RESUMEN
This work comprehends the development and characterization of a carbon black-based electrode modified with Au microflowers to increase its effect as a capacitance biosensor for the determination of PARK7/DJ-1. Due to its high surface-to-volume ratio and biocompatibility, Au particles are suitable for antibody binding, and by monitoring surface capacitance, it is possible to identify the immune-pair interaction. Au microflowers allowed the adequate immobilization of Parkinsonian-related proteins: PARK7/DJ-1 and its antibody. The protein is associated with several antioxidant mechanisms, but its abnormal concentrations or mutations can be the cause of the loss of dopaminergic neurons, leading to Parkinson's disease. The device was characterized by scanning electron microscopy and cyclic voltammetry, revealing the flower-like structures and the electrochemically-interest enhancements they provide, such as increased heterogeneous electron transfer rate coefficient and electroactive area. The self-assembled monolayers of different molecules were optimized with the aid of 22 central composite experiments and a linear calibration curve was obtained between 0.700 and 120 ng mL-1 of PARK7/DJ-1, with a limit of detection of 0.207 ng mL-1. The data confirms that the addition of Au microflowers enhanced the electrochemical signal of the device, as well as allowed for the determination of an early stage Parkinson's disease biomarker with appreciable analytical performance.
Asunto(s)
Técnicas Biosensibles , Capacidad Eléctrica , Técnicas Electroquímicas , Oro , Enfermedad de Parkinson , Proteína Desglicasa DJ-1 , Oro/química , Técnicas Biosensibles/métodos , Enfermedad de Parkinson/diagnóstico , Técnicas Electroquímicas/métodos , Técnicas Electroquímicas/instrumentación , Humanos , Inmunoensayo/métodos , Biomarcadores/análisis , Anticuerpos Inmovilizados/inmunología , Límite de Detección , ElectrodosRESUMEN
Mesial temporal lobe epilepsy (MTLE) is a chronic neurological disorder affecting almost 40% of adult patients with epilepsy. Hippocampal sclerosis (HS) is a common histopathological abnormality found in patients with MTLE. HS is characterised by extensive neuronal loss in different hippocampus sub-regions. In this study, we used laser microdissection-based microproteomics to determine the protein abundances in different regions and layers of the hippocampus dentate gyrus (DG) in an electric stimulation rodent model which displays classical HS damage similar to that found in patients with MTLE. Our results indicate that there are differences in the proteomic profiles of different layers (granule cell and molecular), as well as different regions, of the DG (ventral and dorsal). We have identified new signalling pathways and proteins present in specific layers and regions of the DG, such as PARK7, RACK1, and connexin 31/gap junction. We also found two major signalling pathways that are common to all layers and regions: inflammation and energy metabolism. Finally, our results highlight the utility of high-throughput microproteomics and spatial-limited isolation of tissues in the study of complex disorders to fully appreciate the large biological heterogeneity present in different cell populations within the central nervous system.
Asunto(s)
Conexinas/metabolismo , Epilepsia del Lóbulo Temporal/metabolismo , Hipocampo/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Proteómica/métodos , Receptores de Cinasa C Activada/metabolismo , Animales , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/etiología , Regulación de la Expresión Génica , Humanos , Captura por Microdisección con Láser , Especificidad de Órganos , Mapas de Interacción de Proteínas , Ratas , Transducción de SeñalRESUMEN
In this study, platinum electrodes were fabricated on the bio-based poly(ethylene terephthalate) (Bio-PET) substrates for the development of flexible electrochemical sensors for the detection of Parkinson's disease biomarkers. Dopamine was detected by voltammetric measurements, displaying a 3.5 × 10-5 mol L-1 to 8.0 × 10-4 mol L-1 linear range with a limit of detection of 5.1 × 10-6 mol L-1. Parkinson's disease protein 7 (PARK7/DJ-1) was successfully detected by electrochemical impedance spectroscopy after electrode functionalization with specific anti-PARK7/DJ-1 antibodies. In this case, analytical curves presented a linear behavior from 40 ng mL-1 to 150 ng mL-1 of PARK7/DJ-1 with a limit of detection of 7.5 ng mL-1. Besides, the electrodes did not suffer any change in the electrochemical response after manual tests of mechanical tension. The proposed sensor and immunosensor were applied for the determination of Parkinson's disease biomarkers concentrations found in the human body, being adequate as an alternative method to diagnose this disease.
