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1.
EBioMedicine ; 59: 102980, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32862101

RESUMO

BACKGROUND: Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease as well as Lou Gehrig's disease, is a progressive neurological disorder selectively affecting motor neurons with no currently known cure. Around 20% of the familial ALS cases arise from dominant mutations in the sod1 gene encoding superoxide dismutase1 (SOD1) enzyme. Aggregation of mutant SOD1 in familial cases and of wild-type SOD1 in at least some sporadic ALS cases is one of the known causes of the disease. Riluzole, approved in 1995 and edaravone in 2017 remain the only drugs with limited therapeutic benefits. METHODS: We have utilised the ebselen template to develop novel compounds that redeem stability of mutant SOD1 dimer and prevent aggregation. Binding modes of compounds have been visualised by crystallography. In vitro neuroprotection and toxicity of lead compounds have been performed in mouse neuronal cells and disease onset delay of ebselen has been demonstrated in transgenic ALS mice model. FINDING: We have developed a number of ebselen-based compounds with improvements in A4V SOD1 stabilisation and in vitro therapeutic effects with significantly better potency than edaravone. Structure-activity relationship of hits has been guided by high resolution structures of ligand-bound A4V SOD1. We also show clear disease onset delay of ebselen in transgenic ALS mice model holding encouraging promise for potential therapeutic compounds. INTERPRETATION: Our finding established the new generation of organo-selenium compounds with better in vitro neuroprotective activity than edaravone. The potential of this class of compounds may offer an alternative therapeutic agent for ALS treatment. The ability of these compounds to target cysteine 111 in SOD may have wider therapeutic applications targeting cysteines of enzymes involved in pathogenic and viral diseases including main protease of SARS-Cov-2 (COVID-19). FUNDING: Project funding was supported by the ALS Association grant (WA1128) and Fostering Joint International Research (19KK0214) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Compostos Organosselênicos/uso terapêutico , Superóxido Dismutase-1/metabolismo , Esclerose Amiotrófica Lateral/mortalidade , Esclerose Amiotrófica Lateral/patologia , Animais , Azóis/química , Azóis/metabolismo , Azóis/uso terapêutico , Betacoronavirus/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Dimerização , Modelos Animais de Doenças , Estabilidade Enzimática , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Compostos Organosselênicos/química , Compostos Organosselênicos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Superóxido Dismutase-1/genética , Taxa de Sobrevida , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/metabolismo
2.
Nat Commun ; 11(1): 3848, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737286

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper and lower motor neurons (MNs). We find a significant reduction of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases, a pathological hallmark in ALS, we design, synthesize and characterize an array of retromer stabilizers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. We select compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuates locomotion impairment and increases MNs survival. Moreover, compound 2a increases VPS35 in iPSCs-derived MNs and shows brain bioavailability. Our results clearly suggest the retromer as a valuable druggable target in ALS.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Hidrazonas/farmacologia , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Proteínas de Transporte Vesicular/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Hidrazonas/síntese química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/síntese química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Relação Estrutura-Atividade , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Proteínas de Transporte Vesicular/metabolismo
3.
Nat Commun ; 11(1): 3753, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719333

RESUMO

Reactive astrocytes have been implicated in the pathogenesis of neurodegenerative diseases, including a non-cell autonomous effect on motor neuron survival in ALS. We previously defined a mechanism by which microglia release three factors, IL-1α, TNFα, and C1q, to induce neurotoxic astrocytes. Here we report that knocking out these three factors markedly extends survival in the SOD1G93A ALS mouse model, providing evidence for gliosis as a potential ALS therapeutic target.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Astrócitos/metabolismo , Complemento C1q/metabolismo , Progressão da Doença , Interleucina-1alfa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Complemento C3/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia , Superóxido Dismutase-1/metabolismo
4.
PLoS One ; 15(6): e0234076, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32520965

