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1.
Cell ; 184(3): 655-674.e27, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33497611

Resumo

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , DNA Helicases/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Esclerose Tuberosa/metabolismo , Sequência de Aminoácidos , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , DNA Helicases/química , Evolução Molecular , Feminino , Humanos , Insulina/farmacologia , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/química , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/metabolismo
2.
Cell ; 180(2): 311-322.e15, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31883793

Resumo

The propagation of electrical impulses along axons is highly accelerated by the myelin sheath and produces saltating or "jumping" action potentials across internodes, from one node of Ranvier to the next. The underlying electrical circuit, as well as the existence and role of submyelin conduction in saltatory conduction remain, however, elusive. Here, we made patch-clamp and high-speed voltage-calibrated optical recordings of potentials across the nodal and internodal axolemma of myelinated neocortical pyramidal axons combined with electron microscopy and experimentally constrained cable modeling. Our results reveal a nanoscale yet conductive periaxonal space, incompletely sealed at the paranodes, which separates the potentials across the low-capacitance myelin sheath and internodal axolemma. The emerging double-cable model reproduces the recorded evolution of voltage waveforms across nodes and internodes, including rapid nodal potentials traveling in advance of attenuated waves in the internodal axolemma, revealing a mechanism for saltation across time and space.


Assuntos
Potenciais de Ação/fisiologia , Bainha de Mielina/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Nós Neurofibrosos/fisiologia , Animais , Axônios/metabolismo , Axônios/fisiologia , Masculino , Modelos Neurológicos , Fibras Nervosas Mielinizadas/metabolismo , Técnicas de Patch-Clamp/métodos , Células Piramidais/fisiologia , Ratos , Ratos Wistar
3.
Cell ; 183(2): 335-346.e13, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33035452

Resumo

Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.


Assuntos
Músculo Esquelético/metabolismo , Miosinas de Músculo Esquelético/efeitos dos fármacos , Miosinas de Músculo Esquelético/genética , Adulto , Animais , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Linhagem Celular , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Masculino , Camundongos , Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Espasticidade Muscular/genética , Espasticidade Muscular/fisiopatologia , Músculo Esquelético/fisiologia , Miosinas/efeitos dos fármacos , Miosinas/genética , Miosinas/metabolismo , Isoformas de Proteínas , Ratos , Ratos Wistar , Miosinas de Músculo Esquelético/metabolismo
4.
Cell ; 182(3): 713-721.e9, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32778225

Resumo

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. Here, we report the pilot-scale production of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2 µg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addition, BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manufacture. These results support the further evaluation of BBIBP-CorV in a clinical trial.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Avaliação Pré-Clínica de Medicamentos/métodos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Vacinas de Produtos Inativados/uso terapêutico , Vacinas Virais/uso terapêutico , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/genética , COVID-19 , Vacinas contra COVID-19 , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Feminino , Cobaias , Imunogenicidade da Vacina , Macaca fascicularis , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Filogenia , Pneumonia Viral/virologia , Coelhos , Ratos , Ratos Wistar , SARS-CoV-2 , Vacinas de Produtos Inativados/efeitos adversos , Células Vero , Vacinas Virais/efeitos adversos
5.
Cell ; 168(1-2): 73-85.e11, 2017 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-27916274

Resumo

The recent discovery that genetically modified α cells can regenerate and convert into ß-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-ß-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into ß-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated ß-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in ß-like cell counts, suggestive of α-to-ß-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated ß-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Células Secretoras de Glucagon/citologia , Células Secretoras de Insulina/citologia , Ácido gama-Aminobutírico/administração & dosagem , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Diferenciação Celular/efeitos dos fármacos , Diabetes Mellitus/induzido quimicamente , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Células Secretoras de Glucagon/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Proteínas do Tecido Nervoso , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/farmacologia
6.
Immunity ; 54(5): 1002-1021.e10, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33761330

Resumo

Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.


