RESUMO
OBJECTIVE: Genetic loss of Tsc1/Tsc2 function in tuberous sclerosis complex (TSC) results in altered mammalian target of rapamycin (mTOR) signaling and abnormal brain development. Although earlier studies have focused on characterization of cortical tubers, in this study we sought to examine the unique cellular and molecular features of the perituberal cortex in order to better understand its contribution to epileptogenesis, cognitive dysfunction, and autism. METHODS: Standard histologic and immunohistochemical labeling was used to assess structural abnormalities and cell-specific pattern of mTORC1 activation in surgically resected cortical tubers and perituberal cortex. Western blotting was performed to quantify the expression of the mTORC1 and mTORC2 biomarkers phospho-S6 (Ser235/236), phospho-S6 (Ser240/244), and phospho-Akt (Ser473), in addition to evaluating the differential expression levels of several neuronal and glial-specific proteins in tubers and peritubers, as compared to non-TSC epilepsy specimens. RESULTS: Tubers demonstrated mild to severe disruption of cortical lamination, the presence of pS6-positive dysplastic neurons and giant cells, an overall increase in mTORC1 and a decrease in mTORC2 activity, increased axonal connectivity and growth, and hypomyelination. Perituberal cortex presented similar histologic, immunohistochemical, and molecular features; however, they were overall milder. Axonal growth was specific for TSC and was negatively correlated with deficient myelination. SIGNIFICANCE: Our results show an extension of cellular dysplasia and dysregulated mTOR signaling in the perituberal tissue, and demonstrate for the first time aberrant connectivity in human TSC brain. This study provides new insights into the pathophysiology of neurologic dysfunction associated with TSC and supports the intrinsic epileptogenicity of normal-appearing perituberal cortex. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.
Assuntos
Encefalopatias/diagnóstico , Encéfalo/anormalidades , Esclerose Tuberosa/diagnóstico , Encéfalo/crescimento & desenvolvimento , Encefalopatias/metabolismo , Encefalopatias/patologia , Córtex Cerebral/anormalidades , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/patologia , Feminino , Humanos , Masculino , Estudos Prospectivos , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologiaRESUMO
Interferon-gamma (IFN-gamma) has potent antiviral activity in neurons which is affected by the production of nitric oxide (NO). This study examines the interactions between cannabinoid receptor-1 (CB(1)), IFNgamma-induced pathways, and inhibition of vesicular stomatitis virus (VSV) replication in neuronal cells. CB(1) is abundantly expressed in neurons of the CNS and the NB41A3 neuroblastoma cell line. CB(1) activation of NB41A3 cells by the synthetic cannabinoid, WIN55,212-2, is associated with an inhibition of Ca(2+) mobilization, leading to diminished nitric oxide synthase (NOS)-1 activity and the production of NO, in vitro. This ultimately results in antagonism of IFN-gamma-mediated antiviral activity and enhanced viral replication. Therefore, activation of cells expressing CB(1) by endogenous (or exogenous) ligands may contribute to decreased inflammation and to increased viral replication in neurons and disease in the CNS.
Assuntos
Canabinoides/farmacologia , Fatores Imunológicos/farmacologia , Interferon gama/antagonistas & inibidores , Neurônios/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Linhagem Celular , Sobrevivência Celular , Humanos , Interferon gama/imunologia , Camundongos , Neurônios/virologia , Óxido Nítrico/biossíntese , Receptores de Canabinoides/metabolismo , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimento , Ensaio de Placa Viral , Replicação Viral/imunologiaRESUMO
In this study we investigate the role of the protein inhibitor of NOS-1 (PIN) in the interferon-gamma (IFN-gamma)-mediated posttranscriptional accumulation of nitric oxide synthase-1 (NOS-1) and the anti-vesicular stomatitis virus response in neuronal cells. IFN-gamma-induced enhancement of NOS-1 activity is crucial for its antiviral activity in the central nervous system. IFN-gamma treatment of neuronal cells results in an increase of total NOS-1 and decrease of total PIN proteins without alteration in their respective mRNA levels. PIN/NOS-1 complexes decreased after IFN-gamma treatment. Transfection of cells with small interfering RNA (siRNA) for PIN results in a higher constitutive activity of NOS-1 and inhibition of viral replication. IFN-gamma treatment did not change the amount of NOS-1 detectable by Western blot, when PIN is diminished by RNAi treatment. Overexpression of PIN results in lower constitutive NOS-1 expression and activity, and diminishes activation of NOS-1 by IFN-gamma. Our findings indicate that in neurons, IFN-gamma upregulates NOS-1 through proteasomal degradation of PIN.
