RESUMEN
This paper aims to solve the problems of complicated-unstable test solution preparation process and insufficient extraction of the active ingredient astragaloside â £ in the legal method for the determination of astragaloside â £ in Astragali Radix. The continuous single-factor analysis of seven main factors affecting the content of astragaloside â £ was carried out by HPLC-ELSD, and then the pre-paration method of test solution was optimized. This optimized method exhibited excellent performance in precision, repeatability and stability. The average recovery rate of astragaloside â £ was 99.65% with RSD 2.2%. Astragaloside â £ showed a good linearity between the logarithm of peak area and the logarithm of injection quantity in the range of 0.46-9.1 µg(r=0.999 6). The contents of astragaloside â £ in 29 batches of Astragali Radix were determined by the new and the legal methods. The results showed that the average content of astragaloside â £ in these Astragali Radix samples determined by the former method was 1.458 times than that of the latter one, indicating the new method was simple, reliable and more adequate to extract target compound. According to the results, it is suggested to improve the content standard of astragaloside â £ in Astragali Radix in the new edition of Chinese Pharmacopeia.
Asunto(s)
Planta del Astrágalo , Medicamentos Herbarios Chinos , Saponinas , Triterpenos , Cromatografía Líquida de Alta Presión , Triterpenos/análisisRESUMEN
BACKGROUND: The underlying mechanism of viral infection as a risk factor for Parkinson's disease (PD), the second most common neurodegenerative disease, remains unclear. OBJECTIVE: We used Mac-1-/- and gp91phox-/- transgene animal models to investigate the mechanisms by which poly I:C, a mimic of virus double-stranded RNA, induces PD neurodegeneration. METHOD: Poly I:C was stereotaxically injected into the substantia nigra (SN) of wild-type (WT), Mac-1-knockout (Mac-1-/-) and gp91 phox-knockout (gp91 phox-/-) mice (10 µg/µl), and nigral dopaminergic neurodegeneration, α-synuclein accumulation and neuroinflammation were evaluated. RESULT: Dopaminergic neurons in the nigra and striatum were markedly reduced in WT mice after administration of poly I:C together with abundant microglial activation in the SN, and the expression of α-synuclein was also elevated. However, these pathological changes were greatly dampened in Mac-1-/- and gp91 phox-/- mice. CONCLUSIONS: Our findings demonstrated that viral infection could result in the activation of microglia as well as NADPH oxidase, which may lead to neuron loss and the development of Parkinson's-like symptoms. Mac-1 is a key receptor during this process.
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Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Antígeno de Macrófago-1/metabolismo , NADPH Oxidasa 2/metabolismo , Enfermedades Neurodegenerativas/metabolismo , ARN Bicatenario/toxicidad , Sustancia Negra/metabolismo , Animales , Muerte Celular/genética , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Neuronas Dopaminérgicas/citología , Inflamación/metabolismo , Antígeno de Macrófago-1/genética , Masculino , Ratones , Ratones Noqueados , Microglía/metabolismo , NADPH Oxidasa 2/genética , NADPH Oxidasas/metabolismo , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/genética , ARN Bicatenario/metabolismo , Sustancia Negra/citología , Sustancia Negra/patología , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Exposure to benzo(a)pyrene (BaP) was associated with cognitive impairments and some Alzheimer's disease (AD)-like pathological changes. However, it is largely unknown whether BaP exposure participates in the disease progression of AD. OBJECTIVES: To investigate the effect of BaP exposure on AD progression and its underlying mechanisms. METHODS: BaP or vehicle was administered to 4-month-old APPswe/PS1dE9 transgenic (APP/PS1) mice and wildtype (WT) mice for 2 months. Learning and memory ability and exploratory behaviors were evaluated 1 month after the initiation/termination of BaP exposure. AD-like pathological and biochemical alterations were examined 1 month after 2-month BaP exposure. Levels of soluble beta-amyloid (Aß) oligomers and the number of Aß plaques in the cortex and the hippocampus were quantified. Gene expression profiling was used to evaluate alternation of genes/pathways associated with AD onset and progression. Immunohistochemistry and Western blot were used to demonstrate neuronal loss and neuroinflammation in the cortex and the hippocampus. Treatment of primary neuron-glia cultures with aged Aß (a mixture of monomers, oligomers, and fibrils) and/or BaP was used to investigate mechanisms by which BaP enhanced Aß-induced neurodegeneration. RESULTS: BaP exposure induced progressive decline in spatial learning/memory and exploratory behaviors in APP/PS1 mice and WT mice, and APP/PS1 mice showed severer behavioral deficits than WT mice. Moreover, BaP exposure promoted neuronal loss, Aß burden and Aß plaque formation in APP/PS1 mice, but not in WT mice. Gene expression profiling showed most robust alteration in genes and pathways related to inflammation and immunoregulatory process, Aß secretion and degradation, and synaptic formation in WT and APP/PS1 mice after BaP exposure. Consistently, the cortex and the hippocampus of WT and APP/PS1 mice displayed activation of microglia and astroglia and upregulation of inducible nitric oxide synthase (iNOS), glial fibrillary acidic protein (GFAP), and NADPH oxidase (three widely used neuroinflammatory markers) after BaP exposure. Furthermore, BaP exposure aggravated neurodegeneration induced by aged Aß peptide in primary neuron-glia cultures through enhancing NADPH oxidase-derived oxidative stress. CONCLUSION: Our study showed that chronic exposure to environmental pollutant BaP induced, accelerated, and exacerbated the progression of AD, in which elevated neuroinflammation and NADPH oxidase-derived oxidative insults were key pathogenic events.
