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1.
Mol Psychiatry ; 28(7): 2857-2871, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37365239

RESUMEN

Chemogenetic approaches using Designer Receptors Exclusively Activated by Designer Drugs (DREADD, a family of engineered GPCRs) were recently employed in microglia. Here, we used Cx3cr1CreER/+:R26hM4Di/+ mice to express Gi-DREADD (hM4Di) on CX3CR1+ cells, comprising microglia and some peripheral immune cells, and found that activation of hM4Di on long-lived CX3CR1+ cells induced hypolocomotion. Unexpectedly, Gi-DREADD-induced hypolocomotion was preserved when microglia were depleted. Consistently, specific activation of microglial hM4Di cannot induce hypolocomotion in Tmem119CreER/+:R26hM4Di/+ mice. Flow cytometric and histological analysis showed hM4Di expression in peripheral immune cells, which may be responsible for the hypolocomotion. Nevertheless, depletion of splenic macrophages, hepatic macrophages, or CD4+ T cells did not affect Gi-DREADD-induced hypolocomotion. Our study demonstrates that rigorous data analysis and interpretation are needed when using Cx3cr1CreER/+ mouse line to manipulate microglia.


Asunto(s)
Microglía , Neuronas , Ratones , Animales , Neuronas/metabolismo , Macrófagos
2.
Toxicol Appl Pharmacol ; 455: 116258, 2022 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-36174671

RESUMEN

A characteristic of cytochrome P450 (CYP) enzymes is their ability to generate H2O2, either directly or indirectly via superoxide anion, a reaction referred to as "NADPH oxidase" activity. H2O2 production by CYPs can lead to the accumulation of cytotoxic reactive oxygen species which can compromise cellular functioning and contribute to tissue injury. Herein we determined if form selective CYP inhibitors could distinguish between the activities of the monooxygenase and NADPH oxidase activities of rat recombinant CYP1A2, CYP2E1, CYP3A1 and CYP3A2 and CYP1A1/2-enriched ß-naphthoflavone-induced rat liver microsomes, CYP2E1-enriched isoniazide-induced rat liver microsomes and CYP3A subfamily-enriched dexamethasone-induced rat liver microsomes. In the presence of 7,8-benzoflavone (2.0 µM) for CYP1A2 and 4-methylpyrazole (32 µM) or DMSO (16 mM) for CYP2E1, monooxygenase activity was blocked without affecting NADPH oxidase activity for both the recombinant enzymes and microsomal preparations. Ketoconazole (1.0 µM), a form selective inhibitor for CYP3A subfamily enzymes, completely inhibited monooxygenase activity of rat recombinant CYP3A1/3A2 and CYP3A subfamily in rat liver microsomes; it also partially inhibited NADPH oxidase activity. 7,8-benzoflavone is a type I ligand, which competes with substrate binding, while 4-methylpyrazole and DMSO are type II heme binding ligands. Interactions of heme with these type II ligands was not sufficient to interfere with oxygen activation, which is required for NADPH oxidase activity. Ketoconazole, a type II ligand known to bind multiple sites on CYP3A subfamily enzymes in close proximity to heme, also interfered, at least in part, with oxygen activation. These data indicate that form specific inhibitors can be used to distinguish between monooxygenase reactions and H2O2 generating NADPH oxidase of CYP1A2 and CYP2E1. Mechanisms by which ketoconazole inhibits CYP3A NADPH oxidase remain to be determined.


Asunto(s)
Citocromo P-450 CYP1A2 , Inhibidores Enzimáticos del Citocromo P-450 , Ratas , Animales , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Peróxido de Hidrógeno/metabolismo , NADP/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Cetoconazol/farmacología , Superóxidos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , beta-naftoflavona/farmacología , Fomepizol , Ligandos , Dimetilsulfóxido , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/metabolismo , Hemo/metabolismo , Dexametasona/farmacología , Oxígeno/metabolismo
3.
J Toxicol Environ Health B Crit Rev ; 25(4): 162-209, 2022 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-35676826

