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1.
Sci Adv ; 5(12): eaay2670, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31897430

RESUMO

The cellular and the molecular mechanisms by which long noncoding RNAs (lncRNAs) may regulate presynaptic function and neuronal activity are largely unexplored. Here, we established an integrated screening strategy to discover lncRNAs implicated in neurotransmitter and synaptic vesicle release. With this approach, we identified neuroLNC, a neuron-specific nuclear lncRNA conserved from rodents to humans. NeuroLNC is tuned by synaptic activity and influences several other essential aspects of neuronal development including calcium influx, neuritogenesis, and neuronal migration in vivo. We defined the molecular interactors of neuroLNC in detail using chromatin isolation by RNA purification, RNA interactome analysis, and protein mass spectrometry. We found that the effects of neuroLNC on synaptic vesicle release require interaction with the RNA-binding protein TDP-43 (TAR DNA binding protein-43) and the selective stabilization of mRNAs encoding for presynaptic proteins. These results provide the first proof of an lncRNA that orchestrates neuronal excitability by influencing presynaptic function.


Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Longo não Codificante/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Movimento Celular/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Hipocampo/citologia , Humanos , Camundongos , Camundongos Transgênicos , Neurogênese/genética , Neurônios/metabolismo , Neurotransmissores/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transfecção
2.
Acta Physiol (Oxf) ; 221(1): 59-73, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28218996

RESUMO

AIM: Traffic between the plasma membrane and the endomembrane compartments is an essential feature of eukaryotic cells. The secretory pathway sends cargoes from biosynthetic compartments to the plasma membrane. This is counterbalanced by a retrograde endocytic route and is essential for cell homoeostasis. Cells need to adapt rapidly to environmental challenges such as the reduction of pO2 which, however, has not been analysed in relation to membrane trafficking in detail. Therefore, we determined changes in the plasma membrane trafficking in normoxia, hypoxia, and after reoxygenation. METHODS: Membrane trafficking was analysed by using the bulk membrane endocytosis marker FM 1-43, the newly developed membrane probe mCLING, wheat germ agglutinin as well as fluorescently labelled cholera toxin subunit B. Additionally, the uptake of specific membrane proteins was determined. In parallel, a non-biased SILAC screen was performed to analyse the abundance of membrane proteins in normoxia and hypoxia. RESULTS: Membrane trafficking was increased in hypoxia and quickly reversed upon reoxygenation. This effect was independent of the hypoxia-inducible factor (HIF) system. Using SILAC technology, we identified that the actin-bundling protein T-plastin is recruited to the plasma membrane in hypoxia. By the use of T-plastin knockdown cells, we could show that T-plastin mediates the hypoxia-induced membrane trafficking, which was associated with an increased actin density in the cells as determined by electron microscopy. CONCLUSION: Membrane trafficking is highly dynamic upon hypoxia. This phenotype is quickly reversible upon reoxygenation, which suggests that this mechanism participates in the cellular adaptation to hypoxia.


Assuntos
Membrana Celular/metabolismo , Hipóxia/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Transporte Proteico/fisiologia , Animais , Linhagem Celular , Humanos , Ratos
3.
Mol Psychiatry ; 21(12): 1752-1767, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-26809838

RESUMO

Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of ~20%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a 15N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated 15N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration.


Assuntos
Eritropoetina/metabolismo , Neurogênese/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Sistema Nervoso Central/metabolismo , Cognição/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Neurônios/metabolismo , Oligodendroglia/metabolismo , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Proteínas Recombinantes/metabolismo
4.
J Physiol ; 557(Pt 1): 77-91, 2004 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15004214

RESUMO

Phosphoinositides are key regulators of synaptic vesicle cycling and endocytic traffic; the actin cytoskeleton also seems to be involved in modulating these processes. We investigated the effects of perturbing phosphoinositide signalling and actin dynamics on vesicle cycling in frog motor nerve terminals, using fluorescence and electron microscopy, and electrophysiology. Antibody staining for beta-actin revealed that actin surrounds but does not overlap with synaptic vesicle clusters. Latrunculin A, which disrupts actin filaments by binding actin monomers, and wortmannin, an inhibitor of phosphatidyl inositol-3-kinase (PI3-kinase), each disrupted the pattern of presynaptic actin staining, but not vesicle clusters in resting terminals. Latrunculin A, but not wortmannin, also reduced vesicle mobilization and exocytosis. Both drugs inhibited the stimulation-induced uptake of the styryl dye FM1-43 and blocked vesicle reformation from internalized membrane objects after tetanic stimulation. These results are consistent with a role of PI3-kinase and the actin cytoskeleton in the slow pathway of vesicle endocytosis, used primarily by reserve pool vesicles.


Assuntos
Androstadienos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Tiazóis/farmacologia , Acetilcolina/metabolismo , Actinas/efeitos dos fármacos , Actinas/metabolismo , Animais , Citoesqueleto/efeitos dos fármacos , Eletrofisiologia , Exocitose/efeitos dos fármacos , Corantes Fluorescentes , Cobaias , Técnicas In Vitro , Potenciais da Membrana/fisiologia , Microeletrodos , Microscopia Eletrônica , Microscopia de Fluorescência , Placa Motora/efeitos dos fármacos , Placa Motora/ultraestrutura , Junção Neuromuscular/ultraestrutura , Inibidores de Fosfoinositídeo-3 Quinase , Terminações Pré-Sinápticas/efeitos dos fármacos , Rana pipiens , Transdução de Sinais/efeitos dos fármacos , Tiazolidinas , Wortmanina
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