β2 ar antagonist propranolol  (MedChemExpress)

Journal: Aging Cell

Article Title: Aerobic Exercise Attenuates Autophagy‐Lysosomal Flux Deficits via β2‐AR‐Mediated ESCRT‐III Subunit CHMP4B in Mice With Human MAPT P301L

doi: 10.1111/acel.70184

Figure Lengend Snippet: AE upregulates CHMP4B expression through β2‐AR‐RXRα signaling in MAPT P301L mice. (A and B) The mRNA and protein levels of β2‐AR in the hippocampus of WT, MAPT and AE‐treated MAPT mice ( n = 6 mice per group). (C and D) The level of HT7, β2‐AR, pSβ2‐AR, SQSTM1 and LC3‐II in N2a cells with overexpression of MAPT and treated with Teb ( n = 6 independent experiments for each group). (E) Representative images of autophagic flux and (F) quantification of LC3 puncta in N2a cells with overexpression of MAPT and treated with Teb (8 images from n = 4 independent experiments for each group). (G and H) The protein levels of HT7, CHMP4B, SQSTM1 and LC3‐II in the N2a cells with overexpression of MAPT and CHMP4B and treated with si Adrb 2 and si Chmp4b or Teb detected by western blotting ( n = 6 independent experiments for each group). (I‐L) The mRNA levels of the Chmp4b transcription factors Thap11 , Nr2f6 , Prdm15 and Rxrα in the hippocampus of WT, MAPT and MAPT+AE mice ( n = 6 mice per group). (M and N) The protein levels of RXRα in the hippocampus of WT, MAPT and MAPT+AE mice ( n = 6 mice per group). (O) Schematic diagram revealing the mouse CHMP4B promoter region and Rxrα potential binding Site1 and Site2. (P) ChIP assay results determined the enrichment of RXRα at potential binding site 1 and site 2 of the CHMP4B promoter region. (Q) The direct binding capacity of RXRα at Site2 to the CHMP4B promoter was validated using qPCR of the ChIP products ( n = 6 mice per group). (R) Diagram of the principle of Dual‐luciferase experiment. (S) Dual‐luciferase reporter gene assays the binding level of RXRα to the CHMP4B promoter at Site2 in N2a cells with overexpression of RXRα and treated with si Rxrα ( n = 6 independent experiments for each group). (T and U) The protein levels of CHMP4B in the N2a cells with overexpression of MAPT and treated with si Rxrα or Teb were detected by western blotting ( n = 6 mice per group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. The data are presented as the means ± SEMs. One‐way ANOVA with Bonferroni post hoc test was used to analyze the data.

Article Snippet: Mice were treated with the systemic β2‐AR antagonist propranolol (MedChemExpress, HY‐B0573B) at a dose of 30 mg/kg by intragastric administration 1 h prior to the AE (Tsai et al. ).

Techniques: Expressing, Over Expression, Western Blot, Binding Assay, Luciferase

Journal: Aging Cell

Article Title: Aerobic Exercise Attenuates Autophagy‐Lysosomal Flux Deficits via β2‐AR‐Mediated ESCRT‐III Subunit CHMP4B in Mice With Human MAPT P301L

doi: 10.1111/acel.70184

Figure Lengend Snippet: The β2‐AR inhibitor propranolol reverses the ameliorative effects on cognition and pathology in MAPT P301L mice after AE. (A) Escape latency to the hidden platform during the 6‐day training trials in the MWM test ( n = 8 mice per group). * p < 0.05, (MAPT+AE vs. MAPT); # p < 0.05, ## p < 0.01 (MAPT+AE + Prop vs. MAPT+AE). (B) The swimming pathway traveled to locate the platform on day 7. (C) Escape latency, (D) number of crossings of the hidden platform area, (E) the time in target quadrants and (F) swimming speed on day 7. (G and H) The protein levels of SQSTM1 and LC3‐II in the hippocampus of the AE and propranolol‐inhibited groups ( n = 6 mice per group). (I and J) Electron microscopy analysis of autophagosomes shows arrows indicating autophagosomes ( n = 9 images from 3 mice per group; scale bar: 0.5 μm). (K and L) The protein levels of soluble Tau of (pS396, pS404, pT231 and Tau5) in the hippocampus were detected using western blotting and quantitatively analyzed. (M) Representative confocal images of LC3 and Tau5 immunofluorescence co‐labeling in the DG region of the hippocampus of the mice. (N) Quantification of the ratio of LC3 + Tau5 + :Tau5 + . (O and P) Nissl staining and quantification of intact neurons in the hippocampal CA1 region of mice. ( n = 6 mice per group). (Q and R) The protein levels of RXRα and CHMP4B in the hippocampus of the AE and propranolol‐inhibited groups ( n = 6 mice per group). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. The data are presented as the means ± SEMs. Two‐way ANOVA with Bonferroni post hoc test was used for (A), and one‐way ANOVA with Bonferroni post hoc test was used to analyze the data in (C–F) and unpaired t ‐test for the other data.

Article Snippet: Mice were treated with the systemic β2‐AR antagonist propranolol (MedChemExpress, HY‐B0573B) at a dose of 30 mg/kg by intragastric administration 1 h prior to the AE (Tsai et al. ).

Techniques: Electron Microscopy, Western Blot, Immunofluorescence, Labeling, Staining