RESUMO
In the absence of appropriate medical care, exposure to organophosphorus nerve agents, such as VX, can lead to respiratory failure, and potentially death by asphyxiation. Despite the critical role of respiratory disturbances in organophosphorus-induced toxicity, the nature and underlying mechanisms of respiratory failure remain poorly understood. This study aimed to characterize respiratory alterations by determining their type and duration in mice exposed to a subcutaneous sublethal dose of VX. Respiratory ventilation in Swiss mice was monitored using dual-chamber plethysmography for up to 7 days post-exposure. Cholinesterase activity was assessed via spectrophotometry, and levels of inflammatory biomarkers were quantified using Luminex technology in blood and tissues involved in respiration (diaphragm, lung, and medulla oblongata). Additionally, a histological study was conducted on these tissues to ensure their structural integrity. Ventilatory alterations appeared 20-25â¯minutes after the injection of 0.9 LD50 VX and increased until the end of the recording, i.e., 40â¯minutes after intoxication. Concurrent with the occurrence of apnea, increased inspiratory and expiratory times resulted in a significant decrease in respiratory rate in exposed mice compared to controls. Ventilatory amplitude and, consequently, minute volume were reduced, while specific airway resistance significantly increased, indicating bronchoconstriction. These ventilatory effects persisted up to 24 or even 72â¯hours post-intoxication, resolving on the 7th day. They were correlated with a decrease in acetylcholinesterase activity in the diaphragm, which persisted for up to 72â¯hours, and with the triggering of an inflammatory reaction in the same tissue. No significant histologic lesions were observed in the examined tissues. The ventilatory alterations observed up to 72â¯hours post-VX exposure appear to result from a functional failure of the respiratory system rather than tissue damage. This comprehensive characterization contributes to a better understanding of the respiratory effects induced by VX exposure, which is crucial for developing specific medical countermeasures.
Assuntos
Substâncias para a Guerra Química , Compostos Organotiofosforados , Animais , Substâncias para a Guerra Química/toxicidade , Camundongos , Masculino , Compostos Organotiofosforados/toxicidade , Acetilcolinesterase/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Diafragma/efeitos dos fármacosRESUMO
Despite the international convention on the prohibition of chemical weapons ratified in 1997, the threat of conflicts and terrorist attacks involving such weapons still exists. Among these, organophosphorus-nerve agents (OPs) inhibit cholinesterases (ChE) causing cholinergic syndrome. The reactivation of these enzymes is therefore essential to protect the poisoned people. However, these reactivating molecules, mainly named oximes, have major drawbacks with limited efficacy against some OPs and a non-negligible ChE inhibitor potential if administered at an inadequate dose, an effect that they are precisely supposed to mitigate. As a result, this project focused on assessing therapeutic efficacy, in mice, up to the NOAEL dose, the maximum dose of oxime that does not induce any observable toxic effect. NOAEL doses of HI-6 DMS, a reference oxime, and JDS364. HCl, a candidate reactivator, were assessed using dual-chamber plethysmography, with respiratory ventilation impairment as a toxicity criterion. Time-course modeling parameters and pharmacodynamic profiles, reflecting the interaction between the oxime and circulating ChE, were evaluated for treatments at their NOAEL and higher doses. Finally, the therapeutic potential against OPs poisoning was determined through the assessment of protective indices. For JDS364. HCl, the NOAEL dose corresponds to the smallest dose inducing the most significant therapeutic effect without causing any abnormality in ChE activity. In contrast, for HI-6 DMS, its therapeutic benefit was observed at doses higher than its NOAEL, leading to alterations in respiratory function. These alterations could not be directly correlated with ChE inhibition and had no adverse effects on survival. They are potentially attributed to the stimulation of non-enzymatic cholinergic targets by HI-6 DMS. Thus, the NOAEL appears to be an optimal dose for evaluating the efficacy of oximes, particularly when it can be linked to respiratory alterations effectively resulting from ChE inhibition.
