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1.
Viruses ; 12(9)2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32911874

RESUMO

Since the global outbreak of SARS-CoV-2 (COVID-19), infections of diverse human organs along with multiple symptoms continue to be reported. However, the susceptibility of the brain to SARS-CoV-2, and the mechanisms underlying neurological infection are still elusive. Here, we utilized human embryonic stem cell-derived brain organoids and monolayer cortical neurons to investigate infection of brain with pseudotyped SARS-CoV-2 viral particles. Spike-containing SARS-CoV-2 pseudovirus infected neural layers within brain organoids. The expression of ACE2, a host cell receptor for SARS-CoV-2, was sustained during the development of brain organoids, especially in the somas of mature neurons, while remaining rare in neural stem cells. However, pseudotyped SARS-CoV-2 was observed in the axon of neurons, which lack ACE2. Neural infectivity of SARS-CoV-2 pseudovirus did not increase in proportion to viral load, but only 10% of neurons were infected. Our findings demonstrate that brain organoids provide a useful model for investigating SARS-CoV-2 entry into the human brain and elucidating the susceptibility of the brain to SARS-CoV-2.


Assuntos
Betacoronavirus/fisiologia , Neurônios/virologia , Organoides/virologia , Prosencéfalo/virologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Axônios/enzimologia , Diferenciação Celular , Células Cultivadas , Córtex Cerebral/citologia , Células-Tronco Embrionárias/virologia , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/fisiologia , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/virologia , Neurônios/enzimologia , Peptidil Dipeptidase A/fisiologia , Prosencéfalo/citologia , Receptores Virais/fisiologia , Carga Viral , Tropismo Viral , Internalização do Vírus
2.
Life Sci ; 258: 118204, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32763296

RESUMO

AIMS: Liver kinase B1 (LKB1) is a serine/threonine kinase. Although many biological functions of LKB1 have been identified, the role of hypothalamic LKB1 in the regulation of central energy metabolism and susceptibility to obesity is unknown. Therefore, we constructed POMC neuron-specific LKB1 knockout mice (PomcLkb1 KO) and studied it at the physiological, morphological, and molecular biology levels. MAIN METHODS: Eight-week-old male PomcLkb1 KO mice and their littermates were fed a standard chow fat diet (CFD) or a high-fat diet (HFD) for 3 months. Body weight and food intake were monitored. Dual-energy X-ray absorptiometry was used to measure the fat mass and lean mass. Glucose and insulin tolerance tests and serum biochemical markers were evaluated in the experimental mice. In addition, the levels of peripheral lipogenesis genes and central energy metabolism were measured. KEY FINDINGS: PomcLkb1 KO mice did not exhibit impairments under normal physiological conditions. After HFD intervention, the metabolic phenotype of the PomcLkb1 KO mice changed, manifesting as increased food intake and an enhanced obesity phenotype. More seriously, PomcLkb1 KO mice showed increased leptin resistance, worsened hypothalamic inflammation and reduced POMC neuronal expression. SIGNIFICANCE: We provide evidence that LKB1 in POMC neurons plays a significant role in regulating energy homeostasis. LKB1 in POMC neurons emerges as a target for therapeutic intervention against HFD-induced obesity and metabolic diseases.


Assuntos
Deleção de Genes , Neurônios/enzimologia , Obesidade/enzimologia , Pró-Opiomelanocortina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tecido Adiposo/patologia , Animais , Dieta Hiperlipídica , Epididimo/patologia , Comportamento Alimentar , Regulação da Expressão Gênica , Glucose/metabolismo , Hipotálamo/patologia , Inflamação/patologia , Leptina/metabolismo , Fígado/enzimologia , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Obesidade/sangue , Obesidade/patologia , Pró-Opiomelanocortina/genética , Ganho de Peso
3.
Ecotoxicol Environ Saf ; 201: 110799, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32544743

