sim-a9 wild type  (Absolute Biotech Inc)

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A SNAP29, SNAP23, STX3, SEC22B, and FKBP5 co-immunoprecipitation (SKA2 IP) and whole cell extract (WCE) in hippocampus (HIP), prefrontal cortex (PFC) and amygdala (AMY) samples of mice ( n = 8). B HIS pull down assay (replicated in 3 independent in vitro experiments). DDK(Flag)-tagged SNAP23, SNAP29, Syntaxin3 or Syntaxin4 was incubated with purified magnetic beads-HIS-tagged SKA2 or magnetic beads-HIS protein alone. After incubation, bead bound proteins were eluted at room temperature (RT) or at 95 °C and subjected to western blot analysis using antibodies against HIS and FLAG. Input lane contains HIS alone (left) or HIS-tagged SKA2 (right). C – M SIM-A9 cells transfected with SKA2, FKBP5 or their respective controls, were harvested 24 h later. After immunoprecipitation (IP) of protein complexes, input and co-IP proteins were quantified by western blotting. C , F , I , K Representative blots of ( D , E , G , H , J , L , M ). Graphs display quantification of SNAP29/SEC22B, STX3/SEC22B, SKA2/SNAP29, FKBP5/SEC22B protein association after SEC22B or SNAP29 IP (unpaired two tailed t-test: ( D ) t 6 = 8.945, p < 0.0001, ( E ) t 6 = 12.94, p < 0.0001, ( G ) t 6 = 6.056, p = 0.0009, ( H ) t 6 = 5.554, p = 0.0014; one-way ANOVA: ( J ) F 2, 9 = 17.28, p = 0.0008, Tukey’s post hoc test: ctrl vs. FKBP5-OE, p = 0.0743, ctrl vs. FKBP5-KO, p = 0.0218, FKBP5-OE vs. FKBP5-KO, p = 0.0006; unpaired two tailed t-test: ( L ) t 6 = 10.27, p < 0.0001, ( M ) t 6 = 8.140, p = 0.0002; n = mean derived from four independent in vitro experiments). * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: The murine microglia cell lines SIM-A9 wild type (Kerafast, END001), SIM-A9 Sec22b KO and SIM-A9 Fkbp5 KO were cultured at 37 °C, 6% CO 2 in DMEM high glucose with GlutaMAX (Thermo Fisher Scientific, 10566016), supplemented with 10% FBS (Thermo Fisher, 10270-106), 5% horse serum (Thermo Fisher Scientific, 16050-122) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, 15240-062).

Techniques: Immunoprecipitation, Pull Down Assay, In Vitro, Incubation, Purification, Magnetic Beads, Western Blot, Transfection, Co-Immunoprecipitation Assay, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A , B IL-1β release measured via ELISA from supernatants of SIM-A9 cells 24 h after manipulation of SKA2 and/or FKBP5 expression, and following overnight LPS (100 ng/mL) and treatment with LLOMe (0.25 mM) for 3 h (unpaired two tailed t-test: (A) t 4 = 11.99, p = 0.0003; one-way ANOVA: B F 3, 8 = 158.6, p < 0.0001; Tukey’s post hoc test: ctrl vs. SKA2-OE, p = 0.0384, ctrl vs. FKBP5-OE, p < 0.0001, SKA2-OE vs. FKBP5-OE, p < 0.0001, FKBP5-OE vs. SKA2 + FKBP5 OE, p < 0.0001; n = mean derived from three independent in vitro experiments). C Schematic overview of the SA pathway with SKA2 and FKBP5. The cargo receptor TRIM16, together with SEC22B, transfers molecular cargo (e.g., IL-1β) to the autophagy-related LC3B-positive membrane carriers. SEC22B, now acting as an R-SNARE on the delimiting membrane facing the cytosol, carries out fusion at the plasma membrane in conjunction with the Q bc -SNAREs, SNAP23 and SNAP29 (SNAP23/29), and one of the plasma membrane Q a -SNAREs, STX3 or STX4 (STX3/4), thus delivering IL-1β to the extracellular milieu, where it exerts its biological functions. FKBP5 acts as a positive regulator of SA by enhancing TRIM16-SEC22B complex formation as well as autophagosome-plasma membrane fusion via the SNARE-protein complex assembly. In contrast, SKA2 inhibits the SNARE-protein complex formation during vesicle-plasma membrane fusion, thereby acting as gatekeeper of SA. D , E Schematic overview of in vivo microdialysis and the experimental design and timeline; each sample was collected over 30 min indicated by the light gray lines. Quantifications of IL-1β, determined by capillary-based immunoblotting from in vivo medioprefrontal cortex microdialysis of C57Bl/6NCrl mice injected intraperitoneally with ULK1 inhibitor (ULK1i, an autophagy inhibitor) or saline ( F ; repeated measures two-way ANOVA, time × treatment interaction: F 5, 30 = 7.064, p = 0.0002; Šidák’s multiple comparisons post hoc test, post-FS-1: p = 0.0084; n = 4 mice per group) as well as of wild type (WT) and global Fkbp5 knockout mice ( G ; repeated measures two-way ANOVA, time × genotype interaction: F 5, 30 = 34.15, p < 0.0001; Šidák’s multiple comparisons post hoc test: FS: p = 0.009, post-FS-1: p = 0.0163, post-FS-2: p = 0.0294; n = 4 mice per group). FS foot shock. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: The murine microglia cell lines SIM-A9 wild type (Kerafast, END001), SIM-A9 Sec22b KO and SIM-A9 Fkbp5 KO were cultured at 37 °C, 6% CO 2 in DMEM high glucose with GlutaMAX (Thermo Fisher Scientific, 10566016), supplemented with 10% FBS (Thermo Fisher, 10270-106), 5% horse serum (Thermo Fisher Scientific, 16050-122) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, 15240-062).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, Derivative Assay, In Vitro, Membrane, In Vivo, Western Blot, Injection, Saline, Knock-Out

