# NEW TECHNOLOGIES FOR DETECTION, MONITORING AND TREATMENT OF PARKINSON'S DISEASE

EDITED BY : Martin J. McKeown, Xun Chen, Meeko Oishi and Aiping Liu PUBLISHED IN : Frontiers in Aging Neuroscience, Frontiers in Neuroscience and Frontiers in Neurology

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ISSN 1664-8714 ISBN 978-2-88963-886-4 DOI 10.3389/978-2-88963-886-4

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# NEW TECHNOLOGIES FOR DETECTION, MONITORING AND TREATMENT OF PARKINSON'S DISEASE

Topic Editors:

Martin J. McKeown, University of British Columbia, Canada Xun Chen, University of Science and Technology of China, China Meeko Oishi, University of New Mexico, United States Aiping Liu, University of British Columbia, Canada

Citation: McKeown, M. J., Chen, X., Oishi, M., Liu, A., eds. (2020). New Technologies for Detection, Monitoring and Treatment of Parkinson's Disease. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-886-4

# Table of Contents

*04 Increased DJ-1 and* a*-Synuclein in Plasma Neural-Derived Exosomes as Potential Markers for Parkinson's Disease*

Zhen-Hua Zhao, Zhi-Ting Chen, Rui-Ling Zhou, Xu Zhang, Qin-Yong Ye and Yin-Zhou Wang

*12 N1 and P1 Components Associate With Visuospatial-Executive and Language Functions in Normosmic Parkinson's Disease: An Event-Related Potential Study*

Yi-Qi Lin, Shi-Shuang Cui, Juan-Juan Du, Gen Li, Yi-Xi He, Ping-Chen Zhang, Yang Fu, Pei Huang, Chao Gao, Bin-Yin Li and Sheng-Di Chen

*21 Progressive Gait Deficits in Parkinson's Disease: A Wearable-Based Biannual 5-Year Prospective Study*

Markus A. Hobert, Susanne Nussbaum, Tanja Heger, Daniela Berg, Walter Maetzler and Sebastian Heinzel

Paul B. Camacho, Ronald Carbonari, Sa Shen, Cindy Zadikoff, Arthur F. Kramer and Citlali López-Ortiz

*55 Use of Overlapping Group LASSO Sparse Deep Belief Network to Discriminate Parkinson's Disease and Normal Control*

Ting Shen, Jiehui Jiang, Wei Lin, Jingjie Ge, Ping Wu, Yongjin Zhou, Chuantao Zuo, Jian Wang, Zhuangzhi Yan and Kuangyu Shi

*67 Regional High Iron in the Substantia Nigra Differentiates Parkinson's Disease Patients From Healthy Controls*

Kiarash Ghassaban, Naying He, Sean Kumar Sethi, Pei Huang, Shengdi Chen, Fuhua Yan and Ewart Mark Haacke

*77 Altered Global Synchronizations in Patients With Parkinson's Disease: A Resting-State fMRI Study*

Mengyan Li, Yanjun Liu, Haobo Chen, Guihe Hu, Shaode Yu, Xiuhang Ruan, Zhenhang Luo, Xinhua Wei and Yaoqin Xie

*87 Alterations of Regional Homogeneity in Parkinson's Disease Patients With Freezing of Gait: A Resting-State fMRI Study* Yanjun Liu, Mengyan Li, Haobo Chen, Xinhua Wei, Guihe Hu, Shaode Yu,

Xiuhang Ruan, Jin Zhou, Xiaoping Pan, Ze Li, Zhenhang Luo and Yaoqin Xie

*99 Motion Biomarkers Showing Maximum Contrast Between Healthy Subjects and Parkinson's Disease Patients Treated With Deep Brain Stimulation of the Subthalamic Nucleus. A Pilot Study*

Andreas Kuhner, Isabella Katharina Wiesmeier, Massimo Cenciarini, Timo Leon Maier, Stefan Kammermeier, Volker Arnd Coenen, Wolfram Burgard and Christoph Maurer

## Increased DJ-1 and α-Synuclein in Plasma Neural-Derived Exosomes as Potential Markers for Parkinson's Disease

Zhen-Hua Zhao1† , Zhi-Ting Chen2† , Rui-Ling Zhou1 , Xu Zhang1 , Qin-Yong Ye2 \* † and Yin-Zhou Wang1 \* †

1 Department of Neurology, Fujian Provincial Hospital, Provincial Clinical College of Fujian Medical University, Fuzhou, China, 2 Department of Neurology, Fujian Medical University Union Hospital, Fuzhou, China

#### Edited by:

Xun Chen, University of Science and Technology of China, China

#### Reviewed by:

Liuqing Yang, Johns Hopkins Medicine, United States Deep R. Sharma, SUNY Downstate Medical Center, United States En Huang, Temple University, United States

#### \*Correspondence:

Qin-Yong Ye<EMAIL_ADDRESS>Yin-Zhou Wang<EMAIL_ADDRESS>
†These authors have contributed equally to this work

Received: 03 September 2018 Accepted: 21 December 2018 Published: 14 January 2019

#### Citation:

Zhao Z-H, Chen Z-T, Zhou R-L, Zhang X, Ye Q-Y and Wang Y-Z (2019) Increased DJ-1 and α-Synuclein in Plasma Neural-Derived Exosomes as Potential Markers for Parkinson's Disease. Front. Aging Neurosci. 10:438. doi: 10.3389/fnagi.2018.00438 The diagnosis of PD might be in difficulty, especially in the early stages. Therefore, the identification of novel biomarkers is imperative for the diagnosis and monitoring disease progression in PD. DJ-1 and α-synuclein, are two proteins that are critically involved in the pathogenesis of PD, and they have been examined as disease biomarkers in studies. However, no study exists regarding DJ-1 in plasma neural-derived exosomes. In the present study, the levels of DJ-1 and α-synuclein in plasma neural-derived exosomes were studied together in order to investigate novel biomarkers for PD. DJ-1 and α-synuclein in plasma and plasma neural-derived exosomes of the patients with PD and controls were quantified by ELISAs. The data revealed that the levels of DJ-1 and α-synuclein in plasma neural-derived exosomes and the ratio of plasma neural-derived exosomal DJ-1 to total DJ-1 were significantly higher in patients with PD, compared with controls, while levels of the two proteins in plasma exhibited no difference between the patients with PD and controls. However, no relationship was identified between biomarkers and disease progression. In addition, significant positive correlations between DJ-1 and α-synuclein in plasma neural-derived exosomes were found in the patients with PD and in healthy individuals. We hypothesize that DJ-1 in plasma neural-derived exosomes may be used as a potential biomarker as α-synuclein in PD and they might participate in the mechanism of PD together.

Keywords: α-synuclein, DJ-1, biomarker, plasma, exosome, Parkinson disease

#### INTRODUCTION

Parkinson's disease (PD), characterized by a set of extrapyramidal motor features, is the second most common neurodegenerative disease worldwide. The beginning of disease-specific pathology occurs before the onset of classical clinical manifestations, and the symptoms of PD are not apparent until almost 60% of the dopamine neurons have died (Berardelli et al., 2013). PD in the early stages is prone to be misdiagnosed, due to a lack of suitable disease biomarkers and overt clinical symptoms. Consequently, there is still a substantial misdiagnosis rate.

Gene mutations of α-synuclein culminate in familial PD and α-synuclein has a close association with the pathogenic mechanism of heredofamilial and sporadic PD (Lucking and Brice, 2000; Braak and Del Tredici, 2017). DJ-1 is another gene product that has previously been

associated with both heredofamilial and sporadic PD, and appears to be associated with oxidative stress, which is an acritical process related to PD (Choi et al., 2006). Consequently, DJ-1 and α-synuclein have been considered to be leading biomarkers for PD (Hong et al., 2010). However, it is presently unknown whether the combination of DJ-1 and α-synuclein will be a more suitable combination for the diagnosis of PD.

Cerebrospinal fluid (CSF) has been reported to function as an accurate and reliable source for biomarkers in PD (Farotti et al., 2017). However, obtaining CSF is a cumbersome process compared to blood sampling in a regular clinical setting. Blood is an accessible source for application in a clinical setting (Mehta and Adler, 2016). However, a principal disadvantage of blood biomarkers is that they do not directly reflect the conditions in the central nerve system and are more easily affected by the peripheral environment.

Exosomes containing proteins and other constituents of their cellular origin are a means of communication between cells. Exosomes might be a pathway for neurons to divert proteins from neurons into the CSF or into the peripheral blood via blood– brain barrier. These materials associated with PD, including α-synuclein, maybe excessive and/or in a toxic or insoluble structure. To confirm this hypothesis, Shi and colleagues injected mice intracerebroventricularly with 125I-labeled α-synuclein or tau. And, they observed that 125I-labeled α-synuclein or tau could be detected in neural-derived blood exosomes which expressed the neuronal adhesion molecule L1CAM (Shi et al., 2014, 2016). Exosomal α-synuclein in neural-derived blood exosomes was increased in patients with PD (Shi et al., 2014). However, to the best of our knowledge, there are no relevant studies on exosomal DJ-1 in neural-derived blood exosomes.

In the present study, a combination of chemical and immunochemical methods were used to harvest and enrich neural-derived exosomes from small volumes of plasma in quantities that was enough to detect the implicated proteins in the pathogenic mechanism of PD. The levels of DJ-1 and α-synuclein in plasma neural-derived exosomes were analyzed in order to evaluate any potential associations with PD, and to assess their suitability as biomarkers for the disease. In addition, the levels of α-synuclein in plasma neural-derived exosomes were analyzed to investigate any associations between DJ-1 and α-synuclein, and the relationship between these proteins and the progression of PD were further investigated.

### MATERIALS AND METHODS

#### Participants

This study was performed in the Department of Neurology of Fujian Provincial Hospital, Fuzhou, China, and was approved by the Ethics Committees of Fujian Provincial Hospital. Laboratorians and statistical analyst were blinded to any clinical information and the grouping. All individuals provided written informed consents.

From January 2016 to June 2017, a total of 39 PD patients were recruited. All patients with PD were diagnosed according to the United Kingdom Parkinson's Disease Society Brain Bank criteria (Hughes et al., 1992). The healthy controls were the patients' husbands or wifes, or healthy community volunteers. The individuals in the control group did not exhibit any signs or symptoms indicative of neurological disease. All individuals were excluded on the following criteria: the presence of (1) tumors, (2) essential tremors, secondary parkinsonism, or Parkinson-plus syndrome, (3) severe craniocerebral trauma, (4) inflammatory, infectious, or autoimmune diseases in the peripheral and central systems, (5) severe systemic diseases, such as anemia, hepatosis, heart failure, pulmonary disorders, and chronic renal failure, and (6) other neurodegenerative diseases.

All patients with PD and control subjects received an assessment which contained medical history, physical and neurological examinations, laboratory tests, and neuropsychological assessment. The laboratory evaluation consisted of routine blood parameters, serum electrolytes, blood urea nitrogen, creatinine, fasting blood glucose, vitamin B12 and thyroid stimulating hormone. The above results for all study participants were normal. All patients with PD were assessed using Hoehn-Yahr (H-Y) and Unified Parkinson's Disease Rating Scale-III (UPDRS-III) scores. The duration of disease since the time of initial diagnosis was available and used for subsequent analysis. Blood samples were collected at fasting state in the morning, after patients and controls were enrolled in the present study, and all plasmas were stored at −80◦C.

### Isolation of Plasma Neural-Derived Exosomes

Exosomes were isolated according to the experimental protocol described in a previous report (Goetzl et al., 2015). Briefly, 0.8 ml of plasma was incubated with 0.15 ml of thromboplastin-D (Thermo Fisher Scientific, Inc., Waltham, MA, United States) for 60 min. The plasma was then added to 0.35 ml of calciumand magnesium-free Dulbecco's balanced salt solution (DBS-2) with protease inhibitor cocktail (Roche Applied Science, Inc., Madison, WI, United States) and phosphatase inhibitor cocktail (Thermo Scientific, Inc.). Following centrifugation at 1,500 × g for 20 min, the supernatant was mixed with 252 µl of ExoQuick exosome precipitation solution (EXOQ; System Biosciences, Inc., Palo Alto, CA, United States), and incubated for 1 h at 4◦C. The resulting exosome suspensions were centrifuged at 1,500 × g for 30 min at 4◦C, and each precipitation was re-suspended in 150 µl of DBS-2 with inhibitor cocktails and 100 µl of 3% bovine serum albumin. The suspensions were subsequently incubated for 1 h at 4◦C with 1 µg of mouse anti-human CD171 (L1CAM neural adhesion protein) biotinylated antibody (clone 5G3, eBioscience; Thermo Fisher Scientific, Inc.) and 25 µl of streptavidin-agarose resin (Thermo Scientific, Inc.) with 50 µl of 3% BSA. Following being centrifuged at 200 × g for 10 min at 4◦C and removal of the supernatant, each precipitation was suspended in 50 µl of 0.05 M glycine-HCl (pH 3.0) by vortexing for 10 s. Subsequently, 0.45 ml of DBS-2 with 2 g/100 ml of BSA, 0.10% Tween 20 and the inhibitor cocktails, was added to each suspension. After vortex-mixing, each suspension was incubated for 10 min at 37◦C with. Samples were then stored at −80◦C prior to ELISAs. The protein concentration of exosomes from the neural source was

determined using the Bradford protein assay (Tiangen Biotech, Co., Ltd., Beijing, China), with bovine serum albumin as a standard.

