RESUMO
BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.
Assuntos
Modelos Animais de Doenças , Microglia , Neurônios , Receptores Purinérgicos P2X7 , Convulsões , Animais , Microglia/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genética , Masculino , Camundongos , Convulsões/metabolismo , Convulsões/genética , Neurônios/metabolismo , Feminino , Camundongos Endogâmicos C57BL , Ácido Caínico , Epilepsias Mioclônicas/metabolismo , Epilepsias Mioclônicas/genética , Hipocampo/metabolismo , Estado Epiléptico/metabolismo , Estado Epiléptico/genética , Camundongos Knockout , Pentilenotetrazol , Transdução de Sinais , Neurônios GABAérgicos/metabolismo , Epilepsia/metabolismo , Epilepsia/genética , Encéfalo/metabolismoRESUMO
María Teresa Miras Portugal devoted most of her scientific life to the study of purinergic signalling. In an important part of her work, she used a model system: the chromaffin cells of the adrenal medulla. It was in these cells that she identified diadenosine polyphosphates, from which she proceeded to the study of adrenomedullary purinome: nucleotide synthesis and degradation, adenosine transport, nucleotide uptake into chromaffin granules, exocytotic release of nucleotides and autocrine regulation of chromaffin cell function via purinoceptors. This short review will focus on the current state of knowledge of the purinoceptors of adrenal chromaffin cells, a subject to which María Teresa made seminal contributions and which she continued to study until the end of her scientific life.
Assuntos
Medula Suprarrenal , Células Cromafins , Portugal , Medula Suprarrenal/metabolismo , Receptores Purinérgicos/metabolismo , Nucleotídeos/metabolismoRESUMO
Neuropathic pain is a form of chronic pain arising from damage of the neural cells that sense, transmit or process sensory information. Given its growing prevalence and common refractoriness to conventional analgesics, the development of new drugs with pain relief effects constitutes a prominent clinical need. In this respect, drugs that reduce activity of sensory neurons by modulating ion channels hold the promise to become effective analgesics. Here, we evaluated the mechanical antinociceptive effect of IQM-PC332, a novel ligand of the multifunctional protein downstream regulatory element antagonist modulator (DREAM) in rats subjected to chronic constriction injury of the sciatic nerve as a model of neuropathic pain. IQM-PC332 administered by intraplantar (0.01-10 µg) or intraperitoneal (0.02-1 µg/kg) injection reduced mechanical sensitivity by ≈100% of the maximum possible effect, with ED50 of 0.27 ± 0.05 µg and 0.09 ± 0.01 µg/kg, respectively. Perforated-patch whole-cell recordings in isolated dorsal root ganglion (DRG) neurons showed that IQM-PC332 (1 and 10 µM) reduced ionic currents through voltage-gated K+ channels responsible for A-type potassium currents, low, T-type, and high voltage-activated Ca2+ channels, and transient receptor potential vanilloid-1 (TRPV1) channels. Furthermore, IQM-PC332 (1 µM) reduced electrically evoked action potentials in DRG neurons from neuropathic animals. It is suggested that by modulating multiple DREAM-ion channel signaling complexes, IQM-PC332 may serve a lead compound of novel multimodal analgesics.