Assuntos
Proteínas do Sistema Complemento/imunologia , Fibroblastos/imunologia , Inflamação/imunologia , Membrana Sinovial/imunologia , Imunidade Adaptativa/imunologia , Animais , Artrite Reumatoide/imunologia , Linhagem Celular , Cães , Humanos , Mediadores da Inflamação/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Ratos Wistar , Transdução de Sinais/imunologia
7.
Cell ; 163(2): 456-92, 2015 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-26451489

Resumo

We present a first-draft digital reconstruction of the microcircuitry of somatosensory cortex of juvenile rat. The reconstruction uses cellular and synaptic organizing principles to algorithmically reconstruct detailed anatomy and physiology from sparse experimental data. An objective anatomical method defines a neocortical volume of 0.29 ± 0.01 mm(3) containing ~31,000 neurons, and patch-clamp studies identify 55 layer-specific morphological and 207 morpho-electrical neuron subtypes. When digitally reconstructed neurons are positioned in the volume and synapse formation is restricted to biological bouton densities and numbers of synapses per connection, their overlapping arbors form ~8 million connections with ~37 million synapses. Simulations reproduce an array of in vitro and in vivo experiments without parameter tuning. Additionally, we find a spectrum of network states with a sharp transition from synchronous to asynchronous activity, modulated by physiological mechanisms. The spectrum of network states, dynamically reconfigured around this transition, supports diverse information processing strategies. PAPERCLIP: VIDEO ABSTRACT.


Assuntos
Simulação por Computador , Modelos Neurológicos , Neocórtex/citologia , Neurônios/classificação , Neurônios/citologia , Córtex Somatossensorial/citologia , Algoritmos , Animais , Membro Posterior/inervação , Masculino , Neocórtex/fisiologia , Rede Nervosa , Neurônios/fisiologia , Ratos , Ratos Wistar , Córtex Somatossensorial/fisiologia
8.
Nature ; 629(8014): 1133-1141, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38750368

Resumo

The N-methyl-D-aspartate (NMDA) receptor is a glutamate-activated cation channel that is critical to many processes in the brain. Genome-wide association studies suggest that glutamatergic neurotransmission and NMDA receptor-mediated synaptic plasticity are important for body weight homeostasis1. Here we report the engineering and preclinical development of a bimodal molecule that integrates NMDA receptor antagonism with glucagon-like peptide-1 (GLP-1) receptor agonism to effectively reverse obesity, hyperglycaemia and dyslipidaemia in rodent models of metabolic disease. GLP-1-directed delivery of the NMDA receptor antagonist MK-801 affects neuroplasticity in the hypothalamus and brainstem. Importantly, targeting of MK-801 to GLP-1 receptor-expressing brain regions circumvents adverse physiological and behavioural effects associated with MK-801 monotherapy. In summary, our approach demonstrates the feasibility of using peptide-mediated targeting to achieve cell-specific ionotropic receptor modulation and highlights the therapeutic potential of unimolecular mixed GLP-1 receptor agonism and NMDA receptor antagonism for safe and effective obesity treatment.


Assuntos
Maleato de Dizocilpina , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Obesidade , Receptores de N-Metil-D-Aspartato , Animais , Humanos , Masculino , Camundongos , Ratos , Tronco Encefálico/metabolismo , Tronco Encefálico/efeitos dos fármacos , Modelos Animais de Doenças , Maleato de Dizocilpina/efeitos adversos , Maleato de Dizocilpina/farmacologia , Maleato de Dizocilpina/uso terapêutico , Dislipidemias/tratamento farmacológico , Dislipidemias/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores
9.
Cell ; 156(1-2): 291-303, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24439383

Resumo

Neural stem cells (NSCs) exist in germinal centers of the adult brain and in the carotid body (CB), an oxygen-sensing organ that grows under chronic hypoxemia. How stem cell lineage differentiation into mature glomus cells is coupled with changes in physiological demand is poorly understood. Here, we show that hypoxia does not affect CB NSC proliferation directly. Rather, mature glomus cells expressing endothelin-1, the O2-sensing elements in the CB that secrete neurotransmitters in response to hypoxia, establish abundant synaptic-like contacts with stem cells, which express endothelin receptors, and instruct their growth. Inhibition of glomus cell transmitter release or their selective destruction markedly diminishes CB cell growth during hypoxia, showing that CB NSCs are under the direct "synaptic" control of the mature O2-sensitive cells. Thus, glomus cells not only acutely activate the respiratory center but also induce NSC-dependent CB hypertrophy necessary for acclimatization to chronic hypoxemia.


