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1.
Nat Commun ; 11(1): 4949, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009388

RESUMO

Electron microscopy (EM) is widely used for studying cellular structure and network connectivity in the brain. We have built a parallel imaging pipeline using transmission electron microscopes that scales this technology, implements 24/7 continuous autonomous imaging, and enables the acquisition of petascale datasets. The suitability of this architecture for large-scale imaging was demonstrated by acquiring a volume of more than 1 mm3 of mouse neocortex, spanning four different visual areas at synaptic resolution, in less than 6 months. Over 26,500 ultrathin tissue sections from the same block were imaged, yielding a dataset of more than 2 petabytes. The combined burst acquisition rate of the pipeline is 3 Gpixel per sec and the net rate is 600 Mpixel per sec with six microscopes running in parallel. This work demonstrates the feasibility of acquiring EM datasets at the scale of cortical microcircuits in multiple brain regions and species.


Assuntos
Processamento de Imagem Assistida por Computador , Microscopia Eletrônica de Transmissão , Rede Nervosa/ultraestrutura , Neurônios/ultraestrutura , Animais , Automação , Camundongos , Neocórtex/diagnóstico por imagem , Software
2.
PLoS Biol ; 18(8): e3000820, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866173

RESUMO

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.


Assuntos
Ácido Glutâmico/metabolismo , Cinesina/metabolismo , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo , Neurônios/metabolismo , Espastina/deficiência , Tubulina (Proteína)/metabolismo , Potenciais de Ação , Animais , Membrana Celular/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Atividade Motora , Neurônios/patologia , Neurônios/ultraestrutura , Transporte Proteico , Espastina/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismo
3.
PLoS One ; 15(8): e0237230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764808

RESUMO

In vivo two-photon microscopy utilizing a nonlinear optical process enables, in living mouse brains, not only the visualization of morphologies and functions of neural networks in deep regions but also their optical manipulation at targeted sites with high spatial precision. Because the two-photon excitation efficiency is proportional to the square of the photon density of the excitation laser light at the focal position, optical aberrations induced by specimens mainly limit the maximum depth of observations or that of manipulations in the microscopy. To increase the two-photon excitation efficiency, we developed a method for evaluating the focal volume in living mouse brains. With this method, we modified the beam diameter of the excitation laser light and the value of the refractive index in the immersion liquid to maximize the excitation photon density at the focal position. These two modifications allowed the successful visualization of the finer structures of hippocampal CA1 neurons, as well as the intracellular calcium dynamics in cortical layer V astrocytes, even with our conventional two-photon microscopy system. Furthermore, it enabled focal laser ablation dissection of both single apical and single basal dendrites of cortical layer V pyramidal neurons. These simple modifications would enable us to investigate the contributions of single cells or single dendrites to the functions of local cortical networks.


Assuntos
Encéfalo/ultraestrutura , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Animais , Desenho de Equipamento , Feminino , Masculino , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/ultraestrutura , Fótons
4.
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
5.
Proc Natl Acad Sci U S A ; 117(25): 14503-14511, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513712

RESUMO

The nanoscale co-organization of neurotransmitter receptors facing presynaptic release sites is a fundamental determinant of their coactivation and of synaptic physiology. At excitatory synapses, how endogenous AMPARs, NMDARs, and mGluRs are co-organized inside the synapse and their respective activation during glutamate release are still unclear. Combining single-molecule superresolution microscopy, electrophysiology, and modeling, we determined the average quantity of each glutamate receptor type, their nanoscale organization, and their respective activation. We observed that NMDARs form a unique cluster mainly at the center of the PSD, while AMPARs segregate in clusters surrounding the NMDARs. mGluR5 presents a different organization and is homogenously dispersed at the synaptic surface. From these results, we build a model predicting the synaptic transmission properties of a unitary synapse, allowing better understanding of synaptic physiology.


