RESUMEN
Manganese (Mn) is an essential heavy metal in the human body, while excess Mn leads to neurotoxicity, as observed in this study, where 100 µM of Mn was administered to the human neuroblastoma (SH-SY5Y) cell model of dopaminergic neurons in neurodegenerative diseases. We quantitated pathway and gene changes in homeostatic cell-based adaptations to Mn exposure. Utilizing the Gene Expression Omnibus, we accessed the GSE70845 dataset as a microarray of SH-SY5Y cells published by Gandhi et al. (2018) and applied statistical significance cutoffs at p < 0.05. We report 74 pathway and 10 gene changes with statistical significance. ReactomeGSA analyses demonstrated upregulation of histones (5 out of 10 induced genes) and histone deacetylases as a neuroprotective response to remodel/mitigate Mn-induced DNA/chromatin damage. Neurodegenerative-associated pathway changes occurred. NF-κB signaled protective responses via Sirtuin-1 to reduce neuroinflammation. Critically, Mn activated three pathways implicating deficits in purine metabolism. Therefore, we validated that urate, a purine and antioxidant, mitigated Mn-losses of viability in SH-SY5Y cells. We discuss Mn as a hypoxia mimetic and trans-activator of HIF-1α, the central trans-activator of vascular hypoxic mitochondrial dysfunction. Mn induced a 3-fold increase in mRNA levels for antioxidant metallothionein-III, which was induced 100-fold by hypoxia mimetics deferoxamine and zinc.
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Manganeso , Neuroblastoma , Humanos , Manganeso/toxicidad , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuroblastoma/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Biomarcadores/metabolismoRESUMEN
Background: The G2019S mutation of LRRK2, which enhances kinase activity of the protein, confers a substantial risk of developing Parkinson's disease (PD). However, the mutation demonstrates incomplete penetrance, suggesting the involvement of other genetic or environmental modulating factors. Here, we investigated whether LRRK2 G2019S knock-in (KI) mice treated with the inflammogen lipopolysaccharide (LPS) could model LRRK2 PD. Results: We found that short-term (2 weeks) treatment with LPS did not result in the loss of dopaminergic neurons in either LRRK2 G2019S KI or wild-type (WT) mice. Compared with WT mice, LRRK2 G2019S-KI mice showed incomplete recovery from LPS-induced weight loss. In LRRK2 G2019S KI mice, LPS treatment led to upregulated phosphorylation of LRRK2 at the autophosphorylation site Serine 1292, which is known as a direct readout of LRRK2 kinase activity. LPS treatment caused a greater increase in the activated astrocyte marker glial fibrillary acidic protein (GFAP) in the striatum and substantia nigra of LRRK2 G2019S mice than in those of WT mice. The administration of caffeine, which was recently identified as a biomarker of resistance to developing PD in individuals with LRRK2 mutations, attenuated LPS-induced astrocyte activation specifically in LRRK2 G2019S KI mice. Conclusions: Our findings suggest that 2 weeks of exposure to LPS is not sufficient to cause dopaminergic neuronal loss in LRRK2 G2019S KI mice but rather results in increased astrocyte activation, which can be ameliorated by caffeine.
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Chronic neuroinflammation is implicated in the pathogenesis of Parkinson's disease (PD), one of the most common neurodegenerative diseases. Itaconate, an endogenous metabolite derived from the tricarboxylic acid cycle via immune-responsive gene 1 activity, may mediate anti-inflammatory responses by activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway. This study investigates the neuroprotective potential of 4-octyl itaconate (OI), a cell-permeable derivative of itaconate, in cellular models of PD. OI not only suppressed lipopolysaccharide-induced proinflammatory cascades of inducible nitric oxide synthase, cyclooxygenase-2, and cytokines release in mouse BV2 microglial cells but also activated the Nrf2 signaling pathway and its downstream targets in these cells. Conditioned medium derived from OI-treated BV2 cells protected against rotenone- and MPP+-induced neurotoxicity in Neuro 2A cells. Overall, our findings support the anti-inflammatory neuroprotective potential of OI in PD.