Asunto(s)
Enfermedad de Alzheimer/patología , Benzo(a)pireno/toxicidad , Disfunción Cognitiva/inducido químicamente , Neuronas/efectos de los fármacos , Placa Amiloide/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Conducta Animal/efectos de los fármacos , Disfunción Cognitiva/patología , Modelos Animales de Enfermedad , Conducta Exploratoria/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Neuronas/patología , Presenilina-1/genética , Memoria Espacial/efectos de los fármacosRESUMEN
Chronic neuroinflammation contributes to the pathogenesis of Parkinson's disease (PD). However, cellular and molecular mechanisms by which chronic neuroinflammation is formed and maintained remain elusive. This study aimed to explore detailed mechanisms by which anti-inflammatory cytokine interleukin-10 (IL-10) prevented chronic neuroinflammation and neurodegeneration. At 24 h after an intranigral injection of lipopolysaccharide (LPS), levels of NLRP3, pro-caspase-1, pro-IL-1ß, active caspase-1, and mature IL-1ß in the midbrain were much higher in IL-10-/- mice than wildtype mice. Mechanistically, IL-10-/- microglia produced more intracellular reactive oxygen species (iROS) and showed more profound activation of NADPH oxidase (NOX2) than wildtype microglia. Meanwhile, suppression of NOX2-derived iROS production blocked LPS-elicited caspase-1 activation and IL-1ß maturation in IL-10-/- microglia in vitro and in vivo. One month after intranigral LPS injection, IL-10-/- mice revealed more profound microglial activation and dopaminergic neurodegeneration in the substantia nigra than wildtype mice. Importantly, such PD-like pathological changes were prevented by IL-1ß neutralization. Collectively, IL-10 inhibited LPS-elicited production of NOX2-derived iROS thereby suppressing synthesis of NLRP3, pro-caspase-1 and pro-IL-1ß and their activation and cleavage. By this mechanism, IL-10 prevented chronic neuroinflammation and neurodegeneration. This study suggested boosting anti-inflammatory effects of IL-10 and suppressing NLRP3 inflammasome activation could be beneficial for PD treatment.
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Caspasa 1/metabolismo , Neuronas Dopaminérgicas/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Células Cultivadas , Neuronas Dopaminérgicas/efectos de los fármacos , Femenino , Interleucina-10/genética , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , NADPH Oxidasa 2/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Sustancia Negra/citología , Sustancia Negra/metabolismoRESUMEN
BACKGROUND: Metabolic dysfunction and neuroinflammation are increasingly implicated in Parkinson's disease (PD). The pentose phosphate pathway (PPP, a metabolic pathway parallel to glycolysis) converts glucose-6-phosphate into pentoses and generates ribose-5-phosphate and NADPH thereby governing anabolic biosynthesis and redox homeostasis. Brains and immune cells display high activity of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP. A postmortem study reveals dysregulation of G6PD enzyme in brains of PD patients. However, spatial and temporal changes in activity/expression of G6PD in PD remain undetermined. More importantly, it is unclear how dysfunction of G6PD and the PPP affects neuroinflammation and neurodegeneration in PD. METHODS: We examined expression/activity of G6PD and its association with microglial activation and dopaminergic neurodegeneration in multiple chronic PD models generated by an intranigral/intraperitoneal injection of LPS, daily subcutaneous injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 6 days, or transgenic expression of A53T α-synuclein. Primary microglia were transfected with G6PD siRNAs and treated with lipopolysaccharide (LPS) to examine effects of G6PD knockdown on microglial activation and death of co-cultured neurons. LPS alone or with G6PD inhibitor(s) was administrated to mouse substantia nigra or midbrain neuron-glia cultures. While histological and biochemical analyses were conducted to examine microglial activation and dopaminergic neurodegeneration in vitro and in vivo, rotarod behavior test was performed to evaluate locomotor impairment in mice. RESULTS: Expression and activity of G6PD were elevated in LPS-treated midbrain neuron-glia cultures (an in vitro PD model) and the substantia nigra of four in vivo PD models. Such elevation was positively associated with microglial activation and dopaminergic neurodegeneration. Furthermore, inhibition of G6PD by 6-aminonicotinamide and dehydroepiandrosterone and knockdown of microglial G6PD attenuated LPS-elicited chronic dopaminergic neurodegeneration. Mechanistically, microglia with elevated G6PD activity/expression produced excessive NADPH and provided abundant substrate to over-activated NADPH oxidase (NOX2) leading to production of excessive reactive oxygen species (ROS). Knockdown and inhibition of G6PD ameliorated LPS-triggered production of ROS and activation of NF-кB thereby dampening microglial activation. CONCLUSIONS: Our findings indicated that G6PD-mediated PPP dysfunction and neuroinflammation exacerbated each other mediating chronic dopaminergic neurodegeneration and locomotor impairment. Insight into metabolic-inflammatory interface suggests that G6PD and NOX2 are potential therapeutic targets for PD.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Mediadores de Inflamación/metabolismo , Degeneración Nerviosa/metabolismo , Vía de Pentosa Fosfato/fisiología , Animales , Células Cultivadas , Técnicas de Cocultivo , Neuronas Dopaminérgicas/patología , Femenino , Técnicas de Silenciamiento del Gen , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Embarazo , Ratas , Ratas Endogámicas F344 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Atmospheric ultrafine particles (UFPs) and pesticide rotenone were considered as potential environmental risk factors for Parkinson's disease (PD). However, whether and how UFPs alone and in combination with rotenone affect the pathogenesis of PD remains largely unknown. METHODS: Ultrafine carbon black (ufCB, a surrogate of UFPs) and rotenone were used individually or in combination to determine their roles in chronic dopaminergic (DA) loss in neuron-glia, and neuron-enriched, mix-glia cultures. Immunochemistry using antibody against tyrosine hydroxylase