Assuntos
Dineínas/fisiologia , Interferon gama/farmacologia , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Western Blotting , Células Cultivadas , Cricetinae , Cricetulus , Dineínas do Citoplasma , Primers do DNA/química , Ensaio de Imunoadsorção Enzimática , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Neurônios/enzimologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/genética , Plasmídeos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de Interferon/metabolismo , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Interferon (IFN)-gamma, is not only a marker of T(H)1 CD4, CD8 and natural killer (NK) cells, it is also a critical antiviral mediator which is central to the elimination of viruses from the CNS. In this review, we describe IFN-gamma, its receptor, signal transduction from receptor engagement, and antiviral downstream mediators. We demonstrate that although neurons are post-mitotic and non-renewing, they respond to IFN-gamma in a fashion similar to peripheral fibroblasts or lymphocytes. We have illustrated this review with details about studies on the role(s) of IFN-gamma in the pathogenesis of measles virus (MV), herpes simplex virus (HSV) type 1, and vesicular stomatitis virus (VSV) infections of the CNS. For VSV infection, IFN-gamma signals through Jaks 1 and 2 and STAT1 to activate (interferon regulatory factor) IRF-1; although viral protein synthesis is inhibited, PKR is not a critical mediator in the antiviral response to VSV in murine neurons. In contrast, induction of nitric oxide synthase (NOS) type 1 and its production of nitric oxide is essential in the elimination of viruses from neurons.
Assuntos
Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Interferon gama/fisiologia , Animais , Humanos , Modelos Biológicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Transdução de SinaisRESUMO
Pharmacologic inhibition of the proteasome resulted in increased NOS-1 protein levels and increased NO production by neuronal cells. This correlated with an increased antiviral effect of IFN-gamma against the replication of vesicular stomatitis virus (VSV) replication in vitro. We also observed that a regulatory protein, Protein Inhibitor of NOS-1 (PIN) was down-regulated by IFN-gamma treatment, and more ubiquitinated PIN accumulated in IFN-gamma treated neurons. In cells of the reticuloendothelial system, IFN-gamma treatment induces the expression of a set of low molecular weight MHC-encoded proteins (LMPs), which replace the beta-subunit of the proteasome complex during the proteasome neosynthesis, resulting in a complex termed the immunoproteasome. LMP2, -7, and -10 were induced and the immunoproteasome was generated by IFN-gamma treatment in neuronal cells. Importantly, we observed that IFN-gamma induced inhibition of VSV protein synthesis was not dependent on ubiquitination.
Assuntos
Antivirais/imunologia , Interferon gama/imunologia , Neurônios/virologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Infecções por Rhabdoviridae/imunologia , Ubiquitina/metabolismo , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Antivirais/farmacologia , Linhagem Celular Tumoral , Dineínas/metabolismo , Fibroblastos , Interferon gama/farmacologia , Camundongos , Camundongos Transgênicos , Neuroblastoma , Neurônios/citologia , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Inibidores de Proteassoma , RNA Mensageiro/metabolismo , Infecções por Rhabdoviridae/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologia , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimento , Replicação Viral/imunologiaRESUMO
During acute Vesicular Stomatitis Virus (VSV) infection of the mouse central nervous system, neutrophils, natural killer (NK) cells, macrophages, and CD4+ and CD8+ T cells are recruited from the circulation in response to chemokines and cytokines. This study elucidated the production of these factors and infiltration of these peripheral cells. Chemokines that were observed included CCL1, CXCL10 (IP-10), CCL5 (RANTES), CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CXCL1 (MIP-2), CCL2 (MCP-1), and CCL11 (eotaxin). Cytokines produced in response to the infection include IL-1 and interferon-gamma, but not type I interferons. Neutrophils are the first recruited cell type, appearing as early as 24 h after intranasal application of the virus. NK cells follow, but T cells are not detected until 6 days postinfection.
