The Role Of Complement Receptor 3-regulated Microglial Activation In Cognitive Impairment In Parkinson’s Disease

Parkinson’s disease(PD)is a progressive neurodegenerative disease.In addition to characteristic motor disorders,patients with PD exhibit a variety of non-motor symptoms,including cognitive impairment,which severely influence quality of life of patients.Previous studies on PD concentrated on the nigrostriatal dopaminergic system.While studies have gradually found that the cortex,hippocampus and locus coeruleus norepinephrine(LC-NE)system play important roles in cognitive impairment in PD.The pathogenic mechanisms of cognitive impairment in PD remain unclear.Microglia-mediated chronic neuroinflammation is one of the pathogenic mechanisms of PD.Complement receptor 3(CR3)is expressed in microglia and plays a role in regulating inflammatory response.Synapse loss is associated with cognitive decline in Alzheimer’s disease(AD).Inhibition of CR3 reduced the number of phagocytic microglia and the extent of early synapse loss in an AD mouse model,suggesting that CR3 was involved in cognitive impairment.However,it is not clear whether CR3 is involved in the development of cognitive impairment in PD.Nuclear factor-kappa B(NF-κB)and Janus kinase/signal transducer and activator of transcription(JAK/STAT)pathways play important roles in regulating microglial activation and associated neuroinflammation.High levels of proinflammatory cytokines were detected in the brain tissue,cerebrospinal fluid and serum of patients with PD,which may be related to cognitive impairment of PD.While the expression levels of CR3 in patients with PD have not been reported.We speculated that CR3 might be involved in the cognitive impairment in PD by regulating inflammatory response,while the specific mechanisms remain to be further explored.In this study,rotenone(Rot)-induced PD mouse model and BV2 microglia were used to investigate the role of CR3 in cognitive impairment in PD and the related molecular mechanism.The levels of CR3 and proinflammatory cytokines in serum and gray matter volume(GMV)were detected in patients with PD to further explore the mechanism of CR3 in the development of PD,providing evidences for the molecular mechanism research of cognitive impairment in PD and the search for novel biomarkers and therapeutic targets.This study is mainly divided into two parts:PartⅠThe role of CR3-regulated microglial activation in cognitive impairment in a Rot-induced PD mouse modelObjective:To investigate the role of CR3 in cognitive impairment in PD and whether CR3 is involved in cognitive impairment and neurodegeneration in PD by regulating microglial activation.Methods:1.PD animal model:Adult male C57BL/6 mice were randomly divided into control(Con)group,0.75 mg/kg Rot(Rot-0.75)group and 1.5 mg/kg Rot(Rot-1.5)group.The mice in Rot groups were daily intraperitoneally injected with Rot for 3consecutive weeks.Wild type(WT)mice and CR3 knockout(CR3 KO)mice were selected.Each strain was randomly divided into two groups,i.e.,WT-Con group,WT-Rot group,CR3 KO-Con group,CR3 KO-Rot group.The mice in Rot groups were daily intraperitoneally injected with Rot at a dose of 1.5 mg/kg for 3 consecutive weeks.2.Behavioral tests for assessing cognitive abilities:Morris water maze test,novel object recognition test and passive avoidance test were carried out.3.Immunohistochemical staining:Immunohistochemical staining was performed on hippocampal,cortical and LC-NE neurons,synapses and microglial cells in mice with anti-neuron-specific nuclear protein(Neu-N),postsynaptic density-95(PSD-95),tyrosine hydroxylase(TH)and ionized calcium binding adaptor molecule-1(Iba-1)antibodies.4.Western blot:The protein levels of Neu-N,PSD-95 and the phosphorylation levels of p65 and IκBα,two key proteins of nuclear factor-kappa B(NF-κB),as well as signal transducer and activator of transcription(STAT)1 and STAT3 signaling pathways andα-synuclein at serine 129(Ser129-α-Syn)in the hippocampus and cortex of