Fig 2: Immune signaling in neurons recruits and activates microglia.(A) Schematic of spatial transcriptomics experiment. CK (n = 3) and CK-p25 (n = 4) were induced for 2 weeks. Coronal brain sections were stained and imaged for ?H2AX and then sequenced. (B) UMAP of capture areas from all samples. Leiden clusters are indicated by color and number. Each dot represents one capture area. (C) Leiden clusters superimposed onto a CK-p25 brain slice used for spatial transcriptomics. (D) UMAP indicating the density of ?H2AX+ capture areas. (E) ?H2AX+ capture areas identified in one CK-p25 brain slice. (F) Spatial clusters, ?H2AX+ capture areas, and reactive microglia signature gene expression in one CK-p25 sample. (G) Schematic of neuronal p65 knockdown experiment. CK-p25 mice received retro-orbital injections of scramble shRNA–red fluorescent protein (RFP) adeno-associated virus (AAV) or shp65-RFP AAV. CK mice received retro-orbital injections of phosphate-buffered saline (PBS). Mice recovered for 2 weeks before being taken off dox. Brains were collected at the 2-week time point. (H) Reverse transcription quantitative polymerase chain reaction of immune genes in sorted RFPhi ?H2AXhi nuclei. (I) Representative images of Iba1 in CK, scramble, and p65kd cortex. (J) Quantification of (left to right) Iba1+ soma area, number Iba1+ per image, average branch length per Iba1+ cell, and number end points per Iba1+ cell. Each data point represents one image. Two images were taken per mouse. (K) Heatmap of differentially expressed genes from p65 versus scramble contrast. (L) Up-regulated and down-regulated Gene Ontology (biological pathway) terms identified through GSEA of p65kd versus scramble contrast. (L) Heatmap of significantly up-regulated and down-regulated genes in PU.1hi nuclei from p65kd cortex compared to PU.1hi nuclei from scramble cortex. Error bars represent SEM; ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05. Student’s t test (H). One-way ANOVA followed by Holm-Sidak’s test for multiple comparisons (I). (H) Scramble (n = 5), p65kd (n = 5). (I) CK (n = 7), scramble (n = 5), p65kd (n = 6).

Fig 3: ECRG4 inhibits the NF-?B pathway via the upregulation of OGN in BCa cells. (A) NF-?B transcriptional activity was detected using an NF-?B luciferase reporter gene system. (B) Western blotting detected the protein levels of ECRG4, OGN, p65, and p-p65 (S536) in BCa cells after transfection with ECRG4-overexpressing or silencing plasmids. (C,D) Quantification of the protein levels of ECRG4, OGN, p65, and p-p65 (S536) in panel (B). (E) The NF-?B luciferase reporter gene system was used to detect the NF-?B transcriptional activity of BCa cells after transfection with indicated plasmids. (F) Western blotting detected the protein levels of p65 and p-p65 (S536) in BCa cells after transfection with the indicated plasmids. (G) Quantification of the protein levels of p65 and p-p65 (S536) in panel (F). (H) The proposed mechanism of ECRG4 inhibition of the carcinogenesis of BCa cells via NFIC/OGN/NF-?B signaling. (A–G) ANOVA followed by Bonferroni’s post hoc test. All data were replicated three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: Induction of DNA DSBs is sufficient to elicit immune pathway signaling in neuron primary culture.(A) Left: Representative images of NeuN and ?H2AX immunostaining in etoposide (ETP) and vehicle-treated primary cultures. Each data point represents ?H2AX mean gray value for one nucleus. Right: Representative images of p65 immunostaining in ETP- and vehicle-treated primary cultures. Each data point represents p65 mean gray value for one nucleus. (B) Schematic of ETP treatment. DIV13 neuron primary cultures were treated with either 50 µM ETP or vehicle control (DMSO) for 6 hours. (C) Differential Gene Ontology (biological pathway) terms identified through GSEA from ETP versus DMSO contrast. (D) Left: Heatmap of stage 1 and stage 2 signature enrichment in DMSO- and ETP-treated neurons. Right: Quantification of stage 1 and stage 2 signature enrichment in DMSO- and ETP-treated neurons. Each data point represents one biological replicate. (E) Venn diagram of significantly up-regulated protein-coding genes from ETP-treated neurons and stage 1 and stage 2 gene signatures. Percentages are in reference to the total number of unique genes from all three gene sets. Genes overlapping in ETP and stage 2 are in turquoise. Genes overlapping in ETP, stage 2, and stage 1 are in magenta. The area of the circles is in proportion to the size of the gene sets. Error bars represent SEM; ****P < 0.0001. Student’s t test (A). Wilcoxon test (D). Data are representative of three independent experiments (A).

Fig 5: Inflammatory signaling in DSB-bearing neurons is positively correlated with Alzheimer’s disease pathology.(A) Schematic of the stage 2 signature analysis in the AD snRNA-seq dataset (37). (B) Quantification of stage 2 signature correlation with global pathology in cell type clusters (37). Stage 2 genes were tested for significant and positive correlation with the global pathology metric for each major cell type. The -log10 P value for these tests is shown in the histogram. The dashed line indicates a P value of 0.01 after Bonferroni correction for multiple testing. Ex, excitatory neurons; In, inhibitory neurons; Ast, astrocytes; Oli, oligodendrocytes; Opc, oligodendrocyte precursor cells; Mic, microglia. (C) Stage 2 signature genes ranked by their correlation with global pathology in excitatory neurons. Example stage 2 genes are shown with red circles. (D) Gene Ontology of stage 2 signature genes positively correlated with global pathology. (E) Schematic of ?H2AXhi nuclei sorting from AD and non-AD brain tissue. (F) Heatmap of stage 1 and stage 2 signature enrichment in ?H2AXhi and ?H2AXlo human NeuNhi nuclei. (G to I) Quantification of stage 1 and stage 2 signature enrichment in ?H2AXhi and ?H2AXlo human NeuNhi nuclei samples by FANS gate (G), as well as FANS gate and disease status (H and I). (J) Representative image of ?H2AX, p65, and NeuN in the AD brain. Left: Two NeuN+ nuclei are outlined (white dashed line). Right: Magnification of the two outlined nuclei. Top nucleus (A) represents low DSB burden; bottom nucleus (B) represents high DSB burden. (K) Quantification of p65 nuclear enrichment in low and high DSB-burdened neurons. Each dot represents the average of 23 to 41 NeuN+ nuclei per individual. Error bars represent SEM; *P < 0.05. Student’s t test (K). Wilcoxon test (G to I).