Assuntos
Modelos Neurológicos , Neurônios/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Ácido Glutâmico/metabolismo , Hipocampo/citologia , Hipocampo/diagnóstico por imagem , Hipocampo/fisiologia , Microscopia Intravital , Neurônios/ultraestrutura , Técnicas de Patch-Clamp , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Imagem Individual de Molécula
6.
Neuron ; 107(2): 368-382.e8, 2020 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-32442399

RESUMO

The ventral tegmental area (VTA) has dopamine, GABA, and glutamate neurons, which have been implicated in reward and aversion. Here, we determined whether VTA-glutamate or -GABA neurons play a role in innate defensive behavior. By VTA cell-type-specific genetic ablation, we found that ablation of glutamate, but not GABA, neurons abolishes escape behavior in response to threatening stimuli. We found that escape behavior is also decreased by chemogenetic inhibition of VTA-glutamate neurons and detected increases in activity in VTA-glutamate neurons in response to the threatening stimuli. By ultrastructural and electrophysiological analysis, we established that VTA-glutamate neurons receive a major monosynaptic glutamatergic input from the lateral hypothalamic area (LHA) and found that photoinhibition of this input decreases escape responses to threatening stimuli. These findings indicate that VTA-glutamate neurons are activated by and required for innate defensive responses and that information on threatening stimuli to VTA-glutamate neurons is relayed by LHA-glutamate neurons.


Assuntos
Agressão/fisiologia , Ácido Glutâmico/fisiologia , Neurônios/fisiologia , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/fisiologia , Animais , Reação de Fuga , Humanos , Região Hipotalâmica Lateral/citologia , Região Hipotalâmica Lateral/fisiologia , Hipotálamo/citologia , Hipotálamo/fisiologia , Camundongos , Neurônios/ultraestrutura , Optogenética , Estimulação Luminosa , Reflexo Monosináptico/fisiologia , Área Tegmentar Ventral/ultraestrutura , Ácido gama-Aminobutírico/fisiologia
7.
Toxicology ; 438: 152461, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32278788

RESUMO

The α-Synuclein (α-syn) and tau have synergistic effects on neurodegenerative diseases induced by environmental factors or genetic mutation. Thus, we investigated the role of α-syn and tau in neurodegeneration induced by chronic methamphetamine (METH) exposure (1.0∼20.0 mg/kg/d body weight, for 14 consecutive days). Here, we present a mice model with evidences of α-syn and tau participating in toxicology in chronic METH. METH increased α-syn level in the stratum oriens, pyramidal layer, stratum radiatum and stratum moleculare of hippocampal CA1, CA2 and CA3, polymorph layer of hippocampal dentate gyrus (DG), and substantia nigra (SN). The subcellular locations of the upregulated α-syn were mainly found in mitochondria and axons. The METH upregulated α-syn may directly induce mitochondrial damage, myelin sheath destruction, and synaptic failure. Also, the excess α-syn might indirectly promote tau phosphorylation through tau kinase GSK3ß and CDK5, leading to microtubule depolymerization and eventually fusion deficit of autophagosome and lysosome. In the in vitro experiment, the autophagic vacuoles failed to fuse with the lysosome. The neuropathology induced by both the direct and indirect effects of α-syn could be alleviated by α-syn knockout. Taking together, these results indicate that the α-syn mediates the neurodegenerative process induced by chronic METH and that reducing α-syn might be a potential approach to protect the toxic effects of METH and also be, to a broader view, of therapeutic value in neurodegenerative diseases.


Assuntos
Hipocampo/metabolismo , Degeneração Neural , Neurônios/metabolismo , Síndromes Neurotóxicas/prevenção & controle , Substância Negra/metabolismo , alfa-Sinucleína/deficiência , Animais , Autofagia , Axônios/metabolismo , Axônios/ultraestrutura , Células Cultivadas , Quinase 5 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipocampo/ultraestrutura , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Masculino , Metanfetamina , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Neurônios/ultraestrutura , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/patologia , Fosforilação , Substância Negra/ultraestrutura , Fatores de Tempo , alfa-Sinucleína/genética , Proteínas tau/metabolismo
8.
J Neurosci ; 40(11): 2228-2245, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32001612

