples were treated with 1% Triton X-100 and 1X Protease Inhibitor Cocktail Set III (Gendepot, Barker, TX, USA) and centrifuged at 18,000× g for 30 min at 4°C. After centrifugation, the supernatant was concentrated by centrifugation of 10,000× g for 30 min at 4°C using a NANOSEP 3 kDa OMEGA filter (Pall Life sciences, Ann Arbor, MI, USA) prewashed with DBPS until the samples were concentrated 5-fold.

Sandwich
Enzyme-Linked Immunosorbent Assay (ELISA). Recombinant His DJ-1 (His-DJ-1) was purchased RBC ELISA OxiDJ-1 levels in unmedicated PD were higher than medicated PD or healthy control. [13] CSF Luminex assay DJ-1 levels were higher in PD than in control or AD.
[18] Plasma Luminex assay DJ-1 was not a suitable biomarker of PD.
[20] CSF Luminex assay DJ-1 levels in PD were lower than in controls.
[20] Whole blood 2D-PAGE, WB DJ-1 levels were changed in the late stage of PD. [21] CSF ELISA DJ-1 levels did not change among Parkinsonian syndromes. [22] CSF Luminex assay ere was no correlation between DJ-1 and striatal dopaminergic function. [23] Urine WB DJ-1 levels in PD males were significantly higher than those in controls. [24] Saliva WB DJ-1 was increased in PD and correlated with UPDRS score. [25] Urine ELISA OxiDJ-1 levels were higher in PD. is study a Western blot analysis; b enzyme-linked immunosorbent assay.   from Sino biological (Beijing, China). To generate OxiDJ-1, His-DJ-1 was incubated with 5 mM H 2 O 2 or equivalent vehicle (sterile distilled water (DW)) for 1.5 h at 37°C. e generated OxiDJ-1 was confirmed by Western blot assay and standards of His-DJ-1 with H 2 O 2 which were freshly made just before ELISA. For ELISA to detect OxiDJ-1, we coated each well of a MaxiSorp flat-bottom 96 well plate (44-2404-21, Nunc, Roskilde, Denmark) with OxiDJ-1 antibody (0.5 μg/ml) and 50 mM carbonate buffer overnight at 4°C. Each well was then washed 3 times with 200 μl of 1X PBST and blocked with 150 μl of SuperBlock T20 ( ermo Fisher Scientific, Waltham, MA, USA) for 1 h at RT on a shaker. After blocking, each well was washed 4 times with 200 μl of PBST. Next, standard OxiDJ-1, patient and control urine samples were loaded into duplicate wells of the plate and incubated overnight at 4°C on a shaker. e wells were washed 4 times and then incubated in 100 μl of DJ-1 antibody conjugated with HRP (0.7 μg/ml, 60R-2218, Fitzgerald Industries International, Concord, MA, USA) in SuperBlock T20 for 1.5 h at RT on a shaker. After that, the plate was washed 5 times with 200 μl of PBST. We then added 100 μl of 3, 3′5, 5′-tetramethylbenzidine (Sigma-Aldrich) into each well and incubated them for 10 min in a dark container at RT on a shaker. Lastly, 50 μl of 2 N HCl was added to each well, and the absorbance was measured by Synergy 2 (Biotek Instrument, Winooski, VT, USA) at a 450 nm wavelength.

Statistical Analysis.
e graphs are presented as the mean ± SEM. e data were analyzed using Prism6 (GraphPad software, La Jolla, CA, USA). Each statistical analysis is described in detail in the figure legends.  decrease in DJ-1 levels when DJ-1 concentration was gradually increased by Western blot analysis (Figures 1(a) and 1(b)). However, His-DJ-1 treated with DW, a vehicle control, had decreased or undetectable OxiDJ-1 levels (Figures 1(a) and  1(b)), while increased DJ-1 levels were detected. ese data clearly confirm the activity of the OxiDJ-1 antibody. In addition, the formation of high-molecular weight DJ-1 aggregates seems to be facilitated by H 2 O 2 treatment, as expected (Figure 1(a)). We then designed a sandwich ELISA for OxiDJ-1, which was composed of oxidized DJ-1 and HRP-conjugated DJ-1 antibodies. Using the developed ELISA, we obtained an elaborate standard curve which had a gradual increase in H 2 O 2 -treated His-DJ-1 concentration, but not in DW-treated His-DJ-1 which had fluctuating absorbance (Figure 1(c)).

