Journal: Frontiers in aging neuroscience

Article Title: NLRP3 inflammasome activation and pyroptosis are dispensable for tau pathology.

doi: 10.3389/fnagi.2024.1459134

Figure Lengend Snippet: FIGURE 1 NLRP3 pathway activation in mouse organotypic slice cultures. (A) IL-1β measurement in the supernatant of OSCs stimulated with lipopolysaccharides (LPS) and nigericin and treated with NLRP3 small molecule inhibitor (MCC950 or Compound 1). Elevated IL-1β levels in LPS + nigericin condition, which are significantly reduced by increasing concentration of NLRP3 inhibitors. Figure represents two independent experiments combined to increase the replicates per condition. Quantitative data are shown as individual datapoint, mean with standard error. PBS and LPS n = 16, Nigericin: n = 10, 10 nM, Compound 1: 100 nM (n = 3), 300 nM (n = 3), 10 μM (n = 4); MCC950: 10 nM (n = 3), 300 nM (n = 4), 10 μM (n = 2). (B) OSCs supernatant shows elevated IL-1β levels after K18 seeding, which are significantly decreased by addition of 1 μM NLRP3 small molecule inhibitor called JNJ81977038. Data are shown as individual datapoint, mean with standard error. Significance ****p < 0.0001 by one-way ANOVA with multiple comparisons test.

Article Snippet: Slices were treated with 1 μM of a NLRP3 small molecule inhibitor [Compound 1; example 49 of WO2020234715A1 (Novartis AG)].

Techniques: Activation Assay, Concentration Assay

Journal: Frontiers in aging neuroscience

Article Title: NLRP3 inflammasome activation and pyroptosis are dispensable for tau pathology.

doi: 10.3389/fnagi.2024.1459134

Figure Lengend Snippet: FIGURE 2 No effect of Nlrp3 knockout on tau pathology by IHC. (A) AT100 immunohistochemistry demonstrated no significant effect on tau phosphorylation/ aggregation in all analyzed regions of late-stage Nlrp3−/−xP301S vs. Nlrp3+/+xP301S mice. Brain stem, spinal cord, and midbrain showed a trend in reduced pathology for Nlrp3 deficiency, which was not significant. Early stage P301S mice displayed much lower aggregated tau levels compared to late-stage animals and no effect of Nlrp3 deletion on tauopathy was observed at this age in any of the regions analyzed. Image analysis data are shown as mean + individual values for each animal (average of two sections/animal). Early stage Nlrp3+/+xP301S: n = 24, Nlrp3−/−xP301S: n = 26; Late-stage Nlrp3+/+xP301S: n = 18, Nlrp3−/−xP301S: n = 19. No significance by unpaired t-test of age-matched groups. (B) Representative images of the AT00 IHC staining are demonstrated. Scale bars: 2.5 mm (overview images), 100 μm (higher magnification images). (C) Biochemical analysis of tau pathology in brainstem of Nlrp3xP301S mice. Aggregated tau levels were not significantly reduced in the sarcosyl-insoluble fraction or total homogenate of the brainstem of late-stage Nlrp3−/− vs. Nlrp3+/+xP301S mice. Early stage Nlrp3+/+xP301S: n = 23, Nlrp3−/−xP301S: n = 25; Late-stage Nlrp3+/+xP301S: n = 16, Nlrp3−/−xP301S: n = 16. Data are expressed as mean + individual values of each animal tested (values = average of two sections/mouse).

Article Snippet: Slices were treated with 1 μM of a NLRP3 small molecule inhibitor [Compound 1; example 49 of WO2020234715A1 (Novartis AG)].

Techniques: Knock-Out, Immunohistochemistry, Phospho-proteomics

Journal: Frontiers in aging neuroscience

Article Title: NLRP3 inflammasome activation and pyroptosis are dispensable for tau pathology.

doi: 10.3389/fnagi.2024.1459134

Figure Lengend Snippet: FIGURE 4 AT100 immunohistochemistry was not altered in Gsdmd deficient mice. (A) Immunohistochemistry with phospho-tau AT100 antibody showed no significant effect on tau pathology in GsdmdxP301S mice. No significant difference is detected between Nlrp3−/−xP301S vs. Nlrp3+/+xP301S mice at early and late stage. Quantitative data are shown as mean + individual values for each animal. Early stage Nlrp3+/+xP301S: n = ±10, Nlrp3−/−xP301S: n = ±15; Late-stage Nlrp3+/+xP301S: n = ±25, Nlrp3−/−xP301S: n = ±20. No significance by unpaired t-test of age-matched groups. (B) Representative images of brain stem, midbrain, spinal cord, and cortex are shown. The whole brain sections are displayed at 2.5 mm and the spinal cord sections are displayed at 100 μm.

