RESUMEN
Stress response pathways protect the lung from the damaging effects of environmental toxicants. Here we investigate the role of the fragile X mental retardation protein (FMRP), a multifunctional protein implicated in stress responses, in the lung. We report that FMRP is expressed in murine and human lungs, in the airways and more broadly. Analysis of airway stress responses in mice and in a murine cell line ex vivo, using the well-established naphthalene injury model, reveals that FMRP-deficient cells exhibit increased expression of markers of oxidative and genotoxic stress and increased cell death. Further inquiry shows that FMRP-deficient cells fail to actuate the integrated stress response pathway (ISR) and upregulate the transcription factor ATF4. Knockdown of ATF4 expression phenocopies the loss of FMRP. We extend our analysis of the role of FMRP to human bronchial BEAS-2B cells, using a 9,10-phenanthrenequinone air pollutant model, to find that FMRP-deficient BEAS-2B cells also fail to actuate the ISR and exhibit greater susceptibility. Taken together, our data suggest that FMRP has a conserved role in protecting the airways by facilitating the ISR. This article has an associated First Person interview with the first author of the paper.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Xenobióticos , Animales , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Regulación de la Expresión Génica , Humanos , Pulmón/metabolismo , Ratones , Factores de Transcripción/metabolismoRESUMEN
Neuronal activity is responsible for the high energy consumption in the brain. However, the cellular mechanisms draining ATP upon the arrival of a stimulus are yet to be explored systematically at the post-synapse. Here, we provide evidence that a significant fraction of ATP is consumed upon glutamate stimulation to energize mGluR-induced protein synthesis. We find that both mGluR and NMDAR alter protein synthesis and ATP consumption with distinct kinetics at the synaptic-dendritic compartments. While mGluR activation leads to a rapid and sustained reduction in neuronal ATP levels, NMDAR activation has no immediate impact on the same. ATP consumption correlates inversely with the kinetics of protein synthesis for both receptors. We observe a persistent elevation in protein synthesis within 5 minutes of mGluR activation and a robust inhibition of the same within 2 minutes of NMDAR activation, assessed by the phosphorylation status of eEF2 and metabolic labeling. However, a delayed protein synthesis-dependent ATP expenditure ensues after 15 minutes of NMDAR stimulation. We identify a central role for AMPK in the correlation between protein synthesis and ATP consumption. AMPK is dephosphorylated and inhibited upon mGluR activation, while it is phosphorylated upon NMDAR activation. Perturbing AMPK activity disrupts receptor-specific modulations of eEF2 phosphorylation and protein synthesis. Our observations, therefore, demonstrate that the regulation of the AMPK-eEF2 signaling axis by glutamate receptors alters neuronal protein synthesis and bioenergetics.
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Receptores de N-Metil-D-Aspartato , Sinapsis , Metabolismo Energético , Factor 2 de Elongación Peptídica , Fosforilación , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismoRESUMEN
Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR2), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR2-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR2, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR2, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR2-dependent hyperexcitability of nociceptors, and PAR2 association with ß-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR2 A cholestanol-conjugated PAR2 antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR2 signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR2 antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.
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Dolor Crónico/etiología , Endosomas/fisiología , Síndrome del Colon Irritable/fisiopatología , Receptor PAR-2/fisiología , Transducción de Señal/fisiología , Animales , Endocitosis , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Humanos , Nocicepción , Nociceptores/fisiología , Tripsina/farmacologíaRESUMEN
Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.
