RESUMEN
The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES+/CD133+) and their differentiated (diff) progeny cells (NES-/CD133-). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFß1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53ß in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.
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Astrocitos/metabolismo , Neoplasias Encefálicas/metabolismo , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Anciano , Diferenciación Celular , Línea Celular Tumoral , Senescencia Celular , Vesículas Extracelulares/ultraestructura , Gelatina/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Nanopartículas/ultraestructura , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Tamaño de la Partícula , Fenotipo , Podosomas/metabolismo , Isoformas de Proteínas/metabolismo , Proteolisis , Proteoma/metabolismoRESUMEN
Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.
Asunto(s)
Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Quinurenina 3-Monooxigenasa/antagonistas & inhibidores , Cromatografía Líquida de Alta Presión/métodos , FluorescenciaRESUMEN
The unfolded protein response (UPR) is a highly evolutionarily conserved response to endoplasmic reticulum (ER) stress, which functions to return cells to homeostasis or send them into apoptosis, depending on the degree of cellular damage. ß-N-methylamino-L-alanine (L-BMAA) has been shown to induce ER stress in a variety of models and has been linked to several types of neurodegenerative disease including Guamanian amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC). L-Serine, an amino acid critical for cellular metabolism and neurological signaling, has been shown to be protective against L-BMAA-induced neurotoxicity in both animal and cell culture models. While the mechanisms of L-BMAA neurotoxicity have been well characterized, less is known about L-serine neuroprotection. We recently reported that L-serine and L-BMAA generate similar differential expression profiles in a human ER stress/UPR array, despite L-serine being neuroprotective and L-BMAA being linked to neurodegenerative disease. Here, we further investigate the mechanism(s) of L-serine-induced UPR dysregulation by examining key genes and proteins in the ER stress/UPR pathways. We report that L-serine selectively increased protein disulfide isomerase (PDI) protein translation, an ER chaperone involved in refolding misfolded proteins, suggesting it may be modulating the UPR to favor recovery from ER stress. This constitutes a new mechanism for L-serine-mediated neuroprotection and has implications for its use as a therapy for neurodegenerative illnesses.
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Estrés del Retículo Endoplásmico/efectos de los fármacos , Neuroprotección/efectos de los fármacos , Proteína Disulfuro Isomerasas/metabolismo , Serina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Aminoácidos Diaminos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Toxinas de Cianobacterias , Humanos , L-Lactato Deshidrogenasa/metabolismo , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/prevención & control , Neuroblastoma/patología , Proteína Disulfuro Isomerasas/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 1 de Unión a la X-Box/genética , Proteína 1 de Unión a la X-Box/metabolismoRESUMEN
ß-N-methylamino-L-alanine (L-BMAA) is a neurotoxic non-protein amino acid produced by cyanobacteria. Recently, chronic dietary exposure to L-BMAA was shown to trigger neuropathology in nonhuman primates consistent with Guamanian ALS/PDC, a paralytic disease that afflicts Chamorro villagers who consume traditional food items contaminated with L-BMAA. However, the addition of the naturally occurring amino acid L-serine to the diet of the nonhuman primates resulted in a significant reduction in ALS/PDC neuropathology. L-serine is a dietary amino acid that plays a crucial role in central nervous system development, neuronal signaling, and synaptic plasticity and has been shown to impart neuroprotection from L-BMAA-induced neurotoxicity both in vitro and in vivo. We have previously shown that L-serine prevents the formation of autofluorescent aggregates and death by apoptosis in human cell lines and primary cells. These effects are likely imparted by L-serine blocking incorporation of L-BMAA into proteins hence preventing proteotoxic stress. However, there are likely other mechanisms for L-serine-mediated neuroprotection. Here, we explore the molecular mechanisms of L-serine neuroprotection using a human unfolded protein response real-time PCR array with genes from the ER stress and UPR pathways, and western blotting. We report that L-serine caused the differential expression of many of the same genes as L-BMAA, even though concentrations of L-serine in the culture medium were ten times lower than that of L-BMAA. We propose that L-serine may be functioning as a small proteostasis regulator, in effect altering the cells to quickly respond to a possible oxidative insult, thus favoring a return to homeostasis.
