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1.
Nat Commun ; 11(1): 5073, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033238

RESUMO

Brain cells continuously produce and release protons into the extracellular space, with the rate of acid production corresponding to the levels of neuronal activity and metabolism. Efficient buffering and removal of excess H+ is essential for brain function, not least because all the electrogenic and biochemical machinery of synaptic transmission is highly sensitive to changes in pH. Here, we describe an astroglial mechanism that contributes to the protection of the brain milieu from acidification. In vivo and in vitro experiments conducted in rodent models show that at least one third of all astrocytes release bicarbonate to buffer extracellular H+ loads associated with increases in neuronal activity. The underlying signalling mechanism involves activity-dependent release of ATP triggering bicarbonate secretion by astrocytes via activation of metabotropic P2Y1 receptors, recruitment of phospholipase C, release of Ca2+ from the internal stores, and facilitated outward HCO3- transport by the electrogenic sodium bicarbonate cotransporter 1, NBCe1. These results show that astrocytes maintain local brain extracellular pH homeostasis via a neuronal activity-dependent release of bicarbonate. The data provide evidence of another important metabolic housekeeping function of these glial cells.


Assuntos
Astrócitos/metabolismo , Bicarbonatos/metabolismo , Encéfalo/metabolismo , Espaço Extracelular/metabolismo , Acetazolamida/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Anidrases Carbônicas/metabolismo , Células Cultivadas , Estimulação Elétrica , Fluorescência , Hipocampo/metabolismo , Concentração de Íons de Hidrogênio , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Antagonistas Purinérgicos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptores Purinérgicos/metabolismo , Transdução de Sinais , Simportadores de Sódio-Bicarbonato/metabolismo
2.
Nat Commun ; 11(1): 4966, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009404

RESUMO

Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults, with an unknown etiology. A hallmark of TLE is the characteristic loss of layer 3 neurons in the medial entorhinal area (MEA) that underlies seizure development. One approach to intervention is preventing loss of these neurons through better understanding of underlying pathophysiological mechanisms. Here, we show that both neurons and glia together give rise to the pathology that is mitigated by the amino acid D-serine whose levels are potentially diminished under epileptic conditions. Focal administration of D-serine to the MEA attenuates neuronal loss in this region thereby preventing epileptogenesis in an animal model of TLE. Additionally, treatment with D-serine reduces astrocyte counts in the MEA, alters their reactive status, and attenuates proliferation and/or infiltration of microglia to the region thereby curtailing the deleterious consequences of neuroinflammation. Given the paucity of compounds that reduce hyperexcitability and neuron loss, have anti-inflammatory properties, and are well tolerated by the brain, D-serine, an endogenous amino acid, offers new hope as a therapeutic agent for refractory TLE.


Assuntos
Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/patologia , Serina/uso terapêutico , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Comportamento Animal , Encéfalo/patologia , Córtex Entorrinal/efeitos dos fármacos , Córtex Entorrinal/patologia , Gliose/patologia , Inflamação/patologia , Microglia/efeitos dos fármacos , Microglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos Sprague-Dawley , Serina/administração & dosagem , Serina/farmacologia
3.
Nat Commun ; 11(1): 4640, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934232

RESUMO

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.


Assuntos
Microtúbulos/química , Paclitaxel/química , Linhagem Celular Tumoral , Citoesqueleto/química , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Humanos , Isomerismo , Microtúbulos/metabolismo , Neurônios/química , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Paclitaxel/farmacologia
4.
Nat Commun ; 11(1): 4448, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895370

RESUMO

Substance abuse disorders are linked to alteration of circadian rhythms, although the molecular and neuronal pathways implicated have not been fully elucidated. Addictive drugs, such as cocaine, induce a rapid increase of dopamine levels in the brain. Here, we show that acute administration of cocaine triggers reprogramming in circadian gene expression in the striatum, an area involved in psychomotor and rewarding effects of drugs. This process involves the activation of peroxisome protein activator receptor gamma (PPARγ), a nuclear receptor involved in inflammatory responses. PPARγ reprogramming is altered in mice with cell-specific ablation of the dopamine D2 receptor (D2R) in the striatal medium spiny neurons (MSNs) (iMSN-D2RKO). Administration of a specific PPARγ agonist in iMSN-D2RKO mice elicits substantial rescue of cocaine-dependent control of circadian genes. These findings have potential implications for development of strategies to treat substance abuse disorders.