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Alquinos , Azidas , Química Clic , Reacción de Cicloadición , Biosíntesis de ProteínasRESUMEN
The ribosomal p70 S6 Kinase 1 (S6K1) has been implicated in the etiology of complex neurological diseases including autism, depression and dementia. Though no major gene disruption has been reported in humans in RPS6KB1, single nucleotide variants (SNVs) causing missense mutations have been identified, which have not been assessed for their impact on protein function. These S6K1 mutations have the potential to influence disease progression and treatment response. We mined the Simon Simplex Collection (SSC) and SPARK autism database to find inherited SNVs in S6K1 and characterized the effect of two missense SNVs, Asp14Asn (allele frequency = 0.03282%) and Glu44Gln (allele frequency = 0.0008244%), on S6K1 function in HEK293, human ES cells and primary neurons. Expressing Asp14Asn in HEK293 cells resulted in increased basal phosphorylation of downstream targets of S6K1 and increased de novo translation. This variant also showed blunted response to the specific S6K1 inhibitor, FS-115. In human embryonic cell line Shef4, Asp14Asn enhanced spontaneous neural fate specification in the absence of differentiating growth factors. In addition to enhanced translation, neurons expressing Asp14Asn exhibited impaired dendritic arborization and increased levels of phosphorylated ERK 1/2. Finally, in the SSC families tracked, Asp14Asn segregated with lower IQ scores when found in the autistic individual rather than the unaffected sibling. The Glu44Gln mutation showed a milder, but opposite phenotype in HEK cells as compared to Asp14Asn. Although the Glu44Gln mutation displayed increased neuronal translation, it had no impact on neuronal morphology. Our results provide the first characterization of naturally occurring human S6K1 variants on cognitive phenotype, neuronal morphology and maturation, underscoring again the importance of translation control in neural development and plasticity.
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Hipocampo/metabolismo , Neuronas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/fisiología , Alelos , Animales , Forma de la Célula/genética , Frecuencia de los Genes , Células HEK293 , Hipocampo/citología , Humanos , Mutación , Neurogénesis/fisiología , Neuronas/citología , Fosforilación , Ratas , Ratas Sprague-Dawley , Proteínas Quinasas S6 Ribosómicas 70-kDa/genéticaRESUMEN
Stress is known to cause contrasting patterns of morphological and physiological plasticity in the hippocampus and amygdala. An obligatory cellular process underlying such neural changes is de novo translation and alterations in protein expression. Yet the nature of the translational response to stress in neurons remains largely unexplored. Even less is known about how glia are affected. Using a click-chemistry-based method to label the de novo proteome in live brain slices, we monitored translation in neurons and astrocytes of the basolateral amygdala (BLA) and dorsal hippocampal area CA3 (dCA3) in rats at different time-points after a single 2-hr exposure to immobilization stress. We observed enhancements in neuronal translation in both brain regions 1 hour after stress. This initial increase persisted in the BLA up to 10 days afterwards. In contrast, dCA3 neuronal translation gradually decreased to below control levels 10 days later. Translation profiles of dCA3 astrocytes followed timelines similar to neurons, but in BLA astrocytes translation peaked 1 day later and remained elevated 10 days later. Together our results demonstrate that stress causes an immediate upregulation of protein synthesis in both amygdalar and hippocampal neurons and astrocytes. However, these two areas eventually exhibit opposite temporal profiles of protein expression well after the end of stress. These findings identify new metrics of stress-induced plasticity at the level of cell-type specific proteomic landscape that may provide important insights into the molecular basis of the divergent temporal effects of stress across brain regions and biological scales.
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Complejo Nuclear Basolateral/fisiología , Región CA3 Hipocampal/fisiología , Plasticidad Neuronal/fisiología , Estrés Psicológico/metabolismo , Animales , Astrocitos/fisiología , Complejo Nuclear Basolateral/fisiopatología , Región CA3 Hipocampal/fisiopatología , Masculino , Neuronas/fisiología , Biosíntesis de Proteínas/fisiología , Ratas , Ratas Wistar , Restricción Física , Estrés Psicológico/fisiopatologíaRESUMEN
The proper regulation of translation is required for the expression of long-lasting synaptic plasticity. A major site of translational control involves the phosphorylation of eukaryotic initiation factor 2 α (eIF2α) by PKR-like endoplasmic reticulum (ER) kinase (PERK). To determine the role of PERK in hippocampal synaptic plasticity, we used the Cre-lox expression system to selectively disrupt PERK expression in the adult mouse forebrain. Here, we demonstrate that in hippocampal area CA1, metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) is associated with increased eIF2α phosphorylation, whereas stimulation of early- and late-phase long-term potentiation (E-LTP and L-LTP, respectively) is associated with decreased eIF2α phosphorylation. Interesting, although PERK-deficient mice exhibit exaggerated mGluR-LTD, both E-LTP and L-LTP remained intact. We also found that mGluR-LTD is associated with a PERK-dependent increase in eIF2α phosphorylation. Our findings are consistent with the notion that eIF2α phosphorylation is a key site for the bidirectional control of persistent forms of synaptic LTP and LTD and suggest a distinct role for PERK in mGluR-LTD.
