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1.
Anal Chem ; 93(32): 11108-11115, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34348022

RESUMO

Studies of the metal content of metalloproteins in tissues from the human central nervous system (CNS) can be compromised by preparative techniques which alter levels of, or interactions between, metals and the protein of interest within a complex mixture. We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and either proton or synchrotron X-ray fluorescence within electrophoresis gels to analyze the endogenous metal content of copper-zinc superoxide dismutase (SOD1) purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis and Parkinson's disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. Validating our novel approach, we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein versus confounding metalloproteins. We analyzed tissues from nine healthy individuals and five CNS regions (occipital cortex, substantia nigra, locus coeruleus, dorsal spinal cord, and ventral spinal cord). We found that Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28, a ratio very close to the expected value of 1. Our methodological workflow can be applied to the study of endogenous native SOD1 in a pathological context and adapted to a range of metalloproteins from human tissues and other sources.


Assuntos
Esclerose Amiotrófica Lateral , Zinco , Sistema Nervoso Central , Cobre , Humanos , Mutação , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Fluxo de Trabalho
2.
Nat Commun ; 12(1): 4742, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362891

RESUMO

The ancestors of cyanobacteria generated Earth's first biogenic molecular oxygen, but how they dealt with oxidative stress remains unconstrained. Here we investigate when superoxide dismutase enzymes (SODs) capable of removing superoxide free radicals evolved and estimate when Cyanobacteria originated. Our Bayesian molecular clocks, calibrated with microfossils, predict that stem Cyanobacteria arose 3300-3600 million years ago. Shortly afterwards, we find phylogenetic evidence that ancestral cyanobacteria used SODs with copper and zinc cofactors (CuZnSOD) during the Archaean. By the Paleoproterozoic, they became genetically capable of using iron, nickel, and manganese as cofactors (FeSOD, NiSOD, and MnSOD respectively). The evolution of NiSOD is particularly intriguing because it corresponds with cyanobacteria's invasion of the open ocean. Our analyses of metalloenzymes dealing with reactive oxygen species (ROS) now demonstrate that marine geochemical records alone may not predict patterns of metal usage by phototrophs from freshwater and terrestrial habitats.


Assuntos
Antioxidantes/metabolismo , Cianobactérias/enzimologia , Cianobactérias/metabolismo , Evolução Molecular , Teorema de Bayes , Coenzimas , Cobre , Cianobactérias/genética , Água Doce , Ferro , Manganês , Níquel/química , Estresse Oxidativo , Filogenia , Espécies Reativas de Oxigênio , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxidos , Zinco
3.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209958

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins-and of secreted metabolites-involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome-lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte-MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Astrócitos/citologia , Metabolômica/métodos , Proteômica/métodos , Superóxido Dismutase/genética , Esclerose Amiotrófica Lateral/genética , Animais , Astrócitos/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Feminino , Glutationa/metabolismo , Humanos , Metabolismo dos Lipídeos , Masculino , Camundongos , Cultura Primária de Células , Mapas de Interação de Proteínas , Transdução de Sinais , Medula Espinal/citologia , Medula Espinal/metabolismo
4.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209129

RESUMO

Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and plays a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). It has been implicated as driver of disease progression and is observed in ALS patients, as well as in the transgenic SOD1G93A mouse model. Here, we explore and validate the therapeutic potential of the d-enantiomeric peptide RD2RD2 upon oral administration in SOD1G93A mice. Transgenic mice were treated daily with RD2RD2 or placebo for 10 weeks and phenotype progression was followed with several behavioural tests. At the end of the study, plasma cytokine levels and glia cell markers in brain and spinal cord were analysed. Treatment resulted in a significantly increased performance in behavioural and motor coordination tests and a decelerated neurodegenerative phenotype in RD2RD2-treated SOD1G93A mice. Additionally, we observed retardation of the average disease onset. Treatment of SOD1G93A mice led to significant reduction in glial cell activation and a rescue of neurons. Analysis of plasma revealed normalisation of several cytokines in samples of RD2RD2-treated SOD1G93A mice towards the levels of non-transgenic mice. In conclusion, these findings qualify RD2RD2 to be considered for further development and testing towards a disease modifying ALS treatment.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Neurônios Motores/enzimologia , Superóxido Dismutase/metabolismo , Administração Oral , Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Peptídeos , Superóxido Dismutase/genética
5.
Int J Mol Sci ; 22(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208517