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Retículo Endoplásmico/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Proteostasis/efectos de los fármacos , Serina/farmacología , Aminoácidos Diaminos/toxicidad , Análisis de Varianza , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Toxinas de Cianobacterias , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Agonistas de Aminoácidos Excitadores/toxicidad , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neuroblastoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pliegue de Proteína/efectos de los fármacos , Proteolisis/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genéticaRESUMEN
We have previously demonstrated that the kynurenine pathway (KP), the major biochemical pathway for tryptophan metabolism, is dysregulated in many inflammatory disorders that are often associated with sexual dimorphisms. We aimed to identify a potential functional interaction between the KP and gonadal hormones. We have treated primary human macrophages with progesterone in the presence and absence of inflammatory cytokine interferon-gamma (interferon-γ) that is known to be a potent inducer of regulating the KP enzyme. We found that progesterone attenuates interferon-γ-induced KP activity, decreases the levels of the excitotoxin quinolinic acid, and increases the neuroprotective kynurenic acid levels. We also showed that progesterone was able to reduce the inflammatory marker neopterin. These results may shed light on the gender disparity in response to inflammation.
RESUMEN
Emerging evidence suggests that inflammation has a key role in depression and suicidal behavior. The kynurenine pathway is involved in neuroinflammation and regulates glutamate neurotransmission. In the cerebrospinal fluid (CSF) of suicidal patients, levels of inflammatory cytokines and the kynurenine metabolite quinolinic acid (QUIN), an N-methyl-d-aspartate receptor agonist, are increased. The enzyme amino-ß-carboxymuconate-semialdehyde-decarboxylase (ACMSD) limits QUIN formation by competitive production of the neuroprotective metabolite picolinic acid (PIC). Therefore, decreased ACMSD activity can lead to excess QUIN. We tested the hypothesis that deficient ACMSD activity underlies suicidal behavior. We measured PIC and QUIN in CSF and plasma samples from 137 patients exhibiting suicidal behavior and 71 healthy controls. We used DSM-IV and the Montgomery-Åsberg Depression Rating Scale and Suicide Assessment Scale to assess behavioral changes. Finally, we genotyped ACMSD tag single-nucleotide polymorphisms (SNPs) in 77 of the patients and 150 population-based controls. Suicide attempters had reduced PIC and a decreased PIC/QUIN ratio in both CSF (P<0.001) and blood (P=0.001 and P<0.01, respectively). The reductions of PIC in CSF were sustained over 2 years after the suicide attempt based on repeated measures. The minor C allele of the ACMSD SNP rs2121337 was more prevalent in suicide attempters and associated with increased CSF QUIN. Taken together, our data suggest that increased QUIN levels may result from reduced activity of ACMSD in suicidal subjects. We conclude that measures of kynurenine metabolites can be explored as biomarkers of suicide risk, and that ACMSD is a potential therapeutic target in suicidal behavior.
Asunto(s)
Carboxiliasas/genética , Ácidos Picolínicos/líquido cefalorraquídeo , Ácido Quinolínico/líquido cefalorraquídeo , Conducta Autodestructiva/genética , Ideación Suicida , Intento de Suicidio , Adolescente , Adulto , Anciano , Alelos , Estudios de Casos y Controles , Niño , Femenino , Humanos , Inflamación , Quinurenina/metabolismo , Masculino , Persona de Mediana Edad , Ácidos Picolínicos/sangre , Polimorfismo de Nucleótido Simple , Ácido Quinolínico/sangre , Conducta Autodestructiva/sangre , Conducta Autodestructiva/líquido cefalorraquídeo , Adulto JovenRESUMEN
Schizophrenia has a clear sexual dimorphism in age of onset and progression. The underlying mechanisms of this dimorphism are not known, but may be found in the interactions of sex hormones with the tryptophan catabolising kynurenine pathway. Schizophrenia is associated with general inflammation and disruption of glutamatergic and dopaminergic signalling. Metabolites of the kynurenine pathway have been shown to be immunomodulatory and have effects on glutamatergic and dopaminergic signalling. This review discusses the currently available literature on sex hormones and their effect on the kynurenine pathway in the context of the glutamatergic, dopaminergic and immunological features of schizophrenia.