Assuntos
Substâncias para a Guerra Química , Reativadores da Colinesterase , Agentes Neurotóxicos , Humanos , Camundongos , Animais , Reativadores da Colinesterase/farmacologia , Reativadores da Colinesterase/uso terapêutico , Reativadores da Colinesterase/química , Agentes Neurotóxicos/toxicidade , Nível de Efeito Adverso não Observado , Substâncias para a Guerra Química/toxicidade , Oximas/farmacologia , Oximas/uso terapêutico , Oximas/química , Compostos de Piridínio/farmacologia , Inibidores da Colinesterase/toxicidade , Inibidores da Colinesterase/química , Colinesterases , Acetilcolinesterase , Antídotos/farmacologia , Antídotos/uso terapêuticoRESUMO
Recent events demonstrated that organophosphorus nerve agents are a serious threat for civilian and military populations. The current therapy includes a pyridinium aldoxime reactivator to restore the enzymatic activity of acetylcholinesterase located in the central nervous system and neuro-muscular junctions. One major drawback of these charged acetylcholinesterase reactivators is their poor ability to cross the blood-brain barrier. In this study, we propose to evaluate glucoconjugated oximes devoid of permanent charge as potential central nervous system reactivators. We determined their in vitro reactivation efficacy on inhibited human acetylcholinesterase, the crystal structure of two compounds in complex with the enzyme, their protective index on intoxicated mice, and their pharmacokinetics. We then evaluated their endothelial permeability coefficients with a human in vitro model. This study shed light on the structural restrains of new sugar oximes designed to reach the central nervous system through the glucose transporter located at the blood-brain barrier.
Assuntos
Intoxicação por Organofosfatos , Acetilcolinesterase , Animais , Antídotos/farmacologia , Antídotos/uso terapêutico , Inibidores da Colinesterase/farmacologia , Camundongos , Intoxicação por Organofosfatos/tratamento farmacológico , Compostos Organofosforados/farmacologia , Oximas/química , Oximas/farmacologia , Oximas/uso terapêutico , AçúcaresRESUMO
This article relates the life, career and main scientific achievements of a pioneer in neuroendocrinology and French cell biology research, Mrs Andrée Tixier-Vidal, who passed away in December 2021. After her first works on hypophyseal-thyroid neuroendocrine axis, in birds then in mammals, Andrée Tixier-Vidal devoted herself then her group at the College of France to the histophysiological study of adenohypophysis and namely of prolactin (PRL) cells. Using in vitro models of organotypic cultures and cultures of GH3 cells, she described up to ultrastructural level the secretory process of PRL and its regulation by TRH. Furthermore, she extended her study to the TRH neurons themselves thanks to original models of in vitro cultures of hypothalamic neurons. Her fundamental and methodological achievements have largely contributed to major knowledge advances in cell biology of the secretion during the last century.
Title: De la neuroendocrinologie à la biologie cellulaire : Andrée Tixier-Vidal (19232021). Abstract: Cet article relate la vie, la carrière et l'Åuvre scientifique de Mme Andrée Tixier-Vidal, disparue en décembre 2021. Il montre comment, après avoir développé une approche histophysiologique originale de la neuroendocrinologie et tout particulièrement de l'axe hypophyso-thyroïdien, elle a réalisé des travaux pionniers qui ont complètement renouvelé les connaissances sur les neurones hypothalamiques à thyréolibérine (TRH) qui interviennent dans la régulation des cellules à thyréostimuline (TSH), mais également de celles à prolactine (PRL). Le fil conducteur de ses recherches a été la biologie cellulaire de la sécrétion abordée par les techniques morphologiques et cytochimiques sur des modèles originaux de cultures organotypiques d'hypophyse mais aussi de cellules tumorales GH3 et enfin de neurones hypothalamiques. Le rayonnement scientifique de Mme Tixier-Vidal et de son équipe se prolonge encore à travers les multiples générations de chercheurs qui ont eu le privilège de profiter de son dynamisme intellectuel et de son enthousiasme pour la recherche en biologie.
