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Data points represent one image from one mouse. Right: Quantification of number γH2AX + and γH2AX − cells with ≥2 Ccl2 puncta. ( G) Left: Representative images of Ccl2 γH2AX staining for 1-, 2-, and 6-week CK-p25 cortex. ( F) Heatmap of differentially expressed genes associated with inflammation from bulk RNA-seq. FDR, false discovery rate IFN, interferon NES, normalized enrichment score. ( E) Differential Gene Ontology terms from bulk RNA-seq. snRNA-seq: n = 3 per genotype at 1- and 2-week time points. Bulk RNA-seq: n = 2 per genotype for bulk RNA-seq, 2-week time point. ( C) Representative dot plot of γH2AX and NeuN immunoreactivity in 2-week CK-p25 cortex. Each data point represents percent γH2AX hi nuclei from one mouse. ( B) Quantification of percent γH2AX hi for 1 to 6 weeks. ( A) Flow cytometry dot plots of γH2AX hi nuclei (turquoise) from CK and CK-p25 cortex. These findings highlight a previously unidentified role for neurons in the mechanism of disease-associated neuroinflammation. In conclusion, DSBs activate immune pathways in neurons, which in turn adopt a senescence-associated secretory phenotype to elicit microglia activation. Inhibition of NFκB in DSB-bearing neurons also reduces microglia activation in organotypic mouse brain slice culture. Spatial transcriptomics reveal that regions of CK-p25 brain tissue dense with DSB-bearing neurons harbor signatures of inflammatory microglia, which is ameliorated by NFκB knockdown in neurons. In humans, Alzheimer's disease pathology is closely associated with immune activation in excitatory neurons. DSB-bearing neurons enter a late-stage DNA damage response marked by nuclear factor κB (NFκB)-activated senescent and antiviral immune pathways. Here, we characterize DSB-bearing neurons from the CK-p25 mouse model of neurodegeneration using single-nucleus, bulk, and spatial transcriptomic techniques. However, it is not clear how DSB-bearing neurons influence neuroinflammation associated with neurodegeneration. DNA double-strand breaks (DSBs) are linked to neurodegeneration and senescence.
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