Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Autophagy ; 16(2): 347-370, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30983487

RESUMO

Parkinson disease (PD) is an age-related neurodegenerative disorder associated with misfolded SNCA/α-synuclein accumulation in brain. Impaired catabolism of SNCA potentiates formation of its toxic oligomers. LRRK2 (leucine-rich repeat kinase-2) mutations predispose to familial and sporadic PD. Mutant LRRK2 perturbs chaperone-mediated-autophagy (CMA) to degrade SNCA. We showed greater age-dependent accumulation of oligomeric SNCA in striatum and cortex of aged LRRK2R1441G knockin (KI) mice, compared to age-matched wildtype (WT) by 53% and 31%, respectively. Lysosomal clustering and accumulation of CMA-specific LAMP2A and HSPA8/HSC70 proteins were observed in aged mutant striatum along with increased GAPDH (CMA substrate) by immunohistochemistry of dorsal striatum and flow cytometry of ventral midbrain cells. Using our new reporter protein clearance assay, mutant mouse embryonic fibroblasts (MEFs) expressing either SNCA or CMA recognition 'KFERQ'-like motif conjugated with photoactivated-PAmCherry showed slower cellular clearance compared to WT by 28% and 34%, respectively. However, such difference was not observed after the 'KFERQ'-motif was mutated. LRRK2 mutant MEFs exhibited lower lysosomal degradation than WT indicating lysosomal dysfunction. LAMP2A-knockdown reduced total lysosomal activity and clearance of 'KFERQ'-substrate in WT but not in mutant MEFs, indicating impaired CMA in the latter. A CMA-specific activator, AR7, induced neuronal LAMP2A transcription and lysosomal activity in MEFs. AR7 also attenuated the progressive accumulation of both intracellular and extracellular SNCA oligomers in prolonged cultures of mutant cortical neurons (DIV21), indicating that oligomer accumulation can be suppressed by CMA activation. Activation of autophagic pathways to reduce aged-related accumulation of pathogenic SNCA oligomers is a viable disease-modifying therapeutic strategy for PD.Abbreviations: 3-MA: 3-methyladenine; AR7: 7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; CMA: chaperone-mediated autophagy; CQ: chloroquine; CSF: cerebrospinal fluid; DDM: n-dodecyl ß-D-maltoside; DIV: days in vitro; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GWAS: genome-wide association studies; HSPA8/HSC70: heat shock protein 8; KFERQ: CMA recognition pentapeptide; KI: knockin; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LDH: lactate dehydrogenase; LRRK2: leucine-rich repeat kinase 2; MEF: mouse embryonic fibroblast; NDUFS4: NADH:ubiquinone oxidoreductase core subunit S4; NE: novel epitope; PD: Parkinson disease; RARA/RARα: retinoic acid receptor, alpha; SNCA: synuclein, alpha; TUBB3/TUJ1: tubulin, beta 3 class III; WT: wild-type.

2.
Transl Neurodegener ; 8: 23, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428316

RESUMO

Background: Parkinson's disease (PD) is characterized by dopaminergic neuronal loss in the substantia nigra pars compacta and intracellular inclusions called Lewy bodies (LB). During the course of disease, misfolded α-synuclein, the major constituent of LB, spreads to different regions of the brain in a prion-like fashion, giving rise to successive non-motor and motor symptoms. Etiology is likely multifactorial, and involves interplay among aging, genetic susceptibility and environmental factors. Main body: The prevalence of PD rises exponentially with age, and aging is associated with impairment of cellular pathways which increases susceptibility of dopaminergic neurons to cell death. However, the majority of those over the age of 80 do not have PD, thus other factors in addition to aging are needed to cause disease. Discovery of neurotoxins which can result in parkinsonism led to efforts in identifying environmental factors which may influence PD risk. Nevertheless, the causality of most environmental factors is not conclusively established, and alternative explanations such as reverse causality and recall bias cannot be excluded. The lack of geographic clusters and conjugal cases also go against environmental toxins as a major cause of PD. Rare mutations as well as common variants in genes such as SNCA, LRRK2 and GBA are associated with risk of PD, but Mendelian causes collectively only account for 5% of PD and common polymorphisms are associated with small increase in PD risk. Heritability of PD has been estimated to be around 30%. Thus, aging, genetics and environmental factors each alone is rarely sufficient to cause PD for most patients. Conclusion: PD is a multifactorial disorder involving interplay of aging, genetics and environmental factors. This has implications on the development of appropriate animal models of PD which take all these factors into account. Common converging pathways likely include mitochondrial dysfunction, impaired autophagy, oxidative stress and neuroinflammation, which are associated with the accumulation and spread of misfolded α-synuclein and neurodegeneration. Understanding the mechanisms involved in the initiation and progression of PD may lead to potential therapeutic targets to prevent PD or modify its course.

