RESUMO
HIV-1 Associated Neurocognitive Disorder (HAND) is a common and clinically detrimental complication of HIV infection. Viral proteins, including Tat, released from infected cells, cause neuronal toxicity. Substance abuse in HIV-infected patients greatly influences the severity of neuronal damage. To repurpose small molecule inhibitors for anti-HAND therapy, we employed MOLIERE, an AI-based literature mining system that we developed. All human genes were analyzed and prioritized by MOLIERE to find previously unknown targets connected to HAND. From the identified high priority genes, we narrowed the list to those with known small molecule ligands developed for other applications and lacking systemic toxicity in animal models. To validate the AI-based process, the selective small molecule inhibitor of DDX3 helicase activity, RK-33, was chosen and tested for neuroprotective activity. The compound, previously developed for cancer treatment, was tested for the prevention of combined neurotoxicity of HIV Tat and cocaine. Rodent cortical cultures were treated with 6 or 60 ng/ml of HIV Tat and 10 or 25 µM of cocaine, which caused substantial toxicity. RK-33 at doses as low as 1 µM greatly reduced the neurotoxicity of Tat and cocaine. Transcriptome analysis showed that most Tat-activated transcripts are microglia-specific genes and that RK-33 blocks their activation. Treatment with RK-33 inhibits the Tat and cocaine-dependent increase in the number and size of microglia and the proinflammatory cytokines IL-6, TNF-α, MCP-1/CCL2, MIP-2, IL-1α and IL-1ß. These findings reveal that inhibition of DDX3 may have the potential to treat not only HAND but other neurodegenerative diseases. Graphical Abstract RK-33, selective inhibitor of Dead Box RNA helicase 3 (DDX3) protects neurons from combined Tat and cocaine neurotoxicity by inhibition of microglia activation and production of proinflammatory cytokines.
Assuntos
Azepinas/farmacologia , Cocaína/toxicidade , RNA Helicases DEAD-box/antagonistas & inibidores , Imidazóis/farmacologia , Microglia/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Complexo AIDS Demência/tratamento farmacológico , Complexo AIDS Demência/enzimologia , Animais , Azepinas/uso terapêutico , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , Inibidores da Captação de Dopamina/toxicidade , Relação Dose-Resposta a Droga , Feminino , Imidazóis/uso terapêutico , Masculino , Microglia/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
We recently demonstrated direct evidence of increased monoamine oxidase (MAO) activity in the brain of a simian immunodeficiency virus (SIV) model of human immunodeficiency virus (HIV)-associated central nervous system (CNS) disease, consistent with previously reported dopamine deficits in both SIV and HIV infection. In this study, we explored potential mechanisms behind this elevated activity. MAO B messenger RNA was highest in macaques with the most severe SIV-associated CNS lesions and was positively correlated with levels of CD68 and GFAP transcripts in the striatum. MAO B messenger RNA also correlated with viral loads in the CNS of SIV-infected macaques and with oxidative stress. Furthermore, in humans, striatal MAO activity was elevated in individuals with HIV encephalitis, compared with activity in HIV-seronegative controls. These data suggest that the neuroinflammation and oxidative stress caused by SIV infection in the CNS may provide the impetus for increased transcription of MAO B and that MAO, and more broadly, oxidative stress, have significant potential as therapeutic targets in CNS disease due to HIV.
Assuntos
Complexo AIDS Demência/enzimologia , Encéfalo/enzimologia , Monoaminoxidase/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/enzimologia , Adulto , Animais , Química Encefálica , Corpo Estriado/enzimologia , Feminino , Perfilação da Expressão Gênica , Glutationa/análise , Humanos , Macaca nemestrina/virologia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Reação em Cadeia da Polimerase em Tempo Real , Carga ViralRESUMO
Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest.
