RESUMEN
Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Nucleares/genética , Proteínas Portadoras/metabolismo , Cromatina/metabolismo , ADN , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Femenino , Inestabilidad Genómica , Mutación de Línea Germinal , Recombinación Homóloga , Humanos , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Masculino , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Cultivo Primario de Células , Reparación del ADN por RecombinaciónRESUMEN
Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron-specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high-content microscopy, and live-imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome. This function of ATG5 is independent of bulk turnover of synaptic proteins and requires the targeting of PKA inhibitory R1 subunits to autophagosomes. Neuronal loss of ATG5 causes synaptic accumulation of PKA-R1, which sequesters the PKA catalytic subunit and diminishes cAMP/PKA-dependent phosphorylation of postsynaptic cytoskeletal proteins that mediate AMPAR trafficking. Furthermore, ATG5 deletion in glutamatergic neurons augments AMPAR-dependent excitatory neurotransmission and causes the appearance of spontaneous recurrent seizures in mice. Our findings identify a novel role of autophagy in regulating PKA signaling at glutamatergic synapses and suggest the PKA as a target for restoration of synaptic function in neurodegenerative conditions with autophagy dysfunction.
Asunto(s)
Neuronas , Sinapsis , Ratones , Animales , Sinapsis/metabolismo , Neuronas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Transducción de Señal , AutofagiaRESUMEN
BACKGROUND: The identification of novel toxins from overlooked and taxonomically exceptional species bears potential for various pharmacological applications. The remipede Xibalbanus tulumensis, an underwater cave-dwelling crustacean, is the only crustacean for which a venom system has been described. Its venom contains several xibalbin peptides that have an inhibitor cysteine knot (ICK) scaffold. RESULTS: Our screenings revealed that all tested xibalbin variants particularly inhibit potassium channels. Xib1 and xib13 with their eight-cysteine domain similar to spider knottins also inhibit voltage-gated sodium channels. No activity was noted on calcium channels. Expanding the functional testing, we demonstrate that xib1 and xib13 increase PKA-II and Erk1/2 sensitization signaling in nociceptive neurons, which may initiate pain sensitization. Our phylogenetic analysis suggests that xib13 either originates from the common ancestor of pancrustaceans or earlier while xib1 is more restricted to remipedes. The ten-cysteine scaffolded xib2 emerged from xib1, a result that is supported by our phylogenetic and machine learning-based analyses. CONCLUSIONS: Our functional characterization of synthesized variants of xib1, xib2, and xib13 elucidates their potential as inhibitors of potassium channels in mammalian systems. The specific interaction of xib2 with Kv1.6 channels, which are relevant to treating variants of epilepsy, shows potential for further studies. At higher concentrations, xib1 and xib13 activate the kinases PKA-II and ERK1/2 in mammalian sensory neurons, suggesting pain sensitization and potential applications related to pain research and therapy. While tested insect channels suggest that all probably act as neurotoxins, the biological function of xib1, xib2, and xib13 requires further elucidation. A novel finding on their evolutionary origin is the apparent emergence of X. tulumensis-specific xib2 from xib1. Our study is an important cornerstone for future studies to untangle the origin and function of these enigmatic proteins as important components of remipede but also other pancrustacean and arthropod venoms.
