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1.
Mol Med Rep ; 19(6): 5169-5176, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059107

RESUMO

The aim of the present study was to probe the mechanism of apoptosis induced by endoplasmic reticulum stress (ERS) in manganese­induced rats. A total of 60 Sprague­Dawley rats were randomly divided into a Vehicle group, LoMag group, HiMag group, and HiMag + 4­phenylbutyrate (PBA) group. Manganese content was measured by Inductively Coupled Plasma­Atomic Emission Spectrometry. Pathogenic morphology, the cellular structure of the striatum and ER were observed by hematoxylin and eosin staining and electron microscopy. The TUNEL method was used to examine neuronal apoptosis in the rat striatum. The expression levels of glucose­regulated protein 78KD (GRP78), C/EBP homologous protein (CHOP), c­Jun N­terminal kinase (JNK) and caspase­12 were analyzed by western blot analysis. The results revealed that striatal manganese concentrations in the LoMag and HiMag groups were higher than that in the Vehicle group (P<0.01). Rat striatal neuronal structure and apoptotic rates in the LoMag and HiMag groups were higher than those in the Vehicle group (P<0.05). 4­PBA treatment effectively reduced the apoptotic cell number (P<0.05). In addition, ER swelling and vacuolization in the HiMag + PBA group was reduced compared with that in the HiMag group. In addition, the protein expression levels of GRP78, CHOP, JNK and caspase­12 in the LoMag and HiMag groups were higher than those in the Vehicle group (P<0.05). However, the expression of these four proteins was reduced by 4­PBA treatment (P<0.05). In conclusion, 4­PBA significantly reduced the damage and apoptosis induced by manganese exposure in rats.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Manganês/toxicidade , Animais , Apoptose/efeitos dos fármacos , Caspase 12/metabolismo , Corpo Estriado/química , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Fenilbutiratos/farmacologia , Ratos , Ratos Sprague-Dawley , Espectrofotometria Atômica , Fator de Transcrição CHOP/metabolismo
2.
Int J Mol Sci ; 20(9)2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31060255

RESUMO

GSK3 (glycogen synthase kinase 3) is a conserved protein kinase governing numerous regulatory pathways. In Drosophila melanogaster, GSK3 is encoded by shaggy (sgg), which forms 17 annotated transcripts corresponding to 10 protein isoforms. Our goal was to demonstrate how differential sgg transcription affects lifespan, which GSK3 isoforms are important for the nervous system, and which changes in the nervous system accompany accelerated aging. Overexpression of three sgg transcripts affected the lifespan in a stage- and tissue-specific way: sgg-RA and sgg-RO affected the lifespan only when overexpressed in muscles and in embryos, respectively; the essential sgg-RB transcript affected lifespan when overexpressed in all tissues tested. In the nervous system, only sgg-RB overexpression affected lifespan, causing accelerated aging in a neuron-specific way, with the strongest effects in dopaminergic neurons and the weakest effects in GABAergic neurons. Pan-neuronal sgg-RB overexpression violated the properties of the nervous system, including the integrity of neuron bodies; the number, distribution, and structure of mitochondria; cytoskeletal characteristics; and synaptic activity. Such changes observed in young individuals indicated premature aging of their nervous system, which paralleled a decline in survival. Our findings demonstrated the key role of GSK3 in ensuring the link between the pathology of neurons and lifespan.


Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Estágios do Ciclo de Vida/genética , Longevidade/genética , Animais , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Especificidade de Órgãos/genética , Fenótipo
3.
J Stroke Cerebrovasc Dis ; 28(7): 1832-1840, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31078389