Assuntos
Ácido Glutâmico/metabolismo , Glutaminase/antagonistas & inibidores , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Complexo AIDS Demência/enzimologia , Animais , Bioensaio , Isquemia Encefálica/enzimologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Camundongos , Microglia/enzimologia , Microglia/metabolismo , Esclerose Múltipla/enzimologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/isolamento & purificação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Receptores Toll-Like/agonistas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Microglia are critical cells in mediating the pathophysiology of neurodegenerative disorders such as HIV-associated neurocognitive disorders. We hypothesize that HIV-1 glycoprotein 120 (gp120) activates microglia by enhancing outward K(+) currents, resulting in microglia secretion of neurotoxins, consequent neuronal dysfunction, and death. To test this hypothesis, we studied the effects of gp120 on outward K(+) current in cultured rat microglia. Application of gp120 enhanced outward K(+) current in a dose-dependent manner, which was blocked by voltage-gated K(+) (K(v) ) channel blockers. Western blot analysis revealed that gp120 produced an elevated expression of K(v) channel proteins. Examination of activation and inactivation of outward K(+) currents showed that gp120 shifted membrane potentials for activation and steady-state inactivation. The gp120-associated enhancement of outward K(+) current was blocked by either a CXCR4 receptor antagonist T140 or a specific protein kinase A (PKA) inhibitor H89, suggesting the involvement of chemokine receptor CXCR4 and PKA in gp120-mediated enhancement of outward K(+) current. Biological significance of gp120-induced enhancement of microglia outward K(+) current was demonstrated by experimental results showing the neurotoxic activity of gp120-stimulated microglia, evaluated by TUNEL staining and MTT assay, significantly attenuated by K(v) channel blockers. Taken together, these results suggest that gp120 induces microglia neurotoxic activity by enhancing microglia outward K(+) current and that microglia K(v) channels may function as a potential target for the development of therapeutic strategies.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteína gp120 do Envelope de HIV/fisiologia , Microglia/metabolismo , Microglia/virologia , Canais de Potássio/metabolismo , Receptores CXCR4/fisiologia , Transdução de Sinais/fisiologia , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/metabolismo , Complexo AIDS Demência/virologia , Animais , Células Cultivadas , Técnicas de Cocultura , Potenciais da Membrana/fisiologia , Microglia/enzimologia , Degeneração Neural/enzimologia , Degeneração Neural/metabolismo , Degeneração Neural/virologia , Ratos , Ratos Sprague-DawleyRESUMO
Histone deacetylases (HDACs) play a pivotal role in epigenetic regulation of transcription and homeostasis of protein acetylation in histones and other proteins involved in chromatin remodeling. Histone hypoacetylation and transcriptional dysfunction have been shown to be associated with a variety of neurodegenerative diseases. More recently, neuron specific overexpression of HDAC2 has been shown to modulate synaptic plasticity and learning behavior in mice. However, the role of HDAC2 in development of HIV-associated neurocognitive disorders (HAND) is not reported. Herein we report that HIV-1 Tat protein upregulate HDAC2 expression in neuronal cells leading to transcriptional repression of genes involved in synaptic plasticity and neuronal function thereby contributing to the progression of HAND. Our results indicate upregulation of HDAC2 by Tat treatment in dose and time dependant manner by human neuroblastoma SK-N-MC cells and primary human neurons. Further, HDAC2 overexpression was associated with concomitant downregulation in CREB and CaMKIIa genes that are known to regulate neuronal activity. These observed effects were completely blocked by HDAC2 inhibition. These results for the first time suggest the possible role of HDAC2 in development of HAND. Therefore, use of HDAC2 specific inhibitor in combination with HAART may be of therapeutic value in treatment of neurocognitive disorders observed in HIV-1 infected individuals.
Assuntos
Complexo AIDS Demência/enzimologia , Produtos do Gene tat/fisiologia , HIV-1/metabolismo , Histona Desacetilase 2/metabolismo , Neurônios/enzimologia , Linhagem Celular Tumoral , Citometria de Fluxo , Produtos do Gene tat/antagonistas & inibidores , Humanos , Ácidos Hidroxâmicos/farmacologia , Reação em Cadeia da Polimerase , Regulação para CimaRESUMO
Human immunodeficiency virus-associated neurological disease (HAND) still causes significant morbidity, despite success reducing viral loads with combination antiretroviral therapy. The dopamine (DA) system is particularly vulnerable in HAND. We hypothesize that early, "reversible" DAergic synaptic dysfunction occurs long before DAergic neuron loss. As such, aging human immunodeficiency virus (HIV)-infected individuals may be vulnerable to other age-related neurodegenerative diseases like Parkinson's disease (PD), underscoring the need to understand shared molecular targets in HAND and PD. Previously, we reported that the neurotoxic HIV-1 transactivating factor (Tat) acutely disrupts mitochondrial and endoplasmic reticulum calcium homeostasis via ryanodine receptor (RyR) activation. Here, we further report that Tat disrupts DA transporter (DAT) activity and function, resulting in increased plasma membrane (PM) DAT and increased DAT V(max), without changes in K(m) or total DAT protein. Tat also increases calpain protease activity at the PM, demonstrated by total internal reflection fluorescence microscopy of a cleavable fluorescent calpain substrate. Tat-increased PM DAT and calpain activity are blocked by the RyR antagonists ryanodine and dantrolene, the calpain inhibitor calpastatin, and by a specific inhibitor of GSK-3ß. We conclude that Tat activates RyRs via a calcium- and calpain-mediated mechanism that upregulates DAT trafficking to the PM, and is independent of DAT protein synthesis, reinforcing the feasibility of RyR and GSK-3ß inhibition as clinical therapeutic approaches for HAND. Finally, we provide key translational relevance for these findings by highlighting published human data of increased DAT levels in striata of HAND patients and by demonstrating similar findings in Tat-expressing transgenic mice.