Asunto(s)
Quinurenina/metabolismo , Esquizofrenia/inmunología , Caracteres Sexuales , Animales , Humanos , Neuroinmunomodulación/fisiologíaRESUMEN
MAPK-activated protein kinase 2 (MK2) is a checkpoint kinase involved in the DNA damage response. MK2 inhibition enhances the efficacy of chemotherapeutic agents; however, whether MK2 inhibition alone, without concurrent chemotherapy, would attenuate survival of cancer cells has not been investigated. CMPD1 is a widely used non-ATP competitive inhibitor that prevents MK2 phosphorylation. We employed CMPD1 together with MK2 knock-down and ATP-competitive MK2 inhibitor III (MK2i) in a panel of glioblastoma cells to assess whether MK2 inhibition could induce cancer cell death. While CMPD1 was effective at selective killing of cancer cells, MK2i and MK2 knock-down had no effect on viability of glioblastoma cells. CMPD1 treatment induced a significant G2/M arrest but MK2i-treated cells were only minimally arrested at G1 phase. Intriguingly, at doses that were cytotoxic to glioblastoma cells, CMPD1 did not inhibit phosphorylation of MK2 and of its downstream substrate Hsp27. These results suggest that CMPD1 exhibits cytotoxic activity independently of MK2 inhibition. Indeed, we identified tubulin as a primary target of the CMPD1 cytotoxic activity. This study demonstrates how functional and mechanistic studies with appropriate selection of test compounds, combining genetic knock-down and pharmacological inhibition, coordinating timing and dose levels enabled us to uncover the primary target of an MK2 inhibitor commonly used in the research community. Tubulin is emerging as one of the most common non-kinase targets for kinase inhibitors and we propose that potential tubulin-targeting activity should be assessed in preclinical pharmacology studies of all novel kinase inhibitors.
RESUMEN
The microenvironment of glioblastoma (GBM) contains high levels of inflammatory cytokine interleukin 6 (IL-6), which contributes to promote tumour progression and invasion. The common epidermal growth factor receptor variant III (EGFRvIII) mutation in GBM is associated with significantly higher levels of IL-6. Furthermore, elevated IL-1ß levels in GBM tumours are also believed to activate GBM cells and enhance IL-6 production. However, the crosstalk between these intrinsic and extrinsic factors within the oncogene-microenvironment of GBM causing overproduction of IL-6 is poorly understood. Here, we show that EGFRvIII potentiates IL-1ß-induced IL-6 secretion from GBM cells. Importantly, exacerbation of IL-6 production is most effectively attenuated in EGFRvIII-expressing GBM cells with inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) and MAPK-activated protein kinase 2 (MK2). Enhanced IL-6 production and increased sensitivity toward pharmacological p38 MAPK and MK2 inhibitors in EGFRvIII-expressing GBM cells is associated with increased MK2-dependent nuclear-cytoplasmic shuttling and accumulation of human antigen R (HuR), an IL-6 mRNA-stabilising protein, in the cytosol. IL-1ß-stimulated activation of the p38 MAPK-MK2-HuR pathway significantly enhances IL-6 mRNA stability in GBM cells carrying EGFRvIII. Further supporting a role for the p38 MAPK-MK2-HuR pathway in the development of inflammatory environment in GBM, activated MK2 is found in more than 50% of investigated GBM tissues and correlates with lower grade and secondary GBMs. Taken together, p38 MAPK-MK2-HuR signalling may enhance the potential of intrinsic (EGFRvIII) and extrinsic (IL-1ß) factors to develop an inflammatory GBM environment. Hence, further improvement of brain-permeable and anti-inflammatory inhibitors targeting p38 MAPK, MK2 and HuR may combat progression of lower grade gliomas into aggressive GBMs.
Asunto(s)
Neoplasias Encefálicas , Receptores ErbB/farmacología , Glioblastoma , Interleucina-1beta/farmacología , Interleucina-6/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proteínas ELAV/metabolismo , Proteína 1 Similar a ELAV , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Inflamación/genética , Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células Tumorales Cultivadas , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Ciguatoxin (CTX), is a toxic compound produced by microalgae (dinoflagellate) Gambierdiscus spp., and is bio-accumulated and bio-transformed through the marine food chain causing neurological deficits. To determine the mechanism of CTX-mediated cytotoxicity in human neurons, we measured extracellular lactate dehydrogenase (LDH) activity, intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) and H2AX phosphorylation at serine 139 as a measure for DNA damage in primary cultures of human neurons treated with Pacific (P)-CTX-1B and P-CTX-3C. We found these marine toxins can induce a time and dose-dependent increase in extracellular LDH activity, with a concomitant decline in intracellular NAD(+) levels and increased DNA damage at the concentration range of 5-200 nM. We also showed that pre- and post-treatment with rosmarinic acid (RA), the active constituent of the Heliotropium foertherianum (Boraginaceae) can attenuate CTX-mediated neurotoxicity. These results further highlight the potential of RA in the treatment of CTX-induced neurological deficits.