RESUMO

GOAL: The present study aimed to examine whether Am80 (tamibarotene) protects the hippocampus against cerebral ischemia-reperfusion (I/R) injury and whether phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway mediates this effect. MATERIALS AND METHODS: Rats were subjected to 90 minutes of middle cerebral artery occlusion followed by 24 hours of reperfusion. The animals were randomly divided into 7 groups: sham-operated group; I/R group; groups pretreated with 2 mg/kg, 6 mg/kg, and 10 mg/kg of Am80; Am80 (6 mg/kg) combined with the selective PI3K inhibitor wortmannin (0.6 mg/kg), and wortmannin (0.6 mg/kg) only group. After 24 hours of reperfusion, neurological deficits and infarct volume were measured. Pathological changes in hippocampal neurons were analyzed by transmission electron microscopy. Neuronal survival was examined by TUNEL staining. The expression of Bcl-2, Bax, and Akt, and Akt phosphorylation (p-Akt) were measured by Western blotting and quantitative real-time polymerase chain reaction. FINDINGS: The pretreatment with Am80 improved the neurologic deficit score, reduced infarct volume, and decreased the number of TUNEL-positive cells in the hippocampus. Moreover, Am80 pretreatment downregulated the expression of Bax, upregulated the expression of Bcl-2, and increased the level of p-Akt. Wortmannin abolished in part the increase in p-Act and the neuroprotective effect exerted on the ischemic by Am80 pretreatment. CONCLUSIONS: Our results documented that Am80 pretreatment protects ischemic hippocampus after cerebral I/R by regulating the expression of apoptosis-related proteins through the activation of the PI3K/Akt signaling pathway.


Assuntos
Benzoatos/farmacologia , Hipocampo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/prevenção & controle , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Tetra-Hidronaftalenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Hipocampo/enzimologia , Hipocampo/ultraestrutura , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/patologia , Masculino , Neurônios/enzimologia , Neurônios/ultraestrutura , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Sprague-Dawley , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo
4.
Bull Exp Biol Med ; 166(6): 816-819, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31020582

RESUMO

Intraocular neurografts of the septal region of rats were used as the model of deafferentiated brain area where the lack of adequate innervation is compensated for own interneuronal connections. Septum anlage from the brain of a 17-day fetus served as the donor material. The grafts developing in the anterior eye chamber over 3 months represented well-differentiated samples of the nervous tissue. A comparative morphometric study of the tripartite organization of synapses in the grafts and in the septum in situ was conducted. In the grafts, the mean volume and perimeter of synaptic terminals were below the normal. At the same time, postsynaptic densities did not differ from the control. A significant difference was found in the degree of surrounding of presynaptic terminals by astrocytic processes: in the grafts this parameter was higher by 1.8 times. Our results attest to an important role of perisynaptic glia in the formation of functionally active synaptic contacts with unusual neuronal targets.


Assuntos
Segmento Anterior do Olho/ultraestrutura , Astrócitos/ultraestrutura , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Septo do Cérebro/ultraestrutura , Transmissão Sináptica/fisiologia , Animais , Segmento Anterior do Olho/inervação , Comunicação Celular , Embrião de Mamíferos , Masculino , Ratos , Ratos Wistar , Septo do Cérebro/transplante , Transplante de Tecidos , Transplante Heterotópico , Transplante Homólogo
5.
J Therm Biol ; 81: 110-117, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30975407

RESUMO

The hypothalamus is crucial to ensure the functionality of the entire organisms, such as body temperature, feed intake and energy regulation. Exposing broilers to high ambient temperature usually induces lower feed intake and energy imbalance. We investigated the molecular mechanisms by which heat stress impairs the appetite via dysfunction in hypothalamus of the broilers. Broilers were allocated to three groups: the normal control (NC) group, and fed ad libitum; heat-stress (HS) group, and fed ad libitum; pair-fed (PF) group, which received the feed intake equal to HS group. Experiment lasted from the age of 28 to 42 d. The results showed that HS increased the head surface temperature of broiler and changed hypothalamic ultrastructure. HS treatment also increased the serum corticosterone in the broilers after 7 days of heat stress, elevated the FT4 and FT3 after 14 days of heat stress. Heat stress of 14 days showed a tendency to increase the leptin. However, the serum corticosterone in the HS group had no significant difference after 14 days of heat stress. In addition, HS treatment decreased the expression of orexigenic gene neuropeptide Y (NPY) after 14 days of heat stress, while HS treatment had no effect on the reactive oxygen species (ROS), as well as the gene expression of AMPKα1 and LKB1 in the hypothalamus. In conclusion, HS increased the surface temperature of head in broiler, and then altered the integrity of hypothalamus. Meanwhile, HS increased the serum corticosterone which may ascribe to the activation of HPA axis in the broilers. In addition, chronic heat stress decreased the expression of orexigenic gene NPY, which may cause the broiler to reduce feed intake.