Asunto(s)
Técnicas Biosensibles/instrumentación , Espectroscopía Dieléctrica/instrumentación , Inmunoensayo/instrumentación , Enfermedad de Parkinson/diagnóstico , Platino (Metal)/química , Proteína Desglicasa DJ-1/análisis , Anticuerpos Inmovilizados/química , Biomarcadores/análisis , Biomarcadores/sangre , Biomarcadores/orina , Elasticidad , Electrodos , Diseño de Equipo , Humanos , Enfermedad de Parkinson/sangre , Enfermedad de Parkinson/orina , Proteína Desglicasa DJ-1/sangre , Proteína Desglicasa DJ-1/orinaRESUMEN
Epigallocatechin-3-gallate (EGCG) is a polyhydroxyphenol constituent of green tea (e.g., Camellia sinensis) with known antioxidant properties. Due to these properties, others have proposed it as a potential therapeutic agent for the treatment of Parkinson's disease (PD). Previously, we demonstrated that EGCG prolonged the lifespan and locomotor activity in wild-type Canton-S flies exposed to the neurotoxicant paraquat (PQ), suggesting neuroprotective properties. Both gene mutations and environmental neurotoxicants (e.g., PQ) are factors involved in the development of PD. Thus, the first aim of this study was to create a suitable animal model of PD, which encompasses both of these factors. To create the model, we knocked down dj-1-ß function specifically in the dopaminergic neurons to generate TH > dj-1-ß-RNAi/+ Drosophila melanogaster flies. Next, we induced neurotoxicity in the transgenic flies with PQ. The second aim of this study was to validate the model by comparing the effects of vehicle, EGCG, and chemicals with known antioxidant and neuroprotective properties in vivo (e.g., propyl gallate and minocycline) on life-span, locomotor activity, lipid peroxidation, and neurodegeneration. The EGCG treatment provided protection and prevention from the PQ-induced reduction in the life-span and locomotor activity and from the PQ-induced increase in lipid peroxidation and neurodegeneration. These effects were augmented in the EGCG-treated flies when compared to the flies treated with either PG or MC. Altogether, these results suggest that the transgenic TH > dj-1-ß-RNAi/+ flies treated with PQ serve as a suitable PD model for screening of potential therapeutic agents.
Asunto(s)
Catequina/análogos & derivados , Proteínas de Drosophila/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/prevención & control , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Paraquat/toxicidad , Animales , Animales Modificados Genéticamente , Antioxidantes/metabolismo , Catequina/farmacología , Catequina/uso terapéutico , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteínas de Drosophila/genética , Drosophila melanogaster , Femenino , Herbicidas/toxicidad , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/genética , Locomoción/efectos de los fármacos , Locomoción/genética , Masculino , Minociclina/farmacología , Minociclina/uso terapéutico , Proteínas del Tejido Nervioso/genética , Enfermedades Neurodegenerativas/genética , Fármacos Neuroprotectores/uso terapéutico , Estrés Oxidativo/genética , Proteína Desglicasa DJ-1 , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson's disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein.
