RESUMEN
Alzheimer's disease (AD) research started several decades ago and despite the many efforts employed to develop new treatments or approaches to slow and/or revert disease progression, AD treatment remains an unsolved issue. Knowing that mitochondria loss of function is a central hub for many AD-associated pathophysiological processes, there has been renewed interest in exploring mitochondria as targets for intervention. In this perspective, the present study was aimed to investigate the possible beneficial effects of 2,4 dinitrophenol (DNP), a mitochondrial uncoupler agent, in an in vitro model of AD. Retinoic acid-induced differentiated SH-SY5Y cells were incubated with okadaic acid (OA), a neurotoxin often used as an AD experimental model, and/or with DNP. OA caused a decrease in neuronal cells viability, induced multiple mitochondrial anomalies including increased levels of reactive oxygen species, decreased bioenergetics and mitochondria content markers, and an altered mitochondria morphology. OA-treated cells also presented increased lipid peroxidation levels, and overactivation of tau related kinases (GSK3ß, ERK1/2 and AMPK) alongside with a significant augment in tau protein phosphorylation levels. Interestingly, DNP co-treatment ameliorated and rescued OA-induced detrimental effects not only on mitochondria but also but also reinstated signaling pathways homeostasis and ameliorated tau pathology. Overall, our results show for the first time that DNP has the potential to preserve mitochondria homeostasis under a toxic insult, like OA exposure, as well as to reestablish cellular signaling homeostasis. These observations foster the idea that DNP, as a mitochondrial modulator, might represent a new avenue for treatment of AD.
Asunto(s)
2,4-Dinitrofenol , Enfermedad de Alzheimer , Mitocondrias , Ácido Ocadaico , Especies Reactivas de Oxígeno , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Ácido Ocadaico/farmacología , Ácido Ocadaico/toxicidad , Humanos , 2,4-Dinitrofenol/farmacología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Proteínas tau/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Peroxidación de Lípido/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Tretinoina/farmacologíaRESUMEN
Halomonas pacifica CARE-V15 was isolated from the southeastern coast of India to determine its genome sequence. Secondary metabolite gene clusters were identified using an anti-SMASH server. The concentrated crude ethyl acetate extract was evaluated by GC-MS. The bioactive compound from the crude ethyl acetate extract was fractionated by gel column chromatography. HPLC was used to purify the 3,6-diisobutyl-2,5-piperazinedione (DIP), and the structure was determined using FTIR and NMR spectroscopy. Purified DIP was used in an in silico molecular docking analysis. Purified DIP exhibits a stronger affinity for antioxidant genes like glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GSR). Using in silco molecular docking analysis, the protein-ligand binding affinities of GSR (-4.70 kcal/mol), GST (-5.27 kcal/mol), and GPx (-5.37 kcal/mol) were measured. The expression of antioxidant genes were investigated by qRT-PCR. The in vivo reactive oxygen species production, lipid peroxidation, and cell death levels were significantly (p ≤ 0.05) increased in OA-induced group, but all these levels were significantly (p ≤ 0.05) decreased in the purified DIP pretreated group. Purified DIP from halophilic bacteria could thus be a useful treatment for neurological disorders associated with oxidative stress.
Asunto(s)
Acetatos , Antioxidantes , Halomonas , Fármacos Neuroprotectores , Animales , Antioxidantes/farmacología , Antioxidantes/metabolismo , Pez Cebra/metabolismo , Fármacos Neuroprotectores/farmacología , Ácido Ocadaico/metabolismo , Ácido Ocadaico/farmacología , Simulación del Acoplamiento Molecular , Estrés Oxidativo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Dicetopiperazinas/metabolismo , Dicetopiperazinas/farmacología , Glutatión Transferasa/metabolismoRESUMEN
Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6â nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3ß-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Proteínas tau , Humanos , Enfermedad de Alzheimer/metabolismo , Neuronas/metabolismo , Ácido Ocadaico/metabolismo , Ácido Ocadaico/farmacología , Ácido Ocadaico/uso terapéutico , Fosforilación , Proteínas tau/antagonistas & inhibidores , Proteínas tau/metabolismo , Tauopatías/tratamiento farmacológico , Tauopatías/metabolismo , Tauopatías/patología , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacologíaRESUMEN
After a recent total synthesis had resolved all issues surrounding the constitution and stereostructure of prorocentin, it was possible to devise a new approach aiming at an improved supply of this scarce marine natural product; this compound is a cometabolite of the prototypical phosphatase inhibitor okadaic acid but still awaits detailed biological profiling. The revised entry starts from 2-deoxy-d-glucose; keys to success were a telescoped hemiacetal reduction/acetal cleavage and an exquisitely selective gold/Brønsted acid-cocatalyzed spiroacetalization.