RESUMO

Sensory cortex exhibits receptive field plasticity throughout life in response to changes in sensory experience and offers the experimental possibility of aligning functional changes in receptive field properties with underpinning structural changes in synapses. We looked at the effects on structural plasticity of two different patterns of whisker deprivation in male and female mice: chessboard deprivation, which causes functional plasticity; and all deprived, which does not. Using 2-photon microscopy and chronic imaging through a cranial window over the barrel cortex, we found that layer 2/3 neurones exhibit robust structural plasticity, but only in response to whisker deprivation patterns that cause functional plasticity. Chessboard pattern deprivation caused dual-component plasticity in layer 2/3 by (1) increasing production of new spines that subsequently persisted for weeks and (2) enlarging spine head sizes in the preexisting stable spine population. Structural plasticity occurred on basal dendrites, but not apical dendrites. Both components of plasticity were absent in αCaMKII-T286A mutants that lack LTP and experience-dependent potentiation in barrel cortex, implying that αCaMKII autophosphorylation is not only important for stabilization and enlargement of spines, but also for new spine production. These studies therefore reveal the relationship between spared whisker potentiation in layer 2/3 neurones and the form and mechanisms of structural plasticity processes that underlie them.SIGNIFICANCE STATEMENT This study provides a missing link in a chain of reasoning that connects LTP to experience-dependent functional plasticity in vivo We found that increases in dendritic spine formation and spine enlargement (both of which are characteristic of LTP) only occurred in barrel cortex during sensory deprivation that produced potentiation of sensory responses. Furthermore, the dendritic spine plasticity did not occur during sensory deprivation in mice lacking LTP and experience-dependent potentiation (αCaMKII autophosphorylation mutants). We also found that the dual-component dendritic spine plasticity only occurred on basal dendrites and not on apical dendrites, thereby resolving a paradox in the literature suggesting that layer 2/3 neurones lack structural plasticity in response to sensory deprivation.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Espinhas Dendríticas/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/enzimologia , Privação Sensorial/fisiologia , Córtex Somatossensorial/fisiopatologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Tamanho Celular , Espinhas Dendríticas/ultraestrutura , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/ultraestrutura , Fosforilação , Processamento de Proteína Pós-Traducional , Técnica de Janela Cutânea , Córtex Somatossensorial/citologia , Distúrbios Somatossensoriais/fisiopatologia , Vibrissas/lesões , Vibrissas/inervação
9.
Curr Protoc Neurosci ; 91(1): e90, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31971665

RESUMO

Mosaic analysis with a repressible cell marker (MARCM)-related technologies are positive genetic mosaic labeling systems that have been widely applied in studies of Drosophila brain development and neural circuit formation to identify diverse neuronal types, reconstruct neural lineages, and investigate the function of genes and molecules. Two types of MARCM-related technologies have been developed: single-colored and twin-colored. Single-colored MARCM technologies label one of two twin daughter cells in otherwise unmarked background tissues through site-specific recombination of homologous chromosomes during mitosis of progenitors. On the other hand, twin-colored genetic mosaic technologies label both twin daughter cells with two distinct colors, enabling the retrieval of useful information from both progenitor-derived cells and their subsequent clones. In this overview, we describe the principles and usage guidelines for MARCM-related technologies in order to help researchers employ these powerful genetic mosaic systems in their investigations of intricate neurobiological topics. © 2020 by John Wiley & Sons, Inc.


Assuntos
Drosophila melanogaster/genética , Neurônios/ultraestrutura , Animais , Divisão Celular , Linhagem da Célula , Células Clonais/ultraestrutura , Cor , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Expressão Gênica , Genes de Insetos , Genes Reporter , Genes Supressores , Discos Imaginais/ultraestrutura , Mosaicismo , Células-Tronco Neurais/citologia , Interferência de RNA , Recombinases , Recombinação Genética
10.
Biochem Biophys Res Commun ; 523(4): 931-938, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-31964527