Asunto(s)
Esterasas/química , Esterasas/metabolismo , Enfermedad de Parkinson/enzimología , Esterasas/genética , Humanos , Peróxido de Hidrógeno/farmacología , Simulación del Acoplamiento Molecular , Mutación , Nitrofenoles/metabolismo , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/genética , Proteína Desglicasa DJ-1/química , Proteína Desglicasa DJ-1/genética , Proteína Desglicasa DJ-1/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Parkinson's disease (PD) is the second most frequent neurodegenerative disorder. It is characterized by selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). Early-onset familial forms of PD are associated with mutations in several genes, including parkin, pink1 and dj-1. DJ-1 encodes a protein whose neuroprotective function has not been completely clarified yet. We aim to understand the neuroprotective mechanisms of DJ-1, in particular, DJ-1's involvement in the regulation of the PI3K/PTEN/AKT/mTOR pathway and neuronal autophagy in a neurotoxic context induced by C2-ceramide, by using CAD cells, a murine cathecolaminergic cell line. We demonstrated that C2-ceramide induces CAD cell death associated with decreased phosphorylation of PTEN at Ser380, AKT at Ser473, and mTOR at Ser2448; and increased of autophagic flux (increased LC3-II and autophagosome formation). Additionally, we showed that overexpression of DJ-1 protects against C2-ceramide-induced neuronal death and it is not associated with change in the phosphorylation of mTOR at Ser2448. In conclusion, these data suggest that DJ-1 reinforces the PI3K/AKT survival pathway and inhibits autophagy, probably by a mechanism independent from mTOR.
Asunto(s)
Autofagia , Neuronas/citología , Proteínas Oncogénicas/metabolismo , Peroxirredoxinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Esfingosina/análogos & derivados , Animales , Línea Celular , Supervivencia Celular , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteína Desglicasa DJ-1 , Transducción de Señal , Esfingosina/metabolismo , Esfingosina/toxicidad , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
In this study, we investigated the molecular mechanism of doxorubicin (dxr)-induced cytotoxicity on Jurkat cells - a model cell of human acute lymphoblastic leukemia - under normoxic (20% O2) and hypoxic (5% O2) conditions. Using in-cell western analysis, immunofluorescence, flow cytometry analysis, and biochemical inhibitors, we evaluated several oxidative stress (OS) and cell death markers. It was found that dxr (5-100 µmol/l) induced apoptosis by OS mechanisms involving DNA fragmentation (8-48%), loss of mitochondrial membrane potential (ΔΨm, 33-92%), and H2O2 production (15-42%) under normoxia. In addition, dxr (10 µmol/l) induced activation and/or nuclei translocation of NF-κB (6.6, 1.6-fold increase), p53 (4.3, 3.1 f), c-Jun (9.5, 5.0 f), apoptosis-inducing factor (AIF) (1.9, 3.9 f), caspase-3 (3.7, 1.9 f), overexpression of Parkin (2.1, 1.2 f)/PINK-1 (2.1 f) proteins, and reduced DJ-1 levels by half compared with untreated cells under normoxia, according to immunofluorescence and in-cell western analysis, respectively. In contrast, dxr (10 µmol/l) could not induce apoptosis in Jurkat cells under hypoxia. Effectively, dxr significantly reduced DNA fragmentation (6%), expression levels of cell death (e.g. p53, c-Jun, caspase-3, AIF), and OS (e.g. Parkin) markers, whereas it increased ΔΨm, hypoxia-inducible factor 1-α (HIF-1α, 3.1, 2.3 f), NF-κB (6.8, 2.0 f), and DJ-1 (1.3, 1.0 f) levels. This investigation suggests that dxr might efficiently eliminate acute lymphoblastic leukemia cells by OS-induced apoptosis under normoxic conditions through a minimal completeness of cell death signaling (i.e. mitochondria-caspase-3/AIF-dependent pathways) and through a direct DNA damage process. However, hypoxic conditions may reduce the effectiveness of dxr toxicity.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Doxorrubicina/farmacología , Mitocondrias/efectos de los fármacos , Acetilcisteína/metabolismo , Factor Inductor de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Hipoxia de la Célula , Humanos , Peróxido de Hidrógeno/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Proteínas Oncogénicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras , Proteína Desglicasa DJ-1 , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
To establish the effect of low (11 mM) and high (55 mM) glucose concentrations (G11, G55) on Jurkat cells exposed to rotenone (ROT, a class 5 mitocan). We demonstrated that ROT induces apoptosis in Jurkat cells cultured in G11 by oxidative stress (OS) mechanism involving the generation of anion superoxide radical (O2(â-), 68%)/hydrogen peroxide (H2O2, 54%), activation of NF-κB (32%), p53 (25%), c-Jun (17%) transcription factors, and caspase-3 (28%), apoptosis-inducing factor (AIF, 36%) nuclei translocation, c-Jun N-terminal kinase (JNK) activation, and loss of mitochondria transmembrane potential (ΔΨm, 62%) leading to nuclei fragmentation (~10% and ~40% stage I-II fragmented nuclei, resp.). ROT induces massive cytoplasmic aggregates of DJ-1 (93%), and upregulation of Parkin compared to untreated cells, but no effect on PINK-1 protein was observed. Cell death marker detection and DJ-1 and Parkin expression were significantly reduced when cells were cultured in G55 plus ROT. Remarkably, metformin sensitized Jurkat cells against ROT in G55. Our results indicate that a high-glucose milieu promotes resistance against ROT/H2O2-induced apoptosis in Jurkat cells. Our data suggest that combined therapy by using mitochondria-targeted damaging compounds and regulation of glucose (e.g., metformin) can efficiently terminate leukemia cells via apoptosis in hyperglycemic conditions.
