RESUMO
Human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Though the underlying mechanisms remain poorly characterized, mouse models of congenital CMV infection have demonstrated that the neuronal migration process is damaged. In this study, we evaluated the effects of HCMV infection on connexin 43 (Cx43), a crucial adhesion molecule mediating neuronal migration. We show in multiple cellular models that HCMV infection downregulated Cx43 posttranslationally. Further analysis identified the immediate early protein IE1 as the viral protein responsible for the reduction of Cx43. IE1 was found to bind the Cx43 C terminus and promote Cx43 degradation through the ubiquitin-proteasome pathway. Deletion of the Cx43-binding site in IE1 rendered it incapable of inducing Cx43 degradation. We validated the IE1-induced loss of Cx43 in vivo by introducing IE1 into the fetal mouse brain. Noteworthily, ectopic IE1 expression induced cortical atrophy and neuronal migration defects. Several lines of evidence suggest that these damages result from decreased Cx43, and restoration of Cx43 levels partially rescued IE1-induced interruption of neuronal migration. Taken together, the results of our investigation reveal a novel mechanism of HCMV-induced neural maldevelopment and identify a potential intervention target. IMPORTANCE Congenital CMV (cCMV) infection causes neurological sequelae in newborns. Recent studies of cCMV pathogenesis in animal models reveal ventriculomegaly and cortical atrophy associated with impaired neural progenitor cell (NPC) proliferation and migration. In this study, we investigated the mechanisms underlying these NPC abnormalities. We show that Cx43, a critical adhesion molecule mediating NPC migration, is downregulated by HCMV infection in vitro and HCMV-IE1 in vivo. We provide evidence that IE1 interacts with the C terminus of Cx43 to promote its ubiquitination and consequent degradation through the proteasome. Moreover, we demonstrate that introducing IE1 into mouse fetal brains led to neuronal migration defects, which was associated with Cx43 reduction. Deletion of the Cx43-binding region in IE1 or ectopic expression of Cx43 rescued the IE1-induced migration defects in vivo. Our study provides insight into how cCMV infection impairs neuronal migration and reveals a target for therapeutic interventions.
Assuntos
Conexina 43 , Infecções por Citomegalovirus , Citomegalovirus , Proteínas Imediatamente Precoces , Animais , Humanos , Recém-Nascido , Camundongos , Conexina 43/genética , Conexina 43/metabolismo , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Congenital human cytomegalovirus (HCMV) infection causes severe damage to the fetal brain, and the underlying mechanisms remain elusive. Cytokine signaling is delicately controlled in the fetal central nervous system to ensure proper development. Here we show that suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the IL-6 cytokine family signaling, was upregulated during HCMV infection in primary neural progenitor cells (NPCs) with a biphasic expression pattern. From viral protein screening, pUL97 emerged as the viral factor responsible for prolonged SOCS3 upregulation. Further, by proteomic analysis of the pUL97-interacting host proteins, regulatory factor X 7 (RFX7) was identified as the transcription factor responsible for the regulation. Depletion of either pUL97 or RFX7 prevented the HCMV-induced SOCS3 upregulation in NPCs. With a promoter-luciferase activity assay, we demonstrated that the pUL97 kinase activity and RFX7 were required for SOCS3 upregulation. Moreover, the RFX7 phosphorylation level was increased by either UL97-expressing or HCMV-infection in NPCs, suggesting that pUL97 induces RFX7 phosphorylation to drive SOCS3 transcription. We further revealed that elevated SOCS3 expression impaired NPC proliferation and migration in vitro and caused NPCs migration defects in vivo. Taken together, these findings uncover a novel regulatory mechanism of sustained SOCS3 expression in HCMV-infected NPCs, which perturbs IL-6 cytokine family signaling, leads to NPCs proliferation and migration defects, and consequently affects fetal brain development.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Humanos , Citomegalovirus/fisiologia , Interleucina-6/metabolismo , Proteômica , Fatores de Transcrição/metabolismo , Células-Tronco , Proteína 3 Supressora da Sinalização de Citocinas/metabolismoRESUMO
The lateral hypothalamus (LH) is an important brain region mediating sleep-wake behavior. Recent evidence has shown that astrocytes in central nervous system modulate the activity of adjacent neurons and participate in several physiological functions. However, the role of LH astrocytes in sleep-wake regulation remains unclear. Here, using synchronous recording of electroencephalogram/electromyogram in mice and calcium signals in LH astrocytes, we show that the activity of LH astrocytes is significantly increased during non-rapid eye movement (NREM) sleep-to-wake transitions and decreased during Wake-to-NREM sleep transitions. Chemogenetic activation of LH astrocytes potently promotes wakefulness and maintains long-term arousal, while chemogenetic inhibition of LH astrocytes decreases the total amount of wakefulness in mice. Moreover, by combining chemogenetics with fiber photometry, we show that activation of LH astrocytes significantly increases the calcium signals of adjacent neurons, especially among GABAergic neurons. Taken together, our results clearly illustrate that LH astrocytes are a key neural substrate regulating wakefulness and encode this behavior through surrounding GABAergic neurons. Our findings raise the possibility that overactivity of LH astrocytes may be an underlying mechanism of clinical sleep disorders.