Asunto(s)
Canales de Potasio , Animales , Canales de Potasio/metabolismo , Canales de Potasio/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Filogenia , Ratones , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Evolución Molecular , Humanos , Venenos de Artrópodos/químicaRESUMEN
BACKGROUND: The majority of BRCA1-mutant breast cancers are characterized by a triple-negative phenotype and a basal-like molecular subtype, associated with aggressive clinical behavior. Current treatment options are limited, highlighting the need for the development of novel targeted therapies for this tumor subtype. METHODS: Our group previously showed that EZH2 is functionally relevant in BRCA1-deficient breast tumors and blocking EZH2 enzymatic activity could be a potent treatment strategy. To validate the role of EZH2 as a therapeutic target and to identify new synergistic drug combinations, we performed a high-throughput drug combination screen in various cell lines derived from BRCA1-deficient and -proficient mouse mammary tumors. RESULTS: We identified the combined inhibition of EZH2 and the proximal DNA damage response kinase ATM as a novel synthetic lethality-based therapy for the treatment of BRCA1-deficient breast tumors. We show that the combined treatment with the EZH2 inhibitor GSK126 and the ATM inhibitor AZD1390 led to reduced colony formation, increased genotoxic stress, and apoptosis-mediated cell death in BRCA1-deficient mammary tumor cells in vitro. These findings were corroborated by in vivo experiments showing that simultaneous inhibition of EZH2 and ATM significantly increased anti-tumor activity in mice bearing BRCA1-deficient mammary tumors. CONCLUSION: Taken together, we identified a synthetic lethal interaction between EZH2 and ATM and propose this synergistic interaction as a novel molecular combination for the treatment of BRCA1-mutant breast cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Proteínas de la Ataxia Telangiectasia Mutada , Proteína BRCA1 , Neoplasias de la Mama , Proteína Potenciadora del Homólogo Zeste 2 , Indoles , Inhibidores de Proteínas Quinasas , Piridonas , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/deficiencia , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Humanos , Indoles/farmacología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Mutaciones Letales SintéticasRESUMEN
Hyperalgesic priming is characterized by enhanced nociceptor sensitization by pronociceptive mediators, prototypically PGE2 . Priming has gained interest as a mechanism underlying the transition to chronic pain. Which stimuli induce priming and what cellular mechanisms are employed remains incompletely understood. In adult male rats, we present the cytokine Oncostatin M (OSM), a member of the IL-6 family, as an inducer of priming by a novel mechanism. We used a high content microscopy based approach to quantify the activation of endogenous PKA-II and ERK of thousands sensory neurons in culture. Incubation with OSM increased and prolonged ERK activation by agents that increase cAMP production such as PGE2 , forskolin, and cAMP analogs. These changes were specific to IB4/CaMKIIα positive neurons, required protein translation, and increased cAMP-to-ERK signaling. In both, control and OSM-treated neurons, cAMP/ERK signaling involved RapGEF2 and PKA but not Epac. Similar enhancement of cAMP-to-ERK signaling could be induced by GDNF, which acts mostly on IB4/CaMKIIα-positive neurons, but not by NGF, which acts mostly on IB4/CaMKIIα-negative neurons. In vitro, OSM pretreatment rendered baseline TTX-R currents ERK-dependent and switched forskolin-increased currents from partial to full ERK-dependence in small/medium sized neurons. In summary, priming induced by OSM uses a novel mechanism to enhance and prolong coupling of cAMP/PKA to ERK1/2 signaling without changing the overall pathway structure.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hiperalgesia/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Oncostatina M/toxicidad , Animales , Antineoplásicos/toxicidad , Humanos , Hiperalgesia/inducido químicamente , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratas , Ratas Sprague-DawleyRESUMEN
The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t6A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient's dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening.
Asunto(s)
Discapacidades del Desarrollo/genética , Proteínas de Unión al GTP/genética , Progeria/genética , Proteínas de Unión al ARN/genética , Alelos , Consanguinidad , Daño del ADN , Discapacidades del Desarrollo/patología , Genoma Humano , Inestabilidad Genómica , Homocigoto , Humanos , Recién Nacido , Masculino , Mutación , Linaje , Progeria/patología , ARN de Transferencia/genética , Análisis de Secuencia de ARN , Acortamiento del TelómeroRESUMEN
Maturation of nociceptive neurons depends on changes in transcription factors, ion channels and neuropeptides. Mature nociceptors initiate pain in part by drastically reducing the activation threshold via intracellular sensitization signaling. Whether sensitization signaling also changes during development and aging remains so far unknown. Using a novel automated microscopy approach, we quantified changes in intracellular signaling protein expression and in their signaling dynamics, as well as changes in intracellular signaling cascade wiring, in sensory neurons from newborn to senescent (24 months of age) rats. We found that nociceptive subgroups defined by the signaling components protein kinase A (PKA)-RIIß (also known as PRKAR2B) and CaMKIIα (also known as CAMK2A) developed at around postnatal day 10, the time of nociceptor maturation. The integrative nociceptor marker, PKA-RIIß, allowed subgroup segregation earlier than could be achieved by assessing the classical markers TRPV1 and Nav1.8 (also known as SCN10A). Signaling kinetics remained constant over lifetime despite in part strong changes in the expression levels. Strikingly, we found a mechanism important for neuronal memory - i.e. the crosstalk from cAMP and PKA to ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) - to emerge postnatally. Thus, maturation of nociceptors is closely accompanied by altered expression, activation and connectivity of signaling pathways known to be central for pain sensitization and neuronal memory formation.