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Fármacos Neuroprotectores , Síndromes de Neurotoxicidad , Enfermedad de Parkinson , Animales , Ratones , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Rotenona/toxicidad , Microglía , Factor 2 Relacionado con NF-E2RESUMEN
OBJECTIVE: To identify markers of resistance to developing Parkinson disease (PD) among LRRK2 mutation carriers (LRRK2+), we carried out metabolomic profiling in individuals with PD and unaffected controls (UC), with and without the LRRK2 mutation. METHODS: Plasma from 368 patients with PD and UC in the LRRK2 Cohort Consortium (LCC), comprising 118 LRRK2+/PD+, 115 LRRK2+/UC, 70 LRRK2-/PD+, and 65 LRRK2-/UC, and CSF available from 68 of them, were analyzed by liquid chromatography with mass spectrometry. For 282 analytes quantified in plasma and CSF, we assessed differences among the 4 groups and interactions between LRRK2 and PD status, using analysis of covariance models adjusted by age, study site cohort, and sex, with p value corrections for multiple comparisons. RESULTS: Plasma caffeine concentration was lower in patients with PD vs UC (p < 0.001), more so among LRRK2+ carriers (by 76%) than among LRRK2- participants (by 31%), with significant interaction between LRRK2 and PD status (p = 0.005). Similar results were found for caffeine metabolites (paraxanthine, theophylline, 1-methylxanthine) and a nonxanthine marker of coffee consumption (trigonelline) in plasma, and in the subset of corresponding CSF samples. Dietary caffeine was also lower in LRRK2+/PD+ compared to LRRK2+/UC with significant interaction effect with the LRRK2+ mutation (p < 0.001). CONCLUSIONS: Metabolomic analyses of the LCC samples identified caffeine, its demethylation metabolites, and trigonelline as prominent markers of resistance to PD linked to pathogenic LRRK2 mutations, more so than to idiopathic PD. Because these analytes are known both as correlates of coffee consumption and as neuroprotectants in animal PD models, the findings may reflect their avoidance by those predisposed to develop PD or their protective effects among LRRK2 mutation carriers.
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Alcaloides/sangre , Cafeína/sangre , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Fármacos Neuroprotectores/sangre , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/genética , Anciano , Alcaloides/líquido cefalorraquídeo , Cafeína/líquido cefalorraquídeo , Cromatografía Liquida , Estudios de Cohortes , Femenino , Heterocigoto , Humanos , Masculino , Espectrometría de Masas , Metabolómica , Persona de Mediana Edad , Fármacos Neuroprotectores/líquido cefalorraquídeo , Enfermedad de Parkinson/líquido cefalorraquídeo , Teofilina/sangre , Teofilina/líquido cefalorraquídeo , Xantinas/sangre , Xantinas/líquido cefalorraquídeoRESUMEN
Higher serum urate concentration is associated with decreased risk of Parkinson's disease (PD) as well as slower disease progression, but its relationship with severity of PD remains unclear. This study investigated whether changes in serum urate concentration over 5 years were associated with disease progression assessed by MDS-UPDRS Part III score, Hoehn and Yahr stage, or DaTscan imaging. Average serum urate concentration was stable over time and change in serum urate concentration did not correlate with worsening of measures of PD progression. These results suggest that serum urate concentration is not a monitoring biomarker of PD progression in early stages.
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Progresión de la Enfermedad , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/fisiopatología , Ácido Úrico/sangre , Anciano , Biomarcadores/sangre , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Índice de Severidad de la EnfermedadRESUMEN
Two purines, caffeine and urate, have been associated with a reduced risk of idiopathic Parkinson's disease (PD) in multiple cohorts and populations. The Harvard Biomarkers Study (HBS) is a longitudinal study designed to accelerate the discovery and validation of molecular diagnostic and progression markers of early-stage PD. To investigate whether these 'reduced risk' factors are associated with PD within this cohort, we conducted a cross-sectional, case-control study in 566 subjects consisting of idiopathic PD patients and healthy controls. Caffeine intake as assessed by a validated questionnaire was significantly lower in idiopathic PD patients compared to healthy controls in males (mean difference -125âmg/day, pâ<â0.001) but not in females (mean difference -30âmg/day, pâ=â0.29). A strong inverse association was also observed with plasma urate levels both in males (mean difference -0.46âmg/dL, pâ=â0.017) and females (mean difference -0.45âmg/dL, pâ=â0.001). Both analyses stratified for sex and adjusted for age, body mass index, and either urate level or caffeine consumption, respectively. These results highlight the robustness of caffeine intake and urate as factors inversely associated with idiopathic PD.
