Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs impact the response of microglial TREM2 and autophagy to Aβ. A Western blot analysis of TREM2-related protein levels in cortex of APP/PS1 mice and the corresponding quantification results (n = 4). B Relative mRNA levels of DAP12 and CD68 in cortex (n = 5). C Western blot analysis of autophagy-related protein levels in cortex and the corresponding quantification results (n = 4). D Relative mRNA levels of BECLIN1 and LAMP1 in cortex (n = 5). Western blot analysis of TREM2-related E and autophagy-related F Protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 4 h in the presence of Aβ and the corresponding quantification results (n = 3). Western blot analysis of TREM2-related G and autophagy-related H protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 12 h in the presence of Aβ and the corresponding quantification results (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs suppress TREM2 activation through binding to TREM2. A Molecular docking results of L-Leucine and KICA binding to TREM2 extracellular domain. B Biolayer interferometry (BLI) analysis of interaction of BCAAs or BCKAs with TREM2. BCAAs and BCKAs was titrated from 15.63 to 500 μM and 31.25 to 500 μM, respectively. C LC–MS/MS spectra of TREM2 immunoprecipitated L-Leucine- 13 C of BV2 cells after BCAAs isotope treatment for 1 h. D Thermal stabilization of TREM2 protein evaluated by cellular thermal shift assay (CETSA) after treating BV2 cells with BCAAs/ BCKAs for 4 h (n = 3). E Representative images of binding of fluorescein labeled-Aβ to cell surface TREM2 in BV2 cells after treatment of BCAAs/ BCKAs for 2 h (n = 3, Scale bars, 40 μm). F Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of S1P for 4 h (n = 3, Scale bars, 40 μm). G Western blot analysis of phosphorylation levels of SYK in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of sphingosine-1-phosphate (S1P) for 4 h (n = 3). Data in D , E , F and G are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Thermal Shift Assay, Labeling, Western Blot, Phospho-proteomics

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs reduce the expression and recycling of microglial TREM2 via inhibiting autophagy. Western blot analysis of TREM2-related A and autophagy-related B protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3). C Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). D Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). Western blot analysis of TREM2-related E and autophagy-related F protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3). G Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3, Scale bars, 40 μm). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test or Student’s t test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Labeling