Assuntos
Corpo Carotídeo/metabolismo , Células-Tronco Neurais/metabolismo , Oxigênio/metabolismo , Centro Respiratório/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Camundongos , Camundongos Transgênicos , Prolil Hidroxilases/metabolismo , Ratos , Ratos Wistar
10.
PLoS Biol ; 22(8): e3002768, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39163472

Resumo

According to the synaptic homeostasis hypothesis (SHY), sleep serves to renormalize synaptic connections that have been potentiated during the prior wake phase due to ongoing encoding of information. SHY focuses on glutamatergic synaptic strength and has been supported by numerous studies examining synaptic structure and function in neocortical and hippocampal networks. However, it is unknown whether synaptic down-regulation during sleep occurs in the hypothalamus, i.e., a pivotal center of homeostatic regulation of bodily functions including sleep itself. We show that sleep, in parallel with the synaptic down-regulation in neocortical networks, down-regulates the levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) in the hypothalamus of rats. Most robust decreases after sleep were observed at both sites for AMPARs containing the GluA1 subunit. Comparing the effects of selective rapid eye movement (REM) sleep and total sleep deprivation, we moreover provide experimental evidence that slow-wave sleep (SWS) is the driving force of the down-regulation of AMPARs in hypothalamus and neocortex, with no additional contributions of REM sleep or the circadian rhythm. SWS-dependent synaptic down-regulation was not linked to EEG slow-wave activity. However, spindle density during SWS predicted relatively increased GluA1 subunit levels in hypothalamic synapses, which is consistent with the role of spindles in the consolidation of memory. Our findings identify SWS as the main driver of the renormalization of synaptic strength during sleep and suggest that SWS-dependent synaptic renormalization is also implicated in homeostatic control processes in the hypothalamus.


Assuntos
Hipotálamo , Receptores de AMPA , Sono de Ondas Lentas , Sinapses , Animais , Receptores de AMPA/metabolismo , Hipotálamo/metabolismo , Masculino , Sinapses/metabolismo , Sinapses/fisiologia , Ratos , Sono de Ondas Lentas/fisiologia , Sono REM/fisiologia , Privação do Sono/metabolismo , Privação do Sono/fisiopatologia , Sono/fisiologia , Neocórtex/metabolismo , Homeostase , Ratos Sprague-Dawley , Regulação para Baixo , Ratos Wistar
11.
Cell ; 150(3): 633-46, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22863013

Resumo

N-methyl-d-aspartate receptors (NMDARs) are located in neuronal cell membranes at synaptic and extrasynaptic locations, where they are believed to mediate distinct physiological and pathological processes. Activation of NMDARs requires glutamate and a coagonist whose nature and impact on NMDAR physiology remain elusive. We report that synaptic and extrasynaptic NMDARs are gated by different endogenous coagonists, d-serine and glycine, respectively. The regionalized availability of the coagonists matches the preferential affinity of synaptic NMDARs for d-serine and extrasynaptic NMDARs for glycine. Furthermore, glycine and d-serine inhibit NMDAR surface trafficking in a subunit-dependent manner, which is likely to influence NMDARs subcellular location. Taking advantage of this coagonist segregation, we demonstrate that long-term potentiation and NMDA-induced neurotoxicity rely on synaptic NMDARs only. Conversely, long-term depression requires both synaptic and extrasynaptic receptors. Our observations provide key insights into the operating mode of NMDARs, emphasizing functional distinctions between synaptic and extrasynaptic NMDARs in brain physiology.


