The Role Of CCL2 In The Pathogenesis Of Mild Cognitive Impairment In Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder of the elderly after Alzheimer’s disease (AD) and the most common movement disorder. Cognitive dysfunction is a pretty common non-motor symptom of Parkinson’s disease. With China’s aging population, PD and PD cognitive impairment are one of the major diseases which harm the healthy of the elderly population and bring a heavy burden to social and family.Mild cognitive impairment in Parkinson’s disease (PD-MCI) is an intermediate state between cognitively normal Parkinson’s disease (PDCN) and Parkinson’s disease dementia (PDD). The Movement Disorder Society (MDS) formalized diagnostic criteria for PD-MCI in January 2012. The proposed diagnostic criteria defined the presence and status of PD-MCI, and the study of PD-MCI will help us to look insight into the characteristics and evolution of PD cognitive dysfunction.Currently the etiology of PD and PD-MCI remains unclear, may be related to genetic, environmental, aging and other factors. Substantial evidence has indicated the involvement of inflammatory processes in the pathology of PD recently. CC chemokine ligand 2/Monocyte chemoattractant protein 1 (CCL2/MCP-1), a member of the chemokine family produced by neurons and glial cells, and its receptor CC chemokine receptor -2 (CCR2) have been implicated in many neurodegenerative disorders. In the studies of Alzheimer’s disease (Alzheimer’s disease, AD) and HIV dementia it has been found that CCL2 can accelerate the decline of cognitive function, leading to the formation of dementia, indicating that CCL2 may be associated with cognitive dysfunction; on the other hand, in the studies of PD it has been found that the levels of CCL2 in blood and cerebrospinal fluid of PD patients were higher than the normal control group, indicating that CCL2 may be associated with PD. However, there are no researches which study the relation of CCL2 and PD-MCI.The blood brain barrier (BBB) damage, proliferation and activation of microglia play an important role in the pathogenesis of PD and PD-MCI. Studies have found that CCL2 may play an important role in opening the BBB. In the study of AD, it was found that CCL2 can activate microglia and promote inflammatory cytokines, oxygen free radicals, leading to Aβ formation and neuronal death. What is the role of CCL2 in the pathology of PD and PD-MCI? Is it also through the blood-brain barrier damage, promote proliferation of microglia activation and inflammatory immune reaction, leading to die of dopaminergic neurons?Bindarit,2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy] propanoic acid, is a small synthetic indazole derivative that preferentially inhibits transcription of the CCL2. Bindarit has shown clinical efficacy in a broad array of experimental inflammatory, autoimmune and vascular disorders, as well as success in recent clinical trials for diabetic nephropathy and lupus nephritis. Such beneficial effect has been associated with bindarit’s ability of anti-CCL2 activity and to interfere with monocyte recruitment, which is also a critical feature in neuroinflammatory disease. A recent study of AD showed that it can inhibit CCL2 production, reduce the toxicity of the abnormal accumulation of A(3 to neurons. However, there is no report about its role in the treatment of PD.So, the tasks of this study were (1) animal experiments:Preparation of chronic PD mouse model to explore the possible role of CCL2, whether through blood-brain barrier damage and promote inflammation in the brain, leading to the death of dopaminergic neurons, and preliminary observe the therapeutic effect Bindarit; (2) cell experiments:explore whether CCL2 promote microglial proliferation, oxygen free radicals and inflammatory cytokine production, leading to neuronal apoptosis; (3) clinical studies:explore the relationship between CCL2 and cognitive dysfunction in PD patients. This research is designed from cellular level, animal level and the clinical case study to explore the role of CCL2 in the progression of PD-MCI, try to provide new theoretical basis and foundation for the early intervention and treatment of PD-MCI.Part Ⅰ:CCL2 damage the blood-brain barrier and cause neuroinflammation in chronic mouse model of Parkinson’s disease.Objective:To investigate the cognitive function in chronic Parkinson’s disease model of C57BL/6 mice and to explore the possible role of CCL2 in the blood-brain barrier damage and the protective effect of Bindarit on the blood-brain barrier; also investigate the role of CCL2 in the activation of microglia, neuroinflammation and degeneration of dopaminergic neurons.Methods:33 male 8-week-old C57BL/6 mice were randomly divided into three groups, including