Assuntos
Apetite/genética , Galinhas/fisiologia , Resposta ao Choque Térmico , Hipotálamo/patologia , Hipotálamo/fisiopatologia , Animais , Temperatura Corporal , Galinhas/sangue , Galinhas/genética , Cabeça/fisiologia , Temperatura Alta , Hipotálamo/ultraestrutura , Masculino , Neurônios/patologia , Neurônios/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo
6.
Nat Commun ; 10(1): 1549, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948706

RESUMO

Characterizing the precise three-dimensional morphology and anatomical context of neurons is crucial for neuronal cell type classification and circuitry mapping. Recent advances in tissue clearing techniques and microscopy make it possible to obtain image stacks of intact, interweaving neuron clusters in brain tissues. As most current 3D neuronal morphology reconstruction methods are only applicable to single neurons, it remains challenging to reconstruct these clusters digitally. To advance the state of the art beyond these challenges, we propose a fast and robust method named G-Cut that is able to automatically segment individual neurons from an interweaving neuron cluster. Across various densely interconnected neuron clusters, G-Cut achieves significantly higher accuracies than other state-of-the-art algorithms. G-Cut is intended as a robust component in a high throughput informatics pipeline for large-scale brain mapping projects.


Assuntos
Mapeamento Encefálico/métodos , Simulação por Computador , Rede Nervosa , Neurônios/citologia , Algoritmos , Biologia Computacional , Modelos Teóricos , Neurônios/ultraestrutura
7.
Toxicol Lett ; 311: 37-48, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31029751

RESUMO

Polybrominated diphenyl ether-153 (BDE-153) has been demonstrated to induce neuronal apoptosis in rat cerebral cortex and primary neurons, however, the roles of mitochondria and endoplasmic reticulum (ER) remain unclear in the BDE-153-induced neuronal apoptosis. To this purpose, we observed the mitochondria and ER ultrastructure changes in the neuronal apoptosis in rats following BDE-153 treatment, detected the mitochondrial membrane potential (MMP), Ca2+-Mg2+-ATP enzyme activity, and the changes of mitochondria and ER apoptosis related molecules in rat cerebral cortex and in primary neurons following BDE-153 treatment. Results showed that compared to the control group, neuronal apoptosis was significantly increased in a dose-dependent manner in rat cerebral cortex and in primary neurons following BDE-153 treatment. In comparison with control, BDE-153 treatment induced remarkable ultrastructural changes in ER rather than in mitochondria, and the severity of ER damage was worse with the increasing BDE-153 dose. Meanwhile, ER apoptosis related molecules including caspase-12 (at mRNA level), cleaved caspase-12 (at protein level), and Tmem132a (at mRNA and protein levels) were significantly increased in the cerebral cortex in rats following BDE-153 treatment, while procaspase-12 protein was significantly decreased, comparing with control. In contrast, mitochondria apoptosis related molecules (MMP, Ca2+-Mg2+-ATP enzyme activity, cyt-C protein, caspase-3, 8, 9 mRNA, caspase-8, 9 enzyme activities) did not significantly changed in the cerebral cortex of rats or in primary neurons following BDE-153 treatment, except for the elevated caspase-3 mRNA and enzyme activity. Therefore, we conclude that BDE-153 induced neuronal apoptosis was dependent on p53, and mediated more by ER than mitochondria in the cerebral cortex of rats and in primary neurons. The findings suggest that ER is a potential sensitive target of BDE-153 neurotoxicity, providing a scientific evidence for the mechanism and intervention study on PBDE's neurotoxicity.