Assuntos
Região Hipotalâmica Lateral , Vigília , Animais , Camundongos , Vigília/fisiologia , Região Hipotalâmica Lateral/fisiologia , Astrócitos , Cálcio , Sono/fisiologia , Neurônios GABAérgicos/fisiologia , HipotálamoRESUMO
The dorsal raphe nucleus (DRN) has previously been proved to be involved in the regulation of the sleep-wake behavior. DRN contains several neuron types, such as 5-HTergic and GABAergic neurons. GABAergic neurons, which are the second largest cell subtype in the DRN, participate in a variety of neurophysiological functions. However, their role in sleep-wake regulation and the underlying neural circuitry remains unclear. Herein, we used fiber photometry and synchronous electroencephalogram (EEG)/electromyography (EMG) recording to demonstrate that DRN GABAergic neurons exhibit high activities during wakefulness and low activities during NREM sleep. Short-term optogenetic activation of DRN GABAergic neurons reduced the latency of NREM-to-wake transition and increased the probability of wakefulness, while long-term optogenetic activation of these neurons significantly increased the amount of wakefulness. Chemogenetic activation of DRN GABAergic neurons increased wakefulness for almost 2 h and maintained long-lasting arousal. In addition, inhibition of DRN GABAergic neurons with chemogenetics caused a reduction in the amount of wakefulness. Finally, similar to the effects of activating the soma of DRN GABAergic neurons, optogenetic stimulation of their terminals in the ventral tegmental area (VTA) induced instant arousal and promoted wakefulness. Taken together, our results illustrated that DRN GABAergic neurons are vital to the induction and maintenance of wakefulness, which promote wakefulness through the GABAergic DRN-VTA pathway.
Assuntos
Núcleo Dorsal da Rafe , Área Tegmentar Ventral , Área Tegmentar Ventral/metabolismo , Vigília/fisiologia , Sono/fisiologia , Neurônios GABAérgicos/fisiologiaRESUMO
During the long coevolution of human cytomegalovirus (HCMV) and humans, the host has formed a defense system of multiple layers to eradicate the invader, and the virus has developed various strategies to evade host surveillance programs. The intrinsic immunity primarily orchestrated by promyelocytic leukemia (PML) nuclear bodies (PML-NBs) represents the first line of defense against HCMV infection. Here, we demonstrate that microrchidia family CW-type zinc finger 3 (MORC3), a PML-NBs component, is a restriction factor targeting HCMV infection. We show that depletion of MORC3 through knockdown by RNA interference or knockout by CRISPR-Cas9 augmented immediate-early protein 1 (IE1) gene expression and subsequent viral replication, and overexpressing MORC3 inhibited HCMV replication by suppressing IE1 gene expression. To relief the restriction, HCMV induces transient reduction of MORC3 protein level via the ubiquitin-proteasome pathway during the immediate-early to early stage. However, MORC3 transcription is upregulated, and the protein level recovers in the late stages. Further analyses with temporal-controlled MORC3 expression and the major immediate-early promoter (MIEP)-based reporters show that MORC3 suppresses MIEP activity and consequent IE1 expression with the assistance of PML. Taken together, our data reveal that HCMV enforces temporary loss of MORC3 to evade its repression against the initiation of immediate-early gene expression.
