RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive motoneuron degeneration, and effective clinical treatments are lacking. In this study, we evaluated whether intranasal delivery of mesenchymal stem cell-derived small extracellular vesicles (sEVs) is a strategy for ALS therapy using SOD1G93A mice. In vivo tracing showed that intranasally-delivered sEVs entered the central nervous system and were extensively taken up by spinal neurons and some microglia. SOD1G93A mice that intranasally received sEV administration showed significant improvements in motor performances and survival time. After sEV administration, pathological changes, including spinal motoneuron death and synaptic denervation, axon demyelination, neuromuscular junction degeneration and electrophysiological defects, and mitochondrial vacuolization were remarkably alleviated. sEV administration attenuated the elevation of proinflammatory cytokines and glial responses. Proteomics and transcriptomics analysis revealed upregulation of the complement and coagulation cascade and NF-ĸB signaling pathway in SOD1G93A mouse spinal cords, which was significantly inhibited by sEV administration. The changes were further confirmed by detecting C1q and NF-ĸB expression using Western blots. In conclusion, intranasal administration of sEVs effectively delays the progression of ALS by inhibiting neuroinflammation and overactivation of the complement and coagulation cascades and NF-ĸB signaling pathway and is a potential option for ALS therapy.
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Esclerosis Amiotrófica Lateral , Vesículas Extracelulares , FN-kappa B , Transducción de Señal , Animales , Masculino , Ratones , Administración Intranasal , Esclerosis Amiotrófica Lateral/metabolismo , Coagulación Sanguínea , Modelos Animales de Enfermedad , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/metabolismo , FN-kappa B/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
The copper chaperone for Sod1 (Ccs) is a metallochaperone that plays a multifaceted role in the maturation of Cu,Zn superoxide dismutase (Sod1). The Ccs mutation R163W was identified in an infant with fatal neurological abnormalities. Based on a comprehensive structural and functional analysis, we developed the first data-driven model for R163W-related pathogenic phenotypes. The work here confirms previous findings that the substitution of arginine with tryptophan at this site, which is located adjacent to a conserved Zn binding site, creates an unstable Zn-deficient protein that loses its ability to efficiently activate Sod1. Intriguingly, R163W Ccs can reduce copper (i.e., Cu(II) â Cu(I)) bound in its Sod1-like domain (D2), and this novel redox event is accompanied by disulfide bond formation. The loss of Zn binding, along with the unusual ability to bind copper in D2, diverts R163W Ccs toward aggregation. The remarkably high affinity of D2 Cu(I) binding converts R163W from a Cu chaperone to a Cu scavenger that accelerates Sod1 deactivation (i.e., an Anti-chaperone). Overall, these findings present a first-of-its-kind molecular mechanism for Ccs dysfunction that leads to pathogenesis in humans.
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Cobre , Chaperonas Moleculares , Superóxido Dismutasa-1 , Humanos , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/química , Cobre/metabolismo , Zinc/metabolismo , Modelos Moleculares , Sustitución de Aminoácidos , Sitios de Unión , Oxidación-ReducciónRESUMEN
Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons in the central nervous system (CNS). Mutations in the metalloenzyme SOD1 are associated with inherited forms of ALS and cause a toxic gain of function thought to be mediated by dimer destabilization and misfolding. SOD1 binds two Cu and two Zn ions in its homodimeric form. We have applied native ambient mass spectrometry imaging to visualize the spatial distributions of intact metal-bound SOD1G93A complexes in SOD1G93A transgenic mouse spinal cord and brain sections and evaluated them against disease pathology. The molecular specificity of our approach reveals that metal-deficient SOD1G93A species are abundant in CNS structures correlating with ALS pathology whereas fully metalated SOD1G93A species are homogenously distributed. Monomer abundance did not correlate with pathology. We also show that the dimer-destabilizing post-translational modification, glutathionylation, has limited influence on the spatial distribution of SOD1 dimers.