was performed to detect DA neuronal loss. Measurement of extracellular superoxide and intracellular reactive oxygen species (ROS) were performed to examine activation of NADPH oxidase. Genetic deletion and pharmacological inhibition of NADPH oxidase and MAC-1 receptor in microglia were employed to examine their role in DA neuronal loss triggered by ufCB and rotenone. RESULTS: In rodent midbrain neuron-glia cultures, ufCB and rotenone alone caused neuronal death in a dose-dependent manner. In particularly, ufCB at doses of 50 and 100µg/cm2 induced significant loss of DA neurons. More importantly, nontoxic doses of ufCB (10µg/cm2) and rotenone (2nM) induced synergistic toxicity to DA neurons. Microglial activation was essential in this process. Furthermore, superoxide production from microglial NADPH oxidase was critical in ufCB/rotenone-induced neurotoxicity. Studies in mix-glia cultures showed that ufCB treatment activated microglial NADPH oxidase to induce superoxide production. Firstly, ufCB enhanced the expression of NADPH oxidase subunits (gp91phox, p47phox and p40phox); secondly, ufCB was recognized by microglial surface MAC-1 receptor and consequently promoted rotenone-induced p47phox and p67phox translocation assembling active NADPH oxidase. CONCLUSION: ufCB and rotenone worked in synergy to activate NADPH oxidase in microglia, leading to oxidative damage to DA neurons. Our findings delineated the potential role of ultrafine particles alone and in combination with pesticide rotenone in the pathogenesis of PD.
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Neuronas Dopaminérgicas/enzimología , Microglía/enzimología , NADPH Oxidasas/metabolismo , Rotenona/toxicidad , Siliconas/toxicidad , Hollín/toxicidad , Animales , Células Cultivadas , Técnicas de Cocultivo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/patología , Neuronas/efectos de los fármacos , Neuronas/enzimología , Neuronas/patología , Material Particulado , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Endotoxin tolerance (ET) is a reduced responsiveness of innate immune cells like macrophages/monocytes to an endotoxin challenge following a previous encounter with the endotoxin. Although ET in peripheral systems has been well studied, little is known about ET in the brain. The present study showed that brain immune cells, microglia, being different from peripheral macrophages, displayed non-cell autonomous mechanisms in ET formation. Specifically, neurons and astroglia were indispensable for microglial ET. Macrophage colony-stimulating factor (M-CSF) secreted from these non-immune cells was essential for governing microglial ET. Neutralization of M-CSF deprived the neuron-glia conditioned medium of its ability to enable microglia to form ET when microglia encountered two lipopolysaccharide (LPS) treatments. Recombinant M-CSF protein rendered enriched microglia refractory to the second LPS challenge leading to microglial ET. Activation of microglial M-CSF receptor (M-CSFR; also known as CSF1R) and the downstream ERK1/2 signals was responsible for M-CSF-mediated microglial ET. Endotoxin-tolerant microglia in neuron-glia cultures displayed M2-like polarized phenotypes, as shown by upregulation of M2 marker Arg-1, elevated production of anti-inflammatory cytokine interleukin 10, and decreased secretion of pro-inflammatory mediators (tumor necrosis factor α, nitric oxide, prostaglandin E2 and interleukin 1ß). Endotoxin-tolerant microglia protected neurons against LPS-elicited inflammatory insults, as shown by reduced neuronal damages in LPS pre-treatment group compared with the group without LPS pre-treatment. Moreover, while neurons and astroglia became injured during chronic neuroinflammation, microglia failed to form ET. Thus, this study identified a distinct non-cell autonomous mechanism of microglial ET. Interactions of M-CSF secreted by neurons and astroglia with microglial M-CSFR programed microglial ET. Loss of microglial ET could be an important pathogenetic mechanism of inflammation-associated neuronal damages.
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Astrocitos/metabolismo , Endotoxinas , Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Microglía/metabolismo , Neuronas/metabolismo , Neuroprotección/fisiología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BLRESUMEN
During viral infection, extracellular dsRNA is a potent signaling molecule that activates many innate immune cells, including macrophages. TLR3 is a well-known receptor for extracellular dsRNA, and internalization of extracellular dsRNA is required for endosomal TLR3 activation. Preserved inflammatory responses of TLR3-deficient macrophages to extracellular dsRNA strongly support a TLR3-independent mechanism in dsRNA-mediated immune responses. The present study demonstrated that CD11b/CD18 (Mac-1 [macrophage-1 Ag]), a surface integrin receptor, recognized extracellular dsRNA and induced macrophage immune responses. CD11b deficiency reduced inflammatory cytokine induction elicited by polyinosinic:polycytidylic acid (poly I:C; a synthetic dsRNA) in mouse sera and livers, as well as in cultured peritoneal macrophages. dsRNA-binding assay and confocal immunofluorescence showed that Mac-1, especially the CD11b subunit, interacted and colocalized with poly I:C on the surface of macrophages. Further mechanistic studies revealed two distinct signaling events following dsRNA recognition by Mac-1. First, Mac-1 facilitated poly I:C internalization through the activation of PI3K signaling and enhanced TLR3-dependent activation of IRF3 in macrophages. Second, poly I:C induced activation of phagocyte NADPH oxidase in a TLR3-independent, but Mac-1-dependent, manner. Subsequently, phagocyte NADPH oxidase-derived intracellular reactive oxygen species activated MAPK and NF-κB pathways. Our results indicate that extracellular dsRNA activates Mac-1 to enhance TLR3-dependent signaling and to trigger TLR3-independent, but Mac-1-dependent, inflammatory oxidative signaling, identifying a novel mechanistic basis for macrophages to recognize extracellular dsRNA to regulate innate immune responses. This study identifies Mac-1 as a novel surface receptor for extracellular dsRNA and implicates it as a potential therapeutic target for virus-related inflammatory diseases.