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Cafeína/administración & dosificación , Enfermedad de Parkinson/metabolismo , Ácido Úrico/sangre , Anciano , Biomarcadores/metabolismo , Estudios de Casos y Controles , Estudios Transversales , Ingestión de Alimentos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/sangre , Factores SexualesRESUMEN
OBJECTIVE: To investigate whether women and men with Parkinson disease (PD) differ in their biochemical and clinical responses to long-term treatment with inosine. METHODS: The Safety of Urate Elevation in Parkinson's Disease (SURE-PD) trial enrolled 75 people with early PD and baseline serum urate below 6 mg/dL and randomized them to 3 double-blinded treatment arms: oral placebo or inosine titrated to produce mild (6.1-7.0 mg/dL) or moderate (7.1-8.0 mg/dL) serum urate elevation for up to 2 years. Parkinsonism, serum urate, and plasma antioxidant capacity were measured at baseline and repeatedly on treatment; CSF urate was assessed once, at 3 months. Here in secondary analyses results are stratified by sex. RESULTS: Inosine produced an absolute increase in average serum urate from baseline that was 50% greater in women (3.0 mg/dL) than in men (2.0 mg/dL), consistent with expected lower baseline levels in women. Similarly, only among women was CSF urate significantly greater on mild or moderate inosine (+87% [p < 0.001] and +98% [p < 0.001], respectively) than on placebo (in contrast to men: +10% [p = 0.6] and +14% [p = 0.4], respectively). Women in the higher inosine dosing group showed a 7.0 Unified Parkinson's Disease Rating Scale (UPDRS) points/year lower rate of decline vs placebo (p = 0.01). In women, slower rates of UPDRS change were associated with greater increases in serum urate (r = -0.52; p = 0.001), and with greater increases in plasma antioxidant capacity (r = -0.44; p = 0.006). No significant associations were observed in men. CONCLUSIONS: Inosine produced greater increases in serum and CSF urate in women compared to men in the SURE-PD trial, consistent with the study's design and with preliminary evidence for slower clinical decline in early PD among women treated with urate-elevating doses of inosine. CLINICALTRIALSGOV IDENTIFIER: NCT00833690. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that inosine produced greater urate elevation in women than men and may slow PD progression in women.
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Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/diagnóstico , Caracteres Sexuales , Ácido Úrico/sangre , Biomarcadores/sangre , Femenino , Estudios de Seguimiento , Humanos , Inosina/uso terapéutico , Masculino , Pruebas de Estado Mental y Demencia , Enfermedad de Parkinson/tratamiento farmacológico , Resultado del TratamientoRESUMEN
The therapeutic value of inhibiting translation of the amyloid precursor protein (APP) offers the possibility to reduce neurotoxic amyloid formation, particularly in cases of familial Alzheimer's disease (AD) caused by APP gene duplications (Dupâ»APP) and in aging Down syndrome individuals. APP mRNA translation inhibitors such as the anticholinesterase phenserine, and high throughput screened molecules, selectively inhibited the uniquely folded iron-response element (IRE) sequences in the 5'untranslated region (5'UTR) of APP mRNA and this class of drug continues to be tested in a clinical trial as an anti-amyloid treatment for AD. By contrast, in younger age groups, APP expression is not associated with amyloidosis, instead it acts solely as a neuroprotectant while facilitating cellular ferroportin-dependent iron efflux. We have reported that the environmental metallotoxins Lead (Pb) and manganese (Mn) cause neuronal death by interfering with IRE dependent translation of APP and ferritin. The loss of these iron homeostatic neuroprotectants thereby caused an embargo of iron (Fe) export from neurons as associated with excess unstored intracellular iron and the formation of toxic reactive oxidative species (ROS). We propose that APP 5'UTR directed translation activators can be employed therapeutically to protect neurons exposed to high acute Pb and/or Mn exposure. Certainly, high potency APP translation activators, exemplified by the Food and Drug Administration (FDA) pre-approved M1 muscarinic agonist AF102B and high throughput-screened APP 5'UTR translation activators, are available for drug development to treat acute toxicity caused by Pb/Mn exposure to neurons. We conclude that APP translation activators can be predicted to prevent acute metal toxicity to neurons by a mechanism related to the 5'UTR specific yohimbine which binds and targets the canonical IRE RNA stem loop as an H-ferritin translation activator.