Assuntos
Analgésicos/farmacologia , Proteínas Interatuantes com Canais de Kv/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Traumatismos dos Nervos Periféricos/complicações , Animais , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Ligantes , Masculino , Potenciais da Membrana/efeitos dos fármacos , Neuralgia/metabolismo , Traumatismos dos Nervos Periféricos/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition where motor neurons (MNs) degenerate. Most of the ALS cases are sporadic (sALS), whereas 10% are hereditarily transmitted (fALS), among which mutations are found in the gene that codes for the enzyme superoxide dismutase 1 (SOD1). A central question in ALS field is whether causative mutations display selective alterations not found in sALS patients, or they converge on shared molecular pathways. To identify specific and common mechanisms for designing appropriate therapeutic interventions, we focused on the SOD1-mutated (SOD1-ALS) versus sALS patients. Since ALS pathology involves different cell types other than MNs, we generated lymphoblastoid cell lines (LCLs) from sALS and SOD1-ALS patients and healthy donors and investigated whether they show changes in oxidative stress, mitochondrial dysfunction, metabolic disturbances, the antioxidant NRF2 pathway, inflammatory profile, and autophagic flux. Both oxidative phosphorylation and glycolysis appear to be upregulated in lymphoblasts from sALS and SOD1-ALS. Our results indicate significant differences in NRF2/ARE pathway between sALS and SOD1-ALS lymphoblasts. Furthermore, levels of inflammatory cytokines and autophagic flux discriminate between sALS and SOD1-ALS lymphoblasts. Overall, different molecular mechanisms are involved in sALS and SOD1-ALS patients and thus, personalized medicine should be developed for each case.
Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/imunologia , Linfócitos/imunologia , Mutação/genética , Medicina de Precisão , Superóxido Dismutase-1/genética , Ácidos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/genética , Autofagia/genética , Linhagem Celular Transformada , Metabolismo Energético , Feminino , Heterozigoto , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Metabolômica , Pessoa de Meia-Idade , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Consumo de Oxigênio , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
We have investigated whether the stress response mediated by the adrenal medulla in rats subjected to chronic constriction injury of the sciatic nerve (CCI) modulates their nocifensive behavior. Treatment with SK29661 (300 mg/kg; intraperitoneal (I.P.)), a selective inhibitor of phenylethanolamine N-methyltransferase (PNMT) that converts noradrenaline (NA) into adrenaline (A), fully reverted mechanical allodynia in the injured hind paw without affecting mechanical sensitivity in the contralateral paw. The effect was fast and reversible and was associated with a decrease in the A to NA ratio (A/NA) in the adrenal gland and circulating blood, an A/NA that was elevated by CCI. 1,2,3,4-tetrahydroisoquinoline-7-sulfonamide (SKF29661) did not affect exocytosis evoked by Ca2+ entry as well as major ionic conductances (voltage-gated Na+, Ca2+, and K+ channels, nicotinic acetylcholine receptors) involved in stimulus-secretion coupling in chromaffin cells, suggesting that it acted by changing the relative content of the two adrenal catecholamines. Denervation of the adrenal medulla by surgical splanchnectomy attenuated mechanical allodynia in neuropathic animals, hence confirming the involvement of the adrenal medulla in the pathophysiology of the CCI model. Inhibition of PNMT appears to be an effective and probably safe way to modulate adrenal medulla activity and, in turn, to alleviate pain secondary to the injury of a peripheral nerve.
Assuntos
Medula Suprarrenal/fisiologia , Hiperalgesia/fisiopatologia , Neuralgia/metabolismo , Glândulas Suprarrenais/efeitos dos fármacos , Medula Suprarrenal/metabolismo , Animais , Catecolaminas/farmacologia , Células Cromafins/efeitos dos fármacos , Modelos Animais de Doenças , Epinefrina/metabolismo , Hiperalgesia/metabolismo , Masculino , Neuralgia/fisiopatologia , Norepinefrina/metabolismo , Feniletanolamina N-Metiltransferase/antagonistas & inibidores , Feniletanolamina N-Metiltransferase/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
We have tested the hypothesis that neuropathic pain acting as a stressor drives functional plasticity in the sympathoadrenal system. The relation between neuropathic pain and adrenal medulla function was studied with behavioral, immunohistochemical and electrophysiological techniques in rats subjected to chronic constriction injury of the sciatic nerve. In slices of the adrenal gland from neuropathic animals, we have evidenced increased cholinergic innervation and spontaneous synaptic activity at the splanchnic nerveâ»chromaffin cell junction. Likewise, adrenomedullary chromaffin cells displayed enlarged acetylcholine-evoked currents with greater sensitivity to α-conotoxin RgIA, a selective blocker of α9 subunit-containing nicotinic acetylcholine receptors, as well as increased exocytosis triggered by voltage-activated Ca2+ entry. Altogether, these adaptations are expected to facilitate catecholamine output into the bloodstream. Last, but most intriguing, functional and immunohistochemical data indicate that P2X3 and P2X7 purinergic receptors and transient receptor potential vanilloid-1 (TRPV1) channels are overexpressed in chromaffin cells from neuropathic animals. These latter observations are reminiscent of molecular changes characteristic of peripheral sensitization of nociceptors following the lesion of a peripheral nerve, and suggest that similar phenomena can occur in other tissues, potentially contributing to behavioral manifestations of neuropathic pain.