RESUMO

This study investigated the effects of oral administration of ß-glucan 1,3 (pharmaceutical grade 10%) on growth performance and carcass traits in two breeds of weanling rabbits adapted to survive in Egypt, New Zealand White (NZW) and Animal Production Research Institute (APRI) rabbits, with special attention to relative mRNA expression of interleukins and antioxidant enzyme genes, biochemical, and histological alterations. Oral administration of ß-glucan with doses 0.25 and 0.5 ml per one-liter of drinking water significantly accelerated body weight gain (BWG) in both rabbits' breeds, reduced total feed consumption (FC), and reduced feed conversion ratio (FCR), especially the 0.5 ml per one-liter dose in both rabbit breeds. There are remarkable differences in all the growth performance traits due to breed effect. The interaction effect between ß-glucan and breed significantly improved BWG, FC, and FCR. There were non-significant differences in all carcass traits studied due to oral administration of ß-glucan with both doses, except in dressing percentages. The highest of the dressing percentages were observed at doses 0.25 ml per one-liter (51%) and 0.5 ml per one-liter (52%) compared with control (50%). Our findings show significant variations in the final BW, total daily gain, feed consumption, and total feed conversion ratio between NZW and APRI rabbits. Absence of significant differences in the hot carcass weight and dressing percentage between the genetic groups had been reported in this study. Supplementing NZW and APRI rabbits with ß-glucan increased blood total protein and globulin. The duodenal villi dimensions, splenic lymphoid diameter, muscular fiber diameter, and muscular glycogen areas were significantly increased by ß-glucan administration. Expression of intestinal interleukin-18 (IL-18) in NZW rabbits treated with 0.25 and 0.5 doses of ß-glucan was significantly upregulated and enhanced the immune response. ß-glucan upregulated the expression of intestinal occludin mRNA particularly at dose 0.5 ß-glucan as well as upregulated intestinal superoxide dismutase 1 (SOD1) and glutathione peroxidase 1 (GPx1), which modulates anti-inflammatory and antioxidant properties. In conclusion, oral administration of ß-glucan at a dose of 0.25 or 0.5 ml per one-liter drinking water provided beneficial effects in the growth performance and health status of rabbits.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , Ganho de Peso/efeitos dos fármacos , beta-Glucanas/farmacologia , Imunidade Adaptativa/efeitos dos fármacos , Administração Oral , Animais , Duodeno/metabolismo , Duodeno/patologia , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Interleucina-18/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Ocludina/genética , Ocludina/metabolismo , Coelhos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Regulação para Cima/efeitos dos fármacos
5.
PLoS One ; 15(4): e0232408, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353034

RESUMO

Mitochondria are quantitatively the most important sources of reactive oxygen species (ROS) which are formed as by-products during cellular respiration. ROS generation occurs when single electrons are transferred to molecular oxygen. This leads to a number of different ROS types, among them superoxide. Although most studies focus on ROS generation in the mitochondrial matrix, the intermembrane space (IMS) is also important in this regard. The main scavengers for the detoxification of superoxide in the IMS are Cu, Zn superoxide dismutase (SOD1) and cytochrome-c. Similar to ROS, certain reactive carbonyl species are known for their high reactivity. The consequences are deleterious modifications to essential components compromising cellular functions and contributing to the etiology of severe pathological conditions like cancer, diabetes and neurodegeneration. In this study, we investigated the susceptibility of SOD1 and cytochrome-c to in vitro glycation by the dicarbonyl methylglyoxal (MGO) and the resulting effects on their structure. We utilized experimental techniques like immunodetection of the MGO-mediated modification 5-hydro-5-methylimidazolone, differential scanning calorimetry, fluorescence emission and circular dichroism measurements. We found that glycation of cytochrome-c leads to monomer aggregation, an altered secondary structure (increase in alpha helical content) and slightly more compact folding. In addition to structural changes, glycated cytochrome-c displays an altered thermal unfolding behavior. Subjecting SOD1 to MGO does not influence its secondary structure. However, similar to cytochrome-c, subunit aggregation is observed under denaturating conditions. Furthermore, the appearance of a second peak in the calorimetry diagram indirectly suggests de-metallation of SOD1 when high MGO levels are used. In conclusion, our data demonstrate that MGO has the potential to alter several structural parameters in important proteins of energy metabolism (cytochrome-c) and antioxidant defense (cytochrome-c, SOD1).