Assuntos
Relógios Circadianos/efeitos dos fármacos , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Cocaína/efeitos adversos , Núcleo Accumbens/efeitos dos fármacos , PPAR gama/metabolismo , Receptores de Dopamina D2/metabolismo , Administração Oral , Animais , Relógios Circadianos/fisiologia , Cocaína/administração & dosagem , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Dopamina/metabolismo , Injeções Intraperitoneais , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Núcleo Accumbens/fisiopatologia , PPAR gama/agonistas , Pioglitazona/administração & dosagem , Receptores de Dopamina D2/genética , Recompensa , Transdução de Sinais
5.
Nat Commun ; 11(1): 4458, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895383

RESUMO

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.


Assuntos
Diabetes Mellitus Experimental/dietoterapia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Fator 1 de Crescimento de Fibroblastos/administração & dosagem , Hipoglicemiantes/administração & dosagem , Hipotálamo/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Proteína Relacionada com Agouti/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Glicemia/análise , Comunicação Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/administração & dosagem , Sacarose na Dieta/efeitos adversos , Humanos , Hipotálamo/citologia , Hipotálamo/patologia , Injeções Intraventriculares , Leptina/genética , Masculino , Melanocortinas/metabolismo , Hormônios Estimuladores de Melanócitos/administração & dosagem , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , RNA-Seq , Receptor Tipo 4 de Melanocortina/genética , Receptores de Melanocortina/antagonistas & inibidores , Receptores de Melanocortina/metabolismo , Indução de Remissão/métodos , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única , Técnicas Estereotáxicas , Transcriptoma/efeitos dos fármacos
6.
Int J Nanomedicine ; 15: 6339-6353, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32922005

RESUMO

Introduction: Epilepsy is a chronic neurological condition characterized by behavioral, molecular, and neurochemical alterations. Current antiepileptic drugs are associated with various adverse impacts. The main goal of the current study is to investigate the possible anticonvulsant effect of selenium nanoparticles (SeNPs) against pentylenetetrazole (PTZ)-mediated epileptic seizures in mice hippocampus. Sodium valproate (VPA) was used as a standard anti-epileptic drug. Methods: Mice were assigned into five groups (n=15): control, SeNPs (5 mg/kg, orally), PTZ (60 mg/kg, intraperitoneally), SeNPs+PTZ and VPA (200 mg/kg)+PTZ. All groups were treated for 10 days. Results: PTZ injection triggered a state of oxidative stress in the hippocampal tissue as represented by the elevated lipoperoxidation, heat shock protein 70 level, and nitric oxide formation while decreased glutathione level and antioxidant enzymes activity. Additionally, the blotting analysis showed downregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the epileptic mice. A state of neuroinflammation was recorded following the developed seizures represented by the increased pro-inflammatory cytokines. Moreover, neuronal apoptosis was recorded following the development of epileptic convulsions. At the neurochemical level, acetylcholinesterase activity and monoamines content were decreased in the epileptic mice, accompanied by high glutamate and low GABA levels in the hippocampal tissue. However, SeNP supplementation was found to delay the onset and decreased the duration of tonic, myoclonic, and generalized seizures following PTZ injection. Moreover, SeNPs were found to provide neuroprotection through preventing the development of oxidative challenge via the upregulation of Nrf2 and HO-1, inhibiting the inflammatory response and apoptotic cascade. Additionally, SeNPs reversed the changes in the activity and levels of neuromodulators following the development of epileptic seizures. Conclusion: The obtained results suggest that SeNPs could be used as a promising anticonvulsant drug due to its potent antioxidant, anti-inflammatory, and neuromodulatory activities.


Assuntos
Nanopartículas/química , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Convulsões/tratamento farmacológico , Selênio/uso terapêutico , Aminoácidos , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anticonvulsivantes/efeitos adversos , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Apoptose/efeitos dos fármacos , Colinérgicos/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Masculino , Camundongos , Nanopartículas/administração & dosagem , Neurônios/efeitos dos fármacos , Neurotransmissores/metabolismo , Oxirredução , Pentilenotetrazol , Convulsões/induzido quimicamente , Convulsões/prevenção & controle , Selênio/administração & dosagem , Selênio/farmacologia
7.
Nature ; 584(7820): 252-256, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760004