Assuntos
Infecções por Citomegalovirus , Proteínas Imediatamente Precoces , Adenosina Trifosfatases/metabolismo , Citomegalovirus/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Replicação ViralRESUMO
Aflatoxin M1 (AFM1) and ochratoxin A (OTA), which are occasionally detected in milk and commercial baby foods, could easily enter and reach the gastrointestinal tract, posing impairment to the first line of defense and causing dysfunction of the tissue. The objective of this study was to investigate the immunostimulatory roles of individual and combined AFM1 and OTA on the immature intestine. Thus, we used ELISA assays to evaluate the generation of cytokines from ex vivo CD-1 fetal mouse jejunum induced by AFM1 and OTA and explored the related regulatory pathways and pivot genes using RNA-seq analysis. It was found that OTA exhibited much stronger ability in stimulating pro-inflammatory cytokine IL-6 from jejunum tissues than AFM1 (OTA of 4 µM versus AFM1 of 50 µM), whereas the combination of the two toxins seemed to exert antagonistic actions. In addition, transcriptomics also showed that most gene members in the enriched pathway 'cytokine-cytokine receptor interaction' were more highly expressed in OTA than the AFM1 group. By means of PPI network analysis, NFKB1 and RelB were regarded as hub genes in response to OTA but not AFM1. In the human FHs 74 Int cell line, both AFM1 and OTA enhanced the content of reactive oxygen species, and the oxidative response was more apparent in OTA-treated cells in comparison with AFM1. Furthermore, OTA and AFM1 + OTA raised the protein abundance of p50/RelB, and triggered the translocation of the dimer from cytosol to nucleus. Therefore, the experimental data ex vivo and in vitro showed that OTA-induced inflammation was thought to be bound up with the up-regulation and translocation of NF-κB, though AFM1 seemed to have no obvious impact. Since it was the first attempt to uncover the appearances and inner mechanisms regarding inflammation provoked by AFM1 and OTA on immature intestinal models, further efforts are needed to understand the detailed metabolic steps of the toxin in cells and to clarify their causal relationship with the signals proposed from current research.
Assuntos
Aflatoxina M1 , Intestinos , Aflatoxina M1/análise , Animais , Citocinas/genética , Contaminação de Alimentos/análise , Inflamação/induzido quimicamente , Intestinos/química , Camundongos , Leite/química , OcratoxinasRESUMO
Defensive behavior, a group of responses that evolved due to threatening stimuli, is crucial for animal survival in the natural environment. For defensive measures to be timely and successful, a high arousal state and immediate sleep-to-wakefulness transition are required. Recently, the glutamatergic basal forebrain (BF) has been implicated in sleep-wake regulation; however, the associated physiological functions and underlying neural circuits remain unknown. Here, using in vivo fiber photometry, we found that BF glutamatergic neuron is activated by various threatening stimuli, including predator odor, looming threat, sound, and tail suspension. Optogenetic activation of BF glutamatergic neurons induced a series of context-dependent defensive behaviors in mice, including escape, fleeing, avoidance, and hiding. Similar to the effects of activated BF glutamatergic cell body, photoactivation of BF glutamatergic terminals in the ventral tegmental area (VTA) strongly drove defensive behaviors in mice. Using synchronous electroencephalogram (EEG)/electromyogram (EMG) recording, we showed that photoactivation of the glutamatergic BF-VTA pathway produced an immediate transition from sleep to wakefulness and significantly increased wakefulness. Collectively, our results clearly demonstrated that the glutamatergic BF is a key neural substrate involved in wakefulness and defensive behaviors, and encodes these behaviors through glutamatergic BF-VTA pathway. Overexcitation of the glutamatergic BF-VTA pathway may be implicated in clinical psychiatric diseases characterized by exaggerated defensive responses, such as autism spectrum disorders.
