Fig 1: Analysis of inflammasomes and pyroptotic signaling in the lung (left panel) and RV (right panel) tissue. Relative mRNA expression of NLRP3 (A, I) and NLRC4 (F, N), relative protein expression (B-D, G, H, J–L, O, P) and representative immunoblots and total protein staining (E, M) of csp-1, procsp-1 (B, J), IL-1ß, pro-IL-1ß (C, K), N-terminal GSDMD (D, L), AIM2 (G, O) and Iba1 (H, P) in control group, monocrotaline group (MCT) and prematurely terminated monocrotaline group (ptMCT). Data are presented as mean ± SEM; n = 8–10 per group; *p ? 0.05 vs. Control; # p ? 0.05 vs. MCT
Fig 2: Overexpression of AIM2 in vivo contributed to impaired synaptic structure and function. (a) Schematic of lentivirus microinjection into the DG. (b) Representative images showing the location of EGFP (green) expression. The time spent exploring two identical objects during the training phase (c) and the displaced object during the test session (d) by control and AIM2-OE mice in the OPS test was recorded. n = 18–20 for each group. t(36) =3.932, p = 0.0004. Western blotting (e) and quantification of PSD-95 (f) and MAP-2 (g) protein levels in the DG region of control and AIM2-OE mice. n = 6 for each group. t(10) = 4.065, p = 0.0023 for PSD-95; t(10) = 3.490, p = 0.0058 for MAP-2. (h) Overview of the DG region in control and AIM2-OE mice. (i) Neuronal tracing in control and AIM2-OE mice. (j) Quantification of the numbers of neuronal intersections in control (n = 2–3 neurons/mouse, N = 5 mice/group) and AIM2-OE (n = 2–3 neurons/mouse, N = 5 mice/group) mice by Sholl analysis. F(1, 25) = 4.270, p = 0.0493. (k) Representative images of dendrite morphology. (l) Quantitative analysis of the mean spine density in control (n = 2–3 spines/mouse, N = 5 mice/group) and AIM2-OE (n = 2–3 spines/mouse, N = 5 mice/group) mice. t(22) = 7.368, p < 0.0001. (m) The I/O curve of AIM2-OE mice (n = 2–3 slices/mouse, N = 4 mice/group) was decreased compared with that of control mice (n = 1–3 slices/mouse, N = 4 mice/group). F(1,12) = 54.81, p < 0.0001. (n, o) LTP in the DG region was evaluated in hippocampal slices from control (n = 1–3 slices/mouse, N = 4 mice/group) and AIM2-OE (n = 2–3 slices/mouse, N = 4 mice/group) mice. The slope of the regression line decreased markedly after overexpression of AIM2. t(15) = 4.631, p = 0.0003. Mean mEPSC amplitude (p) and frequency (q) in DG neurons of control (n = 3–5 neurons/mouse, N = 3 mice/group) and AIM2-OE (n = 3–5 neurons/mouse, N = 3 mice/group) mice. t(25) = 5.231, p < 0.0001 for amplitude; t(25) = 0.2409, p = 0.8116 for frequency. (r) Representative traces of mEPSC recordings in DG hippocampal acute slices from control and AIM2-OE mice. Data were shown as mean ± SEM. Unpaired two-tailed t-test for d, f, g, l, o, p and q. Two-way ANOVA followed by Bonferroni's post hoc test for j and m. *p < 0.05, **p < 0.01, ***p < 0.001; ns no significance.