Establishment of an
ese results suggest that our developed OxiDJ-1 sandwich ELISA could be a promising tool to measure OxiDJ-1 levels.  [26,27]. To induce the formation of OxiDJ-1 in cells, HEK293T cells were treated with H 2 O 2 for the indicated times and OxiDJ-1 levels of both the cell lysates and culture media were measured by both Western blot and ELISA. In the Western blot assay, increased OxiDJ-1 levels were observed in cell-lysates in a time-dependent manner, though it was not statistically significant (Figures 2(a) and 2(b)). e OxiDJ-1 ELISA of the lysates showed significantly higher OxiDJ-1 levels after the 3 h treatment than after only the 0 or 1 h treatment (Figure 2(c)). Moreover, both Western blot and ELISA of the culture media exhibited significantly higher OxiDJ-1 levels in the 3 h treatment than the 0 or 1 h treatment ese data support that our OxiDJ-1 ELISA is sensitive enough to detect live cell-derived OxiDJ-1. In addition, our results suggest that OxiDJ-1 is secreted in the culture medium.

Investigations of OxiDJ-1 Levels in Urine Samples of Korean PD
Patients. Next, we tested the presence of OxiDJ-1 in human urine. A previous study used proteomic analysis to confirm that DJ-1 is secreted in human urine via exosomes [28]. Another study reported the presence of OxiDJ-1 in RBCs of human PD patients [13]. However, to date, there are no reports on the presence of OxiDJ-1 in human biofluids. In our study, Western blot assays of non-PD control or PD patient urine samples detected OxiDJ-1, but there was no significant difference in the amount of OxiDJ-1 between PD and non-PD participants (Figures 3(a) and 3(b)). Even after looking for gender-specific relevance within the two groups, we did not find any distinct differences (Figure 3(c)). Strikingly, when we used the established OxiDJ-1 ELISA, we found that PD patients had significantly 2-fold higher levels of OxiDJ-1 levels in their urine than non-PD controls (Figure 4(a)). us, our data confirms the presence of OxiDJ-1 in human urine.