Assuntos
Adeno-Hipófise , Hormônio Liberador de Tireotropina , Animais , Humanos , França , Neuroendocrinologia/história , Adeno-Hipófise/metabolismo , Prolactina , Hormônio Liberador de Tireotropina/metabolismoRESUMO
Organophosphorus (OP) cholinesterase inhibitors, which include insecticides and chemical warfare nerve agents, are very potent neurotoxicants. Given that the actual treatment has several limitations, the present study provides a general method, called the zebrafish-OP-antidote test (ZOAT), and basic scientific data, to identify new antidotes that are more effective than the reference pyridinium oximes after acute OP poisoning. The reactivation capacity of a chemical compound can be measured using in vivo and ex vivo acetylcholinesterase (AChE) assays. We demonstrated that it is possible to differentiate between chemical compound protective efficacies in the central and peripheral nervous system via the visual motor response and electric field pulse motor response tests, respectively. Moreover, the ability to cross the brain-blood barrier can be estimated in a physiological context by combining an AChE assay on the head and trunk-tail fractions and the cellular and tissue localization of AChE activity in the whole-mount animal. ZOAT is an innovative method suitable for the screening and rapid identification of chemicals and mixtures used as antidote for OP poisoning. The method will make it easier to identify more effective medical countermeasures for chemical threat agents, including combinatorial therapies.
Assuntos
Reativadores da Colinesterase , Intoxicação por Organofosfatos , Acetilcolinesterase , Animais , Antídotos/farmacologia , Inibidores da Colinesterase/farmacologia , Reativadores da Colinesterase/farmacologia , Larva , Intoxicação por Organofosfatos/tratamento farmacológico , Oximas , Peixe-ZebraRESUMO
(1) Background: Human exposure to organophosphorus compounds employed as pesticides or as chemical warfare agents induces deleterious effects due to cholinesterase inhibition. One therapeutic approach is the reactivation of inhibited acetylcholinesterase by oximes. While currently available oximes are unable to reach the central nervous system to reactivate cholinesterases or to display a wide spectrum of action against the variety of organophosphorus compounds, we aim to identify new reactivators without such drawbacks. (2) Methods: This study gathers an exhaustive work to assess in vitro and in vivo efficacy, and toxicity of a hybrid tetrahydroacridine pyridinaldoxime reactivator, KM297, compared to pralidoxime. (3) Results: Blood-brain barrier crossing assay carried out on a human in vitro model established that KM297 has an endothelial permeability coefficient twice that of pralidoxime. It also presents higher cytotoxicity, particularly on bone marrow-derived cells. Its strong cholinesterase inhibition potency seems to be correlated to its low protective efficacy in mice exposed to paraoxon. Ventilatory monitoring of KM297-treated mice by double-chamber plethysmography shows toxic effects at the selected therapeutic dose. This breathing assessment could help define the No Observed Adverse Effect Level (NOAEL) dose of new oximes which would have a maximum therapeutic effect without any toxic side effects.
Assuntos
Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Compostos de Pralidoxima/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inibidores da Colinesterase/administração & dosagem , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Humanos , Injeções Intraperitoneais , Masculino , Camundongos , Estrutura Molecular , Compostos de Pralidoxima/química , Proteínas Recombinantes/metabolismoRESUMO
Respiration failure during exposure by cholinesterase inhibitors has been widely assumed to be due to inhibition of cholinesterase in the brain. Using a double chamber plethysmograph to measure various respiratory parameters, we observed long "end inspiratory pauses" (EIP) during most exposure that depressed breathing. Surprisingly, Colq KO mice that have a normal level of acetylcholinesterase (AChE) in the brain but a severe deficit in muscles and other peripheral tissues do not pause the breathing by long EIP. In mice, long EIP can be triggered by a nasal irritant. Eucalyptol, an agonist of cold receptor (TRPM8) acting on afferent sensory neurons and known to reduce the EIP triggered by such irritants, strongly reduced the EIP induced by cholinesterase inhibitor. These results suggest that acetylcholine (ACh) spillover from the neuromuscular junction, which is unchanged in Colq KO mice, may activate afferent sensory systems and trigger sensory reflexes, as reversed by eucalyptol. Indeed, the role of AChE at the cholinergic synapses is not only to accurately control the synaptic transmission but also to prevent the spillover of ACh. In the peripheral tissues, the ACh flood induced by cholinesterase inhibition may be very toxic due to interaction with non-neuronal cells that use ACh at low levels to communicate with afferent sensory neurons.
