Hsin-Yu Liu

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.

Mitochondrial targeting of human NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2) and its association with early-onset hypertrophic cardiomyopathy and encephalopathy

BackgroundNADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), containing one iron sulfur cluster ([2Fe-2S] binuclear cluster N1a), is one of the core nuclear-encoded subunits existing in human mitochondrial complex I. Defects in this subunit have been associated with Parkinsons disease, Alzheimers disease, Bipolar disorder, and Schizophrenia. The aim of this study is to examine the mitochondrial targeting of NDUFV2 and dissect the pathogenetic mechanism of one human deletion mutation present in patients with early-onset hypertrophic cardiomyopathy and encephalopathy.MethodsA series of deletion and point-mutated constructs with the c-myc epitope tag were generated to identify the location and sequence features of mitochondrial targeting sequence for NDUFV2 in human cells using the confocal microscopy. In addition, various lengths of the NDUFV2 N-terminal and C-terminal fragments were fused with enhanced green fluorescent protein to investigate the minimal region required for correct mitochondrial import. Finally, a deletion construct that mimicked the IVS2+5_+8delGTAA mutation in NDUFV2 gene and would eventually produce a shortened NDUFV2 lacking 19-40 residues was generated to explore the connection between human gene mutation and disease.ResultsWe identified that the cleavage site of NDUFV2 was located around amino acid 32 of the precursor protein, and the first 22 residues of NDUFV2 were enough to function as an efficient mitochondrial targeting sequence to carry the passenger protein into mitochondria. A site-directed mutagenesis study showed that none of the single-point mutations derived from basic, hydroxylated and hydrophobic residues in the NDUFV2 presequence had a significant effect on mitochondrial targeting, while increasing number of mutations in basic and hydrophobic residues gradually decreased the mitochondrial import efficacy of the protein. The deletion mutant mimicking the human early-onset hypertrophic cardiomyopathy and encephalopathy lacked 19-40 residues in NDUFV2 and exhibited a significant reduction in its mitochondrial targeting ability.ConclusionsThe mitochondrial targeting sequence of NDUFV2 is located at the N-terminus of the precursor protein. Maintaining a net positive charge and an amphiphilic structure with the overall balance and distribution of basic and hydrophobic amino acids in the N-terminus of NDUFV2 is important for mitochondrial targeting. The results of human disease cell model established that the impairment of mitochondrial localization of NDUFV2 as a mechanistic basis for early-onset hypertrophic cardiomyopathy and encephalopathy.

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.

Neuronally-expressed Sarm1 regulates expression of inflammatory and antiviral cytokines in brains:

Sarm1 is the fifth Toll/IL-1 receptor (TIR) domain-containing adaptor protein identified to regulate TLR downstream signaling. Unlike the other TIR domain-containing adaptor proteins, Sarm1 is predominantly expressed in the brain. Our previous study indicated that Sarm1 regulates dendritic growth, axonal extension and neuronal polarity. Here, we investigated whether Sarm1 is involved in innate immunity in the brain. First, regional and cell-type distribution of Sarm1 in mouse brains was revealed using double immunostaining. Sarm1 was widely distributed in different regions of brains, including the cerebral cortex, hippocampus, amygdala, cerebellum and midbrain. Moreover, Sarm1 is present in both projection and inhibitory neurons, but, interestingly, not in microglial cells—the main immune cells in the brain. These results suggest that Sarm1 is unlikely to regulate microglial activity in a cell-autonomous manner. However, compared with wild type littermates, the RNA expression levels of several inflammatory and antiviral cytokines were altered in the embryonic and adult brains of Sarm1 knockdown transgenic mice. These data imply that Sarm1 influences cytokine expression in neurons. In conclusion, our findings suggest that Sarm1 regulates the innate immune responses of the central nervous system through regulating the inflammatory and anti-virus cytokines produced by neurons.

