Fig. 3: Continuous upregulation of pyroptosis at both transcriptional and translational levels in Rnaset2^−/− mice.

A qPCR results for pyroptosis-related markers (Nlrp3, Pycard (protein: ASC), Casp1, Gsdmd, and IL-1b) in Rnaset2^−/− mouse brain samples, normalized to Rnaset2^+/+ brain samples and a housekeeping gene at 3, 6, 17, and 28 weeks of age. Nlrp3 mRNA expression exhibits a substantial increase at 3 weeks (p < 0.05), after which Nlrp3 mRNA levels decline and remain low. The mean expression of Asc (Pycard) mRNA shows a significant increase at 3 weeks (p < 0.0001) and sustained upregulation at 6 weeks (p < 0.0001). Furthermore, Pycard mRNA levels at 3 and 6 weeks are significantly elevated (p < 0.0001) compared to 17 and 28 weeks, indicating an early pyroptotic response. Casp1 and Gsdmd exhibit a comparable pattern, with both mRNAs significantly increased at 3 weeks (p < 0.001), aligning with a peak in pyroptotic activity. Similar to Nlrp3, both markers exhibit diminished expression levels at subsequent time points. IL-1b, a downstream effector of pyroptosis, shows a delayed response, with a significant increase occurring only at 6 weeks (p < 0.05) before declining. This pattern is distinct from the immediate early response of Nlrp3, Pycard, Casp1, and Gsdmd, emphasizing a subsequent cytokine response. Data are depicted as scatter dot plots with bars representing mean ± SEM with ^* = p < 0.05, ^** = p < 0.01, ^*** = p < 0.001, ^**** = p < 0.0001, ns not significant, One-way ANOVA followed by Tukey’s multiple comparison test; n = 4 for Nlrp3, Asc (Pycard), IL-1b for all measured time points; n = 4 for Casp1 and Gsdmd at 3 and 6 weeks of age; n = 5 for Casp1 and Gsdmd for 17 and 28 weeks of age. Nlrp3: NOD-like receptor pyrin domain-containing 3, Asc: Apoptosis-associated speck-like protein containing a CARD, Casp1: Caspase 1, Gsdmd: Gasdermin D, IL-1β: Interleukin-1 beta. (B-D) Western blot analysis of brain tissues from Rnaset2^−/− and Rnaset2^+/+ mice demonstrates elevated ASC protein levels at 3 weeks of age, indicating inflammasome activation (B). Similarly, CASP1 protein levels are increased in Rnaset2^−/− brain specimens (C). GSDMD protein levels exhibit variation, evidenced by an increase in one of the two Rnaset2^−/− brain samples (D). Loading control: β-ACTIN or β-TUBULIN. Mean ± SEM; n = 2 per group and time point. WT1-2: Rnaset2^+/+, KO1-2: Rnaset2^−/−. ASC: Apoptosis-associated speck-like protein containing a CARD, CASP1: Caspase 1, GSDMD: Gasdermin D. (E–G) The 6-week time point reveals increased levels of pyroptotic markers in Rnaset2^−/− brain samples. ASC protein levels are persistently increased in Rnaset2^−/− samples, indicating continuous inflammasome activation (E, p < 0.05). CASP1 protein amounts show a trend toward increased levels in Rnaset2^−/− brain tissues (F). GSDMD protein levels are significantly increased (G, p < 0.05). Loading control: β-ACTIN or β-TUBULIN. Mean ± SEM with ^* = p < 0.05, CASP1: p = 0.0679 ns not significant, Student’s t test; n = 4 per group and time point. WT1-4: Rnaset2^+/+, KO1-4: Rnaset2^−/−. ASC: Apoptosis-associated speck-like protein containing a CARD, CASP1 Caspase 1, GSDMD Gasdermin D. H–J At 17 weeks, Rnaset2^−/− specimens display sustained activation of pyroptotic indicators. ASC protein levels are significantly elevated in Rnaset2^−/− brain samples (H, p < 0.001). Likewise, the protein amount of CASP1 is significantly increased in Rnaset2^−/− brain samples, signifying persistent pyroptotic signaling (I, p < 0.01). Similarly, GSDMD protein levels are significantly increased (J, p < 0.001). Loading control: β-ACTIN or β-TUBULIN. Mean ± SEM with ^** = p < 0.01, ^*** = p < 0.001, Student’s t test; n = 4 per group and time point. WT1-4: Rnaset2^+/+, KO1-4: Rnaset2^−/−. ASC: Apoptosis-associated speck-like protein containing a CARD, CASP1 Caspase 1, GSDMD Gasdermin D. K–M The 28-week time point indicates a discontinuation of pyroptosis. The quantity of ASC protein remains elevated in Rnaset2^−/− brain samples (K, p < 0.01). CASP1 protein amounts exhibit no significant difference between Rnaset2^−/− and Rnaset2^+/+ (L). Likewise, the quantity of GSDMD protein amounts shows no variation between Rnaset2^−/− and Rnaset2^+/+ brain samples. Loading control: β-ACTIN or β-TUBULIN. Mean ± SEM with ^** = p < 0.01, ns not significant, Student’s t test; n = 3 per group and time point. WT1-3: Rnaset2^+/+, KO1-3: Rnaset2^−/−. ASC Apoptosis-associated speck-like protein containing a CARD, CASP1 Caspase 1, GSDMD Gasdermin D.