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The COVID-19 pandemic caused by SARS-CoV-2 highlighted the importance of reliable detection methods for disease control and surveillance. Optimizing detection antibodies by rational screening antigens would improve the sensitivity and specificity of antibody-based detection methods such as colloidal gold immunochromatography. In this study, we screened three peptide antigens with conserved sequences in the N protein of SARS-CoV-2 using bioinformatical and structural biological analyses. Antibodies that specifically recognize these peptides were prepared. The epitope of the peptide that had the highest binding affinity with its antibody was located on the surface of the N protein, which was favorable for antibody binding. Using the optimal antibody that can recognize this epitope, we developed colloidal gold immunochromatography, which can detect the N protein at 10 pg/mL. Importantly, this antibody could effectively recognize both the natural peptide antigen and mutated peptide antigen in the N protein, showing the feasibility of being applied in the large-scale population testing of SARS-CoV-2. Our study provides a platform with reference significance for the rational screening of detection antibodies with high sensitivity, specificity, and reliability for SARS-CoV-2 and other pathogens.
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Anticuerpos Antivirales , COVID-19 , Proteínas de la Nucleocápside de Coronavirus , Epítopos , SARS-CoV-2 , SARS-CoV-2/inmunología , Anticuerpos Antivirales/inmunología , Humanos , Epítopos/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/química , COVID-19/diagnóstico , COVID-19/inmunología , COVID-19/virología , Sensibilidad y Especificidad , Fosfoproteínas/inmunología , Fosfoproteínas/química , Oro Coloide/química , Prueba Serológica para COVID-19/métodos , Antígenos Virales/inmunologíaRESUMEN
Thrombosis is the main cause of catastrophic events including ischemic stroke, myocardial infarction and pulmonary embolism. Acetylsalicylic acid (ASA) therapy offers a desirable approach to antithrombosis through a reduction of platelet reactivity. However, major bleeding complications, severe off-target side effects, and resistance or nonresponse to ASA greatly attenuate its clinical outcomes. Herein, we report a cationic fibrinogen-mimicking nanoparticle, denoted as ASA-RGD-CS@TPP, to achieve activated-platelet-targeted delivery and efficient release of ASA for safer and more effective antithrombotic therapy. This biomimetic antithrombotic system was prepared by one-pot ionic gelation between cationic arginine-glycine-aspartic acid (RGD)-grafted chitosan (RGD-CS) and anionic tripolyphosphate (TPP). The platform exhibited selective binding to activated platelets, leading to efficient release of ASA and subsequent attenuation of platelet functions, including the remarkable inhibition of platelet aggregation through a potent blockage of cyclooxygenase-1 (COX-1). After intravenous administration, ASA-RGD-CS@TPP displayed significantly prolonged circulation time and successful prevention of thrombosis in a mouse model. ASA-RGD-CS@TPP was demonstrated to significantly enhance antithrombotic therapy while showing minimal coagulation and hemorrhagic risks and excellent biocompatibility in vivo as compared to free ASA. This platform provides a simple, safe, effective and targeted strategy for the development of antithrombotic nanomedicines.
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Plaquetas , Quitosano , Fibrinógeno , Fibrinolíticos , Nanopartículas , Quitosano/química , Animales , Nanopartículas/química , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Ratones , Fibrinógeno/química , Fibrinógeno/metabolismo , Fibrinolíticos/farmacología , Fibrinolíticos/química , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Liberación de Fármacos , Activación Plaquetaria/efectos de los fármacos , Aspirina/farmacología , Aspirina/química , Agregación Plaquetaria/efectos de los fármacos , Humanos , Cationes/química , MasculinoRESUMEN
Inhibitor-2 (I-2) is a prototypic inhibitor of protein phosphatase-1 (PP1), a major serine-threonine phosphatase that regulates synaptic plasticity and learning and memory. Although I-2 is a potent inhibitor of PP1 in vitro, our previous work has elucidated that, in vivo, I-2 may act as a positive regulator of PP1. Here we show that I-2 and PP1γ, but not PP1α, positively regulate synaptic transmission in hippocampal neurons. Moreover, we demonstrated that I-2 enhanced PP1γ interaction with its major synaptic scaffold, neurabin, by Förster resonance energy transfer (FRET)/Fluorescence lifetime imaging microscopy (FLIM) studies, while having a limited effect on PP1 auto-inhibitory phosphorylation. Furthermore, our study indicates that the effect of I-2 on PP1 activity in vivo is dictated by I-2 threonine-72 phosphorylation. Our work thus demonstrates a molecular mechanism by which I-2 positively regulates PP1 function in synaptic transmission.