Assuntos
Glicina/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Serina/metabolismo , Sinapses , Animais , Membrana Celular , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Potenciação de Longa Duração , Depressão Sináptica de Longo Prazo , Neuroglia/metabolismo , Neurônios/citologia , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo
12.
EMBO J ; 41(4): e106523, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34935159

Resumo

Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca2+ ) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin-based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca2+ sensor caldendrin, a brain-specific homolog of the well-known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V-dependent pathway. We propose that caldendrin transforms myosin into a stationary F-actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Espinhas Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Miosina Tipo V/metabolismo , Actinas/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Calmodulina/metabolismo , Retículo Endoplasmático Liso/metabolismo , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Espectrometria de Massas , Camundongos Knockout , Miosina Tipo V/genética , Domínios e Motivos de Interação entre Proteínas , Ratos Wistar
13.
Nature ; 586(7828): 287-291, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32728214

Resumo

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.


Assuntos
Transporte de Elétrons , Hipóxia/metabolismo , Mitocôndrias/metabolismo , Sistemas do Segundo Mensageiro , Sódio/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fosfatos de Cálcio/metabolismo , Linhagem Celular Tumoral , Precipitação Química , Humanos , Masculino , Fluidez de Membrana , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Trocador de Sódio e Cálcio/metabolismo
14.
Bioessays ; : e2400094, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39115324

Resumo

Many strands of research by different groups, starting from teratocarcinomas in the laboratory mouse, later moving the corresponding human tumors, contributed to the isolation and description of human pluripotent stem cells (PSCs). In this review, I highlight the contributions from my own research, particularly at the Wistar Institute during the 1980s, when with my colleagues we characterized one of the first clonal lines of pluripotent human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, and identified key features including cell surface antigen markers that have since found a place in the study and exploitation of human PSC. Much of this research depended upon close teamwork with colleagues, many in other laboratories, who contributed different expertise and experience. It was also often driven by circumstance and chance rather than pursuit of a grand design.

15.
Genes Dev ; 32(9-10): 645-657, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29748249

Resumo

Cholesterol is a major constituent of myelin membranes, which insulate axons and allow saltatory conduction. Therefore, Schwann cells, the myelinating glia of the peripheral nervous system, need to produce large amounts of cholesterol. Here, we define a crucial role of the transcription factor Maf in myelination and cholesterol biosynthesis and show that Maf acts downstream from Neuregulin1 (Nrg1). Maf expression is induced when Schwann cells begin myelination. Genetic ablation of Maf resulted in hypomyelination that resembled mice with defective Nrg1 signaling. Importantly, loss of Maf or Nrg1 signaling resulted in a down-regulation of the cholesterol synthesis program, and Maf directly binds to enhancers of cholesterol synthesis genes. Furthermore, we identified the molecular mechanisms by which Nrg1 signaling regulates Maf levels. Transcription of Maf depends on calmodulin-dependent kinases downstream from Nrg1, whereas Nrg1-MAPK signaling stabilizes Maf protein. Our results delineate a novel signaling cascade regulating cholesterol synthesis in myelinating Schwann cells.


Assuntos
Colesterol/biossíntese , Bainha de Mielina/metabolismo , Neuregulina-1/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Células de Schwann/metabolismo , Transdução de Sinais , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Colesterol/genética , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-maf/genética , Ratos , Ratos Wistar
16.
J Neurosci ; 44(17)2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38438259