Assuntos
Citocromos c/química , Mitocôndrias/metabolismo , Aldeído Pirúvico/farmacologia , Superóxido Dismutase-1/química , Animais , Citocromos c/metabolismo , Cavalos , Mitocôndrias/efeitos dos fármacos , Dobramento de Proteína , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase-1/metabolismo
6.
PLoS Comput Biol ; 16(4): e1007773, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32294079

RESUMO

Evolutionarily conserved mechanisms maintain homeostasis of essential elements, and are believed to be highly time-variant. However, current approaches measure elemental biomarkers at a few discrete time-points, ignoring complex higher-order dynamical features. To study dynamical properties of elemental homeostasis, we apply laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) to tooth samples to generate 500 temporally sequential measurements of elemental concentrations from birth to 10 years. We applied dynamical system and Information Theory-based analyses to reveal the longest-known attractor system in mammalian biology underlying the metabolism of nutrient elements, and identify distinct and consistent transitions between stable and unstable states throughout development. Extending these dynamical features to disease prediction, we find that attractor topography of nutrient metabolism is altered in amyotrophic lateral sclerosis (ALS), as early as childhood, suggesting these pathways are involved in disease risk. Mechanistic analysis was undertaken in a transgenic mouse model of ALS, where we find similar marked disruptions in elemental attractor systems as in humans. Our results demonstrate the application of a phenomological analysis of dynamical systems underlying elemental metabolism, and emphasize the utility of these measures in characterizing risk of disease.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Cobre/análise , Dente/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores/metabolismo , Criança , Pré-Escolar , Biologia Computacional , Cobre/sangue , Cobre/urina , Feminino , Homeostase , Humanos , Lactente , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Curva ROC , Risco , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
7.
Mutat Res ; 852: 503167, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32265040

RESUMO

Chronic kidney disease (CKD) is a multifactorial disorder with an important genetic component, and several studies have demonstrated potential associations with allelic variants. In addition, CKD patients are also characterized by high levels of genomic damage. Nevertheless, no studies have established relationships between DNA damage, or genomic instability present in CKD patients, and gene polymorphisms. To fill in this gap, the potential role of polymorphisms in genes involved in base excision repair (OGG1, rs1052133; MUTYH, rs3219489; XRCC1, rs25487), nucleotide excision repair (ERCC2/XPD, rs1799793, rs171140, rs13181; ERCC4, rs3136166); phase II metabolism (GSTP1, rs749174; GSTO1, rs2164624; GSTO2, rs156697), and antioxidant enzymes (SOD1, rs17880135, rs1041740, rs202446; SOD2, rs4880; CAT, rs1001179; GPX1, rs17080528; GPX3, rs870406: GPX4, rs713041) were inquired. In addition, some genes involved in CKD (AGT, rs5050; GLO1, rs386572987; SHROOM3, rs17319721) were also evaluated. The genomic damage, the genomic instability, and oxidative damage were evaluated by using the micronucleus and the comet assay in 589 donors (415 CKD patients and 174 controls). Our results showed significant associations between genomic damage and genes directly involved in DNA repair pathways (XRCC1, and ERCC2), and with genes encoding for antioxidant enzymes (SOD1 and GPX1). GSTO2, as a gene involved in phase II metabolism, and MUTYH showed also an association with genomic instability. Interestingly, the three genes associated with CKD (AGT, GLO1, and SHROOM3) showed associations with both the high levels of oxidatively damaged DNA and genomic instability. These results support our view that genomic instability can be considered a biomarker of the CKD status.


Assuntos
Angiotensinogênio/genética , Reparo do DNA , Instabilidade Genômica , Lactoilglutationa Liase/genética , Proteínas dos Microfilamentos/genética , Insuficiência Renal Crônica/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Angiotensinogênio/metabolismo , Estudos de Casos e Controles , Ensaio Cometa , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Lactoilglutationa Liase/metabolismo , Masculino , Proteínas dos Microfilamentos/metabolismo , Testes para Micronúcleos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/fisiopatologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
8.
Anim Sci J ; 91(1): e13327, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32219959