RESUMO

Superoxide dismutase 3 (SOD3), also known as extracellular superoxide dismutase, is an enzyme that scavenges reactive oxygen species (ROS). It has been reported that SOD3 exerts anti-inflammatory abilities in several immune disorders. However, the effect of SOD3 and the underlying mechanism in inflammatory bowel disease (IBD) have not been uncovered. Therefore, in the present study, we investigated whether SOD3 can protect intestinal cells or organoids from inflammation-mediated epithelial damage. Cells or mice were treated with SOD3 protein or SOD3-transduced mesenchymal stem cells (MSCs). Caco-2 cells or intestinal organoids stimulated with pro-inflammatory cytokines were used to evaluate the protective effect of SOD3 on epithelial junctional integrity. Dextran sulfate sodium (DSS)-induced colitis mice received SOD3 or SOD3-transduced MSCs (SOD3-MSCs), and were assessed for severity of disease and junctional protein expression. The activation of the mitogen-activated protein kinase (MAPK) pathway and elevated expression of cytokine-encoding genes decreased in TNF-α-treated Caco-2 cells or DSS-induced colitis mice when treated with SOD3 or SOD3-MSCs. Moreover, the SOD3 supply preserved the expression of tight junction (ZO-1, occludin) or adherence junction (E-cadherin) proteins when inflammation was induced. SOD3 also exerted a protective effect against cytokine- or ROS-mediated damage to intestinal organoids. These results indicate that SOD3 can effectively alleviate enteritis symptoms by maintaining the integrity of epithelial junctions and regulating inflammatory- and oxidative stress.


Assuntos
Colite/etiologia , Colite/metabolismo , Mucosa Intestinal/metabolismo , Células-Tronco Mesenquimais/metabolismo , Superóxido Dismutase/genética , Junções Íntimas/metabolismo , Animais , Biomarcadores , Células CACO-2 , Colite/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Junções Íntimas/patologia
6.
J Photochem Photobiol B ; 221: 112248, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34192628

RESUMO

Melatonin is mainly secreted by the pineal gland, and it is also produced by various ocular structures such as the lens. It has been recently demonstrated that melatonin ocular synthesis can be induced by blocking the blue component of white light by means of filters. Melatonin exhibits antioxidant properties that can be useful to face light-induced oxidative stress as well as oxidative events associated to ocular pathologies like cataracts. Moreover, as oxidative stress is a main event in cataract development, changes in melatonin levels could happen and be relevant in the progression of this pathology, a subject that remains uncertain. The goal of this work was to analyze the ability of a short wavelength light blocking (yellow) filter to modulate endogenous melatonin concentration and the antioxidant and cytoprotective actions induced by yellow filter's use in lens. Furthermore, we evaluated the potential changes in aqueous humor melatonin concentration from patients with cataracts. In human lens epithelial cells, white light-emitting diode (LED) light challenge reduced melatonin secretion, protein levels of the enzymes involved in melatonin synthesis (hydroxyindole-O-methyltransferase and unphosphorylated and phosphorylated forms of arylalkylamine N-acetyltransferase) and cell viability whereas increased reactive oxygen species production. Yellow filter exposure precluded melatonin secretion reduction and protected cells from oxidative damage. Consistent with cataract patient's results, significantly lower levels of melatonin were observed in aqueous humor of alloxan-induced diabetic cataract rabbits as compared to those of control rabbits. In contrast, aqueous humor melatonin levels of diabetic cataract animals maintaining in cages covered with a yellow filter resembled control values. This recovery seems to be mediated by the induction of melatonin biosynthetic enzymes protein expression. Yellow filter also preserved Nrf2 lens protein expression and superoxide dismutase protein levels and activity in diabetic animals. Modulation of endogenous ocular melatonin concentration using blocking filters might be a promising approach to prevent premature lens opacification.