RESUMO

A fundamental challenge in developing treatments for autism spectrum disorders is the heterogeneity of the condition. More than one hundred genetic mutations confer high risk for autism, with each individual mutation accounting for only a small fraction of cases1-3. Subsets of risk genes can be grouped into functionally related pathways, most prominently those involving synaptic proteins, translational regulation, and chromatin modifications. To attempt to minimize this genetic complexity, recent therapeutic strategies have focused on the neuropeptides oxytocin and vasopressin4-6, which regulate aspects of social behaviour in mammals7. However, it is unclear whether genetic risk factors predispose individuals to autism as a result of modifications to oxytocinergic signalling. Here we report that an autism-associated mutation in the synaptic adhesion molecule Nlgn3 results in impaired oxytocin signalling in dopaminergic neurons and in altered behavioural responses to social novelty tests in mice. Notably, loss of Nlgn3 is accompanied by a disruption of translation homeostasis in the ventral tegmental area. Treatment of Nlgn3-knockout mice with a new, highly specific, brain-penetrant inhibitor of MAP kinase-interacting kinases resets the translation of mRNA and restores oxytocin signalling and social novelty responses. Thus, this work identifies a convergence between the genetic autism risk factor Nlgn3, regulation of translation, and oxytocinergic signalling. Focusing on such common core plasticity elements might provide a pragmatic approach to overcoming the heterogeneity of autism. Ultimately, this would enable mechanism-based stratification of patient populations to increase the success of therapeutic interventions.


Assuntos
Transtorno Autístico/metabolismo , Transtorno Autístico/psicologia , Modelos Animais de Doenças , Ocitocina/metabolismo , Comportamento Social , Animais , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/efeitos dos fármacos
8.
Nat Commun ; 11(1): 3996, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778725

RESUMO

Psychomotor stimulants increase dopamine levels in the striatum and promote locomotion; however, their effects on striatal pathway function in vivo remain unclear. One model that has been proposed to account for these motor effects suggests that stimulants drive hyperactivity via activation and inhibition of direct and indirect pathway striatal neurons, respectively. Although this hypothesis is consistent with the cellular actions of dopamine receptors and received support from optogenetic and chemogenetic studies, it has been rarely tested with in vivo recordings. Here, we test this model and observe that cocaine increases the activity of both pathways in the striatum of awake mice. These changes are linked to a dopamine-dependent cocaine-induced strengthening of upstream orbitofrontal cortex (OFC) inputs to the dorsomedial striatum (DMS) in vivo. Finally, depressing OFC-DMS pathway with a high frequency stimulation protocol in awake mice over-powers the cocaine-induced potentiation of OFC-DMS pathway and attenuates the expression of locomotor sensitization, directly linking OFC-DMS potentiation to cocaine-induced hyperactivity.


Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Hipercinese/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Animais , Comportamento Animal , Modelos Animais de Doenças , Dopamina , Feminino , Hipercinese/induzido quimicamente , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Optogenética
9.
PLoS One ; 15(8): e0237667, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833960

RESUMO

BACKGROUND AND AIMS: This is the first time that obesity and diabetes mellitus (DM) as protein conformational diseases (PCD) are reported in children and they are typically diagnosed too late, when ß-cell damage is evident. Here we wanted to investigate the level of naturally-ocurring or real (not synthetic) oligomeric aggregates of the human islet amyloid polypeptide (hIAPP) that we called RIAO in sera of pediatric patients with obesity and diabetes. We aimed to reduce the gap between basic biomedical research, clinical practice-health decision making and to explore whether RIAO work as a potential biomarker of early ß-cell damage. MATERIALS AND METHODS: We performed a multicentric collaborative, cross-sectional, analytical, ambispective and blinded study; the RIAO from pretreated samples (PTS) of sera of 146 pediatric patients with obesity or DM and 16 healthy children, were isolated, measured by sound indirect ELISA with novel anti-hIAPP cytotoxic oligomers polyclonal antibody (MEX1). We carried out morphological and functional studied and cluster-clinical data driven analysis. RESULTS: We demonstrated by western blot, Transmission Electron Microscopy and cell viability experiments that RIAO circulate in the blood and can be measured by ELISA; are elevated in serum of childhood obesity and diabetes; are neurotoxics and works as biomarkers of early ß-cell failure. We explored the range of evidence-based medicine clusters that included the RIAO level, which allowed us to classify and stratify the obesity patients with high cardiometabolic risk. CONCLUSIONS: RIAO level increases as the number of complications rises; RIAOs > 3.35 µg/ml is a predictor of changes in the current indicators of ß-cell damage. We proposed a novel physio-pathological pathway and shows that PCD affect not only elderly patients but also children. Here we reduced the gap between basic biomedical research, clinical practice and health decision making.


