Neurodevelopment is characterized by rapid rates of neural cell proliferation and differentiation followed by massive cell death in which more than half of all recently generated brain cells are pruned back. Large amounts of DNA damage, cellular debris, and by-products of cellular stress are generated during these neurodevelopmental events, all of which can potentially activate immune signalling. How the immune response to this collateral damage influences brain maturation and function remains unknown. Here we show that the AIM2 inflammasome contributes to normal brain development and that disruption of this immune sensor of genotoxic stress leads to behavioural abnormalities. During infection, activation of the AIM2 inflammasome in response to double-stranded DNA damage triggers the production of cytokines as well as a gasdermin-D-mediated form of cell death known as pyroptosis1,2,3,4. We observe pronounced AIM2 inflammasome activation in neurodevelopment and find that defects in this sensor of DNA damage result in anxiety-related behaviours in mice. Furthermore, we show that the AIM2 inflammasome contributes to central nervous system (CNS) homeostasis specifically through its regulation of gasdermin-D, and not via its involvement in the production of the cytokines IL-1 and/or IL-18. Consistent with a role for this sensor of genomic stress in the purging of genetically compromised CNS cells, we find that defective AIM2 inflammasome signalling results in decreased neural cell death both in response to DNA damage-inducing agents and during neurodevelopment. Moreover, mutations in AIM2 lead to excessive accumulation of DNA damage in neurons as well as an increase in the number of neurons that incorporate into the adult brain. Our findings identify the inflammasome as a crucial player in establishing a properly formed CNS through its role in the removal of genetically compromised cells.
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We thank members of the Lukens laboratory and the Center for Brain Immunology and Glia (BIG) for discussions. This work was supported by The Hartwell Foundation (Individual Biomedical Research Award to J.R.L.), a Rettsyndrome.org grant (22349 to J.R.L.), The Owens Family Foundation (awarded to J.R.L.), and a NARSAD Young Investigator Grant from the Brain & Behavior Research Foundation (27515 to J.R.L.). C.R.L was supported by a NIH National Institute of General Medical Sciences predoctoral training grant (3T32GM008328) and a Wagner Fellowship. A.C.B. was supported by a Medical Scientist Training Program Grant (5T32GM007267-38) and an Immunology Training Grant (5T32AI007496-25). H.E.E. was supported by a Cell and Molecular Biology Training Grant (T32GM008136). E.L.F. was supported by a National Multiple Sclerosis Foundation Postdoctoral Fellowship (FG-1707-28590). C.E.B. was supported by Hutcheson and Stull Undergraduate Research Fellowships.
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Molecular components of the AIM2 inflammasome are abundantly expressed in the brain during neurodevelopment.
a–c, Brains from P5 (a) and P21 (b) wild-type mice were evaluated for the mRNA expression of inflammasome component genes (red) Casp1 (P5 n = 4, P21 n = 2), Aim2 (P5 n = 3, P21 n = 2) and Gsdmd (P5 n = 3, P21 n = 2) using RNAscope. c, Quantification of Casp1 (P5 n = 4, P21 n = 2), Aim2 (P5 n = 3, P21 n = 2) and Gsdmd (P5 n = 3, P21 n = 2) mRNA puncta in the hippocampus per 40× image; from one experiment. n values refer to biological replicates. Data are mean ± s.e.m.
Extended Data Fig. 2 ASC speck formation routinely occurs in the developing brain but is rare in mature lymph nodes under steady-state conditions.
a, Top, sagittal image of ASC speck formation (green) in the brain of P5 ASC-citrine reporter mice. Original magnification, ×10. Bottom, ASC specks are detected throughout the brain using a ×40 objective including in the cerebellum (i), midbrain (ii), hippocampus (iii) and thalamus (iv). Representative images from three independent experiments with similar results. b, c, Adult (8–12 weeks old) ASC-citrine reporter mice were evaluated for peripheral inflammasome activation based on ASC speck formation (green) in the deep cervical lymph node (DCLN) using confocal microscopy with a ×10 objective (b). c, Arrow shows magnified image of ASC speck (green) formed in the DCLN. Representative images from two independent experiments with similar results.