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Esclerosis Amiotrófica Lateral , Encéfalo , Espectrometría de Masas , Ratones Transgénicos , Médula Espinal , Superóxido Dismutasa-1 , Animales , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/química , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Ratones , Médula Espinal/metabolismo , Médula Espinal/patología , Espectrometría de Masas/métodos , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Cobre/metabolismo , Zinc/metabolismo , Humanos , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/química , Mutación , Procesamiento Proteico-Postraduccional , Multimerización de Proteína , Modelos Animales de Enfermedad , MasculinoRESUMEN
INTRODUCTION: Muscle health in diabetes mellitus (DM) is often neglected, which leads to muscle wasting. Increased reactive oxygen species in DM could decrease antioxidant enzymes such as superoxide dismutase-1 (SOD-1) and -2 (SOD-2) and inhibit calcineurin (CN) and PGC-1α signalling pathways. Chlorogenic acid (CGA) is known as a potent antioxidant and activators of CN and PGC-1α. This study aimed to determine the effect of CGA on mRNA expressions of SOD-1, SOD-2, CN and PGC-1α in inhibiting the progression of DM to muscle wasting. MATERIALS AND METHODS: This study was conducted at Department of Anatomy, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada starting on July 20th, 2020. A total of 24 male Wistar rats were randomly divided into six groups (four rats per group), i.e., control, DM 1.5 months (DM1.5), and DM 2 months (DM2); and DM groups treated with CGA in three different doses, namely CGA1 (12.5 mg/kg BW), CGA2 (25 mg/kg BW), and CGA3 (50 mg/kg BW). Control group was only injected with normal saline, while diabetic model was induced by intraperitoneal injection of streptozotocin. Blood glucose levels were measured twice (one week after diabetic induction and before termination). The soleus muscle tissue was harvested to analyse the mRNA expressions of SOD-1, SOD- 2, CN and PGC-1α using RT-PCR. In addition, the tissue samples were stained with immunohistochemistry for CN and haematoxylin-eosin (HE) for morphologic analysis under light microscopy. RESULTS: The mRNA expressions of SOD-1 and SOD-2 in the CGA1 group were relatively higher compared to the DM2 groups. The mRNA expression of CN in the CGA1 group was significantly higher compared to the DM2 group (p = 0.008). The mRNA expression of PGC-1α in the CGA1 group was significantly higher compared to the DM2 group (p = 0.025). Immunohistochemical staining showed that CNimmunopositive expression in the CGA1 group was more evident compared to the other groups. Haematoxylin-eosin staining showed that muscle tissue morphology in the CGA1 group was similar to that in the control group. CONCLUSION: Chlorogenic acid at a dose of 12.5 mg/kg BW shows lower blood glucose level, good skeletal muscle tissue morphology and higher mRNA expressions of SOD-1, SOD-2, CN and PGC-1α compared to the DM groups.
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Calcineurina , Ácido Clorogénico , Diabetes Mellitus Experimental , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , ARN Mensajero , Animales , Masculino , Ratas , Calcineurina/metabolismo , Ácido Clorogénico/farmacología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/tratamiento farmacológico , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/tratamiento farmacológico , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Ratas Wistar , ARN Mensajero/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates.