Assuntos
Acetilcolina/metabolismo , Inibidores da Colinesterase/toxicidade , Reflexo/efeitos dos fármacos , Insuficiência Respiratória/induzido quimicamente , Células Receptoras Sensoriais , Transdução de Sinais/fisiologia , Acetilcolinesterase/metabolismo , Animais , Colágeno/metabolismo , Feminino , Irritantes/toxicidade , Masculino , Camundongos , Camundongos Knockout , Proteínas Musculares/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Fisostigmina/toxicidade , Brometo de Piridostigmina/toxicidade , Respiração/efeitos dos fármacos , Insuficiência Respiratória/fisiopatologiaRESUMO
Posttranslational modifications of tubulin are emerging regulators of microtubule functions. We have shown earlier that upregulated polyglutamylation is linked to rapid degeneration of Purkinje cells in mice with a mutation in the deglutamylating enzyme CCP1. How polyglutamylation leads to degeneration, whether it affects multiple neuron types, or which physiological processes it regulates in healthy neurons has remained unknown. Here, we demonstrate that excessive polyglutamylation induces neurodegeneration in a cell-autonomous manner and can occur in many parts of the central nervous system. Degeneration of selected neurons in CCP1-deficient mice can be fully rescued by simultaneous knockout of the counteracting polyglutamylase TTLL1. Excessive polyglutamylation reduces the efficiency of neuronal transport in cultured hippocampal neurons, suggesting that impaired cargo transport plays an important role in the observed degenerative phenotypes. We thus establish polyglutamylation as a cell-autonomous mechanism for neurodegeneration that might be therapeutically accessible through manipulation of the enzymes that control this posttranslational modification.
Assuntos
Doenças Neurodegenerativas/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Células de Purkinje/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Transporte Biológico Ativo/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Peptídeos/genética , Células de Purkinje/patologia , Tubulina (Proteína)/genéticaRESUMO
In mammals, biological rhythms are driven by a master circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Recently, we have demonstrated that in the camel, the daily cycle of environmental temperature is able to entrain the master clock. This raises several questions about the structure and function of the SCN in this species. The current work is the first neuroanatomical investigation of the camel SCN. We carried out a cartography and cytoarchitectural study of the nucleus and then studied its cell types and chemical neuroanatomy. Relevant neuropeptides involved in the circadian system were investigated, including arginine-vasopressin (AVP), vasoactive intestinal polypeptide (VIP), met-enkephalin (Met-Enk), neuropeptide Y (NPY), as well as oxytocin (OT). The neurotransmitter serotonin (5-HT) and the enzymes tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) were also studied. The camel SCN is a large and elongated nucleus, extending rostrocaudally for 9.55 ± 0.10 mm. Based on histological and immunofluorescence findings, we subdivided the camel SCN into rostral/preoptic (rSCN), middle/main body (mSCN) and caudal/retrochiasmatic (cSCN) divisions. Among mammals, the rSCN is unusual and appears as an assembly of neurons that protrudes from the main mass of the hypothalamus. The mSCN exhibits the triangular shape described in rodents, while the cSCN is located in the retrochiasmatic area. As expected, VIP-immunoreactive (ir) neurons were observed in the ventral part of mSCN. AVP-ir neurons were located in the rSCN and mSCN. Results also showed the presence of OT-ir and TH-ir neurons which seem to be a peculiarity of the camel SCN. OT-ir neurons were either scattered or gathered in one isolated cluster, while TH-ir neurons constituted two defined populations, dorsal parvicellular and ventral magnocellular neurons, respectively. TH colocalized with VIP in some rSCN neurons. Moreover, a high density of Met-Enk-ir, 5-HT-ir and NPY-ir fibers were observed within the SCN. Both the cytoarchitecture and the distribution of neuropeptides are unusual in the camel SCN as compared to other mammals. The presence of OT and TH in the camel SCN suggests their role in the modulation of circadian rhythms and the adaptation to photic and non-photic cues under desert conditions.