Assuntos
Calpaína/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Peptídeo Hidrolases/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/patologia , Animais , Western Blotting , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/fisiologia , Glicogênio Sintase Quinase 3 beta , Cinética , Mesencéfalo/citologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Neurônios/metabolismo , Células PC12 , Inibidores de Proteases/farmacologia , Ratos , Frações Subcelulares/fisiologiaRESUMO
Mononuclear phagocyte (MP, macrophages and microglia) dysfunction plays a significant role in the pathogenesis of HIV-1-associated dementia (HAD) through the production and release of soluble neurotoxic factors including glutamate. Glutamate production is greatly increased following HIV-1 infection of cultured MP, a process dependent upon the glutamate-generating enzyme glutaminase. Glutaminase inhibition was previously found to significantly decrease macrophage-mediated neurotoxicity. Potential mechanisms of glutaminase-mediated excitotoxicity including enzyme up-regulation, increased enzyme activity and glutaminase localization were investigated in this report. RNA and protein analysis of HIV-infected human primary macrophage revealed up-regulation of the glutaminase isoform GAC, yet identified no changes in the kidney-type glutaminase isoform over the course of infection. Glutaminase is a mitochondrial protein, but was found to be released into the cytosol and extracellular space following infection. This released enzyme is capable of rapidly converting the abundant extracellular amino acid glutamine into excitotoxic levels of glutamate in an energetically favorable process. These findings support glutaminase as a potential component of the HAD pathogenic process and identify a possible therapeutic avenue for the treatment of neuroinflammatory states such as HAD.
Assuntos
Ácido Glutâmico/biossíntese , Glutaminase/fisiologia , Infecções por HIV/metabolismo , HIV-1 , Macrófagos/metabolismo , Macrófagos/virologia , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/metabolismo , Complexo AIDS Demência/patologia , Complexo AIDS Demência/virologia , Células Cultivadas , Ácido Glutâmico/efeitos adversos , Glutaminase/metabolismo , Infecções por HIV/enzimologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Macrófagos/enzimologia , Macrófagos/patologiaRESUMO
A significant number of patients infected with human immunodeficiency virus-1 (HIV-1) suffer cognitive impairment ranging from mild to severe HIV-associated dementia (HAD), a result of neuronal degeneration in the basal ganglia, cerebral cortex and hippocampus. Mononuclear phagocyte dysfunction is thought to play an important role in the pathogenesis of HAD. Glutamate neurotoxicity is triggered primarily by massive Ca2+ influx arising from over-stimulation of the NMDA subtype of glutamate receptors. The underlying mechanisms, however, remain elusive. We have tested the hypothesis that mitochondrial glutaminase in HIV-infected macrophages is involved in converting glutamine to glutamate. Our results demonstrate that the concentration of glutamate in HIV-1 infected conditioned media was dependent on glutamine dose, and HIV-1 infected conditioned medium mediated glutamine-dependent neurotoxicity. These results indicate HIV-infection mediates neurotoxicity through glutamate production. In addition, glutamate-mediated neurotoxicity correlated with caspase activation and neuronal cell cycle re-activation. Inhibition of mitochondrial glutaminase diminished the HIV-induced glutamate production, and attenuated NMDA over-stimulation and subsequent neuronal apoptosis. These data implicate mitochondrial glutaminase in the induction of glutamate-mediated neuronal apoptosis during HIV-associated dementia, and provides a possible therapeutic strategy for HAD treatment.
Assuntos
Complexo AIDS Demência/enzimologia , Apoptose/fisiologia , Ácido Glutâmico/biossíntese , Glutaminase/metabolismo , Macrófagos/enzimologia , Degeneração Neural/enzimologia , Complexo AIDS Demência/fisiopatologia , Animais , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Encéfalo/virologia , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Glutamina/metabolismo , Glutamina/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Mitocôndrias/enzimologia , Monócitos/fisiologia , Degeneração Neural/fisiopatologia , Degeneração Neural/virologia , Neurônios/metabolismo , Neurônios/patologia , Neurotoxinas/metabolismo , RatosRESUMO
Activation of macrophages and microglia cells after HIV-1 infection and their production of inflammatory mediators contribute to HIV-associated CNS diseases. The mechanisms that initiate and maintain inflammation after HIV-1 infection in the brain have not been well studied. Furthermore, it is not understood why in HIV-associated CNS disease, macrophages and microglia are biased toward inflammation rather than production of mediators that control inflammation. We have focused on the receptor tyrosine kinase RON, a critical negative regulator of macrophage function and inflammation, to determine whether this receptor regulates HIV-1 expression. Overexpressing RON in monocytes/macrophages demonstrates that RON inhibits HIV-1 proviral transcription in part by decreasing the binding activity of NF-kappaB to the HIV-1 long terminal repeat. Because macrophages and microglia cells are a critical reservoir for HIV-1 in the CNS, we examined brain tissues for RON expression and detected RON in astrocytes, cortical neurons, and monocytoid cells. RON was detected in all control patients who were HIV seronegative (n = 7), whereas six of nine brain samples obtained from AIDS patients exhibited reduced RON protein. These data suggest that RON initiates signaling pathways that negatively regulate HIV-1 transcription in monocytes/macrophages and that HIV-1 suppresses RON function by decreasing protein levels in the brain to assure efficient replication. Furthermore, HIV-1 infection would compromise the ability of RON to protect against inflammation and consequent CNS damage.