Assuntos
Viroses do Sistema Nervoso Central/imunologia , Citocinas/metabolismo , Infecções por Rhabdoviridae/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Encéfalo/virologia , Viroses do Sistema Nervoso Central/virologia , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/imunologia , Infecções por Rhabdoviridae/virologia , Vírus da Estomatite Vesicular Indiana/patogenicidadeRESUMO
Vesicular stomatitis virus (VSV), a negative-sense, single-stranded RNA rhabdovirus, causes acute viral encephalitis when administered intranasally to mice. Interleukin-12 (IL-12) is a key pro-inflammatory cytokine that is produced largely by the antigen presenting cells (APC) and that bridges the innate and acquired immune responses. IL-12 is efficacious in enhancing recovery from VSV infection of the murine central nervous system. This effect is mediated by nitric oxide (NO) produced by the neuronal isoform of nitric oxide synthase (NOS-1), and is independent of the pro-inflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). These data implied a link between IL-12 and NOS-1. Here we investigate the role of the IL-12R during VSV pathogenesis, using IL-12R beta2 and IL-12R beta1-deficient mice. We showed that a deficiency in either IL-12R beta2 or IL-12R beta1 had no effect on the outcome of VSV infection of the CNS or on the clearance of VSV from the CNS. Furthermore, these data indicate that IL-23 is not acting redundantly in the absence of IL-12 during VSV-induced encephalitis.
Assuntos
Encefalite Viral/imunologia , Interleucina-12/metabolismo , Interleucinas/metabolismo , Receptores de Interleucina/deficiência , Vírus da Estomatite Vesicular Indiana/patogenicidade , Animais , Encefalite Viral/virologia , Feminino , Interleucina-23 , Subunidade p19 da Interleucina-23 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina/genética , Receptores de Interleucina-12 , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
In this report, the mechanism through which interferon-gamma (IFN-gamma) regulates the expression of nitric oxide synthase (NOS-1) in neurons was examined. We have shown previously that IFN-gamma treatment of cells results in a two log inhibition of vesicular stomatitis virus (VSV) production. This inhibition of VSV replication is dependent both in vitro and in vivo on nitric oxide (NO) production by NOS-1. Furthermore, this effect is associated with the increased expression and activity of NOS-1 following IFN-gamma treatment. In vitro, exposure to IFN-gamma prior to infection with VSV is a prerequisite to establish an effective antiviral state, indicating the necessity for a priming event. Neuroblastoma cells (NB41A3) were treated with IFN-gamma or medium and examined for changes in NOS-1 protein and mRNA expression. NOS-1 protein expression was found to be increased after IFN-gamma treatment, and this was associated with increases in both neosynthesis and NOS-1 protein stability. NOS-1 transcription and mRNA levels were unaffected by IFN-gamma treatment. These data demonstrate that IFN-gamma regulates NOS-1 expression through posttranscriptional and posttranslational mechanisms.
Assuntos
Interferon gama/farmacologia , Óxido Nítrico Sintase/genética , Processamento Pós-Transcricional do RNA/genética , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cicloeximida/farmacologia , Camundongos , Neuroblastoma , Neurônios/enzimologia , Neurônios/imunologia , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I , Inibidores da Síntese de Proteínas/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Processamento Pós-Transcricional do RNA/imunologia , RNA Mensageiro/genética , Proteínas Recombinantes , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Transcrição Gênica/imunologiaRESUMO
In this report, the role of STAT4 and local production of interleukin (IL)-12 in the central nervous system (CNS) were examined during experimental vesicular stomatitis virus (VSV) encephalitis. We have previously shown that IL-12 treatment is beneficial both in vitro and in vivo during experimental VSV infection. This inhibition of VSV replication was dependent on the production of nitric oxide (NO) by the neuronal isoform of nitric oxide synthase (NOS-1). In vitro, IL-12 induces the phosphorylation and nuclear localization of STAT4 in neuroblastoma cell lines. STAT4 expression was not required for host survival or clearance of virus during experimental VSV encephalitis. Taken together, these data suggest that while neurons can respond directly to IL-12 in vitro by signaling through STAT4, STAT4 is not required for survival. It is likely that redundant innate host inflammatory cytokine responses compensate for the absence of IL-12 signaling.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Encefalite Viral/imunologia , Interleucina-12/fisiologia , Infecções por Rhabdoviridae/imunologia , Transativadores/fisiologia , Vírus da Estomatite Vesicular Indiana , Animais , Células CHO , Sistema Nervoso Central/virologia , Cricetinae , Proteínas de Ligação a DNA/genética , Encefalite Viral/virologia , Interleucina-12/farmacologia , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Infecções por Rhabdoviridae/virologia , Fator de Transcrição STAT4 , Transdução de Sinais , Transativadores/genética , Células Tumorais Cultivadas , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimento , Replicação ViralRESUMO
The complement system is a critical component of both the innate and acquired immune systems. It is important in host defense against viruses, bacteria, and fungi for opsonization and for lysis of pathogens. However, activated complement can also cause tissue damage. There is compelling evidence that complement factors are presented in the central nervous system (CNS). Complement activation (by any of the three pathways: classical, alternate, and lectin) can lead to inflammation and tissue damage, while at the same time may also restrict certain pathogens in the CNS. C5a is formed by proteolytic cleavage C5. C5a is considered the most potent proinflammatory mediator, often called an anaphylotoxin. In this communication, we examine the roles of C5 (C5a) in vesicular stomatitis virus (VSV)-induced encephalitis. We found that C5a is produced during VSV infection, but C5-deficient mice had similar pathology as their controls. We concluded that C5 is not a critical factor in mediating the host response during VSV encephalitis.
