RESUMO
Brain-specific microRNAs (miRs) may be involved in synaptic plasticity through the control of target mRNA translation. Brain-derived neurotrophic factor (BDNF) also contributes to the regulation of synaptic function. However, the possible involvement of miRs in BDNF-regulated synaptic function is poorly understood. Importantly, an increase in glucocorticoid levels and the downregulation of BDNF are supposed to be involved in the pathophysiology of depressive disorders. Previously, we reported that glucocorticoid exposure inhibited BDNF-regulated synaptic function via weakening mitogen-activated protein kinase/extracellular signal-regulated kinase1/2 (MAPK/ERK) and/or phospholipase C-gamma (PLC-gamma) intracellular signaling in cultured neurons [Kumamaru et al (2008) Mol Endocrinol 22:546-558; Numakawa et al (2009) Proc Natl Acad Sci U S A 106:647-652]. Therefore, in this study, we investigate the possible influence of glucocorticoid on BDNF/miRs-stimulated biological responses in cultured cortical neurons. Significant upregulation of miR-132 was caused by BDNF, although miR-9, -124, -128a, -128b, -134, -138, and -16 were intact. Transfection of exogenous ds-miR-132 induced marked upregulation of glutamate receptors (NR2A, NR2B, and GluR1), suggesting that miR-132 has a positive effect on the increase in postsynaptic proteins levels. Consistently, transfection of antisense RNA to inhibit miR-132 function decreased the BDNF-dependent increase in the expression of postsynaptic proteins. U0126, an inhibitor of the MAPK/ERK pathway, suppressed the BDNF-increased miR-132, suggesting that BDNF upregulates miR-132 via the MAPK/ERK1/2 pathway. Interestingly, pretreatment with glucocorticoid (dexamethasone, DEX) reduced BDNF-increased ERK1/2 activation, miR-132 expression, and postsynaptic proteins. We demonstrate that the exposure of neurons to an excess glucocorticoid results in a decrease in the BDNF-dependent neuronal function via suppressing miR-132 expression.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , MicroRNAs/metabolismo , Neurônios/efeitos dos fármacos , Receptores de Glutamato/metabolismo , Animais , Butadienos/farmacologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Inibidores Enzimáticos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neurônios/metabolismo , Nitrilas/farmacologia , RNA Antissenso/metabolismo , Ratos , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The very low density lipoprotein receptor (VLDLR) is a newly described receptor which binds to apolipoprotein E (apoE) specifically. The authors designed a synthetic peptide of 17 amino acids representing the N-terminus of the putative first ligand binding domain of human VLDLR, this being a unique domain for VLDLR. When the synthetic peptide was used as the antigen, two different monoclonal antibodies were obtained (anti-VLDLR1 and anti-VLDLR2). Expressional cloning revealed that anti-VLDLR1 recognized the variant form of VLDLR which lacks 84 bp of O-linked sugar domain and anti-VLDLR2 recognized the full length form of VLDLR. The variant VLDLR was expressed in neuroblasts as well as matrix cells and Cajal-Retzius cells in the early stages of the developing human brain; later its expression was sequentially found in glioblasts, astrocytes, oligodendrocytes and finally in myelin. The expression of a full length form of VLDLR was detected in senile plaques and some neurons and satellite glia in aged and Alzheimer brains. This suggests that the variant VLDLR is important for the developing human brain and the full length VLDLR has modified functions in aged and Alzheimer brains.
Assuntos
Apolipoproteínas E/metabolismo , Encéfalo/embriologia , Neuroglia/metabolismo , Neurônios/metabolismo , Receptores de LDL/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos/genética , Sequência de Bases/genética , Northern Blotting , Encéfalo/citologia , Encéfalo/metabolismo , Epitopos/imunologia , Feminino , Feto , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neuroglia/citologia , Neurônios/citologia , Placa Amiloide/metabolismo , Placa Amiloide/patologia , RNA Mensageiro/metabolismo , Receptores de LDL/genética , Receptores de LDL/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Amida was first isolated from a rat hippocampal cDNA library as an Arc-associated protein. Previous studies showed that Amida is a nuclear protein and overexpression of Amida induces cell apoptosis. In this study, we found that Amida mRNA was expressed predominantly in rat testis by Northern blot analysis. During the development of testis, Amida mRNA was barely detectable until postnatal days 24 to 29 during which it increased to levels found in adults. However, Amida protein was not detected until postnatal day 32. Amida mRNA was found to be enriched in spermatocytes and less in round spermatids, but was undetectable in elongated spermatids by in situ hybridization. In addition, Amida protein was observed in the nucleus of spermatocytes and even in the elongated spermatids by immunohistochemistry. The development and cellular localization differences of Amida mRNA and protein implicates that Amida mRNA may undergo posttranscriptional regulation. Furthermore, Amida mRNA decreased significantly in the 8-day experimental cryptorchid testis when spermatogenesis was disrupted. Taken together, these data suggest that Amida is involved in spermatogenesis and may play an important role in development of testicular germ cells.
