Fig 1.
Peli1 curtails phagocytic efficiency of microglia.
(A-D) Flow cytometry of the phagocytic ability for fluorescent microspheres in Peli1+/− and Peli1−/− primary microglia or astrocyte. The data are presented as representative scatter plots showing the frequencies of the cells containing fluorescent microspheres (A, C) and summary bar graphs (B, D). (E, F) Flow cytometry of the phagocytic ability for Rhodamine B–dyed Aβ1–42 peptide in Peli1+/− and Peli1−/− primary microglia. The data are presented as representative histogram showing the frequency of the cells containing fluorescent Aβ1–42 (E) and summary bar graph (F). (G, H) Microscopic analysis of the phagocytosis of Aβ1–42 peptide for 24 hours in Peli1+/− and Peli1−/− primary microglia. The data are presented as representative images (G) and summary bar graph (H). Scale bar: 100 μm. (I-L) Flow cytometry of the phagocytic ability for fluorescent microspheres in control and Peli1-knockdown BV2 cells. The data are presented as representative scatter plots (I), histogram showing the MFI (K) and summary bar graphs (J, L). (M) Immunoblot of Peli1 and Hsp60 (loading control) in Peli1-knockdown BV2 cells that reconstituted with FL Peli1 or Peli1ΔC, showing the reconstitution efficiency of Peli1. (N, O) Flow cytometric analysis of the phagocytic ability for fluorescent microspheres in Peli1-knockdown BV2 cells that reconstituted with Peli1 FL or Peli1ΔC. The data are presented as representative plots (N) and summary bar graph (O). Data with error bars represent mean ± SEM. Each panel is representative of at least 3 independent experiments. Numerical values for (B, D, F, H, J, L, O) are available in S1 Data. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by unpaired Student t test. Aβ, amyloid-β; FL, full-length; MFI, mean fluorescent intensity; Peli1ΔC, C-terminal deleted Peli1.
Fig 2.
Peli1 deficiency promotes C/EBPβ-mediated Cd36 transcription in microglia.
(A-F) Flow cytometry of CD36 expression on the surface of Peli1+/+ and Peli1−/− primary microglia (A, B), control and Peli1-knockdown BV2 cells (C, D), and microglia isolated from age- and sex-matched adult Peli1+/+ and Peli1−/− mice (E, F). The data are presented as representative histograms (A, C, E) and summary bar graphs (B, D, F). (G) Flow cytometric analysis of the phagocytic ability for fluorescent microspheres in Peli1+/− and Peli1−/− primary microglia and in control and Peli1-knockdown BV2 cells that pretreated with anti-CD36 neutralization antibody and presented as summary bar graphs. (H) Real-time qPCR analysis of Cd36 mRNA expression in Peli1+/− and Peli1−/− primary microglia and in control and Peli1-knockdown BV2 cells. (I) Immunoblot of C/EBPα, C/EBPβ, Peli1, and Hsp60 (loading control) in Peli1+/− and Peli1−/− primary microglia and in control and Peli1-knockdown BV2 cells. (J-K) Flow cytometric analysis of the intracellular C/EBPβ expression in microglia isolated from Peli1+/+ and Peli1−/− adult mice. The data are presented as representative histogram showing MFI of C/EBPβ staining (J) and summary bar graph (K). (L) ChIP-qPCR analysis of the binding activity of C/EBPβ in the promoter of Cd36 gene after immunoprecipitation with anti-C/EBPβ antibody in Peli1+/− and Peli1−/− primary microglia, control and Peli1-knockdown BV2 cells, and microglia isolated from age- and sex-matched adult Peli1+/+ and Peli1−/− mice. (M, N) Flow cytometry of the phagocytic ability for Aβ1–42 peptide in Peli1+/− and Peli1−/− primary microglia that transfected with siCebpb. The data were presented as representative histogram (M) and summary bar graph (N). Data with error bars represent mean ± SEM. Each panel is representative of at least 3 independent experiments. Numerical values for (B, D, F, G, H, K, L, N) are available in S1 Data. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by unpaired Student t test. Aβ, amyloid-β; ChIP, chromatin immunoprecipitation; IgG, immunoglobulin G; IP, immunoprecipitation; MFI, mean fluorescent intensity; qPCR, quantitative PCR.