Assuntos
Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/patologia , Células Secretoras de Insulina/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Obesidade/patologia , Estrutura Quaternária de Proteína , Adolescente , Animais , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Criança , Pré-Escolar , Estudos Transversais , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/sangue , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Polipeptídeo Amiloide das Ilhotas Pancreáticas/ultraestrutura , Microscopia Eletrônica de Transmissão , Neurônios/efeitos dos fármacos , Obesidade/sangue , Obesidade/complicações , Projetos Piloto , Cultura Primária de Células , Multimerização Proteica , Ratos , Testes de Toxicidade Aguda
10.
Proc Natl Acad Sci U S A ; 117(33): 20305-20315, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32737160

RESUMO

Posttranslational modifications (PTMs) of α-synuclein (α-syn), e.g., phosphorylation, play an important role in modulating α-syn pathology in Parkinson's disease (PD) and α-synucleinopathies. Accumulation of phosphorylated α-syn fibrils in Lewy bodies and Lewy neurites is the histological hallmark of these diseases. However, it is unclear how phosphorylation relates to α-syn pathology. Here, by combining chemical synthesis and bacterial expression, we obtained homogeneous α-syn fibrils with site-specific phosphorylation at Y39, which exhibits enhanced neuronal pathology in rat primary cortical neurons. We determined the cryo-electron microscopy (cryo-EM) structure of the pY39 α-syn fibril, which reveals a fold of α-syn with pY39 in the center of the fibril core forming an electrostatic interaction network with eight charged residues in the N-terminal region of α-syn. This structure composed of residues 1 to 100 represents the largest α-syn fibril core determined so far. This work provides structural understanding on the pathology of the pY39 α-syn fibril and highlights the importance of PTMs in defining the polymorphism and pathology of amyloid fibrils in neurodegenerative diseases.


Assuntos
Doença de Parkinson , alfa-Sinucleína/química , Amiloide/química , Amiloide/metabolismo , Animais , Células Cultivadas , Microscopia Crioeletrônica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Moleculares , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação , Conformação Proteica , Ratos , Ratos Sprague-Dawley , alfa-Sinucleína/síntese química , alfa-Sinucleína/metabolismo
11.
Free Radic Res ; 54(7): 477-496, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32842814

RESUMO

Smokeless tobacco (SLT) or chewing tobacco has been a highly addictive practice in India across ages, posing major threat to the systemic health and possibly neurodegeneration. Earlier studies showed components of SLT could be harmful to neuronal health. However, mechanism of SLT in neurodegeneration remained unexplored. This study investigated the detrimental role of SLT on differentiated neuronal cell lines, PC12 and SH-SY5Y by using graded doses of water soluble lyophilised SLT. Reduced cell viability, compromised mitochondrial structure and functions were observed when neuronal cell lines were treated with SLT (6 mg/mL) for 24 h. There was reduction of oxidative phosphorylation and aerobic glycolysis as determined by diminution of ATP production (2.5X) and basal respiration (1.9X). Mitochondrial membrane potential was dropped by 3.5 times. Bid, a pro-apoptotic Bcl-2 family protein, has imperative role in regulating mitochondrial outer membrane permeabilization and subsequent cytochrome c release leading to apoptosis. This article for the first time indicated the involvement of Bid in SLT mediated neurotoxicity and possibly neurodegeneration. SLT treatment enhanced expression of cleaved-Bid in time dependent manner. The involvement of Bid was further confirmed by using Bid specific shRNA which reversed the effects of SLT and conferred significant protection from apoptosis up to 72 h. Thus, our results clearly indicated that SLT induced neuronal cell death occurred via production of ROS, alteration of mitochondrial morphology, membrane potential and oxidative phosphorylation, inactivation of survival pathway and activation of apoptotic markers mediated by Bid. Therefore, Bid could be a potential future therapeutic target for SLT induced neurodegeneration.