Asunto(s)
Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Espacio Extracelular/genética , Mediadores de Inflamación/fisiología , Antígeno de Macrófago-1/metabolismo , ARN Bicatenario/fisiología , Animales , Antígeno CD11b/genética , Antígenos CD18/genética , Línea Celular , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Antígeno de Macrófago-1/genética , Macrófagos Peritoneales/enzimología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Masculino , Ratones , Ratones Noqueados , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 3RESUMEN
The variable patterns of DNA methylation in mammals have been linked to a number of physiological processes, including normal embryonic development and disease pathogenesis. Active removal of DNA methylation, which potentially regulates neuronal gene expression both globally and gene specifically, has been recently implicated in neuronal plasticity, learning and memory processes. Model pathways of active DNA demethylation involve ten-eleven translocation (TET) methylcytosine dioxygenases that are dependent on oxidative metabolites. In addition, reactive oxygen species (ROS) and oxidizing agents generate oxidative modifications of DNA bases that can be removed by base excision repair proteins. These potentially link the two processes of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons. We review the current biochemical understanding of the DNA demethylation process and discuss its potential interaction with oxidative metabolism. We then summarise the emerging roles of both processes and their interaction in neural plasticity and memory formation and the pathophysiology of neurodegeneration. Finally, possible therapeutic approaches for neurodegenerative diseases are proposed, including reprogramming therapy by global DNA demethylation and mitohormesis therapy for locus-specific DNA demethylation in post-mitotic neurons.
Asunto(s)
Metilación de ADN , Mitocondrias/genética , Mitocondrias/metabolismo , Neuronas/metabolismo , Cementos de Resina , Animales , Citosina/metabolismo , Dioxigenasas/metabolismo , Humanos , Aprendizaje/fisiología , Memoria/fisiología , Mitocondrias/efectos de los fármacos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacología , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Parkinson's disease (PD) is a prevalent neurodegenerative disorder, marked by the degeneration of dopamine (DA) neurons in the substantia nigra (SN). Current evidence strongly suggests that neuroinflammation, primarily mediated by microglia, contributes to PD pathogenesis. Triggering receptor expressed on myeloid cells 2 (TREM2) might serve as a promising therapeutic target for PD due to its ability to suppress neuroinflammation. Dihydroquercetin (DHQ) is an important natural dihydroflavone and confers apparent anti-inflammatory, antioxidant and anti-fibrotic effects. Recently, DHQ-mediated neuroprotection was exhibited. However, the specific mechanisms of its neuroprotective effects remain incompletely elucidated. METHODS: In this study, rat models were utilized to induce damage to DA neurons using lipopolysaccharide (LPS) and 6-hydroxydopamine (6-OHDA) to assess the impacts of DHQ on the loss of DA neurons. Furthermore, DA neuronal MN9D cells and microglial BV2 cells were employed to investigate the function of TREM2 in DHQ-mediated DA neuroprotection. Finally, TREM2 knockout mice were used to investigate whether the neuroprotective effects mediated by DHQ through a mechanism dependent on TREM2. RESULTS: The main findings demonstrated that DHQ effectively protected DA neurons against neurotoxicity induced by LPS and 6-OHDA and inhibited microglia-elicited neuroinflammation. Meanwhile, DHQ promoted microglial TREM2 signaling activation. Notably, DHQ failed to reduce inflammatory cytokines release and further present neuroprotection from DA neurotoxicity upon TREM2 silencing. Similarly, DHQ didn't exert DA neuroprotection in TREM2 knockout mice. CONCLUSIONS: These findings suggest that DHQ exerted DA neuroprotection by regulating microglia TREM2 activation.