RESUMEN

Studies of nervous system effects of glyphosate, a widely used herbicide, have not been critically examined. The aim of this paper was to systematically review glyphosate-induced neurotoxicity literature to determine its usefulness in regulatory decision-making. The review was restricted to mammalian studies of behavior, neuropathology, and neuropharmacology; in vitro and other biochemical studies were considered supplementary information. Glyphosate formulation studies were also considered, despite uncertainties regarding toxicities of the formulated products; no studies used a formulation vehicle as the control. Inclusion criteria were developed a priori to ensure consistent evaluation of studies, and in vivo investigations were also ranked using ToxRTool software to determine reliability. There were 27 in vivo studies (open literature and available regulatory reports), but 11 studies were considered unreliable (mostly due to critical methodological deficiencies). There were only seven acceptable investigations on glyphosate alone. Studies differed in terms of dosing scenarios, experimental designs, test species, and commercial product. Limitations included using only one dose and/or one test time, small sample sizes, limited data presentation, and/or overtly toxic doses. While motor activity was the most consistently affected endpoint (10 of 12 studies), there were considerable differences in outcomes. In six investigations, there were no marked neuropathological changes in the central or peripheral nervous system. Other neurological effects were less consistent, and some outcomes were less convincing due to influences including high variability and small effect sizes. Taken together, these studies do not demonstrate a consistent impact of glyphosate on the structure or function of the mammalian nervous system.


Asunto(s)
Glicina , Herbicidas , Animales , Glicina/análogos & derivados , Glicina/toxicidad , Herbicidas/toxicidad , Mamíferos , Reproducibilidad de los Resultados , Glifosato
4.
Int J Mol Sci ; 23(17)2022 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-36077227

RESUMEN

The sex and APOE4 genotype are significant risk factors for Alzheimer's disease (AD); however, the mechanism(s) responsible for this interaction are still a matter of debate. Here, we assess the responses of mixed-sex and sex-specific APOE3 and APOE4 primary microglia (PMG) to lipopolysaccharide and interferon-gamma. In our investigation, inflammatory cytokine profiles were assessed by qPCR and multiplex ELISA assays. Mixed-sex APOE4 PMG exhibited higher basal mRNA expression and secreted levels of TNFa and IL1b. In sex-specific cultures, basal expression and secreted levels of IL1b, TNFa, IL6, and NOS2 were 2−3 fold higher in APOE4 female PMG compared to APOE4 males, with both higher than APOE3 cells. Following an inflammatory stimulus, the expression of pro-inflammatory cytokines and the secreted cytokine level were upregulated in the order E4 female > E4 male > E3 female > E3 male in sex-specific cultures. These data indicate that the APOE4 genotype and female sex together contribute to a greater inflammatory response in PMG isolated from targeted replacement humanized APOE mice. These data are consistent with clinical data and indicate that sex-specific PMG may provide a platform for exploring mechanisms of genotype and sex differences in AD related to neuroinflammation and neurodegeneration.


Asunto(s)
Enfermedad de Alzheimer , Apolipoproteína E4 , Apolipoproteínas E/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteínas E/metabolismo , Citocinas/metabolismo , Femenino , Genotipo , Masculino , Ratones , Ratones Transgénicos , Microglía/metabolismo
5.
FASEB J ; 34(1): 1679-1694, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31914683

RESUMEN

Increased pro-inflammatory cytokine levels and proliferation of activated microglia have been found in Parkinson's disease (PD) patients and animal models of PD, suggesting that targeting of the microglial inflammatory response may result in neuroprotection in PD. Microglial proliferation is regulated by many factors, but colony stimulating factor-1 receptor (CSF1R) has emerged as a primary factor. Using data mining techniques on existing microarray data, we found that mRNA expression of the CSF1R ligand, CSF-1, is increased in the brain of PD patients compared to controls. In two different neurotoxic mouse models of PD, acute MPTP and sub-chronic LPS treatment, mRNA and protein levels of CSF1R and CSF-1 were significantly increased. Treatment with the CSF1R inhibitor GW2580 significantly attenuated MPTP-induced CSF1R activation and Iba1-positive cell proliferation, without a reduction of the basal Iba1-positive population in the substantia nigra. GW2580 treatment also significantly decreased mRNA levels of pro-inflammatory factors, without alteration of anti-inflammatory mediators, and significantly attenuated the MPTP-induced loss of dopamine neurons and motor behavioral deficits. Importantly, these effects were observed in the absence of overt microglial depletion, suggesting that targeting CSF1R signaling may be a viable neuroprotective strategy in PD that disrupts pro-inflammatory signaling, but maintains the beneficial effects of microglia.