Assuntos
Neurônios/patologia , Tabaco sem Fumaça/toxicidade , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Citocromos c/metabolismo , Dano ao DNA , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação Oxidativa , Células PC12 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , Proteína Supressora de Tumor p53/metabolismo
12.
PLoS One ; 15(8): e0237328, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790707

RESUMO

α-Synuclein (αSyn) fibrils spread from one neuronal cell to another. This prion-like phenomenon is believed to contribute to the progression of the pathology in Parkinson's disease and other synucleinopathies. The binding of αSyn fibrils originating from affected cells to the plasma membrane of naïve cells is key in their prion-like propagation propensity. To interfere with this process, we designed polypeptides derived from proteins we previously showed to interact with αSyn fibrils, namely the molecular chaperone Hsc70 and the sodium/potassium pump NaK-ATPase and assessed their capacity to bind αSyn fibrils and/or interfere with their take-up by cells of neuronal origin. We demonstrate here that polypeptides that coat αSyn fibrils surfaces in such a way that they are changed affect αSyn fibrils binding to the plasma membrane components and/or their take-up by cells. Altogether our observations suggest that the rationale design of αSyn fibrils polypeptide binders that interfere with their propagation between neuronal cells holds therapeutic potential.


Assuntos
Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Animais , Linhagem Celular , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSC70/farmacologia , Humanos , Camundongos , Modelos Moleculares , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Peptídeos/química , Príons/antagonistas & inibidores , Príons/metabolismo , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/farmacologia
13.
PLoS One ; 15(7): e0235232, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735618

RESUMO

The tamoxifen-dependent Cre/lox system in transgenic mice has become an important research tool across all scientific disciplines for manipulating gene expression in specific cell types. In these mouse models, Cre-recombination is not induced until tamoxifen is administered, which allows researchers to have temporal control of genetic modifications. Interestingly, tamoxifen has been identified as a potential therapy for spinal cord injury (SCI) and traumatic brain injury patients due to its neuroprotective properties. It is also reparative in that it stimulates oligodendrocyte differentiation and remyelination after toxin-induced demyelination. However, it is unknown whether tamoxifen is neuroprotective and neuroreparative when administration is delayed after SCI. To properly interpret data from transgenic mice in which tamoxifen treatment is delayed after SCI, it is necessary to identify the effects of tamoxifen alone on anatomical and functional recovery. In this study, female and male mice received a moderate mid-thoracic spinal cord contusion. Mice were then gavaged with corn oil or a high dose of tamoxifen from 19-22 days post-injury, and sacrificed 42 days post-injury. All mice underwent behavioral testing for the duration of the study, which revealed that tamoxifen treatment did not impact hindlimb motor recovery. Similarly, histological analyses revealed that tamoxifen had no effect on white matter sparing, total axon number, axon sprouting, glial reactivity, cell proliferation, oligodendrocyte number, or myelination, but tamoxifen did decrease the number of neurons in the dorsal and ventral horn. Semi-thin sections confirmed that axon demyelination and remyelination were unaffected by tamoxifen. Sex-specific responses to tamoxifen were also assessed, and there were no significant differences between female and male mice. These data suggest that delayed tamoxifen administration after SCI does not change functional recovery or improve tissue sparing in female or male mice.


Assuntos
Neurônios/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Tamoxifeno/administração & dosagem , Tempo para o Tratamento , Administração Oral , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Membro Posterior/inervação , Membro Posterior/fisiologia , Humanos , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Fatores Sexuais , Corno Dorsal da Medula Espinal/citologia , Corno Dorsal da Medula Espinal/efeitos dos fármacos , Corno Ventral da Medula Espinal/citologia , Corno Ventral da Medula Espinal/efeitos dos fármacos
14.
Anticancer Res ; 40(7): 3685-3696, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620607