Assuntos
Complexo AIDS Demência/enzimologia , Fármacos Anti-HIV/farmacologia , Encéfalo/patologia , HIV-1/genética , Mediadores da Inflamação/fisiologia , Macrófagos/enzimologia , Monócitos/enzimologia , Receptores Proteína Tirosina Quinases/fisiologia , Complexo AIDS Demência/imunologia , Complexo AIDS Demência/patologia , Adulto , Encéfalo/enzimologia , Encéfalo/virologia , Linhagem Celular , Doença Crônica , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Repressão Enzimática/imunologia , HIV-1/imunologia , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/virologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/biossíntese , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/biossíntese , Proteínas Repressoras/fisiologia , Transcrição Gênica/imunologia , Células U937RESUMO
OBJECTIVE: To identify which proteins are differentially secreted from monocyte/macrophages (M/M phi) of HIV-1 seropositive patients with HIV-1-associated dementia (HAD). DESIGN: To compare profiles of secreted M/M phi proteins from individuals with HAD, HIV-1 infection or controls using surface-enhanced laser desorption/ionization (SELDI)-time of flight (TOF) ProteinChip technology. METHODS: M/M phi were isolated by Percoll gradient centrifugation and cultured from whole blood of 11 patients with HAD, 13 HIV-1 seropositive subjects with no dementia (HIV-1 group) and nine HIV-1 seronegative subjects (controls). M/M phi supernatants were removed after 7 days in culture and analyzed by SELDI-TOF. A 14.6 kDa-secreted protein in control M/M phi supernatants was significantly decreased in patients with HAD. The protein was purified from HIV-1 seronegative controls and identified by peptide mapping. Protein concentration in the supernatants was quantified by enzyme-linked immunosorbent assay. RESULTS: A 14.6 kDa protein was identified as lysozyme. Secreted lysozyme concentrations from M/M phi of patients with HAD (81 +/- 35 ng/ml) were significantly lower than that of the HIV-1 group (326 +/- 303 ng/ml) and controls (764 +/- 211 ng/ml). Intracellular lysozyme was similar in all three groups. All patients with HAD were on highly active antiretroviral therapy (HAART). There was no correlation between lysozyme, viral load, CD4 cell count or use of HAART. CONCLUSIONS: A comparison of protein profiles from M/M phi supernatants of patients with HIV-1 infection indicated a specific protein consistently decreased in HAD. The protein was identified as lysozyme, a major macrophage defense protein. This further demonstrates macrophage dysfunction as a significant consequence of HAD.
Assuntos
Complexo AIDS Demência/enzimologia , HIV-1 , Macrófagos/enzimologia , Muramidase/sangue , Complexo AIDS Demência/tratamento farmacológico , Complexo AIDS Demência/virologia , Terapia Antirretroviral de Alta Atividade , Biomarcadores/sangue , Contagem de Linfócito CD4 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , Infecções por HIV/virologia , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Carga ViralRESUMO
The mechanisms of neurodegeneration that result in human immunodeficiency virus (HIV) type 1 dementia have not yet been identified. Here, we report that HIV-infected macrophages secrete the zymogen matrix metalloproteinase-2 (MMP-2), which is activated by exposure to MT1-MMP on neurons. Stromal cell-derived factor 1 alpha (SDF-1), a chemokine overexpressed by astrocytes during HIV infection, was converted to a highly neurotoxic protein after precise proteolytic processing by active MMP-2, which removed the N-terminal tetrapeptide. Implantation of cleaved SDF-1(5-67) into the basal ganglia of mice resulted in neuronal death and inflammation with ensuing neurobehavioral deficits that were abrogated by neutralizing antibodies to SDF-1 and an MMP inhibitor drug. Hence, this study identifies a new in vivo neurotoxic pathway in which cleavage of a chemokine by an induced metalloproteinase results in neuronal apoptosis that leads to neurodegeneration.
