Abstract
1506a
Introduction: Neuroinflammation is widely regarded as a chronic innate immune response in the brain and apotentially pathogenic factor in a number of neurodegenerative diseases such as Alzheimer’s disease (AD), as well as traumatic brain injury. Recently, due to its key signaling steps in the initiation of immune activation, greater attention has been paid to the potential of neuroinflammation as a therapeutic target. Microglia, which comprise approximately 20% of all glial cells, serve as the principal immune cells and play crucial roles in the central nervous system, responding to neuroinflammation via migration and the execution of phagocytosis. The activation of microglia is characterized by an increase in the expression level of TSPO, which can be detected by PET.Transmembrane protein 59 (Tmem59) is a membrane protein that plays an important role in neural stem cell differentiation, glioma apoptosis, dendritic spine formation, and Alzheimer’s disease (AD), nevertheless, the involvement of the Tmem59 gene in the brain immune response has not yet been reported. In the current work, we investigated the effect of Tmem59 in neuroinflammation.
Methods: 18F-DPA-714 imaging in WT and KO mice were performed using a micro PET/CT. Tmem59 down-expression models and vector controls of BV2 microglia cells were established by psiRNA-Tmem59 and Vehicle plasmid transfection, respectively. The change of gene expression was detected by western blotting analysis. Cell phagocytosis assay was used to study the effect of Dcf1 downregulation on the phagocytic ability of LPS-activated microglia
Results: FIGURE 1 | Tmem59 deletion induces activation of microglial cells in vivo. 18F-DPA-714 (green) was used to trace TSPO (a biomarker of microglia) by PET to observe the activity of microglia in vivo. (A,B) Brain observation by PET in WT (A) and Tmem59-KO (B) mice. White arrowhead denotes activated microglia. (C) Quantitation of the TSPO in WT and Tmem59-KO mouse brain (Supplementary Table S1). (D) Immunohistochemical observation of microglia from WT and Tmem59-KO mouse brain sections. Microglial cells were detected by the Iba1 biomarker (green), and the nuclei were counterstained with DAPI (blue). Scale bars represent 200 µm. Higher magnification of confocal images were shown in right panel. Scale bars, 10 µm. (E) Quantitation of the mean density of Iba1 staining in WT and Tmem59-KO mouse brain sections (mean±SEM). n = 4. ∗p < 0.05. Protein expression of Iba1 (F) and CD68 (G) in WT and Tmem59-KO mouse brain tissue . Quantification of protein expression levels normalized to Gapdh. Data are expressed as the mean±SEM. n = 3. ∗p < 0.05; ∗∗p < 0.01.FIGURE 2 | Tmem59 deletion suppresses the phagocytic ability of BV2 microglia cells. BV2 microglial cells were transfected with the psiRNA-hH1neo plasmid or the psiRNA-Tmem59 plasmid. 24 h post-transfection, BV2 microglia were stimulated with LPS (1000 ng/ml) and incubated for 12 h. (A) Image showing the phagocytic ability of BV2 microglia cells. Quanta were spontaneous green, and the cell skeleton was detected by ActinRed (red). Scale bars represent 20 µm. (B) Analysis of the average quantum absorption in each cell to assess the phagocytic activity of BV2 microglia. Data are expressed as the mean±SEM. n = 4. ∗p < 0.05 (Supplementary Table S12).
Conclusions: In conclusion, our data indicates that Tmem59-deficient microglia induced aberrant proinflammatory cytokines release and subsequent microglial dysfunction, which blocked the phagocytic abilities of activated microglia. Taken together, these observations provide novel insight into the role of Tmem59 in activated microglial cells during the neuroimmune response, and further lay the foundation for the elucidation of the mechanism underlying neuroinflammatory-related diseases.
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