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Precursor de Proteína beta-Amiloide/genética , Ferritinas/genética , Proteínas Reguladoras del Hierro/genética , Intoxicación del Sistema Nervioso por Plomo/tratamiento farmacológico , Intoxicación por Manganeso/tratamiento farmacológico , Agonistas Muscarínicos/uso terapéutico , Quinuclidinas/uso terapéutico , Elementos de Respuesta/fisiología , Tiofenos/uso terapéutico , Regiones no Traducidas 5'/efectos de los fármacos , Enfermedad Aguda , Enfermedad de Alzheimer/metabolismo , Animales , Síndrome de Down/metabolismo , Humanos , Hierro/metabolismo , Ratones , Agonistas Muscarínicos/farmacología , Neuronas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Quinuclidinas/farmacología , ARN Mensajero/genética , Ratas , Tiofenos/farmacologíaRESUMEN
OBJECTIVE: LRRK2 mutations, the most common genetic cause of Parkinson disease (PD), display incomplete penetrance, indicating the importance of other genetic and environmental influences on disease pathogenesis in LRRK2 mutation carriers. The present study investigates whether urate, an antioxidant, Nrf2 activator, and inverse risk factor for idiopathic PD, is one such candidate biomarker of PD risk modulation in pathogenic LRRK2 mutation carriers. METHODS: Banked plasma samples or urate levels were obtained for 3 cohorts of age- and sex-matched subjects with and without a known LRRK2 mutation in PD and unaffected controls to conduct a pilot study of 192 subjects from the LRRK2 Cohort Consortium (LCC) and 2 validation studies of 380 additional subjects from the LCC and 922 subjects from the Parkinson's Progression Markers Initiative. Urate levels were compared by multiple regression between subjects with and without a PD diagnosis conditional on LRRK2 status, controlling for age and sex. RESULTS: Nonmanifesting LRRK2 mutation carriers had significantly higher levels of urate than those who developed PD in each of the 3 independent cohorts. A meta-analysis demonstrated an adjusted mean difference of 0.62 mg/dL (p < 0.001), with similar results for separate assessments of women (p < 0.02) and men (p < 0.001). A 2 mg/dL increment in urate concentration decreased the odds of having PD by approximately 50% (odds ratio = 0.48, p = 0.004). INTERPRETATION: These findings identify and substantiate urate as a biomarker of resistance to PD among LRRK2 mutation carriers. Ann Neurol 2019;85:593-599.
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Resistencia a la Enfermedad/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación/genética , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/genética , Ácido Úrico/sangre , Adulto , Anciano , Biomarcadores/sangre , Estudios de Cohortes , Femenino , Predisposición Genética a la Enfermedad/genética , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/diagnósticoRESUMEN
OBJECTIVE: To test the safety, tolerability, and urate-elevating capability of the urate precursor inosine taken orally or by feeding tube in people with amyotrophic lateral sclerosis (ALS). METHODS: This was a pilot, open-label trial in 25 participants with ALS. Treatment duration was 12 weeks. The dose of inosine was titrated at pre-specified time points to elevate serum urate levels to 7-8 mg/dL. Primary outcomes were safety (as assessed by the occurrence of adverse events [AEs]) and tolerability (defined as the ability to complete the 12-week study on study drug). Secondary outcomes included biomarkers of oxidative stress and damage. As an exploratory analysis, observed outcomes were compared with a virtual control arm built using prediction algorithms to estimate ALSFRS-R scores. RESULTS: Twenty-four out of 25 participants (96%) completed 12 weeks of study drug treatment. One participant was unable to comply with study visits and was lost to follow-up. Serum urate rose to target levels in 6 weeks. No serious AEs attributed to study drug and no AEs of special concern, such as urolithiasis and gout, occurred. Selected biomarkers of oxidative stress and damage had significant changes during the study period. Observed changes in ALSFRS-R did not differ from baseline predictions. INTERPRETATION: Inosine appeared safe, well tolerated, and effective in raising serum urate levels in people with ALS. These findings, together with epidemiological observations and preclinical data supporting a neuroprotective role of urate in ALS models, provide the rationale for larger clinical trials testing inosine as a potential disease-modifying therapy for ALS.