Fig 3.
Peli1 mediates the ubiquitination and degradation of C/EBPβ.
(A) qPCR analysis of Cebpb mRNA expression in control and Peli1-knockdown BV2 cells. (B) Immunoblot of C/EBPβ and Hsp60 (loading control) in control and Peli1-knockdown BV2 cells that pretreated with or without a proteasome inhibitor MG132 for 4 hours. (C) Immunofluorescent images showing the intracellular colocalization of Peli1 and C/EBPβ in primary microglia and BV2 cells. The zoomed images indicated the colocalization of Peli1 and C/EBPβ in the nucleus of the cells. Scale bars: 10 μm. (D) Immunoassays on lysates of BV2 cells after immunoprecipitation with control IgG or anti-C/EBPβ and immunoblot analysis of C/EBPβ associated Peli1. (E, F) Ubiquitination of endogenous C/EBPβ in Peli1+/− and Peli1−/− primary microglia (E) and in control and Peli1-knockdown BV2 cells (F) that were pretreated with MG132 for 4 hours, assessed by immunoblot analysis with anti-ubiquitin and anti-C/EBPβ after immunoprecipitation with anti-C/EBPβ (top), and by immunoblot analysis with input proteins and loading control (below). (G) Ubiquitination of C/EBPβ in 293T cells transfected with the indicated expression vectors, assessed by immunoblot analysis with anti-HA after immunoprecipitation with anti-C/EBPβ (top) or by immunoblot analysis with input proteins in lysates without immunoprecipitation (below). (H) In vitro ubiquitination assay of C/EBPβ ubiquitination after a mixture reaction of ubiquitin-charged E2 UbcH5a, in vitro translated C/EBPβ, and with or without HA-Peli1 or HA-Peli1ΔC proteins. Data with error bars represent mean ± SEM. Each panel is representative of at least 3 independent experiments. Numerical values for (A) are available in S1 Data. C/EBP, CCAAT/enhancer-binding protein; HA, hemagglutinin; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; Peli1ΔC, C-terminal-deleted Peli1; qPCR, quantitative PCR; UbcH5a, ubiquitin-conjugating enzyme H5a.
Fig 4.
Peli1 impairs Aβ clearance in 5×FAD mice.
(A-F) Flow cytometry of intracellular Aβ in control and Peli1-knockdown BV2 cells or in Peli1+/− and Peli1−/− primary microglia that incubated with or without the unfixed brain slices obtained from 5×FAD mice. The data are presented as representative histograms showing cellular Aβ MFI (A, E), scatter plots showing the frequencies of the cells that phagocytized with Aβ (C) and summary bar graphs (B, D, F). (G-I) Immunofluorescent images showing Aβ deposition and Iba1+ microglia in the brains from aged Peli1+/+ 5×FAD or Peli1−/− 5×FAD male mice (n = 4 or 5 mice/group, 2–3 slices per mouse were applied for the staining). The data are presented as representative images (G) and summary bar graph quantifying the brain Aβ deposition (H) and Iba1+ microglia (I). Scale bar: 50 μm. (J, K) Flow cytometry of intracellular C/EBPβ expression in microglia isolated from age- and sex-matched aged Peli1+/+ 5×FAD or Peli1−/− 5×FAD mice. The data are presented as representative histogram (J) and summary bar graph (K). (L, M) Flow cytometry of intracellular Peli1 in microglia isolated from 10-month-old age- and sex-matched adult naive and 5×FAD transgenic mice. The data are presented as histogram (L) and summary bar graphs (M). (N) Model of Peli1 in regulating microglia Aβ phagocytosis during AD pathogenesis. Data with error bars represent mean ± SEM. Each panel is representative of at least 3 independent experiments. Numerical values for (B, D, F, H, I, K, M) are available in S1 Data. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by unpaired Student t test. 5×FAD, AD, five familial Alzheimer’s disease; Aβ, amyloid-β; MFI, mean fluorescent intensity; C/EBP, CCAAT/enhancer-binding protein.