Assuntos
Complemento C5a/imunologia , Encefalite Viral/imunologia , Infecções por Rhabdoviridae/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Animais , Células CHO , Sistema Nervoso Central/imunologia , Cricetinae , Modelos Animais de Doenças , Humanos , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/biossíntese , Infecções por Rhabdoviridae/epidemiologiaRESUMO
Interleukin (IL)-12, a key cytokine bridging innate and acquired immunity, is efficacious in enhancing recovery from experimental vesicular stomatitis virus (VSV) infection of the mouse central nervous system (CNS). This response is associated with the upregulation of neuronal nitric oxide synthase (NOS-1), independent of IFN-gamma and TNF-alpha. We hypothesized that neurons may respond directly IL-12. Our data are consistent with the expression of a functional IL-12 receptor (IL-12R) by neurons in culture and this receptor-ligand interaction results in the induction of an innate antiviral immune response. N18 cells, which did not express IL-12Rbeta2 were transfected with the IL-12Rbeta2 receptor gene; Koch's postulates were fulfilled, as clones derived from this transfection were reconstituted for IL-12 responsiveness.
Assuntos
Neurônios/imunologia , Receptores de Interleucina/análise , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Imuno-Histoquímica , Interleucina-12/farmacologia , Camundongos , Neuroblastoma/imunologia , Neuroblastoma/patologia , Neurônios/química , Neurônios/virologia , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo I , RNA Mensageiro/análise , RNA Mensageiro/química , Receptores de Interleucina/genética , Receptores de Interleucina/fisiologia , Receptores de Interleucina-12 , Fator de Transcrição STAT4 , Transativadores/metabolismo , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
In this report, the contribution of PKR to the IFN-gamma mediated inhibition of VSV replication in neurons was examined. IFN-gamma treatment of NB41A3 murine neuroblastoma cells resulted in the reduced expression of VSV protein during infection. PKR was found to be modestly upregulated in NB41A3 cells following IFN-gamma treatment. The phosphorylation state of PKR and its downstream target, eIF2alpha, were unaffected by either IFN-gamma or VSV infection. Inhibition of PKR through the use of 2-aminopurine or the expression of the Influenza A NS1 gene had no effect on the ability of IFN-gamma to inhibit the replication of VSV in vitro. These data indicate that endogenously expressed PKR is not required for the IFN-gamma mediated inhibition of VSV replication in NB41A3 neuroblastoma cells.
Assuntos
Interferon gama/farmacologia , Neurônios/virologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral/efeitos dos fármacos , eIF-2 Quinase/metabolismo , Animais , Camundongos , Células Tumorais Cultivadas , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Proteínas Virais/metabolismoRESUMO
Cyclooxygenase (COX) is the key enzyme for prostaglandin (PG) synthesis. PGs are mediators of many critical physiological and inflammatory responses. There are two isoforms, COX-1 and COX-2, both of which are constitutively expressed in the central nervous system (CNS). Studies have shown that COX-1 and COX-2 are involved in physiological and pathological conditions of the brain. However, little is known about the role(s) of COX in the host defense system against a viral infection in the CNS. In this report, we used Vesicular Stomatitis Virus (VSV) induced acute encephalitis to distinguish between the contribution(s) of the two isoforms. COX-2 activity was inhibited with a COX-2 selective drug, celecoxib (Celebrex), and COX-1 was antagonized with SC560. We found that inhibition of COX-2 led to decreased viral titers, while COX-1 antagonism did not have the same effect at day 1 post infection. 5-lipooxygenase (5-LO) expression and neutrophil recruitment in the CNS were increased in celecoxib-inhibited mice. Furthermore, mice treated with celecoxib expressed more Nitric Oxide Synthase-1 (NOS-1), a crucial component of the innate immune system in the restriction of VSV propagation. The expression of type 1 cytokines, IFN-gamma and IL-12, were also increased in celecoxib-treated mice.