RESUMEN
Dominant mutations in an antioxidant enzyme superoxide dismutase-1 (SOD1) cause amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease characterized by loss of motor neurons. Oxidative stress has also been linked to many of the neurodegenerative diseases and is likely a central mechanism of motor neuron death in ALS. Astrocytes derived from mutant SOD1G93A mouse models or patients play a significant role in the degeneration of spinal motor neurons in ALS through a non-cell-autonomous process. Here we characterize the neuroprotective effects and mechanisms of urate (a.k.a. uric acid), a major endogenous antioxidant and a biomarker of favorable ALS progression rates, in a cellular model of ALS. Our results demonstrate a significant protective effect of urate against motor neuron injury evoked by mutant astrocytes derived from SOD1G93A mice or hydrogen peroxide induced oxidative stress. Overall, these results implicate astrocyte dependent protective effect of urate in a cellular model of ALS. These findings together with our biomarker data may advance novel targets for treating motor neuron disease.
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Esclerosis Amiotrófica Lateral/metabolismo , Antioxidantes/farmacología , Astrocitos/metabolismo , Neuronas Motoras/metabolismo , Estrés Oxidativo , Superóxido Dismutasa-1/genética , Ácido Úrico/farmacología , Esclerosis Amiotrófica Lateral/genética , Animales , Línea Celular , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Ratones , Neuronas Motoras/efectos de los fármacos , Mutación , Ácido Úrico/metabolismoRESUMEN
The genome annotation and identification of gene function depends on conserved biochemical activity. However, in the cell, proteins with the same biochemical function can participate in different cellular pathways and cannot complement one another. Similarly, two proteins of very different biochemical functions are put in the same class of cellular function; for example, the classification of a gene as an oncogene or a tumour suppressor gene is not related to its biochemical function, but is related to its cellular function. We have taken an approach to identify peptide signatures for cellular function in proteins with known biochemical function. ATPases as a test case, we classified ATPases (2360 proteins) and kinases (517 proteins) from the human genome into different cellular function categories such as transcriptional, replicative, and chromatin remodelling proteins. Using publicly available tool, MEME, we identify peptide signatures shared among the members of a given category but not between cellular functional categories; for example, no motif sharing is seen between chromatin remodelling and transporter ATPases, similarly between receptor Serine/Threonine Kinase and Receptor Tyrosine Kinase. There are motifs shared within each category with significant E value and high occurrence. This concept of signature for cellular function was applied to developmental regulators, the polycomb and trithorax proteins which led to the prediction of the role of INO80, a chromatin remodelling protein, in development. This has been experimentally validated earlier for its role in homeotic gene regulation and its interaction with regulatory complexes like the Polycomb and Trithorax complex. Proteins 2017; 85:682-693. © 2016 Wiley Periodicals, Inc.
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Adenosina Trifosfatasas/genética , ADN Helicasas/genética , Genoma Humano , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas del Grupo Polycomb/genética , Proteínas Quinasas/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/clasificación , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Transporte Biológico/genética , Cromatina/química , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Anotación de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteínas Quinasas/clasificación , Proteínas Quinasas/metabolismoRESUMEN
Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a central regulator of cellular and mitochondrial metabolism. Cellular bioenergetics are critically important in "energy-guzzling" neurons, but the components and wiring of the transcriptional circuit through which PGC-1α regulates the neuronal electron transport chain have not been established. This information may be vital for restoring neuronal bioenergetics gene expression that is compromised during incipient Parkinson's neuropathology and in aging-dependent brain diseases. Here we delineate a neuronal transcriptional circuit controlled by endogenous PGC-1α. We show that a feed-forward circuit of endogenous neuronal PGC-1α and the orphan nuclear estrogen-related receptor α (ERRα) activates the nuclear-encoded mitochondrial electron transport chain. PGC-1α not only trans-activated expression of ERRα, but also coactivated ERRα target genes in complexes I, II, IV, and V of the neuronal electron transport chain via association with evolutionary conserved ERRα promoter binding motifs. Chemical activation of this transcriptional program induced transcription of the neuronal electron transport chain. These data highlight a neuronal transcriptional circuit regulated by PGC-1α that can be therapeutically targeted for Parkinson's and other neurodegenerative diseases.