#### Transmission Electron Microscopy

The exosomes were placed in a droplet of 2.5% glutaraldehyde in PBS buffer at pH 7.2 and fixed for 2 h at 4◦C. The samples were washed in 0.1 M PBS buffer and fixed in 1% osmium tetroxide for 2 h at 4◦C. The samples were then dehydrated for 10 min in increasing concentrations of alcohol (50, 70, 90, and 100%, repeated three times), then infiltrated with alcohol/propylene oxide (1:1) and propylene oxide for 10 min at room temperature, respectively. The samples were embedded in Quetol-812 epoxy resin and polymerized at 40◦C for 2 h, 60◦C for 4 h, and 80◦C for 10 h. Ultrathin sections (100 nm) were cut using a Leica UC6 ultra-microtome, and then were stained with uranyl acetate for 15 min and with lead citrate for 10 min at room temperature. The results were observed in a FEI TecnaiSPIRIT transmission electron microscope (Camcor, Eugene, OR, United States), operated at 120 kV.

### ELISA Quantification of Exosome Proteins and Plasma Protein

The proteins in plasma neural-derived exosomes were quantified by ELISA kits for α-synuclein and human DJ-1 (R&D Systems, Inc., Minneapolis, MN, United States), according to the manufacturer's protocol. Plasma α-synuclein and human DJ-1 were also quantified, and the test for biomarker was repeated 3 times.

#### Data Analyses

In the present study, SPSS Statistics 19.0 (IBM, Corp., Armonk, NY, United States) was used for statistical analyses. p < 0.05 was accepted to be statistically significant in all cases. All continuous variables, including age, UPDRS-III scores, duration of disease, the levels of DJ-1 in exosomes, total DJ-1 in plasma, α-synuclein in exosomes, total α-synuclein in plasma, and their ratio are presented as the mean ± standard deviation (SD). A χ 2 test was used for comparing the differences between sexes. In order to compare the differences between age and biomarkers between the groups, a t-test was used to detect statistically significant differences when the data were normally distributed, and a Mann–Whitney U-test when the data were not normally distributed. In order to evaluate correlations among the biomarkers, Pearson's correlation coefficients for DJ-1 and α-synuclein of plasma were obtained. Spearman's correlation coefficients were also obtained to evaluate the correlations among the biomarkers of which the data do not conform to the normal distribution. Receiver operating characteristic (ROC) curves for the biomarkers were generated to evaluate their sensitivities and specificities in distinguishing PD from healthy controls. The "optimum" cut-off value for a ROC curve was the point associated with the maximal sum of sensitivity and specificity. ROC curves for the combination of DJ-1 and α-synuclein were also calculated using logistic regression analysis.

## RESULTS

### Evaluation of DJ-1 and/or α-Synuclein in Plasma and Plasma Neural-Derived Exosome as Biomarkers for PD

Plasma neural-derived exosomes were extracted and corroborated by transmission electron microscopy (**Figure 1A**). The concentrations of DJ-1 in the plasma neural-derived exosomes and plasma were measured using ELISAs. There was no significant difference in DJ-1 in the total plasma between patients with PD and controls in the total plasma (2.26 ± 0.65 vs. 2.49 ± 0.60 ng/ml, p = 0.104). The concentrations of DJ-1 in the plasma neural-derived exosomes were significantly higher in patients with PD compared with healthy controls (2.94 ± 0.96 vs. 2.34 ± 0.86 ng/mg, p = 0.002). The ratio of plasma neural-derived exosomal DJ-1 to total DJ-1 (exo/total) was also significantly higher in patients with PD, compared with the controls (1.39 ± 0.57 vs. 1.00 ± 0.50, p = 0.001) (**Table 1**).

The concentrations of α-synuclein in plasma neural-derived exosome and in plasma were also measured using ELISAs. Similar to the findings of a previous study (Shi et al., 2014), there was no significant difference in the plasma α-synuclein concentrations between patients with PD and controls (3.01 ± 1.51 vs. 3.01 ± 1.17 ng/ml, p = 0.565). However, the concentrations of α-synuclein in plasma neural-derived exosomes were significantly higher in patients with PD compared with healthy controls (7.75 ± 2.74 vs. 6.50 ± 1.17 ng/mg, p = 0.018 < 0.05) (**Table 1**).

To further evaluate the potential for DJ-1 and α-synuclein in plasma neural-derived exosomes to aid in the diagnosis of PD, ROC analysis was performed to characterize its sensitivity and specificity. The AUC for α-synuclein was 0.654, when the cut off value was 8.14 ng/mg, with a sensitivity of 48.7% and specificity of 85.0% (**Figure 1B**). The ROC analysis performance of DJ-1 in plasma neural-derived exosomes was identified to be moderate (AUC = 0.703, sensitivity = 79.5%, specificity = 57.5%) (**Figure 1C**), as the cut off values was 2.06 ng/mg. The exo/total DJ-1 generated a similar AUC result (0.724) with a sensitivity of 59.0% and a specificity of 82.5% at a cutoff value of 0.415 (**Figure 1D**). The AUC for the combination of DJ-1 and α-synuclein was 0.714 (**Figure 1E**), with a sensitivity of 82.1% and a specificity of 52.5% at a cutoff value of 0.35 on the predicted risk algorithm. The combination of these two proteins did not perform a significant discrimination.

### Evaluating the Relationships Between DJ-1 and α-Synuclein Levels of Plasma and Plasma Neural-Derived Exosomes

To evaluate the relationships between DJ-1 and α-synuclein levels of plasma and plasma neural-derived exosomes, the present study conducted a correlation analysis. A significant positive correlation was identified between DJ-1 and α-synuclein levels of plasma neural-derived exosomes in patients with PD (**Figure 2A**) and controls (**Figure 2B**). No correlation was found between the plasma levels of DJ-1 and α-synuclein in patients with PD

(**Figure 2C**), but a weak correlation was found in controls (**Figure 2D**).

#### Correlation Between DJ-1 or α-Synuclein and the Progression of PD

Patients with PD were classified into H-Y stages 1–5 (Hoehn and Yahr, 1967), and were divided into two groups, early stage PD (H-Y 1 to 2) which included 22 patients and advanced stage PD (H-Y 3 to 5) which included 17 patients. Patients with PD in the advanced stage had a longer disease duration and higher UPDRS-III scores when compared with patients with PD in the early stage. However, no significant differences were identified in the neural-derived exosome and plasma levels of DJ-1 and α-synuclein between patients with PD at the early stage and advanced stages of disease (**Table 1**).

### DISCUSSION

The development of effective biomarkers is an important and urgent task for PD in the early stage. A reliable biomarker can be conductive to identifying patients with PD in the early stage. And, it can also be used for the development of new therapy to treat the disease.

Plasma neural-derived exosomes may serve as a potentially sources that accurately reflects the changes of the central nervous system (CNS) (Shi et al., 2016). Exosomes from CNS which contain jettisoned and potentially toxic forms of α-synuclein or other disease-associated proteins that may be transported into the systemic circulation system (Shi et al., 2014). In addition, these exosomes may avoid the interferences from the blood contamination, system inflammation, and potential tumor. The study of Fiandaca et al. (2015) revealed that the exosomal levels of P-S396-tau, P-T181-tau and Aβ1-42 were significantly higher than in controls at one to 10 years prior to diagnosis with Alzheimer's disease. In a subsequent series of studies, astrocyte-derived exosomes or neural-derived exosomes were obtained from plasma by using the same method of polymerbased precipitation and combination of biotinylated antibody and streptavidin-agarose resin (Goetzl et al., 2015, 2016). In the present study, neural-derived exosomes were obtained using this method, and their origin was confirmed using transmission electron microscopy, and similar results were obtained when compared with a previous study (Shi et al., 2014). The principal

exo, exosomes; α-syn, α-synuclein; aMann–Whitney U-test; bStudent's t-test; P1, compare between control subjects and PD; P2, compare between early stage PD and advanced stage PD. ∗p < 0.05.

advantage of this method using polymer-based precipitation, and combination of biotinylated antibody and streptavidin-agarose resin is that it is simple and can be easily performed in a typical laboratory without a Luminex workstation.

The α-synuclein in the CNS is associated with the progression of PD, and has been hypothesized to be secreted in means of exosomes (Marques and Outeiro, 2012; Gui et al., 2015). Previous studies have reported that plasma α-synuclein was not a suitable biomarker for PD (Lee et al., 2006; Li et al., 2007; Shi et al., 2010), as any peripheral cells containing abundant α-synuclein would affect the result (Barbour et al., 2008). In the present study, plasma α-synuclein levels exhibited no significant differences between patients with PD and controls. However, a higher level of α-synuclein was detected in plasma neural-derived exosomes in patients with PD by ELISA. These results were similar to those obtained by a previous study, in which the levels were detected by Luminex assays (Shi et al., 2014). As the results were free from methodological interference, the present study confirmed that α-synuclein in plasma neural-derived exosomes may function as a reliable biomarker for PD. However, there were limitations to the present study, namely that the sensitivity and specificity were not high enough. Therefore, the sample size needs to be expanded in future studies.

DJ-1 had previously been hypothesized to be associated with the mechanism of PD (Bonifati et al., 2003). Consequently, DJ-1 in biological fluids has garnered much interest as a promising biomarker for PD; however, the inconsistent results obtained by numerous studies regarding plasma DJ-1 suggest its unsuitability to be a reliable biomarker for PD (Waragai et al., 2007; An et al., 2018). Similar to α-synuclein, the plasmic level of DJ-1 is greatly affected by contamination due to its remarkably high content in erythrocytes (Shi et al., 2010). Therefore, researchers have focused on alternative sources of DJ-1. For example, a previous study reported that DJ-1 in saliva was increased in patients with PD compared with controls (Devic et al., 2011), and may present an association with disease progression (Kang et al., 2014). In a Korean study, the levels of DJ-1 in urinary exosomes in males were increased in patients with PD compared with controls and were increased with age in PD (Ho et al., 2014). Other forms of DJ-1, such as oxidized DJ-1 (Ogawa et al., 2014) and DJ-1 isoforms (Lin et al., 2012), have also been studied. To the best of our knowledge, the present study is the first to report that DJ-1 in plasma neural-derived exosomes was significantly increased in patients with PD compared with healthy controls. However, the combination of DJ-1 and α-synuclein in plasma neuralderived exosomes did not significantly enhance the performance of diagnosis.

In the present study, a significant positive correlation between DJ-1 and α-synuclein of plasma neural-derived exosomes was observed in patients with PD. The interaction of α-synuclein and DJ-1 serves an important role in heredofamilial and sporadic PD (Antipova and Bandopadhyay, 2017). A previous study reported that DJ-1 could be observed on the rim of Lewy bodies in patients with sporadic PD (Jin et al., 2005). The total level of DJ-1 protein is significantly increased in the brains of patients with PD compared with controls (Moore et al., 2005; Choi et al., 2006). In addition, DJ-1 can modulate the aggregation of α-synuclein and protect the neurons form cell toxicity of α-synuclein by chaperone-like activity (Sun et al., 2012) and physical interactions (Zondler et al., 2014). The knockdown or knock-out of DJ-1 has been demonstrated to increase α-synuclein toxicity, thus making neurons more vulnerable to the dopaminergic selective neurotoxin 6-hydroxydopamine (Batelli et al., 2015). The results of the present study further confirmed there is a correlation between DJ-1 and α-synuclein. Although the interactions between DJ-1 and α-synuclein have not been completely elucidated, DJ-1 and α-synuclein may participate together in the pathology of PD.

The mechanism for the increased α-synuclein and DJ-1 in the exosome is presently unclear. It has been hypothesized that α-synuclein may be secreted out of cell via exocytosis, exosomes, or other extracellular vesicles (Faure et al., 2006). In addition, there are several potential mechanisms for increasing α-synuclein in the exosome. Increasing α-synuclein synthesis may culminate in its increased secretion to the extracellular space in previous in vitro studies providing evidence for this potential mechanism. When neurons over-expressing α-synuclein would actively secrete an increased amount of α-synuclein into the extracellular space by the pathway of exosomes (Emmanouilidou et al., 2010; Reyes et al., 2015), α-synuclein induced cell death may be prevented by exosomal secretion. However, this self protective mechanism would damage neighboring cells (Lee et al., 2005). Another potential mechanism is the dysfunction of the autophagy-lysosomal pathway or macroautophagy. An example of this being ammonium chloride or bafilomycin A1, which inhibits lysosomal function, which would then increase the release of α-synuclein in exosomes (Alvarez-Erviti et al., 2011). This effect may also be observed in α-synuclein over-expressing SH-SY5Y cells treated with secretory carrier membrane protein 5, which is an autophagy inhibitor (Yang et al., 2017). If macroautophagy function was inhibited by silencing the key element autophagy related gene 5, the releasing of α-synuclein from cells by the pathway of exosomes would be observed as a kind of compensation (Fussi et al., 2018). The dysfunction of the endocytic pathway may be another mechanism for increasing α-synuclein secretion via the exosome. The glucocerebrosidase gene is a pathogenic gene in hereditary PD, and its mutation has been hypothesized to decrease glucocerebrosidase enzymatic activity and attenuate endocytic function. The inhibition of this enzymatic activity or expression of this mutant gene results in release of α-synuclein and its oligomers (Papadopoulos et al., 2018). Research regarding in exosomal DJ-1 is rare; however, we hypothesize that increasing DJ-1 in pathogenicity of α-synuclein maybe partially transferred with the exosome into extracellular space.

#### CONCLUSION

α-Synuclein and DJ-1 in plasma neural-derived exosomes may serve as potential biomarkers for PD. However, further studies should be conducted with larger patient cohorts in order to corroborate the significance of these findings and the relationship of these biomarkers and disease progression.

#### AUTHOR CONTRIBUTIONS

fnagi-10-00438 January 10, 2019 Time: 18:52 # 7

Z-HZ was responsible for the conception and design of the present study, execution of the experimental work, and wrote the first draft of the manuscript, and review and critique of the Manuscript. Z-TC undertook study design, execution of statistical analysis, execution of experimental work, and the review and critique of the manuscript. R-LZ and XZ organized the research project and reviewed and critiqued the manuscript. Y-ZW organized the research project and review and critiqued the statistical analysis and the

#### REFERENCES

manuscript. Q-YY was responsible for the conception of experiments, execution of experimental work, design and execution of statistical analysis, and the review and critique of the manuscript.