Assuntos
Neuralgia/patologia , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Canais de Cátion TRPV/metabolismo , Acetilcolina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Medula Suprarrenal/metabolismo , Medula Suprarrenal/patologia , Animais , Capsaicina/farmacologia , Catecolaminas/metabolismo , Células Cromafins/citologia , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Modelos Animais de Doenças , Potenciais Evocados/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Gânglios Espinais/patologia , Gânglios Espinais/fisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Neuralgia/metabolismo , Neurônios/patologia , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X3/genética , Receptores Purinérgicos P2X7/genética , Canais de Cátion TRPV/genéticaRESUMO
Hypomorphic mutations in the gene encoding the tissue-nonspecific alkaline phosphatase (TNAP) enzyme, ALPL in human or Akp2 in mice, cause hypophosphatasia (HPP), an inherited metabolic bone disease also characterized by spontaneous seizures. Initially, these seizures were attributed to the impairment of GABAergic neurotransmission caused by altered vitamin B6 (vit-B6) metabolism. However, clinical cases in human newborns and adults whose convulsions are refractory to pro-GABAergic drugs but controlled by the vit-B6 administration, suggest that other factors are involved. Here, to evaluate whether neurodevelopmental alterations are underlying the seizures associated to HPP, we performed morphological and functional characterization of postnatal homozygous TNAP null mice, a model of HPP. These analyses revealed that TNAP deficient mice present an increased proliferation of neural precursors, an altered neuronal morphology, and an augmented neuronal activity. We found that these alterations were associated with a partial downregulation of the purinergic P2X7 receptor (P2X7R). Even though deficient P2X7R mice present similar neurodevelopmental alterations, they do not develop neonatal seizures. Accordingly, we found that the additional blockage of P2X7R prevent convulsions and extend the lifespan of mice lacking TNAP. In agreement with these findings, we also found that exogenous administration of ATP or TNAP antagonists induced seizures in adult wild-type mice by activating P2X7R. Finally, our results also indicate that the anticonvulsive effects attributed to vit-B6 may be due to its capacity to block P2X7R. Altogether, these findings suggest that the purinergic signalling regulates the neurodevelopmental alteration and the neonatal seizures associated to HPP.