Asunto(s)
Neuronas Dopaminérgicas , Glicoproteínas de Membrana , Microglía , Fármacos Neuroprotectores , Quercetina , Receptores Inmunológicos , Animales , Masculino , Ratones , Ratas , Línea Celular , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Lipopolisacáridos , Glicoproteínas de Membrana/metabolismo , Ratones Noqueados , Microglía/efectos de los fármacos , Microglía/metabolismo , Fármacos Neuroprotectores/farmacología , Oxidopamina , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Quercetina/farmacología , Quercetina/análogos & derivados , Ratas Sprague-Dawley , Receptores Inmunológicos/metabolismo , Ratones Endogámicos C57BLRESUMEN
Strong evidence indicates critical roles of NADPH oxidase (a key superoxide-producing enzyme complex during inflammation) in activated microglia for mediating neuroinflammation and neurodegeneration. However, little is known about roles of neuronal NADPH oxidase in neurodegenerative diseases. This study aimed to investigate expression patterns, regulatory mechanisms and pathological roles of neuronal NADPH oxidase in inflammation-associated neurodegeneration. The results showed persistent upregulation of NOX2 (gp91phox; the catalytic subunit of NADPH oxidase) in both microglia and neurons in a chronic mouse model of Parkinson's disease (PD) with intraperitoneal LPS injection and LPS-treated midbrain neuron-glia cultures (a cellular model of PD). Notably, NOX2 was found for the first time to exhibit a progressive and persistent upregulation in neurons during chronic neuroinflammation. While primary neurons and N27 neuronal cells displayed basal expression of NOX1, NOX2 and NOX4, significant upregulation only occurred in NOX2 but not NOX1 or NOX4 under inflammatory conditions. Persistent NOX2 upregulation was associated with functional outcomes of oxidative stress including increased ROS production and lipid peroxidation. Neuronal NOX2 activation displayed membrane translocation of cytosolic p47phox subunit and was inhibited by apocynin and diphenyleneiodonium chloride (two widely-used NADPH oxidase inhibitors). Importantly, neuronal ROS production, mitochondrial dysfunction and degeneration induced by inflammatory mediators in microglia-derived conditional medium were blocked by pharmacological inhibition of neuronal NOX2. Furthermore, specific deletion of neuronal NOX2 prevented LPS-elicited dopaminergic neurodegeneration in neuron-microglia co-cultures separately grown in the transwell system. The attenuation of inflammation-elicited upregulation of NOX2 in neuron-enriched and neuron-glia cultures by ROS scavenger N-acetylcysteine indicated a positive feedback mechanism between excessive ROS production and NOX2 upregulation. Collectively, our findings uncovered crucial contribution of neuronal NOX2 upregulation and activation to chronic neuroinflammation and inflammation-related neurodegeneration. This study reinforced the importance of developing NADPH oxidase-targeting therapeutics for neurodegenerative diseases.
Asunto(s)
Enfermedades Neuroinflamatorias , Enfermedad de Parkinson , Animales , Ratones , Células Cultivadas , Neuronas Dopaminérgicas/metabolismo , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Lipopolisacáridos/metabolismo , Ratones Endogámicos C57BL , Microglía/metabolismo , NADPH Oxidasas/metabolismo , Enfermedad de Parkinson/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Clinical and pathological evidence revealed that α-synuclein (α-syn) pathology seen in PD patients starts in the gut and spreads via anatomically connected structures from the gut to the brain. Our previous study demonstrated that depletion of central norepinephrine (NE) disrupted brain immune homeostasis, producing a spatiotemporal order of neurodegeneration in the mouse brain. The purpose of this study was 1) to determine the role of peripheral noradrenergic system in the maintenance of gut immune homeostasis and in the pathogenesis of PD and 2) to investigate whether NE-depletion induced PD-like α-syn pathological changes starts from the gut. For these purposes, we investigated time-dependent changes of α-synucleinopathy and neuronal loss in the gut following a single injection of DSP-4 (a selective noradrenergic neurotoxin) to A53T-SNCA (human mutant α-syn) over-expression mice. We found DPS-4 significantly reduced the tissue level of NE and increased immune activities in gut, characterized by increased number of phagocytes and proinflammatory gene expression. Furthermore, a rapid-onset of α-syn pathology was observed in enteric neurons after 2 weeks and delayed dopaminergic neurodegeneration in the substantia nigra was detected after 3-5 months, associated with the appearance of constipation and impaired motor function, respectively. The increased α-syn pathology was only observed in large, but not in the small, intestine, which is similar to what was observed in PD patients. Mechanistic studies reveal that DSP-4-elicited upregulation of NADPH oxidase (NOX2) initially occurred only in immune cells during the acute intestinal inflammation stage, and then spread to enteric neurons and mucosal epithelial cells during the chronic inflammation stage. The upregulation of neuronal NOX2 correlated well with the extent of α-syn aggregation and subsequent enteric neuronal loss, suggesting that NOX2-generated reactive oxygen species play a key role in α-synucleinopathy. Moreover, inhibiting NOX2 by diphenyleneiodonium or restoring NE function by salmeterol (a ß2-receptor agonist) significantly attenuated colon inflammation, α-syn aggregation/propagation, and enteric neurodegeneration in the colon and ameliorated subsequent behavioral deficits. Taken together, our model of PD shows a progressive pattern of pathological changes from the gut to the brain and suggests a potential role of the noradrenergic dysfunction in the pathogenesis of PD.
