STING deletion protects against amyloid β-induced Alzheimer's disease pathogenesis

Abstract

Introduction: While immune dysfunction has been increasingly linked to Alzheimer's disease (AD) progression, many major innate immune signaling molecules have yet to be explored in AD pathogenesis using genetic targeting approaches.

Methods: To investigate a role for the key innate immune adaptor molecule, stimulator of interferon genes (STING), in AD, we deleted Sting1 in the 5xFAD mouse model of AD-related amyloidosis and evaluated the effects on pathology, neuroinflammation, gene expression, and cognition.

Results: Genetic ablation of STING in 5xFAD mice led to improved control of amyloid beta (Aβ) plaques, alterations in microglial activation status, decreased levels of neuritic dystrophy, and protection against cognitive decline. Moreover, rescue of neurological disease in STING-deficient 5xFAD mice was characterized by reduced expression of type I interferon signaling genes in both microglia and excitatory neurons.

Discussion: These findings reveal critical roles for STING in Aβ-driven neurological disease and suggest that STING-targeting therapeutics may offer promising strategies to treat AD.

Highlights: Stimulator of interferon genes (STING) deficiency in the 5xFAD mouse model of Alzheimer's disease-related amyloidosis results in decreased amyloid beta (Aβ) deposition and altered microglial activation status. Protection against amyloidosis in STING-deficient 5xFAD mice is associated with decreased expression of genes involved in type I IFN signaling, improved neuronal health, and reduced levels of oxidative stress. Loss of STING in 5xFAD mice leads to improved spatial learning and memory.

Keywords: Alzheimer's disease; STING; amyloid beta; amyloidosis; innate immunity; microglia; neurodegenerative disease; neuroimmunology.

FIGURE 1

Amyloid beta load is reduced with STING deletion in 5xFAD mice. 5xFAD Sting1 ^−/− (abbreviated as 5xSt^KO) mice and littermate 5xFAD Sting1 ^+/+ (abbreviated as 5xFAD) controls were harvested at 5 months of age to evaluate Aβ load by IF imaging. (A–D) Analysis of total Aβ burden in the cortex, hippocampus, and subiculum (n = 9 mice/group, six to nine images/mouse, two independent experiments). (A) Representative confocal images of brain sections stained for nuclei (DAPI, blue) and total Aβ (D54D2, red; scale bar = 40 µm) in the cortex (abbreviated as 'C'), subiculum (abbreviated as 'S'), and hippocampus (abbreviated as 'H'). Quantification of percent area covered by D54D2 staining in the cortex (B), hippocampus (C), and subiculum (D). (E,F) Analysis of cortical Aβ plaque burden (n = 9, 9–12 images/mouse, three independent experiments). (E) Representative confocal images of brain sections stained for Aβ plaques (ThioS, cyan; scale bar = 30 µm). (F) Quantification of plaques per FOV. (G,H) Analysis of Aβ42 protein concentrations from whole‐brain lysates (n = 13–15 mice/group, three independent experiments). (G) ELISA quantification of Aβ42 per total protein from the soluble fraction extracted with T‐PER and (H) the insoluble fraction extracted with Gu‐HCL. Statistical significance between experimental groups was calculated by linear mixed‐effects models (B–D,F) and unpaired Student's t test (G,H). Data points represent individual mice and error bars represent mean ± SEM. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001. Aβ, amyloid beta; DAPI, 4',6‐diamidino‐2‐phenylindole; ELISA, enzyme‐linked immunosorbent assay; FOV, field of view; IF, immunofluorescence; SEM, standard error of the mean; STING, stimulator of interferon genes.

FIGURE 2

Genetic ablation of STING dampens microgliosis in 5xFAD mice. (A–L) 5xSt^KO mice and 5xFAD littermate controls were harvested at 5 months of age to evaluate microglial numbers and CD68 phagolysosomal marker expression by immunofluorescence staining. (A–G) Analysis of microglial coverage and recruitment to Aβ plaques (n = 9 mice/group, 9–12 images/mouse, three independent experiments). (A) Representative confocal images of brain sections stained for nuclei (DAPI, blue), plaques (ThioS, cyan), and macrophages/microglia (IBA1, green; scale bar = 30 µm). Quantification of percent area covered by IBA1+ staining in the (B) cortex, (C) hippocampus, and (D) subiculum. (E) Representative confocal images of PAMs (IBA1, green) and plaques (ThioS, cyan; scale bar = 15 µm). (F) Enumeration of IBA1+ cells per FOV in the cortex. (G) Number of PAMs within 15 and 30 µm of plaque surfaces. (H,I) Analysis of CD68 phagolysosomal marker expression (n = 8–9 mice/group, 9–12 images/mouse, three independent experiments). (H) Representative confocal images of brain sections stained for microglia (IBA1, green) and the phagolysosomal marker CD68 (magenta; inset FOV; scale bar = 15 µm). White regions denote colocalization of CD68 and IBA1 staining. (I) Quantification of CD68 staining colocalized with IBA1 staining. (J–L) Sholl analysis of cortical microglia (n = 4–5 mice/genotype, 15 cells/mouse from six to nine images each, three independent experiments). (J) Representative 3D rendering of IBA1+ cells (iscale bar = 10 µm). (K) Quantification of total Sholl intersections at various distances away from the cell soma and (L) total number of intersections within 50 µm of the cell soma. Statistical significance between experimental groups was calculated by linear mixed‐effects models. Data points in the column graphs (B–D, F, G, I, and L) represent individual mice, and data points in the line graph (K) represent means for each experimental group. Error bars represent mean ± SEM. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001. Aβ, amyloid beta; DAPI, 4',6‐diamidino‐2‐phenylindole; FOV, field of view; PAMs, plaque‐associated microglia; SEM, standard error of the mean; STING, stimulator of interferon genes.