Asunto(s)
Inhibidores Enzimáticos , Furanos , Ácido Ocadaico/química , Ácido Ocadaico/farmacología , Inhibidores Enzimáticos/química , Acetales/químicaRESUMEN
Since Alzheimer's disease (AD) is a complex and multifactorial neuropathology, the discovery of multi-targeted inhibitors has gradually demonstrated greater therapeutic potential. Neurofibrillary tangles (NFTs), the main neuropathologic hallmarks of AD, are mainly associated with hyperphosphorylation of the microtubule-associated protein Tau. The overexpression of GSK3ß and DYRK1A has been recognized as an important contributor to hyperphosphorylation of Tau, leading to the strategy of using dual-targets inhibitors for the treatment of this disorder. ZDWX-12 and ZDWX-25, as harmine derivatives, were found good inhibition on dual targets in our previous study. Here, we firstly evaluated the inhibition effect of Tau hyperphosphorylation using two compounds by HEK293-Tau P301L cell-based model and okadaic acid (OKA)-induced mouse model. We found that ZDWX-25 was more effective than ZDWX-12. Then, based on comprehensively investigations on ZDWX-25 in vitro and in vivo, 1) the capability of ZDWX-25 to show a reduction in phosphorylation of multiple Tau epitopes in OKA-induced neurodegeneration cell models, and 2) the effect of reduction on NFTs by 3xTg-AD mouse model under administration of ZDWX-25, an orally bioavailable, brain-penetrant dual-targets inhibitor with low toxicity. Our data highlight that ZDWX-25 is a promising drug for treating AD.
Asunto(s)
Enfermedad de Alzheimer , Ratones , Animales , Humanos , Enfermedad de Alzheimer/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Proteínas tau/metabolismo , Fosforilación , Ácido Ocadaico/metabolismo , Ácido Ocadaico/farmacología , Ácido Ocadaico/uso terapéutico , Modelos Animales de EnfermedadRESUMEN
Protein phosphatase 2A (PP2A) is an important Ser/Thr phosphatase that participates in the regulation of multiple cellular processes. This implies that any deficient activity of PP2A is the responsible of severe pathologies. For instance, one of the main histopathological features of Alzheimer's disease is neurofibrillary tangles, which are mainly comprised by hyperphosphorylated forms of tau protein. This altered rate of tau phosphorylation has been correlated with PP2A depression AD patients. With the goal of preventing PP2A inactivation in neurodegeneration scenarios, we have aimed to design, synthesize and evaluate new ligands of PP2A capable of preventing its inhibition. To achieve this goal, the new PP2A ligands present structural similarities with the central fragment C19-C27 of the well-established PP2A inhibitor okadaic acid (OA). Indeed, this central moiety of OA does not exert inhibitory actions. Hence, these compounds lack PP2A-inhibiting structural motifs but, in contrast, compete with PP2A inhibitors, thus recovering phosphatase activity. Proving this hypothesis, most compounds showed a good neuroprotective profile in neurodegeneration models related to PP2A impairment, highlighting derivative 10, named ITH12711, as the most promising one. This compound (1) restored in vitro and cellular PP2A catalytic activity, measured on a phospho-peptide substrate and by western-blot analyses, (2) proved good brain penetration measured by PAMPA, and (3) prevented LPS-induced memory impairment of mice in the object recognition test. Thus, the promising outcomes of the compound 10 validate our rational approach to design new PP2A-activating drugs based on OA central fragment.