Assuntos
Complexo AIDS Demência/enzimologia , Quimiocinas CXC/toxicidade , Metaloproteinase 2 da Matriz/metabolismo , Degeneração Neural/enzimologia , Neurotoxinas/toxicidade , Complexo AIDS Demência/etiologia , Complexo AIDS Demência/fisiopatologia , Animais , Anticorpos/farmacologia , Astrócitos/metabolismo , Linhagem Celular , Quimiocina CXCL12 , Quimiocinas CXC/antagonistas & inibidores , Quimiocinas CXC/metabolismo , Modelos Animais de Doenças , Encefalite/induzido quimicamente , Encefalite/enzimologia , Encefalite/fisiopatologia , Inibidores Enzimáticos/farmacologia , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Macrófagos/enzimologia , Macrófagos/metabolismo , Inibidores de Metaloproteinases de Matriz , Camundongos , Neostriado/efeitos dos fármacos , Neostriado/patologia , Neostriado/fisiopatologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/virologia , Neurotoxinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidadeRESUMO
Proteinase-activated receptor 1 (PAR-1) is a G protein-coupled receptor that is activated by thrombin and is implicated in the pathogenesis of inflammation. Although PAR-1 is expressed on immunocompetent cells within the brain such as astrocytes, little is known about its role in the pathogenesis of inflammatory brain diseases. Herein, we investigated PAR-1 regulation of brain inflammation by stimulating human astrocytic cells with thrombin or the selective PAR-1-activating peptide. Activated cells expressed significantly increased levels of IL-1 beta, inducible NO synthase, and PAR-1 mRNA. Moreover, supernatants of these same cells were neurotoxic, which was inhibited by an N-methyl-D-aspartate receptor antagonist. Striatal implantation of the PAR-1-activating peptide significantly induced brain inflammation and neurobehavioral deficits in mice compared with mice implanted with the control peptide or saline. Since HIV-related neurological disease is predicated on brain inflammation and neuronal injury, the expression of PAR-1 in HIV encephalitis (HIVE) was investigated. Immunohistochemical analysis revealed that PAR-1 and (pro)-thrombin protein expression was low in control brains, but intense immunoreactivity was observed on astrocytes in HIVE brains. Similarly, PAR-1 and thrombin mRNA levels were significantly increased in HIVE brains compared with control and multiple sclerosis brains. These data indicated that activation and up-regulation of PAR-1 probably contribute to brain inflammation and neuronal damage during HIV-1 infection, thus providing new therapeutic targets for the treatment of HIV-related neurodegeneration.
Assuntos
Complexo AIDS Demência/metabolismo , Astrócitos/metabolismo , Receptores de Trombina/biossíntese , Regulação para Cima , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/fisiopatologia , Sequência de Aminoácidos , Animais , Astrócitos/enzimologia , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Sistema Livre de Células/fisiologia , Corpo Estriado/imunologia , Corpo Estriado/metabolismo , Corpo Estriado/fisiologia , Implantes de Medicamento , Feto , HIV-1/fisiologia , Humanos , Interleucina-1/biossíntese , Masculino , Camundongos , Dados de Sequência Molecular , Esclerose Múltipla/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neurotoxinas/metabolismo , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Peptídeos/administração & dosagem , Peptídeos/fisiologia , Receptor PAR-1 , Receptores de N-Metil-D-Aspartato/fisiologia , Receptores de Trombina/administração & dosagem , Receptores de Trombina/agonistas , Receptores de Trombina/fisiologia , Trombina/farmacologia , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
Many patients infected with human immunodeficiency virus-1 (HIV-1) develop a syndrome of neurologic deterioration known as HIV-associated dementia (HAD). Neurons are not productively infected by HIV-1; thus, the mechanism of HIV-induced neuronal injury remains incompletely understood. Several investigators have observed evidence of neuronal injury, including dendritic degeneration, and apoptosis in CNS tissue from patients with HAD. Caspase enzymes, proteases associated with the process of apoptosis, are synthesized as inactive proenzymes and are activated in a proteolytic cascade after exposure to apoptotic signals. Here we demonstrate that HAD is associated with active caspase-3-like immunoreactivity that is localized to the soma and dendrites of neurons in affected regions of the human brain. Additionally, the cascade of caspase activation was studied using an in vitro model of HIV-induced neuronal apoptosis. Increased caspase-3 proteolytic activity and mitochondrial release of cytochrome c were observed in cerebrocortical cultures exposed to the HIV coat protein gp120. Specific inhibitors of both the Fas/tumor necrosis factor-alpha/death receptor pathway and the mitochondrial caspase pathway prevented gp120-induced neuronal apoptosis. Caspase inhibition also prevented the dendrite degeneration observed in vivo in transgenic mice with CNS expression of HIV/gp120. These findings suggest that pharmacologic interventions aimed at the caspase enzyme pathways may be beneficial for the prevention or treatment of HAD.