Assuntos
Proteínas Nucleares/genética , Testículo/metabolismo , Animais , Criptorquidismo/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Masculino , Proteínas Nucleares/análise , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Maturidade Sexual/genética , Espermátides/metabolismo , Espermatócitos/metabolismo , Testículo/fisiologiaRESUMO
Puralpha, a single-stranded DNA binding protein, recognizes a PUR element (GGN repeat). We have reported that Puralpha binds to a single-stranded oligonucleotide probe containing the cAMP response element (CRE) of rat somatostatin gene using a gel mobility shift assay. Here, we showed that Puralpha binds to the probe only in the presence of a PUR element by a more detailed characterization. We also examined the effects of Puralpha on the enhancer activity of the somatostatin CRE in PC12 cells using the reporter gene assay. Transfected Puralpha suppressed the CRE enhancer activity stimulated by forskolin (which increases intracellular cAMP), but suppression was not observed when the PUR element was deleted. The neurite extension induced by forskolin was inhibited by the transfection of Puralpha, but that by NGF was not suppressed. The c-fos mRNA induced by forskolin, but not by NGF, was also suppressed by Puralpha transfection. These results indicate that Puralpha suppresses the biological activities induced by forskolin, but not by NGF, in PC12 cells and that Puralpha could interfere with a cAMP-CRE signal pathway.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Elementos Facilitadores Genéticos , Somatostatina/genética , Animais , Sequência de Bases , Sítios de Ligação , Colforsina/farmacologia , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos/efeitos dos fármacos , Genes Reporter , Cinética , Luciferases/genética , Sondas de Oligonucleotídeos , Células PC12 , Ratos , Supressão Genética , Fatores de Transcrição , Transfecção , Repetições de TrinucleotídeosRESUMO
BACKGROUND: A very low-density lipoprotein receptor (VLDLR) was recently identified. This receptor reportedly binds specifically to very low-density lipoproteins; however, its distribution and functions in vivo have yet to be elucidated. We investigated the expression and regulation of VLDLR in fetal and carcinoma cells. OBJECTIVE: The expression of VLDLR was examined by immunohistochemistry and reverse-transcriptase polymerase chain reaction using several specimens, including a fetus of 12 to 15 weeks' gestation, various tumors, AGS cells, and INT407 cells. RESULTS: Immunoreactive VLDLR was abundantly present in human fetal intestinal epithelial and gastric adenocarcinoma cells. This receptor was also noted in the intestinal cell line, INT407, and gastric cancer cell line, AGS. In addition, the VLDLR that was expressed in INT407 cells, AGS cells, and gastric adenocarcinoma tissue was present mainly in a variant form lacking the O-linked sugar domain. CONCLUSIONS: These data suggest that an important function of VLDLR may be the mediation of cell growth in developing tissues, such as fetal intestinal and cancer cells. The INT407 and AGS cell lines appear to be useful for examining the regulation of VLDLR expression.
Assuntos
Adenocarcinoma/metabolismo , Feto/metabolismo , Mucosa Intestinal/metabolismo , Lipoproteínas VLDL/metabolismo , Receptores de LDL/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/patologia , Adulto , Animais , Anticorpos Monoclonais/análise , Primers do DNA/química , Feminino , Feto/citologia , Mucosa Gástrica/citologia , Mucosa Gástrica/metabolismo , Regulação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Recém-Nascido , Intestinos/embriologia , Lipoproteínas VLDL/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores de LDL/genética , Receptores de LDL/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/patologia , Células Tumorais CultivadasRESUMO
We have found that both the synaptic vesicles (SV) and synaptic plasma membrane vesicles (SPM) have an activity to fuse with phosphatidylcoline/phosphatidylserine liposomes in a pH-dependent manner. The activity increases with decreases in extravesicular pH. At a pH lower than 4.0, the activity is almost steady at its maximum value, and there was a rapid drop around pH 5.5. The pH-dependent fusion was inhibited by proteolysis with trypsin; hence, at least in part, some membrane proteins play an important role in these pH-dependent fusion processes. To find specific markers, we screened various protein modifiers and found that anion channel blockers, stilbene derivatives (DIDS and SITS) and glibenclamide, affected the fusion process. DIDS and SITS decreased the fusion activity with an IC50 of 180 and 300 microM, respectively, whereas glibenclamide, on the contrary, increased it. From the results of an autoradiogram using 3H-tagged DIDS, a 30 kDa DIDS-binding protein was identified in the synaptic plasma membrane, which is possible to be responsible for the pH-dependent fusion.
Assuntos
Fusão de Membrana , Membranas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestrutura , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico , Animais , Fluorescência , Concentração de Íons de Hidrogênio , Lipossomos , Ratos , Ratos Sprague-Dawley , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/fisiologiaRESUMO
The very low density lipoprotein receptor (VLDLR) was considered to specifically bind to VLDL rich apolipoprotein E (apoE). However, its distribution and functions in vivo have yet to be elucidated. In human and rat VLDLR, a variant form lacking 84 base pairs (bp) in O-linked sugar domain was noted but its significance was not initially understood. This study shows that the variant form of VLDLR coexists with full-length VLDLR in majority of tissues but is a major component in the white matter of human brain. The tissue distribution of a variant VLDLR was detected in myelin as well as in other tissues except for the liver with immunohistochemistry using a monoclonal antibody. This variant VLDLR is proposed to be functionally important for internalizing apoE in human brain. ApoE is associated with beta-amyloid in senile plaques and plays a role in the transport of the beta-amyloid. The presence of VLDLR in myelin may be one explanation as to why beta-amyloid does not accumulate in the white matter which is rich in VLDLR. Recently, evidences on VLDLR obtained mainly using knock-out or transfected mice suggest this receptor to be neither specific for VLDL nor functionally important in mammals. However, no variant form of VLDLR was found in any tissues of mouse. This variant form of VLDLR should thus be studied in greater detail using human tissues or cells.