Assuntos
Apoptose/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Degeneração Neural , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Bifenil Polibromatos/toxicidade , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
8.
Int J Mol Med ; 43(4): 1879-1887, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816425

RESUMO

The aim of the present study was to investigate the protective effects of curcumin and its effect on the methyl ethyl ketone/extracellular signal regulated kinase/cAMP­response element binding protein (MEK/ERK/CREB) pathway. The study was conducted in vivo and in vitro as follows: In vivo: Focal cerebral ischemia­reperfusion (IR) models of rats were made with the plug­line method. Adult male Sprague­Dawley rats were divided into four groups: Sham operation control group, IR and curcumin­treatment groups (100 mg/kg and IC, 300 mg/kg). The effects of curcumin on neurological deficit scores, brain water content and infarct volumes were identified. Transmission electron microscope was utilized to observe morphological changes of hippocampal neurons; hematoxylin and eosin staining was used to observe morphological changes of brain tissue; and the terminal deoxynucleotidyl transferase (TdT)­mediated dUTP nick end labeling method detected neurons apoptosis of hippocampal CA1. Finally, western blot analysis detected the expression of phosphorylated (p)­MEK, p­ERK, p­CREB, B­cell lymphoma­2 (Bcl­2) and Bcl­2 associated X protein (Bax). In vitro: An oxygen­glucose deprivation/reoxygenation method was used on primary cultured astrocytes to make cerebral ischemia­reperfusion models in vitro. Astrocytes were randomly divided into five groups: Normoxia, oxygen­glucose deprivation/reoxygenation (OGD/Reoxy), OGD/Reoxy + curcumin (5, 10, 20 µmol/l). The cell viability and toxicity were assessed by MTT and lactate dehydrogenase release assay, and levels of p­MEK, p­ERK and p­CREB proteins were analyzed by the western blotting method. Curcumin was demonstrated to improve nerve damage symptoms and infarct volume, reduce brain water content, relieve neuronal apoptosis and also increase the expression of p­MEK, p­ERK, p­CREB, Bcl­2 and reduce Bax levels in vivo and in vitro. In conclusion curcumin can mitigate focal cerebral ischemia­reperfusion injuries and this effect may be carried out through the MEK/ERK/CREB pathway.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Curcumina/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Isquemia Encefálica/complicações , Sobrevivência Celular/efeitos dos fármacos , Curcumina/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , L-Lactato Desidrogenase/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Fármacos Neuroprotetores/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/complicações , Água
9.
Nat Neurosci ; 22(5): 828-839, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30886406

RESUMO

Electron microscopy (EM) is a powerful tool for circuit mapping, but identifying specific cell types in EM datasets remains a major challenge. Here we describe a technique enabling simultaneous visualization of multiple genetically identified neuronal populations so that synaptic interactions between them can be unequivocally defined. We present 15 adeno-associated virus constructs and 6 mouse reporter lines for multiplexed EM labeling in the mammalian nervous system. These reporters feature dAPEX2, which exhibits dramatically improved signal compared with previously described ascorbate peroxidases. By targeting this enhanced peroxidase to different subcellular compartments, multiple orthogonal reporters can be simultaneously visualized and distinguished under EM using a protocol compatible with existing EM pipelines. Proof-of-principle double and triple EM labeling experiments demonstrated synaptic connections between primary afferents, descending cortical inputs, and inhibitory interneurons in the spinal cord dorsal horn. Our multiplexed peroxidase-based EM labeling system should therefore greatly facilitate analysis of connectivity in the nervous system.


Assuntos
Córtex Cerebral/ultraestrutura , Microscopia Eletrônica/métodos , Neurônios/ultraestrutura , Células do Corno Posterior/ultraestrutura , Sinapses/ultraestrutura , Adenoviridae/fisiologia , Animais , Genes Reporter , Vetores Genéticos , Imuno-Histoquímica/métodos , Camundongos Transgênicos , Microscopia Confocal , Neurônios Aferentes/ultraestrutura , Peroxidases/química , Razão Sinal-Ruído
10.
Nat Commun ; 10(1): 1144, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850633