Assuntos
Prosencéfalo Basal , Vigília , Animais , Prosencéfalo Basal/fisiologia , Eletroencefalografia/métodos , Mesencéfalo , Camundongos , Sono/fisiologia , Vigília/fisiologiaRESUMO
Human cytomegalovirus (HCMV) has a large (â¼235 kb) genome with more than 200 predicted open reading frames that exploits numerous cellular factors to facilitate its replication. A key feature of HCMV-infected cells is the emergence of a distinctive membranous cytoplasmic compartment termed the virion assembly compartment (vAC). Here, we report that host protein WD repeat domain 11 (WDR11) plays a key role in vAC formation and virion morphogenesis. We found that WDR11 was upregulated at both mRNA and protein levels during HCMV infection. At the late stage of HCMV replication, WDR11 relocated to the vAC and colocalized with markers of the trans-Golgi network (TGN) and vAC. Depletion of WDR11 hindered HCMV-induced membrane reorganization of the Golgi and TGN, altered vAC formation, and impaired HCMV secondary envelopment and virion morphogenesis. Further, motifs critical for the localization of WDR11 in TGN were identified by alanine-scanning mutagenesis. Mutation of these motifs led to WDR11 mislocation outside the TGN and loss of vAC formation. Taken together, these data indicate that host protein WDR11 is required for efficient viral replication at the stage of virion assembly, possibly by facilitating the remodeling of the endomembrane system for vAC formation and virion morphogenesis. IMPORTANCE During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood. In this report, we identified a host factor, WDR11, that plays an important role in vAC formation. Our findings argue that WDR11 contributes to the relocation of the Golgi and trans-Golgi network to the vAC, a membrane reorganization process that appears to be required for efficient virion maturation. The present work provides new insights into the vAC formation and HCMV virion morphogenesis and a potential novel target for antiviral treatment.
Assuntos
Infecções por Citomegalovirus , Citomegalovirus , Interações entre Hospedeiro e Microrganismos , Repetições WD40 , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/fisiopatologia , Infecções por Citomegalovirus/virologia , Humanos , Morfogênese , Vírion/metabolismo , Montagem de Vírus/genética , Replicação Viral/genética , Repetições WD40/genética , Rede trans-Golgi/metabolismoRESUMO
Congenital cytomegalovirus (cCMV) infection is the leading infectious cause of neurodevelopmental disorders. However, the neuropathogenesis remains largely elusive due to a lack of informative animal models. In this study, we developed a congenital murine CMV (cMCMV) infection mouse model with high survival rate and long survival period that allowed long-term follow-up study of neurodevelopmental disorders. This model involves in utero intracranial injection and mimics many reported clinical manifestations of cCMV infection in infants, including growth restriction, hearing loss, and impaired cognitive and learning-memory abilities. We observed that abnormalities in MRI/CT neuroimaging were consistent with brain hemorrhage and loss of brain parenchyma, which was confirmed by pathological analysis. Neuropathological findings included ventriculomegaly and cortical atrophy associated with impaired proliferation and migration of neural progenitor cells in the developing brain at both embryonic and postnatal stages. Robust inflammatory responses during infection were shown by elevated inflammatory cytokine levels, leukocyte infiltration, and activation of microglia and astrocytes in the brain. Pathological analyses and CT neuroimaging revealed brain calcifications induced by cMCMV infection and cell death via pyroptosis. Furthermore, antiviral treatment with ganciclovir significantly improved neurological functions and mitigated brain damage as shown by CT neuroimaging. These results demonstrate that this model is suitable for investigation of mechanisms of infection-induced brain damage and long-term studies of neurodevelopmental disorders, including the development of interventions to limit CNS damage associated with cCMV infection.
