The Association Between TREM2 Polymorphisms And Parkinson’s Disease And TREM2 Expression In MPTP-treated Mice

Parkinson’s disease (PD) is a common neurodegenerative disease among the elderly people, which is characterized by progressive loss of dopaminergic neurons, aggregation of a-synuclein (a-syn) and formation of Lewy body (LB) in the substantia nigra of the midbrain. When patients with PD progress to an advanced stage, the motor symptoms as well as the non-motor symptoms such as dementia and depression can cause severe disability and declined life quality, putting heavy burden on both caregivers and the society. Nevertheless, all current anti-Parkinson drugs are symptomatic therapies and there are not enough evidences to promise the neuroprotective or disease-modifying benefits of current pharmacotherapies.The etiology and pathogenesis of PD remains not fully understood. PD was used to be considered as a sporadic disease but now it’s well established that PD results from a complex interaction among genetic, environmental factors and aging, since pathogenic mutations in several genes are demonstrated to cause familial PD. Specific mutations in SNCA (a-synuclein), LRRK2 (Leucine-rich repeat kinase 2), GBA (Glucocerebrosidase), parkin, PINK1 (PTEN induced putative kinase 1) and DJ-1 gene have been confirmed to cause familial PD in the past two decades. However, with few notable exceptions in some populations,< 5% of all PD cases are caused by known single-gene mutations, suggesting that additional genes may contribute to the pathogenesis of PD in other ways in most sporadic cases.In recent years, some large-scale genome-wide association study (GWAS) have found that single nucleotide polymorphisms (SNP) in a multitude of genes are associated with increased risk for sporadic PD. Polymorphisms of SNCA, GBA, MAPT (Microtubule-associated protein tau), LRRK2 and RIT2 (Ras-like without CAAX 2) have been demonstrated to be associated with the risk for sporadic PD. These polymorphisms may not cause PD directly, but they may have an effect on the biological function of related proteins either by changing the coding sequence of the protein or by posttranscriptional modification, thereby influencing the susceptibility of PD. Hence, it makes great sense to search for potential associated SNPs of PD in order to reveal the genetic contribution in the pathogenesis of sporadic PD.It is generally recognized that neuroinflammation, oxidative stress, mitochondrial dysfunction and the prion-like spreading of α-syn, and so forth, may play a role of their own in degeneration of dopaminergic neurons, among which neuroinflammation mediated by over-activated microglia plays a role that can not be neglected. Microglia is the resident macrophage in the central nerve system (CNS), acting as a double-faced immune cell in neuroinflammation. In one way, microglias can monitor the internal environment of the CNS, so that they can recognize the dead neurons and cell debris, also promote the phagocytosis and clearance of them, thereby preventing neuroinflammation resulting from tissue damage. In the other way, when over-activated, microglia can release proinflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) as well as inducible nitric oxide synthase (iNOS), which will induce neuronal death directly or indirectly. Massive activated microglias were found in the substantia nigra of both PD patients and MPTP-treated PD mice models. What’s more, evidence shows that the activation of microglias may even occur before the degeneration and death of dopaminergic neurons, indicating that neuroinflammtion contributes to the process of neuron degeneration rather than just a concomitant phenomenon of neuronal death. Neuroinflammation mediated by microglia may directly induce neuronal death or cause neuron degeneration via oxidative stress, accelerating the progression of PD. Therefore, timely blocking of the inflammation process seems to be a promising potential target for neuroprotective intervention in PD.Triggering receptor expressed on myeloid cells-2 (TREM2) is an immunoreceptor expressed on the membrane of a variety of myeloid cells such as macrophages, dendritic cells, osteoclasts, and microglias, etc. TREM2 can promote phagocytosis of apoptotic cells and inhibit inflammatory cytokine like IL-1β, IL-6 and TNF-a, production in response to apoptotic cells and debris. In other words, Stimulation of TREM2 receptor may trigger the microglia to scavenge the dead neurons and cell debris in the CNS, thus preventing and inhibiting neuroinflammation.Recently, the role of TREM2 in a variety of neurodegenerative diseases like PD and Alzheimer’s disease (AD) has been a hot concern of researchers. In 2013, two independent GWAS studies both demonstrated that a missense mutation rs75932628-T (p.R47H) on exon2 of TREM2 gene increases the risk of AD by nearly threefold. Later on, Rayaprolu et.al reported that rs75932628-T was associated with increased risk for PD in an American cohort (OR= 2.67). Soon the positive association between rs75932628-T and PD was replicated in Spanish and another American cohort. In addition, various studies have demonstrated that rs75932628-T of TREM2 gene is also a genetic risk factor for frontotemporal dementia and amyotrophic lateral sclerosis, suggesting that TREM2 polymorphisms may be a general risk factor for neurodegenerative diseases and TREM2 may play a significant role in neurodegeneration.The mRNA expression of TREM2 gene is heterogeneous among different brain regions in healthy human brains, with the highest in the microglias