Asunto(s)
Anisoles/farmacología , Antiinflamatorios/farmacología , Proliferación Celular/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Microglía/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Pirimidinas/farmacología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Microglía/patología , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Transducción de Señal/efectos de los fármacos , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patología
6.
Drug Metab Dispos ; 48(6): 459-480, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32193359

RESUMEN

Multidrug resistance protein 1 (MDR1, ABCB1, P-glycoprotein) and breast cancer resistance protein (BCRP, ABCG2) are key efflux transporters that mediate the extrusion of drugs and toxicants in cancer cells and healthy tissues, including the liver, kidneys, and the brain. Altering the expression and activity of MDR1 and BCRP influences the disposition, pharmacodynamics, and toxicity of chemicals, including a number of commonly prescribed medications. Histone acetylation is an epigenetic modification that can regulate gene expression by changing the accessibility of the genome to transcriptional regulators and transcriptional machinery. Recently, studies have suggested that pharmacological inhibition of histone deacetylases (HDACs) modulates the expression and function of MDR1 and BCRP transporters as a result of enhanced histone acetylation. This review addresses the ability of HDAC inhibitors to modulate the expression and the function of MDR1 and BCRP transporters and explores the molecular mechanisms by which HDAC inhibition regulates these transporters. While the majority of studies have focused on histone regulation of MDR1 and BCRP in drug-resistant and drug-sensitive cancer cells, emerging data point to similar responses in nonmalignant cells and tissues. Elucidating epigenetic mechanisms regulating MDR1 and BCRP is important to expand our understanding of the basic biology of these two key transporters and subsequent consequences on chemoresistance as well as tissue exposure and responses to drugs and toxicants. SIGNIFICANCE STATEMENT: Histone deacetylase inhibitors alter the expression of key efflux transporters multidrug resistance protein 1 and breast cancer resistance protein in healthy and malignant cells.


Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Epigénesis Genética/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Proteínas de Neoplasias/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Acetilación/efectos de los fármacos , Animales , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Histonas/metabolismo , Humanos , Modelos Animales
7.
Brain Behav Immun ; 89: 245-255, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32621847

RESUMEN

Microglia are the resident immune cells of the center nervous system and participate in various neurological diseases. Here we determined the function of microglia in epileptogenesis using microglial ablation approaches. Three different microglia-specific genetic tools were used, CX3CR1CreER/+:R26iDTA/+, CX3CR1CreER/+:R26iDTR/+, and CX3CR1CreER/+:Csf1rFlox/Flox mice. We found that microglial depletion led to worse kainic acid (KA)-induced status epilepticus, higher mortality rate, and increased neuronal degeneration in the hippocampus. In KA-induced chronic spontaneous recurrent seizures, microglial depletion increased seizure frequency, interictal spiking, and seizure duration. Therefore, microglial depletion aggravates the severity of KA-induced acute and chronic seizures. Interestingly, microglial repopulation reversed the effects of depletion upon KA-induced status epilepticus. Our results demonstrate a beneficial role of microglia in suppressing both acute and chronic seizures, suggesting that microglia are a potential therapeutic target for the management of epilepsy.