Assuntos
Humor Aquoso/metabolismo , Melatonina/metabolismo , Substâncias Protetoras/metabolismo , Idoso , Animais , Catarata/metabolismo , Catarata/patologia , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Feminino , Humanos , Cristalino/citologia , Luz , Masculino , Melatonina/farmacologia , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Regulação para Cima/efeitos dos fármacos
7.
Am J Physiol Cell Physiol ; 321(1): C176-C186, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34106788

RESUMO

Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, patients with MELAS treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in patients with MELAS are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared with control, healthy individuals. We observed that mitochondria from patients were fragmented, had a threefold decline in the average speed of motility, a twofold reduced mitochondrial membrane potential, and a 1.5- to 2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts exhibited increased MnSOD levels and lysosomal function when compared with healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 h resulted in increased mitochondrial respiration compared with control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.


Assuntos
Fibroblastos/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Síndrome MELAS/genética , Mitocôndrias/efeitos dos fármacos , Sirolimo/farmacologia , Estudos de Casos e Controles , Pré-Escolar , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Lactente , Lisossomos/metabolismo , Síndrome MELAS/tratamento farmacológico , Síndrome MELAS/metabolismo , Síndrome MELAS/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mutação , Fosforilação Oxidativa/efeitos dos fármacos , Cultura Primária de Células , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Adulto Jovem
8.
Environ Health ; 20(1): 66, 2021 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-34090432

RESUMO

BACKGROUND: Prenatal exposure to heavy metals is implicated in the etiology of birth defects. We investigated whether concentrations of cadmium (Cd) and lead (Pb) in umbilical cord tissue are associated with risk for neural tube defects (NTDs) and whether selected genetic variants of the fetus modify their associations. METHODS: This study included 166 cases of NTD fetuses/newborns and 166 newborns without congenital malformations. Umbilical cord tissue was collected at birth or elective pregnancy termination. Cd and Pb concentrations were assessed by inductively coupled plasma-mass spectrometry, and 20 single-nucleotide polymorphisms (SNPs) in 9 genes were genotyped. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to estimate the risk for NTDs in association with metal concentrations or genotype using logistic regression. Multiplicative-scale interactions between the metals and genotypes on NTD risk were assessed with logistic regression, and additive-scale interactions were estimated with a non-linear mixed effects model. RESULTS: Higher concentrations of Cd were observed in the NTD group than in the control group, but no difference was found for Pb. Concentrations of Cd above the median level showed a risk effect, while the association between Pb and NTD risk was not significant in univariate analyses. The association of Cd was attenuated after adjusting for periconceptional folic acid supplementation. Fetuses with the AG and GG genotypes of rs4880 in SOD2 (superoxide dismutase 2) tended to have a lower risk, but fetuses with the CT and TT genotypes of rs1801133 in MTHFR (5,10-methylenetetrahydrofolatereductase) have a higher risk for NTDs when compared to their respective wild-type. rs4880 and Cd exhibited a multiplicative-scale interaction on NTD risk: the association between higher Cd and the risk for NTDs was increased by over fourfold in fetuses carrying the G allele [OR 4.43 (1.30-15.07)] compared to fetuses with the wild-type genotype. rs1801133 and Cd exposure showed an additive interaction, with a significant relative excess risk of interaction [RERI 0.64 (0.02-1.25)]. CONCLUSIONS: Prenatal exposure to Cd may be a risk factor for NTDs, and the risk effect may be enhanced in fetuses who carry the G allele of rs4880 in SOD2 and T allele of rs1801133 in MTHFR.