Asunto(s)
Envejecimiento/genética , AMP Cíclico/genética , Nociceptores/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Animales Recién Nacidos/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Ganglios Espinales/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Canal de Sodio Activado por Voltaje NAV1.8/genética , Ratas , Canales Catiónicos TRPV/genéticaRESUMEN
The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies.
Asunto(s)
Proteínas de Unión al ADN/genética , Respuesta al Choque Térmico/genética , Proteínas Nucleares/genética , Empalme del ARN , ARN Mensajero/genética , ARN no Traducido/genética , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Exones , Células HeLa , Factores de Transcripción del Choque Térmico , Histona Acetiltransferasas , Chaperonas de Histonas , Calor , Humanos , Intrones , Proteínas Nucleares/metabolismo , Dominios Proteicos , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Contribution of the small intestine to systemic inflammation after cardiac arrest (CA) is poorly understood. The objective was to evaluate whether an in vivo rat model of 6 min CA is suitable to initiate intestinal ischaemia-reperfusion-injury and to evaluate histomorphological changes and inflammatory processes in the small intestinal mucosa resp. in sera. METHODS: Adult male Wistar rats were subjected to CA followed by cardio-pulmonary resuscitation. Proximal jejunum and serum was collected at 6 h, 24 h, 72 h and 7 d post return of spontaneous circulation (ROSC) and from a control group. The small intestine was evaluated histomorphologically. Cytokine concentrations were measured in jejunum lysates and sera. RESULTS: Histomorphological evaluation revealed a significant increase in mucosal damage in the jejunum at all timepoints compared to controls (p < 0.0001). In jejunal tissues, concentrations of IL-1α, IL-1ß, IL-10, and TNF-α showed significant peaks at 24 h and were 1.5- to 5.7-fold higher than concentrations at 6 h and in the controls (p < 0.05). In serum, a significant higher amount of cytokine was detected only for IL-1ß at 24 h post-ROSC compared to controls (15.78 vs. 9.76 pg/ml). CONCLUSION: CA resulted in mild small intestinal tissue damage but not in systemic inflammation. A rat model of 6 min CA is not capable to comprehensively mimic a post cardiac arrest syndrome (PCAS). Whether there is a vital influence of the intestine on the PCAS still remains unclear.
Asunto(s)
Modelos Animales de Enfermedad , Paro Cardíaco/patología , Mucosa Intestinal/patología , Intestino Delgado/patología , Daño por Reperfusión/patología , Animales , Citocinas/metabolismo , Paro Cardíaco/complicaciones , Paro Cardíaco/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Masculino , Ratas , Ratas Wistar , Daño por Reperfusión/etiología , Daño por Reperfusión/metabolismo , Factores de TiempoRESUMEN
Knowledge about the molecular structure of protein kinase A (PKA) isoforms is substantial. In contrast, the dynamics of PKA isoform activity in living primary cells has not been investigated in detail. Using a high content screening microscopy approach, we identified the RIIß subunit of PKA-II to be predominantly expressed in a subgroup of sensory neurons. The RIIß-positive subgroup included most neurons expressing nociceptive markers (TRPV1, NaV1.8, CGRP, IB4) and responded to pain-eliciting capsaicin with calcium influx. Isoform-specific PKA reporters showed in sensory-neuron-derived F11 cells that the inflammatory mediator PGE2 specifically activated PKA-II but not PKA-I. Accordingly, pain-sensitizing inflammatory mediators and activators of PKA increased the phosphorylation of RII subunits (pRII) in subgroups of primary sensory neurons. Detailed analyses revealed basal pRII to be regulated by the phosphatase PP2A. Increase of pRII was followed by phosphorylation of CREB in a PKA-dependent manner. Thus, we propose RII phosphorylation to represent an isoform-specific readout for endogenous PKA-II activity in vivo, suggest RIIß as a novel nociceptive subgroup marker, and extend the current model of PKA-II activation by introducing a PP2A-dependent basal state.