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Ácido Ocadaico/farmacología , Ácido Ocadaico/metabolismo , Neuroprotección , Tauopatías/tratamiento farmacológico , Tauopatías/metabolismo , Proteínas tau/metabolismo , Proteína Fosfatasa 2/metabolismo , FosforilaciónRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory loss and cognitive decline, with hallmark pathologies related to amyloid beta (Aß) and TAU. Natural phytochemicals show promise for drug discovery to fill the current therapeutic innovation gap in AD. This study investigated the effect of cucurbitacin E (CuE), one of the bioactive components of Ecballium elaterium, on TAU fibril formation in okadaic acid-induced AD in rats. In a randomized design, we assigned 30 female Sprague Dawley rats to one of five experimental groups: (1) control, (2) stereotaxic surgery, (3) stereotaxic surgery + artificial cerebrospinal fluid, (4) stereotaxic surgery + okadaic acid (AD model), and (5) stereotaxic surgery + okadaic acid + CuE treatment. For experimental groups 4 and 5, rats were administered OKA-ICV (200 ng/kg) followed by CuE (4 mg/[kg·day], intraperitoneally) for 20 days. Expression of the MAPK1/3 and MAPK14 genes associated with TAU metabolism, hippocampal protein levels of these genes, cognitive functions of the rats, and histological accumulation of TAU in the brain were evaluated. Our findings in this preclinical model collectively suggest that phytochemical CuE contributes to memory gain by reducing TAU protein accumulation, which warrants further evaluation in future in vitro and in vivo studies.
Asunto(s)
Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Ratas , Femenino , Animales , Proteínas tau/metabolismo , Ácido Ocadaico/farmacología , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Ratas Sprague-Dawley , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Enfermedades Neurodegenerativas/metabolismo , Encéfalo/metabolismo , Modelos Animales de EnfermedadRESUMEN
Post-translational modification (PTM) is crucial for many biological events, such as the modulation of bone metabolism. Phosphorylation and O-GlcNAcylation are two examples of PTMs that can occur at the same site in the protein: serine and threonine residues. This phenomenon may cause crosstalk and possible interactions between the molecules involved. Protein phosphatase 2 A (PP2A) is widely expressed throughout the body and plays a major role in dephosphorylation. At the same location where PP2A acts, O-GlcNAc transferase (OGT) can introduce uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) molecules and mediates O-GlcNAc modifications. To examine the effects of PP2A inhibition on OGT localization and expression, osteoblastic MC3T3-E1 cells were treated with Okadaic Acid (OA), a potent PP2A inhibitor. In the control cells, OGT was strictly localized in the nucleus. However, OGT was observed diffusely in the cytoplasm of the OA-treated cells. This change in localization from the nucleus to the cytoplasm resulted from an increase in mitochondrial OGT expression and translocation of the nucleocytoplasmic isoform. Furthermore, knockdown of PP2A catalytic subunit α isoform (PP2A Cα) significantly affected OGT expression (p < 0.05), and there was a correlation between PP2A Cα and OGT expression (r = 0.93). These results suggested a possible interaction between PP2A and OGT, which strengthens the notion of an interaction between phosphorylation and O-GlcNAcylation.