RESUMO

Excessive lipid peroxidation negatively affects the physiological response and meat quality of chickens. Delaying post-hatch feeding was previously found to increase lipid peroxidation in the skeletal muscle of finishing broiler chickens. The aims of this study were to investigate the effects of delayed post-hatch feeding on lipid peroxidation and the mRNA expressions of antioxidant enzymes in the pectoralis major muscle of broiler chicks during the post-hatching period. Newly hatched chicks either had immediate free access to feed (freely-fed chicks) or had no access to feed from 0 to 2 days old (delayed-fed chicks), after which both groups were fed ad libitum until 4 or 13 days old. The lipid peroxidation level was higher in the delayed-fed than freely-fed chicks at 2, 4, and 13 days old. At 2 days old, the mRNA expressions of Cu/Zn-SOD, Mn-SOD, and GPX7 were lower in the delayed-fed than freely-fed chicks, while catalase mRNA levels did not differ. Furthermore, at 4 and 13 days old, lower mRNA expressions of Cu/Zn-SOD and Mn-SOD were observed in the delayed-fed than freely-fed chicks. These results suggest that delaying post-hatch feeding reduces the mRNA levels of Cu/Zn-SOD and Mn-SOD, consequently affecting muscle lipid peroxidation in chicks during subsequent growth.


Assuntos
Ração Animal , Galinhas/metabolismo , Métodos de Alimentação/veterinária , Peroxidação de Lipídeos/fisiologia , Músculo Esquelético/metabolismo , Peroxidases/metabolismo , RNA Mensageiro/metabolismo , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase/metabolismo , Fenômenos Fisiológicos da Nutrição Animal , Animais , Expressão Gênica , Peroxidases/genética , RNA Mensageiro/genética , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética , Fatores de Tempo
9.
Mol Cell Biochem ; 466(1-2): 117-128, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32056106

RESUMO

Aberrant structural formations of Cu/Zn superoxide dismutase enzyme (SOD1) are the probable mechanism by which circumscribed mutations in the SOD1 gene cause familial amyotrophic lateral sclerosis (ALS1). SOD1 forms aberrant structures which can proceed by nucleation to insoluble aggregates. Here, the SOD1 aggregation reaction was investigated predominantly by time-course studies on ALS1 variants G85R, G37R, D101G, and D101N in human embryonic kidney cells (HEK293FT), with analysis by detergent ultracentrifugation extractions and high-resolution PAGE methodologies. Nucleation was found to be pseudo-zeroth order and dependent on time and concentration at constant 37.0 °C and pH 7.4. The predominant subsets of the total SOD1 expression set which comprised the nucleation phase were both soluble and insoluble inactive monomers, trimers, and hexamers with reduced intra-disulfide bonds. Superoxide exposure via paraquat initiated the formation of SOD1 trimers in untransfected SH-SY5Y cells and increased the aggregation propensity of G85R in HEK293FT. These data show the kinetic formation of aberrant SOD1 subsets implicated in ALS1 and indicate that superoxide substrate may initiate its radical polymerization. In an instance of the utility of methodological reductionism in molecular theory: though many ALS1 variants retain their global enzymatic activity, the SOD1 subsets most implicated in causing ALS1 do not retain their specific activity.


Assuntos
Mutação de Sentido Incorreto , Agregação Patológica de Proteínas/metabolismo , Superóxido Dismutase-1/metabolismo , Substituição de Aminoácidos , Esclerose Amiotrófica Lateral/genética , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Agregação Patológica de Proteínas/genética , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genética
10.
Oxid Med Cell Longev ; 2020: 6325378, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32064027