Asunto(s)
Capsaicina/farmacología , Nocicepción/efectos de los fármacos , Proteína Fosfatasa 2/genética , Células Receptoras Sensoriales/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Péptido Relacionado con Gen de Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/metabolismo , Calcio/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/genética , Proteína Quinasa Tipo I Dependiente de AMP Cíclico/metabolismo , Ciclosporina/farmacología , Dinoprostona/farmacología , Regulación de la Expresión Génica , Masculino , Canal de Sodio Activado por Voltaje NAV1.8/genética , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Fosforilación , Cultivo Primario de Células , Proteína Fosfatasa 2/metabolismo , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Transducción de Señal , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
The antimetabolite 5-fluorouracil is a widely used chemotherapeutic for the treatment of several solid cancers. However, resistance to 5-fluorouracil remains a major drawback in its clinical use. In this study we report that treatment of HeLa cells with 5-fluorouracil resulted in de novo assembly of stress granules. Moreover, we revealed that stress granule assembly under stress conditions as well as disassembly is altered in cells treated with 5-fluorouracil. Notably, we discovered that RACK1, a protein mediating cell survival and apoptosis, is a component of 5-fluorouracil-induced stress granules. To explore the mode of action of 5-fluorouracil accountable for de novo stress granule assembly, we analyzed 5-fluorouracil metabolites and noticed that stress granule assembly is caused by RNA, not DNA incorporating 5-fluorouracil metabolites. Interestingly, we observed that other RNA incorporating drugs also cause assembly of stress granules. Thus, our results suggest that incorporation of chemotherapeutics into RNA may result in stress granule assembly with potential significance in chemoresistance.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Fluorouracilo/farmacología , ARN/metabolismo , Ribonucleoproteínas/metabolismo , Estrés Fisiológico , Antimetabolitos Antineoplásicos/metabolismo , Azacitidina/metabolismo , Línea Celular , ADN/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Fluorouracilo/metabolismo , Proteínas de Unión al GTP/análisis , Células HeLa , Humanos , Proteínas de Neoplasias/análisis , Estrés Oxidativo , Receptores de Cinasa C Activada , Receptores de Superficie Celular/análisis , Ribonucleoproteínas/análisis , Tioguanina/metabolismoRESUMEN
Functional cell-to-cell variability is ubiquitous in multicellular organisms as well as bacterial populations. Even genetically identical cells of the same cell type can respond differently to identical stimuli. Methods have been developed to analyse heterogeneous populations, e.g., mixture models and stochastic population models. The available methods are, however, either incapable of simultaneously analysing different experimental conditions or are computationally demanding and difficult to apply. Furthermore, they do not account for biological information available in the literature. To overcome disadvantages of existing methods, we combine mixture models and ordinary differential equation (ODE) models. The ODE models provide a mechanistic description of the underlying processes while mixture models provide an easy way to capture variability. In a simulation study, we show that the class of ODE constrained mixture models can unravel the subpopulation structure and determine the sources of cell-to-cell variability. In addition, the method provides reliable estimates for kinetic rates and subpopulation characteristics. We use ODE constrained mixture modelling to study NGF-induced Erk1/2 phosphorylation in primary sensory neurones, a process relevant in inflammatory and neuropathic pain. We propose a mechanistic pathway model for this process and reconstructed static and dynamical subpopulation characteristics across experimental conditions. We validate the model predictions experimentally, which verifies the capabilities of ODE constrained mixture models. These results illustrate that ODE constrained mixture models can reveal novel mechanistic insights and possess a high sensitivity.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Modelos Neurológicos , Células Receptoras Sensoriales/fisiología , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Factor de Crecimiento Nervioso/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The genetic variants leading to impairment of intellectual performance are highly diverse and are still poorly understood. ST3GAL3 encodes the Golgi enzyme ß-galactoside-α2,3-sialyltransferase-III that in humans predominantly forms the sialyl Lewis a epitope on proteins. ST3GAL3 resides on chromosome 1 within the MRT4 locus previously identified to associate with nonsyndromic autosomal recessive intellectual disability. We searched for the disease-causing mutations in the MRT4 family and a second independent consanguineous Iranian family by using a combination of chromosome sorting and next-generation sequencing. Two different missense changes in ST3GAL3 cosegregate with the disease but were absent in more than 1000 control chromosomes. In cellular and biochemical test systems, these mutations were shown to cause ER retention of the Golgi enzyme and drastically impair ST3Gal-III functionality. Our data provide conclusive evidence that glycotopes formed by ST3Gal-III are prerequisite for attaining and/or maintaining higher cognitive functions.