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: Primary antibodies used : FKBP5 (1:1000, Bethyl, A301-430A), FKBP5 (1:1000, Cell Signaling Technology, #12210), ACTIN (1:5000, Santa Cruz Biotechnology, sc-1616), GAPDH (1:8000, Millipore CB1001), SNAP29 (1:1000, Sigma, SAB1408650), SNAP23 (1:1000, Sigma, SAB2102251), STX3 (1:1000, Sigma, SAB2701366), STX4 (1:1000, Cell Signaling Technology, #67657), SEC22B (1:1000, Abcam, ab181076), SKA2 (1:1000, Thermo Fisher, PA5-20818), SKA2 (1:500, Millipore-Sigma, SAB3500102), SKA1 (1:1000, Thermo Fisher, PA5-20817), SKA3 (1:1000, Thermo Fisher, PA5-20819), GSDMD (1:1000, Cell Signaling Technology, 39754), VCP (1:10000, Abcam, Ab11433), NEK7 (1:50, Abcam, Ab133514), NLRP3 (1:50, Cell Signaling Technology, 15101), HIS (1:5000, Cell Signaling Technology, #12698), FLAG M2 (1:10000, Sigma, A8592).

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: Primary antibodies used : FKBP5 (1:1000, Bethyl, A301-430A), FKBP5 (1:1000, Cell Signaling Technology, #12210), ACTIN (1:5000, Santa Cruz Biotechnology, sc-1616), GAPDH (1:8000, Millipore CB1001), SNAP29 (1:1000, Sigma, SAB1408650), SNAP23 (1:1000, Sigma, SAB2102251), STX3 (1:1000, Sigma, SAB2701366), STX4 (1:1000, Cell Signaling Technology, #67657), SEC22B (1:1000, Abcam, ab181076), SKA2 (1:1000, Thermo Fisher, PA5-20818), SKA2 (1:500, Millipore-Sigma, SAB3500102), SKA1 (1:1000, Thermo Fisher, PA5-20817), SKA3 (1:1000, Thermo Fisher, PA5-20819), GSDMD (1:1000, Cell Signaling Technology, 39754), VCP (1:10000, Abcam, Ab11433), NEK7 (1:50, Abcam, Ab133514), NLRP3 (1:50, Cell Signaling Technology, 15101), HIS (1:5000, Cell Signaling Technology, #12698), FLAG M2 (1:10000, Sigma, A8592).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, Derivative Assay, In Vitro, Membrane, Clinical Proteomics, 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 , 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: Primary antibodies used : FKBP5 (1:1000, Bethyl, A301-430A), FKBP5 (1:1000, Cell Signaling Technology, #12210), ACTIN (1:5000, Santa Cruz Biotechnology, sc-1616), GAPDH (1:8000, Millipore CB1001), SNAP29 (1:1000, Sigma, SAB1408650), SNAP23 (1:1000, Sigma, SAB2102251), STX3 (1:1000, Sigma, SAB2701366), STX4 (1:1000, Cell Signaling Technology, #67657), SEC22B (1:1000, Abcam, ab181076), SKA2 (1:1000, Thermo Fisher, PA5-20818), SKA2 (1:500, Millipore-Sigma, SAB3500102), SKA1 (1:1000, Thermo Fisher, PA5-20817), SKA3 (1:1000, Thermo Fisher, PA5-20819), GSDMD (1:1000, Cell Signaling Technology, 39754), VCP (1:10000, Abcam, Ab11433), NEK7 (1:50, Abcam, Ab133514), NLRP3 (1:50, Cell Signaling Technology, 15101), HIS (1:5000, Cell Signaling Technology, #12698), FLAG M2 (1:10000, Sigma, A8592).

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