Mechanism Of TREM2-regulated Microglia Polarization In Benzo[a]pyrene-induced Cognitive Impairment In Mice

Objective:To investigate the role of TREM2 in microglia(MC)polarization-mediated neuronal synaptic remodeling in the cognitive impairment induced by benzo[a]pyrene(B[a]P)in vivo and in vitro,so as to provide new evidence for the mechanism study of B[a]P neurotoxicity.Methods:1.Changes of neuronal synaptic remodeling in B[a]P-induced cognitive impairment in miceForty specific pathogen-free(SPF)male ICR mice(8 weeks old)were randomly categorized into 4 groups based on their weights,including solvent control group(vegetable oil),low,middle,and high dose B[a]P(0.5mg/kg,2mg/kg,10mg/kg)treated groups,10 per group.The treated groups were administrated B[a]P solution by intraperitoneal injection(2.5 m L/kg),once every other day(9:00 am),total 30 times.The solvent control group was injected with the same volume of vegetable oil.After exposure,we used water maze test and open field test to detect the spatial learning and memory ability and spontaneous behavior in mice,respectively.The pathomorphological changes of cerebral cortex were observed under a light microscope and the synaptic ultrastructural changes in hippocampal neurons of mice were observed under a transmission electron microscope.Using real-time fluorescence quantitative PCR(QPCR)and Western blot(WB),we detected the gene and protein expression levels of synaptic functional proteins including SYP,PSD95,NLGN1,NLGN2,MDGA1,MDGA2,NRXN1,and SNAP25 in cerebral cortex in mice.HT22 hippocampal neuronal cell line derived from mouse were cultured and randomly divided into 5 groups:blank group,solvent control group(DMSO),treated groups at low,middle,and high dose of B[a]P(0.2μM,2μM,20μM).Cells were treated when the medium was replaced with DMEM medium containing 3% fetal bovine serum,the blank control or solvent control group were only added medium or 0.5%(v/v)DMSO,respectively,instead of B[a P solution.After 24 hours of treatment,the morphological changes of the cells were observed under an inverted microscope,and the cell viability was detected using the CCK-8 assay kit.Next,HT22 hippocampal neuronal cell was co-cultured with BV2 microglia cell line in a transwell co-culture plate,with BV2 in the well above and HT22 in the well below,and then were treated with different concentrations of B[a]P for 24 hours.We observed the morphological changes in HT22 cells,determined the cell viability and the leakage rate of lactate dehydrogenase(LDH)in the supernatant,and detected the gene and protein levels of synaptic functional proteins in co-cultured HT22 cells using QPCR and WB methods,respectively.2.Changes of MC polarization and TREM2 in synaptic remodeling induced by B[a]PAfter B[a]P treatment,the MC polarization markers and TREM2 were detected the gene and protein levels in cerebral cortex in mice using QPCR and WB,respectively.The levels of IL-1β and TNF-α in cerebral cortex were detected using ELISA.After BV2 cell were treated with different concentrations of B[a]P for 24 hours,the morphological changes were observed,the cell viability and LDH leakage rate were detected,and the MC polarization markers and TREM2 were assayed their gene and protein levels using QPCR,immunofluorescence,and WB methods,and also detected the levels of inflammatory factors in BV2 cells using ELISA.The MC polarization type and TREM2 expression were conprehensively judged based on the above results.3.The changes of PI3K/Akt and NFκB pathway in synaptic remodeling induced by B[a]PAfter the B[a]P treatment mentioned above,the protein levels of PI3K/Akt and NFκB were detected in the cerebral cortex in mice and cultured BV2 cells using WB,and to explore the potential impact of B[a]P on the above pathways.4.Mechanism of TREM2 regulated MC polarization imediated the synaptic remodeling induced by B[a]PIn order to verify whether the TREM2-regulated MC polarization mediated B[a]P-induced synaptic remodeling via PI3K/Akt and NFκB pathway,we transfected BV2 cells using the pc DNA-TREM2 plasmid to overexpress TREM2 gene or TREM2-sh RNA lentivirus vector to inhibit TREM2 gene,respectively.BV2 cells were cultured alone or co-cultured with HT22 together according to the experiment demand.In each culture mode,the cells were