RESUMO

BACKGROUND: Brain damage in premature infants often occurs in very low birth weight infants (VLBW) as a result of hypoxia-ischemia and can lead to cognitive impairment and movement disorders. Many miRNAs have been demonstrated to participate in hypoxia-ischemic brain damage (HIBD). This study was designed to investigate the roles of miR-200b-3p in brain damage of neonatal rats induced by hypoxia-ischemia. METHODS AND RESULTS: Three-day-old SD rats were used to establish the model of hypoxia-ischemic brain injury mimicking premature infants. RT-qPCR showed that miR-200b-3p was up-regulated in rat brains at the early stage following hypoxia-ischemic treatment. Bioinformatics analysis identified that Slit2 is a target gene of miR-200b-3p and luciferase reporter gene assay confirmed that miR-200b-3p can interact with and target Slit2 mRNA. Inhibition of miR-200b-3p by antagomir increased Slit2 expression at both the mRNA and protein levels in rat brains. TUNEL assay and transmission electron microscopy (TEM) analysis showed decreased numbers of apoptotic neurons in the hypoxia-ischemia-treated animals as a result of administration of miR-200b-3p antagomir. Administration of miR-200b-3p antagomir attenuated spatial and learning memory loss in the animals induced by hypoxia-ischemia as compared to controls. CONCLUSION: Our study has demonstrated that Slit2 is a target gene of miR-200b-3p and that the hypoxia-ischemic brain damage in neonatal rats was alleviated by inhibiting miR-200b-3p via Slit2. miR-200b-3p may be a potential therapeutic target of HIBD for further investigation.


Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Hipóxia-Isquemia Encefálica/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Animais , Animais Recém-Nascidos , Antagomirs/farmacologia , Sequência de Bases , Encéfalo/fisiopatologia , Encéfalo/ultraestrutura , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/fisiopatologia , Memória/efeitos dos fármacos , MicroRNAs/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Ratos Sprague-Dawley , Aprendizagem Espacial/efeitos dos fármacos , Regulação para Cima/genética
11.
Cell Mol Life Sci ; 77(1): 161-177, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31161284

RESUMO

Peripheral nervous system development involves a tight coordination of neuronal birth and death and a substantial remodelling of the myelinating glia cytoskeleton to achieve myelin wrapping of its projecting axons. However, how these processes are coordinated through time is still not understood. We have identified engulfment and cell motility 1, Elmo1, as a novel component that regulates (i) neuronal numbers within the Posterior Lateral Line ganglion and (ii) radial sorting of axons by Schwann cells (SC) and myelination in the PLL system in zebrafish. Our results show that neuronal and myelination defects observed in elmo1 mutant are rescued through small GTPase Rac1 activation. Inhibiting macrophage development leads to a decrease in neuronal numbers, while peripheral myelination is intact. However, elmo1 mutants do not show defective macrophage activity, suggesting a role for Elmo1 in PLLg neuronal development and SC myelination independent of macrophages. Forcing early Elmo1 and Rac1 expression specifically within SCs rescues elmo1-/- myelination defects, highlighting an autonomous role for Elmo1 and Rac1 in radial sorting of axons by SCs and myelination. This uncovers a previously unknown function of Elmo1 that regulates fundamental aspects of PNS development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bainha de Mielina/metabolismo , Neurogênese , Neurônios/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Apoptose , Axônios/metabolismo , Axônios/ultraestrutura , Movimento Celular , Neurônios/metabolismo , Neurônios/ultraestrutura , Nervos Periféricos/crescimento & desenvolvimento , Nervos Periféricos/ultraestrutura , Células de Schwann/citologia , Células de Schwann/metabolismo , Células de Schwann/ultraestrutura
12.
Methods Mol Biol ; 2041: 209-221, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646491

RESUMO

Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.