Asunto(s)
Proteína Fosfatasa 2 , Procesamiento Proteico-Postraduccional , Proteína Fosfatasa 2/metabolismo , Ácido Ocadaico/farmacología , N-Acetilglucosaminiltransferasas/metabolismo , Isoformas de Proteínas/metabolismo , Acetilglucosamina/metabolismoRESUMEN
The search for strategies for strengthening the synaptic efficiency in Aß25-35-treated slices is a challenge for the compensation of amyloidosis-related pathologies. Here, we used the recording of field excitatory postsynaptic potentials (fEPSPs), nitric oxide (NO) imaging, measurements of serine/threonine protein phosphatase (STPP) activity, and the detection of the functional mitochondrial parameters in suspension of brain mitochondria to study the Aß25-35-associated signaling in the hippocampus. Aß25-35 aggregates shifted the kinase-phosphatase balance during the long-term potentiation (LTP) induction in the enhancement of STPP activity. The PP1/PP2A inhibitor, okadaic acid, but not the PP2B blocker, cyclosporin A, prevented Aß25-35-dependent LTP suppression for both simultaneous and delayed enzyme blockade protocols. STPP activity in the Aß25-35-treated slices was upregulated, which is reverted relative to the control values in the presence of PP1/PP2A but not in the presence of the PP2B blocker. A selective inhibitor of stress-induced PP1α, sephin1, but not of the PP2A blocker, cantharidin, is crucial for Aß25-35-mediated LTP suppression prevention. A mitochondrial Na+/Ca2+ exchanger (mNCX) blocker, CGP37157, also attenuated the Aß25-35-induced LTP decline. Aß25-35 aggregates did not change the mitochondrial transmembrane potential or reactive oxygen species (ROS) production but affected the ion transport and Ca2+-dependent swelling of organelles. The staining of hippocampal slices with NO-sensitive fluorescence dye, DAF-FM, showed stimulation of the NO production in the Aß25-35-pretreated slices at the dendrite-containing regions of CA1 and CA3, in the dentate gyrus (DG), and in the CA1/DG somata. NO scavenger, PTIO, or nNOS blockade by selective inhibitor 3Br-7NI partly restored the Aß25-35-induced LTP decline. Thus, hippocampal NO production could be another marker for the impairment of synaptic plasticity in amyloidosis-related states, and kinase-phosphatase balance management could be a promising strategy for the compensation of Aß25-35-driven deteriorations.
Asunto(s)
Amiloidosis , Potenciación a Largo Plazo , Proteínas Amiloidogénicas , Cantaridina , Ciclosporina , Hipocampo/fisiología , Humanos , Potenciación a Largo Plazo/fisiología , Mitocondrias , Óxido Nítrico , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas , Especies Reactivas de Oxígeno , Serina , Intercambiador de Sodio-Calcio , TreoninaRESUMEN
The pathological characteristics of Alzheimer's disease (AD) include formation of senile plaques resulting from amyloid-ß (Aß) deposition and neurofibrillary tangles caused by tau hyperphosphorylation. Reducing tau hyperphosphorylation is crucial for treatment of AD. Network pharmacology analysis showed that CTS may reduce tau hyperphosphorylation by regulating the phosphatidylinositol 3 kinases/protein kinase B/ glycogen synthase kinase-3ß (PI3K/Akt/GSK3ß) pathway. We investigated the ability of cryptotanshinone (CTS) to reduce Aß-induced tau hyperphosphorylation and characterized the underlying mechanisms. Amyloid-ß42 oligomers (AßO) were used to establish an AD model in HT22 cells. The expression levels of tau and related proteins in PI3K/Akt/GSK3ß pathway were measured using western blot and immunofluorescence staining. The above-mentioned proteins were then evaluated in an okadaic acid (OKA)-induced AD cell model to verify the results. Synapse-associated proteins including post-synaptic density protein-95 (PSD95) and synaptophysin were also evaluated. We found that CTS significantly reduced tau hyperphosphorylation at Ser202, Ser404, Thr181, and Thr231 in AßO- and OKA-induced cell models. Moreover, we also found that CTS reversed AßO-induced reductions in the levels of PSD95 and synaptophysin. We used LY294002 to block PI3K and the results showed that CTS exerted neuroprotective effects through regulation of the PI3K/Akt/GSK3ß signaling pathway. In summary, we showed for the first time that CTS inhibited AD-related tau hyperphosphorylation and reduced the effects of AßO on the expression levels of PSD95 and synaptophysin via the PI3K/Akt/GSK3ß pathway in HT22 cells.