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Solventes , Superóxido Dismutasa-1 , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/genética , Humanos , Solventes/química , Desplegamiento Proteico , Unión Proteica , Pliegue de Proteína , Modelos Moleculares , Gránulos de Estrés/metabolismo , Gránulos de Estrés/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Conformación Proteica , Espectroscopía de Resonancia MagnéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal disease that causes degeneration of motor neurons (MNs) and paralysis. ALS can be caused by mutations in the gene that encodes copper/zinc superoxide dismutase (SOD1). SOD1 is known mostly as a cytosolic antioxidant protein, but SOD1 is also in the nucleus of non-transgenic (tg) and human SOD1 (hSOD1) tg mouse MNs. SOD1's nuclear presence in different cell types and subnuclear compartmentations are unknown, as are the nuclear functions of SOD1. We examined hSOD1 nuclear localization and DNA damage in tg mice expressing mutated and wildtype variants of hSOD1 (hSOD1-G93A and hSOD1-wildtype). We also studied ALS patient-derived induced pluripotent stem (iPS) cells to determine the nuclear presence of SOD1 in undifferentiated and differentiated MNs. In hSOD1-G93A and hSOD1-wildtype tg mice, choline acetyltransferase (ChAT)-positive MNs had nuclear hSOD1, but while hSOD1-wildtype mouse MNs also had nuclear ChAT, hSOD1-G93A mouse MNs showed symptom-related loss of nuclear ChAT. The interneurons had preserved parvalbumin nuclear positivity in hSOD1-G93A mice. hSOD1-G93A was seen less commonly in spinal cord astrocytes and, notably, oligodendrocytes, but as the disease emerged, the oligodendrocytes had increased mutant hSOD1 nuclear presence. Brain and spinal cord subcellular fractionation identified mutant hSOD1 in soluble nuclear extracts of the brain and spinal cord, but mutant hSOD1 was concentrated in the chromatin nuclear extract only in the spinal cord. Nuclear extracts from mutant hSOD1 tg mouse spinal cords had altered protein nitration, footprinting peroxynitrite presence, and the intact nuclear extracts had strongly increased superoxide production as well as the active NADPH oxidase marker, p47phox. The comet assay showed that MNs from hSOD1-G93A mice progressively (6-14 weeks of age) accumulated DNA single-strand breaks. Ablation of the NCF1 gene, encoding p47phox, and pharmacological inhibition of NADPH oxidase with systemic treatment of apocynin (10 mg/kg, ip) extended the mean lifespan of hSOD1-G93A mice by about 25% and mitigated genomic DNA damage progression. In human postmortem CNS, SOD1 was found in the nucleus of neurons and glia; nuclear SOD1 was increased in degenerating neurons in ALS cases and formed inclusions. Human iPS cells had nuclear SOD1 during directed differentiation to MNs, but mutant SOD1-expressing cells failed to establish wildtype MN nuclear SOD1 levels. We conclude that SOD1 has a prominent nuclear presence in the central nervous system, perhaps adopting aberrant contexts to participate in ALS pathobiology.
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Esclerosis Amiotrófica Lateral , Núcleo Celular , Daño del ADN , Células Madre Pluripotentes Inducidas , Neuronas Motoras , Estrés Oxidativo , Superóxido Dismutasa-1 , Animales , Humanos , Ratones , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Células Madre Pluripotentes Inducidas/metabolismo , Ratones Transgénicos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , NADPH Oxidasas/metabolismo , NADPH Oxidasas/genética , Fenotipo , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Understanding the role of iron in ethanol-derived hepatic stress could help elucidate the efficacy of dietary or clinical interventions designed to minimize liver damage from chronic alcohol consumption. We hypothesized that normal levels of iron are involved in ethanol-derived liver damage and reduced dietary iron intake would lower the damage caused by ethanol. We used a pair-fed mouse model utilizing basal Lieber-DeCarli liquid diets for 22 weeks to test this hypothesis. In our mouse model, chronic ethanol exposure led to mild hepatic stress possibly characteristic of early-stage alcoholic liver disease, seen as increases in liver-to-body weight ratios. Dietary iron restriction caused a slight decrease in non-heme iron and ferritin (FeRL) expression while it increased transferrin receptor 1 (TfR1) expression without changing ferroportin 1 (FPN1) expression. It also elevated protein lysine acetylation to a more significant level than in ethanol-fed mice under normal dietary iron conditions. Interestingly, iron restriction led to an additional reduction in nicotinamide adenine dinucleotide (NAD+) and NADH levels. Consistent with this observation, the major mitochondrial NAD+-dependent deacetylase, NAD-dependent deacetylase sirtuin-3 (SIRT3), expression was significantly reduced causing increased protein lysine acetylation in ethanol-fed mice at normal and low-iron conditions. In addition, the detection of superoxide dismutase 1 and 2 levels (SOD1 and SOD2) and oxidative phosphorylation (OXPHOS) complex activities allowed us to evaluate the changes in antioxidant and energy metabolism regulated by ethanol consumption at normal and low-iron conditions. We observed that the ethanol-fed mice had mild liver damage associated with reduced energy and antioxidant metabolism. On the other hand, iron restriction may exacerbate certain activities of ethanol further, such as increased protein lysine acetylation and reduced antioxidant metabolism. This metabolic change may prove a barrier to the effectiveness of dietary reduction of iron intake as a preventative measure in chronic alcohol consumption.