## FUNDING

This work was supported by the Natural Science Foundation of Fujian Province (Grant No. 2015J01426) and Outstanding Youth Foundation of Fujian Provincial Hospital (Grant No. 2014060).

in Parkinson's disease. Neurosci. Lett. 480, 78–82. doi: 10.1016/j.neulet.2010. 06.009

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zhao, Chen, Zhou, Zhang, Ye and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# N1 and P1 Components Associate With Visuospatial-Executive and Language Functions in Normosmic Parkinson's Disease: An Event-Related Potential Study

Yi-Qi Lin, Shi-Shuang Cui, Juan-Juan Du, Gen Li, Yi-Xi He, Ping-Chen Zhang, Yang Fu, Pei Huang, Chao Gao, Bin-Yin Li\* and Sheng-Di Chen\*

Department of Neurology and Institute of Neurology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China

Background: Hyposmia is one of the most important clinical markers of Parkinson's disease (PD) with a prevalence ranging from 50 to 96% of PD patients. A significant association was found between hyposmia and cognitive impairment of PD. However, there were no reports of event-related potentials (ERP) performance in PD patients with and without hyposmia for cognitive functions assessment.

#### Edited by:

Aiping Liu, The University of British Columbia, Canada

#### Reviewed by:

Yi Li, New York University, United States Niels Janssen, Universidad de La Laguna, Spain

\*Correspondence:

Bin-Yin Li<EMAIL_ADDRESS>Sheng-Di Chen<EMAIL_ADDRESS>
Received: 06 September 2018 Accepted: 21 January 2019 Published: 05 February 2019

#### Citation:

Lin Y-Q, Cui S-S, Du J-J, Li G, He Y-X, Zhang P-C, Fu Y, Huang P, Gao C, Li B-Y and Chen S-D (2019) N1 and P1 Components Associate With Visuospatial-Executive and Language Functions in Normosmic Parkinson's Disease: An Event-Related Potential Study. Front. Aging Neurosci. 11:18. doi: 10.3389/fnagi.2019.00018 Purpose: The aim of our study was to compare ERP performance and its association with cognitive domains between PD with and without hyposmia.

Methods: Olfactory functions were assessed by Sniffin' Sticks test-16 (SS-16). Twentyfour subjects were included in PD with hyposmia group and nineteen were in PD without hyposmia group. ERP measures were recorded during a delayed match to sample (DMS) task with Chinese characters. The parameters of ERP components including N1, N2, P1, P2, and P3 in retrieval epoch were compared between the two groups and the correlation between ERP results and MOCA item score was also analyzed.

Results: No significant difference was found in ERP performance between PD with and without hyposmia. Among all participants, N1 latency was significantly negatively related to visuospatial-executive item score of Montreal Cognitive Assessment (MOCA) (rs = −0.381, P = 0.012) and P1 amplitude was positively associated with language item score of MOCA (rs = 0.302, P = 0.049). Within the normosmic group, a significant association was found between N1 latency and visuospatial-executive item score (rs = −0.619, P = 0.005) and there was also a correlation between language score and P1 amplitude (rs = 0.537, P = 0.018). In the hyposmic group, only a significant correlation was found between N1 latency and clock drawing test performance (rs = −0.413, P = 0.045) rather than visuospatial-executive item score. Furthermore, SS-16 score was not found to be significantly associated with either visuospatial-executive or language item score of MOCA.

**12**

Conclusion: No significant difference was found in ERP components between PD with and without hyposmia. N1 latency and P1 amplitude were respectively associated with visuospatial-executive and language functions in the normosmic group while in the hyposmic group, only a significant correlation was found between N1 latency and clock drawing test performance rather than visuospatial-executive item score in MOCA.

Keywords: hyposmia, Parkinson's disease, event-related potentials, working memory, visuospatial function, language

#### INTRODUCTION

fnagi-11-00018 February 2, 2019 Time: 18:20 # 2

Hyposmia is one of the most important non-motor symptoms of Parkinson's disease (PD) with a prevalence ranging from 50 to 96% of PD patients (Doty et al., 1988; Wenning et al., 1995; Hawkes et al., 1997; Boesveldt et al., 2008; Duda, 2010). It generally predates motor symptoms and the olfactory testing is useful in differentiating PD from non-PD patients with a sensitivity ranging from 79 to 100% and a specificity from 80 to 89% (Doty, 2012). Therefore, hyposmia is reported to be a critical clinical marker of PD.

In recent years olfactory dysfunction is found to be associated with other non-motor symptoms in PD patients such as chronic constipation, clinical possible rapid eye movement behavior disorder (RBD) (Chen et al., 2015) and psychotic symptoms (Morley et al., 2011). Notably, increasing evidence suggests that hyposmia is strongly associated with cognitive impairment in PD patients and may be a risk factor for PD dementia (Bohnen et al., 2010; Stephenson et al., 2010; Morley et al., 2011; Parrao et al., 2012; Domellof et al., 2017). In fact, the association between olfaction and cognition has been long reported in mild cognitive impairment and Alzheimer's disease which was evidenced by prominent atrophy in the primary olfactory cortex and hippocampus (Vasavada et al., 2015) as well as a strong correlation between tau pathology in the olfactory bulb and limbic systems (Attems et al., 2005). However, the role of hyposmia in cognitive impairment in PD patients is not clear.

Event-related potentials (ERP) have been widely used for assessing cognitive functions and brain ability. ERP wave latency and amplitude represent respectively the length of time spent and the amount of neural resources participating during information processing. There were increasing studies of ERP in PD patients owning to its independence of motor speed and disability. The abnormal P300 was reported to be associated with cognitive impairment in PD (Katsarou et al., 2004; Matsui et al., 2007) while other ERP measures were barely studied in PD. It remained unclear whether the ERP components were altered in PD with hyposmia, especially the early ERP components reflecting visuospatial processing which was one of the mostly impaired cognitive domains in PD.

The delayed match to sample (DMS) task, one of the most popular tasks during ERP records in PD patients (Seer et al., 2016), was used in our study to test working memory which related to several cognitive abilities including storage capacity, retrieval strategies (Unsworth and Engle, 2007) and visuospatial attention (Bleckley et al., 2003; Giuliano et al., 2014). And Chinese characters applied in our DMS task were more acceptable for Chinese participants and more dependable on visual working memory (Opitz et al., 2014). What's more, we focused on retrieval epoch rather than encoding epoch in view of impaired retrieval process and reserved encoding ability of memory deficit in PD (Mahurin et al., 1993).

To our knowledge, there was no research focusing on cognitive ERP measures in PD patients with hyposmia. The aim of this study was to improve our understanding of the role of hyposmia in cognitive impairment in PD using the ERP technique with DMS task. Our study investigated difference in ERP components and its association with cognitive domains between PD patients with and without hyposmia.

### MATERIALS AND METHODS

#### Subject

Idiopathic PD patients aging from 50 to 80 were recruited from movement disorder clinic of the Department of Neurology, Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine from September 2016 to October 2017. PD was diagnosed according to the clinical Movement Disorder Society (MDS) diagnostic criteria (Postuma et al., 2015) by senior Movement Disorder Specialist. Exclusion criteria included history of head injury, stroke, psychiatric disorder, poor vision, nasal and paranasal disease or other factors affecting olfactory function. Patients with less than 6 years of education or Mini-Mental State Examination (MMSE) score less than 24 were also excluded.

Finally 43 PD patients were enrolled in our study. Hyposmia was defined with score of Sniffin' Sticks test-16 (SS-16) (Burghart Messtechnik, Wedel, Germany) less than 8.3 (Chen et al., 2012) which was consistent with previous researches in our department (Chen et al., 2015). There were 24 subjects in PD with hyposmia group and 19 in PD without hyposmia group. All participants were informed of the research protocol and this study was carried out in accordance with the recommendations of Ethical Review of Biomedical Research Involving Human Subjects by China's Ministry of Health with written informed consent from all subjects. The protocol was approved by Ethics Committee of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.

#### Neuropsychological Assessment

fnagi-11-00018 February 2, 2019 Time: 18:20 # 3

Demographic characters including age, sex, education years and disease duration were recorded (**Table 1**). The disease progression was assessed by modified Hoehn and Yahr (H-Y) scale (Hoehn and Yahr, 1967) and levodopa equivalent dose (LED) which was calculated as reported (Tomlinson et al., 2010). Motor subtypes were divided into tremor dominant type and non-tremor dominant type. Montreal Cognitive Assessment (MOCA) Beijing version was used to evaluate cognitive performance (Yu et al., 2012). Night sleep quality was assessed by Parkinson's disease Sleep Scale (PDSS) (Wang et al., 2008). Rapid Eye Movement Sleep Behavior Disorder Questionnaire-Hong Kong (RBD-HK) (Shen et al., 2014) and Epworth Sleepiness Scale (ESS) (Kumar et al., 2003) were used to evaluate RBD and excessive daytime sleepiness (EDS). The anxiety symptoms were evaluated by Hamilton Anxiety Rating

TABLE 1 | Demographics and clinical characteristics in PD with and without hyposmia groups (mean ± standard deviation).

Scale (HAMA) (Thompson, 2015) and 17-item Hamilton Rating Scale for Depression (HAMD-17) (Hamilton, 1960) was used for depression screening.

#### The Delayed Match to Sample Task

Each participant was required to participate in a practice block before the test block during the DMS task (**Figure 1**). In each trial, after a single of "+" in the screen of 500 ms, a set of six visually similar and easily confused Chinese characters (sample stimulus) presented for 2000 ms which should be remembered by subjects. After a blank for 3000 ms, a character (probe stimulus) demonstrated in the middle of the screen could either be a member of the previous set of characters (answer "yes") or be another similar-looking one (answer "no"). The character presented until a response before 2000 ms or until 2000 ms without response. Subjects were asked to answer "yes" by pressing the button of "1" or "no" by pressing the button of "2" on a keyboard as soon as the response was ensured. The "yes" and "no" answers were randomized and 50% of the correct answers were "yes" and the rest was "no" in each block. The inter-trial interval was 5000 ms. Patients were allowed to begin the test until the accuracy of the practice block was higher than 50%. Each test contained 100 trials. This DMS task was programmed by E-Prime 2.0 software (Psychology Software Tools, Inc., Pittsburgh, PA, United States) in which reaction time and accuracy of each subject were also recorded. The retrieval period of our study was defined from 200 ms before probe stimulus onset to 1000 ms after.

#### EEG Recordings and Data Analysis

ERP was recorded from 32 Ag-AgCl electrodes (Fp1, Fp2, F3, F4, C3, C4, P3, P4, O1, O2, F7, F8, T7, T8, P7, P8, Fz, FCz, Cz, Pz, FC1, FC2, CP1, CP2, FC5, FC6, CP5, CP6, FT9, FT10, TP9, TP10) which were placed according to the international 10–20 system with a 32-channel amplifier (BrainAmp by Brain Products, Munich, Germany).

All channels were digitized at 500 Hz with 200 ms-long prestimulus baseline used for baseline correction. The filter bandpass was 0.1–70 Hz. Electro-oculogram (EOG) was also recorded to correct ocular artifacts. A semi-automatic check was used in artifact rejection. Trials were rejected if EEG voltage step was

higher than 50 µV/ms or if difference of values in intervals was higher than 200 µV or lower than 0.5 µV. The mean rejection rate for all participants was higher than 1%. ERPs were based on correct trials and data not rejected for artifacts.

The five ERP components including N1, N2, P1, P2, and P300 were analyzed in reference of early studies, meanwhile, electrodes with maximal amplitude were chosen, which was consistent with the previous report of our center using the same paradigm (Li et al., 2016). N1 amplitude was measured as the maximal negative peak within the latency window of 150–210 ms on P7, P8 electrodes. P1 amplitude was identified at the most positive peak on O1, O2, P7 and P8 within 84–140 ms after the stimulus onset. N2 amplitude was considered at the maximal negative peak on F3, F4, C3, C4, CZ, FZ and FCz within the latency window of 230–300 ms. P2 amplitude was identified at the maximal positive peak on FC1, FC2, CZ, FZ and FCz within the latency window of 150–250 ms. The P300component amplitude was analyzed as the maximal positive peak within 250–450 ms on O1, O2 and Pz electrodes. The N1P2 amplitude was summed absolute amplitude of the N1 and P2 peaks on P7, P8, FC1, FC2, CZ, FZ and FCz electrodes. The N1P2 amplitude divided by average noise level yielded the signal-to-noise (S/N) ratio.

#### Statistical Analysis

All statistical analysis was performed by SPSS 19.0 (IBM, Armonk, NY). Demographic characteristics and clinical variables were compared between the two groups using the independent sample t-test or chi square analysis. The Spearman's correlation was analyzed between each ERP component parameter and MOCA item score. The association between SS-16 and MOCA item score was also analyzed by Spearman's correlation. All ERP components parameters were compared between the two groups using independent Mann–Whitney U-test. All tests were 2-tailed and P-values < 0.05 were considered statistically significant.

#### RESULTS

### Demographics and Clinical Characteristics

There was no significant group difference in demographic characters including age, sex, and education degree. No significant difference was revealed in disease duration, H-Y stage, tremor subtype and LED between PD with and without hyposmia. Questionnaire results of sleep disorder (PDSS, RBD-HK and ESS) as well as anxiety (HAMA) and depression (HAMD-17) were similar between the two groups. No significant difference was found in total score of MMSE or MOCA (**Table 1**).

