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

Highlights

• TLR7 regulates expression of genes critical for neuronal and glial development and neural activity.

• TLR7 downstream genes are relevant to neurological and psychological diseases.

• Tlr7 deletion reduces anxiety and aggression but sharpens olfaction.

• Tlr7 deletion impairs contextual fear and long-term potentiation of hippocampus.

Abstract

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.

Introduction

The innate immune system acts as an alarm sensor to detect various pattern molecules and consequently induces cellular responses to these molecules. The pattern molecules include danger signals (such as foreign pathogens) as well as damage signals (such as intracellular molecules released from dead cells) (Czirr and Wyss-Coray, 2012). Toll-like receptors are the best-studied receptors for pattern molecule recognition (Kawai and Akira, 2010, Kawai and Akira, 2011). In addition to inducing innate immune responses, the original finding showed that the Drosophila Toll gene, the prototype of Toll-like receptors, controls dorso-ventral patterning during embryogenesis (Anderson et al., 1985, Hashimoto et al., 1988). Recent study also demonstrated that Drosophila Toll-6 and Toll-7 control wiring specificity of Drosophila olfactory circuit assembly (Ward et al., 2015). Accumulated studies evidence a role for Drosophila Tolls in development.

In the mammalian nervous system, endosomal TLR3, TLR7 and TLR8 and AIM2 inflammasomes are expressed in neurons and are involved in differential recognition of nucleic acids (Chen et al., 2017, Hung et al., 2018, Liu et al., 2013, Liu et al., 2015, Wu et al., 2017, Wu et al., 2016). For instance, both TLR7 and TLR8 recognize single-stranded RNA (ssRNA). TLR7 can also bind several different uridine-rich miRNAs (such as Let-7, miR-21 and miR-29a) released from other cells through exosomes (Fabbri et al., 2012, Lehmann et al., 2012, Liu et al., 2015). TLR3 is specific for double-stranded RNA (dsRNA) and can recognize heterologous RNAs released from necrotic cells (Kariko et al., 2004). In the absence of infection, injury-induced acute inflammation requires TLR3 (Cavassani et al., 2008), further supporting a role for TLR3 in recognition of self nucleic acids. Since TLR3 and TLR7 are localized at endosomes to recognize their ligands, they are also able to act as sensors of intraneuronal distress by detecting nucleic acids derived from autophagosomes in the same cells (Czirr and Wyss-Coray, 2012). Thus, dying cells, exosomes and autophagosomes are sources of endogenous ligands that can activate TLR3 and TLR7. Consequently, in the absence of exogenous pathogens, TLR3, TLR7 and TLR8 fine-tune neuronal morphology by detecting endogenous ligands during neuronal development or under intrinsic stress conditions. Reduced expression or deletion of Tlr3, Tlr7 and Tlr8 genes in neurons is sufficient to promote dendritic extension of neurons (Chen et al., 2017, Hung et al., 2018, Liu et al., 2013, Liu et al., 2015). Therefore, deletion of Tlr3, Tlr7 or Tlr8 is expected to impact brain function and activity.

Indeed, Tlr3 deletion in mice alters hippocampus- and amygdala-dependent memory. Tlr3 KO mice performed better in Morris water maze, novel object recognition and contextual fear conditioning assays, which are hippocampus-associated behavioral paradigms. However, amygdala-dependent behaviors, including anxiety, were impaired in these Tlr3 KO mice (Okun et al., 2010). Although it is unclear why Tlr3 deletion has such contrasting effects on hippocampus- and amygdala-dependent behaviors, these behavioral assays support the hypothesis that TLR3 is required for brain function.

TLR7 has been proven to sense endogenous ssRNA, and TLR7 activation restricts axonal and dendritic growth of neurons (Liu et al., 2013, Liu et al., 2015). Tlr7 knockdown via in utero electroporation at embryonic day 15.5 results in more complex dendritic arbors of cortical neurons at P7 and P14 but not P21 (Liu et al., 2013), suggesting that neuronal morphology is more sensitive to Tlr7 deletion at earlier developmental stages. To further dissect the role of TLR7 in the nervous system, in this report, we first employed next generation sequencing to investigate the roles of TLR7 in young and mature cultured neurons. A series of behavioral paradigms were then applied to analyze the impact of Tlr7 deletion on brain functions. Our study suggests that young and mature neurons exhibit different transcriptomic profiles upon Tlr7 deletion, which may influence neuronal function and regulate mouse behaviors. Our results strengthen the evidence for a crucial role of TLR7 in brain function.