RESUMO

In addition to the lung, the liver is considered another major target for paraquat (PQ) poisoning. Hydrogen sulfide (H2S) has been demonstrated to be effective in the inhibition of oxidative stress and inflammation. The aim of this study was to investigate the protective effect of exogenous H2S against PQ-induced acute liver injury. The acute liver injury model was established by a single intraperitoneal injection of PQ, evidenced by histological alteration and elevated serum aminotransferase levels. Different doses of NaHS were administered intraperitoneally one hour before exposure to PQ. Analysis of the data shows that exogenous H2S attenuated the PQ-induced liver injury and oxidative stress in a dose-dependent manner. H2S significantly suppressed reactive oxygen species (ROS) generation and the elevation of malondialdehyde content while it increased the ratio of GSH/GSSG and levels of antioxidant enzymes including SOD, GSH-Px, HO-1, and NQO-1. When hepatocytes were subjected to PQ-induced oxidative stress, H2S markedly enhanced nuclear translocation of Nrf2 via S-sulfhydration of Keap1 and resulted in the increase in IDH2 activity by regulating S-sulfhydration of SIRT3. In addition, H2S significantly suppressed NLRP3 inflammasome activation and subsequent IL-1ß excretion in PQ-induced acute liver injury. Moreover, H2S cannot reverse the decrease in SIRT3 and activation of the NLRP3 inflammasome caused by PQ in Nrf2-knockdown hepatocytes. In summary, H2S attenuated the PQ-induced acute liver injury by enhancing antioxidative capability, regulating mitochondrial function, and suppressing ROS-induced NLRP3 inflammasome activation. The antioxidative effect of H2S in PQ-induced liver injury can at least partly be attributed to the promotion of Nrf2-driven antioxidant enzymes via Keap1 S-sulfhydration and regulation of SIRT3/IDH2 signaling via Nrf2-dependent SIRT3 gene transcription as well as SIRT3 S-sulfhydration. Thus, H2S supplementation can form the basis for a promising novel therapeutic strategy for PQ-induced acute liver injury.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hepatócitos/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Inflamação/metabolismo , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Paraquat/toxicidade , Animais , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Glutationa/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Isocitrato Desidrogenase/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Masculino , Mitocôndrias/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/genética , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Sirtuínas/metabolismo , Sulfetos/administração & dosagem , Superóxido Dismutase-1/metabolismo , Transaminases/metabolismo
11.
Oxid Med Cell Longev ; 2020: 7468738, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32064028

RESUMO

Cardiac arrest (CA) yields poor neurological outcomes. Salubrinal (Sal), an endoplasmic reticulum (ER) stress inhibitor, has been shown to have neuroprotective effects in both in vivo and in vitro brain injury models. This study investigated the neuroprotective mechanisms of Sal in postresuscitation brain damage in a rodent model of CA. In the present study, rats were subjected to 6 min of CA and then successfully resuscitated. Either Sal (1 mg/kg) or vehicle (DMSO) was injected blindly 30 min before the induction of CA. Neurological status was assessed 24 h after CA, and the cortex was collected for analysis. As a result, we observed that, compared with the vehicle-treated animals, the rats pretreated with Sal exhibited markedly improved neurological performance and cortical mitochondrial morphology 24 h after CA. Moreover, Sal pretreatment was associated with the following: (1) upregulation of superoxide dismutase activity and a reduction in maleic dialdehyde content; (2) preserved mitochondrial membrane potential; (3) amelioration of the abnormal distribution of cytochrome C; and (4) an increased Bcl-2/Bax ratio, decreased cleaved caspase 3 upregulation, and enhanced HIF-1α expression. Our findings suggested that Sal treatment improved neurological dysfunction 24 h after CPR (cardiopulmonary resuscitation), possibly through mitochondrial preservation and stabilizing the structure of HIF-1α.


Assuntos
Lesões Encefálicas/tratamento farmacológico , Córtex Cerebelar/efeitos dos fármacos , Cinamatos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Parada Cardíaca/fisiopatologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Tioureia/análogos & derivados , Aldeídos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Lesões Encefálicas/complicações , Lesões Encefálicas/metabolismo , Lesões Encefálicas/fisiopatologia , Reanimação Cardiopulmonar , Caspase 3/metabolismo , Córtex Cerebelar/metabolismo , Córtex Cerebelar/fisiopatologia , Córtex Cerebelar/ultraestrutura , Citocromos c/metabolismo , Parada Cardíaca/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Superóxido Dismutase-1/metabolismo , Tioureia/farmacologia
12.
Oxid Med Cell Longev ; 2020: 3071658, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32082477