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Injury to the axons of retinal ganglion cells (RGCs) is a key pathological event in glaucomatous neurodegeneration. The transcription factors JUN (the target of the c-Jun N-terminal kinases, JNKs) and DDIT3/CHOP (a mediator of the endoplasmic reticulum stress response) have been shown to control the majority of proapoptotic signaling after mechanical axonal injury in RGCs and in other models of neurodegeneration. The downstream transcriptional networks controlled by JUN and DDIT3, which are critical for RGC death, however, are not well defined. To determine these networks, RNA was isolated from the retinas of wild-type mice and mice deficient in Jun, Ddit3, and both Jun and Ddit3 three days after mechanical optic nerve crush injury (CONC). RNA-sequencing data analysis was performed and immunohistochemistry was used to validate potential transcriptional signaling changes after axonal injury. This study identified downstream transcriptional changes after injury including both neuronal survival and proinflammatory signaling that were attenuated to differing degrees by loss of Ddit3, Jun, and Ddit3/Jun. These data suggest proinflammatory signaling in the retina might be secondary to activation of pro-death pathways in RGCs after acute axonal injury. These results determine the downstream transcriptional networks important for apoptotic signaling which may be important for ordering and staging the pro-degenerative signals after mechanical axonal injury.
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Traumatismos del Nervio Óptico , Células Ganglionares de la Retina , Animales , Axones/metabolismo , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Traumatismos del Nervio Óptico/metabolismo , ARN/metabolismo , Células Ganglionares de la Retina/metabolismoRESUMEN
Parkinson's disease (PD), as the second most common neurodegenerative disease, is seriously affecting the life quality of the elderly. However, there is still a lack of efficient medical methods to diagnosis PD before apparent symptoms occur. In recent years, clinical biomarkers including genetic, imaging, and tissue markers have exhibited remarkable benefits in assisting PD diagnoses. Due to the advantages of high-throughput detection of metabolites and almost non-invasive sample collection, metabolomics research of PD is widely used for diagnostic biomarker discovery. However, there are also a few shortages for those identified biomarkers, such as the scarcity of verifications regarding the sensitivity and specificity. Thus, reviewing the research progress of PD biomarkers based on metabolomics techniques is of great significance for developing PD diagnosis. To comprehensively clarify the progress of current metabolic biomarker studies in PD, we reviewed 20 research articles regarding the discovery and validation of biomarkers for PD diagnosis from three mainstream academic databases (NIH PubMed, ISI Web of Science, and Elsevier ScienceDirect). By analyzing those materials, we summarized the metabolic biomarkers identified by those metabolomics studies and discussed the potential approaches used for biomarker verifications. In conclusion, this review provides a comprehensive and updated overview of PD metabolomics research in the past two decades and particularly discusses the validation of disease biomarkers. We hope those discussions might provide inspiration for PD biomarker discovery and verification in the future.