Assuntos
Fosfatase Alcalina/genética , Doenças Ósseas Metabólicas/genética , Hipofosfatasia/genética , Receptores Purinérgicos P2X7/genética , Convulsões/genética , Trifosfato de Adenosina/administração & dosagem , Fosfatase Alcalina/antagonistas & inibidores , Animais , Doenças Ósseas Metabólicas/tratamento farmacológico , Doenças Ósseas Metabólicas/metabolismo , Doenças Ósseas Metabólicas/fisiopatologia , Calcinose/genética , Calcinose/metabolismo , Calcinose/fisiopatologia , Cálcio/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipofosfatasia/tratamento farmacológico , Hipofosfatasia/metabolismo , Hipofosfatasia/fisiopatologia , Camundongos , Camundongos Knockout , Mutação , Receptores Purinérgicos P2X7/biossíntese , Convulsões/metabolismo , Convulsões/fisiopatologia , Vitamina B 6/administração & dosagemRESUMO
UNLABELLED: Neuroinflammation is thought to contribute to the pathogenesis and maintenance of temporal lobe epilepsy, but the underlying cell and molecular mechanisms are not fully understood. The P2X7 receptor is an ionotropic receptor predominantly expressed on the surface of microglia, although neuronal expression has also been reported. The receptor is activated by the release of ATP from intracellular sources that occurs during neurodegeneration, leading to microglial activation and inflammasome-mediated interleukin 1ß release that contributes to neuroinflammation. Using a reporter mouse in which green fluorescent protein is induced in response to the transcription of P2rx7, we show that expression of the receptor is selectively increased in CA1 pyramidal and dentate granule neurons, as well as in microglia in mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus. P2X7 receptor levels were increased in hippocampal subfields in the mice and in resected hippocampus from patients with pharmacoresistant temporal lobe epilepsy. Cells transcribing P2rx7 in hippocampal slices from epileptic mice displayed enhanced agonist-evoked P2X7 receptor currents, and synaptosomes from these animals showed increased P2X7 receptor levels and altered calcium responses. A 5 d treatment of epileptic mice with systemic injections of the centrally available, potent, and specific P2X7 receptor antagonist JNJ-47965567 (30 mg/kg) significantly reduced spontaneous seizures during continuous video-EEG monitoring that persisted beyond the time of drug presence in the brain. Hippocampal sections from JNJ-47965567-treated animals obtained >5 d after treatment ceased displayed strongly reduced microgliosis and astrogliosis. The present study suggests that targeting the P2X7 receptor has anticonvulsant and possibly disease-modifying effects in experimental epilepsy. SIGNIFICANCE STATEMENT: Temporal lobe epilepsy is the most common and drug-resistant form of epilepsy in adults. Neuroinflammation is implicated as a pathomechanism, but the upstream mechanisms driving gliosis and how important this is for seizures remain unclear. In our study, we show that the ATP-gated P2X7 receptor is upregulated in experimental epilepsy and resected hippocampus from epilepsy patients. Targeting the receptor with a new centrally available antagonist, JNJ-47965567, suppressed epileptic seizures well beyond the time of treatment and reduced underlying gliosis in the hippocampus. The findings suggest a potential disease-modifying treatment for epilepsy based on targeting the P2X7 receptor.
Assuntos
Epilepsia do Lobo Temporal/complicações , Epilepsia do Lobo Temporal/tratamento farmacológico , Gliose/tratamento farmacológico , Gliose/etiologia , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Convulsões/tratamento farmacológico , Convulsões/etiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Adolescente , Adulto , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Epilepsia do Lobo Temporal/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Piperazinas/metabolismo , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores da Agregação Plaquetária/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Adulto JovemRESUMO
Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by ectopic expression of the four factors Oct4, Klf4, Sox2, and Myc. Here, we investigated the role of Gata4 in the reprogramming process and present evidence for a negative role of this family of transcription factors in the induction of pluripotency. Coexpression of Gata4 with Oct4, Klf4, and Sox2 with or without Myc in mouse embryonic fibroblasts greatly impaired reprogramming and endogenous Nanog expression. The lack of Nanog upregulation was associated with a blockade in the transition from the initiation phase of reprogramming to the full pluripotent state characteristic of iPS cells. Addition of Nanog to the reprogramming cocktail blocked the deleterious effects observed with Gata4 expression. Downregulation of endogenous Gata4 by short hairpin RNAs during reprogramming both accelerated and increased the efficiency of the process and augmented the mRNA levels of endogenous Nanog. Using comparative genomics, we identified a consensus binding site for Gata factors in an evolutionary conserved region located 9 kb upstream of the Nanog gene. Using chromatin immunoprecipitation, gel retardation, and luciferase assays, we found that Gata4 bound to this region and inhibited Nanog transcription in mouse embryonic stem cells. Overall, our results describe for first time the negative effect of Gata4 in the reprogramming of somatic cells and highlight the role of Gata factors in the transcriptional networks that control cell lineage choices in the early embryo.
