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1.
Proc Natl Acad Sci U S A ; 118(45)2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34740966

RESUMO

Cerebellar Purkinje neurons integrate information transmitted at excitatory synapses formed by granule cells. Although these synapses are considered essential sites for learning, most of them appear not to transmit any detectable electrical information and have been defined as silent. It has been proposed that silent synapses are required to maximize information storage capacity and ensure its reliability, and hence to optimize cerebellar operation. Such optimization is expected to occur once the cerebellar circuitry is in place, during its maturation and the natural and steady improvement of animal agility. We therefore investigated whether the proportion of silent synapses varies over this period, from the third to the sixth postnatal week in mice. Selective expression of a calcium indicator in granule cells enabled quantitative mapping of presynaptic activity, while postsynaptic responses were recorded by patch clamp in acute slices. Through this approach and the assessment of two anatomical features (the distance that separates adjacent planar Purkinje dendritic trees and the synapse density), we determined the average excitatory postsynaptic potential per synapse. Its value was four to eight times smaller than responses from paired recorded detectable connections, consistent with over 70% of synapses being silent. These figures remained remarkably stable across maturation stages. According to the proposed role for silent synapses, our results suggest that information storage capacity and reliability are optimized early during cerebellar maturation. Alternatively, silent synapses may have roles other than adjusting the information storage capacity and reliability.


Assuntos
Cerebelo/crescimento & desenvolvimento , Animais , Sinalização do Cálcio , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Células de Purkinje/fisiologia , Sinapses/fisiologia
2.
Neuron ; 107(4): 617-630.e6, 2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32559415

RESUMO

Stable genomic integration of exogenous transgenes is essential in neurodevelopmental and stem cell studies. Despite tools driving increasingly efficient genomic insertion with DNA vectors, transgenesis remains fundamentally hindered by the impossibility of distinguishing integrated from episomal transgenes. Here, we introduce an integration-coupled On genetic switch, iOn, which triggers gene expression upon incorporation into the host genome through transposition, thus enabling rapid and accurate identification of integration events following transfection with naked plasmids. In vitro, iOn permits rapid drug-free stable transgenesis of mouse and human pluripotent stem cells with multiple vectors. In vivo, we demonstrate faithful cell lineage tracing, assessment of regulatory elements, and mosaic analysis of gene function in somatic transgenesis experiments that reveal neural progenitor potentialities and interaction. These results establish iOn as a universally applicable strategy to accelerate and simplify genetic engineering in cultured systems and model organisms by conditioning transgene activation to genomic integration.


Assuntos
Expressão Gênica , Técnicas de Transferência de Genes , Células-Tronco Neurais , Transgenes , Animais , Linhagem da Célula , Vetores Genéticos , Humanos , Camundongos
3.
Mol Neurodegener ; 9: 60, 2014 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-25524049

RESUMO

BACKGROUND: It is suspected that excess of brain cholesterol plays a role in Alzheimer's disease (AD). Membrane-associated cholesterol was shown to be increased in the brain of individuals with sporadic AD and to correlate with the severity of the disease. We hypothesized that an increase of membrane cholesterol could trigger sporadic AD early phenotypes. RESULTS: We thus acutely loaded the plasma membrane of cultured neurons with cholesterol to reach the 30% increase observed in AD brains. We found changes in gene expression profiles that are reminiscent of early AD stages. We also observed early AD cellular phenotypes. Indeed we found enlarged and aggregated early endosomes using confocal and electron microscopy after immunocytochemistry. In addition amyloid precursor protein vesicular transport was inhibited in neuronal processes, as seen by live-imaging. Finally transient membrane cholesterol loading lead to significantly increased amyloid-ß42 secretion. CONCLUSIONS: Membrane cholesterol increase in cultured neurons reproduces most early AD changes and could thus be a relevant model for deciphering AD mechanisms and identifying new therapeutic targets.


Assuntos
Doença de Alzheimer/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Neurônios/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Memória/fisiologia , Fenótipo , Ratos Sprague-Dawley , Transcriptoma
4.
Hum Mol Genet ; 22(9): 1856-66, 2013 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-23390130

RESUMO

Manipulation of the mouse genome by site-specific mutagenesis has been extensively used to study gene function and model human disorders. Mouse models of myotubular myopathy (XLMTM), a severe congenital muscular disorder due to loss-of-function mutations in the MTM1 gene, have been generated by homologous recombination and shown that myotubularin is essential for skeletal muscle. However, since the Mtm1 deletion occurred constitutively or shortly after birth in these mice, it is not known whether myotubularin is required during adulthood, an important issue in the context of not only muscle biology but also therapies. To delete the Mtm1 gene in adult muscle fibers, we constructed a recombinant adeno-associated vector (AAV) that expresses the Cre recombinase under the muscle-specific desmin promoter. We report that a single injection of this vector into muscles of 3-month-old Mtm1 conditional mice leads to a myotubular myopathy phenotype with myofiber atrophy, disorganization of organelle positioning, such as mitochondria and nuclei, T-tubule defects and severe muscle weakness. In addition, our results show that MTM1-related atrophy and dysfunction correlate with abnormalities in satellite cell number and markers of autophagy, protein synthesis and neuromuscular junction transmission. The expression level of atrogenes was also analyzed. Therefore, we provide a valuable tissue model that recapitulates the main features of the disease, and it is useful to study pathogenesis and evaluate therapeutic strategies. We establish the proof-of-concept that myotubularin is required for the proper function of skeletal muscle during adulthood, suggesting that therapies will be required for the entire life of XLMTM patients.


Assuntos
Músculo Esquelético/metabolismo , Mutagênese Sítio-Dirigida , Miopatias Congênitas Estruturais/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Adenoviridae/genética , Animais , Desmina/genética , Desmina/metabolismo , Deleção de Genes , Vetores Genéticos , Masculino , Camundongos , Debilidade Muscular/genética , Debilidade Muscular/patologia , Doenças Musculares , Miopatias Congênitas Estruturais/patologia , Fenótipo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Análise de Sequência de DNA
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