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1.
Mol Genet Metab ; 142(3): 108497, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38763041

RESUMO

Krabbe disease (KD) is a rare inherited demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase. Most patients with KD exhibit fatal cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before the age of 2-4 years. We have previously reported that primary OLs isolated from the brains of twitcher (twi) mice, an authentic mouse model of KD, have cell-autonomous developmental defects and undergo apoptotic death accompanied by abnormal accumulation of psychosine, an endogenous cytotoxic lyso-derivative of GalCer. In this study, we aimed to investigate the effects of the preclinical promyelinating drugs clemastine and Sob-AM2 on KD OL pathologies using primary OLs isolated from the brains of twi mice. Both agents specifically prevented the apoptotic death observed in twi OLs. However, while Sob-AM2 showed higher efficacy in restoring the impaired differentiation and maturation of twi OLs, clemastine more potently reduced the endogenous psychosine levels. These results present the first preclinical in vitro data, suggesting that clemastine and Sob-AM2 can act directly and distinctly on OLs in KD and ameliorate their cellular pathologies associated with myelin degeneration.


Assuntos
Apoptose , Clemastina , Modelos Animais de Doenças , Leucodistrofia de Células Globoides , Oligodendroglia , Psicosina , Animais , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/tratamento farmacológico , Oligodendroglia/patologia , Oligodendroglia/metabolismo , Oligodendroglia/efeitos dos fármacos , Camundongos , Clemastina/farmacologia , Apoptose/efeitos dos fármacos , Psicosina/análogos & derivados , Psicosina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Encéfalo/patologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Células Cultivadas
2.
Neurobiol Dis ; 120: 51-62, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30176352

RESUMO

Krabbe disease (KD), or globoid cell leukodystrophy, is an inherited lysosomal storage disease with leukodystrophy caused by a mutation in the galactosylceramidase (GALC) gene. The majority of patients show the early onset form of KD dominated by cerebral demyelination with apoptotic oligodendrocyte (OL) death. However, the initial pathophysiological changes in developing OLs remain poorly understood. Here, we show that OLs of twitcher mice, an authentic mouse model of KD, exhibited developmental defects and impaired myelin formation in vivo and in vitro. In twitcher mouse brain, abnormal myelination and reduced expression of myelin genes during the period of most active OL differentiation and myelination preceded subsequent progressive OL death and demyelination. Importantly, twitcher mouse OL precursor cells proliferated normally, but their differentiation and survival were intrinsically defective. These defects were associated with aberrant accumulation of endogenous psychosine (galactosylsphingosine) and reduced activation of the Erk1/2 and Akt/mTOR pathways before apoptotic cell death. Collectively, our results demonstrate that GALC deficiency in developing KD OLs profoundly affects their differentiation and maturation, indicating the critical contribution of OL dysfunction to KD pathogenesis.


Assuntos
Modelos Animais de Doenças , Leucodistrofia de Células Globoides/metabolismo , Oligodendroglia/metabolismo , Psicosina/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligodendroglia/patologia , Psicosina/genética
3.
J Neurosci ; 32(17): 6032-42, 2012 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-22539863

RESUMO

During development, neurons migrate from their site of origin to their final destinations. Upon reaching this destination, the termination of their migration is crucial for building functional architectures such as laminated structures and nuclei. How this termination is regulated, however, is not clear. Here, we investigated the contribution of cell-intrinsic mechanisms and extrinsic factors. Using GAD67-GFP knock-in mice and in utero electroporation cell labeling, we visualized GABAergic neurons and analyzed their motility in vitro. We find that the motility of GABAergic neurons in cortical slices gradually decreases as development proceeds and is almost abolished by the end of the first postnatal week. Consistent with this, a reduction of embryonic interneuron motility occurred in dissociated cultures. This is in part due to cell-intrinsic mechanisms, as a reduction in motility is observed during long-term culturing on glial feeder cells. Cell-intrinsic regulation is further supported by observations that interneurons labeled in early stages migrated more actively than those labeled in late stages in the same cortical explant. We found evidence suggesting that upregulation of the potassium-chloride cotransporter KCC2 underlies this intrinsic regulation. Reduced motility is also observed when embryonic interneurons are plated on postnatal cortical feeder cells, suggesting extrinsic factors derived from the postnatal cortex too contribute to termination. These factors should include secreted molecules, as cultured postnatal cortical cells could exercise this effect without directly contacting the interneuron. These findings suggest that intrinsic mechanisms and extrinsic factors coordinate to reduce the motility of migrating neurons, thereby leading to the termination of migration.