Asunto(s)
Microglía , Estado Epiléptico , Animales , Modelos Animales de Enfermedad , Hipocampo , Ácido Kaínico , Ratones , Ratones Transgénicos , Convulsiones
8.
Glia ; 67(8): 1434-1448, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31179602

RESUMEN

Microglial activation has been recognized as a major contributor to inflammation of the epileptic brain. Seizures are commonly accompanied by remarkable microgliosis and loss of neurons. In this study, we utilize the CX3CR1GFP/+ CCR2RFP/+ genetic mouse model, in which CX3CR1+ resident microglia and CCR2+ monocytes are labeled with GFP and RFP, respectively. Using a combination of time-lapse two-photon imaging and whole-cell patch clamp recording, we determined the distinct morphological, dynamic, and electrophysiological characteristics of infiltrated monocytes and resident microglia, and the evolution of their behavior at different time points following kainic acid-induced seizures. Seizure activated microglia presented enlarged somas with less ramified processes, whereas, infiltrated monocytes were smaller, highly motile cells that lacked processes. Moreover, resident microglia, but not infiltrated monocytes, proliferate locally in the hippocampus after seizure. Microglial proliferation was dependent on the colony-stimulating factor 1 receptor (CSF-1R) pathway. Pharmacological inhibition of CSF-1R reduced seizure-induced microglial proliferation, which correlated with attenuation of neuronal death without altering acute seizure behaviors. Taken together, we demonstrated that proliferation of activated resident microglia contributes to neuronal death in the hippocampus via CSF-1R after status epilepticus, providing potential therapeutic targets for neuroprotection in epilepsy.


Asunto(s)
Proliferación Celular , Gliosis/fisiopatología , Microglía/fisiología , Monocitos/fisiología , Estado Epiléptico/fisiopatología , Animales , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Proteínas de Unión al Calcio/metabolismo , Muerte Celular , Modelos Animales de Enfermedad , Gliosis/etiología , Hipocampo/fisiopatología , Ácido Kaínico , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Neuronas/fisiología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptores CCR2/genética , Receptores CCR2/metabolismo , Estado Epiléptico/complicaciones , Técnicas de Cultivo de Tejidos
9.
Acta Neuropathol ; 138(3): 343-362, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31197504

RESUMEN

Pesticides are unique environmental contaminants that are specifically introduced into the environment to control pests, often by killing them. Although pesticide application serves many important purposes, including protection against crop loss and against vector-borne diseases, there are significant concerns over the potential toxic effects of pesticides to non-target organisms, including humans. In many cases, the molecular target of a pesticide is shared by non-target species, leading to the potential for untoward effects. Here, we review the history of pesticide usage and the neurotoxicity of selected classes of pesticides, including insecticides, herbicides, and fungicides, to humans and experimental animals. Specific emphasis is given to linkages between exposure to pesticides and risk of neurological disease and dysfunction in humans coupled with mechanistic findings in humans and animal models. Finally, we discuss emerging techniques and strategies to improve translation from animal models to humans.


Asunto(s)
Síndromes de Neurotoxicidad/etiología , Plaguicidas/toxicidad , Animales , Humanos
10.
J Biochem Mol Toxicol ; 33(6): e22318, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30897286

RESUMEN

Multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) protect the brain by restricting the passage of chemicals across the blood-brain barrier. Prior studies have demonstrated the epigenetic regulation of MDR1 and BCRP in cancer cells treated with histone deacetylase (HDAC) inhibitors that enhance histone acetylation and gene transcription. In the present study, we tested the in vivo effects of two HDAC inhibitors, valproic acid (VPA; 400 mg/kg) and apicidin (5 mg/kg), on Mdr1 and Bcrp transporter expression in brain regions of adult male mice injected intraperitoneally daily for 7 days. VPA increased Mdr1 protein expression in the striatum (70%) and Bcrp protein in the midbrain (30%). Apicidin enhanced striatal Mdr1 protein (30%) and hippocampal Bcrp protein (20%). Transporter induction correlated with increased histone H3 acetylation in discrete brain regions. In conclusion, HDAC inhibitors upregulate transporter proteins in vivo, which may be important in regulating regional xenobiotic disposition within the brain.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/biosíntesis , Encéfalo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Péptidos Cíclicos/farmacología , Ácido Valproico/farmacología , Acetilación/efectos de los fármacos , Animales , Histona Desacetilasas/metabolismo , Masculino , Ratones
11.
Biochim Biophys Acta Mol Basis Dis ; 1864(2): 432-443, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29113750

