RESUMO
Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impairments in social interaction and restricted/repetitive behaviors. The neurotransmitter γ-aminobutyric acid (GABA) through GABAA receptor signaling in the immature brain plays a key role in the development of neuronal circuits. Excitatory/inhibitory imbalance in the mature brain has been investigated as a pathophysiological mechanism of ASD. However, whether and how disturbances of GABA signaling in embryos that are caused by GABAA receptor inhibitors cause ASD-like pathophysiology are poorly understood. The present study examined whether exposure to the GABAA receptor antagonist picrotoxin causes ASD-like pathophysiology in offspring by conducting behavioral tests from the juvenile period to adulthood and performing gene expression analyses in mature mouse brains. Here, we found that male mice that were prenatally exposed to picrotoxin exhibited a reduction of active interaction time in the social interaction test in both adolescence and adulthood. The gene expression analyses showed that picrotoxin-exposed male mice exhibited a significant increase in the gene expression of odorant receptors. Weighted gene co-expression network analysis showed a strong correlation between social interaction and enrichment of the "odorant binding" pathway gene module. Our findings suggest that exposure to a GABAA receptor inhibitor during the embryonic period induces ASD-like behavior, and impairments in odorant function may contribute to social deficits in offspring.
RESUMO
The mammalian target of rapamycin (mTOR) signaling pathway plays a crucial role in cell metabolism, growth, and proliferation. The overactivation of mTOR has been implicated in the pathogenesis of syndromic autism spectrum disorder (ASD), such as tuberous sclerosis complex (TSC). Treatment with the mTOR inhibitor rapamycin improved social interaction deficits in mouse models of TSC. Prenatal exposure to valproic acid (VPA) increases the incidence of ASD. Rodent pups that are exposed to VPA in utero have been used as an animal model of ASD. Activation of the mTOR signaling pathway was recently observed in rodents that were exposed to VPA in utero, and rapamycin ameliorated social interaction deficits. The present study investigated the effect of rapamycin on social interaction deficits in both adolescence and adulthood, and gene expressions in mice that were exposed to VPA in utero. We subcutaneously injected 600 mg/kg VPA in pregnant mice on gestational day 12.5 and used the pups as a model of ASD. The pups were intraperitoneally injected with rapamycin or an equal volume of vehicle once daily for 2 consecutive days. The social interaction test was conducted in the offspring after the last rapamycin administration at 5-6 weeks of ages (adolescence) or 10-11 weeks of age (adulthood). Whole brains were collected after the social interaction test in the adulthood, and microarray and Western blot analyses were performed. Mice that were exposed to VPA and treated with vehicle exhibited a decrease in social interaction compared with control mice that were treated with vehicle. Rapamycin treatment in VPA-exposed mice improved social deficits. Mice that were exposed to VPA and treated with vehicle exhibited the aberrant expression of genes in the mTOR signaling pathway, and rapamycin treatment recovered changes in the expression of some genes, including Fyb and A330094K24Rik. Rapamycin treatment suppressed S6 phosphorylation in VPA-exposed mice. Aberrant gene expression was associated with social interaction deficits in VPA-exposed mice. Rapamycin may be an effective treatment for non-syndromic ASD in adolescent and adult patients who present impairments in the mTOR signaling pathway.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Relações Interpessoais , Efeitos Tardios da Exposição Pré-Natal/genética , Sirolimo/farmacologia , Ácido Valproico/efeitos adversos , Animais , Peso Corporal/efeitos dos fármacos , Feminino , Redes Reguladoras de Genes/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Proteína S6 Ribossômica/genética , Proteína S6 Ribossômica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