Assuntos
Movimento Celular/fisiologia , Córtex Cerebral , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Interneurônios/fisiologia , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Movimento Celular/genética , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Eletroporação , Embrião de Mamíferos , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Neuroglia/fisiologia , Técnicas de Cultura de Órgãos , Estatísticas não Paramétricas , Simportadores/genética , Simportadores/metabolismo , Fatores de Tempo , Cotransportadores de K e Cl-
4.
Genesis ; 50(5): 424-8, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-21953949

RESUMO

Mature oligodendrocytes are critical for myelin maintenance. To understand the molecular basis for this, genetic manipulation of mature oligodendrocytes is needed. Here we generated a mature oligodendrocyte tTA (tetracycline-controlled transcriptional activator) mouse line which, in combination with a tTA-dependent promoter line driving the expression of the desired transgene, can be used for gain-of-function studies. We used an oligodendrocyte promoter, the mouse proteolipid protein (PLP) promoter, to express mammalianized tTA, and generated a PLP-mtTA mouse line. In adults, mtTA mRNA was predominantly detected in brain white matter where it co-localized with PLP mRNA. mtTA-mediated gene induction was confirmed by crossing to mice with a tTA-dependent promoter driving expression of yellow fluorescent protein (tetO-YFP mice). YFP induction in PLP-mtTA::tetO-YFP mice was consistent with PLP expression in adult mature oligodendrocytes and premyelinating-stage myelinating oligodendrocytes. This PLP-mtTA mouse line is the first to enable gain-of-function studies in mature oligodendrocytes with the tet system.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteína Proteolipídica de Mielina/genética , Oligodendroglia/metabolismo , Animais , Sequência de Bases , Primers do DNA , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Transgenes
5.
JMA J ; 5(1): 44-54, 2022 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-35224259

RESUMO

Cancer patients in adolescents and young adults (AYA) generation aged 15-39 years have various psychosocial needs during their treatment course such as school enrollment, finding employment, marriage, and fertility. It is difficult for medical professionals to gain experience related to providing medical care and consultation support to these kinds of AYA generation cancer patients. There is a need to provide information and establish both support and medical care systems that are able to meet the diverse needs unique to this generation. This review will explain how to launch an AYA support team (AST). We have worked and established the AST since 2016, which is medical care teams that provide support according to the life stage of each individual patient and build a multidisciplinary AYA generation patient support system. The team-building process consisted of two main projects: building and enlarging multidisciplinary team and establishing screening process of psychosocial needs of AYA generation patients. Multidisciplinary healthcare professionals got involved in the AST with already-existing patient support functions in our center: the patient support center, which is an outpatient department and the palliative care team, which is an inpatient interdepartmental team. The AST systematically finds patients in need of assistance and offers them support as a multidisciplinary team. The AST also established a procedure that systematically gathers information about the needs of patients by using a screening tool. In addition, the AST provides the following specialized services: reproductive medicine, supporting cancer patients with children, employment support, and peer support. The AST has been established and sophisticatedly worked. It can flexibly provide various psychosocial support services. This review will explain how to launch an AST.

6.
Dev Neurosci ; 33(2): 118-29, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21865661

RESUMO

Neuronal differentiation is a crucial event during neural development. Recent studies have characterized the development of the diencephalon; however, the origins of the primarily GABAergic prethalamic nuclei, including the zona incerta (ZI), ventral lateral geniculate nucleus (vLG) and reticular thalamic nucleus (RT), remain unclear. Here we characterize Olig2 lineage cells in the developing prethalamus using mice in which tamoxifen-induced recombination permanently labels Olig2-expressing cells. We show that GABAergic neurons in the prethalamic nuclei, including the RT, ZI and vLG, originate from prethalamic Olig2 lineage cells. Based on these data and on those derived from short-term lineage-tracing data using Olig3-lacZ mice and previous reports, we suggest that vLG cells originate from the ventricular zone of the thalamus, zona limitans intrathalamica and prethalamus.