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Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Anciano , Biomarcadores/metabolismo , Humanos , Metabolómica/métodos , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/metabolismo , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: Genome-wide association studies (GWAS) for late onset Alzheimer's disease (AD) may miss genetic variants relevant for delineating disease stages when using clinically defined case/control as a phenotype due to its loose definition and heterogeneity. METHODS: We use a transfer learning technique to train three-dimensional convolutional neural network (CNN) models based on structural magnetic resonance imaging (MRI) from the screening stage in the Alzheimer's Disease Neuroimaging Initiative consortium to derive image features that reflect AD progression. RESULTS: CNN-derived image phenotypes are significantly associated with fasting metabolites related to early lipid metabolic changes as well as insulin resistance and with genetic variants mapped to candidate genes enriched for amyloid beta degradation, tau phosphorylation, calcium ion binding-dependent synaptic loss, APP-regulated inflammation response, and insulin resistance. DISCUSSION: This is the first attempt to show that non-invasive MRI biomarkers are linked to AD progression characteristics, reinforcing their use in early AD diagnosis and monitoring.
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Genetic and genome-wide association studies suggest a central role for microglia in Alzheimer's disease (AD). However, single-cell RNA sequencing (scRNA-seq) of microglia in mice, a key preclinical model, has shown mixed results regarding translatability to human studies. To address this, scRNA-seq of microglia from C57BL/6J (B6) and wild-derived strains (WSB/EiJ, CAST/EiJ, and PWK/PhJ) with and without APP/PS1 demonstrates that genetic diversity significantly alters features and dynamics of microglia in baseline neuroimmune functions and in response to amyloidosis. Results show significant variation in the abundance of microglial subtypes or states, including numbers of previously identified disease-associated and interferon-responding microglia, across the strains. For each subtype, significant differences in the expression of many genes are observed in wild-derived strains relative to B6, including 19 genes previously associated with human AD including Apoe, Trem2, and Sorl1. This resource is critical in the development of appropriately targeted therapeutics for AD and other neurological diseases.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Microglía/metabolismo , RNA-Seq , Animales , Modelos Animales de Enfermedad , Variación Genética , Estudio de Asociación del Genoma Completo , Ratones , Especificidad de la EspecieRESUMEN
The interaction between anticancer drugs and HSA may have a significant impact on the pharmacology and efficacy of drugs. Drugs change the binding properties of HSA by regulating the quenching mechanism, binding mode and binding affinity. In this study, the interactions of cisplatin (cDDP), HSA, and daphnoretin were elucidated by multi-spectroscopic analyses and docking simulation. Fluorescence quenching showed that cDDP could not change the static quenching mechanism of HSA-daphnoretin, but could enhance their binding affinity. Site competition experiments revealed that daphnoretin and cDDP both bound to site I, which was consistent with the results of molecular docking. Thermodynamic date indicated that cDDP and daphnoretin formed a more stable complex with HSA via hydrophobic, van der Waals interaction and hydrogen bond. Three-dimensional fluorescence and circular dichroism spectra showed that cDDP changed the conformation and micro-environment of HSA induced by daphnoretin. This work could provide valuable information for the binding properties and interaction among cDDP, daphnoretin and HSA, and put forward the possibility of using HSA as a multidrug carrier.
