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1.
iScience ; 25(2): 103757, 2022 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-35118365

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder, in which an abnormal and toxic protein called progerin, accumulates in cell nuclei, leading to major cellular defects. Among them, chromatin remodeling drives gene expression changes, including miRNA dysregulation. In our study, we evaluated miRNA expression profiles in HGPS and control fibroblasts. We identified an enrichment of overexpressed miRNAs belonging to the 14q32.2-14q32.3 miRNA cluster. Using 3D FISH, we demonstrated that overexpression of these miRNAs is associated with chromatin remodeling at this specific locus in HGPS fibroblasts. We then focused on miR-376b-3p and miR-376a-3p, both overexpressed in HGPS fibroblasts. We demonstrated that their induced overexpression in control fibroblasts decreases cell proliferation and increases senescence, whereas their inhibition in HGPS fibroblasts rescues proliferation defects and senescence and decreases progerin accumulation. By targeting these major processes linked to premature aging, these two miRNAs may play a pivotal role in the pathophysiology of HGPS.

2.
Sci Rep ; 8(1): 16059, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30375447

RESUMO

Prior to sensory experience spontaneous activity appears to play a fundamental role in the correct formation of prominent functional features of different cortical regions. The use of anaesthesia during pregnancy such as ketamine is largely considered to negatively affect neuronal development by interfering with synaptic transmission. Interestingly, the characteristics of spontaneous activity as well as the acute functional effects of maternal anaesthesia remain largely untested in the embryonic cortex in vivo. In the present work, we performed in vivo imaging of spontaneous calcium activity and cell motility in the marginal zone of the cortex of E14-15 embryos connected to the mother. We made use of a preparation where the blood circulation from the mother through the umbilical cord is preserved and fluctuations in intracellular calcium in the embryonic frontal cortex are acquired using two-photon imaging. We found that spontaneous transients were either sporadic or correlated in clusters of neuronal ensembles at this age. These events were not sensitive to maternal isoflurane anaesthesia but were strongly inhibited by acute in situ or maternal application of low concentration of the anaesthetic ketamine (a non-competitive antagonist of NMDA receptors). Moreover, simultaneous imaging of cell motility revealed a correlated strong sensitivity to ketamine. These results show that anaesthetic compounds can differ significantly in their impact on spontaneous early cortical activity as well as motility of cells in the marginal zone. The effects found in this study may be relevant in the etiology of heightened vulnerability to cerebral dysfunction associated with the use of ketamine during pregnancy.


Assuntos
Anestesia/efeitos adversos , Cálcio/metabolismo , Córtex Cerebral/embriologia , Receptores de N-Metil-D-Aspartato/genética , Anestésicos/efeitos adversos , Animais , Movimento Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Ketamina/administração & dosagem , Ketamina/efeitos adversos , Neurônios/metabolismo , Neurônios/patologia , Gravidez , Transmissão Sináptica/efeitos dos fármacos
3.
J Vis Exp ; (130)2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29286390

RESUMO

Birth defects that involve the cerebral cortex - also known as malformations of cortical development (MCD) - are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.


Assuntos
Encéfalo/patologia , Hibridização Genômica Comparativa/métodos , Eletroporação/métodos , Sequenciamento do Exoma/métodos , Malformações do Desenvolvimento Cortical/genética , Animais , Química Encefálica , DNA/sangue , DNA/genética , DNA/isolamento & purificação , Modelos Animais de Doenças , Feminino , Humanos , Malformações do Desenvolvimento Cortical/sangue , Malformações do Desenvolvimento Cortical/patologia , Gravidez , Ratos
4.
Cytoskeleton (Hoboken) ; 73(10): 566-576, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-26994401

RESUMO

Over the past two decades, substantial progress has been made in visualizing and understanding neuronal cell migration and morphogenesis during brain development. Distinct mechanisms have evolved to support migration of the various cell types that compose the developing neocortex. A specific subset of molecular motors, so far consisting of cytoplasmic dynein 1, Kif1a and myosin II, are responsible for cytoskeletal and nuclear transport in these cells. This review focuses on the emerging roles for each of these motor proteins in the migratory mechanisms of neocortical cell types. We discuss how migration can be cell cycle regulated and how coordination of motor activity is required to ensure migratory direction. © 2016 Wiley Periodicals, Inc.


