Analysis of Six Bone-derived Factors in Plasma and Csf of Chinese Patients With Parkinson's Disease

Background Parkinson's disease (PD) has a close relationship with osteoporosis and bone secretory proteins may be involved in disease progress. Objectives To detect the six bone-derived factors in plasma and CSFof patient swith PD and evaluate their correlations with CRP level, motor impairment and HY stage of the disease. Methods We included 250 PD patients and 250 controls. Levels of OCN, OPN,OPG, SO, BMP2 andDKK-1 in Plasma and CSF were measured by custom protein antibody arrays.Data were analyzed using Mann-Whitney U-testand Spearman’s rank correlation. Results Plasma levels of OCN and OPN were correlated with CRP level and HY stage and motor impairment of PD.Furthermore, the plasma assessment with CSF detection may enhance their potential prediction on PD. Conclusions of PD patients remain unknown. In this study, we aimed to clarify the levels of OCN, OPN, OPG, Sclerostin (SO), Bone morphogenetic protein 2 (BMP2) and Dickkopf-1 (DKK-1) in the plasma and CSF of PD patients and identify candidate biomarkers for detection of PD. We also investigated the relevance of these bone-derived factors with C-reaction protein (CRP) and motor dysfunction and disease progression.


Introduction
Parkinson's disease (PD) is one of the most common neurodegenerative disorders 1 . Patients with PD are at a high risk of osteoporotic fractures as a result of falls and reduced bone mass 2 . Osteoporosis may be a hidden non-motor syndrome of PD and fractures can also be found more commonly in the prodromal period of PD compared to controls 3,4 . This indicates that bone metabolism has a close relationship with the developing of PD 4 . Bone has traditionally been considered a structural organ that supports movement of the body and protects the internal organs. However, an increasing number of studies have shown that the skeleton could also be an active endocrine organ which secretes many kinds of bone-derived factors and contributes to pathophysiology of many diseases, such as Alzheimer's disease, diabetes mellitus, cardiovascular, chronic kidney disease [5][6][7][8] . Osteocalcin (OCN), Osteoprotegerin (OPG) and osteopontin (OPN) were reported upregulated in the plasma of PD patients 9,10 . However, we found that these case-control studies had relatively small sample sizes and the levels of these factors in cerebrospinal uid (CSF) of PD patients remain unknown. In this study, we aimed to clarify the levels of OCN, OPN, OPG, Sclerostin (SO), Bone morphogenetic protein 2 (BMP2) and Dickkopf-1 (DKK-1) in the plasma and CSF of PD patients and identify candidate biomarkers for detection of PD. We also investigated the relevance of these bonederived factors with C-reaction protein (CRP) and motor dysfunction and disease progression.

Subjects
This study enrolled 500 participants, including 250 with PD, and 250 healthy controls. All patients included in this study were tested negative for diabetes mellitus, thyroid disease or kidney disease. All participants were recruited from the First A liated Hospital of Guangzhou Medical University. This study was approved by the institutional Ethics Board Committee of the First A liated Hospital of Guangzhou Medical University and all participants provided written informed consent.

Plasma and CSF
Blood samples were collected from 200 healthy controls and 200 patients with PD and were centrifuged (2,500 g for 15 minutes) within 1 hour of collection. CSF samples were collected by lumbar puncture from 50 healthy controls and 50 patients with PD. CSF samples had no blood contamination (leukocyte number count fewer than 5 cells/µL and erythrocyte number fewer than 200 cells/µL). Sample aliquots were stored in cryotubes at − 80℃ before testing.
The levels of bone-derived factors were measured using custom protein antibody arrays (RayBiotech, www.raybiotech.com). CRP was measured using immunotubidometric assays.

Statistical analysis
Data statistics were carried out by SPSS, version 21 and GraphPad Prism version 6. Student's t tests or Mann-Whitney U test was used to assess the difference of continuous variables between PD and control groups. Differences between groups for categorical variables were assessed using chi-square tests. Spearman coe cient calculation and Kruskal-Wallis H test were used to analyze possible correlations between parameters of interest.
Receiver operating characteristics (ROC) curves were used to determine the diagnostic performance of studied bonederived factors in differentiating PD patients from controls. The accuracy of a biomarker in predicting PD was assessed by calculating the area under the ROC curve (AUC). Values of P < 0.05 were considered signi cant.