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Cisplatino , Albúmina Sérica Humana , Sitios de Unión , Dicroismo Circular , Cumarinas , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Albúmina Sérica Humana/metabolismo , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
BACKGROUND: The risk of glaucoma increases significantly with age and exposure to elevated intraocular pressure, two factors linked with neuroinflammation. The complement cascade is a complex immune process with many bioactive end-products, including mediators of inflammation. Complement cascade activation has been shown in glaucoma patients and models of glaucoma. However, the function of complement-mediated inflammation in glaucoma is largely untested. Here, the complement peptide C3a receptor 1 was genetically disrupted in DBA/2J mice, an ocular hypertensive model of glaucoma, to test its contribution to neurodegeneration. METHODS: A null allele of C3ar1 was backcrossed into DBA/2J mice. Development of iris disease, ocular hypertension, optic nerve degeneration, retinal ganglion cell activity, loss of RGCs, and myeloid cell infiltration in C3ar1-deficient and sufficient DBA/2J mice were compared across multiple ages. RNA sequencing was performed on microglia from primary culture to determine global effects of C3ar1 on microglia gene expression. RESULTS: Deficiency in C3ar1 lowered the risk of degeneration in ocular hypertensive mice without affecting intraocular pressure elevation at 10.5 months of age. Differences were found in the percentage of mice affected, but not in individual characteristics of disease progression. The protective effect of C3ar1 deficiency was then overcome by additional aging and ocular hypertensive injury. Microglia and other myeloid-derived cells were the primary cells identified that express C3ar1. In the absence of C3ar1, microglial expression of genes associated with neuroinflammation and other immune functions were differentially expressed compared to WT. A network analysis of these data suggested that the IL10 signaling pathway is a major interaction partner of C3AR1 signaling in microglia. CONCLUSIONS: C3AR1 was identified as a damaging neuroinflammatory factor. These data help suggest complement activation causes glaucomatous neurodegeneration through multiple mechanisms, including inflammation. Microglia and infiltrating myeloid cells expressed high levels of C3ar1 and are the primary candidates to mediate its effects. C3AR1 appeared to be a major regulator of microglia reactivity and neuroinflammatory function due to its interaction with IL10 signaling and other immune related pathways. Targeting myeloid-derived cells and C3AR1 signaling with therapies is expected to add to or improve neuroprotective therapeutic strategies.
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Degeneración Nerviosa/metabolismo , Nervio Óptico/metabolismo , Receptores de Complemento/biosíntesis , Receptores de Complemento/deficiencia , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Redes Reguladoras de Genes/fisiología , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Nervio Óptico/patología , Receptores de Complemento/genéticaRESUMEN
BACKGROUND: The incidence of dementia and cognitive decline is increasing with no therapy or cure. One of the reasons treatment remains elusive is because there are various pathologies that contribute to age-related cognitive decline. Specifically, with Alzheimer's disease, targeting to reduce amyloid beta plaques and phosphorylated tau aggregates in clinical trials has not yielded results to slow symptomology, suggesting a new approach is needed. Interestingly, exercise has been proposed as a potential therapeutic intervention to improve brain health and reduce the risk for dementia, however the benefits throughout aging are not well understood. RESULTS: To better understand the effects of exercise, we preformed transcriptional profiling on young (1-2 months) and midlife (12 months) C57BL/6 J (B6) male mice after 12 weeks of voluntary running. Data was compared to age-matched sedentary controls. Interestingly, the midlife running group naturally broke into two cohorts based on distance ran - either running a lot and more intensely (high runners) or running less and less intensely (low runners). Midlife high runners had lower LDL cholesterol as well as lower adiposity (%fat) compared to sedentary, than midlife low runners compared to sedentary suggesting more intense running lowered systemic markers of risk for age-related diseases including dementias. Differential gene analysis of transcriptional profiles generated from the cortex and hippocampus showed thousands of differentially expressed (DE) genes when comparing young runners to sedentary controls. However, only a few hundred genes were DE comparing either midlife high runners or midlife low runners to midlife sedentary controls. This indicates that, in our study, the effects of running are reduced through aging. Gene set enrichment analyses identified enrichment of genes involved in extracellular matrix (ECM), vascular remodeling and angiogenesis in young runners but not midlife runners. These genes are known to be expressed in multiple vascular-related cell types including astrocytes, endothelial cells, pericytes and smooth muscle cells. CONCLUSIONS: Taken together these results suggest running may best serve as a preventative measure to reduce risk for cerebrovascular decline. Ultimately, this work shows that exercise may be more effective to prevent dementia if introduced at younger ages.