RESUMO

Mercury (Hg) is an environmental contaminant that poses great risk to human health. However, it is still widely used in artisanal gold-mining enterprises around the world, especially in developing countries. Methylmercury (MeHg) is produced environmentally by biomethylation of inorganic Hg present in water sediments, leading to its subsequent accumulation in the aquatic food chain. Due to its high metabolic rate, the Central Nervous System (CNS) is one of the main targets of MeHg. In the present study, we investigate the impact of chronic MeHg intoxication on NADPH diaphorase (NADPH-d) activity and astrocyte mobilization in the visual cortex of the rat. After 60 days of MeHg administration by oral gavage (0.04 mg/kg/day), tissue samples containing the visual cortex were submitted to measurements of Hg levels, NADPH-d activity, and GFAP immunohistochemistry for identification of astrocytes. MeHg intoxication was associated with increased Hg deposits and with reduced NADPH-d neuropil reactivity in the visual cortex. A morphometric analysis suggested that NADPH-d-positive neurons were mostly spared from MeHg harmful action and intoxicated animals had astrocytic activation similar to the control group. The decrease in NADPH-d neuropil reactivity may be due to the negative effect of chronic MeHg poisoning on both the synthesis and transport of this enzyme in afferent pathways to the visual cortex. The relative resistance of NADPH-d-reactive neurons to chronic MeHg intoxication may be associated with peculiarities in cell metabolism or to a protective role of nitric oxide, safeguarding those neurons from Hg deleterious effects.


Assuntos
Astrócitos/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Compostos de Metilmercúrio/toxicidade , NADPH Desidrogenase/metabolismo , Neurônios/efeitos dos fármacos , Córtex Visual/efeitos dos fármacos , Animais , Astrócitos/enzimologia , Comportamento Animal/efeitos dos fármacos , Poluentes Ambientais/metabolismo , Ouro , Humanos , Masculino , Compostos de Metilmercúrio/metabolismo , Mineração , Neurônios/enzimologia , Ratos , Ratos Wistar , Córtex Visual/enzimologia , Córtex Visual/patologia
4.
Life Sci ; 254: 117755, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32437792

RESUMO

AIMS: Efficient memory formation in rodents depends on adult neurogenesis in the subgranular zone of the hippocampus, and mounting evidence suggests that deficiencies in initiating repair of oxidatively induced DNA damage may impair neurogenesis. Hence, we aimed to determine whether loss of the DNA glycosylase, endonuclease VIII-like 1 (Neil1), affects hippocampal neurogenesis and memory performance in young-adult mice. MAIN METHODS: Eight-week-old male wild-type and Neil1-deficient (Neil1-/-) mice were treated with bromodeoxyuridine to track neuronal proliferation and differentiation. A neurosphere formation assay was further used to measure neuroprogenitor proliferative capacity. Hippocampus-related memory functions were assessed with Y-maze spontaneous alternation and novel object recognition tests. KEY FINDINGS: Young-adult male Neil1-/- mice exhibited diminished adult hippocampal neurogenesis in the dentate gyrus, probably as a result of poor survival of newly proliferated neurons. Furthermore, the Y-maze spontaneous alternation and novel object recognition tests respectively revealed that Neil1 deficiency impairs spatial and non-spatial hippocampus-related memory functions. We also found that expression of p53, a central regulator of apoptosis, was upregulated in the dentate gyrus of Neil1-/- mice, while the level of ß-catenin, a key cell survival molecule, was downregulated. SIGNIFICANCE: The DNA glycosylase, Neil1, promotes successful hippocampal neurogenesis and learning and memory in young-adult mice.


Assuntos
Cognição/fisiologia , DNA Glicosilases/deficiência , Hipocampo/enzimologia , Memória/fisiologia , Neurônios/enzimologia , Animais , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Disfunção Cognitiva/enzimologia , Disfunção Cognitiva/patologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Giro Denteado/citologia , Giro Denteado/enzimologia , Hipocampo/citologia , Hipocampo/metabolismo , Aprendizagem/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese/fisiologia , Neurônios/citologia
5.
Nat Commun ; 11(1): 2501, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427844

RESUMO

Anxiety is common in patients suffering from chronic pain. Here, we report anxiety-like behaviors in mouse models of chronic pain and reveal that nNOS-expressing neurons in ventromedial prefrontal cortex (vmPFC) are essential for pain-induced anxiety but not algesia, using optogenetic and chemogenetic strategies. Additionally, we determined that excitatory projections from the posterior subregion of paraventricular thalamic nucleus (pPVT) provide a neuronal input that drives the activation of vmPFC nNOS-expressing neurons in our chronic pain models. Our results suggest that the pain signal becomes an anxiety signal after activation of vmPFC nNOS-expressing neurons, which causes subsequent release of nitric oxide (NO). Finally, we show that the downstream molecular mechanisms of NO likely involve enhanced glutamate transmission in vmPFC CaMKIIα-expressing neurons through S-nitrosylation-induced AMPAR trafficking. Overall, our data suggest that pPVT excitatory neurons drive chronic pain-induced anxiety through activation of vmPFC nNOS-expressing neurons, resulting in NO-mediated AMPAR trafficking in vmPFC pyramidal neurons.