Assuntos
Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Movimento Celular/fisiologia , Neurônios GABAérgicos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Feminino , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Corpos Geniculados/citologia , Corpos Geniculados/embriologia , Corpos Geniculados/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Modelos Animais , Proteínas do Tecido Nervoso , Fator de Transcrição 2 de Oligodendrócitos , Células-Tronco/fisiologia , Subtálamo/citologia , Subtálamo/embriologia , Subtálamo/metabolismo , Tamoxifeno , Núcleos Ventrais do Tálamo/citologia , Núcleos Ventrais do Tálamo/embriologia , Núcleos Ventrais do Tálamo/metabolismo
7.
Brain Pathol ; 31(5): e12951, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33822434

RESUMO

Krabbe disease (KD), also known as globoid cell leukodystrophy, is an inherited demyelinating disease caused by the deficiency of lysosomal galactosylceramidase (GALC) activity. Most of the patients are characterized by early-onset cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before 2 years of age. However, the mechanisms of molecular pathogenesis in the developing OLs before death and the exact causes of white matter degeneration remain largely unknown. We have recently reported that OLs of twitcher mouse, an authentic mouse model of KD, exhibit developmental defects and endogenous accumulation of psychosine (galactosylsphingosine), a cytotoxic lyso-derivative of galactosylceramide. Here, we show that attenuated expression of microRNA (miR)-219, a critical regulator of OL differentiation and myelination, mediates cellular pathogenesis of KD OLs. Expression and functional activity of miR-219 were repressed in developing twitcher mouse OLs. By using OL precursor cells (OPCs) isolated from the twitcher mouse brain, we show that exogenously supplemented miR-219 effectively rescued their cell-autonomous developmental defects and apoptotic death. miR-219 also reduced endogenous accumulation of psychosine in twitcher OLs. Collectively, these results highlight the role of the reduced miR-219 expression in KD pathogenesis and suggest that miR-219 has therapeutic potential for treating KD OL pathologies.


Assuntos
Leucodistrofia de Células Globoides/patologia , MicroRNAs/genética , Oligodendroglia/patologia , Psicosina/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Modelos Animais de Doenças , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Camundongos Transgênicos , Oligodendroglia/metabolismo
8.
J Biol Chem ; 284(39): 26340-8, 2009 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-19625250

RESUMO

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Neurônios/efeitos dos fármacos , Fator 2 de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/genética , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Citocinas/farmacologia , Quinase do Fator 2 de Elongação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Mutação , Neurônios/citologia , Neurônios/metabolismo , Fator 2 de Elongação de Peptídeos/genética , Fosforilação/efeitos dos fármacos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/metabolismo , Fatores de Tempo
9.
PLoS One ; 12(3): e0174780, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28350885

RESUMO

The neurotrophic factor neuregulin 1 (NRG1) regulates neuronal development, glial differentiation, and excitatory synapse maturation. NRG1 is synthesized as a membrane-anchored precursor and is then liberated by proteolytic processing or exocytosis. Mature NRG1 then binds to its receptors expressed by neighboring neurons or glial cells. However, the molecular mechanisms that govern this process in the nervous system are not defined in detail. Here we prepared neuron-enriched and glia-enriched cultures from embryonic rat neocortex to investigate the role of neurotransmitters that regulate the liberation/release of NRG1 from the membrane of neurons or glial cells. Using a two-site enzyme immunoassay to detect soluble NRG1, we show that, of various neurotransmitters, glutamate was the most potent inducer of NRG1 release in neuron-enriched cultures. NRG1 release in glia-enriched cultures was relatively limited. Furthermore, among glutamate receptor agonists, N-Methyl-D-Aspartate (NMDA) and kainate (KA), but not AMPA or tACPD, mimicked the effects of glutamate. Similar findings were acquired from analysis of the hippocampus of rats with KA-induced seizures. To evaluate the contribution of members of a disintegrin and metalloproteinase (ADAM) families to NRG1 release, we transfected primary cultures of neurons with cDNA vectors encoding NRG1 types I, II, or III precursors, each tagged with the alkaline phosphatase reporter. Analysis of alkaline phosphatase activity revealed that the NRG1 type II precursor was subjected to tumor necrosis factor-α-converting enzyme (TACE) / a Disintegrin And Metalloproteinase 17 (ADAM17) -dependent ectodomain shedding in a protein kinase C-dependent manner. These results suggest that glutamatergic neurotransmission positively regulates the ectodomain shedding of NRG1 type II precursors and liberates the active NRG1 domain in an activity-dependent manner.