Assuntos
Trifosfato de Adenosina/metabolismo , Imunofluorescência/métodos , Neurônios/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Retina/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Transporte Biológico , Exocitose , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Neurônios/ultraestrutura , Retina/ultraestrutura
13.
Environ Toxicol ; 35(1): 55-65, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31497924

RESUMO

Overexposure to manganese (Mn) is widely known to induce alpha-synuclein (α-Syn) oligomerization, which has been attributed to the oxidative damage of α-Syn protein. Trehalose has been shown to induce autophagy and serve as a chemical chaperone, but little information has been reported about its effect on Mn-induced α-Syn oligomerization. In this study, we investigate whether trehalose can effectively interfere with Mn-induced α-Syn oligomerization, using different concentrations of trehalose (2% and 4% (g/vol [mL])) in a mouse model of manganism. After 6 weeks of exposure to Mn, both oxidative stress and autophagy were activated and resulted in α-Syn oligomerization and neuronal cell damage in the mouse brain tissue. Our results also revealed that pretreatment with trehalose significantly reduced the oxidative damage to α-Syn protein and increased autophagy activation. These findings clearly demonstrated that trehalose can relieve Mn-induced α-Syn oligomerization and neuronal cell damage through its anti-oxidative and autophagy-inducing effects.


Assuntos
Autofagia/efeitos dos fármacos , Manganês/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Trealose/farmacologia , alfa-Sinucleína/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Multimerização Proteica
14.
Micron ; 130: 102799, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31846724

RESUMO

The telencephalon of adult Scincella tsinlingensis was detected by light and electron microscopy, which will be used as the basis for further neurobiological comparative studies. The telencephalon of S. tsinlingensis was consisted of paired olfactory bulbs, paired cerebral hemispheres, and a telencephalon medium or impar. Main-olfactory bulb can be classified into six layers such as olfactory nerve fibers layer, glomerular layer, external plexiform layer, mitral layer, internal plexiform layer, granular layer and ependyma layer. The dorsal part of telencephalon contained the cortex and dorsal ventricular ridge. The cerebral cortex of S. tsinlingens was relatively thin, while the dorsal cortex was the thinnest, but gradually thickened as it extended to the medial and lateral cortex. The neural cells, glial cells and ependymal cells widely distributed in the cerebral cortex represented similar ultrastructural characteristics to those described in other vertebrates. Golgi staining revealed multipolar cell, bitufted cell and monotufted cell in three cortical layers of medial cortex. The results indicated that the cytoarchitectonic characteristics of telencephalon in S. tsinlingensis resembled those found in other lizards.


Assuntos
Lagartos/anatomia & histologia , Telencéfalo/ultraestrutura , Animais , Córtex Cerebral/citologia , Córtex Cerebral/ultraestrutura , Feminino , Masculino , Neurônios/citologia , Neurônios/ultraestrutura , Bulbo Olfatório/citologia , Bulbo Olfatório/ultraestrutura , Telencéfalo/citologia
15.
IET Nanobiotechnol ; 13(9): 891-895, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31811756

RESUMO

Conductive atomic force indentation (CAFI) was proposed to study the self-repair behaviour of the neuronal cell membrane here. CAFI was used to detect the changes of membrane potentials by performing the mechanical indentation on neurons with a conductive atomic force microscope. In the experiment, a special insulation treatment was made on the conductive probe, which turned out to be a conductive nanoelectrode, to implement the CAFI function. The mechanical properties of the neuronal cell membrane surface were tested and the membrane potential changes of neurons cultured in vitro were detected. The self-repair behaviour of the neuronal cell membrane after being punctured was investigated. The experiment results show that CAFI provides a new way for the study of self-repair behaviours of neuronal cell membranes and mechanical and electrical properties of living cells.


Assuntos
Microscopia de Força Atômica/métodos , Neurônios/fisiologia , Animais , Membrana Celular/ultraestrutura , Células Cultivadas , Camundongos , Neurônios/ultraestrutura
16.
Fluids Barriers CNS ; 16(1): 39, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31849332

