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Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast Saccharomyces cerevisiae (Sc) Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus Trichoderma reesei to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity. We applied multidisciplinary approaches (next- and third-generation sequencing technology, genetics, cytology, bioinformatics, biochemistry, and single-molecule biophysics) to show that T. reesei Rad51 (TrRad51) is indispensable for interhomolog recombination during meiosis and, like ScDmc1, TrRad51 possesses better mismatch tolerance than ScRad51 during homologous recombination. Our results also indicate that the ancestral TrRad51 evolved to acquire ScDmc1-like properties by creating multiple structural variations, including via amino acid residues in the L1 and L2 DNA-binding loops.
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Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Recombinación Homóloga , Hypocreales/metabolismo , Meiosis , Recombinasa Rad51/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , ADN de Cadena Simple , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Hypocreales/genética , Recombinasa Rad51/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
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Encéfalo/fisiología , Inmunidad Innata/genética , Neurogénesis , Neuronas/fisiología , Transducción de Señal/genética , Receptores Toll-Like/genética , Animales , Humanos , RatonesRESUMEN
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.
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Regulación de la Expresión Génica , Factor 88 de Diferenciación Mieloide/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Receptor Toll-Like 3/metabolismo , Animales , Biomarcadores , Línea Celular , Células Cultivadas , Citocinas/metabolismo , Dendritas/genética , Dendritas/metabolismo , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Noqueados , Morfogénesis/genética , Factor 88 de Diferenciación Mieloide/genética , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal , Receptor Toll-Like 3/genéticaRESUMEN
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.
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Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Memoria/fisiología , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Agresión/fisiología , Animales , Ansiedad/metabolismo , Conducta Animal/fisiología , Depresión/metabolismo , Miedo/fisiología , Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurogénesis , Neuroglía/fisiología , Neuronas/fisiología , Olfato/genética , Olfato/fisiología , TranscriptomaRESUMEN
The efficacy of thyroxine suppressive therapy in reducing nodular growth and its effect to bone mineral density (BMD) in postmenopausal women is still debated. This study aimed to evaluate the therapeutic effect of thyroxine and its influence on BMD. Postmenopausal women with nodular or multinodular goiter during 2013-2015 at Chang Gung Memorial Hospital were enrolled and retrospectively traced back to the first date of visit or treatment. Ninety-four eligible patients were enrolled, of whom 45 were thyroxine-treated (LT-4 group) and 49 were treatment-naïve (control group). Data, including volume of nodules, were analyzed retrospectively. BMD was measured in each LT-4 group patient since the year of enrollment. Nodular volumes were reduced in both LT-4 (from 4.89 ± 4.46 to 4.10 ± 4.57 mL, p = 0.033) and control group (3.48 ± 4.36 to 3.09 ± 2.88 mL, p = 0.239) at initial 2-year follow-up. Nodular volume in LT-4 group increased insignificantly (from 4.89 ± 4.46 to 4.91 ± 5.40 mL, p = 0.711) at the end of 7-year follow-up. The best cut-off predictive nodular volume that may have responded to thyroxine is 2.6 mL (AUC, 0.740; sensitivity, 0.750; specificity, 0.733) during first 2 year. Lumbar spine, total hip and femoral neck BMD were not significantly changed during 2 year's thyroxine suppression therapy. In conclusion, thyroxine suppressive therapy in postmenopausal women had significant reduction in nodule volume at initial 2 years of treatment, especially in volume larger than 2.6 mL. Prolonged thyroxine treatment did not benefit nodular size reduction and may affect BMD minimally in postmenopausal women.
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Densidad Ósea/efectos de los fármacos , Bocio Nodular/tratamiento farmacológico , Tiroxina/uso terapéutico , Absorciometría de Fotón , Anciano , Femenino , Cuello Femoral/diagnóstico por imagen , Cuello Femoral/efectos de los fármacos , Humanos , Vértebras Lumbares/diagnóstico por imagen , Vértebras Lumbares/efectos de los fármacos , Persona de Mediana Edad , Posmenopausia , Estudios Retrospectivos , Tiroxina/farmacología , Resultado del TratamientoRESUMEN
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 â¼2 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.
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Dendritas/fisiología , Regulación hacia Abajo/fisiología , Inhibidores de Crecimiento/fisiología , Interleucina-6/fisiología , Glicoproteínas de Membrana/fisiología , Factor 88 de Diferenciación Mieloide/fisiología , Proteínas Proto-Oncogénicas c-fos/fisiología , Transducción de Señal/fisiología , Receptor Toll-Like 7/fisiología , Animales , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , EmbarazoRESUMEN
Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.