Assuntos
Glutamatos/farmacologia , Neuregulina-1/metabolismo , Neurônios/efeitos dos fármacos , Precursores de Proteínas/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Proteína ADAM17/metabolismo , Acetilcolina/farmacologia , Animais , Western Blotting , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Dipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Ácido Caínico/farmacologia , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Prosencéfalo/citologia , Proteína Quinase C/metabolismo , Proteólise/efeitos dos fármacos , Ratos Sprague-Dawley , Serotonina/farmacologia
10.
J Neurosci ; 24(44): 9760-9, 2004 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-15525761

RESUMO

In neurons, perisynaptic or dendritic translation is implicated in synapse-wide alterations of function and morphology triggered by neural activity. The molecular mechanisms controlling local translation activation, however, have yet to be elucidated. Here, we show that local protein synthesis and translational activation in neuronal dendrites are upregulated by brain-derived neurotrophic factor (BDNF) in a rapamycin and small interfering RNA specific for mammalian target of rapamycin (mTOR)-sensitive manner. In parallel, BDNF induced the phosphorylation of tuberin and the activation of mTOR in dendrites and the synaptoneurosome fraction. mTOR activation stimulated translation initiation processes involving both eIF4E/4E-binding protein (4EBP) and p70S6 kinase/ribosomal S6 protein. BDNF induced phosphorylation of 4EBP in isolated dendrites. Moreover, local puff application of BDNF to dendrites triggered S6 phosphorylation in a restricted area. Taken together, these data indicate that mTOR-dependent translation activation is essential for the upregulation of local protein synthesis in neuronal dendrites.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Dendritos/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas Quinases/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Dendritos/efeitos dos fármacos , Regulação da Expressão Gênica , Proteínas Monoméricas de Ligação ao GTP/análise , Proteínas do Tecido Nervoso/análise , Plasticidade Neuronal/fisiologia , Neuropeptídeos/análise , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Proteínas Quinases/análise , RNA Interferente Pequeno , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Ratos , Proteínas Repressoras/análise , Sirolimo/farmacologia , Sinaptossomos/metabolismo , Serina-Treonina Quinases TOR , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/análise
11.
Brain Res Mol Brain Res ; 111(1-2): 165-74, 2003 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-12654516

RESUMO

Novel protein synthesis in the brain has been suggested to contribute to the formation of synapses and neural circuits during development and the modulation of long-term synaptic plasticity through life. However, cellular and subcellular distribution of neuronal translation machinery and regulator molecules has not yet been extensively characterized in rat brain. In this report, the distribution of translation factors in the developing hippocampus, a region which is highly plastic, was analyzed by immunohistochemistry and Western blotting. Western blot analysis revealed that the hippocampus expresses the factors in all three steps of translation, initiation factors, elongation factors and a release factor. Immunochemical studies of hippocampal slices and culture showed that all translation factors were observed not only in cell bodies but also in dendrites of hippocampal neurons. In addition, the levels of the individual translation factors differed between hippocampal subregions. The differential distribution of translation factors was also confirmed by Western blotting. These results suggest that regulated protein synthesis occurs in the hippocampus, with differences existing between different subregions such as CA1, CA3 and dentate gyrus.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Hipocampo/metabolismo , Vias Neurais/metabolismo , Plasticidade Neuronal/genética , Neurônios/metabolismo , Biossíntese de Proteínas/genética , Sinapses/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Dendritos/metabolismo , Dendritos/ultraestrutura , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Imuno-Histoquímica , Masculino , Vias Neurais/citologia , Vias Neurais/crescimento & desenvolvimento , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética
12.
Neurosci Lett ; 346(1-2): 117-9, 2003 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-12850562