Discussion
Oxidative stress is a highly evaluated risk factor of PD progression [1]. Increased ROS in dopaminergic neurons can modify cytosolic proteins via chemical conjugation [4]. HNE is produced from oxidation of lipids with polyunsaturated omega-6 acyl groups, such as arachidonic or linoleic acid, by lipid peroxidase [2,29]. It has been implicated that HNE is conjugated with α-syn, thereby generating HNE-modified α-syn oligomers [30,31]. Furthermore, PD patients showed higher levels of HNE-modified α-syn oligomers in the substantia nigra (SN) [32,33]. Previous studies have reported that extensive oxidation of lipofuscin could aggravate proteasome activity and cause cytosolic aggregates [34,35]. Inhibited proteasome activity is associated with α-synucleinopathy [36]. Other studies have demonstrated that DAQ can conjugate with α-syn to form unstructured adducts, and further, increased DAQ can exacerbate the survival of PC12, a rat dopaminergic neuronal cell line, via mitochondrial dysfunction [37,38]. is evidence suggests that removal of oxidative stress via ROS-sensing molecules, such as chaperones, heat shock proteins, and DJ-1, is the most adequate therapeutic strategy for PD. In addition, cellular or extracellular OxiDJ-1 levels could be utilized as an indicator of oxidative stress levels.
As summarized in Table 1, numerous studies have investigated the use of DJ-1 as a PD biomarker. Several studies showed that DJ-1 levels in CSF [15,18], plasma [16], and saliva [25] were increased in PD than the comparative groups. But other studies reported that DJ-1 levels were not different in serum samples from PD and control cases [17] and in CSF samples from parkinsonian syndrome and control groups [22]. On the other hand, contradictory levels of DJ-1 in CSF and saliva of PD were reported [20]. X. Lin et al. suggested that two types of HNE-modified DJ-1 isoform showed a distinguishable level in whole blood of PD [21]. Oxidative stressmediated HNE might be associated with oxidation of DJ-1. OxiDJ-1 increased in unmedicated PD than medicated PD or healthy group [13]. Considering the molecular function of DJ-1 against oxidative stress, unmodified total DJ-1 might not be a suitable biomarker for PD, because the DJ-1 level showed inconsistent results depending on applications of biofluids or analytical methods. We assumed that oxidative stress-mediated modification of DJ-1 would be a hopeful approach for a diagnostic tool of PD.
Elimination of ROS by DJ-1 is accompanied by oxidation of DJ-1, itself [8]. A number of studies have shown that DJ-1 forms aggregates in the cytoplasm [39,40]. We also observed increased OxiDJ-1 aggregates after treatment of His-DJ-1 with H 2 O 2 (Figure 1(a)). Analyzing OxiDJ-1 levels in urine using Western blot assay also showed that most OxiDJ-1 in urine formed aggregates whereas monomeric OxiDJ-1 was barely detected after extreme long exposure (Figure 3(a)). We did not see any aggregated OxiDJ-1 or DJ-1 in cell lysates or media in the Western blot assay (data not shown). In HEK293T, OxiDJ-1 levels detected by the ELISA were consistent with those from the Western blot, but the differences were clearer in the ELISA (Figure 2). We only observed significant differences in OxiDJ-1 levels between PD and non-PD participants in the ELISA, not in the Western blot assay (Figures 3 and 4), suggesting that our ELISA is more sensitive than the Western blot assay.
Our previous study demonstrated that there were no significant differences in DJ-1 levels in urine exosomes during different times of day [24]. Further, only Korean male PD patients showed increased DJ-1 protein levels in their urine exosomes. However, DJ-1 proteins existed in both heated-soluble washing fractions, which are used for our analysis, and in retentate fractions, which were not analyzed in our previous study because of their low protein concentrations [24]. erefore, in this study, we used the whole urine sample after incubating it with Triton X-100 detergent and concentrating it by filtration to acquire all proteins in the exosomes. is approach enabled us to detect  Figure 4: Detection of OxiDJ-1 from urine samples using ELISA. (a) OxiDJ-1 levels in urine were significantly increased in PD patients compared to non-PD controls. Student's t-test was used for this analysis, and p value is described in the graph. (b) OxiDJ-1 levels in urine were divided by gender. One-way ANOVA was used for statistical analysis.
the exact levels of total OxiDJ-1 in the urine sample of male and female patients. We were able to observe the presence of OxiDJ-1 in human urine and significant differences in OxiDJ-1 concentration between PD and non-PD participants. Although DJ-1 levels of Korean male PD patients were higher than those of female PD patients in our previous study, OxiDJ-1 levels exhibited no such difference (Supplementary Figure 2). Consistent with previous reports, our study demonstrates that DJ-1 and OxiDJ-1 levels could be used as PD biomarkers in various materials from human. Using an OxiDJ-1 ELISA, we tried to observe differences in OxiDJ-1 levels in urine samples between PD patients and non-PD controls. However, it is important to note that in non-PD controls, OxiDJ-1 levels might be affected by other undiagnosed clinical conditions, including diabetes, hypertension, hyperlipidemia, and stroke. To verify the increase of OxiDJ-1 in PD patients, a future study with more PD patients and healthy, aged non-PD controls as well as healthy, young cohorts is necessary. Our finding might provide a key step towards finding an efficient diagnostic tool for PD.

Conclusion
We observed a 2-fold increase of OxiDJ-1 levels in the urine of Korean PD-patients compared to those in non-PD controls using our OxiDJ-1 ELISA. us, total OxiDJ-1 levels in human urine could be a feasible biomarker to diagnose PD.

Conflicts of Interest
e authors declare that they have no conflicts of interest.