Extended Data Fig. 3 Lack of AIM2 inflammasome signalling results in an increase in anxiety-related behaviours but not depressive-related behaviours.
Adult (8–12 weeks old) wild-type, Ice−/−, Nlrp3−/− and Aim2−/− mice were assessed for behavioural abnormalities. a, b, Number of urinations (WT n = 26, Ice−/− n = 17, Nlrp3−/− n = 11, Aim2−/− n = 24; from three independent experiments) (a) and number of faecal pellets (WT n = 20, Ice−/− n = 17, Nlrp3−/− n = 11, Aim2−/− n = 21; from three independent experiments) (b) were measured during 10 min of open-field testing. c, d, Depressive behaviours were evaluated in adult male mice using the tail suspension test for escape behaviour (WT n = 19, Ice−/− n = 10, Nlrp3−/− n = 5, Aim2−/− n = 14; from two independent experiments) (c) and sucrose preference test (WT n = 3, Ice−/− n = 3, Aim2−/− n = 4; from one independent experiment) (d). All n values refer to the number of mice used. Data are mean ± s.e.m. P values were determined by one-way ANOVA with Tukey’s post hoc tests.
a, Brains from wild-type P5 mice were evaluated for localization of ASC specks (green) in relation to DAPI+ nuclei (blue) containing DNA damage (γH2AX (red), 53BP1 (grey)) in the cerebellum. i, ii, Magnified images in regions showing ASC specks formed in close proximity to nuclei containing DNA damage. Representative images from four mice with similar results, from one experiment. Original magnifications, ×40. Differences in the size of nuclei probably reflect specific stages in replication, DNA repair, differentiation, or cell death that the individual cells are in, as well as differences seen across CNS cell types. b–e, Wild-type and Aim2−/− P5 mice received either control treatment or 14 Gy of ionizing radiation (IR) to induce DNA damage. Brains were obtained 6 h later, and immunostained to measure DNA damage induction (γH2AX staining) and inflammasome activation (ASC speck formation) in the cerebellum. b, Representative cerebellar images of γH2AX staining; from two independent experiments with similar results. Original magnification, ×20. c, Quantification of γH2AX staining in the cerebellum (untreated: WT n = 3, Aim2−/− n = 3; IR-treated: WT n = 8, Aim2−/− n = 8; from two independent experiments). d, Representative cerebellar images of ASC speck formation; from two independent experiments with similar results. Original magnification, ×20. e, Quantification of ASC speck formation in the cerebellum (untreated: WT n = 7, Aim2−/− n = 5; IR-treated: WT n = 6, Aim2−/− n = 8; from two independent experiments). All n values refer to the number of mice used. Data are mean ± s.e.m. P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 5 Anxiety phenotypes do not develop in mice that lack IL-1R, IL-18R, MYD88 or caspase-11.
All behavioural testing was conducted on adult (8–12 weeks old) mice. a–c, Behaviours for anxiety were evaluated by bouts into (a) and time spent in (b) the centre of the open-field arena, and the total distance travelled (c) (WT n = 11, Il1r−/− n = 19, Il18r−/− n = 22, Myd88−/− n = 12; from three independent experiments). d, Anxiety-related behaviours were assessed in wild-type and Myd88−/− mice using the elevated plus maze. Representative heat maps from four independent experiments with similar results depicting path of travel through open and closed arms of the maze. e, Representative heat maps from four independent experiments with similar results of the path travelled by adult wild-type and Myd88−/− mice in the open-field arena. f–h, Quantification of bouts into (f) and time spent in (g) the centre of the open-field arena, and the total distance travelled (h) (WT n = 14, Casp11−/− n = 10; from two independent experiments). All n values refer to the number of mice used. Data are mean ± s.e.m. No statistically significant differences were determined by one-way ANOVA with Tukey’s post hoc tests (a–c) or unpaired two-tailed Student’s t-test (f–h).
Extended Data Fig. 6 Genetic ablation of the AIM2 inflammasome or gasdermin-D in CNS cells limits cell death in response to DNA insults.