#### Task Performance and Brain Activity

There was no significant difference between PD patients with and without hyposmia regarding number of valid trials, reaction time and accuracy. No significant difference was found in parameters of ERP components including N1, N2, P1, P2 and P300 between the two groups (**Table 2** and **Figure 2**). Notably, the N1P2 amplitude did not differ between the two groups. And the mean TABLE 2 | Task performance and parameters of ERP components in PD with and without hyposmia groups (mean ± standard deviation).

S/N ratio was 4.90 in the normosmic group and 5.17 in the hyposmic group and no difference was found between the two groups (**Table 2**).

### Correlation Between ERPs and Cognitive Ability

Correlation was analyzed between ERP component parameter and cognitive performance in all participants (**Tables 3**, **4**). N1 latency was significantly negatively related to visuospatialexecutive item score of MOCA (rs = −0.381, P = 0.012). Among three visuospatial-executive tests, a significant negative correlation between N1 latency and test score was found in cube copy test (rs = −0.401, P = 0.008) and clock drawing (rs = −0.451, P = 0.002) but not in trail making test. What's more, a significant positive correlation was found between P1 amplitude and language item score of MOCA (rs = 0.302, P = 0.049). There was no correlation between MOCA total score and N1 or P1 parameters. Parameters of other ERP components including N2, P2 and P300 were not found significantly associated with MOCA score or MOCA item score. Then the correlations between N1 latency and P1 amplitude and visuospatial-executive and language item score were also analyzed in separate groups (**Table 5**). There was a significant negative correlation between N1 latency and visuospatial-executive item score within the normosmic group (rs = −0.619, P = 0.005) but not within the hyposmic group. Among three visuospatial tests, a significant negative correlation with N1 latency was found in both cube copy test (rs = −0.585,

P = 0.008) and clock drawing test (rs = −0.523, P = 0.022) within the normosmic group and only in clock drawing test (rs = −0.413, P = 0.045) within the hyposmic group. In addition, a significant correlation between P1 amplitude and language item score was found within the normosmic group (rs = 0.537, P = 0.018) but not within the hyposmic group. Furthermore, SS-16 score was not found to be significantly associated with visuospatial-executive or language item score of MOCA (**Table 6**).

Correlation was also analyzed between parameters of N1 and P1 components and clinical characteristics in all subjects (**Table 7**). N1 latency was found to be significantly negatively related to education degree (rs = −0.326, P = 0.033). There was a significant positive correlation between HAMA score and N1 amplitude (rs = 0.360, P = 0.018). No significant correlation was found between other component parameters and clinical characteristics.

TABLE 3 | Correlation analysis between ERP latency and MOCA score in all participants (correlation coefficient rs).

∗P < 0.05; ∗∗P < 0.01.

### DISCUSSION

To our knowledge, our study was the first to compare cognitive ERP measures between PD patients with and without hyposmia. No significant difference in ERP performance was found between the two groups. Among all subjects, N1 latency was significantly negatively related to visuospatial-executive function and there was a significant positive correlation between P1 amplitude and language ability. Within the normosmic group, N1 and P1 also associated with visuospatial-executive and language functions while a significant correlation was only found between N1 latency and clock drawing test performance rather than visuospatialexecutive item score in the hyposmic group. What's more, odor identification ability was not found to be significantly related to visuospatial-executive function or language ability.

TABLE 4 | Correlation analysis between ERP amplitude and MOCA score in all participants (correlation coefficient rs).

TABLE 5 | Correlation analysis between parameters of ERP components (N1 and P1) and MOCA item (visuospatial-executive and language items) score in PD with and without hyposmia groups (correlation coefficient rs).

TABLE 6 | Correlation analysis between SS-16 score and MOCA item (visuospatial-executive and language items) score in all participants (correlation coefficient rs).

Hyposmia is an important clinical biomarker of PD and there are four subtypes of olfactory dysfunction reported in PD including impairment of odor identification, odor discrimination, odor threshold detection and odor recognition memory (Mesholam et al., 1998; Haehner et al., 2009). Odor identification deficit is the most prevalent form of hyposmia and is tested mainly by University of Pennsylvania Smell Identification Test (UPSIT) in PD patients (Doty et al., TABLE 7 | Correlation analysis between parameters of ERP components (N1 and P1) and clinical characteristics in all participants (correlation coefficient rs).

1988) and SS-16 (Haehner et al., 2009) which was used in our study considering its advantage in cross-cultural application.

Previous studies found a significant association between hyposmia and cognitive impairment in PD patients. The percentage of cognitive decline was significantly higher in PD patients with hyposmia than those without (Bohnen et al., 2010; Stephenson et al., 2010; Morley et al., 2011; Parrao et al., 2012). And olfactory dysfunction significantly increased the risk of dementia in PD patients regardless the cognitive functions at baseline according to a 10-year follow up study (Domellof et al., 2017). The underlying mechanism remained unclear. The olfactory bulb was known to be one of the onset sites with appearance of Lewy body pathology during PD progression in Braak's stage (Braak et al., 2003). And over expression of alphasynuclein in the olfactory bulb was reported to initiate hyposmia and other prodromal symptoms of PD in rats (Niu et al., 2018). A hypothesis was proposed that severe olfactory dysfunction might be related to abundant cortical Lewy body deposition (Morley et al., 2011) which was found to be associated with PD dementia (Tsuboi et al., 2007). What's more, the relation between hyposmia and cognitive impairment could also be explained by cholinergic denervation of the limbic archicortex (Bohnen et al., 2010) and dopaminergic denervation of the hippocampus (Bohnen et al., 2008). In our study, no significant difference was found in MMSE or MOCA score between PD with and without hyposmia, which may be associated with limits of our sample size and sensitivities of MMSE and MOCA in cognitive impairment of PD patients (Burdick et al., 2014; Wyman-Chick et al., 2017). Interestingly, no significant difference was found in parameters of ERP components including N1, N2, P1, P2 and P300 recorded during DMS tasks between these two groups. Therefore, the association between cognitive domains and ERP measures in PD with and without hyposmia was important to be investigated.

N1 and P1 were found to respectively associate with visuospatial-executive functions and language in our study while no significant correlation was found between the other components including P2, N2, and P300 and specific cognitive

functions. N1 and P1 are the earliest components in ERP representing visual sensory input. The generator of visual N1 is probably located in lateral extrastriate cortex (Gomez Gonzalez et al., 1994), dorsal occipito-parietal and ventral occipitotemporal areas (Yamazaki et al., 2000) and the visual P1 may be generated in fusiform gyrus (Heinze et al., 1994). N1 and P1 were reported to be associated with visual perceptual processing (Vogel and Luck, 2000; Taylor, 2002) especially in visuospatial attention according to previous reports (Naatanen and Picton, 1987; Luck et al., 1990). Luck et al. (1990) found that N1 was related to attention orienting to relevant stimulus and P1 possibly reflected a facilitation of early sensory processing with attention already focused. On the other hand, several studies demonstrated the role of N1 and P1 in language perception. Selpien et al. (2015) found that N1 and P1 were significantly related to language lateralization. In an auditory ERP study, the association between P1 amplitude and attention was found in linguistic probes stimulation but not in non-linguistic probes (Giuliano et al., 2014).

Abnormal N1 and P1 components in PD patients were observed in several studies. Wright et al. (1996) found attenuated N1 amplitudes during Auditory Oddball Task in 17 PD patients. However, enlarged N1 and P1 amplitudes as well as shortened N1 latency in 34 PD patients were demonstrated in a visual ERP study with oddball and S1–S2 tasks (Li et al., 2003). The discrepancy may be explained by the differences of tasks used in ERP, sample size and analytical approach of component parameters. Unfortunately, the clinical significance of N1 and P1 components in PD patients was investigated in very few studies. Wang et al. (2001) reported the association between N1 and cognitive visual processing in PD patients evidenced by its correlation with the regional cerebral blood flow. Consistent to previous studies, our study found a significant negative correlation between N1 latency and visuospatial-executive function in PD patients and P1 amplitude was significantly positively related to language ability. Therefore N1 latency and P1 amplitude during DMS task were possible to be used for assessing visuospatial-executive functions and language abilities in PD patients. Interestingly, the significant association between these cognitive functions and N1 and P1 components were also found in the normosmic group while in the hyposmic group, there was only a significant correlation between N1 and clock drawing test score rather than visuospatial-executive item score. To explain difference in clinical significance of N1 and P1 components between the two groups, one hypothesis could be that there exists different cognitive processing between these normosmic and hyposmic participants. Increasing evidence shows that PD with and without hyposmia may be two subtypes of PD patients. Lee et al. (2015) found that normosmic PD represented a unique clinical phenotype with a more benign with fewer motor deficits and higher dopamine transporter activity. On the other hand, hyposmia in PD was reported to be associated with tremor-dominant type (Iijima et al., 2011), SNCA rs11931074 and specific non-motor symptoms such as RBD and chronic constipation (Chen et al., 2015). From the aspect of cognition, a good olfactory performance in PD was found to compensate gray matter volume loss (Lee et al., 2014) and there were cholinergic and dopaminergic denervations in PD with hyposmia (Bohnen et al., 2008, 2010). Therefore, some components underlying a cognitive process such as visuospatialexecutive and language functions could be different between PD with and without hyposmia.

The impaired cognitive domains in PD with hyposmia were under debate. Bohnen et al. (2010) found that UPSIT scores were significantly related to episodic verbal learning in 58 PD patients but not other cognitive performance including visuospatial function, visual non-verbal memory, attention and executive function. However, poorer memory and executive functions were reported to be associated with worse olfactory identification in other studies (Morley et al., 2011; Parrao et al., 2012). Our study demonstrated no significant association between SS-16 score and visuospatial-executive or language item score of MOCA in PD patients. The discrepancy is possibly explained by difference in sample size and neurocognitive test batteries which may be avoided by using ERP measures.

There were several limitations of our study. A larger sample size is expected in further studies to confirm our results. In addition, only MOCA with relatively high reliability and validation reported (Nie et al., 2012) was used for cognitive assessment in our study as the psychometric properties of neurocognitive test batteries were rarely studied in Chinese PD patients. What's more, only odor identification was tested which was the mostly impaired olfactory function in PD (Doty et al., 1988). However, in view of possibly different mechanisms underlying four subtypes of hyposmia (Kareken et al., 2003; Hedner et al., 2010), the association between cognitive domains and the other three olfactory functions in PD warrants further investigation.

In conclusion, our study compared ERP measures recorded during DMS tasks at retrieval period and its association with cognitive domains between PD patients with and without hyposmia. No significant difference in ERP components was found between the two groups in the present study. Among all participants, N1 latency was significantly negatively related to visuospatial-executive functions and there was also a significantly positive correlation between P1 amplitude and language ability. In PD without hyposmia, N1 latency and P1 amplitude were respectively associated with visuospatial-executive and language functions while in PD with hyposmia, only a significant correlation was found between N1 latency and clock drawing test performance rather than visuospatial-executive item score in MOCA. What's more, no significant association was found between odor identification ability and visuospatial-executive or language item score of MOCA.

#### AUTHOR CONTRIBUTIONS

All the authors cooperated and contributed to the design and plan of the present study. Y-QL, S-SC, J-JD, GL, Y-XH, P-CZ, YF, PH, and CG performed the research. S-DC was in charge of clinical diagnosis. Y-QL and B-YL analyzed the data. Y-QL wrote the original article. S-DC and B-YL were in charge of manuscript verifying.

### FUNDING

fnagi-11-00018 February 2, 2019 Time: 18:20 # 8

This work was supported by grants from the National Natural Science Foundation of China (Nos. 81430022 and 81771374).

#### REFERENCES

#### ACKNOWLEDGMENTS

The authors thank the patients for their participation in the project.

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controlled search from secondary memory. Psychol. Rev. 114, 104–132. doi: 10.1037/0033-295X.114.1.104

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Lin, Cui, Du, Li, He, Zhang, Fu, Huang, Gao, Li and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Progressive Gait Deficits in Parkinson's Disease: A Wearable-Based Biannual 5-Year Prospective Study

Markus A. Hobert1,2,3, Susanne Nussbaum2,3, Tanja Heger2,3, Daniela Berg1,2,3 , Walter Maetzler1,2,3 and Sebastian Heinzel1 \*

1 Department of Neurology, Christian-Albrechts-University zu Kiel, Kiel, Germany, 2 Department of Neurodegenerative, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany, 3 German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany

Background: Gait changes occur during all Parkinson's disease (PD) stages and wearable sensor-derived gait parameters may quantify PD progression. However, key aspects that may qualify quantitative gait parameters as progression markers in PD remain elusive.

Objectives: Longitudinal changes in gait parameters from a lower-back sensor under convenient and challenging walking conditions in early- and mid-stage PD patients (E-PD, M-PD) compared to controls were investigated.

Methods: Normal- and fast-pace parameters (step: number, time, velocity, variability) were assessed every 6 months for up to 5 years in 22 E-PD (<4 years baseline disease duration), 18 M-PD (>5 years) and 24 controls. Parameter trajectories and associations with MDS-UPDRS-III were tested using generalized estimating equations.

Results: Normal-pace step number (annual change in E-PD: 2.1%, Time∗Group: p = 0.001) and step time variability (8.5%, p < 0.05) longitudinally increased in E-PD compared to controls (0.7%, −12%). For fast pace, no significant progression differences between groups were observed. Longitudinal changes in M-PD did not differ significantly from controls. MDS-UPDRS-III was largely associated with normal-pace parameters in M-PD.

Conclusion: Wearables can quantify progressive gait deficits indicated by increasing step number and step time variability in E-PD. In M-PD, and for fast-pace, gait parameters possess limited potential as PD progression markers.