RESUMEN

Epigenetic mechanisms control various functions throughout the body, from cell fate determination in development to immune responses and inflammation. Neuroinflammation is one of the prime contributors to the initiation and progression of neurodegeneration in a variety of diseases, including Alzheimer's and Parkinson's diseases. Because astrocytes are the largest population of glial cells, they represent an important regulator of CNS function, both in health and disease. Only recently have studies begun to identify the epigenetic mechanisms regulating astrocyte responses in neurodegenerative diseases. These epigenetic mechanisms, along with the epigenetic marks involved in astrocyte development, could elucidate novel pathways to potentially modulate astrocyte-mediated neuroinflammation and neurotoxicity. This review examines the known epigenetic mechanisms involved in regulation of astrocyte function, from development to neurodegeneration, and links these mechanisms to potential astrocyte-specific roles in neurodegenerative disease with a focus on potential opportunities for therapeutic intervention.


Asunto(s)
Astrocitos/citología , Epigénesis Genética , Inflamación/patología , Enfermedades Neurodegenerativas/patología , Acetilación , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Astrocitos/inmunología , Diferenciación Celular , Metilación de ADN , Histonas/metabolismo , Humanos , Metilación , MicroARNs/metabolismo , Enfermedades Neurodegenerativas/genética , Neuroglía/metabolismo , Neuronas/inmunología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología
12.
J Neuroinflammation ; 15(1): 73, 2018 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-29519253

RESUMEN

BACKGROUND: Neuroinflammation is one of the hallmarks of neurodegenerative diseases, such as Parkinson's disease (PD). Activation of glial cells, including microglia and astrocytes, is a characteristic of the inflammatory response. Glycoprotein non-metastatic melanoma protein B (GPNMB) is a transmembrane glycoprotein that releases a soluble signaling peptide when cleaved by ADAM10 or other extracellular proteases. GPNMB has demonstrated a neuroprotective role in animal models of ALS and ischemia. However, the mechanism of this protection has not been well established. CD44 is a receptor expressed on astrocytes that can bind GPNMB, and CD44 activation has been demonstrated to reduce NFκB activation and subsequent inflammatory responses in macrophages. GPNMB signaling has not been investigated in models of PD or specifically in astrocytes. More recently, genetic studies have linked polymorphisms in GPNMB with risk for PD. Therefore, it is important to understand the role this signaling protein plays in PD. METHODS: We used data mining techniques to evaluate mRNA expression of GPNMB and its receptor CD44 in the substantia nigra of PD and control brains. Immunofluorescence and qPCR techniques were used to assess GPNMB and CD44 levels in mice treated with MPTP. In vitro experiments utilized the immortalized mouse astrocyte cell line IMA2.1 and purified primary mouse astrocytes. The effects of recombinant GPNMB on cytokine-induced astrocyte activation was determined by qPCR, immunofluorescence, and measurement of nitric oxide and reactive oxygen production. RESULTS: Increased GPNMB and CD44 expression was observed in the substantia nigra of human PD brains and in GFAP-positive astrocytes in an animal model of PD. GPNMB treatment attenuated cytokine-induced levels of inducible nitric oxide synthase, nitric oxide, reactive oxygen species, and the inflammatory cytokine IL-6 in an astrocyte cell line and primary mouse astrocytes. Using primary mouse astrocytes from CD44 knockout mice, we found that the anti-inflammatory effects of GPNMB are CD44-mediated. CONCLUSIONS: These results demonstrate that GPNMB may exert its neuroprotective effect through reducing astrocyte-mediated neuroinflammation in a CD44-dependent manner, providing novel mechanistic insight into the neuroprotective properties of GPNMB.