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Antioxidantes , Metabolismo Energético , Etanol , Animales , Ratones , Acetilación/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Antioxidantes/metabolismo , Masculino , Hierro/metabolismo , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa/metabolismo , Lisina/metabolismo , Hígado/metabolismo , Hígado/efectos de los fármacos , Receptores de Transferrina/metabolismo , Sirtuina 3/metabolismo , Sirtuina 3/genética , NAD/metabolismo , Ferritinas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Proteínas de Transporte de Catión/genética , Estrés Oxidativo/efectos de los fármacos , Ratones Endogámicos C57BL , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/patología , Hepatopatías Alcohólicas/etiologíaRESUMEN
As a nutraceutical, bovine lactoferrin (bLf), an iron-binding glycoprotein involved in innate immunity, is gaining elevated attention for its ability to exert pleiotropic functions and to be exceptionally tolerated even at high dosages. Some of bLf's activities, including its anti-inflammatory and antioxidant, are tightly linked to its ability to both chelate iron and enter inside the cell nucleus. Here, we present data about Valpalf®, a new formulation containing bLf, sodium citrate, and sodium bicarbonate at a molar ratio of 10-3. In the present study, Valpalf® exhibits superior iron-binding capacity, resistance to tryptic digestion, and a greater capacity to accumulate into the nucleus over time when compared to the native bLf alone. In agreement, Valpalf® effectively reduces interleukin(IL)-6 levels in lipopolysaccharide-stimulated macrophages and modulates the expression of antioxidant enzymes, such as superoxide dismutase 1 and 2, in phorbol-12-myristate-13-acetate-stimulated monocytes. Of note, this potentiated bioactivity was corroborated in a retrospective study on the treatment of anemia of inflammation in hereditary thrombophilic pregnant and non-pregnant women, demonstrating that Valpalf® improves hematological parameters and reduces serum IL-6 levels to a higher extent than bLf alone.
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Suplementos Dietéticos , Interleucina-6 , Lactoferrina , Superóxido Dismutasa , Lactoferrina/farmacología , Lactoferrina/química , Animales , Bovinos , Humanos , Femenino , Superóxido Dismutasa/metabolismo , Interleucina-6/metabolismo , Antioxidantes/farmacología , Antioxidantes/química , Ratones , Citrato de Sodio/farmacología , Superóxido Dismutasa-1/metabolismo , Bicarbonato de Sodio/farmacología , Bicarbonato de Sodio/química , Embarazo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/química , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Hierro/metabolismo , Lipopolisacáridos , Anemia/tratamiento farmacológicoRESUMEN
Post-translational modifications (PTMs) affecting proteins during or after their synthesis play a crucial role in their localization and function. The modification of these PTMs under pathophysiological conditions, i.e., their appearance, disappearance, or variation in quantity caused by a pathological environment or a mutation, corresponds to post-translational variants (PTVs). These PTVs can be directly or indirectly involved in the pathophysiology of diseases. Here, we present the PTMs and PTVs of four major amyotrophic lateral sclerosis (ALS) proteins, SOD1, TDP-43, FUS, and TBK1. These modifications involve acetylation, phosphorylation, methylation, ubiquitination, SUMOylation, and enzymatic cleavage. We list the PTM positions known to be mutated in ALS patients and discuss the roles of PTVs in the pathophysiological processes of ALS. In-depth knowledge of the PTMs and PTVs of ALS proteins is needed to better understand their role in the disease. We believe it is also crucial for developing new therapies that may be more effective in ALS.