RESUMO

BACKGROUND/AIM: Although chemotherapy agents, such as oxaliplatin, cisplatin, paclitaxel and bortezomib frequently cause severe peripheral neuropathy, very few studies have reported the effective strategy to prevent this side effect. In this study, we first investigated whether these drugs show higher neuropathy compared to a set of 15 other anticancer drugs, and then whether antioxidants, such as sodium ascorbate, N-acetyl-L-cysteine, and vitamin B12 have any protective effect against them. MATERIALS AND METHODS: Rat PC12 cells were induced to differentiate into neuronal cells by repeated overlay of serum-free medium supplemented with nerve growth factor. The cytotoxic levels of anticancer drugs against four human oral squamous cell carcinoma cell lines, three normal oral cells, and undifferentiated and differentiated PC12 cells were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Cells were sorted for apoptotic cells (distributed into subG1 phase) and cells at different stages of cell cycle (G1, S and G2/M). RESULTS: All 19 anticancer drugs showed higher cytotoxicity against PC12 compared to oral normal cells. Among them, bortezomib showed the highest cytotoxicity against both undifferentiated and differentiated PC12 cell and, committed them to undergo apoptosis. Sodium ascorbate and N-acetyl-L-cysteine, but not vitamin B12, completely reversed the cytotoxicity of bortezomib. CONCLUSION: Bortezomib-induced neuropathy might be ameliorated by antioxidants.


Assuntos
Antioxidantes/farmacologia , Bortezomib/efeitos adversos , Síndromes Neurotóxicas/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Fator de Crescimento Neural/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Síndromes Neurotóxicas/metabolismo , Células PC12 , Doenças do Sistema Nervoso Periférico/metabolismo , Ratos
15.
Life Sci ; 257: 118037, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32622942

RESUMO

Palmitoylethanolamide (PEA) is an endogenous lipid mediator that, also by blunting astrocyte activation, demonstrated beneficial properties in several in vitro and in vivo models of Alzheimer's disease (AD). In the present study, we used astrocyte-neuron co-cultures from 3xTg-AD mouse (i.e. an animal model of AD) cerebral cortex to further investigate on the role of astrocytes in PEA-induced neuroprotection. To this aim, we evaluated the number of viable cells, apoptotic nuclei, microtubule-associated protein-2 (MAP2) positive cells and morphological parameters in cortical neurons co-cultured with cortical astrocytes pre-exposed, or not, to Aß42 (0.5 µM; 24 h) or PEA (0.1 µM; 24 h). Pre-exposure of astrocytes to Aß42 failed to affect the viability, the number of neuronal apoptotic nuclei, MAP2 positive cell number, neuritic aggregations/100 µm, dendritic branches per neuron, the neuron body area, the length of the longest dendrite and number of neurites/neuron in 3xTg-AD mouse astrocyte-neuron co-cultures. Compared to neurons from wild-type (non-Tg) mouse co-cultures, 3xTg-AD mouse neurons co-cultured with astrocytes from this mutant mice displayed higher number of apoptotic nuclei, lower MAP2 immunoreactivity and several morphological changes. These signs of neuronal suffering were significantly counteracted when the 3xTg-AD mouse cortical neurons were co-cultured with 3xTg-AD mouse astrocytes pre-exposed to PEA. The present data suggest that in astrocyte-neuron co-cultures from 3xTg-AD mice, astrocytes contribute to neuronal damage and PEA, by possibly counteracting reactive astrogliosis, improved neuronal survival. These findings further support the role of PEA as a possible new therapeutic opportunity in AD treatment.


Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Etanolaminas/farmacologia , Ácidos Palmíticos/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Modelos Animais de Doenças , Etanolaminas/metabolismo , Gliose , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Ácidos Palmíticos/metabolismo , Proteínas tau/metabolismo
16.
Life Sci ; 257: 118050, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634425

RESUMO

BACKGROUND AND PURPOSE: Early brain injury is an essential pathological process after subarachnoid hemorrhage (SAH), with many cell death modalities. Ferroptosis is a newly discovered regulated cell death caused by the iron-dependent accumulation of lipid peroxidation, which can be prevented by glutathione peroxidase 4 (GPX4). Our study aimed to investigate the role of GPX4 in neuronal cell death after experimental SAH. METHODS: In vivo experimental SAH was induced by injecting autologous arterial blood into the prechiasmatic cistern in male Sprague-Dawley rats. Meanwhile, the in vitro SAH model was performed with primary rat cortical neurons cultured in medium containing hemoglobin (Hb). Adenovirus was used to overexpress GPX4 before experimental SAH. GPX4 expression was detected by western blot and immunofluorescence experiments. Malondialdehyde (MDA) was measured to evaluate the level of lipid peroxidation. Nissl staining was employed to assess cell death in vivo, whereas lactate dehydrogenase (LDH) release was used to evaluate cell damage in vitro. The brain water content and neurological deficits were evaluated to determine brain injury. RESULTS: Endogenous GPX4 was mainly expressed in neurons, and its expression decreased at 24 h after experimental SAH. Overexpression of GPX4 significantly reduced lipid peroxidation and cell death in the experimental SAH models both in vivo and in vitro. Moreover, overexpression of GPX4 ameliorated brain edema and neurological deficits at 24 h after SAH. CONCLUSIONS: The decrease of GPX4 expression potentially plays an important role in ferroptosis during early brain injury after SAH. Overexpression of GPX4 has a neuroprotective effect after SAH.