Assuntos
Dor Crônica/enzimologia , Dor Crônica/psicologia , Núcleos da Linha Média do Tálamo/enzimologia , Neurônios/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Córtex Pré-Frontal/enzimologia , Animais , Ansiedade , Comportamento Animal , Dor Crônica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleos da Linha Média do Tálamo/citologia , Neurônios/citologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Córtex Pré-Frontal/citologia
6.
Nat Commun ; 11(1): 1962, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327659

RESUMO

Topoisomerase 1 (TOP1) relieves torsional stress in DNA during transcription and facilitates the expression of long (>100 kb) genes, many of which are important for neuronal functions. To evaluate how loss of Top1 affected neurons in vivo, we conditionally deleted (cKO) Top1 in postmitotic excitatory neurons in the mouse cerebral cortex and hippocampus. Top1 cKO neurons develop properly, but then show biased transcriptional downregulation of long genes, signs of DNA damage, neuroinflammation, increased poly(ADP-ribose) polymerase-1 (PARP1) activity, single-cell somatic mutations, and ultimately degeneration. Supplementation of nicotinamide adenine dinucleotide (NAD+) with nicotinamide riboside partially blocked neurodegeneration, and increased the lifespan of Top1 cKO mice by 30%. A reduction of p53 also partially rescued cortical neuron loss. While neurodegeneration was partially rescued, behavioral decline was not prevented. These data indicate that reducing neuronal loss is not sufficient to limit behavioral decline when TOP1 function is disrupted.


Assuntos
DNA Topoisomerases Tipo I/deficiência , Instabilidade Genômica , Doenças Neurodegenerativas/enzimologia , Neurônios/enzimologia , Animais , Apoptose/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Dano ao DNA , DNA Topoisomerases Tipo I/genética , Hipocampo/enzimologia , Hipocampo/patologia , Inflamação , Camundongos , Camundongos Knockout , Mortalidade Prematura , Atividade Motora , Mutação , NAD/administração & dosagem , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Niacinamida/administração & dosagem , Niacinamida/análogos & derivados , Poli(ADP-Ribose) Polimerase-1/metabolismo
7.
J Stroke Cerebrovasc Dis ; 29(6): 104801, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32249206

RESUMO

BACKGROUND: Ischemic stroke is the leading cause of disability and death globally. Micro-RNAs (miRNAs) have been reported to play important roles in the development and pathogenesis of the nervous system. However, the exact function and mechanism of miRNAs have not been fully elucidated about brain damage caused by cerebral ischemia/reperfusion (I/R). METHODS: In this study, we explored the neuroprotective effects of miR-219a-5p on brain using an in vitro ischemia model (mouse neuroblastoma N2a cells treated with oxyglucose deprivation and reperfusion), and in vivo cerebral I/R model in mice. Western blot assay and Reverse Transcription-Polymerase Chain Reaction were used to check the expression of molecules involved. Flow cytometry and cholecystokinin were used to examine cell apoptosis, respectively. RESULTS: Our research shows that miR-219a-5p gradually decreases in cerebral I/R models in vivo and in vitro. In vitro I/R, we find that miR-219a-5p mimics provided evidently protection for cerebral I/R damage, as shown by increased cell viability and decreased the release of LDH and cell apoptosis. Mechanically, our findings indicate that miR-219a-5p binds to cAMP specific 3', 5'-cyclic phosphodiesterase 4D (PDE4D) mRNA in the 3'-UTR region, which subsequently leads to a decrease in Pde4d expression in I/R N2a cells. CONCLUSIONS: Our results provide new ideas for the study of the mechanism of cerebral ischemia/reperfusion injury, and lay the foundation for further research on the treatment of brain I/R injury. Upregulation of miR-219a-5p decreases cerebral ischemia/reperfusion injury by targeting Pde4d in vitro.