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Discapacidad Intelectual/enzimología , Sialiltransferasas/genética , Análisis Mutacional de ADN , Retículo Endoplásmico/metabolismo , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Discapacidad Intelectual/genética , Irán , Masculino , Mutación Missense/genética , Linaje , Plásmidos/genética , Sialiltransferasas/metabolismo , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
Introduction and Objectives: Lipedema is a widespread severe chronic disease affecting mostly women. Characterized by painful bilateral fat accumulation in extremities sparing hands and feet, objective measurement-based diagnosis is currently missing. We tested for characteristic psychometric and/or sensory alterations including pain and for their potential for medical routine diagnosis. Methods: Pain psychometry was assessed using the German Pain Questionnaire. Sensory sensitivity toward painful and nonpainful stimuli was characterized in non-obese lipedema patients and matched controls using the validated quantitative sensory testing (QST) protocol of the German Research Network on Neuropathic Pain. Results: Lipedema patients showed no overt psychometric abnormalities. Pain was reported as somatic rather than psychosomatic aversive. All QST measurements were normal, but the z-score of pressure pain thresholds (PPT) was twofold reduced and the z-score of vibration detection thresholds (VDT) was two and a half times increased. Both thresholds were selectively altered at the affected thigh but not the unaffected hand. Receiver operating characteristic analysis of the combination of PPT and VDT of thigh vs hand into a PVTH score (PPT, VDT, thigh, hand-score) shows high sensitivity and specificity, categorizing correctly 95.8% of the participants as lipedema patients or healthy controls. Bayesian inference analysis corroborated the diagnostic potential of such a combined PVTH score. Conclusion: We propose to assess PPT and VDT at the painful thigh and the pain-free hand. Combination in a PVTH score may allow a convenient lipedema diagnosis early during disease development.
RESUMEN
Pain, which is a central characteristic of lipedema, allows differentiation from other fat tissue diseases. The analysis of the multiple aspects of pain beyond a quantification of pain scale scores could make molecular disease and therapy mechanisms accessible. Lipedema pain is causally linked to lipedema fat. First robust data show peripheral sensory changes. Tissue weight and systemic inflammation are becoming less likely as causes for the experianced pain. Furthermore, genetics and hormonal influences need to be investigated. Lipedema pain cannot currently be treated with drugs. Physical therapy shows transient relief. Liposuction has been shown to have a long-term effect on pain. The potential of modulating the perception of pain with psychotherapeutic approaches is emerging as a potentially effective new therapeutic approach.
Asunto(s)
Lipectomía , Lipedema , Humanos , Lipedema/diagnóstico , Dolor/diagnóstico , Lipectomía/efectos adversos , Tejido AdiposoRESUMEN
Peripheral neuropathy is a common side effect of cancer treatment with paclitaxel. The mechanisms by which paclitaxel is transported into neurons, which are essential for preventing neuropathy, are not well understood. We studied the uptake mechanisms of paclitaxel into neurons using inhibitors for endocytosis, autophagy, organic anion-transporting polypeptide (OATP) drug transporters, and derivatives of paclitaxel. RT-qPCR was used to investigate the expression levels of OATPs in different neuronal tissues and cell lines. OATP transporters were pharmacologically inhibited or modulated by overexpression and CRISPR/Cas9-knock-out to investigate paclitaxel transport in neurons. Through these experiments, we identified OATP1A1 and OATP1B2 as the primary neuronal transporters for paclitaxel. In vitro inhibition of OATP1A1 and OAT1B2 by glycyrrhizic acid attenuated neurotoxicity, while paclitaxel's antineoplastic effects were sustained in cancer cell lines. In vivo, glycyrrhizic acid prevented paclitaxel-induced toxicity and improved behavioral and electrophysiological measures. This study indicates that a set of OATPs are involved in paclitaxel transport into neurons. The inhibition of OATP1A1 and OATP1B2 holds a promising strategy to prevent paclitaxel-induced peripheral neuropathy.