Assuntos
Complexo AIDS Demência/enzimologia , Apoptose , Caspases/metabolismo , Adulto , Animais , Apoptose/efeitos dos fármacos , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Caspase 1/genética , Caspase 3 , Caspase 8 , Caspase 9 , Inibidores de Caspase , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Dendritos/enzimologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/toxicidade , Humanos , Camundongos , Camundongos Transgênicos , Microglia/citologia , Microglia/efeitos dos fármacos , Pessoa de Meia-Idade , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Estudos Prospectivos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Receptor fas/efeitos dos fármacosRESUMO
Human immunodeficiency virus type-1 (HIV-1)-associated dementia (HAD) is a neurodegenerative disease characterized by HIV infection and replication in brain tissue. HIV-1-infected monocytes overexpress inflammatory molecules that facilitate their entry into the brain. Prostanoids are lipid mediators of inflammation that result from cyclooxygenase-2 (COX-2) activity. Because COX-2 is normally induced during inflammatory processes, the aim of this study was to investigate whether COX-2 expression is up-regulated during monocyte-brain endothelium interactions. In vitro cocultures of HIV-infected macrophages and brain endothelium showed an up-regulation of COX-2 expression by both cell types. This up-regulation occurs via an interleukin-1beta (IL1beta)-dependent mechanism in macrophages and via an IL-1beta-independent mechanism in endothelial cells. Thus, interactions between HIV-infected monocytes and brain endothelium result in COX-2 expression and, as such, might contribute to the neuropathogenesis of HIV infection.
Assuntos
Complexo AIDS Demência/enzimologia , Encéfalo/irrigação sanguínea , Comunicação Celular/fisiologia , Endotélio Vascular/enzimologia , HIV-1 , Isoenzimas/biossíntese , Macrófagos/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Complexo AIDS Demência/sangue , Complexo AIDS Demência/patologia , Encéfalo/virologia , Técnicas de Cocultura , Ciclo-Oxigenase 2 , Endotélio Vascular/citologia , Humanos , Interleucina-1/biossíntese , Isoenzimas/genética , Macrófagos/citologia , Macrófagos/virologia , Proteínas de Membrana , Monócitos/citologia , Monócitos/enzimologia , Monócitos/virologia , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
Infection of the brain by lentiviruses, including human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV), causes inflammation and results in neurodegeneration. Molecular diversity within the lentivirus envelope gene has been implicated in the regulation of cell tropism and the host response to infection. Here, we examine the hypothesis that envelope sequence diversity modulates the expression of host molecules implicated in lentivirus-induced brain disease, including matrix metalloproteinases (MMP) and related transcription factors. Infection of primary macrophages by chimeric HIV clones containing brain-derived envelope fragments from patients with HIV-associated dementia (HAD) or nondemented AIDS patients (HIV-ND) showed that MMP-2 and -9 levels in conditioned media were significantly higher for the HAD clones. Similarly, STAT-1 and JAK-1 levels were higher in macrophages infected by HAD clones. Infections of primary feline macrophages by the neurovirulent FIV strain (V(1)CSF), the less neurovirulent strain (Petaluma), and a chimera containing the V(1)CSF envelope in a Petaluma background (FIV-Ch) revealed that MMP-2 and -9 levels were significantly higher in conditioned media from V(1)CSF- and FIV-Ch-infected macrophages, which was associated with increased intracellular STAT-1 and JAK-1 levels. The STAT-1 inhibitor fludarabine significantly reduced MMP-2 expression, but not MMP-9 expression, in FIV-infected macrophages. Analysis of MMP mRNA and protein levels in brain samples from HIV-infected persons or FIV-infected cats showed that MMP-2 and -9 levels were significantly increased in lentivirus-infected brains compared to those of uninfected controls. Elevated MMP expression was accompanied by significant increases in STAT-1 and JAK-1 mRNA and protein levels in the same brain samples. The present findings indicate that two lentiviruses, HIV and FIV, have common mechanisms of MMP-2 and -9 induction, which is modulated in part by envelope sequence diversity and the STAT-1/JAK-1 signaling pathway.