RESUMO

Despite intense interests in developing blood measurements of Alzheimer's disease (AD), the progress has been confounded by limited sensitivity and poor correlation to brain pathology. Here, we present a dedicated analytical platform for measuring different populations of circulating amyloid ß (Aß) proteins - exosome-bound vs. unbound - directly from blood. The technology, termed amplified plasmonic exosome (APEX), leverages in situ enzymatic conversion of localized optical deposits and double-layered plasmonic nanostructures to enable sensitive, multiplexed population analysis. It demonstrates superior sensitivity (~200 exosomes), and enables diverse target co-localization in exosomes. Employing the platform, we find that prefibrillar Aß aggregates preferentially bind with exosomes. We thus define a population of Aß as exosome-bound (Aß42+ CD63+) and measure its abundance directly from AD and control blood samples. As compared to the unbound or total circulating Aß, the exosome-bound Aß measurement could better reflect PET imaging of brain amyloid plaques and differentiate various clinical groups.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Encéfalo/patologia , Exossomos/química , Neurônios/patologia , Fragmentos de Peptídeos/química , Placa Amiloide/patologia , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/sangue , Técnicas Biossensoriais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Exossomos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas Analíticas Microfluídicas , Neurônios/metabolismo , Neurônios/ultraestrutura , Fragmentos de Peptídeos/sangue , Placa Amiloide/diagnóstico por imagem , Placa Amiloide/metabolismo , Tomografia por Emissão de Pósitrons , Agregados Proteicos , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Ressonância de Plasmônio de Superfície , Células THP-1 , Tetraspanina 30/química , Tetraspanina 30/metabolismo
11.
Nat Commun ; 10(1): 1285, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894537

RESUMO

Dendritic spines are the postsynaptic sites that receive most of the excitatory synaptic inputs, and thus provide the structural basis for synaptic function. Here, we describe an accurate method for measurement and analysis of spine morphology based on structured illumination microscopy (SIM) and computational geometry in cultured neurons. Surface mesh data converted from SIM images were comparable to data reconstructed from electron microscopic images. Dimensional reduction and machine learning applied to large data sets enabled identification of spine phenotypes caused by genetic mutations in key signal transduction molecules. This method, combined with time-lapse live imaging and glutamate uncaging, could detect plasticity-related changes in spine head curvature. The results suggested that the concave surfaces of spines are important for the long-term structural stabilization of spines by synaptic adhesion molecules.


Assuntos
Espinhas Dendríticas/ultraestrutura , Hipocampo/ultraestrutura , Microscopia/estatística & dados numéricos , Neurônios/ultraestrutura , Imagem com Lapso de Tempo/estatística & dados numéricos , Animais , Carbocianinas/química , Conjuntos de Dados como Assunto , Espinhas Dendríticas/fisiologia , Embrião de Mamíferos , Corantes Fluorescentes/química , Ácido Glutâmico/metabolismo , Hipocampo/fisiologia , Aprendizado de Máquina , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia/métodos , Redução Dimensional com Múltiplos Fatores , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Cultura Primária de Células , Coloração e Rotulagem/métodos , Imagem com Lapso de Tempo/métodos
12.
Cell Mol Life Sci ; 76(10): 1967-1985, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30840087

RESUMO

Mitochondria are essential components of eukaryotic cells, carrying out critical physiological processes that include energy production and calcium buffering. Consequently, mitochondrial dysfunction is associated with a range of human diseases. Fundamental to their function is the ability to transition through fission and fusion states, which is regulated by several GTPases. Here, we have developed new methods for the non-subjective quantification of mitochondrial morphology in muscle and neuronal cells of Caenorhabditis elegans. Using these techniques, we uncover surprising tissue-specific differences in mitochondrial morphology when fusion or fission proteins are absent. From ultrastructural analysis, we reveal a novel role for the fusion protein FZO-1/mitofusin 2 in regulating the structure of the inner mitochondrial membrane. Moreover, we have determined the influence of the individual mitochondrial fission (DRP-1/DRP1) and fusion (FZO-1/mitofusin 1,2; EAT-3/OPA1) proteins on animal behaviour and lifespan. We show that loss of these mitochondrial fusion or fission regulators induced age-dependent and progressive deficits in animal movement, as well as in muscle and neuronal function. Our results reveal that disruption of fusion induces more profound defects than lack of fission on animal behaviour and tissue function, and imply that while fusion is required throughout life, fission is more important later in life likely to combat ageing-associated stressors. Furthermore, our data demonstrate that mitochondrial function is not strictly dependent on morphology, with no correlation found between morphological changes and behavioural defects. Surprisingly, we find that disruption of either mitochondrial fission or fusion significantly reduces median lifespan, but maximal lifespan is unchanged, demonstrating that mitochondrial dynamics play an important role in limiting variance in longevity across isogenic populations. Overall, our study provides important new insights into the central role of mitochondrial dynamics in maintaining organismal health.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Longevidade/genética , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Mutação , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Estimativa de Kaplan-Meier , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/ultraestrutura , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura
13.
Geroscience ; 41(1): 51-67, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30729413