Asunto(s)
Enfermedad de Alzheimer , Fosfatidilinositol 3-Quinasas , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Ácido Ocadaico/farmacología , Fenantrenos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sinaptofisina/metabolismo , Proteínas tau/metabolismoRESUMEN
This paper examined the toxins naturally produced by marine dinoflagellates and their effects on increases in ß-amyloid plaques along with tau protein hyperphosphorylation, both major drivers of Alzheimer's disease (AD). This approach is in line with the demand for certain natural compounds, namely those produced by marine invertebrates that have the potential to be used in the treatment of AD. Current advances in AD treatment are discussed as well as the main factors that potentially affect the puzzling global AD pattern. This study focused on yessotoxins (YTXs), gymnodimine (GYM), spirolides (SPXs), and gambierol, all toxins that have been shown to reduce ß-amyloid plaques and tau hyperphosphorylation, thus preventing the neuronal or synaptic dysfunction that ultimately causes the cell death associated with AD (or other neurodegenerative diseases). Another group of toxins described, okadaic acid (OA) and its derivatives, inhibit protein phosphatase activity, which facilitates the presence of phosphorylated tau proteins. A few studies have used OA to trigger AD in zebrafish, providing an opportunity to test in vivo the effectiveness of new drugs in treating or attenuating AD. Constraints on the production of marine toxins for use in these tests have been considered. Different lines of research are anticipated regarding the action of the two groups of toxins.
Asunto(s)
Enfermedad de Alzheimer , Dinoflagelados , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides , Animales , Dinoflagelados/metabolismo , Toxinas Marinas/farmacología , Ácido Ocadaico/farmacología , Placa Amiloide , Pez Cebra/metabolismo , Proteínas tau/metabolismoRESUMEN
The actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR) in several mammalian species. Here, we demonstrate that in mouse sperm, within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the occurrence of AR. Moreover, we show that the transient phosphorylation of COFILIN is also influenced by the Slingshot family of protein phosphatases (SSH1). The activity of SSH1 is regulated by the dual action of two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by okadaic acid-sensitive phosphatases. This regulatory mechanism is independent of the commonly observed activating mechanisms involving PP2B and emerges as a new finely tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or okadaic acid did not altered actin polymerization and the AR. Altogether, our results highlight the role of small GTPases in modulating actin dynamics required for AR.
Asunto(s)
Factores Despolimerizantes de la Actina , Capacitación Espermática , Factores Despolimerizantes de la Actina/metabolismo , Actinas/metabolismo , Animales , Cofilina 1/metabolismo , Exocitosis , Masculino , Mamíferos/metabolismo , Ratones , Ácido Ocadaico/metabolismo , Ácido Ocadaico/farmacología , Fosforilación , Semen/metabolismoRESUMEN
This study was performed to investigate whether okadaic acid (200 ng/kg) produces a protective effect in experimental liver injury induced by intragastric administration of 1000, 2000, and 4000 mg/kg amoxicillin in Sprague-Dawley rats (n=50). The control group was given carboxymethylcellulose sodium solution. Liver injury was assessed by the relative organ weight index, serum ALT activity, and liver malondialdehyde content. Histopathological examination of the liver from rats treated with amoxicillin revealed cell swelling, congestion, apoptosis, and necrosis. The relative liver weight index, ALT activity, and liver malondialdehyde content in these animals were higher than in the control. Administration of okadaic acid reduced the degree of amoxicillin-induced damage to hepatocytes. Thus, okadaic acid can alleviate liver injury caused by amoxicillin.