Assuntos
Encéfalo/virologia , Genes env/genética , Variação Genética , Infecções por Lentivirus/enzimologia , Lentivirus/genética , Metaloproteinases da Matriz/metabolismo , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/virologia , Animais , Encéfalo/enzimologia , Gatos , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , HIV/genética , HIV/metabolismo , HIV/fisiologia , Infecções por HIV/enzimologia , Infecções por HIV/virologia , Humanos , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Lentivirus/metabolismo , Lentivirus/fisiologia , Infecções por Lentivirus/virologia , Macrófagos/enzimologia , Macrófagos/virologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT1 , Transdução de Sinais , Transativadores/metabolismo , Proteínas do Envelope Viral/metabolismo , Replicação ViralRESUMO
Matrix metalloproteinases (MMPs) have been identified as mediators of brain injury in HIV-associated neurological diseases. The activity of the 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) was detected by zymography in the cerebrospinal fluid (CSF) of 138 HIV-infected patients (40 with AIDS dementia, 83 with brain opportunistic infections and 15 neurologically asymptomatic), 26 HIV-seronegative individuals with inflammatory neurological diseases (IND) and 12 HIV-seronegative subjects with noninflammatory neurological diseases (NIND). MMP-2 was present in all CSF samples from HIV-seropositive and HIV-seronegative individuals, including those of subjects with NIND. On the contrary, MMP-9 was absent in the CSF of NIND controls, whereas the activity of this MMP was found in the 77 - 100% of CSF samples from HIV-infected patients, including those with HIV dementia, central nervous system (CNS) opportunistic infections or neurologically asymptomatic subjects. The highest levels of MMP-9 were found in the CSF of patients with cryptococcosis, cytomegalovirus encephalitis and tuberculous meningitis and were comparable with those found in the CSF of HIV-negative patients with multiple sclerosis or meningitis. A significant correlation between CSF MMP-9 activity and CSF cell count was found only in patients with HIV dementia. The increased CSF activity of MMPs capable to degrade components of the extracellular matrix of blood-brain barrier may contribute to the transendothelial migration of virus-infected cells into the CNS and development of HIV-associated neurologic damage.
Assuntos
Complexo AIDS Demência/líquido cefalorraquidiano , Infecções Oportunistas Relacionadas com a AIDS/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/análise , Infecções por HIV/líquido cefalorraquidiano , HIV-1 , Metaloproteinase 2 da Matriz/líquido cefalorraquidiano , Metaloproteinase 9 da Matriz/líquido cefalorraquidiano , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Complexo AIDS Demência/enzimologia , Infecções Oportunistas Relacionadas com a AIDS/enzimologia , Adolescente , Adulto , Barreira Hematoencefálica , Contagem de Linfócito CD4 , Contagem de Células , Movimento Celular , Líquido Cefalorraquidiano/citologia , Criança , Pré-Escolar , Infecções por Citomegalovirus/líquido cefalorraquidiano , Infecções por Citomegalovirus/enzimologia , Progressão da Doença , Encefalite Viral/líquido cefalorraquidiano , Encefalite Viral/enzimologia , Feminino , Infecções por HIV/enzimologia , Soronegatividade para HIV , Humanos , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/genética , Meningites Bacterianas/líquido cefalorraquidiano , Meningites Bacterianas/enzimologia , Meningite Criptocócica/líquido cefalorraquidiano , Meningite Criptocócica/enzimologia , Pessoa de Meia-Idade , Doença dos Neurônios Motores/líquido cefalorraquidiano , Doença dos Neurônios Motores/enzimologia , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/enzimologia , Doenças do Sistema Nervoso/enzimologia , Toxoplasmose Cerebral/líquido cefalorraquidiano , Toxoplasmose Cerebral/enzimologia , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/enzimologiaRESUMO
In the human immunodeficiency virus type 1 (HIV-1)-infected brain, the virus does not replicate in astrocytes, but a synthesis of viral regulatory proteins occurs in these cells, leading to accumulation of Nef. As an approach to understand the effects of Nef on astrocyte functional activity, we analyzed whether intracellular Nef interferes with the expression and activation of the enzyme protein kinase C (PKC), which is an important regulator of astroglial functions and HIV-1 replication. Astrocytoma clones (U251 MG) not expressing Nef (Neo), or expressing wild-type Nef (Bru) or nonmyristoylated Nef (TH) were used to monitor the expression and activation of 10 PKC isoforms. The same clones were used to evaluate the effect of Nef on the viral long terminal repeat (LTR) promoter after activation of PKC with the phorbol ester 12-myristate 13-acetate (PMA). PKC intracellular distribution and activation were evaluated by Western blot analysis of cytosolic and membrane fractions of control and Nef-expressing clones. PMA-induced LTR activation was analyzed in clones transfected with a plasmid encoding for the CAT reporter gene controlled by the LTR promoter, by using an enzyme-linked immunosorbent assay to measure CAT expression. Nef selectively downregulated the expression and activation of betaII and epsilon PKC isoforms in astrocytoma cells. Such downregulation correlated with an inhibition of LTR activation after PMA stimulation. The myristoylation of Nef and its membrane localization were essential for these effects. These results suggest that Nef may alter astrocytic functions by interfering with PKC expression and activation and contribute to the restriction of HIV-1 replication in astrocytes.