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Esclerosis Amiotrófica Lateral , Procesamiento Proteico-Postraduccional , Proteína FUS de Unión a ARN , Superóxido Dismutasa-1 , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Humanos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Mutación , Animales , Fosforilación , AcetilaciónRESUMEN
A structural alteration in copper/zinc superoxide dismutase (SOD1) is one of the common features caused by amyotrophic lateral sclerosis (ALS)-linked mutations. Although a large number of SOD1 variants have been reported in ALS patients, the detailed structural properties of each variant are not well summarized. We present SoDCoD, a database of superoxide dismutase conformational diversity, collecting our comprehensive biochemical analyses of the structural changes in SOD1 caused by ALS-linked gene mutations and other perturbations. SoDCoD version 1.0 contains information about the properties of 188 types of SOD1 mutants, including structural changes and their binding to Derlin-1, as well as a set of genes contributing to the proteostasis of mutant-like wild-type SOD1. This database provides valuable insights into the diagnosis and treatment of ALS, particularly by targeting conformational alterations in SOD1. Database URL: https://fujisawagroup.github.io/SoDCoDweb/.
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Esclerosis Amiotrófica Lateral , Mutación , Superóxido Dismutasa-1 , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/enzimología , Humanos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/metabolismo , Bases de Datos de Proteínas , Conformación Proteica , Bases de Datos Genéticas , Superóxido Dismutasa/genética , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismoRESUMEN
Context The overproduction of reactive oxygen species (ROS) during in vitro culture of ovarian tissues impairs follicular development and survival. Aims To evaluate the effects of punicalagin on the development and survival of primordial follicles, stromal cell and collagen fibres, as well as on the levels of mRNA for nuclear factor erythroid 2-related factor 2 (NRF2 ), superoxide dismutase 1 (SOD1 ), catalase (CAT ), glutathione peroxidase 1 (GPX1 ) and perirredoxin 6 (PRDX6 ), and activity of antioxidant enzymes in cultured bovine ovarian tissues. Methods Bovine ovarian cortical tissues were cultured for 6days in α-MEM+ alone or with 1.0, 10.0, or 100.0µM punicalagin at 38.5°C with 5% CO2 . Follicle morphology and growth, stromal cell density, and collagen fibres were evaluated by classical histology, while the expression of mRNA was evaluated by real-time PCR. The activity of enzymes was analysed by the Bradford method. Key results Punicalagin improved follicle survival and development, reduced mRNA expression for SOD1 and CAT , but did not influence stromal cells or collagen fibres. Punicalagin (10.0µM) increased the levels of thiol and activity of SOD1, CAT , and GPX1 enzymes. Conclusions Punicalagin (10.0µM) promotes follicle survival and development and activates SOD1, CAT , and GPX1 enzymes in bovine ovarian tissues. Implications Punicalagin improves follicle development and survival in cultured ovarian tissues.
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Catalasa , Glutatión Peroxidasa GPX1 , Glutatión Peroxidasa , Taninos Hidrolizables , Folículo Ovárico , Animales , Femenino , Bovinos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Folículo Ovárico/enzimología , Taninos Hidrolizables/farmacología , Glutatión Peroxidasa/metabolismo , Glutatión Peroxidasa/genética , Catalasa/metabolismo , Catalasa/genética , Ovario/efectos de los fármacos , Ovario/enzimología , Ovario/metabolismo , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/genética , Antioxidantes/farmacología , Antioxidantes/metabolismo , Técnicas de Cultivo de Tejidos , Superóxido Dismutasa/metabolismoRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of upper and lower motor neurons with an unknown etiology. The difficulty of recovering biological material from patients led to employ lymphoblastoid cell lines (LCLs) as a model for ALS because many pathways, typically located in neurons, are also activated in these cells. METHODS: To investigate the expression of coding and long non-coding RNAs in LCLs, a transcriptomic profiling of sporadic ALS (SALS) and mutated patients (FUS, TARDBP, C9ORF72 and SOD1) and matched controls was realized. Thus, differentially expressed genes (DEGs) were investigated among the different subgroups of patients. Peripheral blood mononuclear cells (PBMCs) were isolated and immortalized into LCLs via Epstein-Barr virus infection; RNA was extracted, and RNA-sequencing analysis was performed. RESULTS: Gene expression profiles of LCLs were genetic-background-specific; indeed, only 12 genes were commonly deregulated in all groups. Nonetheless, pathways enriched by DEGs in each group were also compared, and a total of 89 Kyoto Encyclopedia of Genes and Genomes (KEGG) terms were shared among all patients. Eventually, the similarity of affected pathways was also assessed when our data were matched with a transcriptomic profile realized in the PBMCs of the same patients. CONCLUSIONS: We conclude that LCLs are a good model for the study of RNA deregulation in ALS.