Keywords: Parkinson's disease, progression marker, gait, wearable sensor, prospective study

## INTRODUCTION

Progression markers in Parkinson's disease (PD) are key to advances in PD prognosis and novel treatment efficacy measures. Yet, objective, reliable and quantitative markers of progressive motor deficits are still largely missing. Commonly, semiquantitative rating scales such as the MDS-UPDRS (Goetz et al., 2007) are used to assess motor symptoms and effects of disease modifyers. However,

#### Edited by:

Martin James McKeown, The University of British Columbia, Canada

Reviewed by:

Richard Camicioli, University of Alberta, Canada Antonio Suppa, Sapienza University of Rome, Italy

\*Correspondence: Sebastian Heinzel<EMAIL_ADDRESS>
Received: 29 October 2018 Accepted: 28 January 2019 Published: 13 February 2019

#### Citation:

Hobert MA, Nussbaum S, Heger T, Berg D, Maetzler W and Heinzel S (2019) Progressive Gait Deficits in Parkinson's Disease: A Wearable-Based Biannual 5-Year Prospective Study. Front. Aging Neurosci. 11:22. doi: 10.3389/fnagi.2019.00022

**21**

such clinical ratings are to some extent subjective, substantially placebo-responsive (Shin et al., 2016), partly rater-dependent (Post et al., 2005) and therefore prone to bias. Previously, stopwatch-based motor performance measures have been suggested as progression markers, specifically "turning pegs" and "inserting pegs" in functional dexterity/pegboard tests as measures of upper extremity brady- and hypokinesia (Haaxma et al., 2010). These timed measures have been shown to worsen significantly in early-stage (E-PD) but not mid-stage PD (M-PD) patients over 4 years compared to controls (Heinzel et al., 2017). For timed axial measures, including gait speed and timed-up-and-go-test, progression differences were not significant. However, using wearable sensors (so-called "wearables") gait can be quantified more specifically and more precisely suggesting promising potential of wearables-based progression markers. Previously, quantitative gait parameters have been prospectively assessed in de-novo PD patients (Galna et al., 2015). While not compared to healthy controls (HC), step length and swing time during convenient gait significantly decreased in PD from baseline to month 18. A subsequent 36-month analysis also including HC showed significant group differences between time points regarding step time, length and width variability (Rochester et al., 2017). One other study with unstandardized follow-up intervals compared gait parameters with the change of the item "gait" of the MDS-UPDRS-III and found an association between the worsening in the item "gait" and a decrease of stride length (Schlachetzki et al., 2017). However, key aspects that may qualify these quantitative gait parameters as progression markers in PD remain elusive. In particular, short interval progression characteristics over longer periods, progression in M-PD, and unspecific longitudinal changes in HC need further investigation. Moreover, whether the assessment of gait under convenient or challenging conditions best reveals progressive gait deficits in PD is still unknown.

The present prospective longitudinal study therefore investigated normal- and fast-pace gait as assessed with a lower-back wearable and a validated algorithm deriving gait parameters in E-PD, M-PD, and HC. Assessments were performed 6-monthly for up to 5 years. Differences in longitudinal changes of gait parameters in the PD groups relative to HC were analyzed.

## MATERIALS AND METHODS

### Prospective Study Design and Participants

Prospective data of the MODEP study (MODeling Epidemiological data to study Parkinson's disease progression) (Heinzel et al., 2016, 2017) with standardized biannual clinical and gait assessments over up to 5 years (10 visits) were analyzed. Forty patients with PD according United Kingdom Brain Bank criteria (Hughes et al., 1993), and 24 age- and sex-matched HC were included. Since symptom progression can depend on PD duration (Haaxma et al., 2010), patients were recruited as and a priori stratified into E-PD (<4 years baseline disease duration, n = 22) and M-PD (>5 years, n = 18) as suggested by neuropathological findings (Kordower et al., 2013). The study was approved by the local ethical committee (University of Tübingen; No 46/2010). All participants gave written informed consent.

### Clinical Assessment

Clinical assessments comprised current medication, height, weight, clinical ratings of PD motor symptoms (MDS-UPDRS-III) (Goetz et al., 2007), and Hoehn and Yahr stage. For axial scores, MDS-UPDRS-III axial items (3.9/3.10/3.12/3.13/3.14) were summed (Levy et al., 2000). Levodopa equivalent daily dose [LEDD; mg/day] was calculated (Tomlinson et al., 2010). Visits differed in ON/OFF medication state (E-PD: 18%; M-PD: 25% of visits in ON state) which was accounted for in statistical analyses. Moreover, the freezing of gait (FOG) questionnaire (Giladi et al., 2009) and the Montreal Cognitive Assessment (MoCA) (Nasreddine et al., 2005) were assessed. For FOG a score of 3 or higher in item #3, and for mild cognitive impairment a MoCA score of 22 or lower scores were considered (Carson et al., 2018).

### Gait Assessment

Participants were instructed to walk 20 m, first with normal (convenient) pace and then with fast pace (individual maximum), along a 2 m-wide straight corridor. Both conditions were performed twice, and gait parameters were averaged for the two trials. The wearable (Dynaport Hybrid, McRoberts B.V., The Hague, Netherlands) was fixed with a belt to the participants' lower back. The Dynaport Hybrid is an inertial measurement unit containing a 3D-accelerometer and a 3D-gyroscope with 100 Hz sampling frequency. Reliability of the sensor system and derived movement parameters has been shown previously using a instrumented Timed Up and Go (TUG) tests (van Lummel et al., 2016). After discarding acceleration and deceleration periods of walks (first and last 15% of the data; about 3 m each) (Lindemann et al., 2008), the company-provided validated gait analysis algorithm (Zijlstra and Hof, 2003; Brandes et al., 2006; Dijkstra et al., 2008; Houdijk et al., 2008; Hobert et al., 2017) was applied to extract the following gait parameters: step number, step time, step velocity, and measures of gait variability, i.e., step time variability (calculated as coefficient of variation), gait asymmetry (Yogev et al., 2007; Plotnik et al., 2009) and phase coordination index (PCI) (Plotnik et al., 2007).

#### Statistical Approach

Longitudinal data of gait parameters were analyzed using generalized estimating equations (GEE) with identity-link functions with normal distributions and exchangeable working correlation structure (Zeger et al., 1988; Hardin and Hilbe, 2003). GEE models comprised the subject ID, the within-subject variable Time (visit 1 to 10; centered), the factor Group (E-PD vs. HC; M-PD vs. HC), the interaction term Time∗Group (i.e., group difference in progression) and the covariates age (at baseline), ON/OFF medication state, weight, height, body-mass-index were considered. Parameters for normal- and fast-pace gait were selected as dependent variables. Group effects are related to the median of the observational period. The significance

level was α = 5% (two-sided). Bonferroni-corrections for multiple testing were applied considering two group comparisons and two gait conditions (p < 0.0125, significance threshold; 0.0125 < p < 0.05, statistical trend). Moreover, gait parameters were tested for associations with clinical PD parameters (MDS-UPDRS-III total score, axial score, Hoehn and Yahr stage, LEDD). Here, GEE models were calculated for the overall PD sample, and separately for E-PD and M-PD, comprising the respective clinical parameter and aforementioned covariates (except Group). For these exploratory analyses no correction for multiple testing was considered (p < 0.05). We used IBM SPSS Statistics, V22.0 (Armonk, NY, IBM Corp.) for statistical analyses.

#### RESULTS

E-PD and M-PD did not differ significantly in age and sex-ratio compared to HC. M-PD differed significantly in disease duration, but also in LEDD, MDS-UPDRS-III scores and Hoehn and Yahr stage (**Supplementary Table S1**).

PD groups showed a significantly larger step number and lower velocity compared to HC for normal-pace (E-PD: p = 0.001; M-PD: p < 0.001) and for fast-pace gait (p < 0.001). For GEE analyses, see **Supplementary Tables S2**, **S3**. Moreover, normal-pace step time CoV was significantly higher in M-PD compared to HC (p = 0.002). None of the other gait parameters showed significant differences between the PD groups and HC (p > 0.05), neither for normal- nor for fast-pace gait.

Longitudinal changes of the quantitative gait parameters are shown in **Figure 1**. Significant differences in longitudinal changes (p < 0.0125; Bonferroni-corrected) were only observed between HC and E-PD, and only for normal pace. Specifically, the progression in normal-pace step number differed significantly between E-PD and HC (Time∗Group, p = 0.001), with a significant increase over time only in E-PD (Time: p < 0.001), but not HC (Time: p = 0.070). Moreover, E-PD and HC differed regarding the longitudinal changes in normal-pace step time CoV (Time∗Group, p = 0.002), gait asymmetry (p = 0.009), and for trend, PCI (p = 0.028). However, while HC showed a decrease over time in gait variability parameters (step time CoV, p < 0.001; gait asymmetry, p = 0.003; PCI, p = 0.015), no significant change over time was observed in E-PD (p > 0.05). For fast-pace, none of the longitudinal changes of gait parameters differed significantly between groups.

Excluding individuals who at least once fulfilled the criteria for mild cognitive impairment (3 HC, 5 E-PD, 2 M-PD) or entering MoCA scores as additional factor into GEE analyses largely did not change the main results. Similarly, excluding individuals who reported freezing of gait (2 E-PD, 4 M-PD) or excluding visits in ON medication state resulted in the main findings of progression group differences in normal-pace step number and step time CoV, which remained significant and showed similar effect sizes. To further test the reliability of the results, we analyzed the performances in single gait trials instead of the average of the two trials. For each of the two single trials the interaction effect (Time∗Group) was observed (p < 0.05) for normal-pace step number and step time CoV in E-PD versus HC comparisons. Analyses of the ratio of normal/fast-pace gait parameters showed the longitudinal changes of ratio values to be less pronounced and with larger variability between visits and individuals compared to normalpace parameters alone.

Associations between clinical parameters and wearables-based gait parameters partly differed between gait conditions (normal/fast-pace) and PD groups (**Table 1**). For normal pace, gait variability in E-PD, and in M-PD additionally step number and velocity showed significant associations with clinical parameters. For fast pace, E-PD but not M-PD showed associations of gait variability with clinical parameters. In M-PD, axial scores showed larger associations with fast-pace compared to normal-pace step number and velocity.

### DISCUSSION

The present prospective observational study with 6-month intervals over a period of up to 5 years investigated the potential of quantitative gait parameters for the assessment of changes in normal- and fast-pace gait, respectively, in early- and mid-stage PD patients.

This study supports and extends previous findings suggesting longitudinal changes of normal-pace step number as a potential progression marker in E-PD (Galna et al., 2015; Schlachetzki et al., 2017). Changes were significantly larger in E-PD (2.1%/year) than in HC (0.7%/year), and, importantly, showed linear progression over the 5-year observation period. These findings remained robust when also accounting for further potential confounders and when excluding individuals with mild cognitive impairment and freezing of gait. This makes step number during normal pace a very promising and robust progression parameter for routine diagnostics and clinical trials.

Gait variability parameters may also possess potential as a progression marker in early phases of clinical PD. Significant differences in longitudinal changes were observed between E-PD and HC, however, this finding was not (only) due to a (non-significant) increase in PD (8.5%/year) but partly driven by HC decreasing in gait variability over time. Our finding supports our clinical impression that variability of gait shows relevant changes in short time periods in many PD patients, and may be one of the best predictors of disease milestones like falls (Hausdorff et al., 2003; Callisaya et al., 2011; Henderson et al., 2016). Indeed, previous cross-sectional analyses showed increased gait variability to be associated with PD duration (Hausdorff et al., 2003). Thus, the potential as PD progression marker should be further evaluated in future studies.

M-PD did not show any significant gait changes compared to HC during the relatively long observation period. With increasing PD duration between-patient differences in PD severity and phenotypes might become more apparent, thus single PD progression markers might not be valid for all PD patients (Fereshtehnejad et al., 2015). Overall, our results argue

differences in longitudinal changes compared to controls.

TABLE 1 | Associations of wearables-based and clinical parameters of gait deficits.

Significant associations (p < 0.05) between clinical measures of Parkinson's disease and wearables-based gait parameters are shown. Upper part: normal-pace parameters; lower part: fast-pace parameters. The parameter name and the relative (in %) and the absolute change of the parameter that occurred with one unit change of the respective clinical measure (shown on the left) are indicated. In columns, analysis results are shown for the entire PD group (left), and when only considering E-PD or M-PD groups. H and Y, Hoehn and Yahr stage; CoV, coefficient of variance; E-PD, early-stage Parkinson's disease; gait asym., gait asymmetry; LEDD, levodopa equivalent daily dose; MDS-UPDRS-III, Movement Disorder Society sponsored Unified Parkinson's Disease Rating Scale, part 3, motor examination; M-PD, mid-stage Parkinson's disease; PCI, phase coordination index; S.E., standard error.

against a relevant potential of quantitative gait analysis for the evaluation of disease progression in mid-stage PD.

Interestingly, normal-pace walking was more sensitive to gait-related changes than fast-pace walking. Compared to normal-pace, fast-pace gait might be more challenging and stressful, which may increase (behavioral) variability and confounding with unspecific age-related factors. Therefore, we recommend normal-pace walking to be included in, e.g., clinical trials to assess PD progression, and to omit challenging walking conditions and normal/fast-pace comparisons.

Established clinical PD parameters showed associations with the quantitative wearables-based gait parameters thereby supporting their value and clinical relevance. Associations were largely observed in M-PD, and for normal-pace conditions. Differences in associations might be due to lower variance of the clinical data in E-PD than in M-PD. Moreover, MDS-UPDRS-III total and axial sum scores may less specifically indicate motor symptoms compared to quantitative gait parameters, and in E-PD more specific quantification of motor symptoms may be required. Fast-pace conditions might introduce unspecific (error) variance into gait variability parameter data, thus compared to normal-pace their associations with clinical measures might be weaker.

