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

Abstract DNA double strand breaks (DSBs) are linked to aging, neurodegeneration, and senescence 1,2 . However, the role played by neurons burdened with DSBs in disease-associated neuroinflammation is not well understood. Here, we isolate neurons harboring DSBs from the CK-p25 mouse model of neurodegeneration through fluorescence-activated nuclei sorting (FANS), and characterize their transcriptomes using single-nucleus, bulk, and spatial sequencing techniques. We find that neurons harboring DSBs enter a late-stage DNA damage response marked by the activation of senescent and antiviral-like immune pathways. We identify the NFkB transcription factor as a master regulator of immune gene expression in DSB-bearing neurons, and find that the expression of cytokines like Cxcl10 and Ccl2 develop in DSB-bearing neurons before glial cell types. Alzheimer’s Disease pathology is significantly associated with immune activation in excitatory neurons, and direct purification of DSB-bearing neurons from Alzheimer’s Disease brain tissue further validates immune gene upregulation. Spatial transcriptomics reveal that regions of brain tissue dense with DSB-bearing neurons also harbor signatures of inflammatory microglia, which is ameliorated by NFkB knock down in neurons. Inhibition of NFkB or depletion of Ccl2 and Cxcl10 in DSB-bearing neurons also reduces microglial activation in organotypic brain slice culture. In conclusion, we find that in the context of age-associated neurodegenerative disease, 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 age-associated neuroinflammation. Summary It is unclear how age-associated DNA double strand break (DSB) accumulation in neurons influences the progression of cellular senescence and neurodegenerative disease. Here, we leverage mouse models of neurodegeneration, single-nucleus, bulk, and spatial transcriptomics from Alzheimer’s disease patients, mouse models, and primary neuron cultures to dissect the immune signaling pathways initiated by DSB-bearing neurons that trigger neuroinflammation.