M cells in follicle-associated epithelium (FAE) are specialized antigen-sampling cells that take up intestinal luminal antigens. Transcription factor Spi-B regulates M-cell maturation, but the molecules that promote transcytosis within M cells are not fully identified. Here we show that mouse allograft inflammatory factor 1 (Aif1) is expressed by M cells and contributes to M-cell transcytosis. FAE in Aif1-/- mice has suppressed uptake of particles and commensal bacteria, compared with wild-type mice. Translocation of Yersinia enterocolitica, but not of Salmonella enterica serovar Typhimurium, leading to the generation of antigen-specific IgA antibodies, is also diminished in Aif1-deficient mice. Although ß1 integrin, which acts as a receptor for Y. enterocolitica via invasin protein, is expressed on the apical surface membranes of M cells, its active form is rarely found in Aif1-/- mice. These findings show that Aif1 is important for bacterial and particle transcytosis in M cells.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Transcitose , Animais , Antígenos/metabolismo , Translocação Bacteriana , Proteínas de Ligação ao Cálcio/deficiência , Contagem de Células , Membrana Celular/metabolismo , Forma Celular , Enterócitos/metabolismo , Células Epiteliais/ultraestrutura , Imunidade nas Mucosas , Imunoglobulina A/metabolismo , Integrina beta1/metabolismo , Lactobacillus/fisiologia , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/deficiência , Yersinia/fisiologiaRESUMO
SHANK3 is a synaptic scaffolding protein and plays an important role in neuronal development. SHANK3 interacts with various synaptic molecules, including post-synaptic density-95 (PSD-95), homer and GluR1 AMPA receptor. SHANK3 gene is a causable gene of the Phelan- McDermid syndrome (also known as the 22q13.3 deletion syndrome), whose manifestation is global developmental delay and autistic behavior, especially shows severe speech and language deficit. Additionally since cumulative gene analysis in autistic subjects identified several mutations in SHANK3 gene, including deletion and duplication in a particular region, abnormality of SHANK3 gene is thought the be related with the neuropathology of autism spectrum disorder (ASD). We here review the recent findings in regard to the roles of SHANK3 in higher brain functions, molecular-biologic studies of the complex expression of Shank3 transcripts and production of SHANK3 isoforms, and behavioral studies of Shank3-mutant mice, including our recent findings, and discuss a novel therapeutic approach for ASD.
Assuntos
Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Psicotrópicos/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Psicotrópicos/uso terapêuticoRESUMO
Adult hippocampal neurogenesis has been suggested to play modulatory roles in learning and memory. Importantly, previous studies have shown that newborn neurons in the adult hippocampus are integrated into the dentate gyrus circuit and are recruited more efficiently into the hippocampal memory trace of mice when they become 3 weeks old. Interestingly, a single high-dose treatment with the N-methyl-d-aspartate receptor antagonist memantine (MEM) has been shown to increase hippocampal neurogenesis dramatically by promoting cell proliferation. In the present study, to understand the impact of increased adult neurogenesis on memory performance, we examined the effects of a single treatment of MEM on hippocampus-dependent memory in mice. Interestingly, mice treated with MEM showed an improvement of hippocampus-dependent spatial and social recognition memories when they were trained and tested at 3-6 weeks, but not at 3 days or 4 months, after treatment with MEM. Importantly, we observed a significant positive correlation between the scores for spatial memory (probe trial in the Morris water maze task) and the number of young mature neurons (3 weeks old) in MEM-treated mice, but not saline-treated mice. We also observed that the young mature neurons generated by treatment with MEM were recruited into the trace of spatial memory similarly to those generated through endogenous neurogenesis. Taken together, our observations suggest that treatment with MEM temporally improves hippocampus-dependent memory formation and that the newborn neurons increased by treatment with MEM contribute to this improvement when they become 3 weeks old.
Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Memantina/farmacologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Reconhecimento Psicológico/efeitos dos fármacos , Memória Espacial/efeitos dos fármacos , Células-Tronco Adultas/citologia , Animais , Divisão Celular/efeitos dos fármacos , Senescência Celular , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Neurônios/fisiologia , Reconhecimento Psicológico/fisiologia , Comportamento Social , Memória Espacial/fisiologiaRESUMO
SHANK3 is a synaptic scaffolding protein enriched in the post-synaptic density of excitatory synapses. Since several SHANK3 mutations have been identified in a particular phenotypic group of patients with autism spectrum disorder (ASD), SHANK3 is strongly suspected of being involved in the pathogenesis and neuropathology of ASD. Several SHANK3 isoforms are known to be produced in the developing brain, but they have not been fully investigated. Here, we identified two different amino-terminus truncated Shank3 transcripts. One transcript, designated as Shank3c-3, produces an isoform that contains the entire carboxyl-terminus, but the other transcript, designated as Shank3c-4, produces a carboxyl-terminus truncated isoform. During development, expression of the novel Shank3 transcripts increased after birth, transiently decreased at P14 and then gradually increased again thereafter. We also determined that methyl CpG-binding protein 2 (MeCP2) is involved in regulating expression of the novel Shank3 transcripts. MeCP2 is a transcriptional regulator that has been identified as the causative molecule of Rett syndrome, a neurodevelopmental disorder that includes autistic behavior. We demonstrated a difference between the expression of the novel Shank3 transcripts in wild-type mice and Mecp2-deficient mice. These findings suggest that the SHANK3 isoforms may be implicated in the synaptic abnormality in Rett syndrome. SHANK3 is a synaptic scaffolding protein and is suspected of being implicated in the pathogenesis and neuropathology of ASD. We here identified two different amino-terminus truncated Shank3 transcripts, Shank3c-3 and Shank3c-4, expressed from the intron 10 of the Shank3 gene, and also suggested the epigenetic regulation of their expression via methyl CpG-binding protein 2 (MeCP2) that has been identified as the causative molecule of Rett syndrome.
Assuntos
Neocórtex/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Linhagem Celular , Ilhas de CpG , Feminino , Humanos , Íntrons , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Proteínas dos Microfilamentos , Neocórtex/embriologia , Neocórtex/crescimento & desenvolvimento , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses, and plays important roles in the formation, maturation, and maintenance of synapses. Haploinsufficiency of the SHANK3 gene causes a developmental disorder, 22q13.3 deletion syndrome (known as Phelan-McDermid syndrome), that is characterized by severe expressive language and speech delay, hypotonia, global developmental delay, and autistic behavior. Since several SHANK3 mutations have been identified in a particular phenotypic group in patients with autism spectrum disorder (ASD), the SHANK3 is strongly suspected of being involved in the pathogenesis and neuropathology of ASD. Five CpG-islands have been identified in the SHANK3 gene, and tissue-specific expression of SHANK3 is regulated by DNA methylation in an epigenetic manner. Cumulative evidence has shown that several SHANK3 variants are expressed in the developing rodent brain and that their expression is regulated by DNA methylation of intragenic promoters. We identified novel SHANK3 transcripts whose transcription started at the vicinity of the CpG-island 2 in the mouse brain. Shank3 mutant mice exhibit autistic-like behaviors, including impaired social interaction and repetitive behaviors. In this article we review recent findings in regard to higher brain functions of SHANK3, epigenetic regulation of SHANK3 expression, and SHANK3-related ASD that were obtained from genetic analyses in ASD patients, molecular biological studies using developing mouse brains, and studies of Shank3 mutant mice.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Proteínas do Tecido Nervoso/genética , Animais , Criança , Pré-Escolar , Epigenômica , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Lactente , Camundongos , Camundongos MutantesRESUMO
Laminar organization is a key feature of the mammalian cerebral cortex, but the mechanisms by which final positioning and "inside-out" distribution of neurons are determined remain largely unknown. Here, we demonstrate that Robo1, a member of the family of Roundabout receptors, regulates the correct positioning of layers II/III pyramidal neurons in the neocortex. Specifically, we used RNA interference in mice to suppress the expression of Robo1 in a subset of layers II/III neurons, and observed the positions of these cells at distinct developmental stages. In contrast to control neurons that migrated toward the pial surface by P1, Robo1-suppressed neurons exhibited a delay in entering the cortical plate at respective stages. Unexpectedly, after the first postnatal week, these neurons were predominantly located in the upper part of layers II/III, in contrast to control cells that were distributed throughout these layers. Sequential electroporation studies revealed that Robo1-suppressed cells failed to establish the characteristic inside-out neuronal distribution and, instead, they accumulated beneath the marginal zone regardless of their birthdate. These results demonstrate that Robo receptors play a crucial role in neocortical lamination and particularly in the positioning of layers II/III pyramidal neurons.