Asunto(s)
Ciguatoxinas/toxicidad , Cinamatos/farmacología , Daño del ADN/efectos de los fármacos , Depsidos/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Supervivencia Celular , Células Cultivadas , Feto , Humanos , Neuronas/enzimología , Ácido RosmarínicoRESUMEN
BACKGROUND: Quinolinic acid (QUIN) excitotoxicity is mediated by elevated intracellular Ca2+ levels, and nitric oxide (NOâ¢) mediated oxidative stress leading to DNA damage, and cell death due to energy restriction. METHODS: We evaluated the effect of a series of pomegranate juice extracts (PJE), Helow, Malasi, Qusum, and Hamedh, with antioxidant properties on QUIN induced excitotoxicity on primary cultures of human neurons. RESULTS: We showed that Helow and Malasi can attenuate QUIN-induced excitotoxicity to a greater extent than Qusum and Hamedh from Oman. Similarly, both Helow and Malasi were able to attenuate QUIN-induced Ca2+ influx and nNOS activity to a greater extent compared to Qusum, and Hamedh. All extracts reduced the oxidative effects of increased NO⢠production, and hence preventing NAD+ depletion and cell death. CONCLUSION: In addition to the well-known antioxidant properties of these natural phytochemicals, the inhibitory effect of some of these compounds on specific excitotoxic processes such as calcium influx provides additional evidence for the beneficial health effects of PJE in excitable tissue, particularly within the CNS.
RESUMEN
Autism is a debilitating neurodevelopment disorder characterised by stereotyped interests and behaviours, and abnormalities in verbal and non-verbal communication. It is a multifactorial disorder resulting from interactions between genetic, environmental and immunological factors. Excitotoxicity and oxidative stress are potential mechanisms, which are likely to serve as a converging point to these risk factors. Substantial evidence suggests that excitotoxicity, oxidative stress and impaired mitochondrial function are the leading cause of neuronal dysfunction in autistic patients. Glutamate is the primary excitatory neurotransmitter produced in the CNS, and overactivity of glutamate and its receptors leads to excitotoxicity. The over excitatory action of glutamate, and the glutamatergic receptors NMDA and AMPA, leads to activation of enzymes that damage cellular structure, membrane permeability and electrochemical gradients. The role of excitotoxicity and the mechanism behind its action in autistic subjects is delineated in this review.
Asunto(s)
Trastorno Autístico/etiología , Trastorno Autístico/metabolismo , Ácido Glutámico/metabolismo , Estrés Oxidativo/fisiología , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Trastorno Autístico/patología , Señalización del Calcio/fisiología , HumanosRESUMEN
As many as 2000 IEQs (islet equivalent) of encapsulated human islets are required to normalize glucose levels in diabetic mice. To reduce this number, encapsulated islets were exposed to 100 µM desferrioxamine (DFO) prior to transplantation. Cell viability, glucose-induced insulin secretion, VEGF (Vascular endothelial growth factor), HIF-1α (Hypoxia inducible factor-1 alpha), caspase-3 and caspase-8 levels were assessed after exposure to DFO for 12, 24 or 72 h. Subsequently, 1000, 750 or 500 encapsulated IEQs were infused into peritoneal cavity of diabetic mice after 24 h exposure to DFO. Neither viability nor function in vitro was affected by DFO, and levels of caspase-3 and caspase-8 were unchanged. DFO significantly enhanced VEGF secretion by 1.6- and 2.5-fold at 24 and 72 h, respectively, with a concomitant increase in HIF-1α levels. Euglycemia was achieved in 100% mice receiving 1000 preconditioned IEQs, as compared to only 36% receiving unconditioned IEQs (p < 0.001). Similarly, with 750 IEQ, euglycemia was achieved in 50% mice receiving preconditioned islets as compared to 10% receiving unconditioned islets (p = 0.049). Mice receiving preconditioned islets had lower glucose levels than those receiving unconditioned islets. In summary, DFO treatment enhances HIF-1α and VEGF expression in encapsulated human islets and improves their ability to function when transplanted.