These present results might also provide future clinical and scientific perspectives. Parameters that allow to quantify disease progression, such as kinetic gait parameters derived from wearable devices, could serve as objective outcome markers of pharmacological treatment and other interventions. In this regard reliable quantitative markers of PD motor symptoms and their progression are highly promising as (at least secondary) outcomes in the clinical routine as well as in clinical intervention trials (Moreau et al., 2018). These measures have the potential of increasing sensitivity and objectivity while reducing costs of these clinical trials (Merchant et al., 2018). Importantly,

wearable-based motor measures possess a high relevance for daily living and quality of life (Van Uem et al., 2016), and studies based on such measures may therefore yield results that benefit PD patients.

This study faces some limitations. First, the sample was relatively small. However, the thorough and high-frequency evaluation of participants may partially compensate for this limitation, especially as parameters are needed that are robust even in smaller samples. Second, PD is a heterogeneous disease and further subgroups and non-motor symptoms potentially influencing gait, e.g., depression, were not accounted for. Also, multi-facetted interventions were not controlled for in this observational study. Longitudinal differences in pharmacological and non-pharmacological treatments and their efficacy over time may additionally contribute to the heterogeneity in PD, particularly in advanced PD. While accounting for ON/OFF medication state in statistical analyses modeling of individual treatments is complex. Thus, subtle gait changes and factors contributing to progression differences between PD patients might have remained undetected.

To conclude, number of steps and possibly also gait variability measures as assessed during normal-pace walking with a lower-back wearable are robust and promising progression markers in the early phase of PD.

#### AUTHOR CONTRIBUTIONS

DB and WM conceived the study. DB, WM, TH, and SN organized the study. WM, TH, SN, and MH executed the study. SH and MH designed the statistical analysis. SH executed the statistical analysis. DB, WM, and MH critically reviewed the

#### REFERENCES

results. MH and SH wrote the first draft of the manuscript. DB, WM, and MH critically reviewed the manuscript.

#### FUNDING

The MODEP study was initially supported by Solvay Pharmaceuticals and received financial support by Teva Pharmaceutical Industries Ltd. The analysis of the MODEP data was supported by Lundbeck. The funders were not involved in study design, data collection and analysis, decision to publish, and preparation of the manuscript. None of the authors reports any financial interests or potential conflicts of interest related to this study.

#### ACKNOWLEDGMENTS

We thank the participants for participation in the study. We would also like to thank Dr. Maren Ellerbork, Dr. Jana Godau, Dr. Sandra E. Hasmann, Dr. Miriam Maechtel and Irénée Kanyiki for performing assessments during the active data collection phase. Moreover, we acknowledge financial support by Land Schleswig-Holstein within the funding program Open Access Publikationsfonds.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnagi. 2019.00022/full#supplementary-material

unified parkinson's disease rating scale (MDS-UPDRS): process, format, and clinimetric testing plan. Mov. Disord. 22, 41–47. doi: 10.1002/mds.21198

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Hobert, Nussbaum, Heger, Berg, Maetzler and Heinzel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Hydralazine Protects Nigrostriatal Dopaminergic Neurons From MPP+ and MPTP Induced Neurotoxicity: Roles of Nrf2-ARE Signaling Pathway

Xingfang Guo1 , Chao Han2 , Kai Ma1 , Yun Xia1 , Fang Wan1 , Sijia Yin1 , Liang Kou1 , Yadi Sun1 , Jiawei Wu1 , Junjie Hu1 , Jinsha Huang1 , Nian Xiong1 and Tao Wang1 \*

*1 Department of Neurology, Union Hospital,Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 2 Department of Neurology, The First Affiliated Hospital of USTC and Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China*

#### Edited by:

*Martin James McKeown, University of British Columbia, Canada*

### Reviewed by:

*Margarida Castro-Caldas, New University of Lisbon, Portugal Petr A. Slominsky, Institute of Molecular Genetics (RAS), Russia*

> \*Correspondence: *Tao Wang<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology*

Received: *18 September 2018* Accepted: *28 February 2019* Published: *20 March 2019*

#### Citation:

*Guo X, Han C, Ma K, Xia Y, Wan F, Yin S, Kou L, Sun Y, Wu J, Hu J, Huang J, Xiong N and Wang T (2019) Hydralazine Protects Nigrostriatal Dopaminergic Neurons From MPP*+ *and MPTP Induced Neurotoxicity: Roles of Nrf2-ARE Signaling Pathway. Front. Neurol. 10:271. doi: 10.3389/fneur.2019.00271* Although the pathogenic mechanisms of Parkinson's disease (PD) remain unclear, ample empirical evidence suggests that oxidative stress is involved in the pathogenesis of this disease. The nuclear factor E2-related factor 2 (Nrf2) is known to activate several antioxidant response element (ARE)-driven antioxidative genes that prevents oxidative stress *in vitro* and *in vivo*. Moreover, it was documented that hydralazine is a potent Nrf2 activator. In this study, we tested whether hydralazine can attenuate 1-Methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced neurotoxicity *in vitro* and *in vivo* by activating Nrf2 and its downstream network of antioxidative genes. We found that treatment with hydralazine attenuated MPP+ or H2O2-induced loss of cell viability in human neuroblastoma cell line (SH-SY5Y). In addition, hydralazine significantly promoted the nuclear translocation of Nrf2, and upregulated the expression of its downstream antioxidative genes. Further, knockout of Nrf2 abolished the protection conferred by hydralazine on MPP+ -induced cell death. Similar findings were observed *in vivo*. Before, during, and after MPTP 30 mg/kg (i.p.) administration for 7 days, the mice were given hydralazine (Hyd) 51.7 mg/kg per day by oral gavage for 3 weeks. Oral administration of hydralazine ameliorated oxidative stress, MPTP-induced behavioral disorder, and loss of neurons of dopaminergic system in the substantia nigra (SN) and striatum, all of which were attributed to its ability to activate the Nrf2-ARE pathway. Hydralazine increased the migration of Nrf2 to the nucleus in dopaminergic neurons, enhanced the expression of its downstream antioxidative genes. Together, these datasets show that the Nrf2-ARE pathway mediates the protective effects of hydralazine on Parkinson's disease.

Keywords: Parkinson's disease, hydralazine, neuroprotection, Nrf2-ARE signaling pathway, MPTP, MPP+

### HIGHLIGHTS

#### INTRODUCTION

Currently, the pathogenesis of Parkinson's disease (PD) is elusive. It has been postulated that prolonged increased in reactive oxygen species (ROS) plays important role in modulating the occurrence of the disease (1, 2). This is because ROS compromises the mechanisms that balances oxidant and antioxidant systems (3). One of the mechanisms that prevent cell damage caused by oxidative stress is the antioxidant defense system. By increasing the level of antioxidant enzymes, phase II detoxifying enzymes, quinone oxidoreductase 1 (NQO1), NAD(P)H, heme oxygenase-1 (HMOX1), glutamate-cysteine ligase subunits (GCLC and GCLM), the nuclear factor E2 related factor 2 (Nrf2) modulates the pathophysiological and physiological processes of various diseases (4). These enzymes are involved in glutathione (GSH) synthesis and maintains the form of GSH by suppressing its oxidized form GSSG (5, 6). Several structural and molecular studies on Nrf2 revealed that Nrf2 is expressed in the cytoplasm in physiological conditions via a Kelch-like ECH-associated protein 1 (Keap1)-dependent ubiquitination-proteasomal degradation, and this process is enhanced by electrophiles and oxidants (7, 8). It was revealed that the N terminal region and double glycine repeat domain (DGR) of Keap1 contains the bric-a-brac, tramtrack, broad-complex (BTB) domain, termed as Kelch repeats in the C terminal region. Keap1 keeps the Nrf2 within the cytoplasm in normal cellular conditions by binding to a scaffold protein of Nrf2 ubiquitin ligase (E3), Cul3 through its BTB domain, and binding to the substrate of Nrf2 through its DGR domain, which causes Nrf2 degradation (9) and ubiquitination (10). When stimulated by oxidants or electrophiles, Nrf2 undergoes modifications by some mechanisms which compromise Keap1/Nrf2 interactions, which leads to the dissociation of Nrf2 from Keap1 complex, become stabilized, and then moves into the nucleus. This is followed by binding to the antioxidant response elements (AREs), a consensus gene sequence located in the promoter region of several genes that encode antioxidant enzymes (11). Several studies have provided evidence that Nrf2-ARE signal transduction participates in PD (12, 13). Downregulation of Nrf2 renders the dopaminergic neurons susceptible to oxidative stress damage (14–16), while activation of Nrf2 confers neuroprotection (17–21). Based on this background, the Nrf2-ARE interaction is thought to play important role in neurodegenerative conditions including PD.

Administration of pharmacological antioxidants seem to be the most direct approach to suppress oxidative damage. However, clinical trials on antioxidants e.g., N-acetylcysteine, vitamin E, glutathione, and vitamin C have revealed rather disappointing results for PD (22–25). One possible reason is that the efficacy of these antioxidants is largely based on their ability to stoichiometrically scavenge for oxidants. An alternative promising therapeutic strategy of restoring redox homeostasis in PD by activating the transcription factor Nrf2 may have significant advantages over conventional strategies. Hydralazine (Hyd), an FDA approved treatment for hypertension, is a watersoluble carbonyl-scavenger due to its nucleophilic hydrazine group (26). Because of this, hydralazine has been found to be a powerful antioxidant properties (27–29), Moreover, it was reported that hydralazine could activate the Nrf2 signaling pathway in vitro [human neuroblastoma cell line (SH-SY5Y)] and in vivo (Caenorhabditis elegans) model system (30), and possesses anti-aging properties (31). As mentioned above, oxidative stress and the deregulation of Nrf2-ARE signaling pathways are both involved in PD. Thus, we hypothesized that hydralazine may provide strong neuroprotective effects in Parkinson's disease in both in vitro and in vivo settings. To test this possibility, we investigated the mechanisms that orchestrate the neuroprotective effects of hydralazine using an 1-Methyl-4-phenylpyridinium (MPP+)-induced cytotoxicity model and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice model of PD. We found that hydralazine displayed promising therapeutic efficacy toward PD by activating the Nrf2 signaling pathway.

#### EXPERIMENTAL PROCEDURES

#### Preparation of Human Neuroblastoma SH-SY5Y Cells

SHSY5Y cells were obtained from ATCC (ATCCCRL-2266) and grown in DMEM/F12 medium (hyclone) supplemented with 10% FBS (EVERY GREEN, Zhejiang Tianhang Biotechnology Co., Ltd, China), 100µg/mL streptomycin, and 100 U/ml penicillin (Beijing solarbio science & technology co., Ltd) in high humidity condition with 5% CO2 at 37◦C. After culturing the cells in 100 mm dishes to reach a ∼70% confluence, they were subjected to hydralazine, H2O2 or MPP+ treatment. The dose and duration of application of hydralazine, H2O2 or MPP+ are provided in the figures and text. SiRNA interference were performed by treating the cells with Nrf2 SiRNA (sc-37030) or control SiRNA (sc-37007) (Santa Cruz Biotechnology, Santa Cruz, CA) in 6-well plates for 24 h using the Lipofectamine 3000 reagent (Thermo Fisher Scientific Co., Carlsbad, CA, USA) as indicated in the instructions provided by the manufacturer. After transfection for approximately 24 h, SHSY5Y cells were exposed to hydralazine with or without MPP+. After these treatments, cells were used for biochemical analysis.

#### Cell Viability Evaluation by CCK-8 Assay

The cell counting kit-8 solution (CCK-8) assay was performed to determine the cell viability. Briefly, after seeding the SH-SY5Y cells in 96-well plates at a density of 1 × 104 cells/well, they were treated with reagents. This was followed by incubating with 10 µl CCK-8 buffer for 1 h at 37 C following the instructions provided by the kit company. A microplate reader (BioTek, Winooski, VT, USA) was used to measure the absorbance at 450 nm. All samples were assessed in triplicate.

#### Quantitative Real-Time PCR

Total RNA was isolated from SHY-SY5 cells using RNAiso Plus (TaKaRa, Japan). Total RNA (2 µg) was reverse transcribed to cDNA using the PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa, Japan) to determine the mRNA expressions of Nrf2 by qRT-PCR using SYBR Green reagent (TaKaRa, Japan). The PCR condition was as follows: 95◦C for 5 min, 60◦C for 20 s, 40 amplification cycles. Housekeeping gene β-actin served as an internal control. Data analysis is based on the 11Ct method with normalization of raw data to β-actin. Each reaction was run in triplicate. Nrf2 primer: forward, 5′ -CAGTCAGCGACGGAA AGAGT-3′ ; reverse, 5′ -ACGTAGCCGAAGAAACCTCA-3′ ; βactin primer: forward, 5′ - AGCCATGTA CGTAGCCATCC−3 ′ ; reverse, 5′ - CTCTCAGCTGTGGTGGTGAA -3′ .

### Animals and Treatment

The mice used in this study were kept and handled according to the guidelines of the NIH Guide regarding the Use and Care of Laboratory Animals. All animals were given free water and food ad libitum, and the housing conditions were regulated to a 12 h dark-light cycle and temperature of (22 ± 2 ◦C). This study conformed to the guidelines of the Animal Care and Use Committees of Maximum efforts of Huazhong University of Science and Technology (HUST). Care was taken to use few animals and reduce discomfort. Male C57/BL6 mice, 8 weeks old, were used all of which were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. There are four groups (n = 8 or 9 for per group) in this experiment. The first group mice (MPTP group) only received injections of MPTP-HCl (30 mg/kg, i.p., Sigma) in saline for consecutive 7 days, an MPTP model of PD was generated as previously described (32). The second group mice (Hyd+MPTP group) were administered hydralazine (51.7 mg/kg per day in saline, Sigma) (33) by oral gavage for 3 weeks before, during, and after MPTP administration. The third group mice (Hyd group) were administered hydralazine (51.7 mg/kg per day in saline, Sigma) (33) by oral gavage for 3 weeks, and the fourth group (Control group) received vehicle only. Behavioral test of the animals were performed after the last oral gavage, after behavior test, mice were killed (**Figure 3A**).