Asunto(s)
Antiinflamatorios/uso terapéutico , Astrocitos/efectos de los fármacos , Receptores de Hialuranos/metabolismo , Inflamación/tratamiento farmacológico , Glicoproteínas de Membrana/uso terapéutico , Enfermedad de Parkinson/patología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Análisis de Varianza , Animales , Estudios de Casos y Controles , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Bases de Datos de Compuestos Químicos , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inflamación/etiología , Masculino , Ratones , Neurotoxinas/toxicidad , Óxido Nítrico/metabolismo , Enfermedad de Parkinson/complicaciones , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos
13.
Toxicol Appl Pharmacol ; 359: 102-107, 2018 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-30222979

RESUMEN

NADH cytochrome b5 reductase mediates electron transfer from NADH to cytochrome b5 utilizing flavin adenine dinucleotide as a redox cofactor. Reduced cytochrome b5 is an important cofactor in many metabolic reactions including cytochrome P450-mediated xenobiotic metabolism, steroid biosynthesis and fatty acid metabolism, hemoglobin reduction, and methionine and plasmalogen synthesis. Using recombinant human enzyme, we discovered that cytochrome b5 reductase mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity was oxygen-dependent and preferentially utilized NADH as a co-substrate; NADH was 5-10 times more active than NADPH in supporting redox cycling. Redox cycling activity was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione), nitrofurantoin and 2-hydroxyestradiol. Using menadione as the substrate, quinone redox cycling was found to inhibit reduction of cytochrome b5 by cytochrome b5 reductase, as measured by heme spectral changes in cytochrome b5. Under anaerobic conditions where redox cycling is inhibited, menadione had no effect on the reduction of cytochrome b5. Chemical redox cycling by cytochrome b5 reductase may be important in generating cytotoxic reactive oxygen species in target tissues. This activity, together with the inhibition of cytochrome b5 reduction by redox-active chemicals and consequent deficiencies in available cellular cytochrome b5, are likely to contribute to tissue injury following exposure to quinones and related redox active chemicals.


Asunto(s)
Benzoquinonas/metabolismo , Citocromo-B(5) Reductasa/metabolismo , Nitrofurantoína/metabolismo , Radicales Libres/metabolismo , Humanos , Cinética , Microsomas Hepáticos , NADP/metabolismo , Oxidación-Reducción , Consumo de Oxígeno , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo
14.
Neurobiol Dis ; 108: 115-127, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28823928

RESUMEN

Inflammation arising from central and/or peripheral sources contributes to the pathogenesis of multiple neurodegenerative diseases including Parkinson's disease (PD). Emerging data suggest that differential activation of glia could lead to the pathogenesis and progression of PD. Here, we sought to determine the relationship between lipopolysaccharide (LPS) treatment, loss of dopaminergic neurons and differential activation of glia. Using a model of repeated injections with LPS (1mg/kg, i.p. for 4days), we found that LPS induced a 34% loss of dopamine neurons in the substantia nigra 19days after initiation of treatment, but no further cell loss was observed at 36days. LPS induced a strong pro-inflammatory response with increased mRNA expression of pro-inflammatory markers, including tumor necrosis factor-α (4.8-fold), inducible nitric oxide synthase (2.0-fold), interleukin-1 beta (8.9-fold), interleukin-6 (10.7-fold), and robust glial activation were observed at 1day after final dose of LPS. These pro-inflammatory genes were then reduced at 19days after treatment, when there was a rise in the anti-inflammatory genes Ym1 (1.8-fold) and arginase-1 (2.6-fold). Additionally, 36days after the last LPS injection there was a significant increase in interleukin-10 (2.1-fold) expression. The qPCR data results were supported by protein data, including cytokine measurements, western blotting, and immunofluorescence in brain microglia. Taken together, these data demonstrate that progressive neurodegeneration in the substantia nigra following LPS is likely arrested by microglia shifting to an anti-inflammatory phenotype. Thus, strategies to promote resolution of neuroinflammation may be a promising avenue to slow the progressive loss of dopamine neurons in PD.