Asunto(s)
Glucosa/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Biológicos , Proteínas Oncogénicas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Quinasas/metabolismo , Rotenona/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Caspasa 3/metabolismo , Forma del Núcleo Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/metabolismo , Células Jurkat , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Metformina/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteína Desglicasa DJ-1 , Transducción de Señal/efectos de los fármacos , Superóxidos/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Few studies have focused on experimental testosterone deprivation in immature animals. Therefore, this study used sexually immature rats aiming to evaluate the testes and epididymis histology and proteins expression in these organs on PND50 and 75, after premature antiandrogen exposure, from PND21 to 44. Although the androgen deprivation from pre-puberty up to peripuberty did not alter the histological organization of the testes and epididymis either at puberty or at adulthood, the treatment impaired the expression of specific proteins in epididymal tissue at puberty and adulthood (androgen receptor, calmodulin, Rab11A). These changes may be related to impaired epididymal function, sperm quality and fertility capacity as observed in a previous study. Further studies are necessary to better investigate the molecular mechanisms involved in the impairment on reproductive competence of male rats after precocious hormonal injury.
Asunto(s)
Antagonistas de Andrógenos/farmacología , Epidídimo/efectos de los fármacos , Flutamida/farmacología , Maduración Sexual/efectos de los fármacos , Testículo/efectos de los fármacos , Animales , Calmodulina/metabolismo , Epidídimo/anatomía & histología , Epidídimo/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Desglicasa DJ-1 , Ratas , Ratas Wistar , Receptores Androgénicos/metabolismo , Testículo/anatomía & histología , Testículo/metabolismo , Testosterona/antagonistas & inhibidores , Testosterona/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Parkinson's disease is one of the most common neurodegenerative disorders associated with aging, reaching ⼠2% of individuals over 65 years. Knowledge achieved in the last decade about the genetic basis of Parkinson's disease clearly shows that genetic factors play an important role in the etiology of this disorder. Exon dosage variations account for a high proportion of Parkinson's disease mutations, mainly for PARKIN gene. In the present study, we screened genomic rearrangements in SNCA, PARKIN, PINK1 and DJ-1 genes in 102 Brazilian Parkinson's disease patients with early onset (age of onset ⩽ 50 years), using the multiplex ligation-dependent probe amplification method. Family history was reported by 24 patients, while 78 were sporadic cases. Screening of exon dosage revealed PARKIN and PINK1 copy number variations, but no dosage alteration was found in SNCA and DJ-1 genes. Most of the carriers harbor heterozygous deletions or duplications in the PARKIN gene and only one patient was found to have a deletion in PINK1 exon 1. Data about dosage changes are scarce in the Brazilian population, which stresses the importance of including exon dosage analysis in Parkinson's disease genetic studies.