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INTRODUCTION: Higher serum urate predicts slower progression in PD. The aim of this work was to assess whether oral inosine alters antioxidant capacity of plasma or CSF or urinary markers of oxidative injury in early PD. METHODS: We assayed plasma and CSF antioxidant capacity by ferric-reducing antioxidant power and measured DNA oxidation adduct 8-hydroxydeoxyguanosine from urine in Safety of URate Elevation in PD, a randomized, placebo-controlled trial of oral inosine assessing safety of elevating serum urate from <6 mg/dL to 6.1-7.0 or 7.1-8.0 mg/dL in patients with early PD. RESULTS: At 6 months, antioxidant capacity was 29% higher among mild and 43% higher among moderate group participants compared to placebo and correlated with change in serum urate (r = 0.86) and inversely with rate of clinical decline (r = -0.26). CSF antioxidant capacity and urine 8-hydroxydeoxyguanosine did not differ. CONCLUSIONS: The findings demonstrate a dose-dependent, persistent elevation of plasma antioxidant capacity from oral inosine of potential therapeutic relevance.
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Antioxidantes/uso terapéutico , Inosina/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Antioxidantes/administración & dosificación , Progresión de la Enfermedad , Femenino , Humanos , Inosina/administración & dosificación , Masculino , Persona de Mediana Edad , Estrés Oxidativo/fisiología , Resultado del Tratamiento , Ácido Úrico/sangreRESUMEN
Glutamate excitotoxicity contributes to the development of Parkinson's disease (PD) and pharmacological blockade of metabotropic glutamate receptor 5 (mGluR5) has beneficial anti-akinetic effects in animal models of PD; however, the mechanism by which these antagonists alleviate PD symptoms is largely unknown. In our study, the effects of mGluR5 inhibition on DNA damage were investigated in a rotenone-induced model of PD. We first found that the selective mGluR5 antagonist, 2-methyl-6- (phenylethynyl) pyridine, prevented rotenone-induced DNA damage in MN9D dopaminergic neurons through a mechanism involving the downregulation of intracellular calcium release which was associated with a reduction in endoplasmic reticulum stress and reactive oxygen species (ROS)-related mitochondrial dysfunction. Interestingly, the ROS-related mitochondrial dysfunction was accompanied by an increase in expression of the antioxidant protein, Trx2. Treatment of cells with the calcium chelating agent 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or the ROS scavenger N-acetyl-L-cysteine, also reduced rotenone-induced DNA damage, while transfection of a dominant-negative form of Trx2 increased it. In addition, mGluR5 inhibition altered the expression profiles of proteins involved in DNA repair activity. Specifically, the expression of phosphorylated ERK (p-ERK) and CREB, as well as APE1 and Rad51 were elevated after rotenone stimulation and were subsequently downregulated following blockade of mGluR5. These findings were confirmed in vivo in a rotenone-induced rat model of PD. Inhibition of mGluR5 protected against neurotoxicity by mitigating oxidative stress-related DNA damage associated with 8-hydroxy-2'-deoxyguanosine production and also reduced p-ERK activity and Trx2 expression. These findings provide a novel link between mGluR5 and DNA damage in a model of PD, and reveal a potential mechanism by which mGluR5 mediates DNA damage in neurodegenerative diseases.