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Dictyostelium , Drosophila melanogaster , Animales , Ratones , Codón de Terminación/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Dictyostelium/genética , Proteínas Fúngicas/metabolismo , Glutamina/metabolismoRESUMEN
Efficiently delivering exogenous materials into primary neurons and neural stem cells (NSCs) has long been a challenge in neurobiology. Existing methods have struggled with complex protocols, unreliable reproducibility, high immunogenicity, and cytotoxicity, causing a huge conundrum and hindering in-depth analyses. Here, we establish a cutting-edge method for transfecting primary neurons and NSCs, named teleofection, by a two-step process to enhance the formation of biocompatible calcium phosphate (CaP) nanoparticles. Teleofection enables both nucleic acid and protein transfection into primary neurons and NSCs, eliminating the need for specialized skills and equipment. It can easily fine-tune transfection efficiency by adjusting the incubation time and nanoparticle quantity, catering to various experimental requirements. Teleofection's versatility allows for the delivery of different cargos into the same cell culture, whether simultaneously or sequentially. This flexibility proves invaluable for long-term studies, enabling the monitoring of neural development and synapse plasticity. Moreover, teleofection ensures the consistent and robust expression of delivered genes, facilitating molecular and biochemical investigations. Teleofection represents a significant advancement in neurobiology, which has promise to transcend the limitations of current gene delivery methods. It offers a user-friendly, cost-effective, and reproducible approach for researchers, potentially revolutionizing our understanding of brain function and development.
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Nanopartículas , Células-Madre Neurales , Ácidos Nucleicos , Ácidos Nucleicos/metabolismo , Reproducibilidad de los Resultados , Células-Madre Neurales/metabolismo , Nanopartículas/química , Transfección , Fosfatos de Calcio/químicaRESUMEN
Since SARM1 mutations have been identified in human neurological disease, SARM1 inhibition has become an attractive therapeutic strategy to preserve axons in a variety of disorders of the peripheral (PNS) and central nervous system (CNS). While SARM1 has been extensively studied in neurons, it remains unknown whether SARM1 is present and functional in myelinating glia? This is an important question to address. Firstly, to identify whether SARM1 dysfunction in other cell types in the nervous system may contribute to neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether therapies altering SARM1 function may have unintended deleterious impacts on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore, activation of endogenous SARM1 in cultured oligodendrocytes induces rapid cell death. In contrast, in peripheral glia, SARM1 protein is not detectable in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is detected at very low levels in Schwann cells, in vivo, in zebrafish and mouse. Application of specific SARM1 activators to cultured mouse Schwann cells does not induce cell death and nicotinamide adenine dinucleotide (NAD) levels remain unaltered suggesting Schwann cells likely contain no functionally relevant levels of SARM1. Finally, we address the question of whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for initiation of myelination and myelin sheath maintenance is unaffected in the adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat neurological disease are unlikely to perturb myelination in humans.
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Type I interferons are important antiviral cytokines, but prolonged interferon production is detrimental to the host. The TLR3-driven immune response is crucial for mammalian antiviral immunity, and its intracellular localization determines induction of type I interferons; however, the mechanism terminating TLR3 signaling remains obscure. Here, we show that the E3 ubiquitin ligase ZNRF1 controls TLR3 sorting into multivesicular bodies/lysosomes to terminate signaling and type I interferon production. Mechanistically, c-Src kinase activated by TLR3 engagement phosphorylates ZNRF1 at tyrosine 103, which mediates K63-linked ubiquitination of TLR3 at lysine 813 and promotes TLR3 lysosomal trafficking and degradation. ZNRF1-deficient mice and cells are resistant to infection by encephalomyocarditis virus and SARS-CoV-2 because of enhanced type I interferon production. However, Znrf1-/- mice have exacerbated lung barrier damage triggered by antiviral immunity, leading to enhanced susceptibility to respiratory bacterial superinfections. Our study highlights the c-Src-ZNRF1 axis as a negative feedback mechanism controlling TLR3 trafficking and the termination of TLR3 signaling.
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COVID-19 , Interferón Tipo I , Animales , Ratones , Antivirales , SARS-CoV-2 , Receptor Toll-Like 3 , Genes srcRESUMEN
Cancer patients frequently develop chemotherapy-induced peripheral neuropathy (CIPN), a painful and long-lasting disorder with profound somatosensory deficits. There are no effective therapies to prevent or treat this disorder. Pathologically, CIPN is characterized by a "dying-back" axonopathy that begins at intra-epidermal nerve terminals of sensory neurons and progresses in a retrograde fashion. Calcium dysregulation constitutes a critical event in CIPN, but it is not known how chemotherapies such as paclitaxel alter intra-axonal calcium and cause degeneration. Here, we demonstrate that paclitaxel triggers Sarm1-dependent cADPR production in distal axons, promoting intra-axonal calcium flux from both intracellular and extracellular calcium stores. Genetic or pharmacologic antagonists of cADPR signaling prevent paclitaxel-induced axon degeneration and allodynia symptoms, without mitigating the anti-neoplastic efficacy of paclitaxel. Our data demonstrate that cADPR is a calcium-modulating factor that promotes paclitaxel-induced axon degeneration and suggest that targeting cADPR signaling provides a potential therapeutic approach for treating paclitaxel-induced peripheral neuropathy (PIPN).