Assuntos
Movimento Celular/genética , Córtex Cerebral/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas do Tecido Nervoso/metabolismo , Células Piramidais/fisiologia , Receptores Imunológicos/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Células COS , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Chlorocebus aethiops , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/metabolismo , Indóis/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas Nucleares/metabolismo , Gravidez , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Imunológicos/deficiência , Proteínas Repressoras/metabolismo , Transfecção , Proteínas RoundaboutRESUMO
Activated microglia are observed in various neurodegenerative diseases and are thought to be involved in the processes of neuronal cell death. Motoneuron damage in the facial nuclei after facial nerve avulsion is accelerated in presymptomatic transgenic rats expressing human mutant Cu(2+) /Zn(2+) superoxide dismutase 1 (SOD1), compared with that in wild-type rats. To reveal the functional role of microglia in motoneuronal death, we investigated the microglial response after facial nerve avulsion in presymptomatic mutant SOD1(H46R) (mSOD1(H46R) ) rats. At 3 days after avulsion, microglial clusters were observed in the facial nuclei of both wild-type and mSOD1(H46R) rats. The numbers of microglial clusters, proliferating microglia, and microglial attachments to motoneurons were significantly higher in mSOD1(H46R) rats, compared with those in wild-type rats. Immunopositive signals for the phagocytic marker ED1 were significantly stronger in mSOD1(H46R) rats, compared with that in wild-type rats, at 2 weeks after avulsion. Furthermore, primary microglia prepared from mSOD1(H46R) rats showed enhanced phagocytic activity, compared with that in wild-type rats. The expression of P2Y(12) mRNA was higher in the facial nuclei of mSOD1(H46R) rats, compared with that in wild-type rats. A laser microdissection system revealed that the expression of ATF3 mRNA was higher in the motoneurons of mSOD1(H46R) rats, compared with that in wild-type rats, at 2 days after avulsion. These results indicate that microglial activation in response to early neuronal damage increased in mSOD1(H46R) rats and suggest that the enhanced activation of microglia may lead to an increase in the vulnerability of motoneurons after avulsion in mSOD1(H46R) rats.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Modelos Animais de Doenças , Traumatismos do Nervo Facial/metabolismo , Microglia/metabolismo , Neurônios Motores/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Traumatismos do Nervo Facial/genética , Traumatismos do Nervo Facial/patologia , Humanos , Microglia/patologia , Neurônios Motores/patologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
In the mammalian brain, new neurons are continuously generated throughout life in the dentate gyrus (DG) of the hippocampus. Previous studies have established that newborn neurons migrate a short distance to be integrated into a pre-existing neuronal circuit in the hippocampus. How the migration of newborn neurons is governed by extracellular signals, however, has not been fully understood. Here, we report that NMDA receptor (NMDA-R)-mediated signaling is essential for the proper migration and positioning of newborn neurons in the DG. An intraperitoneal injection of the NMDA-R antagonists, memantine, or 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) into adult male mice caused the aberrant positioning of newborn neurons, resulting in the overextension of their migration in the DG. Interestingly, we revealed that the administration of NMDA-R antagonists leads to a decrease in the expression of Disrupted-In-Schizophrenia 1 (DISC1), a candidate susceptibility gene for major psychiatric disorders such as schizophrenia, which is also known as a critical regulator of neuronal migration in the DG. Furthermore, the overextended migration of newborn neurons induced by the NMDA-R antagonists was significantly rescued by exogenous expression of DISC1. Collectively, these results suggest that the NMDA-R signaling pathway governs the migration of newborn neurons via the regulation of DISC1 expression in the DG.