Assuntos
Apoptose , Encéfalo/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Infarto da Artéria Cerebral Média/enzimologia , MicroRNAs/metabolismo , Neurônios/enzimologia , Traumatismo por Reperfusão/enzimologia , Regiões 3' não Traduzidas , Animais , Sítios de Ligação , Encéfalo/patologia , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neurônios/patologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais
8.
Science ; 367(6484): 1372-1376, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32193327

RESUMO

The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type-specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.


Assuntos
Compostos de Anilina/química , Ascorbato Peroxidases/genética , Engenharia Genética , Neurônios/fisiologia , Nitrocompostos/química , Fenilenodiaminas/química , Polímeros/química , Potenciais de Ação , Animais , Ascorbato Peroxidases/metabolismo , Caenorhabditis elegans , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Condutividade Elétrica , Células HEK293 , Hipocampo , Humanos , Potenciais da Membrana , Camundongos , Neurônios Motores/fisiologia , Células Musculares/fisiologia , Neurônios/enzimologia , Técnicas de Patch-Clamp , Polímeros/metabolismo , Ratos , Transdução Genética
9.
Nucleic Acids Res ; 48(8): 3999-4012, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32201888

RESUMO

In eukaryotic cells, with the exception of the specialized genomes of mitochondria and plastids, all genetic information is sequestered within the nucleus. This arrangement imposes constraints on how the information can be tailored for different cellular regions, particularly in cells with complex morphologies like neurons. Although messenger RNAs (mRNAs), and the proteins that they encode, can be differentially sorted between cellular regions, the information itself does not change. RNA editing by adenosine deamination can alter the genome's blueprint by recoding mRNAs; however, this process too is thought to be restricted to the nucleus. In this work, we show that ADAR2 (adenosine deaminase that acts on RNA), an RNA editing enzyme, is expressed outside of the nucleus in squid neurons. Furthermore, purified axoplasm exhibits adenosine-to-inosine activity and can specifically edit adenosines in a known substrate. Finally, a transcriptome-wide analysis of RNA editing reveals that tens of thousands of editing sites (>70% of all sites) are edited more extensively in the squid giant axon than in its cell bodies. These results indicate that within a neuron RNA editing can recode genetic information in a region-specific manner.


Assuntos
Adenosina Desaminase/metabolismo , Neurônios/enzimologia , Edição de RNA , Adenosina/metabolismo , Animais , Axônios/enzimologia , Citoplasma/enzimologia , Decapodiformes/enzimologia , Células HEK293 , Humanos , Inosina/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sinapses/enzimologia
10.
Mol Cell ; 77(5): 1124-1142.e10, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142685

RESUMO

The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.


Assuntos
Células-Tronco Embrionárias Humanas/enzimologia , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Mitofagia , Células-Tronco Neurais/enzimologia , Neurogênese , Neurônios/enzimologia , Tioléster Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Células HeLa , Células-Tronco Embrionárias Humanas/patologia , Humanos , Cinética , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Células-Tronco Neurais/patologia , Neurônios/patologia , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteômica , Transdução de Sinais , Tioléster Hidrolases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
11.
Nat Commun ; 11(1): 639, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005851

RESUMO

Memories are encoded by memory traces or engrams, represented within subsets of neurons that are synchronously activated during learning. However, the molecular mechanisms that drive engram stabilization during consolidation and consequently ensure its reactivation by memory recall are not fully understood. In this study we manipulate, during memory consolidation, the levels of the de novo DNA methyltransferase 3a2 (Dnmt3a2) selectively within dentate gyrus neurons activated by fear conditioning. We found that Dnmt3a2 upregulation enhances memory performance in mice and improves the fidelity of reconstitution of the original neuronal ensemble upon memory retrieval. Moreover, similar manipulation in a sparse, non-engram subset of neurons does not bias engram allocation or modulate memory strength. We further show that neuronal Dnmt3a2 overexpression changes the DNA methylation profile of synaptic plasticity-related genes. Our data implicates DNA methylation selectively within neuronal ensembles as a mechanism of stabilizing engrams during consolidation that supports successful memory retrieval.