### Rotarod Testing

The general motor deficits were evaluated using the rotarod test. Mice were put in a rod which was 7 cm in diameter, and then tested at a constant speed of 30 rpm. In each test, the mouse was subjected to the rod for 1 min prior to the test. Each animal was put on the roller for 5 min, and then the latency to fall off the rolling rod was calculated. Prior to assessing the behavior of the animals, they were given pre-trials for habituation to the test system for 4 days. An average of five trials were performed for each mouse.

### Pole Testing

Each mouse was placed on the top of a vertical pole (50 cm long and 1 cm in diameter) wrapped with gauze to avoid slip and fall. After pre-trials for habituation to the test system for 4 days, time of the mouse head orient downward (named as Turn) and total time climbing down the pole as an indication of the locomotion activity (TLA) were recorded. Each mouse received five successive trials for average.

#### Tissue Preparation

One day after the behavior test, animals were killed by decapitation under anesthesia. Afterwards, brains were removed immediately was fixed in 4% buffered paraformaldehyde and embedded in paraffin for the next immunofluorescent staining. The ventral midbrain containing the substantia nigra and striatum of the other mice were dissected and frozen immediately in liquid nitrogen and stored at −80◦C for protein extraction.

#### Immunofluorescent Staining

After isolation, the substantia nigra and striatum tissues were embedded in paraffin and sectioned to 5µm thickness. The sections were deparaffinized with xylene, followed by dehydration using ethanol. Finally, EDTA (PH 9.0) antigen retrieval buffer was used for antigen retrieval. These section were put on slices followed by treatment with 3% BSA at room temperature for 30 min, before they were incubated with the following primary antibodies at 4◦C overnight, mouse polyclonal anti-TH (1:200, Proteintech, China), rabbit polyclonal anti-Nrf2 (1:200, Genetex), rabbit monoclonal anti-IBA1(1:200, Abcam), rabbit monoclonal anti-GFAP (1:200, Abcam). Subsequently, secondary antibodies, Alexa Fluor 488-conjugated (1:500, Life Technologies) and/or Alexa Fluor 647-conjugated (1:500, Life Technologies) were added and incubated for 1 h at room temperature. SH-SY5Y cell were fixed with 4% paraformaldehyde for 30 min, washed in PBS, and permeabilized with 0.3% Triton X-100 in PBS for 10 min at room temperature. The cells were treated with rabbit polyclonal anti-Nrf2 (1:200, Genetex) antibody overnight at 4◦C after blocking with 10% normal goat serum for 1 h, and then incubated with Alexa Fluor 488 conjugated (1:500, Life Technologies) secondary antibody for 1 h at room temperature. Images from the sections were examined on an OLYMPUS IX71 fluorescent microscope. Then quantitative analysis of the optical density and of number positive neurons was performed using Image pro plus.

#### Determination of Protein Expression

Nuclear and cytosolic fractions were extracted from the substantia nigra, striatum specimen and human neuroblastoma SH-SY5Y cells using cytoplasmic and nuclear protein extraction kit (Beyotime, Beijing, China) following the instructions given by the manufacturer. Briefly, cells were subjected to various treatments and then suspended in PBS. The next step involved the addition of the cytoplasmic protein isolation reagents A and B. The cytosolic fraction was obtained by centrifugation. The nuclei pellets were suspended in the isolation reagent for nuclear protein. Similarly, the nuclear fraction was obtained by centrifugation. All the isolated proteins from mouse striatum, ventral, and SH-SY5Y cells were treated on ice with RIPA lysis buffer comprising of phosphatase inhibitor A and B, protease inhibitor PMSF (PMSF:RIPA = 1:99), cocktail (Servicebio, Wuhan, China), followed by centrifugation at 12,000 g, for 15 min at 4◦C, to obtain the supernatants. BCA Protein Assay Kit was applied to measure the protein concentration. This was followed by denaturation of the protein specimen using the sodium dodecyl sulfate solution together with boiling for 5 min at 98◦C. About 30 µg sample was loaded onto SDS-PAGE after which electrophoresis was performed to transfer the proteins to a PVDF membrane. Thereafter, 5% milk was using to block the membranes at room temperature for 1 h before primary antibodies, rabbit monoclonal anti-β-actin (1:3000, Antgene, China), rabbit polyclonal anti-Histone H3 (1:1000, Servicebio, China), mouse monoclonal anti-GCLC (1:500, Santa Cruz), rabbit polyclonal anti-Nrf2 (1:1000, Genetex), rabbit monoclonal anti-NQO1 (1:10000, Abcam), rabbit monoclonal anti-HMOX1 (1:2000, Abcam), rabbit monoclonal anti-GCLM (1:5000, Abcam), and rabbit polyclonal tyrosine hydroxylase antibody (1:1000, Proteintech, China) were added and incubated overnight at 4◦C. Finally, HRP-conjugated secondary antimouse antibody (1:5000, Antgene, China) or HRP-conjugated secondary anti-rabbit (1:5000, Antgene, China) was added to the membranes after washing with TBST thrice (5 min/each time) for 1 h at room temperature. After washing, ECL was loaded onto the membrane to detect the immunoreaction using the Bio-Rad imaging system. The branes were analyzed with Imagine J software. Relative band intensities were measured with β-actin or Histone H3 serving as internal control.

#### Determination of the Oxidative Activity by Lipid Peroxidation Assay

The generation of malondialdehyde (MDA) was measured using the lipid peroxidation commercial assay kit following the instructions provided by the manufacturer. Briefly, and tissue were homogenized, sonicated, and centrifuged. The MDA concentration on in the supernatants was measured as well as the protein concentration. The protein concentration was determined using the assay kit (Bio-Rad Laboratories, Hercules, USA). This was followed by performance of the MDA assay following the instructions provided with the Lipid Peroxidation MDA assay kit (Beyotime). The multimode microplate reader was used to quantify the MDA levels in the samples at 532 nm.

### Determination of Cellular Levels of GSSG and GSH Using HPLC

Substantia nigra samples were homogenized, sonicated and centrifuged. In brief, 30 µl supernatant was isocratically eluted through a 4.6 × 150 mm C18 column (ESA, Inc.) with a mobile phase containing 50 mM LiH2PO4, 1.0 mM 1-octanesulfonic acid, and 1.5% (v/v) methanol, and then detected by a 2-channel Coulochem III electrochemical detector (ESA, Inc), set with a guard cell potential 950 mV, Channel 1 potential for GSH detection and Channel 2 potential 880 mV for GSSG detection (34). The quantities of GSSG and GSH were presented as nmol per mg protein.

### Data Analysis

All data are expressed as mean ± SEM and were performed using the SPSS20 software. Between-group comparisons were conducted by Student's t-test, while the one-way ANOVAs was used to compare three or more groups followed by Tukey's multiple comparison test. A P-value of < 0.05 was considered to be statistically significant.

### RESULTS

### Hydralazine Prevents the MPP+ and H2O2-Induced Cell Death in SH-SY5Y Cells, and Activates Nrf2-ARE Signaling in Both Treated and Non-treated MPP + Cells in vitro

Analysis of the influence of MPP+ on SH-SY5Y viability revealed that the cell viability decreased to about 50% or below based on CCK-8 assays when exposed to 1000 µM (1 mM) MPP+ for 24 or 36 h (**Figure 1A**). Therefore, we treated SH-SY5Y cells with MPP+1 mM for 24 h to establish a model of MPP+ induced cytotoxicity. Next, the effects of hydralazine on the survival of cells following exposure to MPP+ were tested. Four doses of hydralazine (2.5, 5, 10, or 20µM) were tested against MPP+. Hydralazine alone at the micromolar range (2.5– 20µM) had no overt effects on these cells. But, hydralazine (10 and 20µM) conferred significant protection against MPP+ according to the CCK-8 assays (**Figure 1B**). Moreover, we chose H2O2 (100µM) as another control of toxicity (30). Similarly, hydralazine protected the cells from H2O2-induced cytotoxicity (**Figure 1C**). Moreover, we observed that hydralazine (10 and 20µM) alone increased the viability of SH-SY5Y in the absence of MPP+ and H2O2 (**Figures 1B,C**). It is wellknown that during cellular metabolism, spontaneous oxidative damage to unsaturated lipids generates many electrophilic carbonyl compounds which are potential threats to cell survival. Since hydralazine is a well-known carbonyl scavenger (35), we speculated that it can improve SH-SY5Y viability by scavenging Carbonyl moieties.

Thereafter, we tested the ability of hydralazine to induce nuclear translocation of Nrf2. SH-SY5Y cells were treated with 10µM hydralazine with or without MPP+, but the control groups were treated with saline following Nrf2 partition quantification and subcellular fractionation. Hydralazine treatment increased the nuclear translocation of Nrf2, which was accompanied by a corresponding decreased in the cytosolic fraction relative to the nuclear Nrf2 fraction (**Figures 1D–F**). The same phenomenon of nuclear translocation was observed based on immunofluorescence (**Figure 1G**). As showed in **Figures 1D–G**, treatment with hydralazine alone triggered a remarkable increase in the nuclear translocation of Nrf2, but the reasons for this effect remain unclear. It is likely that Nrf2 was modified by hydralazine in a way that disrupted the Nrf2:Keap1 interaction, and then, translocated to the nucleus. Further studies are required to answer this question. Next, we determined whether hydralazine (10µM) would increase the downstream protein of the Nrf2-ARE pathway,

FIGURE 1 | Hydralazine prevents the MPP+ and H2O2-induced cell death in SH-SY5Y cells, and activates Nrf2-ARE signaling in both treated and non-treated MPP + cells *in vitro*. SH-SY5Y cells were incubated with various concentrations of MPP+ for 24, 36 h (A). Cell viability was determined by CCK-8 assay. \**p* < 0.05, \*\**p* < 0.01, *n* = 6, compared with the two indicated groups. SH-SY5Y cells were incubated with or without hydralazine (2.5, 5, 10, 20µM) in the absence or presence of 1 mM MPP+ for 24 h (B). The viability of cells under MPP+-mediated cytotoxicity was significantly improved with hydralazine (10 and 20µM) treatment. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, *n* = 6, significant difference between the two indicated groups. SH-SY5Y cells were incubated with or without hydralazine (2.5, 5, 10, 20µM) in the absence or presence of H2O2 (100µM) for 24 h (C). The viability of cells under H2O2 -induced cytotoxicity was significantly improved with hydralazine (10 and 20µM) treatment. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001 *n* = 6, significant difference between the two indicated groups. The results of immunofluorescence and western blot analysis indicated Nrf2 translocated to the nucleus with hydralazine treatment when exposured to MPP+ or not in SH-SY5Y (D–G). Scale bar =500µm. Treated cells were subjected to cell fractionation and western blot analysis. β-actin or Histonen H3 served as a loading control. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, *n* = 6, significant difference between the two indicated groups. Hydralazine induces expression of Nrf2-dependent antioxidant enzymes measured by western blot analysis (H,I). \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, *n* = 6, significant difference between the two indicated groups. Data were presented as mean ± SEM.

GCLC, GCLM, HMOX1, and NQO1 by western blot analysis. All four protein levels of target genes were significantly increased (**Figures 1H,I**). Collectively, the in vitro findings indicate that hydralazine induce nuclear translocation of Nrf2 which activates the ARE genes, thereby conferring protection against MPP+.

#### Hydralazine Activate Nrf2-ARE Signaling and Attenuate MPP+-Mediated Cytotoxicity in an Nrf2-Dependent Pattern

To confirm whether the Nrf2-dependent pathway mediated the neuroprotective effects of hydralazine, the inhibitory effect of hydralazine on MPP+ neurotoxicity was assessed in SH-SY5Y cells after transfection with either control SiRNA or Nrf2 SiRNA. Results from western blotting and real-time PCR indicated that Nrf2 was successfully knocked-down in SH-SY5Y cells by Nrf2 SiRNA compared to control SiRNA (**Figures 2A–C**). Treatment with hydralazine alone triggered a remarkable increase in the nuclear translocation of Nrf2 (**Figures 2D–F**). However, Nrf2 interference abolished the increase in nuclear Nrf2 caused by hydralazine in SH-SY5Y cells. On the other hand, cytosolic Nrf2 displayed a corresponding decrease relative to the control group transfected with control SiRNA. Furthermore, the hydralazineinduced increase in expression of Nrf2-mediated ARE genes, such as GCLC, GCLM, HMOX1, and NQO1 were inhibited following Nrf2 SiRNA in SH-SY5Y cells based on western blot analysis (**Figures 2G,H**). According to these results, we speculate hydralazine activate Nrf2-ARE signaling in an Nrf2 dependent manner.

Meanwhile, our results indicated that hydralazine treatment increased cell viability, which is opposite to the CCK8 assay results recorded in MPP+ exposure alone group in SH-SY5Y cells transfected with control SiRNA (**Figure 2I**, p < 0.01). However, hydralazine failed to increase cell viability following Nrf2 knockdown and MPP+ exposure (**Figure 2I**). Nrf2 SiRNA transfection in SH-SY5Y cells increased their vulnerability to MPP+ neurotoxicity as compared to the control SiRNA transfection group (**Figure 2I**, p < 0.05). These data reveal that the protective effect of hydralazine on SH-SY5Y cells exposed to MPP+ neurotoxicity is dependent on the Nrf2 pathway.