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Circulación Cerebrovascular , Perfilación de la Expresión Génica , Carrera , Transcriptoma , Remodelación Vascular/genética , Factores de Edad , Animales , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/metabolismo , Biología Computacional/métodos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Ontología de Genes , Masculino , Ratones , Modelos Biológicos , Especificidad de Órganos/genéticaRESUMEN
BACKGROUND: Environmental factors are critical in the development of age-related cognitive decline and dementia. A western diet (WD) can cause nutrient deficiency and inflammation that could impact cognition directly. It is increasingly recognized that innate immune responses by brain myeloid cells, such as resident microglia, and infiltrating peripheral monocytes/macrophages may represent an essential link between a WD, cognitive decline, and dementia. Our previous data demonstrated that chronic consumption of a WD induced inflammation through brain myeloid cells in aging mice and a mouse model of Alzheimer's disease (AD). However, the subtypes of myeloid cells that contribute to the WD-induced inflammation remain unclear. METHODS: C57BL/6J (B6), myeloid cell reporter mice (B6.Ccr2RFP/+Cx3cr1GFP/+), and Ccr2-deficient mice (B6.Ccr2RFP/RFP) were fed a WD or a control chow diet (CD) from 2 to 6 or 12 months of age. CD11b+CD45lo and CD11b+CD45hi cells from WD- and CD-fed B6 or Ccr2-deficient mice were characterized using flow cytometry, RNA-sequencing, and immunofluorescence. RESULTS: Ccr2::RFP expressing myeloid cells were significantly increased in brains of WD- compared to CD-fed mice, but were not elevated in Ccr2-deficient WD-fed mice. The percent of CD11b+CD45hi cells was significantly increased in WD- compared to CD-fed mice. Comparison of RNA-sequencing data with immune cell data in ImmGen supports that CD11b+CD45hi cells from WD-fed mice are enriched for peripheral monocytes and neutrophils. Ingenuity pathway analysis predicted these cells elicit proinflammatory responses that may be damaging to the brain. Using stringent criteria for gene expression levels between CD11b+CD45hi and CD11b+CD45lo cells, we identified approximately 70 genes that we predict are uniquely expressed in infiltrating cells, including Itgal, Trem1, and Spp1 (osteopontin, OPN). Finally, we show a significantly greater number of OPN+IBA1- cells in WD- compared to CD-fed mice that we propose are activated neutrophils based on ImmGen data. OPN+IBA1- cells are not significantly increased in Ccr2-deficient WD-fed mice. CONCLUSIONS: These data further support the model that peripheral myeloid cells enter the brain in response to diet-induced obesity. Elucidating their contribution to age-related cognitive decline and age-related neurodegenerative diseases should offer new avenues for therapeutic intervention in Alzheimer's disease and related dementias, where diet/obesity are major risk factors.
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Antígeno CD11a/metabolismo , Dieta Occidental/efectos adversos , Perfilación de la Expresión Génica/métodos , Obesidad/metabolismo , Osteopontina/metabolismo , Receptor Activador Expresado en Células Mieloides 1/metabolismo , Animales , Encéfalo/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/metabolismo , Obesidad/inducido químicamente , Obesidad/genética , Osteopontina/genética , Receptor Activador Expresado en Células Mieloides 1/genéticaRESUMEN
Protein phosphatase-1 (PP1) constrains learning and memory formation in part through its effects on the induction threshold of long-term potentiation (LTP) and depression (LTD). LTD induction requires both the enzymatic activity of PP1 and its proper anchoring to synaptic spines. We have shown previously that neurabin, a major synaptic scaffolding protein, targets PP1 to synapses for LTD induction. Here, we show that PP1 bound on spinophilin, a close homolog of neurabin and another major synaptic PP1 anchoring protein, does not play a role in LTD induction, which suggests that neurabin plays a privileged role in nanodomain targeting of PP1 in LTD induction. We found that protein kinase A can significantly weaken the neurabin-PP1 interaction in neurons via phosphorylation of neurabin at serine 461, a phosphorylation site adjacent to the PP1-binding motif that is not conserved in spinophilin. Finally, we found that a neurabin mutation (S461E), which mimics phosphorylation, blocked AMPA receptor endocytosis and LTD induction. The results indicate the critical importance of nanodomain targeting of PP1 within synaptic spines and its regulation in LTD induction.