Assuntos
Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Fator de Transcrição GATA4/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação para Baixo , Ensaio de Desvio de Mobilidade Eletroforética , Fator de Transcrição GATA4/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Fatores de Transcrição SOXB1/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
The development of hepatoma-based in vitro models to study hepatocyte physiology is an invaluable tool for both industry and academia. Here, we develop an in vitro model based on the HepG2 cell line that produces chenodeoxycholic acid, the main bile acid in humans, in amounts comparable to human hepatocytes. A combination of adenoviral transfections for CCAAT/enhancer-binding protein ß (C/EBPß), hepatocyte nuclear factor 4α (HNF4α), and constitutive androstane receptor (CAR) decreased intracellular glutamate, succinate, leucine, and valine levels in HepG2 cells, suggestive of a switch to catabolism to increase lipogenic acetyl CoA and increased anaplerosis to replenish the tricarboxylic acid cycle. Transcripts of key genes involved in bile acid synthesis were significantly induced by approximately 160-fold. Consistently, chenodeoxycholic acid production rate was increased by more than 20-fold. Comparison between mRNA and bile acid levels suggest that 12-alpha hydroxylation of 7-alpha-hydroxy-4-cholesten-3-one is the limiting step in cholic acid synthesis in HepG2 cells. These data reveal that introduction of three hepatocyte-related transcription factors enhance anabolic reactions in HepG2 cells and provide a suitable model to study bile acid biosynthesis under pathophysiological conditions.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ácido Quenodesoxicólico/biossíntese , Perfilação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/metabolismo , Metabolômica , Receptores Citoplasmáticos e Nucleares/metabolismo , Acetilcoenzima A/metabolismo , Adenoviridae/genética , Aminoácidos/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Receptor Constitutivo de Androstano , Expressão Gênica , Vetores Genéticos , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Humanos , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Receptores Citoplasmáticos e Nucleares/genética , TransfecçãoRESUMO
Although the trigger for the neurodegenerative disease process is unknown, the relevance of aging stands out as a major risk for the development of neurodegeneration. In this review, we highlighted the relationship between the different cellular mechanisms that occur as a consequence of aging and transcription factor nuclear factor erythroid-2-related factor 2 (NRF2) and the connection with the TAU protein. We focused on the relevance of NRF2 in the main processes involved in neurodegeneration and associated with aging, such as genomic instability, protein degradation systems (proteasomes/autophagy), cellular senescence, and stem cell exhaustion, as well as inflammation. We also analyzed the effect of aging on TAU protein levels and its aggregation and spread process. Finally, we investigated the interconnection between NRF2 and TAU and the relevance of alterations in the NRF2 signaling pathway in both primary and secondary tauopathies. All these points highlight NRF2 as a possible therapeutic target for tauopathies.
RESUMO
This work reports the first electroanalytical bioplatform to date for the determination of antibodies against aquaporin-4 (AQP4-Abs), whose serum level is considered as relevant biomarker for certain autoimmune diseases. The bioplatform relies on the use of magnetic microparticles modified with the biotinylated protein for the capture of specific antibodies. The captured IgGs are enzymatically labelled with a secondary antibody conjugated to the horseradish peroxidase (HRP) enzyme. Amperometric transduction is performed using the H2O2/hydroquinone (HQ) system, which results in a cathodic current variation directly proportional to the concentration of the target antibodies. The evaluation of the analytical and operational characteristics of the developed bioplatform shows that it is competitive in terms of sensitivity with the only biosensor reported to date as well as with the commercially available ELISA kits. The achieved limit of detection value is 8.8 pg mL-1. In addition, compared to ELISA kits, the developed bioplatform is advantageous in terms of cost and point of care operation ability. The bioplatform was applied to the analysis of control serum samples with known AQP4-Abs contents as well as of sera from healthy individuals and patients diagnosed with Systemic Lupus Erythematosus (SLE) and Alzheimer (AD) diseases, providing results in agreement with the ELISA methodology.