Asunto(s)
Amoxicilina , Enfermedad Hepática Inducida por Sustancias y Drogas , Alanina Transaminasa , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Hígado , Malondialdehído , Ácido Ocadaico/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Chronic myeloid leukemia (CML) is a cancer type of the white blood cells and because of BCR-ABL translocation it results in increased tyrosine kinase activity. For this purpose, dasatinib is the second-generation tyrosine kinase inhibitor that is used for inhibition of BCR-ABL. Effectively and safetly, dasatinib has been used for imatinib-intolerant/resistant CML patients. Protein phosphatase 2A (PP2A) is the major serine/threonine phosphatase ensuring cellular homeostasis in cells and is associated with many cancer types including leukemias. In this study, we aimed to investigate the effects of dasatinib and okadaic acid (OA), either alone or in combination, on apoptosis and cell cycle arrest and dasatinib effect on enzyme activity and protein-level changes of PP2A in K562 cell line. The cytotoxic effects of dasatinib were evaluated by WST-1 analysis. Apoptosis was determined by Annexin V and Apo-Direct assays by flow cytometry. Cell cycle arrest analysis was performed for the investigation of the cytostatic effect. We also used OA as a PP2A inhibitor to assess apoptosis and cell cycle arrest changes in case of reducing the level of PP2A. PP2A enyzme activity and protein levels of PP2A were examined by serine/threonine phosphatase assay and Western blot analysis, respectively. Apoptosis was increased with dasatinib and OA combination. Cell cycle arrest was determined especially after OA treatment. The enzyme activity was decreased depending on time after dasatinib application. PP2A regulatory and catalytic subunit protein levels were decreased compared to control. Targeting the PP2A by dasatinib and OA has potential for CML treatment.
Asunto(s)
Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Dasatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Ácido Ocadaico/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismoRESUMEN
Regulation of mitochondrial morphology and motility is critical for neurons, but the exact mechanisms are unclear. Here, we demonstrate that these mechanisms may involve collapsin response mediator protein 2 (CRMP2). CRMP2 is attached to neuronal mitochondria and binds to dynamin-related protein 1 (Drp1), Miro 2, and Kinesin 1 light chain (KLC1). Treating neurons with okadaic acid (OA), an inhibitor of phosphatases PP1 and PP2A, resulted in increased CRMP2 phosphorylation at Thr509/514, Ser522, and Thr555, and augmented Drp1 phosphorylation at Ser616. The CRMP2-binding small molecule (S)-lacosamide ((S)-LCM) prevented an OA-induced increase in CRMP2 phosphorylation at Thr509/514 and Ser522 but not at Thr555, and also failed to alleviate Drp1 phosphorylation. The increased CRMP2 phosphorylation correlated with decreased CRMP2 binding to Drp1, Miro 2, and KLC1. (S)-LCM rescued CRMP2 binding to Drp1 and Miro 2 but not to KLC1. In parallel with CRMP2 hyperphosphorylation, OA increased mitochondrial fission and suppressed mitochondrial traffic. (S)-LCM prevented OA-induced alterations in mitochondrial morphology and motility. Deletion of CRMP2 with a small interfering RNA (siRNA) resulted in increased mitochondrial fission and diminished mitochondrial traffic. Overall, our data suggest that the CRMP2 expression level and phosphorylation state are involved in regulating mitochondrial morphology and motility in neurons.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Animales , Células Cultivadas , Cuerpo Estriado/citología , Eliminación de Gen , Cinesinas , Ratones , Dinámicas Mitocondriales/efectos de los fármacos , Neuronas/efectos de los fármacos , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacosRESUMEN
The mussel Mytilus galloprovincialis is one of the most important aquaculture species in Europe. Its main production problem is the accumulation of toxins during coastal blooms, which prevents mussel commercialization. P-glycoprotein (ABCB1/MDR1/P-gp) is part of the multixenobiotic resistance system in aquatic organisms, and okadaic acid, the main DSP toxin, is probably a substrate of the P-gp-mediated efflux. In this study, the presence and possible role of P-gp in the okadaic acid detoxification process was studied in M. galloprovincialis. We identified, cloned, and characterized two complete cDNAs of mdr1 and mdr2 genes. MgMDR1 and MgMDR2 predicted proteins had the structure organization of ABCB full transporters, and were identified as P-gp/MDR/ABCB proteins. Furthermore, the expression of mdr genes was monitored in gills, digestive gland, and mantle during a cycle of accumulation-elimination of okadaic acid. Mdr1 significantly increased its expression in the digestive gland and gills, supporting the idea of an important role of the MDR1 protein in okadaic acid efflux out of cells in these tissues. The expression of M. galloprovincialismrp2, a multidrug associated protein (MRP/ABCC), was also monitored. As in the case of mdr1, there was a significant induction in the expression of mrp2 in the digestive gland, as the content of okadaic acid increased. Thus, P-gp and MRP might constitute a functional defense network against xenobiotics, and might be involved in the resistance mechanisms to DSP toxins.
