Journal: Advanced Science

Article Title: Early‐Life Manganese Overexposure Activates Microglial Phagocytosis through the YY1/TREM2 Axis Leading to Hippocampal Synaptic Remodeling in Mice

doi: 10.1002/advs.78036

Figure Lengend Snippet: Suppression of Yy1 alleviates the excessive of TREM2‐mediated phagocytosis and activation of the phagolysosomal pathway in Mn‐exposed microglia. (A,B) Representative immunofluorescence staining and quantification of YY1 nuclear localization in IBA1 + (red) microglia across different treatment groups. Scale bar, 20 µm, magnification scale bar,10 µm. (C,D) Western blot showing YY1 expression in nuclear and quantification. (E–G) Representative images and 3D reconstructions of latex bead phagocytosis in microglia from various treatment group, with quantitative analysis of phagocytic capacity (relative to Control) and phagocytic microglia rate (%) within the microscopic field. Scale bar, 20 µm. (H) mRNA expression of Trem2 and phagolysosomal pathway–related genes ( Rab7 and Lamp1 ) in different treatment groups determined by RT‐qPCR. (I–L) Western blot analysis showing relative protein expression levels of TREM2 and phagolysosomal pathway (RAB7 and LAMP1) in microglia under different treatments. (M–O) Representative 3D reconstruction images of IBA1 + microglia within the microscopic field illustrating engulfment of PSD95 and VGAT, along with quantification of phagocytic volume. Scale bar, 10 µm. (P,Q) Representative fluorescence Z‐stack projection images reconstructed using Imaris 3D software, showing colocalization of VGLUT1 (green) and PSD95 (red), and VGAT (red) and Gephyrin (green), along with Pearson's correlation coefficient analysis and quantification of ES/IS counts relative to Control. Scale bar, 50 µm. mRNA expressions were normalized to Gapdh and relative to Control, proteins expressions were normalized to Histone H3 or β‐actin and relative to Control. Data were expressed as mean ± SD. For Western blot analysis, n = 3 independent experiments; and for ex vivo brain slice and other experiments, n = 4 independent samples per group. Data were analyzed by two‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01 versus Control group. # p < 0.05, # # p < 0.01 versus Mn group. See also Figures and .

Article Snippet: The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: β‐actin (Proteintech, China), Histone H3 (CST, USA), TREM2 (Abcam, USA), LAMP1 (CST, USA), RAB7 (CST, USA), YY1 (CST, USA), HIF1α (CST, USA), SMURF2 (Invitrogen, USA), and anti‐Ubiquitin (Invitrogen, USA).

Techniques: Activation Assay, Immunofluorescence, Staining, Western Blot, Expressing, Analysis, Control, Quantitative RT-PCR, Fluorescence, Software, Ex Vivo, Slice Preparation

Journal: Advanced Science

Article Title: Early‐Life Manganese Overexposure Activates Microglial Phagocytosis through the YY1/TREM2 Axis Leading to Hippocampal Synaptic Remodeling in Mice

doi: 10.1002/advs.78036

Figure Lengend Snippet: HIF1α regulation and dysfunction of SMURF2‐mediated ubiquitin degradation are responsible for YY1 upregulation. (A) Predicted HIF1α binding sites in the Yy1 promoter region from the JASPAR database. (B) Co‐expression analysis of Hif1a and Yy1 in the hippocampus by GEPIA database. (C) ChIP assay showing the binding between HIF1α and the Yy1 promoter. (D,E) Representative immunofluorescence images and quantitative analysis showing the nuclear expression of YY1 under different treatments. Scale bar, 20 µm, magnification scale bar, 10 µm. (F,G) Western blot analysis of nuclear YY1 protein levels in microglia from different treatment groups. (H) RT‐qPCR analysis of Yy1 , Trem2 , and phagolysosomal pathway‐related genes ( Rab7 and Lamp1 ) under various treatments. (I–M) Western blot and quantification of YY1, TREM2, and phagolysosome (RAB7 and LAMP1) proteins under different treatments. (N,O) Western blot showing YY1 protein degradation dynamics following CHX treatment at different time points under Mn exposure. (P) Immunoprecipitation analysis of YY1 ubiquitination levels in microglia. (Q) Identification of the E3 ubiquitin ligase SMURF2 targeting YY1 by the UbiBrowser 2.0 database. (R) Relative mRNA expression level of Smurf2 in Mn treatment assessed by RT‐qPCR. (S,T) Western blot and quantification of SMURF2 relative protein expression level under Mn treatment. (U) Molecular docking prediction of potential Mn 2 + binding sites within SMURF2. (V,W) Co‐immunoprecipitation assays revealing the interaction strength between SMURF2 and YY1 under different treatments. (X) Analysis of YY1 ubiquitination levels after immunoprecipitation across different experimental groups. mRNA expressions were normalized to Gapdh and relative to Control, proteins expressions were normalized to Histone H3 or β‐actin and relative to Control. Data were expressed as mean ± SD. For in vitro experiments, n = 3 independent experiments for Western blot analysis, and n = 4 independent experiments for all other assays. Data were analyzed by two‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01 versus Control group. ## p < 0.01 versus Mn (25 µ m ) group. See also Figure .

Article Snippet: The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: β‐actin (Proteintech, China), Histone H3 (CST, USA), TREM2 (Abcam, USA), LAMP1 (CST, USA), RAB7 (CST, USA), YY1 (CST, USA), HIF1α (CST, USA), SMURF2 (Invitrogen, USA), and anti‐Ubiquitin (Invitrogen, USA).

Techniques: Ubiquitin Proteomics, Binding Assay, Expressing, Analysis, Immunofluorescence, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Docking Assay, Co-Immunoprecipitation Assay, Control, In Vitro