randomly categorized and treated with Blank(blank control),DMSO(solvent control),B[a]P(20μM),plasmid negative control(B[a]P+pc DNA-control),TREM2 overexpression(B[a]P+pc DNA-TREM2),lentivirus negative control(B[a]P+control-sh RNA),and TREM2 low expression(B[a]P+TREM2-sh RNA).After the B[a]P administration for 24 hours,we observed the cells morphological changes,detected the cell viability and LDH leakage rate,and detected the gene and protein levels of Iba-1,CD206,TREM2,and PI3K/Akt and NFκB pathway in BV2,and the synaptic functional proteins in HT22 cells using methods of QPCR,WB,and immunofluorescence.Results:1.B[a]P induced cognitive dysfunction and abnormal synaptic remodeling in mice.With the increase of the doses of B[a]P,the spatial learning and memory ability and spontaneous behavior were decreased gradually in mice,as manifested by the significantly prolonged escape latency in the treated groups,the significantly decreased duration in the target quadrant,the increased duration in the target opposite quadrant,and the statistic decreased standing times and modification frequencies(P<0.05).With the increase of B[a]P doses,the cerebral cortical nerve cells in the treated group got swollen,degenerated and necrosis,and even demyelination of nerve fibers,displayed the so-called satellite phenomenon and neuronal phagocytosis.The synaptic remodeling was damaged in the B[a]P-treated groups compared to the controls,as exhibted by the significantly decreased synapse numbers,the widened synaptic space,the decreases in interface curvature,the density and length of postsynaptic membrane(P<0.05),and the significant decreases in gene and protein levels of synaptic functional proteins(SYP,PSD95,NRXN1,MDGA2,NLGN2).The results of in vitro study also showed that HT22 cells got gathered into clusters,thicken and less cell connection,was decreased cell viability,and was increased the leakage rate of lactate dehydrogenase(LDH)in a dose-dependent manner.Above findings suggest that B[a]P can induce abnormal neuroanl synaptic remodeling,which may be one of the mechanisms of cognitive dysfunction induced by B[a]P in mice.2.B[a]P induced the MC M1 polarization and inhibited TREM2 gene and protein expressionPrevious studies indicated that the central neuroinflammation caused by MC activation plays an important role in B[a]P’s neurotoxicity.TREM2 is the main molecular switch to regulate the MC polarization phenotype.In this study,it was found that with the increase of the doses of B[a]P,MC cells were activated and predominantly polarizated into M1 type in mice’s cerebral cortex and in BV2 cells in vitro,along with the decreased M2 polarization,and the dose-dependently decreased TREM2 expressions at gene and protein levels(P<0.05).The results suggest that B[a]P exposure can decrease the TREM2 expression(at both gene and protein levels)and induce MC M1 polarization,which result in the inflammatory reaction in central nervous system.3.B[a]P induces the change of PI3K/Akt and NFκB pathway in MCWith the increase of the doses of B[a]P,the protein levels of PI3 K,Akt and p-Akt were significantly decreased in mice’s cerebral cortex and BV2 cells,while NFκB protein level were significantly increased.4.Mechanism of TREM2-regulated MC polarization in synaptic remodeling induced by B[a]POverexpression of TREM2 activated the PI3K/Akt pathway,inhibited NFκB,turned MC M1 into M2 polarization,mitigated the neuroinflammatory response,and alleviated the neurotoxicity of B[a]P,which was characterized as the recovered cells viability in HT22,the increased synaptic functional proteins protein levels,and the improved synaptic remodeling.Low expression of TREM2 inhibited PI3K/Akt pathway,activated NFκB protein,urged MC M1 polarization from MC M2,aggravated the neuroinflammatory response and abnormal synaptic remodeling induced by B[a]P.Conclusions:1.B[a]P exposure can cause cognitive dysfunction in mice.Abnormal synaptic remodeling may be one of the main mechanisms of cognitive dysfunction induced by B[a]P in mice.2.TREM2-regulated the MC M1 polarization was related to the abnormal synaptic remodeling induced by B[a]P in mice.3.MC M1 polarization is potentially the mechanism underlying abnormal synaptic remodeling induced by B[a]P via TREM2/PI3K/Akt and TREM2/NFκB pathways.