Trem2 Knockout Cell Line (BV2)

BACKGROUND AND PURPOSE:Microglial activation plays a role in driving chronic migraine (CM). Triggering receptor expressed on myeloid cells 2 (TREM2) is expressed in brain microglia and impacts neuroinflammation in nervous system diseases. However, its role in CM is unclear. Here, we have investigated the role of microglial TREM2 in the development of CM. EXPERIMENTAL APPROACH:We used male mice receiving repeated intraperitoneal nitroglycerin (NTG) injections as a CM model. Mechanical and thermal hypersensitivity were assessed by mechanical withdrawal threshold and thermal withdrawal latency. TREM2 knockout mice (TREM2) and systemically administered TREM2 agonist COG1410 were evaluated for TREM2's role in CM. TREM2, calcitonin gene-related peptide (CGRP) and c-fos expression in the trigeminal nucleus caudalis (TNC) were measured for central sensitisation assessment. Immunohistochemical analyses and western blots measured protein expression in the TNC and BV-2 microglia. Quantitative real-time polymerase chain reaction (qRT-PCR) detected inflammatory factor expression. KEY RESULTS:Recurrent NTG injection up-regulated TNC protein levels of TREM2, CGRP and c-fos. TREM2 loss accelerated NTG-induced CM development, increased CGRP and c-fos expression, and inhibited TNC autophagy. Conversely, COG1410 prevented hyperalgesia and reduced CGRP/c-fos expression in the TNC after recurrent NTG administration. In vitro, TREM2 knockdown enhanced the expression of inflammation-related genes and the mTOR/p70s6k pathway activation in lipopolysaccharide (LPS)-stimulated BV-2 microglia, whereas COG1410 significantly inhibited LPS-induced mTOR/p70s6k pathway activation and alleviated inflammatory responses. CONCLUSION AND IMPLICATIONS:These data show that TREM2 plays a protective role in CM by modulating microglial activation and autophagy in the TNC via the mTOR/p70s6k pathway.

To investigate the mechanism of TREM2/DAP12 complex in mediating inflammatory responses that affect β-amyloid plaque deposition in Alzheimer's disease (AD) modeled mice. We measured escape latency and platform crossing time using the Morris water maze image automatic acquisition and software analysis system in TREM2 and DAP12 microglia knockout AD model mouse. We monitored the deposition of Aβ plaques in the mouse hippocampus using Congo red staining and measured levels. of inflammatory factors IL-6 and TNF-α by ELISA. Newborn mice with TREM2 knockout were selected for primary microglia isolation and culture, and Aged oligomer Aβ1-42 was added to the microglial culture medium to simulate the AD environment in vivo. Co-immunoprecipitation assay was used to detect the interaction between DAP12 and TREM2, and measured the inflammatory response induced by lipopolysaccharide (LPS) in mice with TREM2 and DAP12 knockdown through adeno-associated virus in BV2 microglia. The escape latency of the AD model mice with TREM2 and DAP12 knockout was higher and the number of crossing platforms lower than in the control group, whereas Aβ deposition and levels of inflammatory factors were higher. In TREM2 knockout microglial cultured with Aβ1-42, levels of IL-6 and TNF-α increased. Immunoprecipation pull-down assays showed that TREM2 binds to the membrane receptor DAP12 to form a complex. Knockout of TREM2 or DAP12 can inhibit LPS-induced microglial inflammatory responses. The TREM2/DAP12 complex inhibits the microglial inflammatory response through the JNK signaling pathway, thereby reducing the deposition of Aβ plaques and attenuation the behavioral manifestation in a mouse AD model.

Methamphetamine (METH) is a highly addictive psychostimulant and one of the most widely abused drugs worldwide. The continuous use of METH eventually leads to neurotoxicity and drug addiction. Studies have shown that neurotoxicity is strongly associated with METH-induced neuroinflammation, and microglia are the key drivers of neuroinflammation. Triggering receptor expressed on myeloid cells 2 (TREM2) is reported to play a key role in activation of microglia and neuroinflammation. Yet, the molecular mechanisms by which METH causes neuroinflammation and neurotoxicity remain elusive. In the current study, we investigated the role of TREM2 in neuroinflammation induced by METH in BV2 cells and the wild-type (WT) C57BL/6J mice, CX3CR1 transgenic mice, and TREM2 knockout (KO) mice. Postmortem samples from the frontal cortex of humans with a history of METH use were also analyzed to determine the levels of TREM2, TLR4, IBA1, and IL-1β. The expression levels of TREM2, TLR4, IBA1, IL-1β, iNOS, and Arg-1 were then assessed in the BV2 cells and frontal cortex of mice and human METH users. Results revealed that the expression levels of TREM2, TLR4, IBA1, and IL-1β were significantly elevated in METH-using individuals and BV2 cells. Microglia were clearly activated in the frontal cortex of WT C57BL/6 mice and CX3CR1 transgenic mice, and the protein levels of IBA1, TREM2, TLR4, and IL-1β were elevated in the METH-induced mouse models. Moreover, TREM2-KO mice showed further increased microglial activation, neuroinflammation, and excitotoxicity induced by METH. Thus, these findings suggest that TREM2 may be a target for regulating METH-induced neuroinflammation.