control group (n=10), MPTP/P model group (n=10), Bindarit treatment group (n=13). MPTP/P group were given 25mg/kg MPTP and 250mg/kg probenecid intraperitoneally; Bindarit treatment group received 25mg/kg MPTP, 250mg/kg probenecid and 100mg/kg Bindarit intraperitoneally; control group was given the same amount of saline. Injections were given twice a week for five weeks. (1) After the treatment, pole test, open field test were used to detect the movement ability and Morris water maze test was used to detect the cognitive function in the mice. (2) Evans Blue experiment were used to observe blood-brain barrier destruction of mice in each group, western blot assay were used to detect tight junction protein ZO-1, occludin expression in brain of mice for each group and electron microscopy was used to detect the ultrastructure of blood-brain barrier in each group. (3)OX42 immunohistochemistry was used to detect microglia activation in each group. (4) TH immunohistochemistry was used to detect the dopaminergic neuronal degeneration in the midbrain of mice in each group. (5) Western blot assay was used to detect the expression of α-synuclein and caspase-3 which is apoptosis-related protein in each group. (6) TUNEL experiment was used to observe neuronal apoptosis in each group. (7) Bio-Plex pro assays were used to detect the levels of TNF-α, IL-1β, IL-17, IFN-γ and CCL2 in serum of mice.Results:(1) The results of pole test showed that time to descend in MPTP/P group were longer than the control group (P<0.05); results of open-field test showed that, compared with the Control group, movement distance was reduced, velocity were slow, time spent in the central region were reduced in MPTP/P group (P <0.05); results of Morris water maze test showed that the escape latency of MPTP/P group was prolonged compared to the control group (P<0.05); (2) The Evans Blue experiment results showed that Evans blue exudation of MPTP/P group increased significantly compared with the control group (P<0.05); Western blot showed that expression of tight junction protein ZO-1 and occludin were reduced in the MPTP/P group compared with the control group (P<0.05), results of transmission electron microscopy showed that microvascular endothelial cell were fractured, perivascular exudation increased, tight junction were damaged in MPTP/P group; (3) Results of OX42 immunohistochemistry showed that the number of OX42 positive cells were increased in the MPTP/P group compared with the control group; (4) Results of TH immunohistochemistry showed that the number of TH positive cells in substantia nigra were reduced in the MPTP/P group compared with the control group (P<0.05); (5) Results of Western blot showed that the protein expression of a-synuclein and caspase-3 in MPTP/P group were increased than the control group (P<0.05); (6) Results of TUNEL showed that the neuronal apoptosis was increased in MPTP/P group compared with control group; (7) Results of Bio-Plex pro assays showed that the level of TNF-α, IL-1β, IL-17, IFN-y and CCL2 in serum of mice was higher in MPTP/P group (P<0.05); The results were all improved in Bindarit treatment group.Conclusions:(1) Stable chronic PD models can be produced by giving C57BL/6 mice with MPTP and probenecid, intraperitoneal injection twice a week for five weeks in which some mice are with cognitive dysfunction. (2) CCL2 may damage the blood-brain barrier in chronic mouse model of PD, and it may play a role in the activation of microglia, neuroinflammation and degeneration of dopaminergic neurons. (3) Bindarit may play a role in the treatment of chronic mouse model of PD.Part Ⅱ:CCL2 promote a-synuclein mediated proliferation and activation of microglia and neuronal apoptosisObjective:To explore the role of CCL2 in proliferation and activation of microglia, whether CCL2 promote a-synuclein mediated proliferation of microglia, leading to oxygen free radicals, inflammatory cytokine production, thereby resulting in increased neuronal apoptosis by culturing primary microglia and neurons.Methods:After separation and purification, the primary cultured microglia was divided into six groups. Method ①0 ng/ml group,12.5ng/ml group,25 ng/ml group, 50 ng/ml group,100 ng/ml group and 200 ng/ml group, which were treating with CCL2 according to the corresponding dose; method ②Control group, CCL2 treated group, α-Syn treated group, CCL2+α-Syn treated group, CCL2+α-SynCCL2 Ab-treated group, CCL2+α-Syn+BIND treatment groups. CCL2 treatment groups: 50ng/ml; α-Syn treatment groups:200ng/ml; CCL2+α-Syn treatment groups: 50ng/ml CCL2+200ng/ml α-Syn; CCL2+α-Syn+CCL2 Ab treatment groups: 50ng/ml CCL2+200ng/ml α-Syn+5ug/mICCL2 Ab; CCL2+α-Syn+BIND treatment groups:50ng/ml CCL2+200ng/ml α-Syn+50nM Bindarit. After 24 hours of treatment:(1) CCK8, Brdu