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Proteínas del Dominio Armadillo/metabolismo , Axones/metabolismo , Calcio/metabolismo , ADP-Ribosa Cíclica/metabolismo , Proteínas del Citoesqueleto/metabolismo , Degeneración Nerviosa/patología , Paclitaxel/efectos adversos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/metabolismo , Animales , Canales de Calcio/metabolismo , ADP-Ribosa Cíclica/antagonistas & inhibidores , Femenino , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratas Sprague-DawleyRESUMEN
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.
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Epítopos/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/fisiología , Neuronas/metabolismo , Receptor Toll-Like 3/fisiología , Receptor Toll-Like 7/fisiología , Animales , Quimiocinas/metabolismo , Citocinas/metabolismo , Epítopos/inmunología , Femenino , Perfilación de la Expresión Génica , Inmunidad Innata , Macrófagos/inmunología , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/fisiología , Transducción de Señal , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismoRESUMEN
While neuronal activity regulates neurite outgrowth and branching, the details of the underlying mechanisms are still largely unclear. This study investigated the effect of the glutamate receptors on neuritogenesis using cultured hippocampal neurons. At 3-4 days in vitro, NMDA treatment promoted axon branching but not primary axon extension. In contrast, blockade of the NMDA receptor (NMDAR) by AP5 treatment enhanced primary axon extension. NMDAR activation also increased dendrite number and total dendrite length. These results suggest that NMDAR controls axon and dendrite morphogenesis. A previous study demonstrated that knockdown of the zinc finger transcription factor B cell lymphoma 11A-long (Bcl11A-L) reduces deleted in colorectal cancer (DCC) and microtubule-associated protein (MAP) 1b expression, thereby promoting axon branching. Here, glutamate stimulation down-regulated the levels of the Bcl11A-L, DCC, MAP1b, and MAP2c proteins. Over-expression of either Bcl11A-L or DCC countered the effect of NMDA or glutamate on axon branching and dendrite outgrowth, indicating that the Bcl11A-L/DCC pathway is an important downstream effector of glutamate receptors in neurite arborization. Because knockdown of Bcl11A-L did not down-regulate MAP2c, our results suggest that glutamate receptors also use a Bcl11A-L-independent pathway to control dendrite outgrowth. To summarize, this study reveals novel pathways downstream of glutamate receptors that regulate axon and dendrite arborization.
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Axones/fisiología , Proteínas Portadoras/fisiología , Dendritas/fisiología , Proteínas Nucleares/fisiología , Receptores de Glutamato/fisiología , Dedos de Zinc/fisiología , Animales , Aumento de la Célula , Células Cultivadas , Receptor DCC , Ácido Glutámico/farmacología , N-Metilaspartato/fisiología , Ratas , Receptores de Superficie Celular/fisiología , Proteínas Represoras , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
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.
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Endosomas/metabolismo , Glicoproteínas de Membrana/fisiología , Neuronas/metabolismo , Receptor Toll-Like 3/fisiología , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 8/fisiología , Animales , Aumento de la Célula , Electroporación , Endosomas/genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/fisiología , Plasticidad Neuronal , Neuronas/ultraestructura , Transducción de Señal , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/genética , Receptor Toll-Like 8/metabolismoRESUMEN
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.
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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.