RESUMO

Regulated protein synthesis is critical for neural development, such as the formation of synapses and neural circuits and the modulation of synaptic plasticity. Protein synthesis is controlled by translation factors, including initiation, elongation and release factors. Here we investigated the developmental changes of eukaryotic initiation factor 2B (eIF2B) subunits in rat hippocampus. The eIF2B beta, gamma, delta and epsilon subunit protein levels were maximal at embryonic day 18 and then decreased during development. Aged hippocampus contained only trace amounts of these subunits. The finding that eIF2B subunit levels are high in developing hippocampus suggests that regulated protein synthesis is active in young, highly plastic brain.


Assuntos
Fator de Iniciação 2B em Eucariotos/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Hipocampo/embriologia , Hipocampo/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Hipocampo/crescimento & desenvolvimento , Masculino , Ratos , Ratos Wistar
13.
Biol Psychiatry ; 67(8): 770-3, 2010 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-20163789

RESUMO

We created the Flexible Accelerated STOP Tetracycline Operator (tetO)-knockin (FAST) system, an efficient method for manipulating gene expression in vivo to rapidly screen animal models of disease. A single gene targeting event yields two distinct knockin mice-STOP-tetO and tetO knockin-that permit generation of multiple strains with variable expression patterns: 1) knockout, 2) Cre-mediated rescue, 3) tetracycline-controlled transcriptional activator (tTA)-mediated misexpression, 4) tetracycline-controlled transcriptional activator (tTA)-mediated overexpression, and 5) tetracycline-controlled transcriptional silencer (tTS)-mediated conditional knockout/knockdown. Using the FAST system, multiple gain-of-function and loss-of-function strains can therefore be generated on a time scale not previously achievable. These strains can then be screened for clinically relevant abnormalities. We demonstrate the flexibility and broad applicability of the FAST system by targeting several genes encoding proteins implicated in neuropsychiatric disorders: Mlc1, neuroligin 3, the serotonin 1A receptor, and the serotonin 1B receptor.


Assuntos
Técnicas de Transferência de Genes , Tetraciclina/farmacologia , Animais , Animais Geneticamente Modificados , Astrócitos/fisiologia , Proteínas de Bactérias/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas de Transporte/genética , Linhagem Celular , Expressão Gênica/genética , Expressão Gênica/fisiologia , Inativação Gênica , Transtornos Mentais/genética , Camundongos , Camundongos Knockout , Doenças do Sistema Nervoso/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
14.
J Neurochem ; 95(5): 1438-45, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16171514

RESUMO

The effects and signaling mechanisms of brain-derived neurotrophic factor (BDNF) on translation elongation were investigated in cortical neurons. BDNF increased the elongation rate approximately twofold, as determined by measuring the ribosomal transit time. BDNF-accelerated elongation was inhibited by rapamycin, implicating the mammalian target of rapamycin (mTOR). To explore the mechanisms underlying these effects, we examined the protein phosphorylation cascades that lead to the activation of translation elongation in neurons. BDNF increased eukaryote elongation factor 1A (eEF1A) phosphorylation and decreased eEF2 phosphorylation. Whereas eEF2 phosphorylation levels altered by BDNF were inhibited by rapamycin, eEF1A phosphorylation was not affected by rapamycin or PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor. BDNF induced phosphorylation of eEF2 kinase (Ser366), as well as decreased its kinase activity. All these events were inhibited by rapamycin. Furthermore, mTOR siRNA, which reduced mTOR levels up to 50%, inhibited the BDNF-induced enhancement in elongation rate and decrease in eEF2 phosphorylation. These results strongly suggest that BDNF enhances translation elongation through the activation of the mTOR-eEF2 pathway.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Neurônios/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Animais , Western Blotting/métodos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/imunologia , Células Cultivadas , Córtex Cerebral/citologia , Interações Medicamentosas , Quinase do Fator 2 de Elongação , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Imuno-Histoquímica/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Isótopos de Fósforo/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Proteínas Quinases/metabolismo , RNA de Cadeia Dupla/farmacologia , Ratos , Ribossomos/metabolismo , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR , Treonina/metabolismo , Fatores de Tempo
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