RESUMO

This study is aimed at investigating the effect of different exercise loads on the reproductive function of obese male mice and the underlying mechanisms. Male mice with high-fat diet-induced obesity were divided into obesity control (OC), obesity moderate-load exercise (OME), and obesity high-load exercise (OHE) groups. The OME and OHE groups were subjected to swimming exercise 5 days per week over a duration of 8 weeks, with the exercise load progressively increased to 2 h per day in the OME group and 2 h twice per day in the OHE group. In the OC group mice without exercise regimen, we observed a decrease in mRNA expression of antioxidant enzymes, increase in free radical products, upregulation of mRNA and protein expression of nuclear factor-κB and proinflammatory cytokines, inhibition of mRNA and protein expression of testosterone synthases, decrease in the serum testosterone level and sperm quality, and increase in sperm apoptosis. Although both moderate-load exercise and high-load exercise reduced body fat, only moderate-load exercise effectively alleviated obesity-induced oxidative stress, downregulated the expression of nuclear factor-κB and proinflammatory cytokines, and reversed the decrease in mRNA and protein expression of testosterone synthases, serum testosterone level, and sperm quality. These changes were not observed in the OHE group mice. Obesity-induced testicular oxidative stress and inflammatory response decreased testosterone synthesis and sperm quality. Moderate-load exercise alleviated the negative effect of obesity on male reproductive function by decreasing testicular oxidative stress and inflammatory responses. Although high-load exercise effectively reduced body fat, its effects on alleviating oxidative stress and improving male reproductive function were limited.


Assuntos
Obesidade/metabolismo , Estresse Oxidativo/fisiologia , Condicionamento Físico Animal/fisiologia , Espermatozoides/metabolismo , Testículo/metabolismo , Testosterona/sangue , Animais , Apoptose/fisiologia , Peso Corporal/fisiologia , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glutationa Peroxidase/metabolismo , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , NF-kappa B/metabolismo , Obesidade/enzimologia , Obesidade/fisiopatologia , Espécies Reativas de Oxigênio/metabolismo , Reprodução/fisiologia , Superóxido Dismutase-1/metabolismo , Natação
13.
J Biol Chem ; 295(10): 3148-3158, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32014991

RESUMO

Cu, Zn superoxide dismutase (SOD1) is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Although the precise mechanisms of SOD1 mutant (SOD1mut)-induced motoneuron toxicity are still unclear, defects in SOD1 proteostasis are known to have a critical role in ALS pathogenesis. We previously reported that the SOD1mut adopts a conformation that exposes a Derlin-1-binding region (DBR) and that DBR-exposed SOD1 interacts with Derlin-1, leading to motoneuron death. We also found that an environmental change, i.e. zinc depletion, induces a conformational change in WT SOD1 (SOD1WT) to the DBR-exposed conformation, suggesting the presence of an equilibrium state between the DBR-masked and DBR-exposed states even with SOD1WT Here, we conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1WT conformational change, and we used a genome-wide siRNA screen to search for regulators of SOD1 proteostasis. This screen yielded 30 candidate genes that maintained an absence of the DBR-exposed SOD1WT conformation. Among these genes was one encoding DDB1- and CUL4-associated factor 4 (DCAF4), a substrate receptor of the E3 ubiquitin-protein ligase complex. Of note, we found that DCAF4 mediates the ubiquitination of an ALS-associated protein and autophagy receptor, optineurin (OPTN), and facilitates autophagic degradation of DBR-exposed SOD1. In summary, our screen identifies DCAF4 as being required for proper proteostasis of DBR-exposed SOD1, which may have potential relevance for the development of therapies for managing ALS.


Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Superóxido Dismutase-1/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Proteostase/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Superóxido Dismutase-1/genética , Ubiquitinação , Wortmanina/farmacologia
14.
Eur J Histochem ; 64(1)2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31988533

RESUMO

Emerging evidence suggests that red blood cells (RBCs) are involved in many functions essential for life. Nuclear factor-kB (NF-kB), nitric oxide synthases (inducible nitric oxide synthase -iNOS-, endothelial nitric oxide synthase -eNOS-) and interleukin-1ß (-IL-1ß-) are all proteins that have been identified in RBCs. In nucleated cells, such as white blood cells (WBCs), these proteins have well investigated roles, linked to stress and inflammation. It is not the same in erythrocytes, for this reason, we considered obese patients for studying the morphology of RBCs. We studied a possible correlation between their morphological changes and several protein expressions. Moreover, we compared the results about the aforementioned proteins and antioxidant markers with those obtained in WBCs from healthy and obese patients before and after omega-3 polyunsaturated fatty acid supplementation. This latter scientific point is important in order to determine whether there are differences in the expression of nucleated and anucleated cells. The morphology of RBCs changed in obese patients, but it is significantly restored after six weeks of supplementation. The expression of antioxidant enzymes changed in RBCs and WBCs in obesity but all proteins restore their positivity after supplementation. We found that: the presence of NF-kB, antioxidant enzymes and eNOS in healthy RBCs could indicate a role of these proteins as regulators of cellular metabolism; obese WBCs showed a higher NF-kB, iNOS and IL-1ß positivity, whereas eNOS presence did not significantly change in these cells. We tried to explain the different positivity of NF-kB, proposing a dual role for this protein, as prolifespan and as proinflammatory processes, depending on examined cells. In conclusion, we have considered the literature that focuses on the omega-6/omega-3 ratio. The ratio changed from the past, especially in people whose diet is strongly westernized worsening the state of health of the patient and leading to an higher incidence of obesity. Our study hypothesizes that the supplementation could help to restore the correct ratio.