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Región CA1 Hipocampal/fisiología , Depresión Sináptica a Largo Plazo/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , eIF-2 Quinasa/metabolismo , Análisis de Varianza , Animales , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteínas de Unión al ADN/metabolismo , Estimulación Eléctrica , Técnicas In Vitro , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Factores de Transcripción/metabolismo , eIF-2 Quinasa/genéticaRESUMEN
Fragile X syndrome (FXS) is the most frequent cause of inherited intellectual disability and autism. It is caused by the absence of the fragile X mental retardation 1 (FMR1) gene product, fragile X mental retardation protein (FMRP), an RNA-binding protein involved in the regulation of translation of a subset of brain mRNAs. In Fmr1 knockout mice, the absence of FMRP results in elevated protein synthesis in the brain as well as increased signaling of many translational regulators. Whether protein synthesis is also dysregulated in FXS patients is not firmly established. Here, we demonstrate that fibroblasts from FXS patients have significantly elevated rates of basal protein synthesis along with increased levels of phosphorylated mechanistic target of rapamycin (p-mTOR), phosphorylated extracellular signal regulated kinase 1/2, and phosphorylated p70 ribosomal S6 kinase 1 (p-S6K1). The treatment with small molecules that inhibit S6K1 and a known FMRP target, phosphoinositide 3-kinase (PI3K) catalytic subunit p110ß, lowered the rates of protein synthesis in both control and patient fibroblasts. Our data thus demonstrate that fibroblasts from FXS patients may be a useful in vitro model to test the efficacy and toxicity of potential therapeutics prior to clinical trials, as well as for drug screening and designing personalized treatment approaches.
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Biomarcadores/metabolismo , Síndrome del Cromosoma X Frágil/genética , Animales , Estudios de Casos y Controles , Células Cultivadas , Evaluación Preclínica de Medicamentos , Fibroblastos/citología , Fibroblastos/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Humanos , Leucina/metabolismo , Masculino , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas Quinasas S6 Ribosómicas/metabolismoRESUMEN
BACKGROUND: Metastasis to the cervical (neck) lymph nodes is one of the most significant clinical factors responsible for death from oral squamous cell carcinoma (SCC). Therefore, the lymph nodes are frequently removed when the tumor is excised (neck dissection), even though the majority of patients will not benefit from the extra surgery. Two subtypes of oral SCC distinguished by the presence of tumor genomic aberrations +3q, -8p, +8q and/or +20 differ in risk for metastasis - high for the 3q8pq20 subtype, harboring one or more of the aberrations and low for the non-3q8pq20 subtype, lacking these alterations. A prior analysis of the literature suggested genes differentially methylated in the two subtypes. Therefore, the goal of this study was to further investigate the methylation status of candidate biomarkers of the non-3q8pq20 subtype, and evaluate their utility for identifying patients at low risk for metastasis. METHODS: Methylation status of genes in a cohort of 52 oral SCC patients with at least five year follow up was determined by pyrosequencing. Gene expression levels were determined by quantitative RT-PCR. Growth following re-expression of HOXA9 in cultured oral SCC cells was assessed by proliferation and colony formation assays. RESULTS: A pilot study evaluating methylation levels of HOXA9, MT1A and HOXA11 promoters in DNA from 12 tumors (six each of the 3q8pq20 and non-3q8pq20 subtypes) revealed that only HOXA9 was differentially methylated. Significant differences in methylation levels of HOXA9 were observed amongst the 52 oral SCCs with respect to genomic subtype and nodal status (p = 0.014, and p = 0.024, respectively, Wilcoxon rank sum test). High levels of HOXA9 methylation and low levels of expression in oral SCC cell lines were observed compared to HaCaT, a non-tumorigenic keratinocyte cell line. Re-expression of HOXA9 in the SCC4 oral cancer cell line resulted in diminished proliferation and colony formation. CONCLUSIONS: HOXA9 methylation is frequent in oral cancers and levels are higher in tumors with greater risk of metastasis. Expression of HOXA9 is low in cells with high levels of methylation and reduced expression appears to confer a growth advantage.