Results
For this study, we obtained plasma samples from 200 PD patients and 200 age-and gender-matched controls, and CSF samples from 50 PD patients and 50 age-and gender-matched controls. More than 90% of plasma samples and 90% of CSF samples were detected in our statistical analyses. SO in CSF analysis was excluded due to low detection rates. In plasma, levels of OCN There was no signi cant difference of OPN, BMP2 or DKK1 levels in CSF between the two groups ( Table 1  To evaluate whether these bone-derived factors could be the potential biomarkers for PD risk, the natural-logarithm values of these levels were analyzed using ROC curves. Compared with the NC group, the AUCs for the plasma and CSF levels of OCN in PD patients were 0.863 (95% CI = 0.771-0.928) and 0.67 (95% CI = 0.559-0.769), respectively.
Moreover, the AUC was higher when combined assessment of plasma and CSF OCN in PD patients, at 0.869 (95% CI = 0.778-0.993) (Fig. 1E) (MDS-UPDRS)-III score (UPDRS III) ( Fig. 1. G, J). In ammation was involved both in the pathology of osteoporosis and PD, we next analyzed the relationship between CRP and these bone-derived factors. We found that signi cant correlations existed between plasma OCN and OPN and CRP levels in PD (Fig. 1I, L). However, no correlation was identi ed between these factors and CRP in CSF. OCN is one of the most abundant bone-speci c non-collagenous protein secreted primarily by osteoblasts and is often used as a biomarker for bone formation. In recent years, OCN has been regarded as a bone-derived hormone that plays important roles in physiological and pathological processes 11 . In peripheral, OCN acts as a regulator of the activity of osteoclasts and also maintains the energy homeostasis 12 . In brain, the uncarboxylated form of OCN can accumulate in the brainstem, thalamus and hypothalamus, and in uencing various neurotransmitters synthesis and signalings 13 . In PD, OCN could correct motor dysfunction and reduce dopaminergic neuronal injury via the AKT/GSK3β signaling pathway in an animal model of PD 14 . In this study, we found that the plasma level of OCN was increased, however the CSF level of OCN was decreased in PD patients. This may be accounted for the different roles of OCN in peripheral circulation and central nervous system. Given the role of OCN in protecting dopaminergic neurons, improving cognition and preventing anxiety and depression,low expression level of OCN in CSF indicates its involvement in motor and non-motor symptoms of PD. The role of OCN in the pathophysiology of PD is obscure.
Plasma level of OCN correlated with CRP and H-Y stage of PD patients indicates in ammation may be a potential bridge between OCN and progression of PD. However, no correlation was identi ed between OCN and CRP in CSF, indicating that other mechanism are involved in PD in central nervous system. Furthermore, compared to analyzing plasma or CSF OCN alone, the combined assessment is more effective in differentiating PD patients from healthy controls and plasma level of OCN was correlated with the disease stage and motor impairment. All these results highlight the important roles of OCN in the pathology of PD.
OPN is a glycosylated phosphoprotein belonging to the small integrin binding ligand, N-linked glycoprotein (SIBLING) family of proteins 15 . It is highly expressed by bone marrow-derived myelomonocytic cells and can act both as a matrix protein and as a cytokine. As a multifunctional protein, OPN plays signi cant roles in regulating reactive oxygen species production, levels of in ammatory cytokines and apoptotic signals 7 . OPN was reported to involve in the pathology of various brain diseases, such as Alzheimer's disease (AD), multiple sclerosis, and traumatic brain injury via neuroprotective and repair-promoting effects 7,16 . OPN is also expressed in substantia nigra and in nigral dopaminergic neurons and its expression is decreased in surviving dopaminergic neurons in PD, suggesting a potential role of OPN in neuroprotection of PD. However, OPN knockout mice displayed less nigral cell death and a decreased glial response in a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced animal model of PD 16,17 . This suggests that OPN may act as a double-edged sword triggering neuronal toxicity or functioning as a neuroprotectant in PD. In ammatory response may be one of the mechanism of OPN in PD as there is signi cant correlation between OPN and CRP. Walter Maetzler have reported that OPN was upregulated both in the plasma and CSF of PD patients 17 , however we only identi ed higher level of OCN in the plasma of PD patients. The incongruence may be due to difference in disease stage, ethnic or environmental factors and more studies are needed to reveal the function of OPN in PD.
In conclusion, we analyzed six bone-derived factors and revealed abnormal expression levels of OCN, OPN, OPG and BMP2 in plasma or CSF of PD. We identi ed that plasma level of OCN and OPN were correlated with CRP, H-Y stage and motor impairment. Our study also suggests that combined assessment of plasma and CSF of OCN or OPN may enhance their potential for predicting PD. In ammation response may be involved in the cross-talks between these two factors and PD. These ndings may contribute to the functional understanding of PD pathophysiology. However, further studies are needed to con rm our ndings and to illustrate the roles of in ammation or immune mechanisms involved in the factors on PD. Availability of data and materials

Abbreviations
The datasets analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests

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