Mixed neural cultures were generated from wild-type, Ice−/−, Aim2−/− and Gsdmd−/− P0 mice. a, Mixed neural cell cultures were left untreated to test for baseline differences in cytotoxicity (WT n = 5, Ice−/− n = 5, Aim2−/− n = 5, Gsdmd−/− n = 2). b, Mixed neural cell cultures were primed with LPS for 4 h followed by transfection with poly(dA:dT) (WT n = 4, Ice−/− n = 4, Aim2−/− n = 4, Gsdmd−/− n = 4). Cell death was measured by LDH release after overnight stimulation. Representative data from three independent experiments. All n values refer to biological replicates from one representative experiment. Data are mean ± s.e.m. P values were determined by one-way ANOVA with Tukey’s post hoc tests.
Extended Data Fig. 7 AIM2 contributes to CNS cell death during neurodevelopment and in response to ionizing radiation.
a, Representative images from wild-type, Ice−/−, Aim2−/− and Gsdmd−/− P5 mice showing TUNEL+ cells (green) in the cerebellum. Images are representative of two independent experiments with similar results. Original magnifications, ×20. b, Representative images of additional markers of cell death (propidium iodide (PI), grey) in wild-type and Aim2−/− P5 mice. Original magnifications, ×20. c, Quantification of propidium iodide-positive cells in the cerebellum of WT (n = 4) and Aim2−/− (n = 4) P5 mice; from one independent experiment. d, e, Wild-type and Aim2−/− P5 mice received either control treatment or 14 Gy of ionizing radiation to induce DNA damage. Brains were obtained 6 h later and the TUNEL assay was conducted on cerebellar sections to evaluate cell death. d, Representative images showing TUNEL staining in the cerebellum of untreated and irradiated wild-type and Aim2−/− P5 mice; from three independent experiments with similar results. Original magnifications, ×20. e, Quantification of TUNEL+ cells in the cerebellums of untreated and irradiated wild-type (n = 11 untreated, n = 9 IR) and Aim2−/− (n = 9 untreated, n = 8 IR) mice; from three independent experiments. All n values refer to the number of mice used. Data are mean ± s.e.m. P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 8 Lack of AIM2 inflammasome components increases the number of Purkinje neurons that are incorporated into the adult brain.
Representative images of cerebellums from adult (8–12 weeks old) wild-type, Ice−/−, Aim2−/− and Gsdmd−/− mice showing an increase in the number of Purkinje cells (calbindin+ cells) in mice lacking inflammasome components. Original magnifications, ×20. Images are representative of three independent experiments with similar results.
Extended Data Fig. 9 Aim2 is expressed by neurons, astrocytes and microglia in the developing brain.
Brains from wild-type P5 mice (n = 3; from 1 experiment) were evaluated for expression of Aim2 using RNAscope. a, Images showing co-expression of Aim2 (green) and CNS cell-specific genes Rbfox3: NeuN (red), Gfap: GFAP (red), and Aif1: Iba1 (red) in the hippocampus. Original magnifications, ×40. b, Quantification of the percentage of CNS cells in images that are positive for Aim2. n values refer to biological replicates. Data are mean ± s.e.m.
Extended Data Fig. 10 Deletion of caspase-1 in CX3CR1-expressing cells does not result in the development of anxiety-related behaviours.
a–c, Adult (8–12 weeks old) Casp1fl/fl (n = 10) and Casp1fl/flCx3cr1cre (n = 11) mice were evaluated for anxiety-related behaviours using the time spent in the open arms (a) and distance travelled (b) in the elevated plus maze, along with total bouts into the centre of the open-field arena (c). Data are from two independent experiments. All n values refer to the number of mice used. d, Schematic of the proposed role that DNA damage surveillance by the AIM2 inflammasome has in neurodevelopment. Data are mean ± s.e.m. P values determined by unpaired two-tailed Student’s t-test show no statistically significant differences. Graphical illustrations were made using BioRender (https://biorender.com/).
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Lammert, C.R., Frost, E.L., Bellinger, C.E. et al. AIM2 inflammasome surveillance of DNA damage shapes neurodevelopment. Nature 580, 647–652 (2020). https://doi.org/10.1038/s41586-020-2174-3
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