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Potenciación a Largo Plazo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteína Fosfatasa 1/metabolismo , Receptores AMPA/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endocitosis , Activación Enzimática , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Neuronas/metabolismo , Fosforilación , Fosfoserina/metabolismo , RatasRESUMEN
Reversible phosphorylation, a fundamental regulatory mechanism required for many biological processes including memory formation, is coordinated by the opposing actions of protein kinases and phosphatases. Type I protein phosphatase (PP1), in particular, has been shown to constrain learning and memory formation. However, how PP1 might be regulated in memory is still not clear. Our previous work has elucidated that PP1 inhibitor-2 (I-2) is an endogenous regulator of PP1 in hippocampal and cortical neurons (Hou et al., 2013). Contrary to expectation, our studies of contextual fear conditioning and novel object recognition in I-2 heterozygous mice suggest that I-2 is a memory suppressor. In addition, lentiviral knock-down of I-2 in the rat dorsal hippocampus facilitated memory for tasks dependent on the hippocampus. Our data indicate that I-2 suppresses memory formation, probably via negatively regulating the phosphorylation of cAMP/calcium response element-binding protein (CREB) at serine 133 and CREB-mediated gene expression in dorsal hippocampus. Surprisingly, the data from both biochemical and behavioral studies suggest that I-2, despite its assumed action as a PP1 inhibitor, is a positive regulator of PP1 function in memory formation. SIGNIFICANCE STATEMENT: We found that inhibitor-2 acts as a memory suppressor through its positive functional influence on type I protein phosphatase (PP1), likely resulting in negative regulation of cAMP/calcium response element-binding protein (CREB) and CREB-activated gene expression. Our studies thus provide an interesting example of a molecule with an in vivo function that is opposite to its in vitro function. PP1 plays critical roles in many essential physiological functions such as cell mitosis and glucose metabolism in addition to its known role in memory formation. PP1 pharmacological inhibitors would thus not be able to serve as good therapeutic reagents because of its many targets. However, identification of PP1 inhibitor-2 as a critical contributor to suppression of memory formation by PP1 may provide a novel therapeutic target for memory-related diseases.
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Memoria/fisiología , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 1/fisiología , Proteínas/fisiología , Animales , Células Cultivadas , Femenino , Hipocampo/fisiología , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , RatasRESUMEN
NMDA receptor signaling plays a complex role in CREB activation and CREB-mediated gene transcription, depending on the subcellular location of NMDA receptors, as well as how strongly they are activated. However, it is not known whether Rac1, the prototype of Rac GTPase, plays a role in neuronal CREB activation induced by NMDA receptor signaling. Here, we report that NSC23766, a widely used specific Rac1 inhibitor, inhibits basal CREB phosphorylation at S133 (pCREB) and antagonizes changes in pCREB levels induced by NMDA bath application in rat cortical neurons. Unexpectedly, we found that NSC23766 affects the levels of neuronal pCREB in a Rac1-independent manner. Instead, our results indicate that NSC23766 can directly regulate NMDA receptors as indicated by their strong effects on both exogenous and synaptically evoked NMDA receptor-mediated currents in mouse and rat neurons, respectively. Our findings strongly suggest that Rac1 does not affect pCREB signaling in cortical neurons and reveal that NSC23766 could be a novel NMDA receptor antagonist.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/antagonistas & inhibidores , Sistemas de Liberación de Medicamentos/métodos , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de Señal/fisiología , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Aminoquinolinas/farmacología , Animales , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Femenino , Masculino , Técnicas de Cultivo de Órganos , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The functional stability of neurons in the face of large variations in both activity and efficacy of synaptic connections suggests that neurons possess intrinsic negative feedback mechanisms to balance and tune excitability. While NMDA receptors have been established to play an important role in glutamate receptor-dependent plasticity through protein dephosphorylation, the effects of synaptic activation on intrinsic excitability are less well characterized. We show that increases in synaptic activity result in dephosphorylation of the potassium channel subunit Kv2.1. This dephosphorylation is induced through NMDA receptors and is executed through protein phosphatase-1 (PP1), an enzyme previously established to play a key role in regulating ligand gated ion channels in synaptic plasticity. Dephosphorylation of Kv2.1 by PP1 in response to synaptic activity results in substantial shifts in the inactivation curve of IK, resulting in a reduction in intrinsic excitability, facilitating negative feedback to neuronal excitability.