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Sinucleinopatías , Humanos , Animales , Ratones , Inflamación/patología , Norepinefrina/metabolismo , Colon/patologíaRESUMEN
What drives the gradual degeneration of dopamine neurons in Parkinson's disease (PD), the second most common neurodegenerative disease, remains elusive. Here, we demonstrated, for the first time, that persistent neuroinflammation was indispensible for such a neurodegenerative process. 1-Methyl-4-phenylpyridinium, lipopolysaccharide (LPS), and rotenone, three toxins often used to create PD models, produced acute but nonprogressive neurotoxicity in neuron-enriched cultures. In the presence of microglia (brain immune cells), these toxins induced progressive dopaminergic neurodegeneration. More importantly, such neurodegeneration was prevented by removing activated microglia. Collectively, chronic neuroinflammation may be a driving force of progressive dopaminergic neurodegeneration. Conversely, ongoing neurodegeneration sustained microglial activation. Microglial activation persisted only in the presence of neuronal damage in LPS-treated neuron-glia cultures but not in LPS-treated mixed-glia cultures. Thus, activated microglia and damaged neurons formed a vicious cycle mediating chronic, progressive neurodegeneration. Mechanistic studies indicated that HMGB1 (high-mobility group box 1), released from inflamed microglia and/or degenerating neurons, bound to microglial Mac1 (macrophage antigen complex 1) and activated nuclear factor-κB pathway and NADPH oxidase to stimulate production of multiple inflammatory and neurotoxic factors. The treatment of microglia with HMGB1 led to membrane translocation of p47(phox) (a cytosolic subunit of NADPH oxidase) and consequent superoxide release, which required the presence of Mac1. Neutralization of HMGB1 and genetic ablation of Mac1 and gp91(phox) (the catalytic submit of NADPH oxidase) blocked the progressive neurodegeneration. Our findings indicated that HMGB1-Mac1-NADPH oxidase signaling axis bridged chronic neuroinflammation and progressive dopaminergic neurodegeneration, thus identifying a mechanistic basis for chronic PD progression.
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Proteína HMGB1/metabolismo , Inflamación/metabolismo , Antígeno de Macrófago-1/metabolismo , Microglía/metabolismo , Degeneración Nerviosa/metabolismo , Análisis de Varianza , Animales , Western Blotting , Recuento de Células , Fraccionamiento Celular , Células Cultivadas , Técnicas de Cocultivo , Dopamina/metabolismo , Inflamación/inducido químicamente , Inflamación/patología , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , Mesencéfalo/citología , Mesencéfalo/metabolismo , Microglía/citología , Microglía/patología , NADPH Oxidasas/metabolismo , Degeneración Nerviosa/patología , Neuronas/citología , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Endogámicas F344 , Transducción de SeñalRESUMEN
Nuclear protein high-mobility group box 1 (HMGB1) can be actively secreted by activated immune cells and functions as a proinflammatory cytokine. Regulation of HMGB1 secretion is critical for treatment of HMGB1-mediated inflammation and related diseases. This study demonstrates that S-nitrosylation (SNO; the covalent binding of nitric oxide [NO] to cysteine thiols) by inducible nitric oxide synthase (iNOS)-derived NO at Cys106 is essential and sufficient for inflammation-elicited HMGB1 secretion. iNOS deletion or inhibition or Cys106Ser mutation prevents lipopolysaccharide (LPS)- and/or poly(I:C)-elicited HMGB1 secretion. NO donors induce SNO of HMGB1 and reproduce inflammogen-triggered HMGB1 secretion. SNO of HMGB1 promotes its proinflammatory and neurodegenerative effects. Intranigral HMGB1 injection induces chronic microglial activation, dopaminergic neurodegeneration, and locomotor deficits, the key features of Parkinson's disease (PD), in wild-type, but not Mac1 (CD11b/CD18)-deficient, mice. This study indicates pivotal roles for SNO modification in HMGB1 secretion and HMGB1-Mac1 interaction for inflammatory neurodegeneration, identifying a mechanistic basis for PD development.
Asunto(s)
Proteína HMGB1/metabolismo , Animales , Inflamación , Lipopolisacáridos/farmacología , Ratones , Óxido Nítrico/metabolismo , Donantes de Óxido NítricoRESUMEN
Ribbon synapses of cochlear hair cells undergo pruning and maturation before the hearing onset. In the central nervous system (CNS), synaptic pruning was mediated by microglia, the brain-resident macrophages, via activation of the complement system. Whether a similar mechanism regulates ribbon synapse pruning is currently unknown. In this study, we report that the densities of cochlear macrophages surrounding hair cells were highest at around P8, corresponding well to the completion of ribbon synaptic pruning by P8-P9. Surprisingly, using multiple genetic mouse models, we found that postnatal pruning of the ribbon synapses and auditory functions were unaffected by the knockout of the complement receptor 3 (CR3) or by ablations of macrophages expressing either LysM or Cx3cr1. Our results suggest that unlike microglia in the CNS, macrophages in the cochlea do not mediate pruning of the cochlear ribbon synapses.
RESUMEN
Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by a progressive loss of dopamine (DA) neurons in the substantia nigra. Accumulating evidence indicates that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for PD. Resveratrol, a nonflavonoid polyphenol naturally found in red wine and grapes, has been known to possess antioxidant, anticancer, and anti-inflammatory properties. Although recent studies have shown that resveratrol provided neuroprotective effects against ischemia, seizure, and neurodegenerative disorders, the mechanisms underlying its beneficial effects on dopaminergic neurodegeneration are poorly defined. In this study, rat primary midbrain neuron-glia cultures were used to elucidate the molecular mechanisms underlying resveratrol-mediated neuroprotection. The results clearly demonstrated that resveratrol protected DA neurons against lipopolysaccharide (LPS)-induced neurotoxicity in concentration- and time-dependent manners through the inhibition of microglial activation and the subsequent reduction of proinflammatory factor release. Mechanistically, resveratrol-mediated neuroprotection was attributed to the inhibition of NADPH oxidase. This conclusion is supported by the following observations. First, resveratrol reduced NADPH oxidase-mediated generation of reactive oxygen species. Second, LPS-induced translocation of NADPH oxidase cytosolic subunit p47 to the cell membrane was significantly attenuated by resveratrol. Third and most importantly, resveratrol failed to exhibit neuroprotection in cultures from NADPH oxidase-deficient mice. Furthermore, this neuroprotection was also related to an attenuation of the activation of mitogen-activated protein kinases and nuclear factor-kappaB signaling pathways in microglia. These findings suggest that resveratrol exerts neuroprotection against LPS-induced dopaminergic neurodegeneration, and NADPH oxidase may be a major player in resveratrol-mediated neuroprotection.
