Increased miR-155 in Microglial Exosomes Following Heat Stress Accelerates Neuronal Autophagy via Their Transfer Into Neurons.

Background: Heat stroke is the outcome of excessive heat stress, which results in core temperatures exceeding 40°C accompanied by a series of complications. The brain is particularly vulnerable to damage from heat stress. In our previous studies, both activated microglia and increased neuronal autophagy were found in the cortices of mice with heat stroke. However, whether activated microglia can accelerate neuronal autophagy under heat stress conditions is still unknown. In this study, we aimed to investigate the underlying mechanism that caused neuronal autophagy upregulation in heat stroke from the perspective of exosome-mediated intercellular communication. Methods: In this study, BV2 and N2a cells were used instead of microglia and neurons, respectively. Exosomes were extracted from BV2 culture supernatants by ultracentrifugation and then characterized via transmission electron microscopy, nanoparticle tracking analysis and Western blotting. N2a cells pretreated with/without miR-155 inhibitor were cocultured with microglial exosomes that were treated with/without heat stress or miR-155 overexpression and subsequently subjected to heat stress treatment. Autophagy in N2a cells was assessed by detecting autophagosomes and autophagy-related proteins through transmission electron microscopy, immunofluorescence, and Western blotting. The expression of miR-155 in BV2 and BV2 exosomes and N2a cells was measured using real-time reverse transcription polymerase chain reaction. Target binding analysis was verified via a dual-luciferase reporter assay. Results: N2a autophagy moderately increased in response to heat stress and accelerated by BV2 cells through transferring exosomes to neurons. Furthermore, we found that neuronal autophagy was positively correlated with the content of miR-155 in microglial exosomes. Inhibition of miR-155 partly abolished autophagy in N2a cells, which was increased by coculture with miR-155-upregulated exosomes. Mechanistic analysis confirmed that Rheb is a functional target of miR-155 and that microglial exosomal miR-155 accelerated heat stress-induced neuronal autophagy mainly by regulating the Rheb-mTOR signaling pathway. Conclusion: Increased miR-155 in microglial exosomes after heat stroke can induce neuronal autophagy via their transfer into neurons. miR-155 exerted these effects by targeting Rheb, thus inhibiting the activity of mTOR signaling. Therefore, miR-155 could be a promising target for interventions of neuronal autophagy after heat stroke.

The involvement of microglial CX3CR1 in heat acclimation-induced amelioration of adult hippocampal neurogenesis impairment in EMF-exposed mice.

Microglial CX3C chemokine receptor 1 (CX3CR1) has been implicated in numerous cellular mechanisms, including signalling pathways that regulate brain homoeostasis and adult hippocampal neurogenesis. Specific environmental conditions can impair hippocampal neurogenesis-related cognition, learning and memory. However, the role of CX3CR1 in the neurogenic alterations resulting from the cross-tolerance protection conferred by heat acclimation (HA) against the effects of electromagnetic field (EMF) exposure is less well understood. Here, we investigated the role of microglial CX3CR1 signalling in adult hippocampal neurogenesis induced by HA in EMF-exposed mice. We found that EMF exposure significantly decreased the number of proliferating and differentiating cells in the dentate gyrus (DG) of the hippocampus, resulting in a reduced neurogenesis rate. Moreover, alterations in the phenotypes of activated microglia and decreased expression levels of CX3CR1, but not sirtuin 1 (SIRT1), were observed in the brains of EMF-exposed mice. Remarkably, HA treatment improved microglial phenotypes, restored the expression of CX3CR1, and ameliorated the decrease in the adult hippocampal neurogenesis rate following EMF exposure. Moreover, pharmacological inhibition of CX3CR1 and SIRT1 failed to restore CX3CR1 expression and ameliorate hippocampal neurogenesis impairment following HA plus EMF stimulation. These results indicate that microglial CX3CR1 is involved in the cross-tolerance protective effect of HA on adult hippocampal neurogenesis upon EMF exposure.

TREM2 Regulates Heat Acclimation-Induced Microglial M2 Polarization Involving the PI3K-Akt Pathway Following EMF Exposure.

The function of triggering receptor expressed on myeloid cells-2 (TREM2) has been described within microglia with a beneficial activated phenotype. However, the role of TREM2 underlying microglial phenotypic alterations in the cross-tolerance protection of heat acclimation (HA) against the inflammatory stimuli electromagnetic field (EMF) exposure is less well known. Here, we investigated the TREM2-related signaling mechanism induced by HA in EMF-stimulated N9 microglial cells (N9 cells). We found that EMF exposure significantly increased the production of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α, IL-1ß, and IL-6), and the expression of M1 markers (CD11b and CD86); meanwhile, decreased the levels of anti-inflammatory cytokines (IL-4 and IL-10) and the expression of M2 markers (CD206 and Arg1) in N9 cells. Clearly, HA treatment decreased the secretion of TNF-α, IL-1ß and IL-6 and the expression of CD11b and CD86, and enhanced the production of IL-4 and IL-10 and the expression of CD206 and Arg1. Moreover, TREM2 esiRNA and selective inhibitor of PI3K clearly decreased anti-inflammatory cytokines production, M2 markers expression, and phosphorylation of PI3K and Akt following HA plus EMF stimulation. These results indicate that TREM2 and PI3K-Akt pathway are involved in the cross-tolerance protective effect of HA in microglial polarization towards the EMF exposure. This finding inspires future studies that aim to explore the non-drug approaches underlying EMF stimulation and other central nervous system (CNS) inflammatory diseases.

Derivatization method for the quantification of lactic acid in cell culture media via gas chromatography and applications in the study of cell glycometabolism.

Lactic acid represents an important metabolite that reflects mitochondria function and may further serve as energy source for cancer cells. In light of this physiological and pathological significance, we developed a novel and sensitive gas chromatography method to detect lactic acid in cell culture media. Here, ethyl chloroformate was selected as derivative reagent and the derivatization process was further optimized in terms of number of reagents and reaction time as well as extraction reagents. Under optimal conditions, good linearity was achieved in the tested calibration range. The limit of detection (LOD) was determined to be 0.67 µmol/L, the recovery rates were 99.6%-106% and the precision rate RSD was <5.49%. Furthermore, this method has been applied to quantify the secretion of lactic acid in cells exposed to mono­2­ethylhexyl phthalate at different doses and in cancer cells over time. Taken in concert, our method proved to be both sensitive and reliable and may be applied for studies on mitochondrial function and cell glycolysis conditions.