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Neuronas/metabolismo , Proteína Fosfatasa 1/metabolismo , Canales de Potasio Shab/metabolismo , Sinapsis/fisiología , Animales , Retroalimentación Fisiológica , Fosforilación , Cultivo Primario de Células , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Protein phosphorylation plays a critical role in neuronal transcription, translation, cell viability, and synaptic plasticity. In neurons, phospho-enzymes and specific substrates directly link glutamate release and post-synaptic depolarization to these cellular functions; however, many of these enzymes and their protein substrates remain uncharacterized or unidentified. In this article, we identify a novel, synaptically driven neuronal phosphoproteome characterized by a specific motif of serine/threonine-glutamine ([S/T]-Q, abbreviated as SQ). These SQ-containing substrates are predominantly localized to dendrites, synapses, the soma; and activation of this SQ phosphoproteome by bicuculline application is induced via calcium influx through L-type calcium channels. On the other hand, acute application of NMDA can inactivate this SQ phosphoproteome. We demonstrate that the SQ motif kinase Ataxia-telangiectasia mutated can also localize to dendrites and dendritic spines, in addition to other subcellular compartments, and is activated by bicuculline application. Pharmacology studies indicate that Ataxia-telangiectasia mutated and its sister kinase ataxia telangiectasia mutated and Rad3-related up-regulate these neuronal SQ substrates. Phosphoproteomics identified over 150 SQ-containing substrates whose phosphorylation is bidirectionally regulated by synaptic activity.
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Neuronas/fisiología , Fosfoproteínas/fisiología , Proteómica , Sinapsis/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Calcio/fisiología , Canales de Calcio Tipo L/fisiología , Corteza Cerebral/citología , Inhibidores Enzimáticos/farmacología , Femenino , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Fosforilación/fisiología , Embarazo , Cultivo Primario de Células , Proteoma/fisiología , Ratas , Bloqueadores de los Canales de Sodio/farmacología , Sinapsis/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown. We find that synaptic N-methyl-D-aspartate (NMDA) receptor stimulation in neurons leads to activation of PP1 through a mechanism involving inhibitory phosphorylation at Thr320 by Cdk5. Synaptic stimulation led to proteasome-dependent degradation of the Cdk5 regulator p35, inactivation of Cdk5, and increased auto-dephosphorylation of Thr320 of PP1. We also found that neither inhibitor-1 nor calcineurin were involved in the control of PP1 activity in response to synaptic NMDA receptor stimulation. Rather, the PP1 regulatory protein, inhibitor-2, formed a complex with PP1 that was controlled by synaptic stimulation. Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons. Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity.
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Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Encéfalo/metabolismo , Calcineurina/metabolismo , Calcio , Células Cultivadas , Depresión Sináptica a Largo Plazo/fisiología , Plasticidad Neuronal , Neuronas/metabolismo , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Transducción de Señal , Transmisión Sináptica/fisiologíaRESUMEN
OBJECTIVE: The present study was aimed to investigate the pharmacological modulatory effects of ropivacaine, an amide-type local anesthetic, on rat Nav1.2 (rNav1.2) and rNav1.5, the two Na(+) channel isoforms heterologously expressed in Xenopus oocytes and in HEK293t cell line, respectively. METHODS: Two-electrode voltage-clamp (TEVC) and whole-cell patch-clamp recordings were employed to record the whole-cell currents. RESULTS: Ropivacaine induced tonic inhibition of peak Na(+) currents of both subtypes in a dose- and frequency-dependent manner. rNav1.5 appeared to be more sensitive to ropivacaine. In addition, for both Na(+) channel subtypes, the steady-state inactivation curves, but not the activation curves, were significantly shifted to the hyperpolarizing direction by ropivacaine. Use-dependent blockade of both rNav1.2 and rNav1.5 channels was induced by ropivacaine through a high frequency of depolarization, suggesting that ropivacaine could preferentially bind to the 2 inactivated Na(+) channel isoforms. CONCLUSION: The results will be helpful in understanding the pharmacological modulation by ropivacaine on Nav1.2 subtype in the central nervous system, and on Nav1.5 subtype abundantly expressed in the heart.