Assuntos
Movimento Celular/fisiologia , Neocórtex/embriologia , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Animais , Dineínas/metabolismo , Humanos , Cinesinas/metabolismo , Miosina Tipo II/metabolismo , Neocórtex/citologia , Células-Tronco Neurais/citologia , Neurônios/citologia
5.
Methods Cell Biol ; 131: 349-63, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26794523

RESUMO

Development of the cerebral cortex is a very dynamic process, involving a series of complex morphogenetic events. Following division of progenitor cells in the ventricular zone, neurons undergo a series of morphological changes and migrate outward toward the cortical plate, where they differentiate and integrate into functional circuits. Errors at several of stages during neurogenesis and migration cause a variety of severe cortical malformations. A number of disease genes encode factors associated with the cytoskeleton, which plays a crucial role throughout cortical development. Methods for regulating gene expression coupled with imaging of subcellular structures have provided important insight into the mechanisms governing normal and abnormal brain development. We describe here a series of protocols for imaging motor protein-dependent processes in real time in the developing rat brain.


Assuntos
Córtex Cerebral/metabolismo , Proteínas Motores Moleculares/genética , Células-Tronco Neurais/metabolismo , Animais , Movimento Celular/fisiologia , Córtex Cerebral/citologia , Eletroporação/métodos , Embrião de Mamíferos/inervação , Células Ependimogliais/citologia , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Microtúbulos/metabolismo , Células-Tronco Neurais/citologia , Transporte Proteico/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Proteína Vermelha Fluorescente
6.
Nat Neurosci ; 19(2): 253-62, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26752160

RESUMO

Brain neural stem cells (radial glial progenitors, RGPs) undergo a mysterious form of cell cycle-entrained interkinetic nuclear migration (INM) that is driven apically by cytoplasmic dynein and basally by the kinesin KIF1A, which has recently been implicated in human brain developmental disease. To understand the consequences of altered basal INM and the roles of KIF1A in disease, we performed constitutive and conditional RNAi and expressed mutant KIF1A in E16 to P7 rat RGPs and neurons. RGPs inhibited in basal INM still showed normal cell cycle progression, although neurogenic divisions were severely reduced. Postmitotic neuronal migration was independently disrupted at the multipolar stage and accompanied by premature ectopic expression of neuronal differentiation markers. Similar effects were unexpectedly observed throughout the layer of surrounding control cells, mimicked by Bdnf (brain-derived neurotrophic factor) or Dcx RNAi, and rescued by BDNF application. These results identify sequential and independent roles for KIF1A and provide an important new approach for reversing the effects of human disease.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Cinesinas/antagonistas & inibidores , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Antígenos de Diferenciação/metabolismo , Ciclo Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proteína Duplacortina , Feminino , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Mitose/efeitos dos fármacos , Gravidez , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Transfecção
7.
Brain ; 136(Pt 11): 3378-94, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24056535

RESUMO

Periventricular nodular heterotopia is caused by defective neuronal migration that results in heterotopic neuronal nodules lining the lateral ventricles. Mutations in filamin A (FLNA) or ADP-ribosylation factor guanine nucleotide-exchange factor 2 (ARFGEF2) cause periventricular nodular heterotopia, but most patients with this malformation do not have a known aetiology. Using comparative genomic hybridization, we identified 12 patients with developmental brain abnormalities, variably combining periventricular nodular heterotopia, corpus callosum dysgenesis, colpocephaly, cerebellar hypoplasia and polymicrogyria, harbouring a common 1.2 Mb minimal critical deletion in 6q27. These anatomic features were mainly associated with epilepsy, ataxia and cognitive impairment. Using whole exome sequencing in 14 patients with isolated periventricular nodular heterotopia but no copy number variants, we identified one patient with periventricular nodular heterotopia, developmental delay and epilepsy and a de novo missense mutation in the chromosome 6 open reading frame 70 (C6orf70) gene, mapping in the minimal critical deleted region. Using immunohistochemistry and western blots, we demonstrated that in human cell lines, C6orf70 shows primarily a cytoplasmic vesicular puncta-like distribution and that the mutation affects its stability and subcellular distribution. We also performed in utero silencing of C6orf70 and of Phf10 and Dll1, the two additional genes mapping in the 6q27 minimal critical deleted region that are expressed in human and rodent brain. Silencing of C6orf70 in the developing rat neocortex produced periventricular nodular heterotopia that was rescued by concomitant expression of wild-type human C6orf70 protein. Silencing of the contiguous Phf10 or Dll1 genes only produced slightly delayed migration but not periventricular nodular heterotopia. The complex brain phenotype observed in the 6q terminal deletion syndrome likely results from the combined haploinsufficiency of contiguous genes mapping to a small 1.2 Mb region. Our data suggest that, of the genes within this minimal critical region, C6orf70 plays a major role in the control of neuronal migration and its haploinsufficiency or mutation causes periventricular nodular heterotopia.