Assuntos
Peróxido de HidrogênioRESUMO
Autologous cell replacement therapy for inherited metabolic disorders requires the correction of the underlying genetic mutation in patient's cells. An unexplored alternative for females affected from X-linked diseases is the clonal selection of cells randomly silencing the X-chromosome containing the mutant allele, without in vivo or ex vivo genome editing. In this report, we have isolated dermal fibroblasts from a female patient affected of ornithine transcarbamylase deficiency and obtained clones based on inactivation status of either maternally or paternally inherited X chromosome, followed by differentiation to hepatocytes. Hepatocyte-like cells derived from these clones display indistinct features characteristic of hepatocytes, but express either the mutant or wild type OTC allele depending on X-inactivation pattern. When clonally derived hepatocyte-like cells were transplanted into FRG® KO mice, they were able to colonize the liver and recapitulate OTC-dependent phenotype conditioned by X-chromosome inactivation pattern. This approach opens new strategies for cell therapy of X-linked metabolic diseases and experimental in vitro models for drug development for such diseases.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Hepatócitos , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Doença da Deficiência de Ornitina Carbomoiltransferase/terapia , Inativação do Cromossomo X/genética , Alelos , Animais , Diferenciação Celular , Células Cultivadas , Células Clonais , Derme/citologia , Feminino , Fibroblastos , Hepatócitos/transplante , Humanos , Camundongos Knockout , Mutação , Cromossomo X/genéticaRESUMO
The voltage-sensitive sodium channel NaV1.1 plays a critical role in regulating excitability of GABAergic neurons and mutations in the corresponding gene are associated to Dravet syndrome and other forms of epilepsy. The activity of this channel is regulated by several protein kinases. To identify novel regulatory kinases we screened a library of activated kinases and we found that AKT1 was able to directly phosphorylate NaV1.1. In vitro kinase assays revealed that the phosphorylation site was located in the C-terminal part of the large intracellular loop connecting domains I and II of NaV1.1, a region that is known to be targeted by other kinases like PKA and PKC. Electrophysiological recordings revealed that activated AKT1 strongly reduced peak Na+ currents and displaced the inactivation curve to more negative potentials in HEK-293 cell stably expressing NaV1.1. These alterations in current amplitude and steady-state inactivation were mimicked by SC79, a specific activator of AKT1, and largely reverted by triciribine, a selective inhibitor. Neurons expressing endogenous NaV1.1 in primary cultures were identified by expressing a fluorescent protein under the NaV1.1 promoter. There, we also observed a strong decrease in the current amplitude after addition of SC79, but small effects on the inactivation parameters. Altogether, we propose a novel mechanism that might regulate the excitability of neural networks in response to AKT1, a kinase that plays a pivotal role under physiological and pathological conditions, including epileptogenesis.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.1/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Animais , Fenômenos Eletrofisiológicos , Epilepsias Mioclônicas/genética , Células HEK293 , Humanos , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Rede Nervosa/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/agonistas , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ribonucleosídeos/farmacologia , Agonistas de Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
The purinergic system is one of the oldest cell-to-cell communication mechanisms and exhibits relevant functions in the regulation of the central nervous system (CNS) development. Amongst the components of the purinergic system, the ionotropic P2X7 receptor (P2X7R) stands out as a potential regulator of brain pathology and physiology. Thus, P2X7R is known to regulate crucial aspects of neuronal cell biology, including axonal elongation, path-finding, synapse formation and neuroprotection. Moreover, P2X7R modulates neuroinflammation and is posed as a therapeutic target in inflammatory, oncogenic and degenerative disorders. However, the lack of reliable technical and pharmacological approaches to detect this receptor represents a major hurdle in its study. Here, we took advantage of the P2rx7-EGFP reporter mouse, which expresses enhanced green fluorescence protein (EGFP) immediately downstream of the P2rx7 proximal promoter, to conduct a detailed study of its distribution. We performed a comprehensive analysis of the pattern of P2X7R expression in the brain of E18.5 mouse embryos revealing interesting areas within the CNS. Particularly, strong labelling was found in the septum, as well as along the entire neural roof plate zone of the brain, except chorioidal roof areas, but including specialized circumventricular roof formations, such as the subfornical and subcommissural organs (SFO; SCO). Moreover, our results reveal what seems a novel circumventricular organ, named by us postarcuate organ (PArcO). Furthermore, this study sheds light on the ongoing debate regarding the specific presence of P2X7R in neurons and may be of interest for the elucidation of additional roles of P2X7R in the idiosyncratic histologic development of the CNS and related systemic functions.
