Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Schematic of the protocol for generation of tdTomato-CD63 + BV-2 cells and lentivirus-induced expression of tGFP-shRNA targeting Sepp1 , created with BioRender. (B) Transduced tdTomato-CD63 + tGFP-shRNA + BV-2 cells were sorted by FACS. (C) Representative images of double-positive (tdTomato + /tGFP + ) BV-2 cells transduced with tGFP-shRNA for downregulation of Sepp1 . Scale bar, 50 μm. (D) Workflow for purification of EVs from the conditioned medium (CM) of tdTomato-CD63 + BV-2 microglia upon stimulation with LPS and ATP; created with BioRender . (E–G) Size distribution and concentration of EVs secreted on CM of BV-2 cells were measured by nanoflow cytometry using a flow nanoanalyzer (NanoFCM); ** p < 0.01 and **** p < 0.0001 by one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 replicates per group). The assessment of tdTomato-CD63 + molecule loading on EVs was performed using the Nanoimager system (ONI). (H) Representative image of single tdTomato-CD63 + EVs. Scale bar, 2 μm. (I and J) Single-molecule fluorescence analysis of tdTomato-CD63 loading on EVs was performed at single-EV level, and the number of tdTomato-CD63 molecules present in single EVs was quantified using the CODI platform from ONI; one-way ANOVA with Holm-Šidák post hoc analysis ( n = 3 replicates per group). * p < 0.05, ** p < 0.01, and **** p < 0.0001 by one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 per group). Scale bar, 50 nm. Data are represented as mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Expressing, shRNA, Transduction, Purification, Concentration Assay, Cytometry, Fluorescence

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Principal-component analysis of transcriptomic profile of tdTomato-CD63 + BV-2 cells transduced with tGFP-shRNA targeting scramble and Sepp1 (three different clones) ( n = 6 replicates per group). (B) Heatmap displaying the Pearson correlation of scramble and Sepp1-shRNA replicates from RNA sequencing (RNA-seq). (C–E) Volcano plots comparing the DEGs of three Sepp1 -shRNA clones versus scramble. Dashed lines indicate a significant threshold using q < 0.05 (−log10 q- value > 1.3) on the y axis and log2 fold change > +0.5 or < −0.5 on the x axis. (F) Venn diagram comparison of up- and downregulated DEGs across different Sepp1 -shRNAs clones using the same criteria as in (D) and (E) ( q < 0.05 and log2 fold change > +0.5 or < −0.5). (G) Common Sepp1 -shRNA DEGs were subjected to Gene Ontology classification, and the top 5 significant ( q < 0.05) for each cellular component (CC), biological process (BP), and molecular function (MF) are displayed as a bar graph based on gene count enrichment.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Transduction, shRNA, Clone Assay, RNA Sequencing, Comparison

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Top 10 enriched cellular processes identified by KEGG pathway analysis of all common regulated DEGs by Sepp1 -shRNA in BV-2 microglia. (B) Heatmap showing the consistent regulation of the components of endocytosis and lysosome pathways from (A) by different Sepp1 -shRNAs in BV-2 microglia. Identified components related to endosome/exosome ESCRT machinery, the retromer complex, and lysosomal activity are highlighted in bold. (C–E) Bar graphs depicting normalized expression levels of highlighted genes from (B) among scramble and Sepp1 -shRNA groups in RNA-seq data. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 replicates per group). Bar graphs indicate mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: shRNA, Activity Assay, Expressing, RNA Sequencing

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Heatmap displaying the top common DEGs ( q < 0.05) regulated in response to Sepp1 downregulation. (B) Enriched pathways associated with the common DEGs in Sepp1 -shRNA were obtained using Metascape software. (C and D) Ingenuity Pathway Analysis (IPA) using all common DEGs regulated in different Sepp1 -shRNA groups predicted LXR/RXR pathway downregulation and identified a network interaction composed mainly of Ptger4 , Npc1 , Trem2 , and Cop1 in response to Sepp1 downregulation (D). (E) Analysis of normalized expression of Ptger4 , Npc1 , Trem2 , and Cop1 in RNA-seq data. * p < 0.05, ** p < 0.01, and **** p < 0.0001 by one-way ANOVA with Holm-Šidák post hoc analysis ( n = 6 replicates per group). Bar graphs indicate mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: shRNA, Software, Expressing, RNA Sequencing