3.
Sci Rep ; 7: 40887, 2017 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-28098219

RESUMO

Aging, genetics and environmental toxicity are important etiological factors in Parkinson's disease (PD). However, its pathogenesis remains unclear. A major obstacle is the lack of an appropriate experimental model which incorporates genetic susceptibility, aging and prolonged environmental toxicity. Here, we explored the interplay amongst these factors using mutant LRRK2R1441G (leucine-rich-repeat-kinase-2) knockin mice. We found that mutant primary cortical and mesencephalic dopaminergic neurons were more susceptible to rotenone-induced ATP deficiency and cell death. Compared with wild-type controls, striatal synaptosomes isolated from young mutant mice exhibited significantly lower dopamine uptake after rotenone toxicity, due to reduced striatal synaptosomal mitochondria and synaptic vesicular proton pump protein (V-ATPase H) levels. Mutant mice developed greater locomotor deficits in open-field tests than wild-type mice following low oral rotenone doses given twice weekly over 50 weeks (half their lifespan). The increased locomotor deficit was associated with specific reduction in striatal mitochondrial Complex-I (NDUFS4) in rotenone-treated mutant but not in similarly treated wild-type mice. Our unique experimental model which incorporates genetic effect, natural aging and prolonged oral environmental toxicity administered to mutant knockin LRRK2 mice over half their life span, with observable and measurable phenotype, is invaluable in further studies of the pathogenic process and therapeutics of PD.


Assuntos
Apoptose/efeitos dos fármacos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/patologia , Rotenona/farmacologia , Administração Oral , Envelhecimento , Animais , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Técnicas de Introdução de Genes , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Rotenona/uso terapêutico , Sinaptossomos/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
4.
PLoS One ; 8(9): e74065, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24040167

RESUMO

Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17ß-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10(-9)-10(-7)M) dose-dependently reduced COMT expression (p<0.05), which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular components, a cell-based COMT assay provides useful initial screening to supplement the current assessments of xenoestrogens for potential estrogenic activity.


Assuntos
Catecol O-Metiltransferase/metabolismo , Ensaio de Imunoadsorção Enzimática , Estrogênios/metabolismo , Receptores Estrogênicos/metabolismo , Compostos Benzidrílicos/farmacologia , Catecol O-Metiltransferase/genética , Dibutilftalato/farmacologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Estrogênios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Fenóis/farmacologia , Ácidos Ftálicos/farmacologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
5.
Free Radic Biol Med ; 53(2): 383-94, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22580300