RESUMO
Selenoprotein T (SelT) is a recently characterized thioredoxin-like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone-producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum-associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT-membrane protein interactions, we sort keratinocyte-associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A-type OST complex which are depleted after SelT knockdown leading to POMC N-glycosylation defects. This study identifies SelT as a novel subunit of the A-type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , Corticotrofos/metabolismo , Degradação Associada com o Retículo Endoplasmático , Hexosiltransferases/genética , Proteínas de Membrana/genética , Selenoproteínas/genética , Transdução de Sinais , Hormônio Adrenocorticotrópico/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Corticotrofos/citologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Glicosilação , Hexosiltransferases/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Microssomos/metabolismo , Hipófise/citologia , Hipófise/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno , Selenoproteínas/antagonistas & inibidores , Selenoproteínas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Organophosphorus nerve agents, like VX, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). AChE inhibited by VX can be reactivated using powerful nucleophilic molecules, most commonly oximes, which are one major component of the emergency treatment in case of nerve agent intoxication. We present here a comparative in vivo study on Swiss mice of four reactivators: HI-6, pralidoxime and two uncharged derivatives of 3-hydroxy-2-pyridinaldoxime that should more easily cross the blood-brain barrier and display a significant central nervous system activity. The reactivability kinetic profile of the oximes is established following intraperitoneal injection in healthy mice, using an original and fast enzymatic method based on the reactivation potential of oxime-containing plasma samples. HI-6 displays the highest reactivation potential whatever the conditions, followed by pralidoxime and the two non quaternary reactivators at the dose of 50 mg/kg bw. But these three last reactivators display equivalent reactivation potential at the same dose of 100 µmol/kg bw. Maximal reactivation potential closely correlates to surviving test results of VX intoxicated mice.
Assuntos
Análise Química do Sangue/métodos , Barreira Hematoencefálica/efeitos dos fármacos , Substâncias para a Guerra Química/toxicidade , Reativadores da Colinesterase/sangue , Compostos Organotiofosforados/toxicidade , Oximas/farmacologia , Compostos de Pralidoxima/farmacologia , Compostos de Piridínio/farmacologia , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Eritrócitos/citologia , Eritrócitos/enzimologia , Meia-Vida , Humanos , Injeções Intraperitoneais , Masculino , Camundongos , Oximas/metabolismo , Compostos de Pralidoxima/metabolismo , Substâncias Protetoras/metabolismo , Substâncias Protetoras/farmacologia , Compostos de Piridínio/metabolismoRESUMO
Glufosinate-ammonium (GLA), the active component of a widely used herbicide, induces convulsions in rodents and humans. In mouse, intraperitoneal treatment with 75 mg/kg GLA generates repetitive tonic-clonic seizures associated with 100% mortality within 72 h after treatment. In this context, we characterized GLA-induced seizures, their histological consequences and the effectiveness of diazepam treatment. Epileptic discharges on electroencephalographic recordings appeared simultaneously in the hippocampus and the cerebral cortex. Diazepam treatment at 6 h immediately stopped the seizures and prevented animal death. However, intermittent seizures were recorded on electroencephalogram from 6 h after diazepam treatment until 24 h, but had disappeared after 15 days. In our model, neuronal activation (c-Fos immunohistochemistry) was observed 6 h after GLA exposure in the dentate gyrus, CA1, CA3, amygdala, piriform and entorhinal cortices, indicating the activation of the limbic system. In these structures, Fluoro-Jade C and Cresyl violet staining did not show neuronal suffering. However, astroglial activation was clearly observed at 24 h and 15 days after GLA treatment in the amygdala, piriform and entorhinal cortices by PCR quantitative, western blot and immunohistochemistry. Concomitantly, glutamine synthetase mRNA expression (PCR quantitative), protein expression (western blot) and enzymatic activity were upregulated. In conclusion, our study suggests that GLA-induced seizures: (a) involved limbic structures and (b) induced astrocytosis without neuronal degeneration as an evidence of a reactive astrocyte beneficial effect for neuronal protection.