Article Snippet: Slices were treated with 1 μM of a NLRP3 small molecule inhibitor [Compound 1; example 49 of WO2020234715A1 (Novartis AG)].

Techniques: Immunohistochemistry

Journal: Frontiers in aging neuroscience

Article Title: NLRP3 inflammasome activation and pyroptosis are dispensable for tau pathology.

doi: 10.3389/fnagi.2024.1459134

Figure Lengend Snippet: FIGURE 5 Late-stage Nlrp3 effect on neurofilament levels and no effect of Gsdmd in P301S mice. (A) Neurofilament light (NF-L) levels were quantified in plasma of Nlrp3xP301S mice as a surrogate marker for neurodegeneration in the CNS. At late stage, NF-L levels were significantly reduced in Nlrp3 knockout mice, compared to age-related Nlrp3 WT mice. No effect was demonstrated in early stage P301S mice. Early stage Nlrp3+/+xP301S: n = 23, Nlrp3−/−xP301S: n = 27; late-stage Nlrp3+/+xP301S: n = 18, Nlrp3−/−xP301S: n = 19. **p < 0.01 by paired t-test of age-matched groups. (B) No effect of GSDMD on plasma NF-L levels were observed in the tau transgenic P301S mouse model. WT: n = 17; Early stage Nlrp3+/+xP301S: n = 9, Nlrp3−/−xP301S: n = 21; Late-stage Nlrp3+/+xP301S: n = 27, Nlrp3−/−xP301S: n = 19. No significance by unpaired t-test of age-matched groups.

Article Snippet: Slices were treated with 1 μM of a NLRP3 small molecule inhibitor [Compound 1; example 49 of WO2020234715A1 (Novartis AG)].

Techniques: Clinical Proteomics, Marker, Knock-Out, Transgenic Assay

Journal: Frontiers in aging neuroscience

Article Title: NLRP3 inflammasome activation and pyroptosis are dispensable for tau pathology.

doi: 10.3389/fnagi.2024.1459134

Figure Lengend Snippet: FIGURE 6 No effect of Nlrp3 or Gsdmd deletion on neuronal count in P301S mice. (A) The number of neurons was calculated in brain regions of Nlrp3xP301S mice at early and late stage. No effect of Nlrp3 on NeuN levels was demonstrated in the different stages nor the different brain regions of P301S mice. Early stage Nlrp3+/+xP301S: n = ±24, Nlrp3−/−xP301S: n = ±26; late-stage Nlrp3+/+xP301S: n = 18, Nlrp3−/−xP301S: n = ±19. No significance by unpaired t-test of age- matched groups. (B) Immunohistochemistry with NeuN antibody was also performed on early and late-stage Gsdmd−/−xP301S vs. Gsdmd+/+xP301S, demonstrating no effect of GSDMD. Early stage Gsdmd+/+xP301S: n = 10, Gsdmd−/−xP301S: n = ±12; Late-stage Gsdmd+/+xP301S: n = ±20, Gsdmd−/−xP301S: n = ±15. No significance by unpaired t-test of age-matched groups.

Article Snippet: Slices were treated with 1 μM of a NLRP3 small molecule inhibitor [Compound 1; example 49 of WO2020234715A1 (Novartis AG)].

Techniques: Immunohistochemistry

Journal: Frontiers in aging neuroscience

Article Title: NLRP3 inflammasome activation and pyroptosis are dispensable for tau pathology.

doi: 10.3389/fnagi.2024.1459134

Figure Lengend Snippet: FIGURE 7 No impact of Gsdmd and Nlrp3 deficiency on plasma IL-18 levels in P301S mice. (A) Mesoscale measurement of pro-inflammatory IL-18 levels in plasma of P301S mice. No significant difference between Nlrp3 knockout mice vs. Nlrp3 WT mice in early and late-stage P301S mice. Early stage Nlrp3+/+xP301S: n = 18, Nlrp3−/−xP301S n = 26; Late-stage Nlrp3+/+xP301S: n = 16, Nlrp3−/−xP301S: n = 18. No significance by paired t-test of age- matched groups. (B) IL-18 concentrations were additionally quantified in plasma of early and late-stage Gsdmd−/−xP301S vs. Gsdmd+/+xP301S, demonstrating no effect of GSDMD. WT: n = 15; Early stage Nlrp3+/+xP301S: n = 8, Nlrp3−/−xP301S: n = 21; Late-stage Nlrp3+/+xP301S: n = 26, Nlrp3−/−xP301S: n = 19. No significance by paired t-test of age-matched groups.

Article Snippet: Slices were treated with 1 μM of a NLRP3 small molecule inhibitor [Compound 1; example 49 of WO2020234715A1 (Novartis AG)].

Techniques: Clinical Proteomics, Knock-Out