of the white matter, followed by substantia nigra and cortex, and the lowest in the cerebellum. Pathological finding shows that LB-like pathological changes were found in two out of five human brains carrying TREM2 mutations, which strongly indicates that TREM2 variants may play an underlying role in the PD pathogenesis. Though the exact ingredient of TREM2 ligands remains unknown, in vitro studies have demonstrated that dopaminergic neurons express TREM2 ligands which binds to TREM2, thereby triggering the phagocytosis of the apoptotic dopaminergic neurons.Taken together, it is rational for us to hypothesize that TREM2 may play a significant role in the pathogenesis of PD, but the underlying mechanisms of TREM2 in PD are unknown. Considering that TREM2 can promote clearance of dead cells and inhibit neuroinflammation, then, will TREM2 be able to boost the phagocytosis of degenerated dopaminergic neurons, cell debris and extracellular a-syn, thereby inhibit cell-damaging neuroinflammation? Studies concerning this issue are scarce by far. In addition, gene polymorphisms are largely influenced by geographic region and ethnic variant The association of TREM2 polymorphisms and PD in Chinese Han population is still absent at present.With this in mind, we directly sequenced exon2 of TREM2 gene and analyzed the association between TREM2 polymorphisms and the risk of PD in a Chinese Han cohort in this study. What is more, we preliminary investigate the mRNA expression of TREM2, α-syn, IL-1β, IL-6, TNF-α and iNOS in the MPTP-treated PD mice model so as to gain insight into the role of TREM2 in microglia-mediated inflammation in the pathogenesis of PD.This study is consisted of two parts:(1) the association study between TREM2 polymorphisms and the risk of PD; (2) The study of TREM2 expression levels in MPTP-treated mice.Part I:The association of TREM2 polymorphisms and Parkinson diseaseObjective:To investigate the association between TREM2 polymorphisms and the risk for Parkinson disease.Methods:476 patients with PD and 432 healthy controls were included in this study. (1) The demographic data and blood samples of all subjects were collected. (2) Genomic DNAs of all subjects were isolated from whole blood and then we directly sequenced exon 2 of TREM2 gene. (3) Fisher exact test was used to compare the distribution of allelic and genotype frequency of rs75932628, rs142232675, rs201258663, rs143332484, p.Y38C and rs104894002 in case-control series. All statistics were 2-tailed, and significance was set at P< 0.05.Results:① There was no statistical difference between the PD group and control group with regard to gender and age (P> 0.05).② From a total of 908 individuals, rs75932628-T was found in 0.2%(1/476) of PD cases and none in controls (0/432)(P=1.00). Rs75932628-T exhibited a minor allele frequency (MAF) of 0.06%. We didn’t find any allelic variants in rs142232675, rs201258663, rs143332484, p.Y38C or rs104894002.③ No significant difference was found in the distribution of allelic and genotype frequency of rs75932628, rs142232675, rs201258663, rs143332484, p.Y38C and rs104894002 (P> 0.05).Conclusions:(1) Our findings suggest that variations in exon2 of TREM2 are much less frequent in southern Han Chinese than that in European-descended populations.(2) Rs75932628-T couldn’t be a genetic risk factor for PD in the Southern Han Chinese population.Parts Ⅱ:The study of TREM2 expression in MPTP-treated MiceObjective:We aim to investigate the gene expression differences of TREM2 and related proinflammatory factors in MPTP-treated mice model of PD and control mice, thus to explore the role of TREM2 in the pathophysiology and pathogenesis of PD.Method:C57BL/6J mice were randomly assigned into MPTP+ probenecid group, probenecid group or saline control group. Low dosage of MPTP was administrated by chronic intraperitoneal injection to induce chronic PD model. Brain tissues were harvested for immunohistochemistry and mRNA level detection. (1) Midbrain paraffin sections were used for immunohistochemical staining of tyrosine hydroxylase (TH) in order to analyze TH positive neuron numbers. (2) Total RNA of brain tissure was extracted and quantitative real-time PCR was performed to detect mRNA levels of TREM2, α-syn, IL-1β, IL-6, TNF-α and iNOS genes. One-way ANOVA was adopted to compare DA neuron degeneration, mRNA relative expression levels of TREM2 and other detected genes. All statistics were 2-tailed, and significance was set at P< 0.05.Results:① The cell counting of TH positive neurons in the midbrain was significantly decreased in the MPTP+ probenecid group in comparison with the probenecid group and saline control group (P< 0.05). TH positive neurons in the MPTP+ probenecid group were decreased by 42.8% in contrast to the saline control group.② The mRNA levels of TREM2, IL-1β, IL-6, TNF-α and iNOS genes were significantly different among the MPTP+ probenecid group, probenecid group and saline control group (P< 0.05), with increased expression levels in MPTP+ probenecid group than the other two groups (P< 0.05). The mRNA levels of SNCA gene was higher than the the probenecid group and saline control group, though the difference didn’t reach statistical significance (P> 0.05).Conclusions:(1) DA neurons degeneration and neuroinflammation in the substantia nigra are prominent in the MPTP+ probenecid intraperitoneal injection induced chronic PD mice model.(2) TREM2 gene expression is increased in the substantia nigra of MPTP- treated chronic PD mice model, suggesting that TREM2 may play a role in the pathophysiology and pathogenesis of PD.