Assuntos
Movimento Celular/fisiologia , Hipocampo/citologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Animais Geneticamente Modificados , Bromodesoxiuridina/metabolismo , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteínas do Domínio Duplacortina , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neuropeptídeos/metabolismo , Fosfopiruvato Hidratase/metabolismo , Gravidez , RNA Mensageiro/metabolismoRESUMO
The 22q13.3 deletion syndrome is characterized by a significant delay in language development, mental retardation, hypotonia, and autistic features. Cumulative evidence has shown that haploinsufficiency of the SHANK3 gene is a major cause of the neurological symptoms of the 22q13.3 deletion syndrome. Shank3, a multidomain protein containing the SH3 and PDZ domains, is thought to play an important role in the formation and function of synapses in the developing brain. In this study, we analyzed the SHANK3 gene in 128 autistic patients with manifestations similar to those seen in the 22q13.3 deletion syndrome. The results showed a 6-amino acid deletion upstream of the SH3 domain, a missense variant (arginine to histidine at amino acid position 656) in the PDZ domain, and the insertion or deletion of a repeated 10-bp GC sequence located 9-bp downstream from the 3' end of exon 11. None of these variants was found in 228 controls.
Assuntos
Povo Asiático/genética , Transtorno Autístico/complicações , Transtorno Autístico/genética , Proteínas de Transporte/genética , Polimorfismo de Nucleotídeo Único/genética , Distúrbios da Fala/complicações , Distúrbios da Fala/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Criança , Pré-Escolar , Feminino , Humanos , Japão , Masculino , Dados de Sequência Molecular , Proteínas do Tecido NervosoRESUMO
Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu(2+)/Zn(2+) superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1(H46R) transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor-alpha (TNFalpha) and monocyte chemoattractant protein-1 (MCP-1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Microglia/patologia , Neurônios Motores/patologia , Fagocitose/genética , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Animais , Modelos Animais de Doenças , Feminino , Gliose/enzimologia , Gliose/genética , Gliose/patologia , Humanos , Masculino , Microglia/enzimologia , Neurônios Motores/enzimologia , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Medula Espinal/enzimologia , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Memantine, which is used clinically for the treatment of Alzheimer's disease (AD), is classified as an N-methyl-d-aspartate (NMDA) receptor antagonist. Since previous studies have shown that NMDA receptor antagonists promote neurogenesis in the adult brain, we examined the effect of memantine on neurogenesis in the adult mouse hippocampus. We intraperitoneally injected 3-month-old mice with memantine (at 10 or 50 mg/kg body weight) followed by 5-bromo-2-deoxyuridine (BrdU) injections (3x) after 3 days. We then examined the number of BrdU+ cells in the dentate gyrus (DG) of the hippocampus at different time points. The number of BrdU+ cells in the 50 mg/kg memantine-injected group increased by 2.1-fold (1 day after BrdU-injection), 3.4-fold (after 7 days), and 6.8-fold (after 28 days), whereas the 10 mg/kg dose of memantine had little effect on labeling compared to the control group. Immunohistochemical staining at 28 days after BrdU-injection revealed that the newly generated cells in the 50 mg/kg memantine-group had differentiated into mature granule neurons. Moreover, when 12-month-old mice were injected with memantine, cell proliferation was promoted in the DG (3.7-fold). These findings demonstrate that memantine promotes the proliferation of neural progenitor cells and the production of mature granule neurons in the adult hippocampus.