Asunto(s)
Transportadores de Anión Orgánico , Enfermedades del Sistema Nervioso Periférico , Humanos , Paclitaxel/efectos adversos , Ácido Glicirrínico/farmacología , Transportadores de Anión Orgánico/metabolismo , Neuronas/metabolismo , Proteínas de Transporte de Membrana , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/prevención & controlRESUMEN
ABSTRACT: Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Analgésicos Opioides , Colforsina/farmacología , Nocicepción , Células Receptoras Sensoriales , Canales IónicosRESUMEN
Many extracellular factors sensitize nociceptors. Often they act simultaneously and/or sequentially on nociceptive neurons. We investigated if stimulation of the protein kinase C epsilon (PKCε) signaling pathway influences the signaling of a subsequent sensitizing stimulus. Central in activation of PKCs is their transient translocation to cellular membranes. We found in cultured nociceptive neurons that only a first stimulation of the PKCε signaling pathway resulted in PKCε translocation. We identified a novel inhibitory cascade to branch off upstream of PKCε, but downstream of Epac via IP3-induced calcium release. This signaling branch actively inhibited subsequent translocation and even attenuated ongoing translocation. A second 'sensitizing' stimulus was rerouted from the sensitizing to the inhibitory branch of the signaling cascade. Central for the rerouting was cytoplasmic calcium increase and CaMKII activation. Accordingly, in behavioral experiments, activation of calcium stores switched sensitizing substances into desensitizing substances in a CaMKII-dependent manner. This mechanism was also observed by in vivo C-fiber electrophysiology corroborating the peripheral location of the switch. Thus, we conclude that the net effect of signaling in nociceptors is defined by the context of the individual cell's signaling history.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Neuronas/metabolismo , Nociceptores/fisiología , Umbral del Dolor/fisiología , Agonistas Adrenérgicos beta/farmacología , Análisis de Varianza , Animales , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Ganglios Espinales/citología , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/fisiopatología , Inositol 1,4,5-Trifosfato/farmacología , Isoproterenol/farmacología , Masculino , Fibras Nerviosas/efectos de los fármacos , Fibras Nerviosas/fisiología , Neuronas/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Proteína Quinasa C-epsilon/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Rianodina/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Canales Catiónicos TRPV/metabolismo , Tionucleótidos/farmacología , Uridina Trifosfato/farmacologíaRESUMEN
Electrophysiological studies demonstrate that transient receptor potential vanilloid subtype 1 (TRPV1) is involved in neuronal transmission. Although it is expressed in the peripheral as well as the central nervous system, the questions remain whether TRPV1 is present in synaptic structures and whether it is involved in synaptic processes. In the present study we gathered evidence that TRPV1 can be detected in spines of cortical neurons, that it colocalizes with both pre- and postsynaptic proteins, and that it regulates spine morphology. Moreover, TRPV1 is also present in biochemically prepared synaptosomes endogenously. In F11 cells, a cell line derived from dorsal-root-ganglion neurons, TRPV1 is enriched in the tips of elongated filopodia and also at sites of cell-cell contact. In addition, we also detected TRPV1 in synaptic transport vesicles, and in transport packets within filopodia and neurites. Using FM4-64 dye, we demonstrate that recycling and/or fusion of these vesicles can be rapidly modulated by TRPV1 activation, leading to rapid reorganization of filopodial structure. These data suggest that TRPV1 is involved in processes such as neuronal network formation, synapse modulation and release of synaptic transmitters.
Asunto(s)
Sinapsis/metabolismo , Canales Catiónicos TRPV/metabolismo , Vesículas Transportadoras/metabolismo , Animales , Línea Celular , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Ratones , Neuritas/metabolismo , Neuronas/metabolismo , Transporte de Proteínas , Seudópodos/genética , Seudópodos/metabolismo , Sinapsis/genética , Canales Catiónicos TRPV/genéticaRESUMEN
Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.