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1.
Proc Natl Acad Sci U S A ; 120(30): e2221413120, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37433002

RESUMO

Effects of micronutrients on brain connectivity are incompletely understood. Analyzing human milk samples across global populations, we identified the carbocyclic sugar myo-inositol as a component that promotes brain development. We determined that it is most abundant in human milk during early lactation when neuronal connections rapidly form in the infant brain. Myo-inositol promoted synapse abundance in human excitatory neurons as well as cultured rat neurons and acted in a dose-dependent manner. Mechanistically, myo-inositol enhanced the ability of neurons to respond to transsynaptic interactions that induce synapses. Effects of myo-inositol in the developing brain were tested in mice, and its dietary supplementation enlarged excitatory postsynaptic sites in the maturing cortex. Utilizing an organotypic slice culture system, we additionally determined that myo-inositol is bioactive in mature brain tissue, and treatment of organotypic slices with this carbocyclic sugar increased the number and size of postsynaptic specializations and excitatory synapse density. This study advances our understanding of the impact of human milk on the infant brain and identifies myo-inositol as a breast milk component that promotes the formation of neuronal connections.


Assuntos
Aleitamento Materno , Leite Humano , Feminino , Lactente , Humanos , Animais , Camundongos , Ratos , Neurônios , Inositol/farmacologia , Açúcares
2.
Proc Natl Acad Sci U S A ; 117(24): 13468-13479, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467162

RESUMO

The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2 However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis.


Assuntos
Exocitose , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins/metabolismo , Humanos , Lipossomos/metabolismo , Fusão de Membrana , Células PC12 , Domínios de Homologia à Plecstrina , Ligação Proteica , Ratos , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Vertebrados , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
3.
EMBO J ; 38(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30733243

RESUMO

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation.


Assuntos
Aciltransferases/fisiologia , Cálcio/metabolismo , Exocitose/fisiologia , Células Ciliadas Auditivas/fisiologia , Terminações Pré-Sinápticas/fisiologia , Sinapses/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Cóclea/citologia , Cóclea/fisiologia , Endocitose , Feminino , Células Ciliadas Auditivas/citologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transmissão Sináptica
4.
Mol Neurobiol ; 55(7): 6050-6062, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29143288

RESUMO

Glycogen synthase kinases-3ß (GSK3ß) is a key regulator of cell homeostasis. In neurons, GSK3ß contributes to control of neuronal transmission and plasticity. Despite extensive studies in non-neuronal cells, crosstalk between GSK3ß and other signaling pathways remains not well defined in neurons. In the present study, we report that GSK3ß positively affected the activity of effectors of mammalian target of rapamycin complex 1 (mTORC1) and complex 2 (mTORC2), in mature neurons in vitro and in vivo. GSK3ß also promoted prosurvival signaling and attenuated kainic acid-induced apoptosis. Our study identified GSK3ß as a positive regulator of prosurvival signaling, including the mTOR pathway, and indicates the possible neuroprotective role of GSK3ß in models of pharmacologically induced excitotoxicity.


Assuntos
Glicogênio Sintase Quinase 3 beta/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Encéfalo/enzimologia , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Isoenzimas/metabolismo , Ácido Caínico , Camundongos Transgênicos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/metabolismo
5.
Mol Neurobiol ; 54(1): 200-211, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-26738851

RESUMO

Changes in the morphology of dendritic spines are prominent during learning and in different neurological and neuropsychiatric diseases, including those in which glycogen synthase kinase-3ß (GSK-3ß) has been implicated. Despite much evidence of the involvement of GSK-3ß in functional synaptic plasticity, it is unclear how GSK-3ß controls structural synaptic plasticity (i.e., the number and shape of dendritic spines). In the present study, we used two mouse models overexpressing and lacking GSK-3ß in neurons to investigate how GSK-3ß affects the structural plasticity of dendritic spines. Following visualization of dendritic spines with DiI dye, we found that increasing GSK-3ß activity increased the number of thin spines, whereas lacking GSK-3ß increased the number of stubby spines in the dentate gyrus. Under conditions of neuronal excitation, increasing GSK-3ß activity caused higher activity of extracellularly acting matrix metalloproteinase-9 (MMP-9), and MMP inhibition normalized thin spines in GSK-3ß overexpressing mice. Administration of the nonspecific GSK-3ß inhibitor lithium in animals with active MMP-9 and animals lacking MMP-9 revealed that GSK-3ß and MMP-9 act in concert to control dendritic spine morphology. Altogether, our data demonstrate that the dysregulation of GSK-3ß activity has dramatic consequences on dendritic spine morphology, implicating MMP-9 as a mediator of GSK-3ß-induced synaptic alterations.


Assuntos
Espinhas Dendríticas/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Animais , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Espinhas Dendríticas/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Transgênicos , Ratos Wistar
6.
PLoS One ; 10(5): e0128285, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26020770