Asunto(s)
Neuronas/efectos de los fármacos , Animales , Antiinflamatorios/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Dopamina/metabolismo , Dopamina/farmacología , Dopamina/fisiología , Embrión de Mamíferos , Femenino , Flavonoides/metabolismo , Flavonoides/farmacología , Flavonoides/uso terapéutico , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Mesencéfalo/citología , Mesencéfalo/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/farmacología , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , NADPH Oxidasas/fisiología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/prevención & control , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Síndromes de Neurotoxicidad/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Fenoles/metabolismo , Fenoles/farmacología , Fenoles/uso terapéutico , Polifenoles , Embarazo , Ratas , Ratas Endogámicas F344 , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Especies Reactivas de Oxígeno/uso terapéutico , ResveratrolRESUMEN
Parkinson's disease (PD) is a progressive neurological disorder characterized by a selective loss of dopamine (DA) neurons in the substantia nigra (SN). Although current therapy can control symptoms of this disorder, there is no effective therapy available to halt its progression. Recently, neuroinflammation has been recognized as an important contributor to the pathogenesis of PD, and nuclear factor-kappaB (NF-kappaB) plays a key role in regulating neuroinflammation. Hence, the modulation of NF-kappaB pathway may have therapeutic potential for PD. Activation of NF-kappaB depends on the phosphorylation of its inhibitor, IkappaB, by the specific IkappaB kinase (IKK) subunit IKK-beta. Compound A (7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1, 4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one hydrochloride), a potent and selective inhibitor of IKK-beta, has recently been reported to provide cardioprotection through specific suppression of NF-kappaB signaling. The present study, for the first time, elucidates neuroprotective effects of compound A. Daily subcutaneous injection of compound A (1 mg/kg) for 7 days inhibited the activation of microglia induced by nigral stereotaxic injection of lipopolysaccharide (LPS) and significantly attenuated LPS-induced loss of DA neurons in the SN. In vitro mechanistic studies revealed that neuroprotective effects of compound A were mediated by 1) suppressing the activity of microglial NADPH oxidase and decreasing the production of reactive oxygen species, and 2) inhibiting NF-kappaB-mediated gene transcription of various proinflammatory mediators in microglia via IKK-beta suppression. These findings indicate that compound A afforded potent neuroprotection against LPS-induced neurodegeneration through selective inhibition of NF-kappaB activation and may be of potential benefit in the treatment of PD.
Asunto(s)
Dopamina/fisiología , Inhibidores Enzimáticos/farmacología , Quinasa I-kappa B/antagonistas & inhibidores , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/toxicidad , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores , Neurotoxinas/antagonistas & inhibidores , Oxazinas/farmacología , Piridinas/farmacología , Animales , Astrocitos/efectos de los fármacos , Western Blotting , Células Cultivadas , Dopamina/metabolismo , Inmunohistoquímica , Indicadores y Reactivos , Interleucina-1beta/metabolismo , Masculino , Microglía/efectos de los fármacos , NADPH Oxidasas/antagonistas & inhibidores , Neuroglía/efectos de los fármacos , Nitritos/metabolismo , Ratas , Ratas Endogámicas F344 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxidos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
alpha-Synuclein (SYN) is the major component of Lewy bodies, the neuropathological hallmarks of Parkinson's disease (PD). Missense mutations and multiplications of the SYN gene cause autosomal dominant inherited PD. Thus, SYN is implicated in the pathogenesis of PD. However, the mechanism whereby SYN promotes neurodegeneration remains unclear. Familial PD with SYN gene mutations are rare because the majority of PD is sporadic and emerging evidence indicates that sporadic PD may result from genetic and environmental risk factors including neuroinflammation. Hence, we examined the relationship between SYN dysfunction and neuroinflammation in mediating dopaminergic neurodegeneration in mice and dopaminergic neuronal cultures derived from wild-type SYN and mutant A53T SYN transgenic mice in a murine SYN-null (SYNKO) background (M7KO and M83KO, respectively). Stereotaxic injection of an inflammagen, lipopolysaccharide, into substantia nigra of these SYN genetically engineered mice induced similar inflammatory reactions. In M7KO and M83KO, but not in SYNKO mice, the neuroinflammation was associated with dopaminergic neuronal death and the accumulation of insoluble aggregated SYN as cytoplasmic inclusions in nigral neurons. Nitrated/oxidized SYN was detected in these inclusions and abatement of microglia-derived nitric oxide and superoxide provided significant neuroprotection in neuron-glia cultures from M7KO mice. These data suggest that nitric oxide and superoxide released by activated microglia may be mediators that link inflammation and abnormal SYN in mechanisms of PD neurodegeneration. This study advances understanding of the role of neuroinflammation and abnormal SYN in the pathogenesis of PD and opens new avenues for the discovery of more effective therapies for PD.