Asunto(s)
Neuronas Dopaminérgicas/inmunología , Lipopolisacáridos/toxicidad , Microglía/inmunología , Degeneración Nerviosa/inmunología , Animales , Astrocitos/inmunología , Astrocitos/patología , Muerte Celular/fisiología , Cuerpo Estriado/inmunología , Cuerpo Estriado/patología , Citocinas/metabolismo , Progresión de la Enfermedad , Neuronas Dopaminérgicas/patología , Inflamación/patología , Inflamación/fisiopatología , Masculino , Ratones Endogámicos C57BL , Microglía/patología , Degeneración Nerviosa/patología , Enfermedades Neurodegenerativas/inmunología , Enfermedades Neurodegenerativas/patología , Neuroinmunomodulación/fisiología , ARN Mensajero/metabolismo , Distribución Aleatoria , Factores de Tiempo
15.
J Neurosci ; 35(6): 2417-22, 2015 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-25673836

RESUMEN

Extracellular calcium concentrations in the brain fluctuate during neuronal activities and may affect the behavior of brain cells. Microglia are highly dynamic immune cells of the brain. However, the effects of extracellular calcium concentrations on microglial dynamics have not been investigated. Here, we addressed this question in mouse brain slices and in vivo using two-photon microscopy. We serendipitously found that extracellular calcium reduction induced microglial processes to converge at distinct sites, a phenomenon we termed microglial process convergence (MPCs). Our studies revealed that MPCs target neuronal dendrites independent of neuronal action potential firing and is mediated by ATP release and microglial P2Y12 receptors. These results indicate that microglia monitor and interact with neurons during conditions of cerebral calcium reduction in the normal and diseased brain.


Asunto(s)
Señalización del Calcio/fisiología , Calcio/fisiología , Dendritas/fisiología , Microglía/fisiología , Neuronas/fisiología , Animales , Espacio Extracelular/fisiología , Femenino , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Purinas/metabolismo , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/fisiología
17.
FASEB J ; 29(5): 1960-72, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25630971

RESUMEN

Attention-deficit hyperactivity disorder (ADHD) is estimated to affect 8-12% of school-age children worldwide. ADHD is a complex disorder with significant genetic contributions. However, no single gene has been linked to a significant percentage of cases, suggesting that environmental factors may contribute to ADHD. Here, we used behavioral, molecular, and neurochemical techniques to characterize the effects of developmental exposure to the pyrethroid pesticide deltamethrin. We also used epidemiologic methods to determine whether there is an association between pyrethroid exposure and diagnosis of ADHD. Mice exposed to the pyrethroid pesticide deltamethrin during development exhibit several features reminiscent of ADHD, including elevated dopamine transporter (DAT) levels, hyperactivity, working memory and attention deficits, and impulsive-like behavior. Increased DAT and D1 dopamine receptor levels appear to be responsible for the behavioral deficits. Epidemiologic data reveal that children aged 6-15 with detectable levels of pyrethroid metabolites in their urine were more than twice as likely to be diagnosed with ADHD. Our epidemiologic finding, combined with the recapitulation of ADHD behavior in pesticide-treated mice, provides a mechanistic basis to suggest that developmental pyrethroid exposure is a risk factor for ADHD.


Asunto(s)
Trastorno por Déficit de Atención con Hiperactividad/inducido químicamente , Trastorno por Déficit de Atención con Hiperactividad/patología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Nitrilos/toxicidad , Piretrinas/orina , Receptores de Dopamina D1/metabolismo , Adolescente , Animales , Trastorno por Déficit de Atención con Hiperactividad/metabolismo , Reacción de Prevención/efectos de los fármacos , Western Blotting , Estudios de Casos y Controles , Niño , Cromatografía Líquida de Alta Presión , Estudios Transversales , Femenino , Humanos , Insecticidas/toxicidad , Locomoción/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/patología , Ratones , Ratones Endogámicos C57BL , Piretrinas/efectos adversos , Piretrinas/toxicidad
18.
Proc Natl Acad Sci U S A ; 110(48): 19561-6, 2013 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-24218591

RESUMEN

Parkinson disease (PD) is the most common movement disorder and, although the exact causes are unknown, recent epidemiological and experimental studies indicate that several environmental agents may be significant risk factors. To date, these suspected environmental risk factors have been man-made chemicals. In this report, we demonstrate via genetic, biochemical, and immunological studies that the common volatile fungal semiochemical 1-octen-3-ol reduces dopamine levels and causes dopamine neuron degeneration in Drosophila melanogaster. Overexpression of the vesicular monoamine transporter (VMAT) rescued the dopamine toxicity and neurodegeneration, whereas mutations decreasing VMAT and tyrosine hydroxylase exacerbated toxicity. Furthermore, 1-octen-3-ol also inhibited uptake of dopamine in human cell lines expressing the human plasma membrane dopamine transporter (DAT) and human VMAT ortholog, VMAT2. These data demonstrate that 1-octen-3-ol exerts toxicity via disruption of dopamine homeostasis and may represent a naturally occurring environmental agent involved in parkinsonism.