Assuntos
Infecções por Citomegalovirus , Modelos Animais de Doenças , Neuroimagem , Animais , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/diagnóstico por imagem , Infecções por Citomegalovirus/fisiopatologia , Infecções por Citomegalovirus/terapia , Feminino , Seguimentos , Camundongos , Camundongos Endogâmicos ICR , GravidezRESUMO
Human cytomegalovirus (HCMV) establishes a persistent/latent infection after primary infection, and the host factor(s) plays a key role in regulating HCMV infection status. The spread of reactivated HCMV via the hematogenous or neural route usually results in severe diseases in newborns and immunocompromised individuals. As the primary reservoirs in vivo, cells of myeloid lineage have been utilized extensively to study HCMV infection. However, the molecular mechanism of HCMV latency/reactivation in neural cells is still poorly understood. We previously showed that HCMV-infected T98G cells maintain a large number of viral genomes and support HCMV reactivation from latency upon cAMP/IBMX treatment. Here, we employed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics to characterize cellular protein changes during HCMV latency and reactivation in T98G cells. A total of 168 differentially expressed proteins (DEPs) were identified, including 89 proteins in latency and 85 proteins in reactivation. Bioinformatics analysis showed that a few biological pathways were associated with HCMV latency or reactivation. Moreover, we validated 16 DEPs by both mRNA and protein expression profiles and further evaluated the effects of ApoE and the phosphatidylinositol 3-kinase (PI3K) pathway on HCMV infection. ApoE knockdown reduced HCMV loads and virus release, whereas overexpressing ApoE hampered HCMV latent infection, indicating a role in HCMV latency establishment/maintenance. Blocking the PI3K pathway by LY294002, a PI3K inhibitor, induced HCMV reactivation from latency in T98G cells. Overall, this comparative proteomics analysis delineates the cellular protein changes during HCMV latency and reactivation and provides a road map to advance our understanding of the mechanism(s) in the context of neural cells. IMPORTANCE Human cytomegalovirus (HCMV) is a highly transmissible betaherpesvirus that has a prevalence of 60% to 90% worldwide. This opportunist pathogen poses a significant threat to newborns and immunosuppressed individuals. One major obstacle for developing effective therapeutics is a poor understanding of HCMV latency/reactivation mechanisms. This study presents, for the first time, a systemic analysis of host cell protein expression changes during HCMV latency establishment and reactivation processes in neural cells. We showed that ApoE was downregulated by HCMV to facilitate latent infection. Also, the proteomics analysis has associated a few PI3K pathway-related proteins with HCMV reactivation. Altogether, this study highlights multiple host proteins and signaling pathways that can be further investigated as potential druggable targets for HCMV-related diseases, especially brain disorders.
Assuntos
Citomegalovirus/fisiologia , Proteômica , Ativação Viral , Latência Viral , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Linhagem Celular Tumoral , Ontologia Genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Mapas de Interação de Proteínas , Proteoma/genética , Proteoma/metabolismo , Transdução de SinaisRESUMO
Hearing loss is one of the most prevalent sensory disabilities worldwide with huge social and economic burdens. The leading cause of sensorineural hearing loss (SNHL) in children is congenital cytomegalovirus (CMV) infection. Though the implementation of universal screening and early intervention such as antiviral or anti-inflammatory ameliorate the severity of CMV-associated diseases, direct and targeted therapeutics is still seriously lacking. The major hurdle for it is that the mechanism of CMV induced SNHL has not yet been well understood. In this review, we focus on the impact of CMV infection on the key players in inner ear development including the Wnt and Notch signaling pathways. Investigations on these interactions may gain new insights into viral pathogenesis and reveal novel targets for therapy.
Assuntos
Infecções por Citomegalovirus/complicações , Citomegalovirus/patogenicidade , Regulação da Expressão Gênica , Perda Auditiva Neurossensorial/virologia , Receptores Notch/genética , Transdução de Sinais , Proteínas Wnt/genética , Animais , Citomegalovirus/genética , Humanos , Camundongos , Receptores Notch/metabolismo , Proteínas Wnt/metabolismoRESUMO
We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WD repeat-containing protein 5 (WDR5) to facilitate capsid nuclear egress. Here, we further show that HCMV infection results in WDR5 localization in a juxtanuclear region, and that its localization to this cellular site is associated with viral replication and late viral gene expression. Furthermore, WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 co-immunoprecipitated with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain WDR5 accumulation in the vAC during infection. WDR5 fractionated with virions either in the presence or absence of Triton X-100 and was present in purified viral particles, suggesting that WDR5 was incorporated into HCMV virions. Thus, WDR5 localized to the vAC and was incorporated into virions, raising the possibility that in addition to capsid nuclear egress, WDR5 could also participate in cytoplasmic HCMV virion morphogenesis.Importance Human cytomegalovirus (HCMV) has a large (â¼235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are reorganized to establish the virion assembly compartment (vAC), which has been shown to necessary for efficient assembly of progeny virions. We previously reported that WDR5 facilitates HCMV nuclear egress. Here, we show that WDR5 is localized to the vAC and incorporated into virions, perhaps contributing to efficient virion maturation. Thus, findings in this study identified a potential role for WDR5 in HCMV assembly in the cytoplasmic phase of virion morphogenesis.