Assuntos
Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/uso terapêutico , Hemorragia Subaracnóidea/tratamento farmacológico , Animais , Antioxidantes/farmacologia , Encéfalo/metabolismo , Edema Encefálico/patologia , Lesões Encefálicas/etiologia , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Ferroptose/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/metabolismo
17.
Nature ; 583(7816): 421-424, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32641825

RESUMO

The suprachiasmatic nucleus (SCN) serves as the body's master circadian clock that adaptively coordinates changes in physiology and behaviour in anticipation of changing requirements throughout the 24-h day-night cycle1-4. For example, the SCN opposes overnight adipsia by driving water intake before sleep5,6, and by driving the secretion of anti-diuretic hormone7,8 and lowering body temperature9,10 to reduce water loss during sleep11. These responses can also be driven by central osmo-sodium sensors to oppose an unscheduled rise in osmolality during the active phase12-16. However, it is unknown whether osmo-sodium sensors require clock-output networks to drive homeostatic responses. Here we show that a systemic salt injection (hypertonic saline) given at Zeitgeber time 19-a time at which SCNVP (vasopressin) neurons are inactive-excited SCNVP neurons and decreased non-shivering thermogenesis (NST) and body temperature. The effects of hypertonic saline on NST and body temperature were prevented by chemogenetic inhibition of SCNVP neurons and mimicked by optogenetic stimulation of SCNVP neurons in vivo. Combined anatomical and electrophysiological experiments revealed that osmo-sodium-sensing organum vasculosum lamina terminalis (OVLT) neurons expressing glutamic acid decarboxylase (OVLTGAD) relay this information to SCNVP neurons via an excitatory effect of γ-aminobutyric acid (GABA). Optogenetic activation of OVLTGAD neuron axon terminals excited SCNVP neurons in vitro and mimicked the effects of hypertonic saline on NST and body temperature in vivo. Furthermore, chemogenetic inhibition of OVLTGAD neurons blunted the effects of systemic hypertonic saline on NST and body temperature. Finally, we show that hypertonic saline significantly phase-advanced the circadian locomotor activity onset of mice. This effect was mimicked by optogenetic activation of the OVLTGAD→ SCNVP pathway and was prevented by chemogenetic inhibition of OVLTGAD neurons. Collectively, our findings provide demonstration that clock time can be regulated by non-photic physiologically relevant cues, and that such cues can drive unscheduled homeostatic responses via clock-output networks.


Assuntos
Relógios Circadianos/fisiologia , Vias Neurais , Neurônios/metabolismo , Sódio/metabolismo , Núcleo Supraquiasmático/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Temperatura Corporal/efeitos dos fármacos , Temperatura Corporal/fisiologia , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Ingestão de Líquidos/efeitos dos fármacos , Glutamato Descarboxilase/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Vias Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Optogenética , Organum Vasculosum/citologia , Organum Vasculosum/efeitos dos fármacos , Organum Vasculosum/enzimologia , Organum Vasculosum/fisiologia , Concentração Osmolar , Solução Salina Hipertônica/administração & dosagem , Solução Salina Hipertônica/metabolismo , Solução Salina Hipertônica/farmacologia , Sódio/administração & dosagem , Sódio/farmacologia , Núcleo Supraquiasmático/citologia , Núcleo Supraquiasmático/efeitos dos fármacos , Vasopressinas/metabolismo
18.
Bratisl Lek Listy ; 121(8): 580-583, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726121