RESUMO

Nicotinamide adenine dinucleotide (reduced form: NADH) serves as a vital redox-energy currency for reduction-oxidation homeostasis and fulfilling energetic demands. While NADH exists as free and bound forms, only free NADH is utilized for complex I to power oxidative phosphorylation, especially important in neurons. Here, we studied how much free NADH remains available for energy production in mitochondria of old living neurons. We hypothesize that free NADH in neurons from old mice is lower than the levels in young mice and even lower in neurons from the 3xTg-AD Alzheimer's disease (AD) mouse model. To assess free NADH, we used lifetime imaging of NADH autofluorescence with 2-photon excitation to be able to resolve the pool of NADH in mitochondria, cytoplasm, and nuclei. Primary neurons from old mice were characterized by a lower free/bound NADH ratio than young neurons from both non-transgenic (NTg) and more so in 3xTg-AD mice. Mitochondrial compartments maintained 26 to 41% more reducing NADH redox state than cytoplasm for each age, genotype, and sex. Aging diminished the mitochondrial free NADH concentration in NTg neurons by 43% and in 3xTg-AD by 50%. The lower free NADH with age suggests a decline in capacity to regenerate free NADH for energetic supply to power oxidative phosphorylation which further worsens in AD. Applying this non-invasive approach, we showed the most explicit measures yet of bioenergetic deficits in free NADH with aging at the subcellular level in live neurons from in-bred mice and an AD model.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/enzimologia , Mitocôndrias/enzimologia , NAD/classificação , NAD/metabolismo , Neurônios/enzimologia , Animais , Modelos Animais de Doenças , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Mitocôndrias/patologia , Neurônios/ultraestrutura , Imagem Óptica , Oxirredução , Fosforilação Oxidativa , Caracteres Sexuais , Proteínas tau/genética
14.
Int J Dev Neurosci ; 74: 1-10, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30753937

RESUMO

The aim of this study was to examine the spatio-temporal appearance of different neuronal cell subtypes by analyzing expression patterns of several neuronal markers (calretinin, neurofilament 200 (NF200), vanilloid receptor 1(VR1) and calcitonin gene-related peptide (CGRP)) of the embryonic human spinal cord (SC). Developing human SCs from 11 human conceptuses beetwen 5-10 developmental weeks (DW) were examined by light and electron microscopy and immunofluorescence. Light and electron microscopy revealed different embryonic stages of recognizable structure of the SC. NF200, CGRP and VR1 positive cells were observed in SCs during 5th-6th DW. NF200 was predominantly expressed in the ventral part, indicating presence of motoneurons. As development advanced, NF200 was mainly expressed in the marginal zone. Expression of CGRP was intense during all of the investigated periods, predominantly during the 5th-6th DW pointing to neural sensory differentiation, as opposed to the last DW when reduced expression of CGRP in the marginal layer indicated the terminations of the sensory afferents. Expression of VR1 was highest in the intermediate zone, at the beginning and at the end of the investigated periods, pointing to VR1 spatial pattern in the visceral afferents in the grey matter, while the first signs of calretinin were found in the 9th-10th DW ventrally. Delineating the relationships between factors involved in processes of neuronal differentiation as well as spatial and temporal arrangement of SC interrelated neurons can provide a useful information about normal SC development as well as the insight in possible causes of anomalies and disorders during embryonic life.