RESUMO

BACKGROUND: A growing body of evidence suggests that the accumulation of amyloid-ß and tau (HPτ) in the brain of patients with the dementia subtype idiopathic normal pressure hydrocephalus (iNPH) is associated with delayed extravascular clearance of metabolic waste. Whether also clearance of intracellular debris is affected in these patients needs to be examined. Hypothetically, defective extra- and intra-cellular clearance of metabolites may be instrumental in the neurodegeneration and dementia characterizing iNPH. This study explores whether iNPH is associated with altered mitochondria phenotype in neurons and astrocytes. METHODS: Cortical brain biopsies of 9 reference (REF) individuals and 30 iNPH patients were analyzed for subcellular distribution and morphology of mitochondria using transmission electron microscopy. In neuronal soma of REF and iNPH patients, we identified normal, pathological and clustered mitochondria, mitochondria-endoplasmic reticulum contact sites and autophagic vacuoles. We also differentiated normal and pathological mitochondria in pre- and post-synaptic nerve terminals, as well as in astrocytic endfoot processes towards vessels. RESULTS: We found a high prevalence of pathological mitochondria in neuronal soma and pre- and post-synaptic terminals, as well as increased mitochondrial clustering, and altered number of mitochondria-endoplasmic reticulum contact sites in iNPH. Non-fused autophagic vacuoles were more abundant in neuronal soma of iNPH patients, suggestive of cellular clearance failure. Moreover, the length of postsynaptic densities was reduced in iNPH, potentially related to reduced synaptic activity. In astrocytic endfoot processes, we also found increased number, area and area fraction of pathological mitochondria in iNPH patients. The proportion of pathological mitochondria correlated significantly with increasing degree of astrogliosis and reduced perivascular expression of aquaporin-4 (AQP4), assessed by light microscopy immunohistochemistry. CONCLUSION: Our results provide evidence of mitochondrial pathology and signs of impaired cellular clearance in iNPH patients. The results indicate that iNPH is a neurodegenerative disease with close similarity to Alzheimer's disease.


Assuntos
Astrócitos/patologia , Encéfalo/patologia , Sistema Glinfático/patologia , Hidrocefalia de Pressão Normal/patologia , Mitocôndrias/patologia , Neurônios/patologia , Astrócitos/ultraestrutura , Autofagia , Encéfalo/ultraestrutura , Retículo Endoplasmático/patologia , Retículo Endoplasmático/ultraestrutura , Sistema Glinfático/ultraestrutura , Humanos , Mitocôndrias/ultraestrutura , Neurônios/ultraestrutura , Sinapses/patologia , Sinapses/ultraestrutura
17.
Int J Mol Sci ; 20(23)2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31757021

RESUMO

Mucopolysaccharidosis type II (MPS II) is a rare lysosomal storage disease (LSD) involving a genetic error in iduronic acid-2-sulfatase (IDS) metabolism that leads to accumulation of glycosaminoglycans within intracellular lysosomes. The primary treatment for MPS II, enzyme replacement therapy, is not effective for central nervous system (CNS) symptoms, such as intellectual disability, because the drugs do not cross the blood-brain barrier. Recently, autophagy has been associated with LSDs. In this study, we examined the morphologic relationship between neuronal damage and autophagy in IDS knockout mice using antibodies against subunit c of mitochondrial adenosine triphosphate (ATP) synthetase and p62. Immunohistological changes suggesting autophagy, such as vacuolation, were observed in neurons, microglia, and pericytes throughout the CNS, and the numbers increased over postnatal development. Oral administration of chloroquine, which inhibits autophagy, did not suppress damage to microglia and pericytes, but greatly reduced neuronal vacuolation and eliminated neuronal cells with abnormal inclusions. Thus, decreasing autophagy appears to prevent neuronal degeneration. These results suggest that an autophagy modulator could be used in addition to conventional enzyme replacement therapy to preserve the CNS in patients with MPS II.


