Formation of memory assemblies through the DNA-sensing TLR9 pathway

The formation of stable and persistent memories relies on the assembly of hippocampal and cortical neurons into interconnected microcircuits. While long-term potentiation (LTP) of synaptic connections is a key mechanism, other factors, such as pre-existing neuronal programs and perineuronal nets (PNNs), contribute to the stability of these assemblies. This research explores whether a unifying process integrates these stimulus-dependent and intrinsic mechanisms, focusing on the potential role of the Toll-like receptor 9 (TLR9) pathway. The hypothesis is that learning-induced DNA damage in specific neuronal populations triggers an inflammatory response mediated by TLR9, leading to the recruitment of these neurons into memory circuits. Understanding this process is crucial because it could explain the link between genomic instability and cognitive decline seen in various neurodegenerative and psychiatric disorders. The study aimed to elucidate the molecular cascade triggered by learning, the specific role of TLR9, and the consequences of its dysfunction on memory formation and neuronal health.

Previous research has established the importance of LTP in memory formation, highlighting its energy-intensive nature and resulting biochemical and morphological adaptations within neurons. Studies have also emphasized the contribution of pre-existing intrinsic neuronal programs, including CREB expression and lineage, and the role of PNNs in stabilizing memory circuits. However, a comprehensive understanding of how these distinct mechanisms integrate during memory formation remains incomplete. This research builds upon these previous findings by investigating a potential overarching process that links stimulus-dependent and pre-existing factors involved in neuronal commitment to memory assemblies. The activation of the NLRP3 inflammasome after contextual fear conditioning (CFC) has also been reported, providing further context for the investigation of immune responses in memory formation.

The study employed several methodologies: 1. **Bulk RNA sequencing (RNA-seq):** RNA was extracted from mouse hippocampi at various time points (96 hours and 21 days) after CFC to identify differentially expressed genes. 2. **Immunofluorescence:** This technique was used to visualize and quantify dsDNA breaks (using γH2AX), nuclear envelope ruptures (using lamin B1), and the co-localization of various proteins, including TLR9, LAMP2, centrin, γ-tubulin, and 53BP1, to investigate the spatial and temporal dynamics of DNA damage and repair. 3. **Extranuclear DNA isolation and sequencing:** To identify the source of DNA activating TLR9, extranuclear DNA was isolated and sequenced. 4. **Neuron-specific TLR9 knockdown:** This was achieved using adeno-associated virus (AAV9) expressing Cre recombinase or TLR9-targeting shRNA in CA1 neurons of Tlr9fl/fl mice. 5. **Behavioral tests:** Contextual fear conditioning (CFC), trace fear conditioning (TFC), and delay fear conditioning (DFC) were used to assess memory function. 6. **Pharmacological manipulations:** TLR9 antagonist ODN2088 and CGAS-STING inhibitors were used to investigate the role of these pathways in memory. 7. **DNase manipulation:** Viral overexpression of DNase2 shRNA or TREX1-GFP was used to explore the role of DNA degradation in memory formation. 8. **Single-nucleus RNA sequencing (snRNA-seq):** This was performed to analyze gene expression changes in different hippocampal cell populations after CFC and TLR9 knockdown. 9. **RNAscope:** This in situ hybridization technique was used to confirm the expression of specific genes in hippocampal cells. The study meticulously controlled for potential confounding factors, such as viral injection-induced inflammation, by using appropriate control groups and validating findings across multiple approaches.

The study's key findings include: 1. **Learning-induced DNA damage:** CFC triggered the formation of dsDNA breaks and nuclear envelope ruptures in distinct CA1 neuron clusters within hours after training. 2. **TLR9 activation:** These events led to the activation of the TLR9 pathway and the accumulation of centrosomal DNA damage repair complexes. 3. **TLR9's role in memory:** Neuron-specific TLR9 knockdown significantly impaired context memory and altered gene expression patterns in specific CA1 neuron clusters. 4. **TLR9's role in centrosome function:** TLR9 played a critical role in centrosome function, including DNA damage repair, ciliogenesis, and PNN formation. 5. **Specificity of TLR9-mediated response:** TLR9, not CGAS-STING, appears responsible for DNA sensing in CFC-related memory formation. 6. **Impact of TLR9 knockdown on gene expression:** snRNA-seq analysis revealed that TLR9 knockdown abolished CFC-induced gene expression changes in multiple neuronal clusters and disrupted normal gene expression patterns. 7. **Impact on neuronal morphology:** TLR9 knockdown also impaired ciliogenesis and PNN formation, indicating disruptions to normal neuronal structure and function. 8. **Genomic instability:** TLR9 knockdown resulted in increased genomic instability, potentially linking it to accelerated senescence and neurodegenerative diseases.

The study provides strong evidence for a novel memory mechanism involving TLR9-mediated DNA damage repair and inflammatory signaling in specific hippocampal CA1 neurons. The findings demonstrate that learning-induced DNA damage, rather than being detrimental, is essential for memory formation, provided the TLR9-mediated repair mechanism is intact. The lack of association between inflammatory responses and immediate early gene (IEG) responses suggests that these processes operate in distinct neuronal populations, potentially contributing to different aspects of memory function. The importance of TLR9 in centrosome function and the observed disruption of ciliogenesis and PNN formation upon TLR9 knockdown further highlight the interconnectedness of various cellular processes in memory formation and maintenance. The study’s findings suggest that targeting the TLR9 pathway could be a promising therapeutic strategy for neurocognitive disorders, but caution is warranted due to the essential role of TLR9 in maintaining genomic stability.

This research reveals a novel mechanism of memory formation involving TLR9-mediated DNA damage repair and inflammatory signaling in hippocampal CA1 neurons. TLR9 activation is crucial for centrosome function, DNA repair, ciliogenesis, and PNN formation, which contribute to memory persistence. Impaired TLR9 function leads to genomic instability and impaired memory, suggesting potential therapeutic targets for neurocognitive disorders. Future studies should investigate the specific DNA sequences that activate TLR9 during learning and the precise mechanisms by which TLR9 regulates centrosome function and neuronal differentiation.

The study primarily focused on mouse models, limiting the direct generalizability to humans. While the researchers addressed several potential confounding factors, the possibility of other unknown mechanisms influencing the results cannot be entirely excluded. Further research is needed to fully understand the molecular details of the TLR9-mediated DNA damage repair process and to explore the clinical implications of these findings in human populations.