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Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/secundario , Metilación de ADN , Proteínas de Homeodominio/genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Regiones Promotoras Genéticas , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Proteínas de Homeodominio/metabolismo , Humanos , Metástasis Linfática , Neoplasias de la Boca/metabolismo , Proyectos Piloto , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , TransfecciónRESUMEN
Persons with pervasive developmental disorders (PDD) exhibit a range of cognitive deficits that hamper their quality of life, including difficulties involving communication, sociability, and perspective-taking. In recent years, a variety of studies in mice that model genetic syndromes with a high risk of PDD have provided insights into the underlying molecular mechanisms associated with these disorders. What is less appreciated is how the molecular anomalies affect neuronal and circuit function to give rise to the cognitive deficits associated with PDD. In this review, we describe genetic mutations that cause PDD and discuss how they alter fundamental social and cognitive processes. We then describe efforts to correct cognitive impairments associated with these disorders and identify areas of further inquiry in the search for molecular targets for therapeutics for PDD.
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Trastornos Generalizados del Desarrollo Infantil/complicaciones , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/genética , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Comunicación Celular , Niño , Preescolar , Trastornos del Conocimiento/psicología , ADN/metabolismo , Metabolismo Energético , Humanos , Calidad de Vida , ARN/metabolismoRESUMEN
INTRODUCTION: Wellbutrin (bupropion hydrochloride; WB), an anti-depressant of the aminoketone class is new highly selective norepinephrine and dopamine reuptake inhibitor; it is effective in the treatment of patients with major depression. MATERIALS AND METHODS: To investigate the in vitro effects of WB in human cultured peripheral blood lymphocytes and human cortical neural (HCN2) cell lines, micronucleus, sister chromatid exchange analysis, cellular viability, and comet assays were employed. The present study is to our knowledge, the first report on WB genotoxicity in cultured human peripheral blood lymphocytes and its cytotoxicity in the HCN2 cell line. We have also investigated the genotoxic potential of WB to induce chromosomal aberrations. RESULTS: WB-induced cytotoxicity (measured as reduction of the nuclear division index) possibly prevented the division of damaged cells. CONCLUSION: We conclude that although, WB exerts potential genotoxic effects in cultured lymphocytes, its cytogenetic effects are very unlikely to occur in blood cultures of WB-administered subjects.
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In this study, we automated the diagnostic procedure of autism spectrum disorder (ASD) with the help of anatomical alterations found in structural magnetic resonance imaging (sMRI) data of the ASD brain and machine learning tools. Initially, the sMRI data was preprocessed using the FreeSurfer toolbox. Further, the brain regions were segmented into 148 regions of interest using the Destrieux atlas. Features such as volume, thickness, surface area, and mean curvature were extracted for each brain region, and the morphological connectivity was computed using Pearson correlation. These morphological connections were fed to XGBoost for feature reduction and to build the diagnostic model. The results showed an average accuracy of 94.16% for the top 18 features. The frontal and limbic regions contributed more features to the classification model. Our proposed method is thus effective for the classification of ASD and can also be useful for the screening of other similar neurological disorders.