Asunto(s)
Dinoflagelados/química , Resistencia a Medicamentos/genética , Expresión Génica , Mytilus/efectos de los fármacos , Ácido Ocadaico/farmacología , Xenobióticos/farmacología , Animales , Perfilación de la Expresión Génica , Mytilus/genéticaRESUMEN
Alterations in prolyl oligopeptidase (PREP) activity have been connected, for example, with bipolar and major depressive disorder, and several studies have reported that lack or inhibition of PREP blocks the effects of lithium on inositol 1,4,5-triphosphate (IP3 ) levels. However, the impact of PREP modulation on other intracellular targets of lithium, such as glycogen synthase kinase 3 beta (GSK3b) or protein kinase B (Akt), has not been studied. We recently found that PREP regulates protein phosphatase 2A (PP2A), and because GSK3b and Akt are PP2A substrates, we studied if PREP-related lithium insensitivity is dependent on PP2A. To assess this, HEK-293 and SH-SY5Y cells with PREP deletion or PREP inhibition (KYP-2047) were exposed to lithium, and thereafter, the phosphorylation levels of GSK3b and Akt were measured by Western blot. As expected, PREP deletion and inhibition blocked the lithium-induced phosphorylation on GSK3b and Akt in both cell lines. When lithium exposure was combined with okadaic acid, a PP2A inhibitor, KYP-2047 did not have effect on lithium-induced GSK3b and Akt phosphorylation. Therefore, we conclude that PREP deletion or inhibition blocks the intracellular effects of lithium on GSK3b and Akt via PP2A activation.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/metabolismo , Fosforilación/efectos de los fármacos , Prolil Oligopeptidasas/antagonistas & inhibidores , Prolil Oligopeptidasas/deficiencia , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Litio/farmacología , Ácido Ocadaico/farmacología , Prolina/análogos & derivados , Prolina/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidoresRESUMEN
BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease, and it manifests as progressive memory loss and cognitive decline. However, there are no effective therapies for AD, which is an urgent problem to solve. Evodiamine, one of the main bioactive ingredients of Evodia rutaecarpa, has been reported to ameliorate blood-brain barrier (BBB) permeability and improve cognitive impairment in ischemia and AD mouse models. However, whether evodiamine alleviates tauopathy remains unclear. This study aimed to examine whether evodiamine ameliorates tau phosphorylation and cognitive deficits in AD models. METHODS: A protein phosphatase 2A inhibitor, okadaic acid (OA), was used to induce tau phosphorylation to mimic AD-like models in neuronal cells. Protein expression and cell apoptosis were detected using Western blotting and flow cytometry, respectively. Spatial memory/cognition was assessed using water maze, passive avoidance tests, and magnetic resonance imaging assay in OA-induced mice models, and brain slices were evaluated further by immunohistochemistry. RESULTS: The results showed that evodiamine significantly reduced the expression of phosphor-tau, and further decreased tau aggregation and neuronal cell death in response to OA treatment. This inhibition was found to be via the inhibition of glycogen synthase kinase 3ß, cyclin-dependent kinase 5, and mitogen-activated protein kinase pathways. In vivo results indicated that evodiamine treatment ameliorated learning and memory impairments in mice, whereas Western blotting and immunohistochemical analysis of the mouse brain also confirmed the neuroprotective effects of evodiamine. CONCLUSIONS: Evodiamine can decrease the neurotoxicity of tau aggregation and exhibit a neuroprotective effect. Our results demonstrate that evodiamine has a therapeutic potential for AD treatment.