Resumo

Oxytocinergic transmission blocks nociception at the peripheral, spinal, and supraspinal levels through the oxytocin receptor (OTR). Indeed, a neuronal pathway from the hypothalamic paraventricular nucleus (PVN) to the spinal cord and trigeminal nucleus caudalis (Sp5c) has been described. Hence, although the trigeminocervical complex (TCC), an anatomical area spanning the Sp5c, C1, and C2 regions, plays a role in some pain disorders associated with craniofacial structures (e.g., migraine), the role of oxytocinergic transmission in modulating nociception at this level has been poorly explored. Hence, in vivo electrophysiological recordings of TCC wide dynamic range (WDR) cells sensitive to stimulation of the periorbital or meningeal region were performed in male Wistar rats. PVN electrical stimulation diminished the neuronal firing evoked by periorbital or meningeal electrical stimulation; this inhibition was reversed by OTR antagonists administered locally. Accordingly, neuronal projections (using Fluoro-Ruby) from the PVN to the WDR cells filled with Neurobiotin were observed. Moreover, colocalization between OTR and calcitonin gene-related peptide (CGRP) or OTR and GABA was found near Neurobiotin-filled WDR cells. Retrograde neuronal tracers deposited at the meningeal (True-Blue, TB) and infraorbital nerves (Fluoro-Gold, FG) showed that at the trigeminal ganglion (TG), some cells were immunopositive to both fluorophores, suggesting that some TG cells send projections via the V1 and V2 trigeminal branches. Together, these data may imply that endogenous oxytocinergic transmission inhibits the nociceptive activity of second-order neurons via OTR activation in CGRPergic (primary afferent fibers) and GABAergic cells.


Assuntos
Estimulação Elétrica , Ocitocina , Núcleo Hipotalâmico Paraventricular , Ratos Wistar , Receptores de Ocitocina , Transmissão Sináptica , Animais , Masculino , Núcleo Hipotalâmico Paraventricular/fisiologia , Núcleo Hipotalâmico Paraventricular/metabolismo , Ocitocina/metabolismo , Ocitocina/análogos & derivados , Ratos , Receptores de Ocitocina/metabolismo , Receptores de Ocitocina/antagonistas & inibidores , Transmissão Sináptica/fisiologia , Nociceptores/fisiologia , Nociceptores/metabolismo , Nociceptividade/fisiologia , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos dos fármacos , Meninges/fisiologia , Inibição Neural/fisiologia
17.
J Neurosci ; 44(30)2024 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-38937101

Resumo

Many neurons including vasopressin (VP) magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus (SON) generate afterhyperpolarizations (AHPs) during spiking to slow firing, a phenomenon known as spike frequency adaptation. The AHP is underlain by Ca2+-activated K+ currents, and while slow component (sAHP) features are well described, its mechanism remains poorly understood. Previous work demonstrated that Ca2+ influx through N-type Ca2+ channels is a primary source of sAHP activation in SON oxytocin neurons, but no obvious channel coupling was described for VP neurons. Given this, we tested the possibility of an intracellular source of sAHP activation, namely, the Ca2+-handling organelles endoplasmic reticulum (ER) and mitochondria in male and female Wistar rats. We demonstrate that ER Ca2+ depletion greatly inhibits sAHPs without a corresponding decrease in Ca2+ signal. Caffeine sensitized AHP activation by Ca2+ In contrast to ER, disabling mitochondria with CCCP or blocking mitochondria Ca2+ uniporters (MCUs) enhanced sAHP amplitude and duration, implicating mitochondria as a vital buffer for sAHP-activating Ca2+ Block of mitochondria Na+-dependent Ca2+ release via triphenylphosphonium (TPP+) failed to affect sAHPs, indicating that mitochondria Ca2+ does not contribute to sAHP activation. Together, our results suggests that ER Ca2+-induced Ca2+ release activates sAHPs and mitochondria shape the spatiotemporal trajectory of the sAHP via Ca2+ buffering in VP neurons. Overall, this implicates organelle Ca2+, and specifically ER-mitochondria-associated membrane contacts, as an important site of Ca2+ microdomain activity that regulates sAHP signaling pathways. Thus, this site plays a major role in influencing VP firing activity and systemic hormonal release.