Assuntos
Proliferação de Células/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Memantina/farmacologia , Neurogênese/efeitos dos fármacos , Neurônios/citologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Fatores Etários , Animais , Bromodesoxiuridina/metabolismo , Contagem de Células , Relação Dose-Resposta a Droga , Hipocampo/citologia , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/fisiologia , Ácidos Siálicos/metabolismo , Fatores de Tempo , Proteínas Supressoras de Tumor/metabolismoRESUMO
New neurons are continuously generated in the hippocampus of the adult mammalian brain, and N-methyl-D-aspartate receptor (NMDA-R) antagonists have been found to increase the number of newly generated neurons in the dentate gyrus (DG) of the adult hippocampus. In this study, we examined the effect of memantine, an NMDA-R antagonist that is clinically used for the treatment of Alzheimer's disease, on primary progenitor cells exhibiting a radial glia-like (RGL) morphology in the DG. We injected 3-month-old mice with memantine (50 mg/kg body weight, intraperitoneally [i.p.]); 3 days later, we injected the mice with 5-bromo-2-deoxyuridine (BrdU; 75 mg/kg body weight, i.p.). We then counted the number of BrdU-labeled RGL progenitor cells in the DG 1 or 7 days after the BrdU-injection. The number of BrdU-labeled RGL progenitor cells had increased significantly by 5.1-fold on day 1 and by 13.7-fold on day 7 after BrdU-injection. Immunohistochemical staining revealed that the BrdU-labeled RGL progenitor cells expressed two primary progenitor cell marker proteins, nestin and Sox2. These results clearly demonstrated that memantine promotes the proliferation of RGL progenitor cells. We also found that memantine increased the ratio of horizontally aligned RGL progenitor cells, which are probably produced by symmetric division. These findings suggest that memantine increases the proliferation of primary progenitor cells and expands the primary progenitor cell pool in the adult hippocampus by stimulating symmetric division.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Memantina/farmacologia , Neurogênese/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Envelhecimento/metabolismo , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Bromodesoxiuridina , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Hipocampo/metabolismo , Proteínas de Filamentos Intermediários/análise , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurogênese/fisiologia , Neuroglia/citologia , Neuroglia/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Fatores de Transcrição SOXB1/análise , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
We report here on two unrelated patients (Patients 1 and 2) with a cryptic microduplication involving a 22q13 segment. Both patients manifested infantile hypotonia, developmental delay, and growth deficiency. In addition, an abnormal signal intensity area was detected in the frontal white matter of Patient 2 by brain MRI. Whole-genome microarray comparative genomic hybridization for Patient 1 and fluorescence in situ hybridization analysis with 22q-subtelomeric probes performed in both patients showed a submicroscopic 22q13 duplication that involved the SHANK3 gene. The duplication in Patient 1 was de novo type, while that in Patient 2 was derived from a familial 17;22 translocation. The presence of common clinical manifestations in the two patients with the common duplicated region led to a conclusion that 22q terminal duplication is a recognizable clinical entity, that is, the 22q13 microduplication syndrome.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 22 , Deficiências do Desenvolvimento/genética , Transtornos do Crescimento/genética , Hipotonia Muscular/genética , Encéfalo/patologia , Proteínas de Transporte/genética , Criança , Pré-Escolar , Feminino , Humanos , Hibridização in Situ Fluorescente , Imageamento por Ressonância Magnética , Proteínas do Tecido Nervoso , SíndromeRESUMO
Recently, neuroligins (NLs)3 and 4X have received much attention as autism-related genes. Here, we identified syntrophin-gamma2 (SNTG2) as a de novo binding partner of NL3. SNTG2 also bound to NL4X and NL4Y. Interestingly, the binding was influenced by autism-related mutations, implying that the impaired interaction between NLs and SNTG2 contributes to the etiology of autism.
Assuntos
Transtorno Autístico/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Mutação , Proteínas do Tecido Nervoso/metabolismo , Transtorno Autístico/patologia , Proteínas de Transporte/genética , Moléculas de Adesão Celular Neuronais , Clonagem Molecular , Humanos , Proteínas de Membrana/genética , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Sinapses/patologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons.