Assuntos
Cádmio/efeitos adversos , Poluentes Ambientais/efeitos adversos , Exposição Materna/efeitos adversos , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Defeitos do Tubo Neural/genética , Superóxido Dismutase/genética , Adulto , Cádmio/análise , Estudos de Casos e Controles , China/epidemiologia , Poluentes Ambientais/análise , Feminino , Feto , Genótipo , Humanos , Recém-Nascido , Chumbo/análise , Troca Materno-Fetal , Defeitos do Tubo Neural/epidemiologia , Polimorfismo de Nucleotídeo Único , Gravidez
9.
Theranostics ; 11(15): 7294-7307, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34158851

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by death of motor neurons in the brain and spinal cord. However, so far, there is no effective treatment for ALS. Methods: In this study, R13, a prodrug of 7,8-dihydroxyflavone, selectively activating tyrosine kinase receptor B (TrkB) signaling pathway, was administered prophylactically to 40-day old SOD1G93A mice for 90 days. The motor performance was investigated by rotarod test, climbing-pole test, grip strength test and hanging endurance test. Afterwards, the spinal cord and medulla oblongata of 130-day old mice were harvested, and the proteomics revealed the effect of R13 on mouse protein expression profile. Astrocytes and microglial proliferation were assessed by immunohistochemical analysis. The number of motor neurons in the spinal cord is determined by Nissl staining. The effect of R13 on gastrocnemius morphology was assessed by HE staining. The effect of R13 on the survival rate was accomplished with worms stably expressing G93A SOD1. Results: Behavioral tests showed that R13 significantly attenuated abnormal motor performance of SOD1G93A mice. R13 reduced the advance of spinal motor neuron pathology and gastrocnemius muscle atrophy. The proliferation of microglia and astrocytes was reduced by R13 treatment. Mitochondriomics analysis revealed that R13 modified the mitochondrial protein expression profiles in the medulla oblongata and spinal cord of SOD1G93A mice, particularly promoting the expression of proteins related to oxidative phosphorylation (OXPHOS). Further study found that R13 activated AMPK/PGC-1α/Nrf1/Tfam, promoted mitochondrial biogenesis and ameliorated mitochondrial dysfunction. Lastly, R13 prolonged the survival rate of worms stably expressing G93A SOD1. Conclusions: These findings suggest oral R13 treatment slowed the advance of motor system disease in a reliable animal model of ALS, supporting that R13 might be useful for treating ALS.


Assuntos
Esclerose Amiotrófica Lateral , Sistema Nervoso Central/enzimologia , Flavonas/farmacologia , Mitocôndrias , Atividade Motora , Superóxido Dismutase-1 , Superóxido Dismutase , Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
10.
ACS Chem Neurosci ; 12(13): 2520-2528, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34138531

RESUMO

A common characteristic of familial (fALS) and sporadic amyotrophic lateral sclerosis (sALS) is the accumulation of aberrant proteinaceous species in the motor neurons and spinal cord of ALS patients-including aggregates of the human superoxide dismutase 1 (hSOD1). hSOD1 is an enzyme that occurs as a stable dimeric protein with several post-translational modifications such as the formation of an intramolecular disulfide bond and the acquisition of metal cofactors that are essential for enzyme activity and further contribute to protein stability. Some mutations and/or destabilizing factors promote hSOD1 misfolding, causing neuronal death. Aggregates containing misfolded wild-type hSOD1 have been found in the spinal cords of sALS as well as in non-hSOD1 fALS patients, leading to the hypothesis that hSOD1 misfolding is a common part of the ALS pathomechanism. Therefore, stabilizing the native conformation of SOD1 may be a promising approach to prevent the formation of toxic hSOD1 species and thus ALS pathogenesis. Here, we present the 16-mer peptide S1VL-21 that interferes with hSOD1 aggregation. S1VL-21 was identified by phage display selection with the native conformation of hSOD1 as a target. Several methods such as microscale thermophoresis (MST) measurements, aggregation assays, and cell viability assays revealed that S1VL-21 has a micromolar binding affinity to native hSOD1 and considerably reduces the formation of hSOD1 aggregates. This present work therefore provides the first important data on a potential lead compound for hSOD1-related drug development for ALS therapy.