RESUMO

Mitochondrial uncoupling protein-4 (UCP4) enhances neuronal survival in 1-methyl-4-phenylpyridinium (MPP(+)) toxicity by suppressing oxidative stress and preserving intracellular ATP and mitochondrial membrane potential (MMP). NF-κB regulates neuronal viability via its complexes, p65 mediating cell death and c-Rel promoting cell survival. We reported previously that NF-κB mediates UCP4 neuroprotection against MPP(+) toxicity. Here, we investigated its link with the NF-κB c-Rel prosurvival pathway in alleviating mitochondrial dysfunction and oxidative stress. We overexpressed a c-Rel-encoding plasmid in SH-SY5Y cells and showed that c-Rel overexpression induced NF-κB activity without affecting p65 level. Overexpression of c-Rel increased UCP4 promoter activity and protein expression. Electrophoretic mobility shift assay showed that H(2)O(2) increased NF-κB binding to the UCP4 promoter and that NF-κB complexes were composed of p50/p50 and p50/c-Rel dimers. Under H(2)O(2)-induced oxidative stress, UCP4 knockdown significantly increased superoxide levels, decreased reduced glutathione (GSH) levels, and increased oxidized glutathione levels, compared to controls. UCP4 expression induced by c-Rel overexpression significantly decreased superoxide levels and preserved GSH levels and MMP under similar stress. These protective effects of c-Rel overexpression in H(2)O(2)-induced oxidative stress were significantly reduced after UCP4 knockdown, indicating that UCP4 is a target effector gene of the NF-κB c-Rel prosurvival pathway to mitigate the effects of oxidative stress.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/metabolismo , Proteínas Nucleares/genética , Fator de Transcrição RelA/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/genética , Proteínas de Desacoplamento Mitocondrial , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-rel , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Superóxidos/antagonistas & inibidores , Superóxidos/metabolismo , Fator de Transcrição RelA/metabolismo
6.
PLoS One ; 7(2): e32810, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22427795

RESUMO

Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4-phenylpyridinium (MPP(+)), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p<0.01), with 20% greater proton leak than vector controls (p<0.01). Increased ATP supply was observed in UCP4-overexpressing cells compared to controls (p<0.05). Although state 4 and state 3 respiration rates of UCP4-overexpressing and control cells were similar, Complex II activity in UCP4-overexpressing cells was 30% higher (p<0.05), associated with protein binding between UCP4 and Complex II, but not that of either Complex I or IV. Mitochondrial ADP consumption by succinate-induced respiration was 26% higher in UCP4-overexpressing cells, with 20% higher ADP:O ratio (p<0.05). ADP/ATP exchange rate was not altered by UCP4 overexpression, as shown by unchanged mitochondrial ADP uptake activity. UCP4 overexpression retained normal mitochondrial morphology in situ, with similar mitochondrial membrane potential compared to controls. Our findings elucidate how UCP4 overexpression increases ATP synthesis by specifically interacting with Complex II. This highlights a unique role of UCP4 as a potential regulatory target to modulate mitochondrial Complex II and ATP output in preserving existing neurons against energy crisis.


Assuntos
Trifosfato de Adenosina/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mitocondriais/metabolismo , Difosfato de Adenosina/metabolismo , Western Blotting , Linhagem Celular Tumoral , Citocromos c/metabolismo , Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial , Proteínas de Membrana Transportadoras/genética , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas Mitocondriais/genética , Proteínas de Desacoplamento Mitocondrial , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Consumo de Oxigênio , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
7.
Free Radic Biol Med ; 49(6): 1023-35, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20600837

RESUMO

We explored the protective mechanisms of human neuronal mitochondrial uncoupling protein-5 (UCP5) in MPP(+)- and dopamine-induced toxicity after its stable overexpression in SH-SY5Y cells. We raised specific polyclonal antibodies. Overexpressed UCP5 localized in mitochondria but not in cytosol. UCP5 overexpression increased proton leak, decreased mitochondrial membrane potential (MMP), reduced ATP production, and increased overall oxygen consumption (demonstrating uncoupling activity). UCP5 overexpression did not affect other neuronal UCP expression (UCP2 and UCP4). Overexpressing UCP5 is protective against MPP(+)- and dopamine-induced toxicity. MPP(+) and dopamine exposure for 6h reduced MMP and increased superoxide levels. ATP levels in UCP5-overexpressing cells were preserved under MPP(+) and dopamine toxicity, comparable to levels in untreated vector controls. At 24h, UCP5 overexpression preserved MMP, ATP levels, and cell survival; attenuated superoxide generation; and maintained oxidative phosphorylation as indicated by lower lactate levels. MPP(+) and dopamine exposure induced UCP5 mRNA transcription but did not decrease transcript degradation, as inhibition of transcription by actinomycin-D abolished induction by either toxin. Compared with our previous studies on UCP4, we observed functional differences between UCP4 and UCP5 in enhancing mitochondrial efficiency. These neuronal UCP homologues may work synergistically to maintain oxidative balance (through uncoupling activities) and ATP production (by modifying MMP).


