Endosomal TLR3, TLR7, and TLR8 control neuronal morphology through different transcriptional programs.

Neuroinflammation is associated with diverse neurological disorders. Endosomal Toll-like receptors (TLRs) including TLR3, TLR7, and TLR8 cell-autonomously regulate neuronal differentiation. However, the mechanisms by which these three TLRs affect neuronal morphology are unclear. In this study, we compare these TLRs in mouse neurons. By combining in vitro neuronal cultures, in utero electroporation, and transcriptomic profiling, we show that TLR8, TLR7, and TLR3 promote dendritic pruning via MYD88 signaling. However, they induce different transcriptomic profiles related to innate immunity, signaling, and neuronal development. The temporal expression patterns and the effects on neuronal morphology are not identical upon activation of these endosomal TLRs. Pathway analyses and in vitro studies specifically implicate mitogen-activated protein kinase signaling in TLR8-mediated dendritic pruning. We further show that TLR8 is more critical for dendritic arborization at a late development stage in vivo. The activation of TLR8, TLR7, or TLR3 results in dendritic shortening, and TLR7 and TLR3 but not TLR8 also control axonal growth. In-depth transcriptomic analyses show that TLRs use different downstream pathways to control neuronal morphology, which may contribute to neuronal development and pathological responses.

The microRNAs Let7c and miR21 are recognized by neuronal Toll-like receptor 7 to restrict dendritic growth of neurons.

Inflammatory responses are known to play critical roles in the regulation of neurodevelopment and neurodegeneration. Although microglial cells are recognized as professional immune cells in brains, recent evidence suggests that neurons also express important receptors and regulators of innate immunity, including Toll-like receptor 7 (TLR7), which is a receptor for single-stranded RNAs (ssRNAs). Here, we report that neuronal TLR7 recognizes endogenous ligands such as the miRNAs Let7c and miR21 and plays a negative role in controlling neuronal growth in a cell-autonomous manner. We show here that hippocampal CA1 neurons in Tlr7(-/Y) mice had more complex dendritic arbors compared with those of wild-type littermates at postnatal (P) day 7, but not at P21. This observation strengthens a role of TLR7 in restricting neuronal growth during development. In cultured neurons, transient knockdown of Tlr7 promoted axonal and dendritic growth, supporting the cell-autonomous effect of TLR7 on neuronal growth. We observed perceptible levels of Let7c and miR21 in the exosomes of the neuronal cultures as well as in developing brains. Treatment with Let7c and miR21 restricted dendritic growth of wild-type neurons but not Tlr7(-/-) neurons. Our study suggests that neuronal TLR7 is activated by endogenous ligands and thus regulates neuronal morphology. Neuronal innate immune responses may influence neurodevelopment and neurodegeneration through the regulation of neuronal morphology.

TLR7 negatively regulates dendrite outgrowth through the Myd88-c-Fos-IL-6 pathway.

Toll-like receptors (TLRs) recognize both pathogen- and danger-associated molecular patterns and induce innate immune responses. Some TLRs are expressed in neurons and regulate neurodevelopment and neurodegeneration. However, the downstream signaling pathways and effectors for TLRs in neurons are still controversial. In this report, we provide evidence that TLR7 negatively regulates dendrite growth through the canonical myeloid differentiation primary response gene 88 (Myd88)-c-Fos-interleukin (IL)-6 pathway. Although both TLR7 and TLR8 recognize single-stranded RNA (ssRNA), the results of quantitative reverse transcription-PCR suggested that TLR7 is the major TLR recognizing ssRNA in brains. In both in vitro cultures and in utero electroporation experiments, manipulation of TLR7 expression levels was sufficient to alter neuronal morphology, indicating the presence of intrinsic TLR7 ligands. Besides, the RNase A treatment that removed ssRNA in cultures promoted dendrite growth. We also found that the addition of ssRNA and synthetic TLR7 agonists CL075 and loxoribine, but not R837 (imiquimod), to cultured neurons specifically restricted dendrite growth via TLR7. These results all suggest that TLR7 negatively regulates neuronal differentiation. In cultured neurons, TLR7 activation induced IL-6 and TNF-α expression through Myd88. Using Myd88-, IL-6-, and TNF-α-deficient neurons, we then demonstrated the essential roles of Myd88 and IL-6, but not TNF-α, in the TLR7 pathway to restrict dendrite growth. In addition to neuronal morphology, TLR7 knockout also affects mouse behaviors, because young mutant mice ∼2 weeks of age exhibited noticeably lower exploratory activity in an open field. In conclusion, our study suggests that TLR7 negatively regulates dendrite growth and influences cognition in mice.