Assuntos
Proteínas de Transporte/metabolismo , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Membranas Sinápticas/metabolismo , Animais , Sítios de Ligação/fisiologia , Células CHO , Células COS , Proteínas de Transporte/química , Proteínas de Transporte/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Córtex Cerebral/ultraestrutura , Chlorocebus aethiops , Cricetinae , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Proteína 4 Homóloga a Disks-Large , Ácido Glutâmico/metabolismo , Guanilato Quinases , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso , Neurônios/ultraestrutura , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Receptores de AMPA/genética , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
The current quantitative study demonstrates that the recruitment of neuronal nitric oxide synthase (nNOS) beneath N-methyl-D-aspartate (NMDA) receptors, via postsynaptic density 95 (PSD-95) proteins significantly enhances nitric oxide (NO) production. Real-time single-cell fluorescence imaging was applied to measure both NO production and Ca(2+) influx in Chinese hamster ovary (CHO) cells expressing recombinant NMDA receptors (NMDA-R), nNOS, and PSD-95. We examined the relationship between the rate of NO production and Ca(2+) influx via NMDA receptors using the NO-reactive fluorescent dye, diaminofluorescein-FM (DAF-FM) and the Ca(2+)-sensitive yellow cameleon 3.1 (YC3.1), conjugated with PSD-95 (PSD-95-YC3.1). The presence of PSD-95 enhanced the rate of NO production by 2.3-fold upon stimulation with 100 microm NMDA in CHO1(+) cells (expressing NMDA-R, nNOS and PSD-95) when compared with CHO1(-) cells (expressing NMDA-R and nNOS lacking PSD-95). The presence of nNOS inhibitor or NMDA-R blocker almost completely suppressed this NMDA-stimulated NO production. The Ca(2+) concentration beneath the NMDA-R, [Ca(2+)](NR), was determined to be 5.4 microm by stimulating CHO2 cells (expressing NMDA-R and PSD-95-YC3.1) with 100 microm NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca(2+) concentration around nNOS, [Ca(2+)](NOS), was obtained over the wide range of [Ca(2+)](NOS). This sigmoidal curve had an EC(50) of approximately 1.2 microm of [Ca(2+)](NOS), implying that [Ca(2+)](NR) = 5.4 microm can activate nNOS effectively.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/biossíntese , Terminações Pré-Sinápticas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Membranas Sinápticas/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Ligação ao Cálcio , Cricetinae , Proteína 4 Homóloga a Disks-Large , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Fluoresceínas , Corantes Fluorescentes , Engenharia Genética , Inibidores do Crescimento/farmacologia , Guanilato Quinases , Substâncias Macromoleculares/metabolismo , Microscopia de Fluorescência , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Membranas Sinápticas/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Fyn-mediated tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D(2) receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D(2) receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D(2) receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.
Assuntos
Catalepsia/induzido quimicamente , Haloperidol/farmacologia , Proteínas Proto-Oncogênicas c-fyn/fisiologia , Animais , Antidiscinéticos/farmacologia , Antipsicóticos/farmacologia , Western Blotting , Cálcio/metabolismo , Catalepsia/patologia , Células Cultivadas , Dopamina/metabolismo , Genótipo , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Indóis/farmacologia , Camundongos , Neurônios/metabolismo , Fosforilação , Piperidinas/farmacologia , Proteínas Tirosina Quinases/química , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de N-Metil-D-Aspartato/química , Transdução de Sinais , Fatores de Tempo , Tirosina/químicaRESUMO
To elucidate the role of N-methyl-D-aspartate (NMDA) receptors during the early stage of cerebral neocortical development, we investigated the effect of an NMDA receptor antagonist, D(-)-2-amino-5-phosphonopentanoic acid (D-APV), on cell migration and proliferation in slice cultures and dissociated primary cultures prepared from rat cerebral neocortex at embryonic Day 17. Pulse-labeling experiments with 5-bromo-2'-deoxyuridine (BrdU) showed that chronic exposure to D-APV in slices delayed neuronal migration. Calcium imaging experiments revealed that functional NMDA receptors were expressed in neurons and the treatment with D-APV delayed neuronal maturation judging from the subunit composition of NMDA receptor subtypes. The results using pulse-labeling with BrdU indicated that exposure to D-APV for 3 days induced a clear increase in the number of proliferating progenitor cells in the ventricular zone in neocortical slices. Exposure to D-APV in primary cultures also increased the proliferation of progenitor cells. The effect of D-APV on progenitor cell proliferation was possibly mediated through neuronal cells. To elucidate the mechanism of enhanced progenitor cell proliferation induced by D-APV, we investigated expression of Hes1 and Hes5 mRNA in the ventricular zone of neocortical slices by reverse transcription-polymerase chain reaction. Tissue exposed to D-APV for 3 days showed higher expression of Hes1 and Hes5 mRNA than did unexposed control tissue. These results suggest that NMDA receptors expressed in neurons function in neuronal migration and maturation and in the proliferation of progenitor cells.