Assuntos
Encéfalo/patologia , Órgãos Circunventriculares/patologia , Epêndima/patologia , Neuroglia/patologia , Animais , Encéfalo/metabolismo , Órgãos Circunventriculares/metabolismo , Epêndima/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Camundongos Transgênicos , Neuroglia/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Receptores Purinérgicos P2X7/metabolismoRESUMO
Prolonged seizures (status epilepticus, SE) may drive hippocampal dysfunction and epileptogenesis, at least partly, through an elevation in neurogenesis, dysregulation of migration and aberrant dendritic arborization of newly-formed neurons. MicroRNA-22 was recently found to protect against the development of epileptic foci, but the mechanisms remain incompletely understood. Here, we investigated the contribution of microRNA-22 to SE-induced aberrant adult neurogenesis. SE was induced by intraamygdala microinjection of kainic acid (KA) to model unilateral hippocampal neuropathology in mice. MicroRNA-22 expression was suppressed using specific oligonucleotide inhibitors (antagomir-22) and newly-formed neurons were visualized using the thymidine analog iodo-deoxyuridine (IdU) and a green fluorescent protein (GFP)-expressing retrovirus to visualize the dendritic tree and synaptic spines. Using this approach, we quantified differences in the rate of neurogenesis and migration, the structure of the apical dendritic tree and density and morphology of dendritic spines in newly-formed neurons.SE resulted in an increased rate of hippocampal neurogenesis, including within the undamaged contralateral dentate gyrus (DG). Newly-formed neurons underwent aberrant migration, both within the granule cell layer and into ectopic sites. Inhibition of microRNA-22 exacerbated these changes. The dendritic diameter and the density and average volume of dendritic spines were unaffected by SE, but these parameters were all elevated in mice in which microRNA-22 was suppressed. MicroRNA-22 inhibition also reduced the length and complexity of the dendritic tree, independently of SE. These data indicate that microRNA-22 is an important regulator of morphogenesis of newly-formed neurons in adults and plays a role in supressing aberrant neurogenesis associated with SE.
RESUMO
Glycine plays two roles in neurotransmission. In caudal areas like the spinal cord and the brainstem, it acts as an inhibitory neurotransmitter, but in all regions of the CNS, it also works as a co-agonist with L-glutamate at N-methyl-D-aspartate receptors (NMDARs). The glycine fluxes in the CNS are regulated by two specific transporters for glycine, GlyT1 and GlyT2, perhaps with the cooperation of diverse neutral amino acid transporters like Asc-1 or SNAT5/SN2. While GlyT2 and Asc-1 are neuronal proteins, GlyT1 and SNAT5 are mainly astrocytic, although neuronal forms of GlyT1 also exist. GlyT1 has attracted considerable interest from the medical community and the pharmaceutical industry since compelling evidence indicates a clear association with the functioning of NMDARs, whose activity is decreased in various psychiatric illnesses. By controlling extracellular glycine, transporter inhibitors might potentiate the activity of NMDARs without activating excitotoxic processes. Physiologically, GlyT1 is a central actor in the cross talk between glutamatergic, glycinergic, dopaminergic, and probably other neurotransmitter systems. Many of these relationships begin to be unraveled by studies performed in recent years using genetic and pharmacological models. These studies are also clarifying the interactions between glycine, glycine transporters, and other co-agonists of the glycine site of NMDARs like D-serine. These findings are also relevant to understand the pathophysiology of devastating diseases like schizophrenia, depression, anxiety, epilepsy, stroke, and chronic pain.