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A SIM-A9 Sec22b −/− cells expressing ASC (apoptosis-associated speck-like protein containing a CARD) -mCerulean (via epifluorescence) show a significantly decreased number of intracellular (white arrows) ASC specks compared to wild type (WT) SIM-A9 cells (unpaired two tailed t-test: t 4 = 3.206, p = 0.0327; n = mean derived from three independent in vitro experiments). B In WT SIM-A9 cells knockdown of Ska2 or LPS treatment leads to a significantly increased number of intracellular ASC specks compared to Scr-shRNA or LPS-treated cells (2-way ANOVA: main LPS treatment effect ($), F 1,31 = 10.60, p = 0.0027, main Ska2 knockdown effect (*), F 1,31 = 5.482, p = 0.0258; n = 9 WT Veh SCR-shRNA, n = 9 WT Veh SKA2-shRNA, n = 9 WT LPS SCR-shRNA, n = 8 WT LPS SKA2-shRNA). C In contrast, knockdown of Ska2 or LPS treatment does not have any effects on the number of ASC specks in SIM-A9 Sec22b −/− cells (2-way ANOVA: n. s. treatment effect F 1,29 = 0.312, p = 0.5804, main Ska2 knockdown effect, F 1,29 = 0.055, p = 0.8157; n = 9 for SEC22B KO Veh SCR-shRNA and SKA2-shRNA, n = 7 SEC22B KO LPS SCR-shRNA, n = 8 SEC22B KO LPS SKA2-shRNA). D , E Knockdown of Ska2 leads to significantly increased SEC22B binding to SNAP29 (unpaired two tailed t-test: t 4 = 4.113, p = 0.0063; n = 4 independent biological replicates) as well as NEK7 binding to NLRP3 in protein lysates of organotypic hippocampal slice cultures (unpaired two tailed t-test: t 4 = 2.998, p = 0.0241; n = 4 independent biological replicates). F IHC images of ASC (green) and DAPI (blue) 2 weeks after viral injection (Scr-shRNA-AAV and Ska2-shRNA-1-AAV) into the hippocampus. Quantification of ASC+ cells (left) and ASC specks (right) 2 weeks after viral injection (paired t-test: ASC+ cells, t 2 = 6.414, p = 0.0235, ASC specks, t 2 = 6.937, p = 0.0202; n = 3 mice). G IHC images of ASC (green) and DAPI (blue) 4 weeks after viral injection (Scr-shRNA-AAV and Ska2-shRNA-1-AAV) into the hippocampus. Quantification of ASC+ cells (left) and ASC specks (right) 4 weeks after viral injection (paired t-test: ASC+ cells, t 2 = 8.511, p = 0.0135; ASC specks, t 2 = 10.99, p = 0.0082; n = 3 mice). H IHC images of CASPASE-1 (CASP-1) (green) and mCherry (red, viral marker) 2 weeks after viral injection (Scr-shRNA-AAV and Ska2-shRNA-1-AAV) into the hippocampus (left). (right) Quantification of CASP-1 expression 2 weeks after viral injection (paired t-test: t 3 = 2.842, p = 0.0655, n = 4 mice). I IHC images of CASP-1 (green) and mCherry (red, viral marker) 4 weeks after viral injection (Scr-shRNA-AAV and Ska2-shRNA-1-AAV) into the hippocampus (left). (right) Quantification of CASP-1 expression 4 weeks after viral injection (paired t-test: t 3 = 3.367, p = 0.0435, n = 4 mice). J Full length Gasdermin D (GSDMD FL) levels as well as the ratio of the cleaved N-terminal form of GSDMD (GSDMD N-term) to GSDMD FL are increased 2 weeks after Ska2 knockdown (unpaired two tailed t-test; GSDMD FL/ β-actin: t 18 = 4.105, p = 0.0007, GSDMD N-term/GSDMD FL: t 18 = 9.259, p < 0.0001; n = 10 independent biological replicates per group). K Examples blots of ( E ). L Schematic overview of the interaction between secretory autophagy (SA) and the GSDMD-mediated IL-1β release. SKA2 depletion results in increased SA-dependent IL-1β release, serving as a molecular vicious feed-forward loop for inflammasome activation. Inflammasome assembly activates CASP-1 enzymatic function. ASC in the inflammasome complex recruits CASP-1. Activation of CASP-1 cleaves GSDMD to release the N-terminal domain, which forms pores in the plasma membrane for uncontrolled IL-1β release. * = p < 0.05; ** = p < 0.01; *** = p < 0.001, **** = p < 0.0001. Data are presented as mean + SEM. Scale bar represents 5 µm in A, 50 µm in ( F , G ) (left), 10 µm in ( B , F , G ) (right), and 250 µm in ( H , I ). Source data are provided as a file.

Article Snippet: The murine microglia cell lines SIM-A9 wild type (Kerafast, END001), SIM-A9 Sec22b KO and SIM-A9 Fkbp5 KO were cultured at 37 °C, 6% CO 2 in DMEM high glucose with GlutaMAX (Thermo Fisher Scientific, 10566016), supplemented with 10% FBS (Thermo Fisher, 10270-106), 5% horse serum (Thermo Fisher Scientific, 16050-122) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, 15240-062).

Techniques: Expressing, Two Tailed Test, Derivative Assay, In Vitro, shRNA, Binding Assay, Injection, Marker, Activation Assay, Membrane