RESUMO

The goal of this study was to determine whether a substantial decrease in adult neurogenesis influences epileptogenesis evoked by the intra-amygdala injection of kainic acid (KA). Cyclin D2 knockout (cD2 KO) mice, which lack adult neurogenesis almost entirely, were used as a model. First, we examined whether status epilepticus (SE) evoked by an intra-amygdala injection of KA induces cell proliferation in cD2 KO mice. On the day after SE, we injected BrdU into mice for 5 days and evaluated the number of DCX- and DCX/BrdU-immunopositive cells 3 days later. In cD2 KO control animals, only a small number of DCX+ cells was observed. The number of DCX+ and DCX/BrdU+ cells/mm of subgranular layer in cD2 KO mice increased significantly following SE (p<0.05). However, the number of newly born cells was very low and was significantly lower than in KA-treated wild type (wt) mice. To evaluate the impact of diminished neurogenesis on epileptogenesis and early epilepsy, we performed video-EEG monitoring of wt and cD2 KO mice for 16 days following SE. The number of animals with seizures did not differ between wt (11 out of 15) and cD2 KO (9 out of 12) mice. The median latency to the first spontaneous seizure was 4 days (range 2-10 days) in wt mice and 8 days (range 2-16 days) in cD2 KO mice and did not differ significantly between groups. Similarly, no differences were observed in median seizure frequency (wt: 1.23, range 0.1-3.4; cD2 KO: 0.57, range 0.1-2.0 seizures/day) or median seizure duration (wt: 51 s, range 23-103; cD2 KO: 51 s, range 23-103). Our results indicate that SE-induced epileptogenesis is not disrupted in mice with markedly reduced adult neurogenesis. However, we cannot exclude the contribution of reduced neurogenesis to the chronic epileptic state.


Assuntos
Tonsila do Cerebelo , Ciclina D2/deficiência , Ácido Caínico/efeitos adversos , Neurogênese , Convulsões , Estado Epiléptico , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/patologia , Tonsila do Cerebelo/fisiopatologia , Animais , Proteína Duplacortina , Eletroencefalografia , Ácido Caínico/farmacologia , Camundongos , Camundongos Knockout , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Convulsões/induzido quimicamente , Convulsões/genética , Convulsões/metabolismo , Convulsões/patologia , Convulsões/fisiopatologia , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/genética , Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Estado Epiléptico/fisiopatologia
7.
Behav Brain Res ; 248: 46-50, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23570858

RESUMO

Glycogen synthase kinase-3 (GSK-3), a multifunctional serine-threonine kinase, is an important regulator in numerous signaling pathways and processes including adult brain neurogenesis. GSK-3 (mal)functioning was implicated in many diseases, in particular neurological and behavioral disorders. We investigated the impact of altered levels of the GSK-3ß isoform on hippocampal size, number of doublecortin-positive cells, and hippocampal-dependent behaviors. Both GSK-3ß transgenic mice (GSK-3ß[S9A] mice) and GSK-3ß neuron-specific knockout (GSK-3ß(n-/-)) mice, showed reduced size of the dentate gyrus (DG) and were impaired in three hippocampal-dependent, species-typical behavioral tasks: digging, marble burying and nest building. We further demonstrate that the number of differentiating, doublecortin-positive new neurons is reduced in GSK-3ß[S9A] mice, but not in GSK-3ß(n-/-) mice. We conclude that GSK-3ß activity must be critically controlled to allow wild type-like volume of the dentate gyrus and for normal execution of hippocampal-dependent, species-typical behavior.


Assuntos
Comportamento Animal/fisiologia , Giro Denteado/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Hipocampo/metabolismo , Camundongos Knockout/metabolismo , Camundongos Transgênicos/metabolismo , Animais , Glicogênio Sintase Quinase 3 beta , Camundongos , Neurogênese/fisiologia , Neurônios/metabolismo , Transdução de Sinais/fisiologia
8.
Dev Biol ; 368(1): 54-62, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22609553

RESUMO

During preimplantation mouse embryo development expression of Cdx2 is induced in outer cells, which are the trophectoderm (TE) precursors. The mechanism of Cdx2 upregulation in these cells remains unclear. However, it has been suggested that the cell position and polarization may play a crucial role in this process. In order to elucidate the role of these two parameters in the formation of TE we analyzed the expression pattern of Cdx2 in the embryos in which either the position of cells and the time of polarization or only the position of cells was experimentally disrupted. Such embryos developed from the blastomeres that were isolated from 8-cell embryos either before or after the compaction, i.e. before or after the cell polarization took place. We found that in the embryos developed from polar blastomeres originated from the 8-cell compacted embryo, the experimentally imposed outer position was not sufficient to induce the Cdx2 in these blastomeres which in the intact embryo would form the inner cells. However, when the polarization at the 8-cell stage was disrupted, the embryos developed from such an unpolarized blastomeres showed the increased number of cells expressing Cdx2. We found that in such experimentally obtained embryos the polarization was delayed until the 16-cell stage. These results suggest that the main factor responsible for upregulation of Cdx2 expression in outer blastomeres, i.e. TE precursors, is their polarity.


Assuntos
Blastômeros/metabolismo , Polaridade Celular , Embrião de Mamíferos/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Blastômeros/citologia , Fator de Transcrição CDX2 , Contagem de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Feminino , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Microscopia Confocal , Fatores de Tempo , Regulação para Cima
9.
Cell ; 138(2): 352-65, 2009 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-19592082

RESUMO

Cyclins are regulatory subunits of cyclin-dependent kinases. Cyclin A, the first cyclin ever cloned, is thought to be an essential component of the cell-cycle engine. Mammalian cells encode two A-type cyclins, testis-specific cyclin A1 and ubiquitously expressed cyclin A2. Here, we tested the requirement for cyclin A function using conditional knockout mice lacking both A-type cyclins. We found that acute ablation of cyclin A in fibroblasts did not affect cell proliferation, but led to prolonged expression of another cyclin, cyclin E, across the cell cycle. However, combined ablation of all A- and E-type cyclins extinguished cell division. In contrast, cyclin A function was essential for cell-cycle progression of hematopoietic and embryonic stem cells. Expression of cyclin A is particularly high in these compartments, which might render stem cells dependent on cyclin A, whereas in fibroblasts cyclins A and E play redundant roles in cell proliferation.


Assuntos
Ciclina A/metabolismo , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Ciclina A/genética , Ciclina E/genética , Ciclina E/metabolismo , Camundongos , Camundongos Knockout
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