Assuntos
Encéfalo/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Animais , Glicina/metabolismo , Humanos , CamundongosRESUMO
BACKGROUND: Several studies have reported the direct conversion of mouse fibroblasts to hepatocyte-like cells with different degrees of maturation by expression of hepatic fate-conversion factors. METHODS: We have used a combination of lentiviral vectors expressing hepatic fate-conversion factors with Oct4, Sox2, Klf4, and Myc to convert mouse embryonic fibroblasts into hepatic cells. RESULTS: We have generated hepatic cells with progenitor-like features (iHepL cells). iHepL cells displayed basic hepatocyte functions but failed to perform functions characteristic of mature hepatocytes such as significant Cyp450 or urea cycle activities. iHepL cells expressed multiple hepatic-specific transcription factors and functional genes characteristic of immature hepatocytes and cholangiocytes, as well as high levels of Foxl1, Cd24a, and Lgr5, specific markers of hepatic progenitor cells. When transplanted into partial hepatectomized and hepatic irradiated mice, they differentiated into hepatocytes and cholangiocytes. However, iHepL cells formed malignant non-teratoma cell aggregations in one out of five engrafted livers and five out of five xenografts assays. All the cells in these tumors had silenced key hepatic fate-conversion factors, and lost hepatic features. CONCLUSIONS: This study highlights the dangers of using pluripotency factors in reprogramming strategies when fate-conversion factors are silenced in vivo, and urges us to perform extensive tumorigenic tests in reprogrammed cells.
Assuntos
Carcinogênese/genética , Reprogramação Celular , Fibroblastos/metabolismo , Inativação Gênica , Teratoma/genética , Animais , Biomarcadores/metabolismo , Antígeno CD24/genética , Antígeno CD24/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Diferenciação Celular , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/transplante , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hepatectomia , Hepatócitos/metabolismo , Hepatócitos/patologia , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/metabolismo , Células-Tronco/patologia , Teratoma/metabolismo , Teratoma/patologia , TransgenesRESUMO
The ATP-gated ionotropic P2X7 receptor (P2X7R) modulates glial activation, cytokine production and neurotransmitter release following brain injury. Levels of the P2X7R are increased in experimental and human epilepsy but the mechanisms controlling P2X7R expression remain poorly understood. Here we investigated P2X7R responses after focal-onset status epilepticus in mice, comparing changes in the damaged, ipsilateral hippocampus to the spared, contralateral hippocampus. P2X7R-gated inward currents were suppressed in the contralateral hippocampus and P2rx7 mRNA was selectively uploaded into the RNA-induced silencing complex (RISC), suggesting microRNA targeting. Analysis of RISC-loaded microRNAs using a high-throughput platform, as well as functional assays, suggested the P2X7R is a target of microRNA-22. Inhibition of microRNA-22 increased P2X7R expression and cytokine levels in the contralateral hippocampus after status epilepticus and resulted in more frequent spontaneous seizures in mice. The major pro-inflammatory and hyperexcitability effects of microRNA-22 silencing were prevented in P2rx7(-/-) mice or by treatment with a specific P2X7R antagonist. Finally, in vivo injection of microRNA-22 mimics transiently suppressed spontaneous seizures in mice. The present study supports a role for post-transcriptional regulation of the P2X7R and suggests therapeutic targeting of microRNA-22 may prevent inflammation and development of a secondary epileptogenic focus in the brain.