Assuntos
Biomarcadores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Medula Espinal/citologia , Medula Espinal/embriologia , Fatores Etários , Calbindina 2/metabolismo , Calbindina 2/ultraestrutura , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Idade Gestacional , Humanos , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/ultraestrutura , Proteínas de Neurofilamentos/metabolismo , Proteínas de Neurofilamentos/ultraestrutura , Neurônios/classificação , Neurônios/ultraestrutura , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/ultraestrutura
15.
Biol Cell ; 111(4): 95-107, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30681171

RESUMO

BACKGROUND INFORMATION: The current consensus on cilia development posits that the ciliary transition zone (TZ) is formed via extension of nine centrosomal microtubules. In this model, TZ structure remains unchanged in microtubule number throughout the cilium life cycle. This model does not however explain structural variations of TZ structure seen in nature and could also lend itself to the misinterpretation that deviations from nine-doublet microtubule ultrastructure represent an abnormal phenotype. Thus, a better understanding of events that occur at the TZ in vivo during metazoan development is required. RESULTS: To address this issue, we characterized ultrastructure of two types of sensory cilia in developing Caenorhabditis elegans. We discovered that, in cephalic male (CEM) and inner labial quadrant (IL2Q) sensory neurons, ciliary TZs are structurally plastic and remodel from one structure to another during animal development. The number of microtubule doublets forming the TZ can be increased or decreased over time, depending on cilia type. Both cases result in structural TZ intermediates different from TZ in cilia of adult animals. In CEM cilia, axonemal extension and maturation occurs concurrently with TZ structural maturation. CONCLUSIONS AND SIGNIFICANCE: Our work extends the current model to include the structural plasticity of metazoan transition zone, which can be structurally delayed, maintained or remodelled in cell type-specific manner.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Cílios/ultraestrutura , Microtúbulos/ultraestrutura , Animais , Caenorhabditis elegans/ultraestrutura , Masculino , Neurônios/ultraestrutura
16.
Neuron ; 101(2): 204-206, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30653934

RESUMO

Two papers in Cell uncover reciprocal crosstalk of local translation and mitochondria in neurons. Rangaraju et al. (2019) observe tethered compartments of stable mitochondria in dendrites that provide a local energy supply for mRNA translation at synapses. Cioni et al. (2019) report a novel association of axonal RNA granules with Rab7a-late endosomes that provides a platform for local translation supporting mitochondria.


Assuntos
Axônios/metabolismo , Dendritos/metabolismo , Mitocôndrias/fisiologia , Neurônios/citologia , Biossíntese de Proteínas/fisiologia , Animais , Transporte Axonal , Axônios/ultraestrutura , Plasticidade Celular , Dendritos/ultraestrutura , Neurônios/ultraestrutura , RNA Mensageiro/metabolismo
17.
G3 (Bethesda) ; 9(3): 737-748, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30635441

RESUMO

The release of neurotransmitters from synaptic vesicles (SVs) at pre-synaptic release sites is the principle means by which information transfer between neurons occurs. Knowledge of the location of SVs within a neuron can thus provide valuable clues about the location of neurotransmitter release within a neuron and the downstream neurons to which a given neuron is connected, important information for understanding how neural circuits generate behavior. Here the development and characterization of four conditional tagged SV markers for Drosophila melanogaster is presented. This characterization includes evaluation of conditionality, specificity for SV localization, and sensitivity of detection in diverse neuron subtypes. These four SV markers are genome-edited variants of the synaptic vesicle-specific protein Rab3. They depend on either the B2 or FLP recombinases for conditionality, and incorporate GFP or mCherry fluorescent proteins, or FLAG or HA epitope tags, for detection.