Assuntos
Esclerose Amiotrófica Lateral , Superóxido Dismutase , Humanos , Ligantes , Neurônios Motores , Mutação/genética , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética
11.
Methods Mol Biol ; 2277: 289-297, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080158

RESUMO

Mitochondrial reactive oxygen species (mtROS) and redox regulation play an important role in stem cell maintenance and cell fate decisions. Although changes in mtROS and redox homeostasis represent a physiological mechanism to drive stem cell commitment and differentiation, dysregulation of this system can lead to defects in stem cell maintenance and regenerative capacity. This chapter explains the methods used to assess mitochondrial superoxide levels and redox regulation in stem cell populations.


Assuntos
Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/análise , Células-Tronco/metabolismo , Animais , Perfilação da Expressão Gênica/métodos , Camundongos , Músculo Esquelético/citologia , Compostos Organofosforados/química , Oxirredução , Fenantridinas/química , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco/fisiologia , Superóxido Dismutase/genética , Superóxidos/análise , Superóxidos/metabolismo , Proteína Desacopladora 2/genética
12.
Stem Cell Res ; 54: 102415, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34118566

RESUMO

Superoxide Dismutase 1 (SOD1) is an antioxidant enzyme that protects the cells from radical oxygen species. To study the behavior of endogenous SOD1 under a microscope, we genetically modified H1 human embryonic stem cells (hESCs) to express SOD1 fused with a SNAP-tag, a protein tag that can be covalently labeled with a variety of synthetic probes. The engineered homozygous clone expressing SOD1-SNAP fusion proteins has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro, providing a versatile platform for imaging-based studies of SOD1.


Assuntos
Células-Tronco Embrionárias Humanas , Linhagem Celular , Células Cultivadas , Humanos , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética
13.
Toxicol Appl Pharmacol ; 426: 115634, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34174261

RESUMO

While Bisphenol A (BPA) has been a requisite plastic additive, as an endocrine disruptor it has been associated with adverse health effects including ovarian disorders. Following implemented restrictions on BPA usage, it is replaced by alternative bisphenols, biological effects of which have not been adequately investigated. Our study examined effects of bisphenols AF (BPAF) and S (BPS), on the human ovarian granulosa cell line COV434, and compared them with BPA, with the focus on cell viability (10-9-10-4 M) and angiogenesis-related factors (10-9-10-5 M), relevant for both the follicle development and ovarian pathologies: vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor AA (PDGF-AA), and matrix metalloproteinase 9 (MMP-9). Each bisphenol impaired cell viability and increased generation of intracellular reactive oxygen species at the highest concentration (10-4 M). While VEGF-A production in BPAF-treated groups did not differ from the control, all doses of BPS and BPA caused a marked reduction in VEGF-A output. Nevertheless, the alterations in VEGF-A production were not caused by the impact on VEGFA gene expression since there were no indications of VEGFA downregulation in the presence of either BPS or BPA. Interestingly, we observed a similar pattern of PDGF-AA output reduction in BPS- and BPA-treated groups to that of VEGF-A production. BPAF and BPS (10-5 M) increased MMP9 expression, however, this effect was not reflected by the increase in MMP-9 production. The results obtained demonstrate that the novel bisphenol analogs are not inert with respect to the ovarian cells, and their effects might contribute to dysregulation of granulosa cells functions.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Células da Granulosa/efeitos dos fármacos , Fenóis/toxicidade , Sulfonas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células da Granulosa/metabolismo , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Neovascularização Fisiológica , Fator de Crescimento Derivado de Plaquetas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073630

RESUMO

Motor neuron diseases (MNDs) are neurodegenerative disorders characterized by upper and/or lower MN loss. MNDs include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). Despite variability in onset, progression, and genetics, they share a common skeletal muscle involvement, suggesting that it could be a primary site for MND pathogenesis. Due to the key role of muscle-specific microRNAs (myomiRs) in skeletal muscle development, by real-time PCR we investigated the expression of miR-206, miR-133a, miR-133b, and miR-1, and their target genes, in G93A-SOD1 ALS, Δ7SMA, and KI-SBMA mouse muscle during disease progression. Further, we analyzed their expression in serum of SOD1-mutated ALS, SMA, and SBMA patients, to demonstrate myomiR role as noninvasive biomarkers. Our data showed a dysregulation of myomiRs and their targets, in ALS, SMA, and SBMA mice, revealing a common pathogenic feature associated with muscle impairment. A similar myomiR signature was observed in patients' sera. In particular, an up-regulation of miR-206 was identified in both mouse muscle and serum of human patients. Our overall findings highlight the role of myomiRs as promising biomarkers in ALS, SMA, and SBMA. Further investigations are needed to explore the potential of myomiRs as therapeutic targets for MND treatment.