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Esclerosis Amiotrófica Lateral , Perfilación de la Expresión Génica , Mutación , Transcriptoma , Humanos , Esclerosis Amiotrófica Lateral/genética , Femenino , Masculino , Persona de Mediana Edad , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Leucocitos Mononucleares/metabolismo , Superóxido Dismutasa-1/genética , Línea Celular , Anciano , Regulación de la Expresión Génica , Proteínas de Unión al ADN , Proteína FUS de Unión a ARNRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder involving motor neuron (MN) loss in the motor cortex, brainstem and spinal cord leading to progressive paralysis and death. Due to the pathogenetic complexity, there are no effective therapies available. In this context the use of mesenchymal stem cells and their vesicular counterpart is an emerging therapeutic strategy to counteract neurodegeneration. The extracellular vesicles derived from adipose stem cells (ASC-EVs) recapitulate and ameliorate the neuroprotective effect of stem cells and, thanks to their small dimensions, makes their use suitable to develop novel therapeutic approaches for neurodegenerative diseases as ALS. Here we investigate a therapeutic regimen of ASC-EVs injection in SOD1(G93A) mice, the most widely used murine model of ALS. Repeated intranasal administrations of high doses of ASC-EVs were able to ameliorate motor performance of injected SOD1(G93A) mice at the early stage of the disease and produce a significant improvement at the end-stage in the lumbar MNs rescue. Moreover, ASC-EVs preserve the structure of neuromuscular junction without counteracting the muscle atrophy. The results indicate that the intranasal ASC-EVs administration acts in central nervous system sites rather than at peripheral level in SOD1(G93A) mice. These considerations allow us to identify future applications of ASC-EVs that involve different targets simultaneously to maximize the clinical and neuropathological outcomes in ALS in vivo models.
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Esclerosis Amiotrófica Lateral , Vesículas Extracelulares , Células Madre Mesenquimatosas , Superóxido Dismutasa-1 , Animales , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/terapia , Esclerosis Amiotrófica Lateral/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Ratones Transgénicos , Modelos Animales de Enfermedad , Tejido Adiposo/metabolismo , Neuronas Motoras/metabolismo , Unión Neuromuscular/metabolismoRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS. METHODS: We used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice. RESULTS: SOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice. CONCLUSIONS: AAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.
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Esclerosis Amiotrófica Lateral , Dependovirus , Modelos Animales de Enfermedad , Terapia Genética , Ratones Transgénicos , Neuronas Motoras , Atrofia Muscular , Animales , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/terapia , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Terapia Genética/métodos , Atrofia Muscular/genética , Atrofia Muscular/terapia , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Dependovirus/genética , Ratones , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Vectores Genéticos/administración & dosificación , Degeneración Nerviosa/genética , Degeneración Nerviosa/terapia , Masculino , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the dysfunction and death of motor neurons through multifactorial mechanisms that remain unclear. ALS has been recognized as a multisystemic disease, and the potential role of skeletal muscle in disease progression has been investigated. Reactive aldehydes formed as secondary lipid peroxidation products in the redox processes react with biomolecules, such as DNA, proteins, and amino acids, resulting in cytotoxic effects. 4-Hydroxy-2-nonenal (HNE) levels are elevated in the spinal cord motor neurons of ALS patients, and HNE-modified proteins have been identified in the spinal cord tissue of an ALS transgenic mice model, suggesting that reactive aldehydes can contribute to motor neuron degeneration in ALS. One biological pathway of aldehyde detoxification involves conjugation with glutathione (GSH) or carnosine (Car). Here, the detection and quantification of Car, GSH, GSSG (glutathione disulfide), and the corresponding adducts with HNE, Car-HNE, and GS-HNE, were performed in muscle and liver tissues of a hSOD1G93A ALS rat model by reverse-phase high-performance liquid chromatography coupled to electrospray ion trap tandem mass spectrometry in the selected reaction monitoring mode. A significant increase in the levels of GS-HNE and Car-HNE was observed in the muscle tissue of the end-stage ALS animals. Therefore, analyzing variations in the levels of these adducts in ALS animal tissue is crucial from a toxicological perspective and can contribute to the development of new therapeutic strategies.