RESUMO
Predatory hunting is an important approach for animals to obtain valuable nutrition and energy, which critically depends on heightened arousal. Yet the neural substrates underlying predatory hunting remain largely undefined. Here, we report that basal forebrain (BF) GABAergic neurons play an important role in regulating predatory hunting. Our results showed that BF GABAergic neurons were activated during the prey (cricket)-hunting and food feeding in mice. Optogenetic activation of BF GABAergic neurons evoked immediate predatory-like actions to both artificial and natural preys, significantly reducing the attack latency while increasing the attack probability and the number of killed natural prey (crickets). Similar to the effect of activating the soma of BF GABAergic neurons, photoactivation of their terminals in the ventral tegmental area (VTA) also strongly promotes predatory hunting. Moreover, photoactivation of GABAergic BF - VTA pathway significantly increases the intake of various food in mice. By synchronous recording of electroencephalogram and electromyogram, we showed that photoactivation of GABAergic BF - VTA pathway induces instant arousal and maintains long-term wakefulness. In summary, our results clearly demonstrated that the GABAergic BF is a key neural substrate for predatory hunting, and promotes this behavior through GABAergic BF - VTA pathway.
Assuntos
Nível de Alerta/fisiologia , Prosencéfalo Basal/metabolismo , Neurônios GABAérgicos/metabolismo , Comportamento Predatório/fisiologia , Animais , Prosencéfalo Basal/química , Eletroencefalografia/métodos , Neurônios GABAérgicos/química , Gryllidae , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Optogenética/métodosRESUMO
The glutamatergic lateral hypothalamus (LH) has been implicated in a variety of behaviors, such as evasion and feeding, while its role in defensive behaviors and relevant neurocircuits remains unclear. Here, we demonstrated that the glutamatergic LH is a critical structure regulating defensive behaviors. Trimethylthiazole (TMT), the odor of mice predator, significantly increased c-Fos expression in the LH. Using fiber photometry technology, we found that TMT exposure increased the activity of LH glutamatergic neurons. Selective activation of LH glutamatergic neurons with optogenetics and chemogenetics promoted a series of defense-related behaviors, including fleeing, avoidance, and hiding, while selective inhibition of LH glutamatergic neurons suppressed the avoidance provoked by TMT. Activation of both the glutamatergic LH terminals in the hypothalamic paraventricular nucleus (PVN) and the glutamatergic projection from the basolateral amygdala (BLA) to the LH elicited defensive behaviors. Finally, by combining the viral-mediated retrograde tracing with anterograde activation, we found that PVN-projecting glutamatergic neurons in the LH were activated by BLA glutamatergic inputs. Taken together, our results illustrate that the glutamatergic LH is a pivotal relay of defensive behaviors and possibly promotes these behaviors through the BLAâLHâPVN pathway.
Assuntos
Aprendizagem da Esquiva/fisiologia , Mecanismos de Defesa , Ácido Glutâmico/metabolismo , Região Hipotalâmica Lateral/metabolismo , Animais , Ácido Glutâmico/análise , Região Hipotalâmica Lateral/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Optogenética/métodosRESUMO
Background: Visceral pain is one of the most common types of pain and particularly in the abdomen is associated with gastrointestinal diseases. Bulleyaconitine A (BAA), isolated from Aconitum bulleyanum, is prescribed in China to treat chronic pain. The present study is aimed at evaluating the mechanisms underlying BAA visceral antinociception. Methods: The rat model of chronic visceral hypersensitivity was set up by colonic perfusion of 2,4,6-trinitrobenzene sulfonic acid (TNBS) on postnatal day 10 with coapplication of heterotypic intermittent chronic stress (HeICS). Results: The rat model of chronic visceral hypersensitivity exhibited remarkable abdominal withdrawal responses and mechanical hyperalgesia in hind paws, which were dose-dependently attenuated by single subcutaneous of administration of BAA (30 and 90 µg/kg). Pretreatment with the microglial inhibitor minocycline, dynorphin A antiserum, and κ-opioid receptor antagonist totally blocked BAA-induced visceral antinociception and mechanical antihyperalgesia. Spontaneous excitatory postsynaptic currents (sEPSCs) in spinal dorsal horn lamina II neurons were recorded by using whole-cell patch clamp. Its frequency (but not amplitude) from TNBS-treated rats was remarkably higher than that from naïve rats. BAA (1 µM) significantly reduced the frequency of sEPSCs from TNBS-treated rats but not naïve rats. BAA-inhibited spinal synaptic plasticity was blocked by minocycline, the dynorphin A antiserum, and κ-opioid receptor antagonist. Dynorphin A also inhibited spinal synaptic plasticity in a κ-opioid receptor-dependent manner. Conclusions: These results suggest that BAA produces visceral antinociception by stimulating spinal microglial release of dynorphin A, which activates presynaptic κ-opioid receptors in afferent neurons and inhibits spinal synaptic plasticity, highlighting a novel interaction mode between microglia and neurons.