Assuntos
Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Produtos do Gene nef/fisiologia , HIV-1/fisiologia , Isoenzimas/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Proteína Quinase C/biossíntese , Sequências Repetidas Terminais , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/virologia , Acilação , Astrocitoma/enzimologia , Neoplasias Encefálicas/enzimologia , Cloranfenicol O-Acetiltransferase/biossíntese , Indução Enzimática/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Produtos do Gene nef/química , Genes Reporter , Humanos , Isoenzimas/genética , Ácido Mirístico/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Proteína Quinase C/genética , Proteína Quinase C beta , Proteína Quinase C-épsilon , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Patients infected with HIV-1 often exhibit cognitive deficits that are related to progressive neuronal degeneration and cell death. The protein Tat, which is released from HIV-1-infected cells, was recently shown to be toxic toward cultured neurons. We now report that Tat induces apoptosis in cultured embryonic rat hippocampal neurons. Tat induced caspase activation, and the caspase inhibitor zVAD-fmk prevented Tat-induced neuronal death. Tat induced a progressive elevation of cytoplasmic-free calcium levels, which was followed by mitochondrial calcium uptake and generation of mitochondrial-reactive oxygen species (ROS). The intracellular calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red protected neurons against Tat-induced apoptosis. zVAD-fmk suppressed Tat-induced increases of cytoplasmic calcium levels and mitochondrial ROS accumulation, indicating roles for caspases in the perturbed calcium homeostasis and oxidative stress induced by Tat. An inhibitor of nitric oxide synthase, and the peroxynitrite scavenger uric acid, protected neurons against Tat-induced apoptosis, indicating requirements for nitric oxide production and peroxynitrite formation in the cell death process. Finally, Tat caused a delayed and progressive mitochondrial membrane depolarization, and cyclosporin A prevented Tat-induced apoptosis, suggesting an important role for mitochondrial membrane permeability transition in Tat-induced apoptosis. Collectively, our data demonstrate that Tat can induce neuronal apoptosis by a mechanism involving disruption of calcium homeostasis, caspase activation, and mitochondrial calcium uptake and ROS accumulation. Agents that interupt this apoptotic cascade may prove beneficial in preventing neuronal degeneration and associated dementia in AIDS patients.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Caspases/metabolismo , Produtos do Gene tat/farmacologia , Neurônios/citologia , Complexo AIDS Demência/enzimologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/fisiologia , Inibidores de Caspase , Células Cultivadas , Quelantes/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Citoplasma/enzimologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Ativação Enzimática/efeitos dos fármacos , HIV-1 , Hipocampo/citologia , Potenciais da Membrana/fisiologia , Mitocôndrias/enzimologia , Neurônios/enzimologia , Neurônios/virologia , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Oxidantes/metabolismo , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Indirect mechanisms are implicated in the pathogenesis of the dementia associated with human immunodeficiency virus-type 1 (HIV-1) infection. Proinflammatory molecules such as tumor necrosis factor alpha and eicosanoids are elevated in the central nervous system of patients with HIV-1-related dementia. Nitric oxide (NO) is a potential mediator of neuronal injury, because cytokines may activate the immunologic (type II) isoform of NO synthase (iNOS). The levels of iNOS in severe HIV-1-associated dementia coincided with increased expression of the HIV-1 coat protein gp41. Furthermore, gp41 induced iNOS in primary cultures of mixed rat neuronal and glial cells and killed neurons through a NO-dependent mechanism. Thus, gp41-induced NO formation may contribute to the severe cognitive dysfunction associated with HIV-1 infection.
Assuntos
Complexo AIDS Demência/enzimologia , Encéfalo/enzimologia , Proteína gp41 do Envelope de HIV/metabolismo , HIV-1 , Óxido Nítrico Sintase/biossíntese , Complexo AIDS Demência/metabolismo , Animais , Encéfalo/metabolismo , Morte Celular , Células Cultivadas , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Indução Enzimática , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp120 do Envelope de HIV/farmacologia , Proteína gp41 do Envelope de HIV/farmacologia , Humanos , Neuroglia/citologia , Neurônios/citologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Reação em Cadeia da Polimerase , RatosRESUMO
Brains from AIDS patients with an HIV-induced encephalopathy but without opportunistic infections or indications for an inflammation were studied by immuno- and enzyme-histochemical methods. It was found that the macrophages of these brains expressed a lysosomal tartrate-resistant acid phosphatase which gave a good immunological cross-reaction with an antibody to the well-characterized iron-containing bovine spleen purple acid phosphatase, belonging to the group of purple phosphatases, which are regarded as a marker for a special phenotype of activated macrophages. It was discussed that the numerous brain macrophages found in AIDS encephalopathy derive from latently infected monocytes which are believed to be drawn to the brain from the bloodstream.