Assuntos
Metilação de DNA , Memória , Neurônios/metabolismo , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Giro Denteado/enzimologia , Giro Denteado/metabolismo , Medo , Aprendizagem , Masculino , Consolidação da Memória , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/enzimologia
12.
Am J Physiol Renal Physiol ; 318(4): F1006-F1016, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32003596

RESUMO

Corticotropin-releasing factor (CRF) regulates diverse physiological functions, including bladder control. We recently reported that Crf expression is under genetic control of Aoah, the locus encoding acyloxyacyl hydrolase (AOAH), suggesting that AOAH may also modulate voiding. Here, we examined the role of AOAH in bladder function. AOAH-deficient mice exhibited enlarged bladders relative to wild-type mice and had decreased voiding frequency and increased void volumes. AOAH-deficient mice had increased nonvoiding contractions and increased peak voiding pressure in awake cystometry. AOAH-deficient mice also exhibited increased bladder permeability and higher neuronal firing rates of bladder afferents in response to stretch. In wild-type mice, AOAH was expressed in bladder projecting neurons and colocalized in CRF-expressing neurons in Barrington's nucleus, an important brain area for voiding behavior, and Crf was elevated in Barrington's nucleus of AOAH-deficient mice. We had previously identified aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptor-γ as transcriptional regulators of Crf, and conditional knockout of AhR or peroxisome proliferator-activated receptor-γ in Crf-expressing cells restored normal voiding in AOAH-deficient mice. Finally, an AhR antagonist improved voiding in AOAH-deficient mice. Together, these data demonstrate that AOAH regulates bladder function and that the AOAH-Crf axis is a therapeutic target for treating voiding dysfunction.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Neurônios/enzimologia , Bexiga Urinária/inervação , Transtornos Urinários/enzimologia , Micção , Urodinâmica , Animais , Compostos Azo/farmacologia , Núcleo de Barrington/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hidrolases de Éster Carboxílico/deficiência , Hidrolases de Éster Carboxílico/genética , Hormônio Liberador da Corticotropina/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Contração Muscular , Neurônios/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Pressão , Pirazóis/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Bexiga Urinária/efeitos dos fármacos , Micção/efeitos dos fármacos , Transtornos Urinários/tratamento farmacológico , Transtornos Urinários/genética , Transtornos Urinários/fisiopatologia , Urodinâmica/efeitos dos fármacos
13.
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
14.
Toxicol Lett ; 322: 66-76, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31945382

RESUMO

Silent Information Regulator 1 (SIRT1), an NAD+-dependent deacetylase, contributes to the neuroprotective effect. However, intracellular signaling pathways that affect SIRT1 function remain unknown. It is well known that N-methyl-D-aspartate (NMDA) receptor activation induces calcium influx which then activates PKC, and SIRT1 is a mRNA target for HuR protein. We hypothesize that Ca2+-PKC-HuR-SIRT1 pathway modulates SIRT1 function. The present study is to investigate the potential pathway of SIRT1 in the SH-SY5Y cell line as an in vitro model of NMDA-induced neurotoxicity. The results showed that: (1) SIRT1 levels were downregulated in NMDA model; (2) NMDA induced an increase in serine phosphorylation of HuR, while inhibition of serine phosphorylation of HuR increased SIRT1 levels, promoting cell survival; (3) PKC inhibitor (Gö 6976) reversed NMDA insults and also suppressed serine phosphorylation of HuR; (4) 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, fully reversed NMDA insults and also inhibited PKC activity evoked by NMDA. These results indicate that intracellular elevated Ca2+ activates PKC, which phosphorylates HuR and then promotes SIRT1 mRNA decay and subsequent neuronal death in NMDA model. Therefore, the study suggests that inhibition of Ca2+-PKC-HuR-SIRT1 pathway could be an effective strategy for preventing certain neurological diseases related to NMDA excitotoxicity.


