Beyond defense: regulation of neuronal morphogenesis and brain functions via Toll-like receptors.

Toll-like receptors (TLRs) are well known as critical pattern recognition receptors that trigger innate immune responses. In addition, TLRs are expressed in neurons and may act as the gears in the neuronal detection/alarm system for making good connections. As neuronal differentiation and circuit formation take place along with programmed cell death, neurons face the challenge of connecting with appropriate targets while avoiding dying or dead neurons. Activation of neuronal TLR3, TLR7 and TLR8 with nucleic acids negatively modulates neurite outgrowth and alters synapse formation in a cell-autonomous manner. It consequently influences neural connectivity and brain function and leads to deficits related to neuropsychiatric disorders. Importantly, neuronal TLR activation does not simply duplicate the downstream signal pathways and effectors of classical innate immune responses. The differences in spatial and temporal expression of TLRs and their ligands likely account for the diverse signaling pathways of neuronal TLRs. In conclusion, the accumulated evidence strengthens the idea that the innate immune system of neurons serves as an alarm system that responds to exogenous pathogens as well as intrinsic danger signals and fine-tune developmental processes of neurons.

Tlr7 deletion alters expression profiles of genes related to neural function and regulates mouse behaviors and contextual memory.

The neuronal innate immune system recognizes endogenous danger signals and regulates neuronal development and function. Toll-like receptor 7 (TLR7), one of the TLRs that trigger innate immune responses in neurons, controls neuronal morphology. To further assess the function of TLR7 in the brain, we applied next generation sequencing to investigate the effect of Tlr7 deletion on gene expression in hippocampal and cortical mixed cultures and on mouse behaviors. Since previous in vivo study suggested that TLR7 is more critical for neuronal morphology at earlier developmental stages, we analyzed two time-points (4 and 18 DIV) to represent young and mature neurons, respectively. At 4 DIV, Tlr7 KO neurons exhibited reduced expression of genes involved in neuronal development, synaptic organization and activity and behaviors. Some of these Tlr7-regulated genes are also associated with multiple neurological and neuropsychiatric diseases. TLR7-regulated transcriptomic profiles differed at 18 DIV. Apart from neuronal genes, genes related to glial cell development and differentiation became sensitive to Tlr7 deletion at 18 DIV. Moreover, Tlr7 KO mice exhibited altered behaviors in terms of anxiety, aggression, olfaction and contextual fear memory. Electrophysiological analysis further showed an impairment of long-term potentiation in Tlr7 KO hippocampus. Taken together, these results indicate that TLR7 regulates neural development and brain function, even in the absence of infectious or pathogenic molecules. Our findings strengthen evidence for the role of the neuronal innate immune system in fine-tuning neuronal morphology and activity and implicate it in neuropsychiatric disorders.

Deletion of the Inflammasome Sensor Aim2 Mitigates Aß Deposition and Microglial Activation but Increases Inflammatory Cytokine Expression in an Alzheimer Disease Mouse Model.

OBJECTIVE: Inflammation is clearly associated with Alzheimer disease (AD). Knockout of Nlrp3, a gene encoding an inflammasome sensor, has been shown to ameliorate AD pathology in a mouse model. Because AIM2 is the most dominant inflammasome sensor expressed in mouse brains, here we investigate whether Aim2 deletion also influences the phenotype of a 5XFAD AD mouse model. METHODS: Quantitative RT-PCR, immunostaining, immunoblotting, and behavioral analyses were applied to compare wild-type, Aim2-/-, 5XFAD, and Aim2-/-;5XFAD mice. RESULTS: We found that Aim2 knockout mitigates Aß deposition in the cerebral cortex and hippocampus of 5XFAD mice. The activation of microglial cells is also reduced in Aim2-/-;5XFAD brains compared with 5XFAD brains. However, Aim2 knockout does not improve memory and anxiety phenotypes of 5XFAD mice in an open field, cued Y-maze, or Barnes maze. Compared with 5XFAD mice, Il-1 expression levels are not reduced in Aim2-/-;5XFAD mice. Unexpectedly, Il-6 and Il-18 expression levels in 5XFAD brains were further increased when Aim2 was deleted. Thus, inflammatory cytokine expression in 5XFAD brains is upregulated by Aim2 deletion through an unknown mechanism. CONCLUSION: Although Aim2 knockout mitigates Aß deposition and microglial activation, Aim2 deletion does not have a beneficial effect on the spatial memory or cytokine expression of 5XFAD mice. Our findings suggest that Aß aggregation and microglial activation may not always be correlated with the expression of inflammatory cytokines or cognitive function of 5XFAD mice. Our study also implies that different inflammasomes likely perform distinct roles in different physiological and/or pathological events.

Involvement of a BH3-only apoptosis sensitizer gene Blm-s in hippocampus-mediated mood control.