Assuntos
Aminobutiratos/toxicidade , Encéfalo/efeitos dos fármacos , Herbicidas/toxicidade , Organofosfatos/toxicidade , Convulsões/induzido quimicamente , Animais , Anticonvulsivantes/administração & dosagem , Astrócitos/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Diazepam/administração & dosagem , Eletroencefalografia , Glutamato-Amônia Ligase/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motoneurons. While the principal cause of the disease remains so far unknown, the onset and progression of the pathology are increasingly associated with alterations in the control of cell metabolism. On the basis of the well-known key roles of 5'-adenosine monophosphate-activated protein kinase (AMPK) in sensing and regulating the intracellular energy status, we hypothesized that mice with a genetic deletion of AMPK would develop locomotor abnormalities that bear similarity with those detected in the very early disease stage of mice carrying the ALS-associated mutated gene hSOD1(G93A). Using an automated gait analysis system (CatWalk), we here show that hSOD1(G93A) mice and age-matched mice lacking the neuronal and skeletal muscle predominant α2 catalytic subunit of AMPK showed an altered gait, clearly different from wild type control mice. Double mutant mice lacking AMPK α2 and carrying hSOD1(G93A) showed the same early gait abnormalities as hSOD1(G93A) mice over an age span of 8 to 16 weeks. Taken together, these data support the concept that altered AMPK function and associated bioenergetic abnormalities could constitute an important component in the early pathogenesis of ALS. Therapeutic interventions acting on metabolic pathways could prove beneficial on early locomotor deficits, which are sensitively detectable in rodent models using the CatWalk system.
Assuntos
Adenilato Quinase/deficiência , Adenilato Quinase/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/psicologia , Transtornos Neurológicos da Marcha/metabolismo , Transtornos Neurológicos da Marcha/psicologia , Envelhecimento/psicologia , Animais , Progressão da Doença , Metabolismo Energético/genética , Transtornos Neurológicos da Marcha/etiologia , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
A hierarchical hormonal cascade along the hypothalamic-pituitary-adrenal axis orchestrates bodily responses to stress. Although corticotropin-releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single-cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH-releasing neuron population reliant on secretagogin's Ca(2+) sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin's loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness.
Assuntos
Corticosterona/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Secretagoginas/metabolismo , Estresse Fisiológico/fisiologia , Animais , Corticosterona/genética , Hormônio Liberador da Corticotropina/genética , Masculino , Camundongos , Neurônios/citologia , Núcleo Hipotalâmico Paraventricular/citologia , Hipófise/citologia , Hipófise/metabolismo , Interferência de RNA , Secretagoginas/genética , Transcriptoma/fisiologiaRESUMO
The therapeutic benefits associated with mesenchymal stem cells (MSCs) largely result from their immunomodulatory and neurotrophic properties. In this study, we evaluated the effects of MSCs on astrocyte cultures exposed to lipopolysaccharide. In response to this inflammatory trigger, astrocytes showed an increased expression of pro-inflammatory genes (IL-1ß, TNFα, IL-6), which was attenuated by pre-exposure to MSC conditioned medium. Furthermore, mediators released by MSCs increased cell proliferation and altered the regulation of intermediate filaments (GFAP, vimentin), pro-inflammatory enzymes (iNOS, COX-2) and receptors (TLR4, CD14, mGluR3, mGluR5). These data demonstrate that MSCs influence diverse cell types participating in the response to neuroinflammation.
Assuntos
Astrócitos/imunologia , Astrócitos/metabolismo , Fatores Imunológicos/metabolismo , Imunomodulação/imunologia , Transplante de Células-Tronco Mesenquimais , Animais , Astrócitos/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Meios de Cultivo Condicionados/farmacologia , Imuno-Histoquímica , Fatores Imunológicos/farmacologia , Imunomodulação/efeitos dos fármacos , Inflamação/induzido quimicamente , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/toxicidade , Masculino , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Urotensin-II-related peptide (URP) is an eight amino-acid neuropeptide recently isolated from rat brain and considered as the endogenous ligand for the GPR14 receptor. Using single and double immunohistochemical labelling, in situ hybridization and ultrastructural immunocytochemistry, we explored the cellular and subcellular localization of URP in the male rat brain. URP peptide was detected in numerous varicose fibres of the median eminence (ME) and organum vasculosum laminae terminalis (OVLT) as well as in neuronal cell bodies of the medial septal nucleus and diagonal band of Broca where corresponding mRNA were also detected. Combining in situ hybridization with immunohistochemistry, we showed that cell bodies of the rat anterior hypothalamus contained both URP mRNA and GnRH peptide. In addition, double ultrastructural immunodetection of URP and GnRH peptides clearly revealed, in the median eminence, the co-localization of both peptides in the same neuronal processes in the vicinity of fenestrated portal vessels. This remarkable cellular and subcellular distribution led us to test the effect of URP on the GnRH-induced gonadotrophins release in the anterior pituitary, and to discuss its putative role at the level of the median eminence.