RESUMO

OBJECTIVES: We investigated the effect of low, medium and high doses of oral vitamin A, on the number of fetal hippocampal neurons. BACKGROUND: High doses of vitamin A during pregnancy may cause embryonic malformations. There are reports about dosages that don't cause macroscopic malformations, but may cause mental and behavioral disorders. Still, quantitative morphological studies explaining this topic are lacking. METHODS: We administered oral vitamin A to pregnant rats on the 10th-12th days of pregnancy at doses of 10000, 20000, 30000, 40000, 50000, 100000 and 200000 IU/kg. We collected the fetuses on the 19th day and removed their brains. After staining with cresyl violet and immunolabeling with Tunel and Ki67 antibody, we examined the hippocampi with stereological methods. RESULTS: Vitamin A decreased hippocampal neuron numbers beginning from 20000 IU/kg. While the number of Ki67 positive cells increased with the dosage, the increase of apoptotic cells begun at the dose of 50000 IU/kg. CONCLUSION: Our study demonstrates that vitamin A, beginning from the dosage of 20000 IU/kg, is decreasing the total hippocampal neuron numbers during the critical period of embryonic brain development and that apoptosis may not be the only factor in this outcome (Tab. 1, Fig. 3, Ref. 27).


Assuntos
Hipocampo , Neurônios , Vitamina A , Vitaminas , Animais , Apoptose , Feminino , Hipocampo/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Neurônios/efeitos dos fármacos , Gravidez , Ratos , Vitamina A/farmacologia , Vitaminas/farmacologia
19.
Mol Cell ; 79(3): 443-458.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32649883

RESUMO

Despite the prominent role of TDP-43 in neurodegeneration, its physiological and pathological functions are not fully understood. Here, we report an unexpected role of TDP-43 in the formation of dynamic, reversible, liquid droplet-like nuclear bodies (NBs) in response to stress. Formation of NBs alleviates TDP-43-mediated cytotoxicity in mammalian cells and fly neurons. Super-resolution microscopy reveals distinct functions of the two RRMs in TDP-43 NB formation. TDP-43 NBs are partially colocalized with nuclear paraspeckles, whose scaffolding lncRNA NEAT1 is dramatically upregulated in stressed neurons. Moreover, increase of NEAT1 promotes TDP-43 liquid-liquid phase separation (LLPS) in vitro. Finally, we discover that the ALS-associated mutation D169G impairs the NEAT1-mediated TDP-43 LLPS and NB assembly, causing excessive cytoplasmic translocation of TDP-43 to form stress granules, which become phosphorylated TDP-43 cytoplasmic foci upon prolonged stress. Together, our findings suggest a stress-mitigating role and mechanism of TDP-43 NBs, whose dysfunction may be involved in ALS pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas de Ligação a DNA/genética , Corpos de Inclusão Intranuclear/metabolismo , Neurônios/metabolismo , RNA Longo não Codificante/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Animais Geneticamente Modificados , Arsenitos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Corpos de Inclusão Intranuclear/efeitos dos fármacos , Corpos de Inclusão Intranuclear/ultraestrutura , Camundongos , Mutação , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Cultura Primária de Células , Transporte Proteico/efeitos dos fármacos , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Estresse Fisiológico
20.
Life Sci ; 257: 118066, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32652135

RESUMO

AIMS: Understanding the underlying molecular mechanisms involved in epileptogenesis is necessary to target the best therapeutic interventions in epilepsy. Recently, it has been postulated that metformin, an old antidiabetic oral drug, has anti-seizure properties mostly due to its antioxidant activities. This study was designed to evaluate the ameliorative effects of metformin on the progression of epilepsy in the temporal lobe epilepsy model in rats. MAIN METHODS: Temporal lobe Epilepsy was induced by intracerebroventricular microinjection of kainic acid. Metformin was orally administered for two weeks before induction of epilepsy. Anti-epileptogenic activity of metformin was evaluated by intracranial electroencepholography (IEEG) recording to detect spontaneous seizures, mossy fiber sprouting by Timm staining, neurogenesis by BrdU staining. KEY FINDINGS: Oral administration of metformin prior to kainite-induced status epilepticus blocked the variant characterizations of epileptogenesis like neuronal cell death, aberrant neurogenesis, mossy fiber sprouting, and spontaneous seizures. SIGNIFICANCE: These findings indicate that metformin has potential anti-epileptogenic properties in temporal lobe epilepsy.


Assuntos
Anticonvulsivantes/farmacologia , Epilepsia do Lobo Temporal/tratamento farmacológico , Metformina/farmacologia , Administração Oral , Animais , Anticonvulsivantes/administração & dosagem , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia do Lobo Temporal/fisiopatologia , Ácido Caínico/toxicidade , Masculino , Metformina/administração & dosagem , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar
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