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1.
Proc Natl Acad Sci U S A ; 120(4): e2213810120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36669113

RESUMO

Reactivation of the inactive X chromosome is a hallmark epigenetic event during reprogramming of mouse female somatic cells to induced pluripotent stem cells (iPSCs). This involves global structural remodeling from a condensed, heterochromatic into an open, euchromatic state, thereby changing a transcriptionally inactive into an active chromosome. Despite recent advances, very little is currently known about the molecular players mediating this process and how this relates to iPSC-reprogramming in general. To gain more insight, here we perform a RNAi-based knockdown screen during iPSC-reprogramming of mouse fibroblasts. We discover factors important for X chromosome reactivation (XCR) and iPSC-reprogramming. Among those, we identify the cohesin complex member SMC1a as a key molecule with a specific function in XCR, as its knockdown greatly affects XCR without interfering with iPSC-reprogramming. Using super-resolution microscopy, we find SMC1a to be preferentially enriched on the active compared with the inactive X chromosome and that SMC1a is critical for the decompacted state of the active X. Specifically, depletion of SMC1a leads to contraction of the active X both in differentiated and in pluripotent cells, where it normally is in its most open state. In summary, we reveal cohesin as a key factor for remodeling of the X chromosome from an inactive to an active structure and that this is a critical step for XCR during iPSC-reprogramming.


Assuntos
Células-Tronco Pluripotentes Induzidas , Feminino , Animais , Camundongos , Reprogramação Celular , Inativação do Cromossomo X/genética , Cromossomo X/genética , Estruturas Cromossômicas , Coesinas
2.
Dev Neurosci ; 43(3-4): 253-261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33940579

RESUMO

Brain development is a complex process that requires a series of precise and coordinated events to take place. When alterations in some of those events occur, neurodevelopmental disorders (NDDs) may appear, with their characteristic symptoms, including cognitive, social motor deficits, and epilepsy. While pharmacologic treatments have been the only therapeutic options for many years, more recently the research is turning to the direct removal of the underlying genetic cause of each specific NDD. This is possible thanks to the increased knowledge of genetic basis of those diseases and the enormous advances in genome-editing tools. Together with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based strategies, there is a great development also of nuclease defective Cas9 (dCas9) tools that, with an extreme flexibility, allow the recruitment of specific protein functions to the desired genomic sites. In this work, we review dCas9-based tools and discuss all the published applications in the setting of therapeutic approaches for NDDs at the preclinical level. In particular, dCas9-based therapeutic strategies for Dravet syndrome, transcallosal dysconnectivity caused by mutations in C11orf46 gene, and Fragile X syndrome are presented and discussed. A direct comparison with other possible therapeutic strategies, such as classic gene replacement or CRISPR/Cas9-based strategies, is provided. We also highlight not only those aspects that constitute a clear advantage compared to previous strategies but also the main technical hurdles related to their applications that need to be overcome.


Assuntos
Sistemas CRISPR-Cas , Transtornos do Neurodesenvolvimento , Sistemas CRISPR-Cas/genética , Edição de Genes , Terapia Genética , Humanos , Mutação , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/terapia
3.
Mol Ther ; 28(1): 235-253, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31607539

RESUMO

Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.


Assuntos
Proteína 9 Associada à CRISPR/genética , Epilepsias Mioclônicas/terapia , Terapia Genética/métodos , Interneurônios/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Convulsões/terapia , Ativação Transcricional , Potenciais de Ação , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Neurônios GABAérgicos/metabolismo , Hipocampo/citologia , Hipocampo/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Resultado do Tratamento
4.
Nanotoxicology ; 9(6): 729-36, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25325157

RESUMO

Amorphous silica nanoparticles (SiO2-NPs) have been studied for their toxic and genotoxic potential. Although contradictory data have been reported and the possible modes of action are not fully elucidated, aneugenic events have been reported, indicating the microtubule (MT) network as a potential target. To investigate this, we examined the effects of 59 nm (10 µg/ml) and 174 nm (7.5 µg/ml) SiO2-NPs on MTs in mitotic and interphase A549 human lung carcinoma cells. No gross morphological changes of the mitotic spindle or induction of multipolar spindles were observed upon SiO2-NPs treatment. The influence of SiO2-NPs on the interphase MTs network dynamics was investigated by in situ depolymerisation/repolymerisation experiments. Results showed a clear increase in MT dynamics after SiO2-NP treatment. Consistent with this, reduced levels of MT acetylation were observed. In addition, live cell microscopy demonstrated that SiO2-NP treatment reduced A549 cell motility. The SiO2-NP doses and conditions (serum-free) used in this study did not induce significant cell toxicity or MN frequencies. Therefore, the effects on MT dynamics, MT acetylation and migration observed, are direct effects of the SiO2-NPs and not a consequence of NP overload or toxic or genotoxic effects.


Assuntos
Movimento Celular/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Nanopartículas/toxicidade , Dióxido de Silício/toxicidade , Acetilação , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência , Microscopia de Vídeo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Nanopartículas/química , Tamanho da Partícula , Dióxido de Silício/química , Fuso Acromático/metabolismo , Propriedades de Superfície
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