Innate immune responses regulate morphogenesis and degeneration: roles of Toll-like receptors and Sarm1 in neurons

The central nervous system is recognized as an immunoprivileged site because peripheral immune cells do not typically enter it. Microglial cells are thought to be the main immune cells in brain. However, recent reports have indicated that neurons express the key players of innate immunity, including Toll-like receptors (TLRs) and their adaptor proteins (Sarm1, Myd88, and Trif), and may produce cytokines in response to pathogen infection. In the absence of an immune challenge, neuronal TLRs can detect intrinsic danger signals and modulate neuronal morphology and function. In this article, we review the recent findings on the involvement of TLRs and Sarm1 in controlling neuronal morphogenesis and neurodegeneration. Abnormal behaviors in TLR- and Sarm1-deficient mice are also discussed.

TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology.

Viral infection during fetal or neonatal stages increases the risk of developing neuropsychiatric disorders such as schizophrenia and autism spectrum disorders. Although neurons express several key regulators of innate immunity, the role of neuronal innate immunity in psychiatric disorders is still unclear. Using cultured neurons and in vivo mouse brain studies, we show here that Toll‐like receptor 3 (TLR3) acts through myeloid differentiation primary response gene 88 (MYD88) to negatively control Disrupted in schizophrenia 1 (Disc1) expression, resulting in impairment of neuronal development. Cytokines are not involved in TLR3‐mediated inhibition of dendrite outgrowth. Instead, TLR3 signaling suppresses expression of several psychiatric disorder‐related genes, including Disc1. The impaired dendritic arborization caused by TLR3 activation is rescued by MYD88 deficiency or DISC1 overexpression. In addition, TLR3 activation at the neonatal stage increases dendritic spine density, but narrows spine heads at postnatal day 21 (P21), suggesting a long‐lasting effect of TLR3 activation on spinogenesis. Our study reveals a novel mechanism of TLR3 in regulation of dendritic morphology and provides an explanation for how environmental factors influence mental health.

Deletion of the Inflammasome Sensor Aim2 Mitigates Aβ 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 Aβ 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 Aβ 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 Aβ 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.

Zebrafish muscleblind-like genes: Identification, structural features and expression

Muscleblind-like (MBNL) proteins are a family of RNA-binding proteins that participate in the regulation of tissue-specific alternative splicing. Misregulation of MBNL activity in humans leads to pathogenesis. Here, we report upon the identification and characterization of three muscleblind-like genes in zebrafish (zmbnl1, zmbnl2 and zmbnl3). Alternative splicing of the three zmbnl primary transcripts gives rise to at least four protein isoforms for zmbnl1, four for zmbnl2 and five for zmbnl3, respectively. All of the zmbnl proteins contain the characteristic CCCH zinc fingers required for RNA binding. In addition, several structural motifs, including a C-terminal Ser/Thr-rich region, are conserved among Mbnl orthologs in vertebrates, but not invertebrates. These genes are broadly expressed in most adult tissues. However, the relative expression levels of specific spliceforms vary across different tissues. During embryogenesis, zmbnl1 and zmbnl2 are both maternally and zygotically expressed. In contrast, zmbnl3 transcripts are not detected until the late pharyngula stage. Our results reveal the expression pattern of various mbnl spliceforms for the first time and suggest that they may play specific roles during fish development.

AIM 2 inflammasomes regulate neuronal morphology and influence anxiety and memory in mice

Inflammasomes are the protein assemblies that consist of inflammasome sensors, adaptor apoptosis-associated speck-like proteins containing a CARD (ASC) and inflammasome caspase. Inflammasomes sense multiple danger signals via various inflammasome sensors and consequently use caspase to trigger proteolytic processing and secretion of IL-1β cytokines. Recent studies have suggested that neurons use their own innate immune system to detect danger signals and regulate neuronal morphology. Here, we investigate whether inflammasomes, the critical components of innate immunity, participate in regulation of neuronal morphology and function. Among various sensors, Absent in melanoma 2 (Aim2) expression in neurons is most prominent. Adding synthetic double-stranded DNA (dsDNA) to cultured neurons induces IL-1β secretion in an AIM2-dependent manner and consequently downregulates dendritic growth but enhances axon extension. The results of Aim2 knockout and knockdown show that AIM2 acts cell-autonomously to regulate neuronal morphology. Behavioral analyses further reveal that Aim2−/− mice exhibit lower locomotor activity, increased anxious behaviors and reduced auditory fear memory. In conclusion, our study suggests that AIM2 inflammasomes regulate neuronal morphology and influence mouse behaviors.

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.