mice were detected.The protein levels of CR3 in the hippocampus,cortex and brain stem of mice were detected.5.Quantitative real-time polymerase chain reaction:The m RNA levels of CR3 and microglial markers of the M1 phenotypes,including inducible nitric oxide synthase(i NOS)and interleukin-1β(IL-1β),and M2 phenotypes,including argininase-1(Arg-1),YM-1 and CD206,were detected in the hippocampus,cortex and brain stem of mice.The m RNA levels of i NOS and IL-1βin BV2 microglia were detected.6.BV2 microglia:A specific si RNA was used to knockdown CR3,and LL-37 was used to activate CR3 in BV2 microglial cells.QNZ,Fludarabine and Stattic were used to inhibit NF-κB,STAT1 and STAT3 pathways in BV2 microglial cells respectively.Results:1.Rot dose-dependently induced cognitive impairment in mice:Compared with the Con group,the learning and memory abilities of mice in the Rot-1.5 group were declined,while the mice in the Rot-0.75 group did not show obvious cognitive impairment.2.Rot dose-dependently induced hippocampal,cortical and LC-NE neurodegeneration and microglial activation in mice:Compared with the Con group,the number of Neu-N~+neurons and staining density of PSD-95 in the hippocampus and cortex in the Rot-1.5 group were decreased,and the number of TH~+neurons in the LC was decreased,while there were no change or weak changes in the Rot-0.75 group.3.Rot dose-dependently induced microglial activation in the hippocampus,cortex and LC in mice:Compared with the Con group,the microglial cells exhibited hypertrophic morphology and increased Iba-1 staining density,while the changes were attenuated or not observed in the Rot-0.75 group.4.Rot induced increased expression levels of CR3 in the hippocampus,cortex and brainstem in mice:Compared with the Con group,Rot induced increased levels of protein and m RNA of CR3 in the hippocampus,cortex and brainstem in mice.5.CR3 deficiency mitigated Rot-induced microglial activation and M1/M2imbalance in hippocampus,cortex,and LC in mice:Compared with the WT-Con group,microglial activation in the hippocampus,cortex and LC were detected in the WT-Rot group,and the m RNA levels of microglial markers of the M1 phenotypes were significantly up-regulated,while the m RNA levels of microglial markers of the M2phenotypes were decreased.While compared with the CR3 KO-Con group,no significant microglial activation was observed in the CR3 KO-Rot group,and the up-regulation of m RNA levels of M1 markers and the decrease of m RNA levels of M2markers were significantly attenuated.6.CR3 deficiency attenuated Rot-induced activation of NF-κB and STAT pathways in the hippocampus and cortex in mice:Compared with the WT-Con group,the phosphorylation levels of p65,IκBαand STAT1,STAT3 signaling pathways in the hippocampus and cortex of the WT-Rot group were increased.Knockout of CR3attenuated the Rot-induced activation of NF-κB and STAT signaling pathways in the mice.7.CR3 mediates Rot-induced microglial M1 activation through NF-κB and STAT pathways:Knockdown or activation of CR3 reduced or enhanced the Rot-induced increase in markers of M1 polarization(i NOS and IL-1β)m RNA in BV2 microglia,respectively.QNZ and Stattic,inhibitors of NF-κB and STAT3,respectively,significantly reduced the Rot-induced increase in i NOS and IL-1βm RNA in BV2microglia.Fludarabine,a STAT1 inhibitor,had no significant effect on Rot-induced increase in i NOS and IL-1βm RNA in BV2 microglia.8.CR3 deficiency attenuated Rot-induced hippocampal,cortical and LC-NE neurodegeneration and phosphorylation of Ser129-α-Syn in mice:Compared with the WT-Con group,hippocampal,cortical and LC-NE neurodegeneration and phosphorylation of Ser129-α-Syn in the hippocampus and cortex were detected in the WT-Rot group,while these changes induced by Rot could be alleviated by knockout of CR3.9.CR3 deficiency alleviated Rot-induced cognitive impairment in mice:Compared with the WT-Con group,mice in the WT-Rot group exhibited impaired learning and memory abilities,which could be attenuated