Assuntos
Aconitina/análogos & derivados , Analgésicos/administração & dosagem , Dinorfinas/metabolismo , Microglia/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Dor Visceral/prevenção & controle , Aconitina/administração & dosagem , Animais , Feminino , Microglia/metabolismo , Células do Corno Posterior/efeitos dos fármacos , Células do Corno Posterior/fisiologia , Ratos Sprague-Dawley , Sinapses/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Dor Visceral/metabolismoRESUMO
The obesity epidemic is a global problem and a great challenge for public health. Overconsumption of food, especially palatable food, is the leading cause of obesity. The precise neural circuits underlying food overconsumption remain unclear and require further characterization. In the present study, we showed that Ca2+ signals of GABAergic neurons within the ventral tegmental area (VTA) increased after the onset of food intake, especially high-fat or high-sugar chow. Optogenetic activation of VTA GABAergic neurons evoked immediate eating of palatable food and significantly increased palatable food intake in satiated mice. Photoinhibition of VTA GABAergic neurons suppressed palatable food intake. Surprisingly, photoactivation of VTA GABAergic neurons suppressed the intake of standard chow in fasted mice, but did not reduce the duration of eating of standard chow. Moreover, we found that photoactivation of these neurons drove a series of anxiety-like behaviors in the open field, elevated plus maze, and marble-burying test. Additionally, we found that VTA GABAergic neurons sent abundant projections to the lateral hypothalamus and photoactivation of GABAergic VTA terminals in the lateral hypothalamus induced overconsumption of palatable food, but not anxiety-like behaviors. Taken together, our results illustrate that GABAergic VTA neurons are a key node in the neural circuitry underlying anxiety-like behavior and over-feeding of palatable food, and that over-excitation of GABAergic VTA neurons may underlie clinical diseases related to anxiety and obesity.
Assuntos
Regulação do Apetite/fisiologia , Comportamento/fisiologia , Neurônios GABAérgicos/fisiologia , Área Tegmentar Ventral/fisiologia , Animais , Ansiedade/fisiopatologia , Comportamento Animal , Cálcio/fisiologia , Ingestão de Alimentos/fisiologia , Região Hipotalâmica Lateral/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/fisiopatologia , OptogenéticaRESUMO
Visceral pain is one of the leading causes for abdominal pain in gastroenterological diseases and is still hard to treat effectively. Bulleyaconitine A (BAA) is an aconitine analog and has been used for the treatment of pain. Our previous work suggested that BAA exerted analgesic effects on neuropathic pain through stimulating the expression of dynorphin A in spinal microglia. Here, we investigated the inhibitory effect of BAA on visceral pain and examined whether the expression of dynorphin A in spinal microglia was responsible for its effects. We found that BAA produced significant antivisceral pain effect induced by acetic acid through stimulating dynorphin A expression in spinal microglia. In addition, anxiety and chronic visceral pain are highly prevalent comorbid conditions in clinical research, which is still a problem to be solved. We also aimed to evaluate the effects of BAA on anxiety. A comorbidity model with characteristics of both chronic visceral pain and anxiety was developed by colorectal injection of 2,4,6-trinitrobenzene sulfonic acid and the induction of heterotypic intermittent chronic stress protocol. In comorbid animals, BAA exerted great antianxiety effects. Meanwhile, the antianxiety mechanism of BAA was different with the antivisceral pain mechanism of BAA. In conclusion, our study demonstrated, for the first time, that BAA exerted marked antivisceral pain and antianxiety effects, which expands the analgesic spectrum and clinical application of BAA. Furthermore, it also it provides a better guidance for the clinical use of BAA.
