Neurons burdened by DNA double strand breaks incite microglia activation through antiviral-like signaling in neurodegeneration.

DNA double strand breaks (DSBs) are linked to neurodegeneration and senescence. However, it is not clear how DSB-bearing neurons influence neuroinflammation associated with neurodegeneration. Here, we characterize DSB-bearing neurons from the CK-p25 mouse model of neurodegeneration using single-nucleus, bulk, and spatial transcriptomic techniques. DSB-bearing neurons enter a late-stage DNA damage response marked by NFκB-activated senescent and antiviral immune pathways. In humans, Alzheimer’s disease pathology is significantly associated with immune activation in excitatory neurons. 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 knock-down in neurons. Inhibition of NFκB in DSB-bearing neurons also reduces microglia activation in organotypic mouse brain slice culture. In conclusion, DSBs activate immune pathways in neurons, which in turn adopt a senescence-associated secretory phenotype to elicit microglia activation. These findings highlight a novel role for neurons in the mechanism of disease-associated neuroinflammation. We used the CK-p25 mouse model of neurodegeneration to study the role of DSB-bearing neurons in neurodegeneration. Two male CK-p25 mice and two male CK mice were used for bulk RNA-sequencing. Stage 1, Stage 2, Baseline neurons, and Glia (so termed by NeuN and gH2AX imunoreactivity) were sorted from each mouse using FANS, then sequenced. For single-nucleus RNA-seq, Stage 1, Stage 2, Baseline neurons, and Glia were sorted and sequenced from three CK-p25 animals and three CK control littermates 1-week and 2-weeks after p25 induction. A total of 1,357 single nucleus libraries were prepared using SMARTseq2 chemistry. Three biological replicates each were used for bulk RNA-seq of etoposide-treated primary neurons (50um, 6hours), and dmso-control primary neurons. Three biological replicates each were used for bulk RNA-seq of FAN-sorted microglia nuclei from CK-p25 p65kd, CK-p25 Scramble, and CK Control PBS mice. Microglia nuclei were sorted based on PU.1 immunoreactivity.