Assuntos
Eritrócitos/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Obesidade/fisiopatologia , Adulto , Catalase/metabolismo , Eritrócitos/patologia , Ácidos Graxos Ômega-3/farmacologia , Feminino , Humanos , Inflamação/fisiopatologia , Interleucina-1beta/metabolismo , Pessoa de Meia-Idade , Obesidade/patologia , Superóxido Dismutase-1/metabolismo
15.
PLoS One ; 15(1): e0227655, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31999698

RESUMO

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis cause the protein to aggregate via a prion-like process in which soluble molecules are recruited to aggregates by conformational templating. These misfolded SOD1 proteins can propagate aggregation-inducing conformations across cellular membranes. Prior studies demonstrated that mutation of a Trp (W) residue at position 32 to Ser (S) suppresses the propagation of misfolded conformations between cells, whereas other studies have shown that mutation of Trp 32 to Phe (F), or Cys 111 to Ser, can act in cis to attenuate aggregation of mutant SOD1. By expressing mutant SOD1 fused with yellow fluorescent protein (YFP), we compared the relative ability of these mutations to modulate the formation of inclusions by ALS-mutant SOD1 (G93A and G85R). Only mutation of Trp 32 to Ser persistently reduced the formation of the amorphous inclusions that form in these cells, consistent with the idea that a Ser at position 32 inhibits templated propagation of aggregation prone conformations. To further test this idea, we produced aggregated fibrils of recombinant SOD1-W32S in vitro and injected them into the spinal cords of newborn mice expressing G85R-SOD1: YFP. The injected mice developed an earlier onset paralysis with a frequency similar to mice injected with WT SOD1 fibrils, generating a strain of misfolded SOD1 that produced highly fibrillar inclusion pathology. These findings suggest that the effect of Trp 32 in modulating the propagation of misfolded SOD1 conformations may be dependent upon the "strain" of the conformer that is propagating.


Assuntos
Príons/química , Príons/genética , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genética , Substituição de Aminoácidos , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Príons/metabolismo , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Superóxido Dismutase-1/metabolismo , Triptofano/química
16.
Sci Rep ; 10(1): 1280, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992746

RESUMO

Increases in axonal sodium currents in peripheral nerves are some of the earliest excitability changes observed in Amyotrophic Lateral Sclerosis (ALS) patients. Nothing is known, however, about axonal sodium channels more proximally, particularly at the action potential initiating region - the axon initial segment (AIS). Immunohistochemistry for Nav1.6 sodium channels was used to investigate parameters of AISs of spinal motoneurones in the G127X SOD1 mouse model of ALS in adult mice at presymptomatic time points (~190 days old). In vivo intracellular recordings from lumbar spinal motoneurones were used to determine the consequences of any AIS changes. AISs of both alpha and gamma motoneurones were found to be significantly shorter (by 6.6% and 11.8% respectively) in G127X mice as well as being wider by 9.8% (alpha motoneurones). Measurements from 20-23 day old mice confirmed that this represented a change during adulthood. Intracellular recordings from motoneurones in presymptomatic adult mice, however, revealed no differences in individual action potentials or the cells ability to initiate repetitive action potentials. To conclude, despite changes in AIS geometry, no evidence was found for reduced excitability within the functional working range of firing frequencies of motoneurones in this model of ALS.