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , 1-Metil-4-fenilpiridínio/toxicidade , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Citoproteção , Dopamina/farmacologia , Glicólise/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/efeitos dos fármacos , Proteínas de Desacoplamento Mitocondrial , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Superóxidos/metabolismo , Transgenes/genética
8.
Free Radic Biol Med ; 49(2): 192-204, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20385226

RESUMO

Mitochondrial uncoupling protein-4 (UCP4) enhances neuronal cell survival in MPP(+)-induced toxicity by suppressing oxidative stress and preserving intracellular ATP and mitochondrial membrane potential. UCP4 expression is increased by MPP(+), but its regulation is unknown. Using serial human UCP4 promoter-luciferase reporter gene constructs, we identified and characterized several cis-acting elements that can regulate UCP4 expression. Core promoter activity exists within 100 bp upstream of the transcription initiation site (TIS=+1). Both CAAT box (-33/-27) and Sp1 (-62/-49) elements are crucial and act synergistically in its transcription. We identified a NF-kappaB putative binding site at -507/-495. Mutation of this site significantly decreased UCP4 promoter activity. Activation of NF-kappaB by TNFalpha or cycloheximide increased, whereas its inhibition by 4-hydroxy-2-nonenal or transfection of pIkappaBalphaM suppressed, UCP4 promoter activity. NF-kappaB inhibition significantly suppressed the MPP(+)-induced increase in UCP4 expression. MPP(+) increased specific binding of NF-kappaB protein complexes to this site in electrophoretic mobility shift assay. Both UCP4 knockdown and NF-kappaB inhibition exacerbated MPP(+)-induced cell death. We present the first direct evidence that UCP4 is regulated by NF-kappaB, mediated via a functional NF-kappaB site in its promoter region, and that UCP4 has a significant role in NF-kappaB prosurvival signaling, mediating its protection against MPP(+) toxicity.


Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Proteínas de Membrana Transportadoras/metabolismo , NF-kappa B/metabolismo , Fármacos Neuroprotetores/metabolismo , Ativação Transcricional , Aldeídos/farmacologia , Sítios de Ligação/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Desacoplamento Mitocondrial , Mutagênese Sítio-Dirigida , Mutação/genética , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/metabolismo , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/genética , Elementos Reguladores de Transcrição/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
9.
Neurotox Res ; 17(4): 332-43, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19763737

RESUMO

Mitochondrial dysfunction is involved in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Uncoupling proteins (UCPs) delink ATP production from biofuel oxidation in mitochondria to reduce oxidative stress. UCP2 is expressed in brain, and has neuroprotective effects under various toxic insults. We observed induction of UCP2 expression by leptin in neuronal cultures, and hypothesize that leptin may preserve neuronal survival via UCP2. We showed that leptin preserved cell survival in neuronal SH-SY5Y cells against MPP+ toxicity (widely used in experimental Parkinsonian models) by maintaining ATP levels and mitochondrial membrane potential (MMP); these effects were accompanied by increased UCP2 expression. Leptin had no effect in modulating reactive oxygen species levels. Stable knockdown of UCP2 expression reduced ATP levels, and abolished leptin protection against MPP+-induced mitochondrial depolarization, ATP deficiency, and cell death, indicating that UCP2 is critical in mediating these neuroprotective effects of leptin against MPP+ toxicity. Interestingly, UCP2 knockdown increased UCP4 expression, but not of UCP5. Our findings show that leptin preserves cell survival by maintaining MMP and ATP levels mediated through UCP2 in MPP+-induced toxicity.


Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Herbicidas/toxicidade , Canais Iônicos/fisiologia , Leptina/farmacologia , Proteínas Mitocondriais/fisiologia , Fármacos Neuroprotetores/farmacologia , 1-Metil-4-fenilpiridínio/metabolismo , Trifosfato de Adenosina/metabolismo , Análise de Variância , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Canais Iônicos/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , MicroRNAs/farmacologia , Proteínas Mitocondriais/genética , Proteínas de Desacoplamento Mitocondrial , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2
10.
Free Radic Biol Med ; 46(6): 810-20, 2009 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-19150400

RESUMO

Mitochondrial uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis. We explored the neuroprotective role of UCP4 with its stable overexpression in SH-SY5Y cells, after exposure to either MPP(+) or dopamine to induce ATP deficiency and oxidative stress. Cells overexpressing UCP4 proliferated faster in normal cultures and after exposure to MPP(+) and dopamine. Differentiated UCP4-overexpressing cells survived better when exposed to MPP(+) with decreased LDH release. Contrary to the mild uncoupling hypothesis, UCP4 overexpression resulted in increased absolute ATP levels (with ADP/ATP ratios similar to those of controls under normal conditions and ADP supplementation) associated with increased respiration rate. Under MPP(+) toxicity, UCP4 overexpression preserved ATP levels and mitochondrial membrane potential (MMP) and reduced oxidative stress; the preserved ATP level was not due to increased glycolysis. Under MPP(+) toxicity, the induction of UCP2 expression in vector controls was absent in UCP4-overexpressing cells, suggesting that UCP4 may compensate for UCP2 expression. UCP4 function does not seem to adhere to the mild uncoupling hypothesis in its neuroprotective mechanisms under oxidative stress and ATP deficiency. UCP4 overexpression increases cell survival by inducing oxidative phosphorylation, preserving ATP synthesis and MMP, and reducing oxidative stress.


Assuntos
1-Metil-4-fenilpiridínio/metabolismo , Trifosfato de Adenosina/metabolismo , Dopamina/metabolismo , Canais Iônicos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Trifosfato de Adenosina/genética , Animais , Anticorpos/imunologia , Apoptose , Fracionamento Celular , Linhagem Celular , Clonagem Molecular , Humanos , Imunização , Epitopos Imunodominantes/química , Epitopos Imunodominantes/imunologia , Canais Iônicos/genética , Potencial da Membrana Mitocondrial , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Mitocôndrias/genética , Mitocôndrias/imunologia , Proteínas Mitocondriais/genética , Proteínas de Desacoplamento Mitocondrial , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo , Peptídeos/administração & dosagem , Peptídeos/síntese química , RNA Interferente Pequeno , Ovinos , Proteína Desacopladora 2
11.
J Magn Reson Imaging ; 27(1): 8-13, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18022844

RESUMO

PURPOSE: To determine whether diffusion abnormalities can be observed in nonsymptomatic family members with a known causative Cu/Zn superoxide dismutase mutation (asymptomatic familial amyotrophic lateral sclerosis; AFALS(+SOD1)) in a family with autosomal dominant familial amyotrophic lateral sclerosis (ALS) using diffusion tensor imaging (DTI). MATERIALS AND METHODS: A total of eight AFALS(+SOD1) subjects (aged 17-43 years) were age-matched with 13 healthy controls (aged 19-45 years) without SOD1 mutations. DTI was carried out on a 1.5T scanner. The diffusion index maps derived were then normalized spatially for voxel-based analysis. region of interest (ROI)-based analysis was also carried out. RESULTS: Our voxel-based and ROI-based analysis showed that AFALS(+SOD1) subjects have decreased fractional anisotropy (FA) (0.5401 vs. 0.5168, P < 0.05) and increased tensor trace (TT) (2.5854 x 10(-3) mm(2)/second vs. 2.6226 x 10(-3) mm(2)/second, P < 0.04) at the posterior limb of the internal capsule compared to the control subjects. Increased radial diffusivity (E((2,3)/2)) was detected on both sides (right = 0.5710 x 10(-3) mm(2)/second vs. 0.5943 x 10(-3) mm(2)/second, P < 0.05; left = 0.5666 x 10(-3) mm(2)/second vs. 0.5872 x 10(-3) mm(2)/second, P < 0.05). No significant change in axial diffusivity (E(1)) was detected. CONCLUSION: Abnormal diffusivity was found at the posterior limb of the internal capsule in AFALS(+SOD1) subjects, hitherto unreported. Our results suggest that DTI may detect diffusion abnormalities in AFALS(+SOD1) subjects before symptoms develop.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Imagem de Difusão por Ressonância Magnética/métodos , Mutação , Superóxido Dismutase/genética , Adolescente , Adulto , Esclerose Amiotrófica Lateral/enzimologia , Anisotropia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Estatísticas não Paramétricas , Superóxido Dismutase-1
12.
J Neurosci Res ; 84(6): 1358-66, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16941493