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Trastorno del Espectro Autista , Mapeo Encefálico , Humanos , Mapeo Encefálico/métodos , Trastorno del Espectro Autista/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Imagen por Resonancia Magnética/métodos , Aprendizaje AutomáticoRESUMEN
Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.
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Dolor en Cáncer , Neoplasias de la Boca , Hormonas Peptídicas , Humanos , Ratones , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Calcitonina , Polipéptido alfa Relacionado con Calcitonina , Receptores de Péptido Relacionado con el Gen de Calcitonina/genética , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Dolor en Cáncer/tratamiento farmacológico , Células Endoteliales/metabolismo , Neoplasias de la Boca/tratamiento farmacológico , Microambiente TumoralRESUMEN
Tuberous sclerosis complex (TSC) and fragile X syndrome (FXS) are caused by mutations in negative regulators of translation. FXS model mice exhibit enhanced metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). Therefore, we hypothesized that a mouse model of TSC, ΔRG transgenic mice, also would exhibit enhanced mGluR-LTD. We measured the impact of TSC2-GAP mutations on the mTORC1 and ERK signaling pathways and protein synthesis-dependent hippocampal synaptic plasticity in ΔRG transgenic mice. These mice express a dominant/negative TSC2 that binds to TSC1, but has a deletion and substitution mutation in its GAP-domain, resulting in inactivation of the complex. Consistent with previous studies of several other lines of TSC model mice, we observed elevated S6 phosphorylation in the brains of ΔRG mice, suggesting upregulated translation. Surprisingly, mGluR-LTD was not enhanced, but rather was impaired in the ΔRG transgenic mice, indicating that TSC and FXS have divergent synaptic plasticity phenotypes. Similar to patients with TSC, the ΔRG transgenic mice exhibit elevated ERK signaling. Moreover, the mGluR-LTD impairment displayed by the ΔRG transgenic mice was rescued with the MEK-ERK inhibitor U0126. Our results suggest that the mGluR-LTD impairment observed in ΔRG mice involves aberrant TSC1/2-ERK signaling.
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Depresión Sináptica a Largo Plazo/genética , Sistema de Señalización de MAP Quinasas/genética , Receptores de Glutamato Metabotrópico/metabolismo , Esclerosis Tuberosa/complicaciones , Esclerosis Tuberosa/genética , Proteínas Supresoras de Tumor/deficiencia , Análisis de Varianza , Animales , Animales Recién Nacidos , Biofisica , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Hipocampo/patología , Técnicas In Vitro , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-Clamp , Receptores de Glutamato Metabotrópico/genética , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Esclerosis Tuberosa/metabolismo , Esclerosis Tuberosa/patología , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Fracture healing is a physiological process resulting in the regeneration of bone defects by the coordinated action of osteoblasts and osteoclasts. Osteoanabolic drugs have the potential to augment the repair of fractures but have constraints like high costs or undesirable side effects. The bone healing potential of a drug can initially be determined by in vitro studies, but in vivo studies are needed for the final proof of concept. Our objective was to develop a femur osteotomy rodent model that could help researchers understand the development of callus formation following fracture of the shaft of the femur and that could help establish whether a potential drug has bone healing properties. Adult male Wistar albino rats were used after Institutional Animal Ethics Committee clearance. The rodents were anesthetized, and under aseptic conditions, complete transverse fractures at the middle one-third of the shafts of the femurs were created using open osteotomy. The fractures were reduced and internally fixed using intramedullary K-wires, and secondary fracture healing was allowed to take place. After surgery, intraperitoneal analgesics and antibiotics were given for 5 days. Sequential weekly x-rays assessed callus formation. The rats were sacrificed based on radiologically pre-determined time points, and the development of the fracture callus was analyzed radiologically and using immunohistochemistry.