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Daño del ADN , Trastornos Parkinsonianos/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Animales , Western Blotting , Línea Celular , Ensayo Cometa , Reparación del ADN/fisiología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Estrés Oxidativo/fisiología , Trastornos Parkinsonianos/patología , Ratas , Ratas Sprague-Dawley , Rotenona/toxicidad , Desacopladores/toxicidadRESUMEN
Urate has emerged as a promising target for neuroprotection based on epidemiological observations, preclinical models, and early clinical trial results in multiple neurologic diseases, including Parkinson's disease (PD). This study investigates the astrocytic mechanism of urate's neuroprotective effect. Targeted biochemical screens of conditioned medium from urate- versus vehicle-treated astrocytes identified markedly elevated glutathione (GSH) concentrations as a candidate mediator of urate's astrocyte-dependent neuroprotective effects. Urate treatment also induced the nuclear translocation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein and transcriptional activation of its key target genes in primary astrocytic cultures. Urate's neuroprotective effect was attenuated when GSH was depleted in the conditioned media either by targeting its synthesis or release by astrocytes. Overall, these results implicate GSH as the extracellular astrocytic factor mediating the protective effect of urate in a cellular model of PD. These results also show that urate can employ a novel indirect neuroprotective mechanism via induction of the Nrf2 signaling pathway, a master regulator of the response to oxidative stress, in astrocytes.
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Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Glutatión/metabolismo , Fármacos Neuroprotectores/farmacología , Ácido Úrico/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Medios de Cultivo Condicionados , Evaluación Preclínica de Medicamentos , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: Mutations in the α-synuclein gene (SNCA) cause autosomal dominant forms of Parkinson's disease, but the substantial risk conferred by this locus to the common sporadic disease has only recently emerged from genome-wide association studies. METHODS: We genotyped a prioritized noncoding variant in SNCA intron 4 in 344 patients with Parkinson's disease and 275 controls from the longitudinal Harvard NeuroDiscovery Center Biomarker Study. RESULTS: The common minor allele of rs2736990 was associated with elevated disease susceptibility (odds ratio, 1.40; P = .0032). CONCLUSIONS: This result increases confidence in the notion that in many clinically well-characterized patients, genetic variation in SNCA contributes to "sporadic" disease.
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Predisposición Genética a la Enfermedad/genética , Enfermedad de Parkinson/genética , Polimorfismo de Nucleótido Simple/genética , alfa-Sinucleína/genética , Anciano , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Intrones/genética , Masculino , Persona de Mediana EdadRESUMEN
Nuclear microenvironments are architecturally organized subnuclear sites where the regulatory machinery for gene expression, replication, and repair resides. This compartmentalization is necessary to attain required stoichiometry for organization and assembly of regulatory complexes for combinatorial control. Combined and methodical application of molecular, cellular, biochemical, and in vivo genetic approaches is required to fully understand complexities of biological control. Here we provide methodologies to characterize nuclear organization of regulatory machinery by in situ immunofluorescence microscopy.
Asunto(s)
Espacio Intracelular/metabolismo , Espacio Intranuclear/metabolismo , Factores de Transcripción/metabolismo , Animales , Adhesión Celular , Técnicas de Cultivo de Célula , Biología Computacional , Recuperación de Fluorescencia tras Fotoblanqueo , Filamentos Intermedios/metabolismo , Metafase , Microscopía , Matriz Nuclear/metabolismo , Transporte de ProteínasRESUMEN
The acute myeloid leukemia 1 (AML1, RUNX1) transcription factor is a key regulator of hematopoietic differentiation that forms multi-protein complexes with co-regulatory proteins. These complexes are assembled at target gene promoters in nuclear microenvironments to mediate phenotypic gene expression and chromatin-related epigenetic modifications. Here, immunofluorescence microscopy and biochemical assays are used to show that RUNX1 associates with the human ATP-dependent SWI/SNF chromatin remodeling complex. The SWI/SNF subunits BRG1 and INI1 bind in vivo to RUNX1 target gene promoters (e.g., GMCSF, IL3, MCSF-R, MIP, and p21). These interactions correlate with histone modifications characteristic of active chromatin, including acetylated H4 and dimethylated H3 lysine 4. Downregulation of RUNX1 by RNA interference diminishes the binding of BRG1 and INI1 at selected target genes. Taken together, our findings indicate that RUNX1 interacts with the human SWI/SNF complex to control hematopoietic-specific gene expression.