RESUMO

Uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis by dissipating proton gradient across mitochondrial inner membrane. The physiological role of neuronal specific UCP5 is unknown. We explored the effects of reduced UCP5 expression on mitochondrial membrane potential (MMP), oxidative stress, ATP levels, and cell viability, under normal and MPP+-induced cytotoxic conditions, in human catecholaminergic SH-SY5Y cells. UCP5 expression was reduced by 56% by siRNA, compared to scrambled-siRNA controls. UCP5 knockdown induced apoptosis but did not affect basal levels of ATP, oxidative stress and MMP in the cells under normal conditions. However, UCP5 knockdown increased MPP+-induced cytotoxicity by 15% and oxidative stress levels by 40%, and partially restored MPP+-induced mitochondrial depolarization by 57%. UCP2 and UCP4 expression were unaffected by UCP5 knockdown. Exacerbation of cytotoxicity, oxidative stress and modification of MMP with reduced UCP5 expression in the face of MPP+ toxicity suggest that UCP5 might be physiologically important in the pathology of oxidative stress-induced neurodegeneration.


Assuntos
1-Metil-4-fenilpiridínio , Trifosfato de Adenosina/deficiência , Proteínas de Membrana Transportadoras/genética , Membranas Mitocondriais/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Apoptose/fisiologia , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/fisiologia , Regulação para Baixo/fisiologia , Humanos , L-Lactato Desidrogenase/metabolismo , Potenciais da Membrana/fisiologia , Proteínas de Membrana Transportadoras/fisiologia , Proteínas de Desacoplamento Mitocondrial , Proteínas do Tecido Nervoso/fisiologia , Oxirredução , Estresse Oxidativo/fisiologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxidos/metabolismo , Transfecção
13.
J Neurosci Res ; 81(2): 261-8, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15948157

RESUMO

Methyl-4-phenylpyridinium ion (MPP(+)), a specific dopaminergic neurotoxin, inhibits mitochondrial complex I activity, generates reactive oxygen species (ROS), reduces ATP production, and induces cell death. We explored changes in expression of uncoupling proteins (UCPs 2, 4, and 5) following MPP(+)-induced toxicity in SK-N-SH cells over 72 hr at the transcriptional (quantification of mRNA by real-time RT-PCR) and translational (Western analysis) levels. UCP5 mRNA and protein were markedly up-regulated (1 mM MPP(+) at 72 hr caused a twofold increase, P < 0.01), as was UCP4 mRNA, albeit to a much lesser extent. Surprisingly, UCP2 mRNA levels decreased at 24 hr (P < 0.05) but thereafter significantly increased to greater than control levels at 72 hr (P < 0.05), although UCP2 protein levels were decreased throughout (1 mM MPP(+) at 72 hr caused a reduction of 50%, P < 0.01). The increase in ROS production may be attenuated by UCP4 and UCP5 up-regulation. The consequence of decreased UCP2 levels is unclear, although this may represent an adaptive response to declines in ATP levels, the subsequent increase in mRNA being a response to further increases in oxidative stress.


Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Linhagem Celular Tumoral , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Canais Iônicos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Desacoplamento Mitocondrial , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , RNA Mensageiro/análise , Proteína Desacopladora 2
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA