The Role Of TREM2 In The Regulation Of Microglia-mediated Neuroinflammation In Parkinson’s Disease

Parkinson’s disease (PD) is a common neurological neurodegenerative disorder characterized by prominent death of dopaminergic neurons in the substantia nigra pars compacta. The dopamine deficiency within the basal ganglia results in classical parkinsonian motor symptoms. Furthermore, numerous non-motor symptoms are also associated with Parkinson’s disease, especially PD mild cognitive impairement may precede the motor symptoms by more than one decade. Up to date, the major management of Parkinson’s disease is symptomatic treatment with drugs that directly stimulate dopamine receptors or increase dopamine concentrations. Disease-modifying treatments could reduce the rate of neurodegeneration or stop the disease process. However, the pathogenesis of Parkinson’s disease remained elusive. Thereby elucidating the pathogenesis of PD might be helpful to identify potential targets for disease modification.Since microglia activation was first characterized in the brains of patients with parkinsonian symptoms 20 years ago, neuroinflammation in Parkinson’s disease has attracted great focus. Microglial activation can be classified into M1 phenotype (overactivated phenotype) and M2 phenotype. Ml microglia could produce multiple proinflammatory cytokines that could promote the clearance of pathogens. However, overactivated microglia are involved in the pathogenesis of PD because excessive proinflammatory cytokines and toxins could damage neighboring neurons in turn. In contrast with M1 phenotype, M2 microglia has an anti-inflammatory effect and and tissue repair function by producing and releasing anti-inflammatory cytokines to inhibit pro-inflammatory responses. Hence, facilitating the switch of microglia phenotype from Ml to M2 by regulating their polarization may provide potential therapy for the treatment of Parkinson’s disease (PD). However, the mechanism that regulates microglia polarization remains unclear.Triggering receptor expressed on myeloid cells 2 (TREM2) is a receptor expressed on the microglia. TREM2 could inhibit the cytokine expression and reduce microglia-mediated pro-inflammatory responses. Furthermore, TREM2 could promotes microglial phagocytosis of neuronal cell debris and apoptotic neurons. A recent large GWAS study has shown that the TREM2 R47H variant increased the risk of PD, AD (Alzheimer’s disease) and FTD (Frontal temporal disease). Therefore, TREM2 may be an important factor regulating M1/M2 microglia balance in PD neuroinflammation.In the present study, we will compare the difference of soluble TREM2 level of peripheral blood between PD patients and healthy controls. And we will investigate the expression of TREM2 and its downstream inflammation cytokines in the PD mouse model. Furthermore, the TREM2-shRNA lentivirus and wtTREM2 lentivirus will be applied to infect microglia cells to knockdown or overexpress TREM2. And then roles of TREM2 in the regulation of microglia polarization of M1/M2 phenotype will be studied. We aim to determine the role of TREM2 in the regulation of microglia-mediated neuroinflammation in Parkinson’s disease.Part I The expression of soluble TREM2 in Parkinson’s disease and Parkinson’s disease-related cognitive impairmentObjectives:To explore the role of TREM2 in the pathogenesis of Parkinson’s disease (PD) and PD-related cognitive dysfunction through evaluating the serum level of sTREM2 in PD patients.Methods:The present study incorporated 114 patients with PD and 120 healthy controls. The demographic data of all subjects were collected. Unified Parkinson Disease Rating Scale part III (UPDRS-Ⅲ) was performed to measure the motor symptom. The Montreal Cognitive Assessment (MoCA) was used to measured global cognitive function. Four cognitive domains including executive function, memory, attention and visuospatial function were assessed by Wechsler Adult Intelligence Scale-Revised (WAIS-R) and Wechsler Memory Scale (WMS) in PD patients. Then the PD patients were further divided into PD patients with normal cognition (PD-CN group, n=66) and PD patients with mild cognitive impairment (PD-MCI group, n=48). The peripheral bloods of both PD patients and healthy controls were collected. We investigated the difference of serum of sTREM2 between PD patients and healthy controls by using enzyme-linked immunosorbent assay (ELISA). And then we investigated the relationship between level of sTREM2 and PD mild cognitive impairment.Results:(1)Among 114 patients with PD,48 patients (42.11%) were defined as PD-MCI patients. The education level was lower in PD-MCI patients compared to PD-CN patients (P<0.05). Motor symptoms (UPDRS part III and Hoehn-Yahr stage) and global cognitive function (MMSE and MoCA) were more severe in PD-MCI patients comparing with PD-CN patients (P<0.05).②Significantly increased expression of sTREM2 in peripheral of PD patients was observed when compared with healthy controls (P<0.05).③The level of sTREM2 was significantly different among the PD-CN patients, PD-MCI patients and healthy controls (P<0.001), with increased expression of sTREM2 in PD-MCI patients than PD-CN patient and healthy controls (P<0.001).④The expression level of sTREM2 in peripheral blood of PD was negatively with MoCA scores (r=-0.464, P<0.001).⑤Increased level of sTREM2 in patients with PD was significantly associated with poorer education (P=0.023), lower similarity subscores (P=0.015) and worse recognition subscores (P=0.018).Conclusions:①Upregulation of soluble TREM2 in peripheral blood of patients with PD is associated with the decline of global cognitive function, memory and executive function.