Asunto(s)
Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Octanoles/toxicidad , Feromonas/toxicidad , Análisis de Varianza , Animales , Cromatografía Líquida de Alta Presión , Drosophila , Microscopía Confocal , Movimiento/efectos de los fármacos
19.
J Pharmacol Exp Ther ; 352(3): 529-40, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25550200

RESUMEN

Sepiapterin reductase (SPR) catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), the precursor for tetrahydrobiopterin (BH4), a cofactor critical for nitric oxide biosynthesis and alkylglycerol and aromatic amino acid metabolism. SPR also mediates chemical redox cycling, catalyzing one-electron reduction of redox-active chemicals, including quinones and bipyridinium herbicides (e.g., menadione, 9,10-phenanthrenequinone, and diquat); rapid reaction of the reduced radicals with molecular oxygen generates reactive oxygen species (ROS). Using recombinant human SPR, sulfonamide- and sulfonylurea-based sulfa drugs were found to be potent noncompetitive inhibitors of both sepiapterin reduction and redox cycling. The most potent inhibitors of sepiapterin reduction (IC50s = 31-180 nM) were sulfasalazine, sulfathiazole, sulfapyridine, sulfamethoxazole, and chlorpropamide. Higher concentrations of the sulfa drugs (IC50s = 0.37-19.4 µM) were required to inhibit redox cycling, presumably because of distinct mechanisms of sepiapterin reduction and redox cycling. In PC12 cells, which generate catecholamine and monoamine neurotransmitters via BH4-dependent amino acid hydroxylases, sulfa drugs inhibited both BH2/BH4 biosynthesis and redox cycling mediated by SPR. Inhibition of BH2/BH4 resulted in decreased production of dopamine and dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 5-hydroxytryptamine. Sulfathiazole (200 µM) markedly suppressed neurotransmitter production, an effect reversed by BH4. These data suggest that SPR and BH4-dependent enzymes, are "off-targets" of sulfa drugs, which may underlie their untoward effects. The ability of the sulfa drugs to inhibit redox cycling may ameliorate ROS-mediated toxicity generated by redox active drugs and chemicals, contributing to their anti-inflammatory activity.


Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/metabolismo , Pterinas/antagonistas & inhibidores , Pterinas/metabolismo , Sulfasalazina/farmacología , Sulfatiazoles/farmacología , Oxidorreductasas de Alcohol/química , Animales , Humanos , Ratones , Oxidación-Reducción/efectos de los fármacos , Células PC12 , Estructura Secundaria de Proteína , Pterinas/química , Ratas , Sulfatiazol
20.
Toxicol Appl Pharmacol ; 288(1): 114-20, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26212258

RESUMEN

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation.


Asunto(s)
Inhibidores de la Colinesterasa/toxicidad , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Insecticidas/toxicidad , Hígado/efectos de los fármacos , Intoxicación por Organofosfatos/prevención & control , Paratión/toxicidad , Vitamina K 3/farmacología , Acetilcolinesterasa/metabolismo , Activación Metabólica , Animales , Inhibidores de la Colinesterasa/metabolismo , Inhibidores Enzimáticos del Citocromo P-450/metabolismo , Citoprotección , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Proteínas Ligadas a GPI/metabolismo , Humanos , Insecticidas/metabolismo , Hígado/enzimología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , NADP/metabolismo , Intoxicación por Organofosfatos/enzimología , Intoxicación por Organofosfatos/etiología , Oxidación-Reducción , Paraoxon/metabolismo , Paraoxon/toxicidad , Paratión/metabolismo , Ratas Long-Evans , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Vitamina K 3/metabolismo
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