by knockout of CR3.Conclusions:1.Rot dose-dependently induced cognitive impairment and hippocampal,cortical and LC-NE neurodegeneration.2.Rot induced microglial activation in brain of mice,and up-regulated the expression level of CR3.3.Knockout of CR3 significantly attenuated Rot-induced microglial activation and M1 polarization.4.CR3 might regulate Rot-induced M1 polarization of microglia through NF-κB and STAT pathways.5.CR3 regulated Rot-induced cognitive impairment in mice.PartⅡThe study of serum levels of CR3 and proinflammatory cytokines,changes of gray matter volume and cognitive impairment in patients with PDObjective:To investigate the changes of serum levels of CR3 and proinflammatory cytokines in patients with PD and their correlation with changes of GMV and cognitive impairment in patients.Methods:1.Subjects:28 healthy control(HC)subjects and 56 patients with PD were recruited.Patients with PD were divided into four groups,i.e.,PD with normal cognition(PD-NC)group,PD with mild cognitive impairment(PD-MCI)group and PD with dementia(PDD)group.2.Scale assessment:Part III of the Unified Parkinson’s Disease Rating Scale was used to assess the motor function of the patients.The modified Hoehn and Yahr(H-Y)scale was used to assess the severity of the disease.Hamilton Anxiety Rating Scale and Hamilton Depression Rating Scale were used to evaluate the emotional state.Mini-Mental State Examination and Montreal Cognitive Assessment were used to evaluate the cognitive function of the subjects.3.Evaluation of blood tests:The serum level of CR3 was detected by enzyme linked immunosorbent assay.Chemiluminescence immunoassay was used to detect the serum levels of interleukin-6(IL-6),IL-1βand tumor necrosis factor-α(TNF-α).The serum level of C-reactive protein(CRP)was detected by immune scattering turbidimetry.4.Imaging examination:High-resolution T1-weighted imaging of head was performed with a 3.0T magnetic resonance imaging scanner.Voxel-based morphometry was used to analyze the GMV.Results:1.Compared with the HC group,cognitive impairment was observed in patients with PD.2.Compared with the HC group,Serum levels of CR3,TNF-αand IL-6 were significantly increased in patients with PD,but there was no significant difference between PD patients with different cognitive levels.3.There was no significant correlation between serum level of CR3 and course of disease,H-Y stage,scores of motor function,cognitive function,anxiety,depression and serum level of TNF-αin patients with PD.4.Compared with the HC group,PD-NC group presented reduction of GMV in the left middle frontal gyrus,triangular part of inferior frontal gyrus and right supramarginal gyrus,superior temporal gyrus,and PD-MCI group mainly showed reduction of GMV in the left middle frontal gyrus,triangular part of inferior frontal gyrus and right supramarginal gyrus,superior temporal gyrus,anterior cingulate and paracingulate gyri,median cingulate and paracingulate gyri,and PDD group mainly presented reduction of GMV in the left middle frontal gyrus,triangular part of inferior frontal gyrus and right thalamus,supramarginal gyrus,superior temporal gyrus,anterior cingulate and paracingulate gyri,median cingulate and paracingulate gyri.5.The levels of cognitive function in PD patients were positively correlated with the volumes of left middle frontal gyrus,triangular part of inferior frontal gyrus and right thalamus,anterior cingulate and paracingulate gyri,median cingulate and paracingulate gyri.6.No significant correlation was found between serum levels of CR3 and volumes of brain regions with reduced GMV in patients with PD.Conclusions:1.Serum levels of CR3,TNF-αand IL-6 were significantly increased in patients with PD.2.PD patients show reduced GMV in frontal lobe,temporal lobe,limbic lobe,thalamus,which is related to the level of cognitive function.3.No significant correlation was found between serum levels of CR3 and cognitive levels or volumes of brain regions with reduced GMV in patients with PD.