#### Hydralazine Confers Protection in Dopaminergic Neurons in the MPTP Model of Parkinson's Disease

To explore the therapeutic effect of hydralazine in PD, the MPTP mice model was used. Mice were divided into four groups (n = 9 for per group) for this experiment. The first group mice (MPTP group) only received injections of MPTP-HCl (30 mg/kg, i.p., Sigma) in saline for consecutive 7 days, an MPTP model of PD was generated as previously described (32). The second group of mice (Hyd+MPTP group) were administered with hydralazine (51.7 mg/kg per day in saline, Sigma) (33) by oral gavage for 3 weeks before, during, and after MPTP administration. The third group mice (Hyd group) were administered with hydralazine (51.7 mg/kg per day in saline, Sigma) (33) by oral gavage for 3 weeks, and the fourth group mice (Control group) received vehicle only. The pretreatment paradigm in the MPTP-induced PD Model was utilized (**Figure 3A**). We observed that the weight of MPTP-treated mice was much lower compared to normal group due to the neurotoxicity of MPTP. But administration of hydralazine alleviated the loss of weight (**Figure 3B**). Behavior testing of the animals was performed after the last oral gavage (**Figure 3A**). The rotarod test was applied to evaluate motor and coordination abilities. In this test, the decrease in latent time on the rod in the MPTP mice compared to the vehicle treated control mice (p < 0.001) was reversed by hydralazine treatment (p < 0.01) (**Figure 3C**). Similar results were obtained in Pole test. The time to orient downward (Turn) and to descend (TLA) was increase in the MPTP mice compared to the control mice (p < 0.05), indicating a motor deficit, and this effect was reversed by hydralazine (p < 0.05) (**Figure 3D**). These results suggested that hydralazine conferred protection in locomotion function.

After the behavioral tests, we tested whether in vivo hydralazine can maintain the integrity of dopaminergic neurons in the SNpc and their terminal fibers in striatum. Thus, we performed immunofluorescence staining using dopaminergic neuronal specific marker, anti-TH antibody. MPTP administration caused remarkable loss of dopaminergic neurons in the SNpc (**Figures 3E,F**) and striatal DA terminal fibers (**Figures 3I,J**) when compared to control. Meanwhile, we found that reduction of TH-positive neurons was abolished in the similar anatomic level sections of SN in the MPTP mice treated with hydralazine than MPTP alone (**Figure 3E**). Quantification analysis using the cell counting kit in a double blind way indicated a statistically significant preservation of TH positive dopaminergic SN neurons in the MPTP-treated mice with hydralazine groups compared with MPTP mice (**Figure 3F**, p < 0.01). This observation was further confirmed by western blotting assay on SN lysates (**Figure 3G**). The ratio of TH/βaction in the MPTP group was significantly lower than in control group (p < 0.001) and MPTP mice with hydralazine groups (**Figure 3H**, p < 0.05). Hydralazine-treated mice in the presence of MPTP showed significant reductions in the loss of the TH positive DA terminals in the striatal region when compared to MPTP alone mice (**Figures 3I,J**, p < 0.01). Consistent with TH-immunofluorescent in the striatal region, western blotting of the striatum lysates rescued the loss of TH positive DA terminals in the hydralazine-treated mice with MPTP compared to MPTP alone mice (**Figures 3K,L**, p < 0.001), Taken together, these findings provide evidence that hydralazine confers protection on nigrostriatal dopaminergic neurons in the subacute model of MPTP neurotoxicity.

### Hydralazine Alleviate Oxidative Stress and Activates Nrf2-Triggered Gene Expression in vivo

Subsequently, we investigated whether hydralazine activates Nrf2-ARE pathway in vivo using mice. Mice were treated as described above to assess the level of MDA, a key product produced from membrane lipid oxidation. Mice treated

FIGURE 2 | with control group transfected with control SiRNA (D,F) by western blot analysis. \**p* < 0.05, \*\**p* < 0.01, *n*=6, significant difference between the two indicated groups. Selective activation of Nrf2 mediated gene transcription by hydralazine in SH-SY5Y cells transfected by control SiRNA, but not in SH-SY5Y cells transfected by Nrf2 SiRNA measured by western blotting (G,H). \**p* < 0.05, *n* = 6, significant difference between the two indicated groups. Hydralazine significantly protected SH-SY5Y cells from MPP+ induced death in an Nrf2-dependent manner measured by CCK-8 assay (I). \**p* < 0.05, \*\**p* < 0.01, *n* = 6, significant difference between the two indicated groups. Data were presented as mean ± SEM.

with MPTP displayed a remarkably increase in MDA levels which were suppressed by hydralazine treatment in the SNpc (**Figure 4A**) and in striatum (**Figure 4B**). Glutathione (GSH) is an antioxidant which has the capacity to prevent damage to important cellular components. Glutathione disulfide (GSSG) is its oxidized form and the ratio of oxidized glutathione to reduced glutathione is frequently used to evaluate oxidative stress. MPTP decreased GSH levels and increased GSSH levels and these effects were reversed by hydralazine in the SNpc, as supported by the increased ratio of GSH/GSSG (**Figures 4C,D**). It can be inferred that Nrf2-ARE signaling suppressed cellular oxidative stress caused by several factors. These experimental results demonstrate that hydralazine suppresses oxidative stress triggered by MPTP.

We further explored whether this effect was due to regulation of the Nrf2-ARE signaling pathway. **Figures 4E–G**, shows that hydralazine induced a dramatic increase in the nuclear translocation of Nrf2 in the SNpc of hydralazine-treated mice with MPTP, compared with the MPTP mice (p < 0.001), corresponding decrease in cytosolic Nrf2 fraction (p < 0.01). Moreover, hydralazine treatment alone enhanced the nuclear Nrf2 levels when compared to control (**Figures 4E,F**, p < 0.01). The cytosolic Nrf2 fraction decreased slightly compared to nuclear Nrf2 fraction (**Figures 4E,G**). This observation was further confirmed by double immunofluorescence (**Figure 4H**) in the SNpc. Consistent with SNpc, western blotting assay on the striatum lysates showed a statistically significant increase in the Nrf2 nuclear translocation, and a corresponding decrease in cytosolic Nrf2 fraction, in both hydralazine-treated mice with MPTP and hydralazine alone mice when compared to control (**Figures 4I,J**). Based on these results, it was observed that hydralazine activated Nrf2-ARE signaling. Moreover, we observed that Nrf2 colocalized with TH (dopaminergic neuron marker) in the SNpc, but not with ionized calcium binding adapter molecule 1 (IBA1, microglial cell marker) or glial fibrillary acidic protein (GFAP, astrocyte marker) as revealed by double immunofluorescence in the SNpc of hydralazine-treated mice with MPTP. Hydralazine increased the nuclear translocation of Nrf2 in the dopaminergic neuron (**Figure 4L**), which explained why hydralazine protected dopaminergic neurons from MPTP induced neurotoxicity to a certain extent.

In the hydralazine treatment group, western blot assay revealed that Nrf2-ARE targeted genes, NQO1, HMOX1, GCLM, and GCLC, were significantly increased in the SNpc (**Figures 4M,N**) and in striatum (**Figures 4O,P**) after compare with MPTP alone. Additionally, treatment with hydralazine alone increased the expression of above ARE genes compared to control group in both SNpc (**Figures 4M,N**) and striatum (**Figures 4O,P**). Based on the above discussion, the mechanism by which hydralazine activated Nrf2-ARE signaling even in the absence of toxicant was not clear. This should be explored in further studies. In conclusion, these data provide in vivo evidence that hydralazine can activate the Nrf2-mediated ARE gene transcription which provides antioxidant effects in vivo.

#### DISCUSSION

Current treatment for PD are primarily designed relief some of the clinical symptoms which hardly translated to improved quality of life in most patients. In addition, there is no known therapy that prevents the pathogenesis and progress of neurodegeneration. Although several studies have been performed to assess the etiology of PD, the associated mechanisms are still not fully understood. Mounting evidence suggests that oxidative stress appears to be involved in the pathogenesis of PD (36). Consequently, we have reasons to believe that increasing the capacity of dopaminergic neurons to confront oxidants could serve as an important strategy to prevent the onset and/or to delay the progression of PD. However, the prevailing knowledge from clinical trials on antioxidants such as ubiquinone, glutathione, N-acetylcysteine, vitamin C, and vitamin E reveals there are conflicting outcomes on their efficacy (22–25). It is likely that oxidants may exert influence by activating death-related pathways rather than directly killing dopaminergic neurons (37), relying only on stoichiometric scavenging of oxidants has proved to be ineffective. The Nrf2 signaling cascade modulates transcriptional expression of oxidative stress to restore redox homeostasis and is a versatile pathway for neurotherapeutics. Several genetic studies have demonstrated that a functional haplotype in the Nrf2 gene promoter, which conferred high transcriptional activity had a protective effect (38), and the deficiency of Nrf2 increased hypersensitivity of the dopaminergic neurons to neurotoxicity caused by MPTP (14, 39, 40) and 6-OHDA (15) in animal models. Moreover, in vitro Nrf2 activation also provides neuroprotective effect against the neurotoxins paraquat (41), 6-OHDA (42, 43), and MPP+ (44). These datasets imply that the Nrf2 pathway may provide neuroprotection which can be exploited to develop drugs that treat PD.

Hydralazine, which was developed by Swiss researchers in the early 1950s (45), was approved drug by FDA for antihypertensive treatment. In this study, we demonstrate that hydralazine displays remarkable neuroprotective effects in a model of PD in vivo and in vitro. Oral administration of hydralazine decreased MDA levels, increased the ratio of GSH/GSSG, alleviated weight loss and the associated motor deficits, attenuated dopaminergic neurons loss and activated Nrf2 signaling pathways, all of which exerted profound neuroprotective activities in MPTP model of PD (**Figure 3**), and these effects were further confirmed in human

FIGURE 3 | Hydralazine confers protection in dopaminergic neurons in the MPTP model of Parkinson's disease. Schematic representation of the MPTP model experimental design (A). Hydralazine alleviated MPTP-induced weight loss (B). \**p* < 0.05, significant difference between the two indicated groups (*n* = 8). Hydralazine ameliorated MPTP-induced behavior disorder (C,D). Performance on the rotarod and pole test was impaired in MPTP-treated group. However, impairment was ameliorated in MPTP model treated with hydralazine. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, significant difference between the two indicated groups (*n* = 8). Tyrosine hydroxylase immunofluorescence of substantia nigra pars compacta (SNpc). Scale bar = 250µm. We found that there were fewer TH positive neurons in the similar anatomic level sections of SN in the MPTP mice than hydralazine- treated mice, MPTP mice with hydralazine and control mice, compared to the MPTP mice group, MPTP mice with hydralazine group significantly attenuated the loss of dopaminergic neurons in SNpc (E,F). \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, *n* = 6, very significant difference between the two indicated groups. The western blotting of TH in SN further reconfirmed the findings (G and H). \**p* < 0.05, \*\*\**p* < 0.001 *n* = 6, significant difference between the two indicated groups. The MPTP neurotoxicity also resulted the decrease of the TH positive DA terminals in the striatal region of the MPTP mice group compared with MPTP mice with hydralazine group. (I,J). Scale bar = 500µm. \*\**p* < 0.01, \*\*\**p* < 0.001, *n* = 6, very significant difference between the two indicated groups. Immunoblotting also validated this observation (K,L). \*\**p* < 0.01, \*\*\**p* < 0.001, *n* = 6, compared with the two indicated groups. Data were presented as mean ± SEM.

(A) and striatum(B) of MPTP mice treated with hydralazine compared with MPTP mice. \**p* < 0.05, *n* = 5, significant difference between the two indicated groups. MPTP administration showed a remarkable decrease in GSH levels and increase in GSSH that was inhibited by hydralazine in the SNpc, corresponding increase in the ratio of GSH/GSSG (C,D). \**p* < 0.05, \*\**p* < 0.01, *n* = 5, significant difference between the two indicated groups. Hydralazine treatment leads to a shift in Nrf2 migration and increases nuclear Nrf2 Protein levels, in the meantime, cytosolic Nrf2 had corresponding decrease in the substantia nigra (E–H) and striatum (I–K) of *(Continued)*

FIGURE 4 | MPTP-treated mice. \*\**p* < 0.01, \*\*\**p* < 0.001. *n* = 6, significant difference between the two indicated groups. Nrf2 colocalized with TH in the SNpc, not with IBA1, GFAP by double immunofluorescence (L) in the SNpc of hydralazine-treated mice with MPTP, hydralazine can increase nuclear translocation of Nrf2 in the dopaminergic neuron (L). Protein levels of Nrf2 were analyzed by Western blot. β-actin and Histonen H3 are used as markers for cytosolic and nuclear fractions, respectively. Hydralazine treatment increased the expression of Nrf2 downstream target in the substantia nigra (M,N) and striatum (O,P) of MPTP-treated mice. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001, *n* = 6, significant difference between the two indicated groups. Data were presented as mean ± SEM.

neuroblastoma SH-SY5Y cells (**Figure 1**). The cell viability was increased following hydralazine treatment when exposed to MPP+ or H2O2. Among the key Nrf2-regulated genes stimulated by hydralazine were NQO1, HMOX1, GCLM, GCLC. It was reported NQO1 ameliorated the baleful effects of DA quinines, and had various antioxidant properties (46, 47). HMOX1 has been implicated in the metabolism of the pro-oxidant heme to form the antioxidant pigment biliverdin which confers resistance to chronic oxidative stress and apoptosis in dopaminergic neurons (48, 49). GCLC and GCLM regulates the production of cellular antioxidant glutathione (50). These proteins are involved in oxidative stress and redox homeostasis. Nrf2 SiRNA transfection was performed to explore the function of Nrf2 in the protective effect of hydralazine against neurotoxicity MPP+ induced in SHSY5Y cells. Nrf2 SiRNA2 transfection treatment abolished the hydralazine-induced ARE genes expression and increased the vulnerability to MPP+ neurotoxicity (**Figure 2**). However, we lacked Nrf2 knockout mice and Nrf2 knockin mutant mice to perform more confirmatory experiments.