Assuntos
Agonistas de Aminoácidos Excitatórios/toxicidade , N-Metilaspartato/toxicidade , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Sirtuína 1/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo , Proteína Semelhante a ELAV 1/metabolismo , Humanos , Neurônios/enzimologia , Neurônios/patologia , Síndromes Neurotóxicas/enzimologia , Síndromes Neurotóxicas/patologia , Fosforilação , Proteína Quinase C/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina , Sirtuína 1/genética
15.
Proc Natl Acad Sci U S A ; 117(4): 2133-2139, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932418

RESUMO

Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. Here, we demonstrate that the Ras→Raf→rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. Our initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. We found that these effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. We conclude that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Consolidação da Memória , Proteínas ras/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Memória , Corpos Pedunculados/enzimologia , Neurônios/enzimologia , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/genética
16.
FASEB J ; 34(2): 2075-2086, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907982

RESUMO

In the free-living nematode Caenorhabditis elegans, the serine/threonine-specific protein kinase, AKT, is known to play a key role in dauer formation, life-span, and stress-resistance through the insulin-like signaling pathway. Although the structure and function of AKT-coding genes of C. elegans are understood, this is not the case for homologous genes in parasitic nematodes. In the present study, we explored a C. elegans akt-1 gene homolog in the parasitic nematode Haemonchus contortus, investigated its transcript isoforms (Hc-akt-1a and Hc-akt-1b), and studied expression and function using both homologous and heterologous functional genomic tools. In C. elegans, we showed that the predicted promoter of Hc-akt-1 drives substantial expression in ASJ neurons of the N2 (wild-type) strain. In H. contortus (Haecon-5 stain), RNAi (soaking) led to a significantly decreased transcript abundance for both Hc-akt-1a and Hc-akt-1b, and reduced larval development in larval stages in vitro. Chemical inhibition was also shown to block larval development. Taken together, the evidence from this study points to a key functional role for Hc-akt-1 in H. contortus.


Assuntos
Regulação Enzimológica da Expressão Gênica , Haemonchus/crescimento & desenvolvimento , Proteínas de Helminto/biossíntese , Neurônios/enzimologia , Proteína Oncogênica v-akt/biossíntese , Animais , Haemonchus/genética , Proteínas de Helminto/genética , Isoenzimas/biossíntese , Isoenzimas/genética , Larva/genética , Larva/crescimento & desenvolvimento , Proteína Oncogênica v-akt/genética
17.
Int J Biochem Cell Biol ; 119: 105680, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31866508

RESUMO

It is usually accepted that prion proteins induce apoptosis in nerve cells. However, the mechanisms of PrPSc-neurotoxicity are not completely clear. Calcineurin is a Ca2+/calmodulin-dependent phosphatase. It activates autophagy, and may represent a link between deregulation of Ca2+ homeostasis and neuronal cell death. In this study, the effect of calcineurin activation mediated by human prion protein induced neuronal cell death via AMPK dephosphorylation and autophagy, was investigated. Synthetic peptides of PrP (PrP 106-126) increased calcineurin activity, without changing the levels of this protein phosphatase. Furthermore, these peptides reduced the levels of AMPK phosphorylation at threonine residue 172 and in autophagy activation. Calcineurin inhibitor, FK506, prevented this effect. The data showed that PrP-treated neurons had lower levels of AMPK than control neurons. This decrease in AMPK levels was matched via activation of autophagy. FK506 prevented the changes in AMPK and autophagy levels induced by PrP peptides. Taken together, the data demonstrated that prion peptides triggered an apoptotic cascade via calcineurin activation, which mediated AMPK dephosphorylation and autophagy activation. Therefore, these data suggest that therapeutic strategies targeting calcineurin inhibition might facilitate the management of neurodegenerative disorders including prion disease.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Calcineurina/metabolismo , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Príons/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Inibidores de Calcineurina/farmacologia , Linhagem Celular Tumoral , Humanos , Neuroblastoma/enzimologia , Neuroblastoma/metabolismo , Neurônios/enzimologia , Neurônios/metabolismo , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Tacrolimo/farmacologia
18.
J Stroke Cerebrovasc Dis ; 29(3): 104531, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31882337