Asunto(s)
Deferoxamina/uso terapéutico , Diabetes Mellitus Experimental/tratamiento farmacológico , Islotes Pancreáticos/efectos de los fármacos , Sideróforos/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Glucemia/metabolismo , Cadáver , Caspasas/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Experimental/fisiopatología , Modelos Animales de Enfermedad , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Insulina/metabolismo , Secreción de Insulina , Trasplante de Islotes Pancreáticos , Ratones , Técnicas de Cultivo de Tejidos , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The kynurenine pathway has been implicated as a major component of the neuroinflammatory response to brain injury and neurodegeneration. We found that the neurotoxic kynurenine pathway intermediate quinolinic acid (QUIN) is rapidly expressed, within 24 h, by reactive microglia following traumatic injury to the rodent neocortex. Furthermore, administration of the astrocytic protein metallothionein attenuated this neuroinflammatory response by reducing microglial activation (by approximately 30%) and QUIN expression. The suppressive effect of MT was confirmed upon cultured cortical microglia, with 1 mug/ml MT almost completely blocking interferon-gamma induced activation of microglia and QUIN expression. These results demonstrate the neuroimmunomodulatory properties of MT, which may have therapeutic applications for the treatment of traumatic brain injury.
Asunto(s)
Lesiones Encefálicas/patología , Regulación de la Expresión Génica/efectos de los fármacos , Metalotioneína/farmacología , Microglía/efectos de los fármacos , Ácido Quinolínico/metabolismo , Análisis de Varianza , Animales , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/metabolismo , Recuento de Células/métodos , Células Cultivadas , Corteza Cerebral/citología , Medios de Cultivo Condicionados/farmacología , Relación Dosis-Respuesta a Droga , Ferritinas/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Proteína Ácida Fibrilar de la Glía/metabolismo , Interferón gamma/farmacología , Microglía/química , Neocórtex/metabolismo , Neocórtex/patología , Neuronas/patología , Ácido Quinolínico/análisis , Ratas , Ratas WistarRESUMEN
BACKGROUND AND PURPOSE: 5-HT (serotonin) is known to be involved in neuroinflammation and immunoregulation. The human immunodeficiency virus (HIV) targets cells such as monocytes/macrophages, which colocalize with 5-HT-releasing cell types, mostly platelets. In this study, we investigated the effects of 5-HT on HIV-1-infected macrophages in vitro. EXPERIMENTAL APPROACH: Human macrophages cultured in serum-free medium were treated over 7 days with 5-HT at three concentrations (0.01, 1 and 100 microM) with or without agonists and antagonists of 5-HT(1A) and 5-HT(2) receptors. After 7 days of treatment, macrophages were infected with HIV-1/Ba-L and virus replication was monitored over 16 days and expression of proviral HIV DNA was investigated by PCR after 24 h of infection. Cell surface expression of HIV-1/Ba-L receptor (CD4) and coreceptor (CCR5) was investigated by flow cytometry. The CCR5 ligand, macrophage inflammatory protein-1alpha (MIP-1alpha), was quantified by ELISA in cell culture supernatants and MIP-1alpha mRNA expression was assessed by reverse transcriptase-PCR. KEY RESULTS: In vitro, 5-HT downregulated the membranous expression of CCR5 and led to a decrease of HIV-1 infection, probably through its action on 5-HT(1A) receptors. 5-HT (100 microM) was also able to induce overexpression of MIP-1alpha mRNA leading to an increase of MIP-1alpha secretion by human macrophages. CONCLUSIONS AND IMPLICATIONS: The effects of 5-HT on HIV infection could be a consequence of the increase in MIP-1alpha concentrations and/or CCR5 receptor downregulation. These results suggest that 5-HT can inhibit the replication of HIV-1 in primary culture of human macrophages through its action on 5-HT(1A) receptors.
Asunto(s)
VIH-1/efectos de los fármacos , Macrófagos/virología , Receptor de Serotonina 5-HT1A/efectos de los fármacos , Serotonina/farmacología , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Antígenos CD4/biosíntesis , Células Cultivadas , ADN Viral/biosíntesis , Humanos , Piperazinas/farmacología , Piridinas/farmacología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores CCR5/metabolismo , Agonistas del Receptor de Serotonina 5-HT1 , Antagonistas del Receptor de Serotonina 5-HT1 , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Replicación Viral/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The neurotransmitter 5-hydroxytryptamine (5-HT), commonly known as serotonin, is released at peripheral sites from activated platelets. At inflammatory sites, macrophages and lymphocytes could be exposed to 5-HT concentrations up to 100 microM. Moreover, 5-HT could modulate cytokine secretion by monocytes/macrophages and immune functions through the uptake of 5-HT at these inflammatory sites from T cells and dendritic cells. HIV infection is also under the control of inflammatory processes (including T cell proliferation and cytokines secretion). On this basis, we studied explored herein the effects of 5-HT on HIV-1/Ba-L (macrophage-tropic virus) replication in primary cultures of human macrophages. This pharmacological study with isotype-selective receptor agonists and antagonist allowed us to show that the 100 microM 5-HT concentration via 5-HT(1A) subtype receptors could decrease HIV replication. This observation was associated with an increase of MIP-1alpha secretion such as an increase of MIP-1alpha mRNA production and with a decrease of HIV-coreceptor CCR5 cell surface expression. Our results point out for the first time the inhibitory effects of 5-HT on HIV replication in primary culture of human macrophages via activation of 5-HT(1A) subtype receptors.