Asunto(s)
Quinazolinas/farmacología , Tauopatías/tratamiento farmacológico , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Animales , Apoptosis/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Cognición/efectos de los fármacos , Cognición/fisiología , Trastornos del Conocimiento/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Neuroprotección/efectos de los fármacos , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Ácido Ocadaico/efectos adversos , Ácido Ocadaico/farmacología , Fosforilación , Quinazolinas/metabolismo , Memoria Espacial/efectos de los fármacos , Proteínas tau/efectos de los fármacos , Proteínas tau/metabolismoRESUMEN
Long-term potentiation (LTP) and long-term depression (LTD) are key forms of synaptic plasticity in the hippocampus. LTP and LTD are believed to underlie the processes occurring during learning and memory. Search of mechanisms responsible for switching from LTP to LTD and vice versa is an important fundamental task. Protein synthesis blockers (PSB) are widely used in models of memory impairment and LTP suppression. Here, we found that blockade of serine/threonine phosphatases 1 (PP1) and 2A (PP2A) with the specific blockers, calyculin A (CalyA) or okadaic acid (OA), and simultaneous blockade of the protein translation by anisomycin or cycloheximide leads to a switch from PSB-impaired LTP to LTD. PP1/PP2A-dependent LTD was extremely sensitive to the intensity of the test stimuli, whose increase restored the field excitatory postsynaptic potentials (fEPSP) to the values corresponding to control LTP in the non-treated slices. PP1/PP2A blockade affected the basal synaptic transmission, increasing the paired-pulse facilitation (PPF) ratio, and restored the PSB-impaired PPF 3 h after tetanus. Prolonged exposure to anisomycin led to the NO synthesis increase (measured using fluorescent dye) both in the dendrites and somata of CA1, CA3, dentate gyrus (DG) hippocampal layers. OA partially prevented the NO production in the CA1 dendrites, as well in the CA3 and DG somas. Direct measurements of changes in serine/threonine phosphatase (STPP) activity revealed importance of the PP1/PP2A-dependent component in the late LTP phase (L-LTP) in anisomycin-treated slices. Thus, serine/threonine phosphatases PP1/PP2A influence both basal synaptic transmission and stimulation-induced synaptic plasticity.
Asunto(s)
Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 2/antagonistas & inhibidores , Inhibidores de la Síntesis de la Proteína/farmacología , Animales , Anisomicina/farmacología , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/fisiología , Cicloheximida/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/fisiología , Masculino , Toxinas Marinas/farmacología , Óxido Nítrico/biosíntesis , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Ratas , Ratas WistarRESUMEN
Norcantharidin (NCTD), the demethylated analog of cantharidin isolated from Mylabris, is known to inhibit renal fibrosis. However, the underlying mechanism is largely unknown. The present study investigates whether NCTD exerts this effect through regulation of the protein phosphatase 2A catalytic subunit (PP2Ac)-Smad3 pathway. HK-2 human renal proximal tubule cells exposed to transforming growth factor (TGF)-ß1 were used as an in vitro model of renal fibrosis. The levels of total Smad3, C-terminal-phosphorylated Smad3 (p-Smad3), PP2Ac, and fibronectin (Fn) were evaluated by Western blotting. A PP2Ac overexpression plasmid and the PP2Ac inhibitor okadaic acid (OA) were used for functional analyses. The subcellular localization of Smad3 was visualized by immunofluorescence labeling. The results showed that PP2Ac overexpression increased Smad3 phosphorylation and nuclear translocation in HK-2 cells, while pharmacologic inhibition of PP2Ac with OA had the opposite effect. NCTD suppressed Fn and p-Smad3 expression and TGF-ß1-induced nuclear entry of Smad3, but these effects were abrogated by inhibition of PP2Ac. Thus, the anti-renal interstitial fibrosis effect of NCTD is exerted through inhibition of PP2Ac-mediated C-terminal phosphorylation of Smad3. These findings highlight the therapeutic potential of NCTD for the treatment of renal interstitial fibrosis.