Asunto(s)
Dopamina/metabolismo , Enfermedades Neurodegenerativas/complicaciones , Enfermedades Neurodegenerativas/genética , Óxido Nítrico Sintasa/metabolismo , Estrés Oxidativo/fisiología , alfa-Sinucleína/genética , Análisis de Varianza , Animales , Recuento de Células , Células Cultivadas , Técnicas de Cocultivo/métodos , Embrión de Mamíferos , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Lipopolisacáridos/efectos adversos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/fisiología , Neuronas/ultraestructura , alfa-Sinucleína/deficienciaRESUMEN
The purpose of this study was to develop a novel therapy for Parkinson's disease (PD). We recently reported that dextromethorphan (DM), an active ingredient in a variety of widely used anticough remedies, protected dopaminergic neurons in rat primary mesencephalic neuron-glia cultures against lipopolysaccharide (LPS)-mediated degeneration and provided potent protection for dopaminergic neurons in a MPTP mouse model. The underlying mechanism for the protective effect of DM was attributed to its anti-inflammatory activity through inhibition of microglia activation. In an effort to develop more potent compounds for the treatment of PD, we have screened a series of analogs of DM, and 3-hydroxymorphinan (3-HM) emerged as a promising candidate for this purpose. Our study using primary mesencephalic neuron-glia cultures showed that 3-HM provided more potent neuroprotection against LPS-induced dopaminergic neurotoxicity than its parent compound. The higher potency of 3-HM was attributed to its neurotrophic effect in addition to the anti-inflammatory effect shared by both DM and 3-HM. First, we showed that 3-HM exerted potent neuroprotective and neurotrophic effects on dopaminergic neurons in rat primary mesencephalic neuron-glia cultures treated with LPS. The neurotrophic effect of 3-HM was glia-dependent since 3-HM failed to show any protective effect in the neuron-enriched cultures. We subsequently demonstrated that it was the astroglia, not the microglia, that contributed to the neurotrophic effect of 3-HM. This conclusion was based on the reconstitution studies, in which we added different percentages of microglia (10-20%) or astroglia (40-50%) back to the neuron-enriched cultures and found that 3-HM was neurotrophic after the addition of astroglia, but not microglia. Furthermore, 3-HM-treated astroglia-derived conditioned media exerted a significant neurotrophic effect on dopaminergic neurons. It appeared likely that 3-HM caused the release of neurotrophic factor(s) from astroglia, which in turn was responsible for the neurotrophic effect. Second, the anti-inflammatory mechanism was also important for the neuroprotective activity of 3-HM because the more microglia were added back to the neuron-enriched cultures, the more significant neuroprotective effect was observed. The anti-inflammatory mechanism of 3-HM was attributed to its inhibition of LPS-induced production of an array of pro-inflammatory and neurotoxic factors, including nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), prostaglandin E2 (PGE2) and reactive oxygen species (ROS). In conclusion, this study showed that 3-HM exerted potent neuroprotection by acting on two different targets: a neurotrophic effect mediated by astroglia and an anti-inflammatory effect mediated by the inhibition of microglial activation. 3-HM thus possesses these two important features necessary for an effective neuroprotective agent. In view of the well-documented very low toxicity of DM and its analogs, this report may provide an important new direction for the development of therapeutic interventions for inflammation-related diseases such as PD.
Asunto(s)
Dextrometorfano/análogos & derivados , Dopamina/fisiología , Lipopolisacáridos/toxicidad , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Antiinflamatorios/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Medios de Cultivo Condicionados , Dextrometorfano/farmacología , Dinoprostona/biosíntesis , Lipopolisacáridos/antagonistas & inhibidores , Mesencéfalo/citología , Microglía/efectos de los fármacos , Microglía/fisiología , Neuroglía/efectos de los fármacos , Neuroglía/fisiología , Neuronas/fisiología , Óxido Nítrico/biosíntesis , Enfermedad de Parkinson/tratamiento farmacológico , Ratas , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Increasing evidence has suggested an important role for environmental toxins such as pesticides in the pathogenesis of Parkinson's disease (PD). Chronic exposure to rotenone, a common herbicide, reproduces features of Parkinsonism in rats. Mechanistically, rotenone-induced dopaminergic neurodegeneration has been associated with both its inhibition of neuronal mitochondrial complex I and the enhancement of activated microglia. Our previous studies with NADPH oxidase inhibitors, diphenylene iodonium and apocynin, suggested that NADPH oxidase-derived superoxide might be a major factor in mediating the microglia-enhanced rotenone neurotoxicity. However, because of the relatively low specificity of these inhibitors, the exact source of superoxide induced by rotenone remains to be further determined. In this study, using primary mesencephalic cultures from NADPH oxidase--null (gp91phox-/-) or wild-type (gp91phox+/+) mice, we demonstrated a critical role for microglial NADPH oxidase in mediating microglia-enhanced rotenone neurotoxicity. In neuron--glia cultures, dopaminergic neurons from gp91phox-/- mice were more resistant to rotenone neurotoxicity than those from gp91phox+/+ mice. However, in neuron-enriched cultures, the neurotoxicity of rotenone was not different between the two types of mice. More importantly, the addition of microglia prepared from gp91phox+/+ mice but not from gp91phox-/- mice to neuron-enriched cultures markedly increased rotenone-induced degeneration of dopaminergic neurons. Furthermore, apocynin attenuated rotenone neurotoxicity only in the presence of microglia from gp91phox+/+ mice. These results indicated that the greatly enhanced neurotoxicity of rotenone was attributed to the release of NADPH oxidase-derived superoxide from activated microglia. This study also suggested that microglial NADPH oxidase may be a promising target for PD treatment.