FIGURE 3

Lack of STING in 5xFAD mice leads to altered microglial activation status. (A–E) Gene expression was assessed in 6‐month‐old 5xFAD and 5xSt^KO mice by pooled snRNA‐seq of cortical tissue (n = 3 per pooled sample). (A) UMAP plot of cells grouped by major brain cell type. Clusters were manually defined based on the expression of cell type‐specific signature genes. (B) Heat map depicting an unbiased list of the 5 most highly expressed genes in each cell cluster. The genes highlighted in red are those that have been previously linked to AD in humans. (C) Quantification of numbers of nuclei sequenced per genotype and visual breakdown by cell type. (D) Volcano plot depicting DEGs (upregulated, pink; downregulated, blue) in the microglia cluster of 5xSt^KO versus 5xFAD cortex. Colored points depict genes with adjusted p‐values less than 0.05 and log₂ fold changes ± 0.25. (E) Bar plots depicting upregulated GO terms enriched in 5xFAD microglia. Functional enrichment analysis was performed using g:Profiler using genes with adjusted p‐values less than 0.05 and log₂ fold changes ± 0.25. Depicted pathways were curated from the top driver terms in GO. (F–M) 5xSt^KO mice and 5xFAD littermate controls were harvested at 5 months of age to evaluate AXL and STAT1 expression by immunofluorescence staining. (F) Representative images of microglia (IBA1, blue), Aβ plaques (ThioS, red), and AXL (yellow; shown in inset) in the cortex (scale bar = 10 µm). White regions depict colocalization of AXL and IBA1 staining. (G) Number of AXL+ IBA1+ cells per FOV, (H) percentage of IBA1+ cells expressing AXL, and (I) number of AXL+ IBA1+ cells per plaque in the cortex (n = 6; 2 independent experiments). (J) Representative images of microglia (IBA1, blue) and STAT1 (yellow; shown in inset) in the cortex (scale bar = 15 µm). White regions denote colocalization of STAT1 and IBA1 staining. (K) Number of STAT1+ IBA1+ cells per FOV, (L) percentage of IBA1+ cells expressing STAT1+, and (M) number of STAT1+ IBA1+ cells per plaque in the cortex (n = 8; two independent experiments). Data points and error bars depict means per animal ± SEM, and statistical testing was performed by linear mixed‐effects models (G–I) and (K–M). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001. Aβ, amyloid beta; AD, Alzheimer's disease; DEG, differentially expressed gene; FOV, field of view; GO, gene ontology; SEM, standard error of the mean; snRNA‐seq, single‐nuclei RNA sequencing; STING, stimulator of interferon genes; UMAP, uniform manifold approximation and projection.

FIGURE 4

Genetic ablation of STING in 5xFAD mice leads to improvements in neuronal health and protection against cognitive decline. (A–C) The impact of STING deletion on neuronal gene expression was assessed in 6‐month‐old 5xFAD and 5xSt^KO mice by pooled snRNA‐seq of cortical tissue (n = 3 per pooled sample). (A) UMAP plot depicting the excitatory neuron cluster. (B) Volcano plot depicting DEGs (upregulated, pink; downregulated, blue) in the excitatory neuron cluster of 5xSt^KO versus 5xFAD cortex. Colored points depict genes with adjusted p‐values less than 0.05 and log₂ fold changes ± 0.25. (C) Bar plots depicting upregulated GO terms enriched in 5xSt^KO (top, pink) and 5xFAD (bottom, blue) excitatory neurons. Functional enrichment analysis was performed using g:Profiler using genes with adjusted p‐values less than 0.05 and log₂ fold changes ± 0.25. Depicted pathways were curated from the top driver terms in GO. (D–I) 5xSt^KO mice and 5xFAD littermate controls were harvested at 5 months of age to evaluate neuritic dystrophy, oxidative stress, and neuronal cell death. (D) Representative images of DNs (APP, yellow) and Aβ plaques (ThioS, cyan) in the cortex (scale bar = 5 µm). (E) Enumeration of APP+ puncta DNs within 15 and 30 µm of Aβ plaques (n = 7–8; two independent experiments). (F) MDA concentration per total protein from whole‐brain tissue lysates (n = 6 mice/group). (G) Representative images of cell death staining (TUNEL, green; shown in inset), neuronal nuclei (NeuN, magenta), and overall nuclei (DAPI, blue) in the CA1 region of the hippocampus (scale bar = 10 µm). (H) TUNEL percent area in the CA1. (I–J) MWM testing was conducted on 4‐month‐old 5xSt^KO mice and 5xFAD littermate controls to evaluate spatial learning and memory (n = 15 mice/group, four independent experiments). (I) Quantification of time spent to reach the platform during the acquisition phase and (J) time spent inside the target quadrant previously containing the old platform on a single MWM probe day. Statistical significance between experimental groups was calculated by linear mixed‐effects models (E,F,H) and two‐way ANOVA with Bonferroni's post‐hoc test (I), and unpaired Student's t test (J). Data points in column graphs (E,F,H,J) represent individual mice, and data points in line graphs (I) represent means for each experimental group. All error bars represent mean ± SEM. ^* p < 0.05, ^** p < 0.01, ^**** p < 0.0001. Aβ, amyloid beta; ANOVA, analysis of variance; APP+, amyloid precursor protein positive; DEGs, differentially expressed gene; DNs, dystrophic neurites; FOV, field of view; GO, gene ontology; MDA, malondialdehyde; MWM, Morris water maze; SEM, standard error of the mean; snRNA‐seq, single‐nuclei RNA sequencing; STING, stimulator of interferon genes; TUNEL, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling; UMAP, uniform manifold approximation and projection.