Assuntos
Esclerose Amiotrófica Lateral , Atrofia Bulboespinal Ligada ao X , MicroRNAs , Mutação de Sentido Incorreto , Superóxido Dismutase-1 , Superóxido Dismutase , Substituição de Aminoácidos , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Atrofia Bulboespinal Ligada ao X/genética , Atrofia Bulboespinal Ligada ao X/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
15.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070927

RESUMO

Citric acid (CA), as an organic chelator, plays a vital role in alleviating copper (Cu) stress-mediated oxidative damage, wherein a number of molecular mechanisms alter in plants. However, it remains largely unknown how CA regulates differentially abundant proteins (DAPs) in response to Cu stress in Brassica napus L. In the present study, we aimed to investigate the proteome changes in the leaves of B. L. seedlings in response to CA-mediated alleviation of Cu stress. Exposure of 21-day-old seedlings to Cu (25 and 50 µM) and CA (1.0 mM) for 7 days exhibited a dramatic inhibition of overall growth and considerable increase in the enzymatic activities (POD, SOD, CAT). Using a label-free proteome approach, a total of 6345 proteins were identified in differentially treated leaves, from which 426 proteins were differentially expressed among the treatment groups. Gene ontology (GO) and KEGG pathways analysis revealed that most of the differential abundance proteins were found to be involved in energy and carbohydrate metabolism, photosynthesis, protein metabolism, stress and defense, metal detoxification, and cell wall reorganization. Our results suggest that the downregulation of chlorophyll biosynthetic proteins involved in photosynthesis were consistent with reduced chlorophyll content. The increased abundance of proteins involved in stress and defense indicates that these DAPs might provide significant insights into the adaptation of Brassica seedlings to Cu stress. The abundances of key proteins were further verified by monitoring the mRNA expression level of the respective transcripts. Taken together, these findings provide a potential molecular mechanism towards Cu stress tolerance and open a new route in accelerating the phytoextraction of Cu through exogenous application of CA in B. napus.


Assuntos
Brassica napus/efeitos dos fármacos , Ácido Cítrico/farmacologia , Cobre/toxicidade , Poluentes Ambientais/toxicidade , Proteínas de Plantas/genética , Proteoma/genética , Adaptação Fisiológica , Brassica napus/genética , Brassica napus/crescimento & desenvolvimento , Brassica napus/metabolismo , Catalase/genética , Catalase/metabolismo , Clorofila/biossíntese , Ácido Cítrico/metabolismo , Cobre/metabolismo , Poluentes Ambientais/antagonistas & inibidores , Poluentes Ambientais/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Peroxidases/classificação , Peroxidases/genética , Peroxidases/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Proteoma/classificação , Proteoma/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Estresse Fisiológico , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
16.
Nat Commun ; 12(1): 3251, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059686

RESUMO

ALS is characterized by progressive inability to execute movements. Motor neurons innervating fast-twitch muscle-fibers preferentially degenerate. The reason for this differential vulnerability and its consequences on motor output is not known. Here, we uncover that fast motor neurons receive stronger inhibitory synaptic inputs than slow motor neurons, and disease progression in the SOD1G93A mouse model leads to specific loss of inhibitory synapses onto fast motor neurons. Inhibitory V1 interneurons show similar innervation pattern and loss of synapses. Moreover, from postnatal day 63, there is a loss of V1 interneurons in the SOD1G93A mouse. The V1 interneuron degeneration appears before motor neuron death and is paralleled by the development of a specific locomotor deficit affecting speed and limb coordination. This distinct ALS-induced locomotor deficit is phenocopied in wild-type mice but not in SOD1G93A mice after appearing of the locomotor phenotype when V1 spinal interneurons are silenced. Our study identifies a potential source of non-autonomous motor neuronal vulnerability in ALS and links ALS-induced changes in locomotor phenotype to inhibitory V1-interneurons.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Interneurônios/patologia , Locomoção/fisiologia , Neurônios Motores/patologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Animais , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fibras Musculares de Contração Rápida/fisiologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Medula Espinal/citologia , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética
17.
Biochem Biophys Res Commun ; 560: 119-125, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33989902