②TREM2 may play an important role in the pathophysiology of PD and PD-MCI. The level of TREM2 in peripheral blood may be helpful to monitor the progression of PD and PD-MCI, which needs to be confirmed in future studies.Part Ⅱ The role of TREM2 in the neuroinflammation of MPTP-intoxicated Parkinson’s disease mouse modelObjectives:This study aims to investigate the role of TREM2 in the pathogenesis of neuroinflammation of PD by comparing the expression levels of TREM2, proinflammatory cytokines and anti-inflammatory factors in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated PD mouse model and saline-injected control mice.Methods:The C57BL/6 mice were randomly assigned into MPTP group, probenecid group and control group. Low dosage of MPTP and probenecid were intraperitoneally injected to induce PD mice model. Immuohistochemical staining of tyrosine hydroxylase (TH) was performed to observe the severity of midbrain dopaminergic neurons dysfunction. The mRNA levels of TREM2 and proinflammatory cytokines including IL-6, IL-1β and TNF-a genes were measured by quantitative real-time PCR. Western blot was performed to evaluate the expression of TREM2 and anti-inflammatory factor Arginase-1. One-way ANOVA analysis and LSD test were used to analyze the expression difference of TREM2, proinflammatory factors, anti-inflammatory factors and loss of dopaminergic neurons among different groups.Results:① The numbers of midbrain TH positive dopaminergic neurons decreased significantly in MPTP group compared to the probenecid group and control group (P<0.001). TH positive dopaminergic neurons were decreased by 39.7% in the MPTP group in comparison with the control group.②The mRNA levels of TREM2, IL-6, IL-1β and TNF-a in midbrain and cortex differs significantly among three groups (P<0.05), with highest levels in the MPTP group (P<0.05).③ TThere were significantly different expression levels of TREM2 and Arginase-1 in midbrain, cortex, hippocampus and striatum among MPTP group, probenecid group and control group (P<0.05), with highest expression levels in MPTP group (P<0.05).Conclusions:TREM2 level is higher in the midbrain, cortex, hippocampus and striatum of MPTP-intoxicated PD mice, which is accompanied by an elevated level of anti-inflammatory factor Arginase-1 and evident dopaminergic neurons loss and neuroinflammation, indicating that TREM2 might be a failure attempt to attenuate the excessive inflammation response in PD, which needs further studies to confirm.Part III The regulation of TREM2 in the polarization of microglia cellsObjectives:We aim to explore the involvement of TREM2 in the polarization of M1/M2 microglia cell.Methods:By using a lentivirus strategy, BV2 microglia were infected with TREM2-shRNA lentivirus to knock down TREM2 expression, while wtTREM2 lentivirus were administered to overexpress TREM2. Scramble TREM2 lentivirus containing a nonsense shRNA sequence and lentiviral particles containing blank vectors were designed as the negative control virus of TREM2-shRNA lentivirus and wtTREM2 lentivirus, respectively. Quantitative real-time PCR, western blot and single staining immunofluorescence techniques were used to verified transfection efficiency in the infected BV2 microglia 96 hours later. Then lOng/ml IL-4 and lOng/ml IL-13 were administered as M1 microglia triggers while 100ng/ml lipopolysaccharide (LPS) and lOng/ml interferon-y (IFN-y) as M2 microglia triggers to stimulate the lentivirus-infected BV2 microglia for 24 hours. Then the expression of TREM2 and M2 marker Arginase-1 were detected by quantitative real-time PCR, western blot and single staining immunofluorescence techniques. And Griess technique was used to evaluated the level of NO as M1 marker.Results:① After transfection, the results of immunofluorescence shows that the transfection effects of TREM2-shRNA, scramble TREM2 and vector lentivirus in BV2 microglia were higher than 90%, while it’s higher than 80% for wtTREM2 lentivirus (P<0.05).② The level of TREM2 and M2 marker Arginase-1 were significantly compromised when TREM2 was knocked down without the stimulation of M1 triggers, while were significantly enhanced after TREM2 overexpression without the stimulation of M2 triggers (P<0.05).③ The expression of TREM2 and Arginase-1 were increased in lentivirus-infected BV2 microglia followed by the treatment with M2 triggers (IL-4/IL-13), while the NO level was downregulated (P<0.05). On the contrary, the expression of TREM2 and Arginase-1 were decreased in lentivirus-infected BV2 microglia followed by the treatment with M1 triggers (LPS/IFN-γ), while NO level was upregulated (P<0.05).④ TREM2 and Arginase-1 expression was significantly decreased in TREM2-knockdown BV2 cells but was increased upon IL-4/IL-13 treatment. On the contrary, TREM2 and Arginase-1 expression was significantly enhanced in TREM2-overexpression BV2 cells whereas they was decreased after LPS/IFN-γ treatment.Conclusions:① Knockdown of TREM2 in the microglia even without treatment of M1 triggers compromised the expression of M2 markers, indicating that TREM2 is essential for M2 microglia polarization, and suggesting that TREM2 plays a pivotal role in the switch of microglia phenotypes from M1 to M2 that may contribute to the immune pathogenesis of PD.② Suppression of TREM2 in microglia inhibits M2 polarization and alternatively exaggerates Ml microglial inflammatory responses. On the contrary, overexpression of TREM2 could enhance the neuroprotective effects of M2 microglia and therefore inhibit M1 microglia-mediate inflammation response and produce anti-inflammatory factors, indicating a neuroprotection role of TREM2 in the neuroinflammation of PD. But the molecular mechanism of TREM2 in the polarization of M1/M2 microglia need to be further investigated in future studies.