Formation of Memory Assemblies through the DNA Sensing Tlr9 Pathway

As hippocampal neurons respond to diverse types of information a subset assembles into stable microcircuits representing a memory. Those neurons typically undergo energy-intensive molecular adaptations, which occasionally results in transient DNA damage. We found, however, discrete clusters of excitatory hippocampal CA1 neurons showing persistent dsDNA breaks, nuclear envelope ruptures, and perinuclear release of histone and dsDNA fragments for hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of Toll-like receptor 9 (Tlr9) signaling and accumulation of centrosomal DNA damage repair (DDR) complexes. Neuron-specific knockdown of Tlr9 impaired memory while blunting CFC-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Most notably, Tlr9 played an essential role in centrosome function, including DDR, ciliogenesis, and build-up of perineuronal nets (PNN). We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and Tlr9-mediated repair, resulting in their recruitment to memory circuits. With compromised Tlr9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments, which have been implicated in accelerated senescence, psychiatric disorders, and neurodegenerative disorders. Maintaining the integrity of Tlr9 inflammatory signaling thus emerges as a promising preventive strategy for neurocognitive deficits. To investigate the effect of contextual fear conditioning (CFC) and the role of Tlr9 signaling in CFC, we knocked out Tlr9 in neurons of the dorsal hippocampus (Tlr9 KO) and performed snRNA-seq to determine cell type specifc effects of CFC, Tlr9 KO, or both. To determine gene expression changes following fear conditioning, we compared mice that underwent CFC to naive mice (home cage controls). To determine Tlr9 driven gene expression changes, we compared fear conditioned Tlr9 KO mice to control mice (viral injection lacking Cre). Lastly, to ensure expression changes were due to Tlr9 KO and CFC rather than the KO alone, we compared naive Tlr9 KO mice to naive control mice.