RESUMO

Amounting evidence suggested that long non coding RNAs (lncRNAs) played vital roles in the progression of various cancers. The aim of this study is to examine the biological roles and underlying mechanisms of lncRNA MAFG-AS1 in the tumorigenesis of breast cancer (BC) cells. Here we showed that downregulation of MAFG-AS1 inhibited the viability, migration, and invasion of BC cells. Mechanism investigation showed that inhibition of MAFG-AS1 induced apoptosis via the intrinsic apoptotic pathway and overexpression of Bcl-2 could inhibited it. Further, MAFG-AS1 acts as a sponge of miR-574-5p which directly binds to SOD2 mRNA. Re-expression of SOD2 using a 3'-UTR mutant SOD2 reversed the effects of silencing of MAFG-AS1 on BC cells. Finally, downregulation of MAFG-AS1 inhibited the growth of tumour in vivo. Together, MAFG-AS1 acts as an oncogene via regulation of miR-574-5p/SOD2 axis in BC cells.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Superóxido Dismutase/genética , Animais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinogênese , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Feminino , Humanos , Camundongos SCID , Invasividade Neoplásica , Superóxido Dismutase/metabolismo
18.
Fish Shellfish Immunol ; 114: 199-206, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33940173

RESUMO

Co-infection with parasites and bacteria is of frequent occurrence in aquaculture, leads to growth impedance otherwise mortality in fish depending on the varying degree of a load of primary pathogen either parasite or bacteria. The mechanistic regulation of immune response during co-infection in fish has merely documented. The aim of this study was to determine the impact of co-infection with Aeromonas hydrophila at three exposure doses of Argulus sp. on the innate immune responses and antioxidative stress enzymes of goldfish (Carassius auratus). The experimental fish were randomly distributed into eight treatment groups viz. T1 (control group without Argulus and A. hydrophila infection), T2 (fish exposed to a sub-lethal dose of A. hydrophila), T3 (low Argulus-infested fish), T4 (T3 + sub-lethal dose of A. hydrophila), T5 (moderate Argulus-infested fish), T6 (T5 + sub-lethal dose of A. hydrophila), T7 (high Argulus-infested fish) and T8 (T7+ sub-lethal dose of A. hydrophila) in duplicates. After distributing experimental fish into their respective treatment group, A. hydrophila was injected to T2, T4, T6 and T8. After the bacterial challenge, four fish from each experimental group were randomly sampled on 24, 72, and 168 h and subjected to the hematological, innate immune parameters and enzymatic analysis. In the co-infection group T8, a high degree of enhanced pathogenicity of A. hydrophila was noticed with increased mortalities (84.2%) in comparison to other groups. The current study shows a declining pattern in RBC, PCV and Hb values with the degree of parasite infestation without co-infection groups. Moreover, in the T8 group, exposure of a sub-lethal dose of bacteria resulted in a drastic reduction of the recorded parameters. Furthermore, a decreased value for WBC, monocyte and neutrophil was found in higher parasite group co-infected with a sub-lethal dose of bacteria relative to other co-infected groups during the experimental period. Also, a decrease in innate immune parameters and antioxidative stress enzymes were observed in the T8 group compared to T7 and T2 groups throughout the trial period. These findings indicate that a rise in the dose of Argulus infection improves A. hydrophila colonization in goldfish and contributes to suppression of the innate immune system and increased mortality.