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Aldehídos , Esclerosis Amiotrófica Lateral , Carnosina , Modelos Animales de Enfermedad , Glutatión , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Aldehídos/metabolismo , Aldehídos/química , Carnosina/metabolismo , Glutatión/metabolismo , Ratas , Músculo Esquelético/metabolismo , Humanos , Superóxido Dismutasa/metabolismo , Masculino , Cromatografía Líquida de Alta Presión , Ratas Transgénicas , Superóxido Dismutasa-1/metabolismo , Ratas Sprague-DawleyRESUMEN
OBJECTIVES: Superoxide dismutase (SOD) is an enzyme that plays a crucial role in protecting cells from oxidative damage. Our study aims to address the lack of papers simultaneously analyzing the immunoreactivity of all three distinct isoforms of SOD in human exocrine pancreas cells. BACKGROUND: Superoxide dismutases (SODs) facilitate the conversion of superoxide radicals into less harmful substances. By neutralizing superoxide radicals, SODs help prevent the formation of highly reactive and destructive species that can adversely affect manifold cellular components. METHODS: The study analyzed immunoreactivity of SODs in samples of six healthy adult human pancreases, while using the indirect immunohistochemical method under a light microscope. RESULTS: SOD1 was predominantly found in centroacinar cells and epithelial cells of the duct system while SOD2 was mainly detected in the epithelial cells of interlobular ducts. Both enzymes were prominently present in the basal region of acinar cells near the cell nucleus. The expression of SOD3 was observed to be rare. CONCLUSION: Understanding the intracellular metabolism of SODs in healthy exocrine pancreas cells serves as a basis for determining the precise role of oxidative damage and SOD signaling in the pathogenesis of various pancreatic diseases, including chronic pancreatitis and pancreatic cancer (Fig. 6, Ref. 24). Text in PDF www.elis.sk Keywords: antioxidants, histology, immunohistochemistry, pancreas, superoxide dismutase.
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Páncreas Exocrino , Superóxido Dismutasa , Humanos , Superóxido Dismutasa/metabolismo , Páncreas Exocrino/enzimología , Páncreas Exocrino/metabolismo , Superóxido Dismutasa-1/metabolismo , Inmunohistoquímica , Adulto , Isoenzimas/metabolismo , Masculino , Femenino , Persona de Mediana EdadRESUMEN
In mitochondria, the detoxification of molar excess H2S as polysulfide proceeded via an oxidation process promoted by Cu/Zn containing superoxide dismutase (SOD1) enzyme, which has been very recently reported as the alternative enzyme for cytosolic H2S oxidation. Herein, we present Ni(II) complexes bearing the terminal SH group as a synthetic functional analogue for the sulfide oxidase function of SOD1. Synthesis, crystal structure and complete spectroscopic characterization of two sets of complexes, [NiLOMe/tBu(PPh3)] (2OMe/tBu) and tetraethyl salt of [NiLOMe/tBu(SH)]-1 (3OMe/tBu), were described (LOMe = (E)-2-methoxy-6-(((2-sulfidophenyl)imino)methyl)phenolate and LtBu = (E)-2,4-di-tert-butyl-6-(((2-sulfidophenyl)imino)methyl)phenolate). Under anaerobic conditions, 3OMe/tBu responded to a catalytic sulfur atom transfer (SAT) reaction with PPh3 to produce SPPh3. The SAT reaction was analyzed using detailed studies of 1H and 31P NMR spectra. Finally, the SAT reactivity pattern was compared with the same in the native enzyme of SOD1.