Fig 3: AIM2-/- mice displayed improved cognitive function and ameliorated synapse loss. The time spent exploring two identical objects during the training phase (a) and the displaced object during the test session (b) by 13-month-old WT and AIM2-/- male mice in the OPS test was recorded. n = 12 for each group. t(22) = 3.351, p = 0.0029. (c) Representative western blot image of PSD-95 and MAP-2 in the DG region in 13-month-old WT and AIM2-/- male mice. Western blot analysis of PSD-95 (d) and MAP-2 (e) in 13-month-old WT and AIM2-/- male mice. ß-Actin was used as a loading control. n = 9 for each group. t(16) = 3.411, p = 0.0036 for PSD-95; t(16) = 3.402, p = 0.0036 for MAP-2. (f) Low-magnification view of the DG region in 13-month-old WT and AIM2-/- male mice. (g) Representative traces of DG neurons. (h) Quantification of the numbers of intersections at different distances from the soma in 13-month-old WT (n = 3 neurons/mouse, N = 3 mice/group) and AIM2-/- male mice (n = 3 neurons/mouse, N = 3 mice/group). F(1,16) = 44.03, p < 0.0001. (i) Representative images of dendrite morphology in DG neurons delineated by Golgi staining. (j) Quantitative analysis of the dendritic spine density in 13-month-old WT (n = 3 spines/mouse, N = 3 mice/group) and AIM2-/- male mice (n = 3 spines/mouse, N = 3 mice/group). t(16) = 10.29, p < 0.0001. Data were shown as mean ± SEM. Unpaired two-tailed t-test for b, d, e and j. Two-way ANOVA followed by Bonferroni's post hoc test for h. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: AIM2 regulated microglial phagocytic activity and synapse engulfment. Double immunofluorescence staining of AIM2 (green) with IBA-1 (red) (a), NeuN (red) (b) and GFAP (red) (c) in the DG of 2- and 13-month-old male mice. (d) High levels of CD68 (green) immunoreactivity in IBA-1+ (red) microglia were detected in 13-month-old male mice. (e) Confocal images showing the presence of PSD-95+ (green) puncta around IBA-1+ (red) microglia and the corresponding 3D reconstructions. (f) Immunostaining for IBA-1 (red) and CD68 (green) in the DG region of control and AIM2-OE mice suggested that AIM2 overexpression induced high levels of CD68 (green) immunoreactivity in IBA-1-positive (red) microglia. (g) Confocal images showed IBA-1 (red) coexpression with PSD-95 (green), and 3D reconstruction demonstrated that IBA-1+ microglia in AIM2-OE mice engulf more synaptic elements. (h) Schematic diagram of microglia and neuron coculture. The overexpression efficiency of the lentivirus was determined through EGFP-positive (green) cells observed via fluorescence microscopy (i) and AIM2 levels via RT–PCR (j). n = 8 for the control group, n = 7 for the AIM2-OE group. t(13) = 4.104, p = 0.0012. Flow cytometric analysis (k) and quantification (l) of microglial phagocytic activity for fluorescent microspheres. n = 7 for each group. t(12) = 4.876, p = 0.0004. (m) Representative confocal images showing the localization of IBA-1 (green), CD68 (gray), MAP-2 (red) and PSD-95 (magenta) in microglia transfected with the control or AIM2-OE lentivirus. Data were shown as mean ± SEM. Unpaired two-tailed t-test for j and l. **p < 0.01, ***p < 0.001.
Fig 5: Aging mice displayed impaired PS behavior associated with elevated AIM2 expression in the DG region. (a) Schematic diagram of the OPS test. The time spent exploring two identical objects during the training phase (b) and the displaced object during the test session (c) by 2- and 13-month-old male mice in the OPS test was recorded. n = 12 for each group. t(22) = 4, p = 0.006. (d) PSD-95 and MAP-2 protein expression levels in the DG of 2- and 13-month-old male mice were analyzed using immunoblot assays. Quantification of the protein expression levels of PSD-95 (e) and MAP-2 (f) normalized to ß-actin as a loading control. n = 6 for each group. t(10) = 6.848, p < 0.0001 for PSD-95; t(10) = 3.485, p = 0.0059 for MAP-2. (g) Low-magnification view of the DG region delineated by Golgi staining. (h) Representative traces of DG neurons. (i) The numbers of intersections at different distances from the soma in 2-month-old (n = 2 neurons/mouse, N = 3 mice/group) and 13-month-old (n = 2–3 neurons/mouse, N = 3 mice/group) male mice were counted. F(1,11) = 35.19, p < 0.0001. (j) Representative images of dendritic spines in DG neurons in 2- and 13-month-old male mice. (k) Quantitative analysis of the dendritic spine density in 2-month-old (n = 3 spines/mouse, N = 3 mice/group) and 13-month-old (n = 3 spines/mouse, N = 3 mice/group) male mice. t(16) = 6.651, p < 0.0001. (l) The mRNA level of AIM2 was measured in 2-month-old (n = 6 mice) and 13-month-old (n = 6 mice) male mice, and the expression of GAPDH mRNA was used for normalization. t(10) = 4.622, p = 0.0009. (m, n) The level of AIM2 in the DG of 2- and 13-month-old male mice was determined by western blotting, and the intensities of the protein bands normalized to those of the ß-actin band are shown. n = 6 for each group. t(10) = 3.437, p = 0.0064. Data were shown as mean ± SEM. Unpaired two-tailed t-test for c, e, f, k, l and n. Two-way ANOVA followed by Bonferroni's post hoc test for i. **p < 0.01, ***p < 0.001.
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