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Fracturas del Fémur , Animales , Fracturas del Fémur/diagnóstico por imagen , Fracturas del Fémur/cirugía , Fémur/diagnóstico por imagen , Fémur/cirugía , Curación de Fractura/fisiología , Masculino , Osteotomía , Ratas , Ratas WistarRESUMEN
BACKGROUND: Mutations in the postsynaptic transmembrane protein neuroligin-3 are highly correlative with autism spectrum disorders (ASDs) and intellectual disabilities (IDs). Fear learning is well studied in models of these disorders, however differences in fear response behaviours are often overlooked. We aim to examine fear behaviour and its cellular underpinnings in a rat model of ASD/ID lacking Nlgn3. METHODS: This study uses a range of behavioural tests to understand differences in fear response behaviour in Nlgn3-/y rats. Following this, we examined the physiological underpinnings of this in neurons of the periaqueductal grey (PAG), a midbrain area involved in flight-or-freeze responses. We used whole-cell patch-clamp recordings from ex vivo PAG slices, in addition to in vivo local-field potential recordings and electrical stimulation of the PAG in wildtype and Nlgn3-/y rats. We analysed behavioural data with two- and three-way ANOVAS and electrophysiological data with generalised linear mixed modelling (GLMM). RESULTS: We observed that, unlike the wildtype, Nlgn3-/y rats are more likely to response with flight rather than freezing in threatening situations. Electrophysiological findings were in agreement with these behavioural outcomes. We found in ex vivo slices from Nlgn3-/y rats that neurons in dorsal PAG (dPAG) showed intrinsic hyperexcitability compared to wildtype. Similarly, stimulating dPAG in vivo revealed that lower magnitudes sufficed to evoke flight behaviour in Nlgn3-/y than wildtype rats, indicating the functional impact of the increased cellular excitability. LIMITATIONS: Our findings do not examine what specific cell type in the PAG is likely responsible for these phenotypes. Furthermore, we have focussed on phenotypes in young adult animals, whilst the human condition associated with NLGN3 mutations appears during the first few years of life. CONCLUSIONS: We describe altered fear responses in Nlgn3-/y rats and provide evidence that this is the result of a circuit bias that predisposes flight over freeze responses. Additionally, we demonstrate the first link between PAG dysfunction and ASD/ID. This study provides new insight into potential pathophysiologies leading to anxiety disorders and changes to fear responses in individuals with ASD.
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Trastorno Autístico , Animales , Trastorno Autístico/metabolismo , Miedo/fisiología , Congelación , Humanos , Neuronas/fisiología , Sustancia Gris Periacueductal/metabolismo , RatasRESUMEN
Oral squamous cell carcinoma (OSCC) patients suffer from poor survival due to metastasis or locoregional recurrence, processes that are both facilitated by perineural invasion (PNI). OSCC has higher rates of PNI than other cancer subtypes, with PNI present in 80% of tumors. Despite the impact of PNI on oral cancer prognosis and pain, little is known about the genes that drive PNI, which in turn drive pain, invasion, and metastasis. In this study, clinical data, preclinical, and in vitro models are leveraged to elucidate the role of neurotrophins in OSCC metastasis, PNI, and pain. The expression data in OSCC patients with metastasis, PNI, or pain demonstrate dysregulation of neurotrophin genes. TrkA and nerve growth factor receptor (NGFR) are focused, two receptors that are activated by NGF, a neurotrophin expressed at high levels in OSCC. It is demonstrated that targeted knockdown of these two receptors inhibits proliferation and invasion in an in vitro and preclinical model of OSCC, and metastasis, PNI, and pain. It is further determined that TrkA knockdown alone inhibits thermal hyperalgesia, whereas NGFR knockdown alone inhibits mechanical allodynia. Collectively the results highlight the ability of OSCC to co-opt different components of the neurotrophin pathway in metastasis, PNI, and pain.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias de la Boca , Carcinoma de Células Escamosas/genética , Humanos , Neoplasias de la Boca/genética , Invasividad Neoplásica/genética , Recurrencia Local de Neoplasia , Procesos Neoplásicos , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso , Dolor , Proteínas Tirosina Quinasas Receptoras , Receptor de Factor de Crecimiento Nervioso , Receptor trkA , Receptores de Factor de Crecimiento Nervioso/genética , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