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Complejos de Coordinación , Níquel , Azufre , Níquel/química , Níquel/metabolismo , Complejos de Coordinación/química , Complejos de Coordinación/metabolismo , Complejos de Coordinación/síntesis química , Azufre/química , Azufre/metabolismo , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/metabolismo , Modelos Moleculares , Catálisis , Anaerobiosis , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/química , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismoRESUMEN
PURPOSE: While reactive oxygen species (ROS) have been identified as key redox signaling agents contributing to aging process, which and how specific oxidants trigger healthy longevity remain unclear. This paper aimed to explore the precise role and signaling mechanism of superoxide (O2â¢-) in health and longevity. METHODS: A tool for precise regulation of O2â¢- levels in vivo was developed based on the inhibition of superoxide dismutase 1 (SOD1) by tetrathiomolybdate (TM) in Caenorhabditis elegans (C. elegans). Then, we examined the effects of TM on lifespan, reproduction, lipofuscin accumulation, mobility, and stress resistance. Finally, the signaling mechanism for longevity induced by TM-O2â¢- was screened by transcriptome analysis and tested in sod-1 and argk-1 RNAi strains, sod-2, sod-3, and daf-16 mutants. RESULTS: TM promoted longevity in C. elegans with a concomitant extension of healthy lifespan as indicated by increasing fertility and mobility and reducing lipofuscin accumulation, as well as enhanced resistance to different abiotic stresses. Mechanically, TM could precisely regulate O2â¢- levels in nematodes via modulating SOD1 activity. An O2â¢- scavenger Mn(III)TBAP abolished TM-induced lifespan extension, while an O2â¢- generator paraquat at low concentration mimicked the life prolongation effects. The longevity in TM-treated worms was abolished by sod-1 RNAi but was not affected in sod-2 or sod-3 mutants. Further transcriptome analysis revealed arginine kinase ARGK-1 and its downstream insulin/insulin-like growth factor 1 signaling (IIS) as potential effectors for TM-O2â¢â¾-induced longevity, and argk-1 RNAi or daf-16 mutant nullified the longevity. CONCLUSIONS: These findings indicate that it is feasible to precisely control specific oxidant in vivo and O2â¢- orchestrates TM-induced health and longevity in C. elegans via ARGK-1-IIS axis.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Longevidad , Molibdeno , Transducción de Señal , Superóxido Dismutasa , Superóxidos , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de los fármacos , Longevidad/efectos de los fármacos , Longevidad/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/genética , Molibdeno/farmacología , Molibdeno/metabolismo , Superóxidos/metabolismo , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/genética , Estrés Oxidativo/efectos de los fármacos , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Especies Reactivas de Oxígeno/metabolismo , Interferencia de ARNRESUMEN
Hepatitis B Virus (HBV) infection significantly elevates the risk of hepatocellular carcinoma (HCC), with the HBV X protein (HBx) playing a crucial role in cancer progression. Sorafenib, the primary therapy for advanced HCC, shows limited effectiveness in HBV-infected patients due to HBx-related resistance. Numerous studies have explored combination therapies to overcome this resistance. Sodium diethyldithiocarbamate (DDC), known for its anticancer effects and its inhibition of superoxide dismutase 1 (SOD1), is hypothesized to counteract sorafenib (SF) resistance in HBV-positive HCCs. Our research demonstrates that combining DDC with SF significantly reduces HBx and SOD1 expressions in HBV-positive HCC cells and human tissues. This combination therapy disrupts the PI3K/Akt/mTOR signalling pathway and promotes apoptosis by increasing reactive oxygen species (ROS) levels. These cellular changes lead to reduced tumour viability and enhanced sensitivity to SF, as evidenced by the synergistic suppression of tumour growth in xenograft models. Additionally, DDC-mediated suppression of SOD1 further enhances SF sensitivity in HBV-positive HCC cells and xenografted animals, thereby inhibiting cancer progression more effectively. These findings suggest that the DDC-SF combination could serve as a promising strategy for overcoming SF resistance in HBV-related HCC, potentially optimizing therapy outcomes.