Assuntos
Autofagia , Mucopolissacaridose II/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cloroquina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mucopolissacaridose II/patologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo
18.
Brain Struct Funct ; 224(9): 3321-3338, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31679085

RESUMO

In both Parkinson's disease (PD) patients and MPTP-treated non-human primates, there is a profound neuronal degeneration of the intralaminar centromedian/parafascicular (CM/Pf) thalamic complex. Although this thalamic pathology has long been established in PD (and other neurodegenerative disorders), the impact of CM/Pf cell loss on the integrity of the thalamo-striatal glutamatergic system and its regulatory functions upon striatal neurons remain unknown. In the striatum, cholinergic interneurons (ChIs) are important constituents of the striatal microcircuitry and represent one of the main targets of CM/Pf-striatal projections. Using light and electron microscopy approaches, we have analyzed the potential impact of CM/Pf neuronal loss on the anatomy of the synaptic connections between thalamic terminals (vGluT2-positive) and ChIs neurons in the striatum of parkinsonian monkeys treated chronically with MPTP. The following conclusions can be drawn from our observations: (1) as reported in PD patients, and in our previous monkey study, CM/Pf neurons undergo profound degeneration in monkeys chronically treated with low doses of MPTP. (2) In the caudate (head and body) nucleus of parkinsonian monkeys, there is an increased density of ChIs. (3) Despite the robust loss of CM/Pf neurons, no significant change was found in the density of thalamostriatal (vGluT2-positive) terminals, and in the prevalence of vGluT2-positive terminals in contact with ChIs in parkinsonian monkeys. These findings provide new information about the state of thalamic innervation of the striatum in parkinsonian monkeys with CM/Pf degeneration, and bring up an additional level of intricacy to the consequences of thalamic pathology upon the functional microcircuitry of the thalamostriatal system in parkinsonism. Future studies are needed to assess the importance of CM/Pf neuronal loss, and its potential consequences on the neuroplastic changes induced in the synaptic organization of the thalamostriatal system, in the development of early cognitive impairments in PD.


Assuntos
Núcleo Caudado/patologia , Neurônios Colinérgicos/patologia , Ácido Glutâmico , Núcleos Intralaminares do Tálamo/patologia , Neurônios/patologia , Transtornos Parkinsonianos/patologia , Putamen/patologia , Animais , Núcleo Caudado/ultraestrutura , Neurônios Colinérgicos/ultraestrutura , Feminino , Interneurônios/patologia , Interneurônios/ultraestrutura , Núcleos Intralaminares do Tálamo/ultraestrutura , Macaca mulatta , Masculino , Vias Neurais/patologia , Vias Neurais/ultraestrutura , Neurônios/ultraestrutura , Putamen/ultraestrutura , Sinapses/patologia , Sinapses/ultraestrutura , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo
19.
Nat Methods ; 16(12): 1323-1331, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31686039

RESUMO

We demonstrate that a deep neural network can be trained to virtually refocus a two-dimensional fluorescence image onto user-defined three-dimensional (3D) surfaces within the sample. Using this method, termed Deep-Z, we imaged the neuronal activity of a Caenorhabditis elegans worm in 3D using a time sequence of fluorescence images acquired at a single focal plane, digitally increasing the depth-of-field by 20-fold without any axial scanning, additional hardware or a trade-off of imaging resolution and speed. Furthermore, we demonstrate that this approach can correct for sample drift, tilt and other aberrations, all digitally performed after the acquisition of a single fluorescence image. This framework also cross-connects different imaging modalities to each other, enabling 3D refocusing of a single wide-field fluorescence image to match confocal microscopy images acquired at different sample planes. Deep-Z has the potential to improve volumetric imaging speed while reducing challenges relating to sample drift, aberration and defocusing that are associated with standard 3D fluorescence microscopy.


Assuntos
Aprendizado Profundo , Microscopia de Fluorescência/métodos , Animais , Caenorhabditis elegans/ultraestrutura , Microscopia Confocal , Neurônios/ultraestrutura
20.
Nat Commun ; 10(1): 4694, 2019 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619676

RESUMO

Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.


Assuntos
Actinas/ultraestrutura , Citoesqueleto/ultraestrutura , DNA/ultraestrutura , Microscopia de Fluorescência/métodos , Microscopia de Polarização/métodos , Miosinas/ultraestrutura , Neurônios/ultraestrutura , Animais , Bacteriófago lambda/genética , Linhagem Celular Tumoral , Hipocampo/citologia , Humanos , Rim , Camundongos
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