Asunto(s)
Exones/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Oncogénicas/genética , Enfermedad de Parkinson/genética , Proteínas Quinasas/genética , Ubiquitina-Proteína Ligasas/genética , alfa-Sinucleína/genética , Adulto , Edad de Inicio , Brasil , Estudios de Casos y Controles , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Femenino , Dosificación de Gen , Duplicación de Gen , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Proteína Desglicasa DJ-1 , Eliminación de SecuenciaRESUMEN
The molecular mechanisms underlying the cellular lost found in the nigrostriatal pathway during the progression of Parkinson's disease (PD) are not completely understood. Human neuroblastoma cell line SH-SY5Y challenged with 6-hydroxydopamine (6-OHDA) has been widely used as an in vitro model for PD. Although this cell line differentiates to dopaminergic neuron-like cells in response to low serum and retinoic acid (RA) treatment, there are few studies investigating the differences between proliferative and RA-differentiated SH-SY5Y cells. Here we evaluate morphological and biochemical changes which occurs during the differentiation of SH-SY5Y cells, and their responsiveness to 6-OHDA toxicity. Exponentially growing SH-SY5Y cells were maintained with DMEM/F12 medium plus 10% of fetal bovine serum (FBS). Differentiation was triggered by the combination of 10 microM RA plus 1% of FBS during 4, 7 and 10 days in culture. We found that SH-SY5Y cells differentiated for 7 days show an increase immunocontent of several relevant neuronal markers with the concomitant decrease in non-differentiated cell marker. Moreover, cells became two-fold more sensitive to 6-OHDA toxicity during the differentiation process. Time course experiments showed loss of mitochondrial membrane potential triggered by 6-OHDA (mitochondrial dysfunction parameter), which firstly occurs in proliferative than neuron-like differentiated cells. This finding could be related to the increase in the immunocontent of the neuroprotective protein DJ-1 during differentiation. Our data suggest that SH-SY5Y cells differentiated by 7 days with the protocol described here represent a more suitable experimental model for studying the molecular and cellular mechanisms underlying the pathophysiology of PD.
Asunto(s)
Adrenérgicos/toxicidad , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Neuroblastoma/patología , Oxidopamina/toxicidad , Enfermedad de Parkinson/patología , Animales , Biomarcadores/análisis , Bovinos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Queratolíticos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neuroblastoma/metabolismo , Proteínas Oncogénicas/análisis , Proteínas Oncogénicas/biosíntesis , Enfermedad de Parkinson/metabolismo , Proteína Desglicasa DJ-1 , Tretinoina/farmacologíaRESUMEN
We describe clinical and molecular findings in a genetic isolate from north-eastern Brazil with early-onset Parkinson's disease (PD) and a novel mutation in the parkin gene. Genealogical studies could connect 255 individuals, of whom 15 had PD. Geographic isolation and multiple consanguineous marriages initially suggested an autosomal recessive inheritance for PD in these patients. The available individuals were personally examined, and DNA was obtained from 26 members: ten early-onset PD patients, one case with likely neuroleptic-induced parkinsonism and 15 unaffected relatives. The average age at onset of PD symptoms was 30.8 years (range 12-46). Haplotype analysis revealed homozygosity in the PD patients for markers across the PARK2 locus. Genomic sequencing identified a novel homozygous splice-site parkin mutation (IVS1 + 1G/T), which completely co-segregated with the early-onset PD phenotype. cDNA analysis confirmed the total loss of parkin transcript in homozygous mutation carriers, delineating this as a loss-of-function mutation. The case with neuroleptic-induced parkinsonism and 13 of 15 healthy relatives were heterozygous carriers of the mutation. The absence of PD in heterozygous carriers indicates a genuinely recessive nature of this mutation, suggesting that parkin haploinsufficiency is not a relevant risk factor for early- or late-onset PD. However, parkin haploinsufficiency could facilitate the emergence of neuroleptic-induced parkinsonism. The cluster reported here, which to our knowledge is the largest described to date with early-onset PD and parkin mutations, also offers a unique opportunity for the search of modifiers of the parkin-related disease.