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Schematic of the experimental design for Sepp1 silencing using siRNA in primary neurons and primary astrocytes. (B) Representative MAP2 and GFAP immunostaining images in primary neurons and astrocytes. Scale bar, 100 μm. (C) Bar graphs showing relative expression of Sepp1 mRNA in primary neurons and astrocytes treated with siRNA targeting the scramble control sequence or Sepp1 ( n = 6 replicates per group from 2 independent experiments). **** p < 0.0001 by two-tailed student t test. (D and E) Size distribution and concentration of EVs secreted in CM of primary neurons (D) and astrocytes (E) measured by nanoflow cytometry (NanoFCM) ( n = 6–8 replicates per group from 2 independent experiments). * p < 0.05 by two-tailed student t-test; ns, no significance. Data are represented as mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Immunostaining, Expressing, Control, Sequencing, Two Tailed Test, Concentration Assay, Cytometry

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: (A) Experimental design for microglia-specific EV labeling and Sepp1 silencing in APP NL-G-F mice. Six-month-old mice received injections into the hippocampus with a lentivirus for microglia-specific expression of mEm-CD9, followed by a lentivirus injection for scramble-shRNA:mCherry or Sepp1 -shRNA:mCherry expression 2 weeks later. (B) Representative images of mEm-CD9 + (green) microglia cells and cells expressing mCherry:shRNA (red) in the APP NL-G-F mouse dentate gyrus in the hippocampus brain region. Scale bar, 100 μm. (C) Confocal microscopy and Imaris 3D rendering of mouse hippocampal tissue. Shown are mEm-CD9 + (green) and P2ry12 + (red). Scale bar, 100 μm. (D) Percentage of the P2ry12 microglial marker overlapped with mEm-CD9 + cells in the hippocampus of APP NL–G-F mice injected with shRNA. (E) Super-resolution confocal z stack images of single microglia co-transduced with mEm-CD9 and shRNA:mCherry in the hippocampus of APP NL-G-F mice. 3D images of shRNA:mCherry (red) and Mac2 (magenta) surfaces overlapped with mEm-CD9 + (green) microglia as well as mEm-CD9 + particles surrounding microglia were generated using Imaris software. Scale bar, 4 μm. (F) Measured Mac2 fluorescence intensity in scramble- and Sepp1-shRNA-transduced microglia in APP NL-G-F mice ( n = 3–4 microglia per mouse, 3–4 mice per group). Two-tailed student t test; ns, no significance. (G) Representative high-magnification images of mEm-CD9 + voxels obtained in areas surrounding Mac2 + MGnD microglia as in (E). Scale bars: total EVs, 1 μm; small EVs, 0.2 μm. (H–K) In situ analysis of mEm-CD9 + voxels surrounding mEm-CD9 + /Mac2 + MGnD microglia. Shown are (H) the size frequency distribution of mEm-CD9 + particles and quantification of the number of mEm-CD9 + voxels per microglia in total EVs (I, total EVs <1,000 nm), small EVs (J, <150 nm), and large EVs (K, >150 nm) among scramble- and Sepp1 -shRNA:mCherry-transduced mEm-CD9 + /Mac2 + MGnD microglia. Two-tailed student t test, * p < 0.05 ( n = 3–4 microglia per mouse, 3–4 mice per group). Data are represented as mean ± SEM.

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Labeling, Expressing, Injection, shRNA, Confocal Microscopy, Marker, Transduction, Generated, Software, Fluorescence, Two Tailed Test, In Situ

Journal: Cell reports

Article Title: Selenoprotein P is a target for regulating extracellular vesicle biogenesis and secretion from activated microglia in vivo

doi: 10.1016/j.celrep.2024.115025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Sepp1 TaqMan probes , Thermo Fischer , Mm00486048_m1.

Techniques: Virus, shRNA, Control, Recombinant, Staining, RNA Sequencing, Software, Saline, Imaging