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The objective of this study was to evaluate the protection conferred by lactoferrin, α-lactalbumin, and ß-lactoglobulin in cerebral ischemia reperfusion (I/R) injury. Rat pheochromocytoma (PC12) cells were used to construct an oxygen and glucose deprivation model in vitro, and ICR mice underwent carotid artery "ligation-relaxation" to construct a cerebral I/R injury model in vivo. The levels of toll-like receptor 4 (TLR4) and downstream factors including nuclear factor-κB, tumor necrosis factor-α, and IL-1ß were measured. Metabonomics detection and data mining were conducted to identify the specific metabolic sponsor of the 3 proteins. The results showed that lactoferrin, α-lactalbumin, and ß-lactoglobulin protected neurons from cerebral I/R injury by increasing the level of bopindolol and subsequently inhibiting the TLR4-related pathway to different degrees; ß-lactoglobulin had the strongest activity of the 3 proteins. In summary, this study is the first to investigate and compare the protective effects of lactoferrin, α-lactalbumin, and ß-lactoglobulin in a cerebral stroke model. The results implicate TLR4 as a novel target of the 3 bioactive proteins to prevent cerebral I/R injury.
Assuntos
Lactalbumina/uso terapêutico , Lactoferrina/uso terapêutico , Lactoglobulinas/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Animais , Glucose/metabolismo , Interleucina-1beta/metabolismo , Lactalbumina/metabolismo , Lactoferrina/metabolismo , Lactoglobulinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Oxigênio/metabolismo , Células PC12 , Ratos , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: MicroRNA (miRNA) therapy, which was widely considered to treat a series of cancer, has been confronted with numerous obstacles to being delivered into target cells because of its easy biodegradation and instability. METHODS: In this research, we successfully constructed 11-mercaptoundecanoic acid modified gold nanocages (AuNCs)/polyethyleneimine (PEI)/miRNA/hyaluronic acid (HA) complexes (abbreviated as AuNCs/PEI/miRNA/HA) using a layer-by-layer method for target-specific intracellular delivery of miRNA by HA receptor mediated endocytosis. RESULTS: The results of UV spectra, hydrodynamic diameter and zeta potential analyses confirmed the formation of AuNCs/PEI/ miRNA/HA complex with its average particle size of ca. 153 nm and surface charge of ca. -9.43 mV. Next, we evaluated the antitumor effect of the nanocomplex mediated by the combination of gene therapy and photothermal therapy (PTT) against hepatocellular carcinoma (HCC) in vitro. CONCLUSION: Our experimental results indicated that the AuNCs/PEI/miRNA/HA complex effectively delivered miRNA to the target cells and its antitumor effect was significantly enhanced by the combination of gene therapy and photothermal therapy. In addition, anti-miR-181b could promote Bel-7402 cell arrest in S phase and improve TIMP-3 mRNA expression. All these results suggested that AuNCs/PEI/miRNA/HA gene delivery system with combination of gene therapy and photothermal therapy might be exploited for HCC treatment.
Assuntos
Carcinoma Hepatocelular/terapia , Terapia Genética , Neoplasias Hepáticas/terapia , MicroRNAs/antagonistas & inibidores , Nanocompostos/administração & dosagem , Fototerapia , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ciclo Celular , Proliferação de Células , Terapia Combinada , Ouro/química , Humanos , Ácido Hialurônico/química , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Nanopartículas Metálicas/química , MicroRNAs/genética , Nanocompostos/química , Polietilenoimina/química , Células Tumorais CultivadasRESUMO
Novel bioactive heterocycles containing a 3,4,5-trimethoxyphenyl fragment as antiproliferative agents by targeting tubulin were synthesized and their preliminary structure activity relationships (SARs) were explored. Among all these chemical agents, 2-(Benzo[d]oxazol-2-ylthio)-N-(4-methoxybenzyl)-N-(3,4,5-trimethoxyphenyl)acetamide (4d) exhibited the potent antiproliferative activity against MGC-803â¯cells with an IC50 value of 0.45 µM by induction of G2/M pahse arrest and cell apoptosis. In addition, 4d could change the membrane potential (ΔΨ) of the mitochondria against MGC-803â¯cells. Importantly, 4d acted as a novel tubulin polymerization inhibitor binding to colchicine site with an IC50 value of 3.35 µM.