Assuntos
Aeromonas hydrophila , Arguloida , Carpa Dourada , Infecções por Bactérias Gram-Negativas/veterinária , Imunidade Inata/fisiologia , Doenças Parasitárias em Animais/parasitologia , Animais , Antioxidantes , Catalase/genética , Catalase/metabolismo , Regulação Enzimológica da Expressão Gênica/imunologia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Infecções por Bactérias Gram-Negativas/complicações , Infecções por Bactérias Gram-Negativas/imunologia , Doenças Parasitárias em Animais/complicações , Doenças Parasitárias em Animais/imunologia , Estresse Fisiológico , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
19.
Neuroscience ; 465: 11-22, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33945797

RESUMO

Amyotrophic lateral sclerosis (ALS) is one of the leading causes of death associated with neurodegenerative diseases worldwide, and the progression of the disease is characteristically accompanied by severe neuroinflammation. Neuroprotective effects of oxymatrine (OMT) were shown to be due to reduced neuroinflammation in the mouse models of Alzheimer's disease and Parkinson's disease. The present study investigated whether OMT has a therapeutic potential in transgenic SOD1-G93A (TgSOD1-G93A) mice. Daily OMT treatment started at the age of 55 days until the end stage of the disease. Body weight and rotarod motor performance were assessed every 3 days starting from 70 days of age. Footprints were recorded to measure the stride length 40 days and 60 days after the initiation of the treatment. Some animals were sacrificed at the age of 115 days, and the lumbar spinal cord was harvested for immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) to evaluate the neuroinflammatory responses. The results indicated that treatment with OMT delayed body weight loss, improved motor performance, and prolonged the survival of SOD1-G93A mice. Mechanistically, OMT treatment enhanced motor neuronal survival and alleviated the activation of microglia and astrocytes compared with those in the vehicle-treated group. Furthermore, the expression of the proinflammatory mediators was downregulated, and the expression of the anti-inflammatory factors was upregulated in the OMT-treated group compared with those in the vehicle-treated group (P < 0.05). Thus, the treatment with OMT had neuroprotective effects, promoting neuronal survival and extending the lifetime of SOD1-G93A mice by suppressing neuroinflammation.


Assuntos
Alcaloides , Esclerose Amiotrófica Lateral , Alcaloides/farmacologia , Esclerose Amiotrófica Lateral/tratamento farmacológico , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Neurônios Motores , Quinolizinas , Medula Espinal , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética
20.
Nat Med ; 27(4): 640-646, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33859435

RESUMO

Apart from well-defined factors in neuronal cells1, only a few reports consider that the variability of sporadic amyotrophic lateral sclerosis (ALS) progression can depend on less-defined contributions from glia2,3 and blood vessels4. In this study we use an expression-weighted cell-type enrichment method to infer cell activity in spinal cord samples from patients with sporadic ALS and mouse models of this disease. Here we report that patients with sporadic ALS present cell activity patterns consistent with two mouse models in which enrichments of vascular cell genes preceded microglial response. Notably, during the presymptomatic stage, perivascular fibroblast cells showed the strongest gene enrichments, and their marker proteins SPP1 and COL6A1 accumulated in enlarged perivascular spaces in patients with sporadic ALS. Moreover, in plasma of 574 patients with ALS from four independent cohorts, increased levels of SPP1 at disease diagnosis repeatedly predicted shorter survival with stronger effect than the established risk factors of bulbar onset or neurofilament levels in cerebrospinal fluid. We propose that the activity of the recently discovered perivascular fibroblast can predict survival of patients with ALS and provide a new conceptual framework to re-evaluate definitions of ALS etiology.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Vasos Sanguíneos/patologia , Fibroblastos/patologia , Esclerose Amiotrófica Lateral/sangue , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Biomarcadores/metabolismo , Colágeno Tipo VI/genética , Colágeno Tipo VI/metabolismo , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Marcadores Genéticos , Humanos , Camundongos Transgênicos , Osteopontina/sangue , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Superóxido Dismutase/genética , Transcrição Genética , Remodelação Vascular
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