Assuntos
Esclerose Amiotrófica Lateral , Axônios/enzimologia , Neurônios Motores , Mutação de Sentido Incorreto , Superóxido Dismutase-1 , Transmissão Sináptica , Substituição de Aminoácidos , Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/patologia , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Axônios/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Neurônios Motores/enzimologia , Neurônios Motores/patologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
17.
Environ Toxicol ; 35(1): 5-14, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31452338

RESUMO

Superoxide dismutase (SOD) acts as the first line of defense against reactive oxygen species (ROS) within cells. In the present study, we determined two novel CuZnSOD genes (designated as CeCSD1 and CeCSD2) from the toxicity-testing freshwater algae Closterium ehrenbergii and examined their structural features, phylogenetic relationships, and gene expression under exposure to different metals. Putative CeCSD1 (204 aa, 20.6 kDa) and CeCSD2 (155 aa, 15.3 kDa) proteins had conserved CuZnSOD family motifs and metal (Cu, Zn) binding sites, but different N-terminus structures, that is, CeCSD1 has a signal peptide to chloroplasts. Phylogenetic analysis of each protein revealed that C. ehrenbergii was well clustered with other green algae and plants. Real-time PCR results showed that the gene expression obviously increased with heavy metal exposure. In addition, excess copper considerably increased the SOD activity and ROS generation but decreased the photosynthetic efficiency in treated cells. These results suggest that CeCSDs are involved in the antioxidant defense system and can be regarded as potential biomarkers for monitoring metal contaminants in aquatic environments.


Assuntos
Closterium/efeitos dos fármacos , Cobre/toxicidade , Água Doce , Expressão Gênica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Zinco/toxicidade , Antioxidantes/metabolismo , Closterium/enzimologia , Cobre/metabolismo , Estresse Oxidativo/genética , Fotossíntese/efeitos dos fármacos , Fotossíntese/genética , Filogenia , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Zinco/metabolismo
18.
Nat Med ; 26(1): 118-130, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31873312

RESUMO

Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.


Assuntos
Esclerose Amiotrófica Lateral/terapia , Dependovirus/metabolismo , Inativação Gênica , Técnicas de Transferência de Genes , Neurônios Motores/patologia , Degeneração Neural/terapia , Pia-Máter/patologia , Medula Espinal/patologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Atrofia , Progressão da Doença , Potencial Evocado Motor , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação/patologia , Interneurônios/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Desenvolvimento Muscular , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Pia-Máter/fisiopatologia , Primatas , Dobramento de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , Medula Espinal/diagnóstico por imagem , Medula Espinal/fisiopatologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Suínos
19.
Oxid Med Cell Longev ; 2019: 3018584, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827671

RESUMO

The extent of oxidative stress and chronic inflammation are closely related events which coexist in a muscle environment under pathologic conditions. It has been generally accepted that the inflammatory cells, as well as myofibers, are sources of reactive species which are, in turn, able to amplify the activation of proinflammatory pathways. However, the precise mechanism underlining the physiopathologic interplay between ROS generation and inflammatory response has to be fully clarified. Thus, the identification of key molecular players in the interconnected pathogenic network between the two processes might help to design more specific therapeutic approaches for degenerative diseases. Here, we investigated whether elevated circulating levels of the proinflammatory cytokine Interleukin-6 (IL-6) are sufficient to perturb the physiologic redox balance in skeletal muscle, independently of tissue damage and inflammatory response. We observed that the overexpression of circulating IL-6 enhances the generation and accumulation of free radicals in the diaphragm muscle of adult NSE/IL-6 mice, by deregulating redox-associated molecular circuits and impinging the nuclear factor erythroid 2-related factor 2- (Nrf2-) mediated antioxidant response. Our findings are coherent with a model in which uncontrolled levels of IL-6 in the bloodstream can influence the local redox homeostasis, inducing the establishment of prooxidative conditions in skeletal muscle tissue.


Assuntos
Interleucina-6/sangue , Músculo Esquelético/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/patologia , NADPH Oxidase 2/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Sirtuína 1/metabolismo , Superóxido Dismutase-1/metabolismo
20.
Proc Natl Acad Sci U S A ; 116(51): 25991-26000, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31796595

RESUMO

Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and in sod1Δ yeast cells. Using Sod1-KD cells we found that the WT-A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Envelhecimento , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Corpos de Inclusão/metabolismo , Peso Molecular , Proteínas Mutantes/química , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Superóxido Dismutase-1/química
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