Asunto(s)
Amidas/farmacología , Anestésicos Locales/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Canales de Sodio/metabolismo , Animales , Fenómenos Biofísicos/efectos de los fármacos , Biofisica , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica/métodos , Humanos , Potenciales de la Membrana/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.5 , Técnicas de Placa-Clamp/métodos , Ratas , Ropivacaína , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/clasificación , Canales de Sodio/genética , Tetrodotoxina/farmacología , Transfección/métodos , Xenopus laevisRESUMEN
BACKGROUND AND PURPOSE: Buthus martensi Karsch (BmK) AS is a scorpion polypeptide toxin, said to target the voltage-gated sodium channels (VGSCs). However, the mechanism of action of BmK AS on the VGSCs has yet to be defined. EXPERIMENTAL APPROACH: We examined the electrophysiological effects of BmK AS in a wide dose range on the rat brain-type VGSC alpha-subunit, rNav1.2a, heterologously expressed in Xenopus oocytes and on the VGSCs endogenously expressed in the dorsal root ganglion neuroblastoma ND7-23 cell line. KEY RESULTS: In the oocytes, BmK AS depolarized the voltage dependence of activation and inactivation of rNav1.2a at 0.1 and 500 nM whereas these parameters were hyperpolarized at 1 nM. In ND7-23 cells, BmK AS hyperpolarized the voltage dependence of activation and inactivation at 0.1, 1 and 100 nM but not 10 nM. BmK AS also hyperpolarized the voltage dependence of recovery from inactivation at 0.1 and 100 nM and slowed the recovery kinetics at all concentrations, but the effects of 1 and 10 nM were relatively smaller than those at 0.1 and 100 nM. Moreover, the inactivation of VGSCs was potentiated by 10 nM BmK AS in both systems, whereas it was inhibited by 0.1 or 100 nM BmK AS in the oocytes or ND7-23 cells respectively. CONCLUSIONS AND IMPLICATIONS: BmK AS modulated the VGSCs in a unique U-shaped dose-dependent manner, which could be due to the opposing effects of binding to two distinct receptor sites on the VGSCs.
Asunto(s)
Neuroblastoma/metabolismo , Péptidos/farmacología , Venenos de Escorpión/farmacología , Canales de Sodio/efectos de los fármacos , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Electrofisiología , Femenino , Ganglios Espinales/metabolismo , Ratones , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas del Tejido Nervioso , Oocitos , Péptidos/administración & dosificación , Unión Proteica , Ratas , Venenos de Escorpión/administración & dosificación , Canales de Sodio/metabolismo , Xenopus laevisRESUMEN
In the present study, using the single fiber recording technique, we found that BmK I, the main toxic component in scorpion Buthus martensi Karsch (BmK) venom, induced dramatic increase in excitability of rapidly adapting (RA) and type I slowly adapting (SAI) low threshold mechanical A fibers of rat. Five micrograms BmK I (691 nmol, in 10 microl saline) administrated to the receptive fields induced spontaneous activity in 80% of RA and SAI fibers, increased the response to 10 g-10 s stimulation at about 20 times and altered the firing pattern to burst mode with maximal NS (number of spikes in burst) averaging from all fibers studied as many as 59. The increase in the excitability of RA and SAI fibers did not recover completely in 2h. Our finding suggests that the gigantic abnormal activity in low threshold mechanical A fibers is involved in BmK scorpion sting pain, and the experimental model of BmK scorpion sting pain can be used to study A-fiber related central pathway which is important for relief of refractory neuropathic pain likewise.