BACKGROUND: Methotrexate (MTX) is widely used in chemotherapy but its associated hepatotoxicity is a major complication, limiting its use. This study evaluates possible therapeutic effect of oral alpha-ketoglutarate (AKG) supplementation against MTX-induced hepatotoxicity. METHODS: HepG2 cells were used to evaluate in-vitro cyto-protection conferred by AKG against MTX induced cytotoxicity. For in-vivo animal study, rats were divided into three groups. Group-I served as control. Group-II animals were administered single intraperitoneal injection of MTX (20 mg/kg/body weight), while Group-III received MTX as in group-II followed by oral AKG (2 gm/kg body weight) for 5 days. 99mTc-Mebrofenin hepatobiliary study was performed under a gamma camera to determine real time functional status of rats' livers. Multiple parameters concerning hepatic mebrofenin uptake and excretion, including Tpeak and T1/2 peak in control and treated animals were determined. Biochemical analysis of the liver homogenate in terms of hepatic enzyme activities in serum, antioxidant status, tissue factor activity, tissue collagen content and histological analysis of the liver tissue were also done. RESULTS: AKG supplementation significantly reversed MTX induced derangement in activities of serum liver enzymes [ALT and ALP (p = 0.003); AST (p = 0.005)], antioxidant status [LPO and GSH (p = 0.005); CAT (p = 0.004)], tissue factor activity (p = 0.005) and tissue collagen content (p = 0.005). Functional imaging confirmed that hepatic retention and fractional biliary excretion were significantly abnormal in MTX treated group (Tpeak: 234 s ± 40 s; T1/2 peak: 846sec ± 32sec) as compared to AKG supplemented group (Tpeak: 144 s ± 35sec; T1/2peak: 468sec ± 27sec). Hepatic extraction fraction (HEF) was 92.2 ± 1.8%, 48.7 ± 2.6% and 69.8 ± 4.3% in control, MTX and AKG supplemented rats respectively. CONCLUSION: 99mTc-mebrofenin imaging strongly suggests therapeutic action of AKG in protecting liver damage by MTX in rats. Functional imaging parameters correlated well with biochemical and histopathological findings.
Asunto(s)
Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Compuestos de Anilina , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Glicina , Ácidos Cetoglutáricos/metabolismo , Hígado/metabolismo , Metotrexato/metabolismo , Metotrexato/toxicidad , Estrés Oxidativo , RatasRESUMEN
In this report, we discuss the design of a novel collagen/pectin (CP) hybrid composite hydrogel (CPBG) containing in-situ mineralized bioactive glass (BG) particles to simulate an integrative 3D cell environment. Systematic analysis of the CP sol revealed collagen and pectin molecules interacted regardless of both possessing similar net negative charge through the mechanism of surface patch binding interaction. Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) confirmed this associative interaction which resulted in the formation of a hybrid crosslinked network with the BG nanoparticles acting as pseudo crosslink junctions. Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Analysis (EDAX) and Transmission Electron Microscopy (TEM) results confirmed uniform mineralization of BG particles, and their synergetic interaction with the network. The in-vitro bioactivity tests on CPBG indicated the formation of bone-like hydroxyapatite (Ca10(PO4)6(OH)2) microcrystals on its surface after interaction with simulated body fluid. This hydrogel was loaded with a model antifungal drug amphotericin-B (AmB) and tested against Candida albicans. The AmB release kinetics from the hydrogel followed the Fickian mechanism and showed direct proportionality to gel swelling behavior. Rheological analysis revealed the viscoelastic compatibility of CPBG for the mechanical load bearing applications. Cell viability tests indicated appreciable compatibility of the hydrogel against U2OS and HaCaT cell lines. FDA/PI on the hydrogel portrayed preferential U2OS cell adhesion on hydrophobic hydroxyapatite layer compared to hydrophilic surfaces, thereby promising the regeneration of both soft and hard tissues.