RESUMO

BACKGROUND: The purpose of this study was to investigate the effects of edaravone on nitric oxide (NO) production, hydroxyl radical (OH-) metabolism, and neuronal nitric oxide synthase (nNOS) expression during cerebral ischemia and reperfusion. METHODS: Edaravone (3 mg/kg) was administered intravenously to 14 C57BL/6 mice just before reperfusion. Eleven additional mice received saline (controls). NO production and OH- metabolism were continuously monitored using bilateral striatal in vivo microdialysis. OH- formation was monitored using the salicylate trapping method. Forebrain ischemia was produced in all mice by bilateral occlusion of the common carotid artery for 10 minutes. Levels of NO metabolites, nitrite (NO2-) and nitrate (NO3-), were determined using the Griess reaction. Brain sections were immunostained with an anti-nNOS antibody and the fractional area density of nNOS-immunoreactive pixels to total pixels determined. RESULTS: Blood pressure and regional cerebral blood flow were not significantly different between the edaravone and control groups. The levels of NO2- did not differ significantly between the 2 groups. The level of NO3- was significantly higher in the edaravone group compared with the control group after reperfusion. 2,3-dihydroxybenzoic acid levels were lower in the edaravone group compared with those in the control group after reperfusion. Immunohistochemistry showed nNOS expression in the edaravone group to be significantly lower than that in the control group 96 hours after reperfusion. CONCLUSIONS: These in vivo data indicate that edaravone may have a neuroprotective effect by reducing levels of OH- metabolites, increasing NO production and decreasing nNOS expression in brain cells.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Edaravone/farmacologia , Depuradores de Radicais Livres/farmacologia , Radical Hidroxila/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Isquemia Encefálica/enzimologia , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Neurônios/patologia , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/patologia , Fatores de Tempo
19.
Proc Natl Acad Sci U S A ; 117(1): 677-688, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871190

RESUMO

A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington's disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization.


Assuntos
Membrana Celular/metabolismo , Doença de Huntington/genética , Neurônios/enzimologia , Diester Fosfórico Hidrolases/genética , Domínios Proteicos/genética , Animais , Autofagia/genética , Corpo Estriado/citologia , Corpo Estriado/patologia , AMP Cíclico/metabolismo , Embrião de Mamíferos , Células HEK293 , Humanos , Doença de Huntington/patologia , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Mutação , Neurônios/citologia , Técnicas de Patch-Clamp , Diester Fosfórico Hidrolases/metabolismo , Cultura Primária de Células , Proteólise , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
J Neuroinflammation ; 16(1): 249, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796106

RESUMO

BACKGROUND: Inflammation can induce cognitive dysfunction in patients who undergo surgery. Previous studies have demonstrated that both acute peripheral inflammation and anaesthetic insults, especially isoflurane (ISO), are risk factors for memory impairment. Few studies are currently investigating the role of ISO under acute peri-inflammatory conditions, and it is difficult to predict whether ISO can aggravate inflammation-induced cognitive deficits. HDACs, which are essential for learning, participate in the deacetylation of lysine residues and the regulation of gene transcription. However, the cell-specific mechanism of HDACs in inflammation-induced cognitive impairment remains unknown. METHODS: Three-month-old C57BL/6 mice were treated with single versus combined exposure to LPS injected intraperitoneally (i.p.) to simulate acute abdominal inflammation and isoflurane to investigate the role of anaesthesia and acute peripheral inflammation in cognitive impairment. Behavioural tests, Western blotting, ELISA, immunofluorescence, qRT-PCR, and ChIP assays were performed to detect memory, the expressions of inflammatory cytokines, HDAC2, BDNF, c-Fos, acetyl-H3, microglial activity, Bdnf mRNA, c-fos mRNA, and Bdnf and c-fos transcription in the hippocampus. RESULTS: LPS, but not isoflurane, induced neuroinflammation-induced memory impairment and reduced histone acetylation by upregulating histone deacetylase 2 (HDAC2) in dorsal hippocampal CaMKII+ neurons. The hyperexpression of HDAC2 in neurons was mediated by the activation of microglia. The decreased level of histone acetylation suppressed the transcription of Bdnf and c-fos and the expressions of BDNF and c-Fos, which subsequently impaired memory. The adeno-associated virus ShHdac2, which suppresses Hdac2 after injection into the dorsal hippocampus, reversed microglial activation, hippocampal glutamatergic BDNF and c-Fos expressions, and memory deficits. CONCLUSIONS: Reversing HDAC2 in hippocampal CaMKII+ neurons exert a neuroprotective effect against neuroinflammation-induced memory deficits.


Assuntos
Disfunção Cognitiva/enzimologia , Regulação Enzimológica da Expressão Gênica , Hipocampo/enzimologia , Histona Desacetilase 2/biossíntese , Microglia/enzimologia , Neurônios/enzimologia , Animais , Células Cultivadas , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/genética , Hipocampo/efeitos dos fármacos , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/genética , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Distribuição Aleatória , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/fisiologia
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