test were used to detect the proliferation and activation of microglia. (2) Griesss reagent was used to detect the content of NO in microglia culture medium. (3) ELISA assays were used to detect the content of TNF-α, IL-1β in microglia culture medium. (4) Immunofluorescence was used to detect the α-Syn expression in microglia. (5) Cultrued primary neurons and flow cytometry was used to detect the neuronal apoptosis after treated with microglia culture medium for 24h.Results:(1) Results of CCK8 and Brdu assays showed that microglia proliferation and activation were the most significant in the 50 ng/ml group, followed by 100 ng/ml group(P<0.05). In the second grouping method, microglia proliferation and activation were the most significant in the CCL2+α-Syn group, followed by CCL2 group (P<0.05), while the results were improved in CCL2+α-Syn+ CCL2 Ab group and CCL2+α-Syn+ BIND group. (2) Results of Griess test showed that the contents of NO in microglia cell culture medium were the highest in 50 ng/ml group, followed by 100 ng/ml group compared with the control group (P<0.05); In the second grouping method, the contents of NO in microglia cell culture medium were the highest in CCL2+α-Syn group (P<0.05), after adding CCL2 Ab or Bindarit, microglia produce relatively less NO. (3) Results of ELISA assay showed that the contents of TNF-α and IL-1β in the microglia cell culture medium were similar compared with the control group (P>0.05); In the second grouping method, the contents of TNF-α and IL-1β in microglia cell culture medium were higher in CCL2 +α-Syn group than the control group (P<0.05), after adding CCL2 Ab or Bindarit, the contents of TNF-α and IL-1β were decreased. (4) Results of immunofluorescence showed that α-Syn expression of microglia were the most in CCL2+α-Syn group, after adding CCL2 Ab or Bindarit, α-Syn expression of microglia were decreased. (5) Results of flow cytometry showed that cultured primary neuron had the highest rate of apoptosis in CCL2+α-Syn group(P<0.05), after adding CCL2 Ab or Bindarit group, the rate of neuronal apoptosis was relative reduced.Conclusions:CCL2 promote α-Synuclein mediated proliferation and activation of microglia, leading to oxygen free radicals, inflammatory cytokine production, thereby resulting in increased neuronal apoptosis.Part Ⅲ:The gene polymorphisms analysis and plasma, cerebrospinal fluid levels of CCL2 with cognitive impairment in Parkinson’s diseaseObjective:(1) To investigate the association between the CCL2 2518A/G (rs1064211) and CCR2 V64I (rs1799864) gene polymorphisms and PD risk in Guangdong Han population. We also analyze the influence of these genotypes on the cognitive function and depression in PD patients (2) The CCL2 levels in plasma and cerebrospinal fluid were detected for patients with PD and PD-MCI to explore the relationship between them.Methods:(1) In this study, a cohort of 521 PD patients and 556 cases of controls were recruited to investigate the association between the CCL2 2518A/G (rs 1064211) and CCR2 V64I (rs1799864) gene polymorphisms and PD risk in the Guangdong Han population. We also analyze the influence of these genotypes on the cognitive function and depression in PD patients by comparing Mini Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCA), Wechsler Adult Intelligence Scale-Chinese Revision (WAIS-RC), Wechsler Memory Scale-Chinese Revision (WMS-RC) and Hamilton Depression Rating Scale (HAMD) ratings in 217 PD patients. (2) We collected plasma from 140 PD patients and 70 controls, cerebrospinal fluid from 45 PD patients and 16 controls, and the PD group was divided into PDCN group and PD-MCI group. The levels of CCL2 in plasma and cerebrospinal fluid were detected by ELISA.Results:(1) Our results showed no significant differences in the genotype frequency between the PD group and the control group (P>0.05). In addition, we also failed to find an influence of the CCL2 and CCR2 genotypes on MMSE scores, MOCA scores, WAIS-RC scores, WMS-RC scores and HAMD scores in PD patients (P>0.05). (2) There were significant differences in plasma levels of CCL2 in PDCN group, PD-MCI group and control group, and plasma levels of CCL2 in PD-MCI were higher than PDCN group(P<0.05). The levels of cerebrospinal fluid CCL2 in PD-MCI group were higher compared with the PDCN group and control group (P<0.05).Conclusions:(1) The CCL2 and CCR2 gene polymorphisms may not be genetic risk factors for PD in Guangdong Han population, and they do not appear to influence cognitive function and depression in PD patients. (2) Plasma CCL2 levels in PD patients are higher than control group, and PD-MCI group is particularly significant. (3) Cerebrospinal fluid CCL2 levels in PD-MCI patients are higher than the control and PDCN group.