Mood disorders are an important public health issue and recent advances in genomic studies have indicated that molecules involved in neurodevelopment are causally related to mood disorders. BLM-s (BCL-2-like molecule, small transcript isoform), a BH3-only proapoptotic BCL-2 family member, mediates apoptosis of postmitotic immature neurons during embryonic cortical development, but its role in the adult brain is unknown. To better understand the physiological role of Blm-s gene in vivo, we generated a Blm-s-knockout (Blm-s-/-) mouse. The Blm-s-/- mice breed normally and exhibit grossly normal development. However, global depletion of Blm-s is highly associated with depression- and anxiety-related behaviors in adult mutant mice with intact learning and memory capacity. Functional magnetic resonance imaging of adult Blm-s-/- mice reveals reduced connectivity mainly in the ventral dentate gyrus (vDG) of the hippocampus with no alteration in the dorsal DG connectivity and in total hippocampal volume. At the cellular level, BLM-s is expressed in DG granule cells (GCs), and Blm-s-/- mice show reduced dendritic complexity and decreased spine density in mature GCs. Electrophysiology study uncovers that mature vGCs in adult Blm-s-/- DG are intrinsically more excitable. Interestingly, certain genetic variants of the human Blm homologue gene (VPS50) are significantly associated with depression traits from publicly resourced UK Biobank data. Taken together, BLM-s is required for the hippocampal mood control function. Loss of BLM-s causes abnormality in the electrophysiology and morphology of GCs and a disrupted vDG neural network, which could underlie Blm-s-null-associated anxiety and depression.

TLR7 and IL-6 differentially regulate the effects of rotarod exercise on the transcriptomic profile and neurogenesis to influence anxiety and memory.

Voluntary exercise is well known to benefit brain performance. In contrast, forced exercise induces inflammation-related stress responses and may cause psychiatric disorders. Here, we unexpectedly found that rotarod testing, a frequently applied assay for evaluating rodent motor coordination, induces anxiety and alters spatial learning/memory performance of mice. Rotarod testing upregulated genes involved in the unfolded protein response and stress responses and downregulated genes associated with neurogenesis and neuronal differentiation. It impacts two downstream pathways. The first is the IL-6-dependent pathway, which mediates rotarod-induced anxiety. The second is the Toll-like receptor 7 (TLR7)-dependent pathway, which is involved in the effect of rotarod exercise on gene expression and its impact on contextual learning and memory of mice. Thus, although rotarod exercise does not induce systemic inflammation, it influences innate immunity-related responses in the brain, controls gene expression and, consequently, regulates anxiety and contextual learning and memory.

Transcriptomic Analysis and C-Terminal Epitope Tagging Reveal Differential Processing and Signaling of Endogenous TLR3 and TLR7.

Toll-like receptor (TLR) signaling is critical for defense against pathogenic infection, as well as for modulating tissue development. Activation of different TLRs triggers common inflammatory responses such as cytokine induction. Here, we reveal differential impacts of TLR3 and TLR7 signaling on transcriptomic profiles in bone marrow-derived macrophages (BMDMs). Apart from self-regulation, TLR3, but not TLR7, induced expression of other TLRs, suggesting that TLR3 activation globally enhances innate immunity. Moreover, we observed diverse influences of TLR3 and TLR7 signaling on genes involved in methylation, caspase and autophagy pathways. We compared endogenous TLR3 and TLR7 by using CRISPR/Cas9 technology to knock in a dual Myc-HA tag at the 3' ends of mouse Tlr3 and Tlr7. Using anti-HA antibodies to detect endogenous tagged TLR3 and TLR7, we found that both TLRs display differential tissue expression and posttranslational modifications. C-terminal tagging did not impair TLR3 activity. However, it disrupted the interaction between TLR7 and myeloid differentiation primary response 88 (MYD88), the Tir domain-containing adaptor of TLR7, which blocked its downstream signaling necessary to trigger cytokine and chemokine expression. Our study demonstrates different properties for TLR3 and TLR7, and also provides useful mouse models for further investigation of these two RNA-sensing TLRs.

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.

RNase A Promotes Proliferation of Neuronal Progenitor Cells via an ERK-Dependent Pathway.

Members of the ribonuclease A (RNase A) superfamily regulate various physiological processes. RNase A, the best-studied member of the RNase A superfamily, is widely expressed in different tissues, including brains. We unexpectedly found that RNase A can trigger proliferation of neuronal progenitor cells (NPC) both in vitro and in vivo. RNase A treatment induced cell proliferation in dissociated neuronal cultures and increased cell mass in neurosphere cultures. BrdU (5-Bromo-2'-Deoxyuridine) labeling confirmed the effect of RNase A on cell proliferation. Those dividing cells were Nestin- and SOX2-positive, suggesting that RNase A triggers NPC proliferation. The proliferation inhibitor Ara-C completely suppressed the effect of RNase A on NPC counts, further supporting that RNase A increases NPC number mainly by promoting proliferation. Moreover, we found that RNase A treatment increased ERK phosphorylation and blockade of the ERK pathway inhibited the effect of RNase A on NPC proliferation. Intracerebroventricular injection of RNase A into mouse brain increased the population of 5-ethynyl-2'-deoxyuridine (EdU) or BrdU-labeled cells in the subventricular zone. Those RNase A-induced NPCs were able to migrate into other brain areas, including hippocampus, amygdala, cortex, striatum, and thalamus. In conclusion, our study shows that RNase A promotes proliferation of NPCs via an ERK-dependent pathway and further diversifies the physiological functions of the RNase A family.

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.