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Proteínas del Dominio Armadillo/metabolismo , Proteínas del Citoesqueleto/metabolismo , Inmunidad Innata , Morfogénesis/inmunología , Neuronas/inmunología , Receptores Toll-Like/metabolismo , Animales , Encéfalo/inmunología , Humanos , Ratones , Microglía/inmunología , Neuronas/patología , Transducción de SeñalRESUMEN
Innate immune responses have been shown to influence brain development and function. Dysregulation of innate immunity is significantly associated with psychiatric disorders such as autism spectrum disorders and schizophrenia, which are well-known neurodevelopmental disorders. Recent studies have revealed that critical players of the innate immune response are expressed in neuronal tissues and regulate neuronal function and activity. For example, Sarm1, a negative regulator that acts downstream of Toll-like receptor (TLR) 3 and 4, is predominantly expressed in neurons. We have previously shown that Sarm1 regulates neuronal morphogenesis and the expression of inflammatory cytokines in the brain, which then affects learning ability, cognitive flexibility, and social interaction. Because impaired neuronal morphogenesis and dysregulation of cytokine expression may disrupt neuronal activity, we investigated whether Sarm1 knockdown affects the synaptic responses of neurons. We here show that reduced Sarm1 expression impairs metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) formation but enhances N-methyl-D-aspartate receptor (NMDAR)-dependent long-term potentiation production in hippocampal CA1 neurons. The expression levels of post-synaptic proteins, including NR2a, NR1, Shank1 and Shank3, are also altered in Sarm1 knockdown mice, suggesting a role for Sarm1 in the maintenance of synaptic homeostasis. The addition of a positive allosteric modulator of mGluR5, CDPPB, ameliorates the LTD defects in slice recording and the behavioral deficits in social interaction and associative memory. These results suggest an important role for mGluR5 signaling in the function of Sarm1. In conclusion, our study demonstrates a role for Sarm1 in the regulation of synaptic plasticity. Through these mechanisms, Sarm1 knockdown results in the impairment of associative memory and social interactions in mice.
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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.
Asunto(s)
Proteínas del Dominio Armadillo/metabolismo , Encéfalo/inmunología , Citocinas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Mediadores de Inflamación/metabolismo , Neuronas/fisiología , Virosis/inmunología , Animales , Antígenos Virales/inmunología , Proteínas del Dominio Armadillo/genética , Encéfalo/patología , Células Cultivadas , Citocinas/genética , Proteínas del Citoesqueleto/genética , Embrión de Mamíferos , Regulación de la Expresión Génica/genética , Inmunidad Innata/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroinmunomodulación , ARN/análisis , TranscriptomaRESUMEN
The neuron-specific transcription factor T-box brain 1 (TBR1) regulates brain development. Disruptive mutations in the TBR1 gene have been repeatedly identified in patients with autism spectrum disorders (ASDs). Here, we show that Tbr1 haploinsufficiency results in defective axonal projections of amygdalar neurons and the impairment of social interaction, ultrasonic vocalization, associative memory and cognitive flexibility in mice. Loss of a copy of the Tbr1 gene altered the expression of Ntng1, Cntn2 and Cdh8 and reduced both inter- and intra-amygdalar connections. These developmental defects likely impair neuronal activation upon behavioral stimulation, which is indicated by fewer c-FOS-positive neurons and lack of GRIN2B induction in Tbr1(+/-) amygdalae. We also show that upregulation of amygdalar neuronal activity by local infusion of a partial NMDA receptor agonist, d-cycloserine, ameliorates the behavioral defects of Tbr1(+/-) mice. Our study suggests that TBR1 is important in the regulation of amygdalar axonal connections and cognition.
Asunto(s)
Amígdala del Cerebelo/patología , Axones/patología , Trastornos del Conocimiento/genética , Trastornos del Conocimiento/patología , Proteínas de Unión al ADN/deficiencia , Animales , Antimetabolitos/uso terapéutico , Axones/metabolismo , Cadherinas/metabolismo , Trastornos del Conocimiento/tratamiento farmacológico , Contactina 2/metabolismo , Cicloserina/uso terapéutico , Modelos Animales de Enfermedad , Conducta Exploratoria/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Factores de Transcripción MEF2/metabolismo , Imagen por Resonancia Magnética , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Netrinas , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas de Dominio T BoxRESUMEN
Cortactin-binding protein 2 (CTTNBP2) interacts with cortactin to regulate cortactin mobility and control dendritic spine formation. CTTNBP2 has also been associated with autistic spectrum disorder. The regulation of dendritic spinogenesis could explain the association of CTTNBP2 with autism. Sequence comparison has indicated that CTTNBP2 N-terminal-like protein (CTTNBP2NL) is a CTTNBP2 homologue. To confirm the specific effect of CTTNBP2 on dendritic spinogenesis, here we investigate whether CTTNBP2NL has a similar function to CTTNBP2. Although both CTTNBP2 and CTTNBP2NL interact with cortactin, CTTNBP2NL is associated with stress fibers, whereas CTTNBP2 is distributed to the cortex and intracellular puncta. We also provide evidence that CTTNBP2, but not CTTNBP2NL, is predominantly expressed in the brain. CTTNBP2NL does not show any activity in the regulation of dendritic spinogenesis. In addition to spine morphology, CTTNBP2 is also found to regulate the synaptic distribution of striatin and zinedin (the regulatory B subunits of protein phosphatase 2A [PP2A]), which interact with CTTNBP2NL in HEK293 cells. The association between CTTNBP2 and striatin/zinedin suggests that CTTNBP2 targets the PP2A complex to dendritic spines. Thus we propose that the interactions of CTTNBP2 and cortactin and the PP2A complex regulate spine morphogenesis and synaptic signaling.