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VIH/fisiología , Macrófagos/virología , Receptor de Serotonina 5-HT1A/fisiología , Serotonina/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/uso terapéutico , VIH/efectos de los fármacos , Humanos , Monocitos/virología , Virosis/diagnóstico , Virosis/tratamiento farmacológicoRESUMEN
The present immunohistochemical study provides evidence that the kynurenine pathway is up-regulated in Alzheimer's disease (AD) brain, leading to increases in the excitotoxin quinolinic acid (QUIN). We show that the regulatory enzyme of the pathway leading to QUIN synthesis, indoleamine 2,3 dioxygenase (IDO) is abundant in AD compared with controls. In AD hippocampus, both IDO- and QUIN-immunoreactivity (-IR) was detected in cortical microglia, astrocytes and neurones, with microglial and astrocytic expression of IDO and QUIN highest in the perimeter of senile plaques. QUIN-IR was present in granular deposits within the neuronal soma of AD cortex and was also seen uniformly labelling neurofibrillary tangles. Our data imply that QUIN may be involved in the complex and multifactorial cascade leading to neuro-degeneration in AD. These results may open a new therapeutic door for AD patients.
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Enfermedad de Alzheimer/metabolismo , Hipocampo/metabolismo , Ácido Quinolínico/metabolismo , Triptófano Oxigenasa/metabolismo , Anciano , Anciano de 80 o más Años , Astrocitos/metabolismo , Humanos , Inmunohistoquímica , Indolamina-Pirrol 2,3,-Dioxigenasa , Quinurenina/metabolismo , Microglía/metabolismo , Persona de Mediana Edad , Neuronas/metabolismo , Placa Amiloide/metabolismoRESUMEN
Quinolinic, picolinic, and nicotinic acids and nicotinamide are end products of the kynurenine pathway from l-tryptophan and are intermediates in the biosynthesis of nicotinamide adenine dinucleotide. These compounds are involved in complex interrelationships with inflammatory and apoptotic responses associated with neuronal cell damage and death in the central nervous system. To facilitate the study of these compounds, we have utilized gas chromatography-mass spectrometry in electron capture negative ionization mode for their concurrent trace quantification in a single sample. Deuterium-labeled quinolinic, picolinic, and nicotinic acids were used as internal standards and the compounds were converted to their hexafluoroisopropyl esters prior to chromatography. Nicotinamide was readily quantified after conversion to nicotinic acid using gas-phase hydrolysis-a process which did not affect the deuterated internal standards. The on-column limit of quantification was less than 1 fmol for each of the analytes and calibration curves were linear. A packed column liner was used in the gas chromatograph inlet to effectively eliminate sample interference effects in the analysis of trace (femtomolar) levels of quinolinic acid. The method enables rapid and specific concurrent quantification of quinolinic, picolinic, and nicotinic acids in tissue extracts and physiological and culture media.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Niacina/análisis , Niacinamida/análisis , Ácidos Picolínicos/análisis , Ácido Quinolínico/análisis , Animales , Femenino , Feto/química , Humanos , Quinurenina/metabolismo , Ovinos/sangreRESUMEN
Activated macrophages produce quinolinic acid (QUIN), a neurotoxin, in several inflammatory brain diseases including AIDS dementia complex. We hypothesized that IL1-beta, IL6, transforming growth factor (TGF-beta2 and platelet activating factor could increase macrophage QUIN production. And that the HIV-1 proteins Nef, Tat and gp41 may also increase synthesis of QUIN by macrophages. At 72 h there were significant increases in QUIN production in the cells stimulated with PAF (914 +/- 50 nM) and Nef (2781 +/- 162 nM), with somewhat less production by Tat stimulation (645 +/- 240 nM). The increases in QUIN production approximated in vitro concentrations of QUIN shown to be neurotoxic and correlated closely with indoleamine 2,3-dioxygenase induction. IL1-beta, IL6, TGF-beta2 and gp41 stimulation produced no significant increase in QUIN production. These results suggest that some of the neurotoxicity of PAF, nef and tat may be mediated by QUIN.