Assuntos
Anormalidades Múltiplas/genética , Encéfalo/anormalidades , Malformações do Desenvolvimento Cortical do Grupo II/genética , Heterotopia Nodular Periventricular/genética , Anormalidades Múltiplas/patologia , Anormalidades Múltiplas/fisiopatologia , Adolescente , Adulto , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Criança , Deleção Cromossômica , Cromossomos Humanos Par 6/genética , Estudos de Coortes , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Exoma/genética , Feminino , Haploinsuficiência/genética , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Malformações do Desenvolvimento Cortical do Grupo II/patologia , Malformações do Desenvolvimento Cortical do Grupo II/fisiopatologia , Mutação/genética , Heterotopia Nodular Periventricular/patologia , Heterotopia Nodular Periventricular/fisiopatologia , Ratos , Ratos Wistar , Síndrome
8.
Neuron ; 74(1): 57-64, 2012 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-22500630

RESUMO

Gamma-frequency oscillations (GFOs, >40 Hz) are a general network signature at seizure onset at all stages of development, with possible deleterious consequences in the immature brain. At early developmental stages, the simultaneous occurrence of GFOs in different brain regions suggests the existence of a long-ranging synchronizing mechanism at seizure onset. Here, we show that hippocamposeptal (HS) neurons, which are GABA long-range projection neurons, are mandatory to drive the firing of hippocampal interneurons in a high-frequency regime at the onset of epileptiform discharges in the intact, immature septohippocampal formation. The synchronized firing of interneurons in turn produces GFOs, which are abolished after the elimination of a small number of HS neurons. Because they provide the necessary fast conduit for pacing large neuronal populations and display intra- and extrahippocampal long-range projections, HS neurons appear to belong to the class of hub cells that play a crucial role in the synchronization of developing networks.


Assuntos
Sincronização Cortical/fisiologia , Epilepsia/fisiopatologia , Neurônios GABAérgicos/fisiologia , Hipocampo/crescimento & desenvolvimento , Células Piramidais/fisiologia , Potenciais de Ação/fisiologia , Fatores Etários , Animais , Hipocampo/citologia , Hipocampo/fisiologia , Técnicas In Vitro , Interneurônios/fisiologia , Camundongos , Rede Nervosa/citologia , Rede Nervosa/fisiologia , Ratos , Septo do Cérebro/citologia , Septo do Cérebro/crescimento & desenvolvimento , Septo do Cérebro/fisiologia
9.
Hum Mol Genet ; 21(5): 1004-17, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22076441

RESUMO

Periventricular nodular heterotopia (PH) is a human brain malformation caused by defective neuronal migration that results in ectopic neuronal nodules lining the lateral ventricles beneath a normal appearing cortex. Most affected patients have seizures and their cognitive level varies from normal to severely impaired. Mutations in the Filamin-A (or FLNA) gene are the main cause of PH, but the underlying pathological mechanism remains unknown. Although two FlnA knockout mouse strains have been generated, none of them showed the presence of ectopic nodules. To recapitulate the loss of FlnA function in the developing rat brain, we used an in utero RNA interference-mediated knockdown approach and successfully reproduced a PH phenotype in rats comparable with that observed in human patients. In FlnA-knockdown rats, we report that PH results from a disruption of the polarized radial glial scaffold in the ventricular zone altering progression of neural progenitors through the cell cycle and impairing migration of neurons into the cortical plate. Similar alterations of radial glia are observed in human PH brains of a 35-week fetus and a 3-month-old child, harboring distinct FLNA mutations not previously reported. Finally, juvenile FlnA-knockdown rats are highly susceptible to seizures, confirming the reliability of this novel animal model of PH. Our findings suggest that the disorganization of radial glia is the leading cause of PH pathogenesis associated with FLNA mutations. Rattus norvegicus FlnA mRNA (GenBank accession number FJ416060).


Assuntos
Córtex Cerebral/metabolismo , Proteínas Contráteis/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neuroglia/fisiologia , Heterotopia Nodular Periventricular/metabolismo , Heterotopia Nodular Periventricular/patologia , Animais , Movimento Celular , Proliferação de Células , Córtex Cerebral/embriologia , Córtex Cerebral/patologia , Ventrículos Cerebrais/patologia , Proteínas Contráteis/genética , Modelos Animais de Doenças , Feminino , Filaminas , Humanos , Lactente , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Neocórtex/embriologia , Neocórtex/metabolismo , Neocórtex/patologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/fisiologia , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Neurônios/fisiologia , Interferência de RNA , Ratos , Convulsões/etiologia
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