Assuntos
Cálcio , Retículo Endoplasmático , Mitocôndrias , Neurônios , Ratos Wistar , Núcleo Supraóptico , Vasopressinas , Animais , Ratos , Vasopressinas/metabolismo , Masculino , Feminino , Neurônios/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Cálcio/metabolismo , Núcleo Supraóptico/metabolismo , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos dos fármacos , Sinalização do Cálcio/fisiologia
18.
J Neurosci ; 44(21)2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38565292

Resumo

Glucagon-like peptide-1 (GLP-1) and its analogs are widely used for diabetes treatment. The paraventricular nucleus (PVN) is crucial for regulating cardiovascular activity. This study aims to determine the roles of GLP-1 and its receptors (GLP-1R) in the PVN in regulating sympathetic outflow and blood pressure. Experiments were carried out in male normotensive rats and spontaneously hypertensive rats (SHR). Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded. GLP-1 and GLP-1R expressions were present in the PVN. PVN microinjection of GLP-1R agonist recombinant human GLP-1 (rhGLP-1) or EX-4 increased RSNA and MAP, which were prevented by GLP-1R antagonist exendin 9-39 (EX9-39) or GLP-1R antagonist 1, superoxide scavenger tempol, antioxidant N-acetylcysteine, NADPH oxidase (NOX) inhibitor apocynin, adenylyl cyclase (AC) inhibitor SQ22536 or protein kinase A (PKA) inhibitor H89. PVN microinjection of rhGLP-1 increased superoxide production, NADPH oxidase activity, cAMP level, AC, and PKA activity, which were prevented by SQ22536 or H89. GLP-1 and GLP-1R were upregulated in the PVN of SHR. PVN microinjection of GLP-1 agonist increased RSNA and MAP in both WKY and SHR, but GLP-1 antagonists caused greater effects in reducing RSNA and MAP in SHR than in WKY. The increased superoxide production and NADPH oxidase activity in the PVN of SHR were augmented by GLP-1R agonists but attenuated by GLP-1R antagonists. These results indicate that activation of GLP-1R in the PVN increased sympathetic outflow and blood pressure via cAMP-PKA-mediated NADPH oxidase activation and subsequent superoxide production. GLP-1 and GLP-1R upregulation in the PVN partially contributes to sympathetic overactivity and hypertension.


Assuntos
Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Hipertensão , Núcleo Hipotalâmico Paraventricular , Ratos Endogâmicos SHR , Sistema Nervoso Simpático , Animais , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular/metabolismo , Masculino , Hipertensão/fisiopatologia , Hipertensão/metabolismo , Ratos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Ratos Endogâmicos WKY , Ratos Sprague-Dawley
19.
J Biol Chem ; 300(3): 105712, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38309509

Resumo

We recently established a method for the isolation of serum-free oligosaccharides, and characterized various features of their structures. However, the precise mechanism for how these glycans are formed still remains unclarified. To further investigate the mechanism responsible for these serum glycans, here, we utilized rat primary hepatocytes to examine whether they are able to secrete free glycans. Our findings indicated that a diverse array of free oligosaccharides such as sialyl/neutral free N-glycans (FNGs), as well as sialyl lactose/LacNAc-type glycans, were secreted into the culture medium by primary hepatocytes. The structural features of these free glycans in the medium were similar to those isolated from the sera of the same rat. Further evidence suggested that an oligosaccharyltransferase is involved in the release of the serum-free N-glycans. Our results indicate that the liver is indeed secreting various types of free glycans directly into the serum.


Assuntos
Hepatócitos , Oligossacarídeos , Animais , Ratos , Hepatócitos/metabolismo , Oligossacarídeos/sangue , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Células Hep G2 , Humanos , Masculino , Ratos Wistar
20.
J Biol Chem ; 300(2): 105597, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38160798

Resumo

Increased expression of angiotensin II AT1A receptor (encoded by Agtr1a) and Na+-K+-Cl- cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension development. However, little is known about their transcriptional control in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wistar-Kyoto rats (WKY). Chromatin immunoprecipitation-quantitative PCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and methyl-CpG binding protein 2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.


Assuntos
Desmetilação do DNA , Hipotálamo , Receptor Tipo 1 de Angiotensina , Membro 2 da Família 12 de Carreador de Soluto , Animais , Ratos , Pressão Sanguínea , DNA/metabolismo , Hipertensão/metabolismo , Hipotálamo/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptor Tipo 1 de Angiotensina/metabolismo , RNA Mensageiro/genética , Sistema Nervoso Simpático/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo
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