Assuntos
Drosophila melanogaster/fisiologia , Proteínas Luminescentes/análise , Neurônios/fisiologia , Transmissão Sináptica , Vesículas Sinápticas , Animais , Biomarcadores/análise , Drosophila melanogaster/ultraestrutura , Neurônios/ultraestrutura , Sensibilidade e Especificidade
18.
Environ Toxicol ; 34(4): 469-475, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30614199

RESUMO

Cadmium (Cd) is considered a possible etiological factor in neurodegenerative diseases. However, the exact mechanism by which Cd induces neurotoxicity is not well elucidated. In this study, Neuro-2a cells were treated with 0, 10, 20, and 40 µM cadmium chloride for 24 hours to investigate the effects of Cd on the cytoskeleton of nerve cells. MTT assay and ELISA assay were used to examine cell viability and release of lactate dehydrogenase (LDH) from cells, respectively. Results showed that Cd reduced cell viability and increased the release of LDH in a dose-dependent manner (P < 0.05). The morphology of treated cell was damaged as indicated by cell collapse and dimensionality reduction. Moreover, the axonal spines and normal features of Cd-treated neurons disappeared. We checked the ultrastructure of Neuro-2a cells and found that Cd-induced swelling, membrane damage, overflow of cytoplasm contents, and cell fragmentation. Damaged mitochondria, expanded endoplasmic reticulum, and abnormal microfilaments were found in Cd-treated cells rather than in untreated cells. Compared with the control group, the relative release of glutamate in the supernatant after Cd treatment was reduced, indicating that Cd exposure could reduce the release of glutamate by inhibiting the function of nerve-2a cells. Cd decreased the mRNA and protein expression levels of cytoskeletal proteins including DBN, SYP, and TAU, which might promote cytoskeleton alterations in Cd-treated cells. In conclusion, Cd-induced actin cytoskeleton alterations and dysfunction of cultured neurons. The results of the present study provide new insights for the investigation of Cd-induced neurotoxicity.


Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Cádmio/toxicidade , Poluentes Ambientais/toxicidade , Neurônios/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Animais , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Neurônios/ultraestrutura , Síndromes Neurotóxicas/patologia
19.
Nat Commun ; 10(1): 342, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30664666

RESUMO

The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. With most studies failing to establish their structural identity and examine whether they are truly open-ended organelles, there is a need to study the anatomy of TNTs at the nanometer resolution. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate that they are composed of a bundle of open-ended individual tunneling nanotubes (iTNTs) that are held together by threads labeled with anti-N-Cadherin antibodies. iTNTs are filled with parallel actin bundles on which different membrane-bound compartments and mitochondria appear to transfer. These results provide evidence that neuronal TNTs have distinct structural features compared to other cell protrusions.


Assuntos
Extensões da Superfície Celular/ultraestrutura , Neurônios/ultraestrutura , Organelas/ultraestrutura , Animais , Transporte Biológico , Catecolaminas/metabolismo , Linhagem Celular , Extensões da Superfície Celular/metabolismo , Microscopia Crioeletrônica/métodos , Humanos , Camundongos , Neurônios/metabolismo , Organelas/metabolismo
20.
Methods Mol Biol ; 1880: 243-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610702

RESUMO

Autophagy is an essential degradative pathway that maintains neuronal homeostasis and prevents axon degeneration. However, the mechanisms of autophagy in neurons are only beginning to be understood. To address this fundamental gap in knowledge, we have established several key methodologies for live-cell imaging and quantitative analysis of autophagy in primary hippocampal neurons. Using these methods, we have defined compartment-specific dynamics of autophagy in real-time under basal versus stress conditions. For example, we have characterized autophagosome biogenesis in the distal axon and subsequent retrograde transport to the soma for degradation. Autophagosomes are also generated locally within the soma. In contrast to the axon, the majority of autophagosomes in dendrites are stationary, while some exhibit bidirectional movement. These studies establish an initial road map for autophagosome dynamics in each compartment of the neuron and set the stage for a more detailed understanding of neuronal autophagy in stress and disease.


Assuntos
Autofagossomos/ultraestrutura , Autofagia , Microscopia de Fluorescência/métodos , Neurônios/citologia , Imagem Óptica/métodos , Animais , Axônios/ultraestrutura , Técnicas de Cultura de Células/métodos , Células Cultivadas , Dendritos/ultraestrutura , Hipocampo/citologia , Camundongos , Camundongos Transgênicos , Microscopia Confocal/métodos , Neuroglia/citologia , Neurônios/ultraestrutura
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