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1.
PLoS One ; 12(9): e0185079, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28945765

RESUMEN

C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.


Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/enzimología , Quinazolinas/farmacología , Animales , Línea Celular , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Atrofia Muscular Espinal/genética , Regiones Promotoras Genéticas , Quinazolinas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/deficiencia , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismo
2.
PLoS One ; 8(2): e56024, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23418498

RESUMEN

We have used a previously unavailable model of pancreatic development, derived in vitro from human embryonic stem cells, to capture a time-course of gene, miRNA and histone modification levels in pancreatic endocrine cells. We investigated whether it is possible to better understand, and hence control, the biological pathways leading to pancreatic endocrine formation by analysing this information and combining it with the available scientific literature to generate models using a casual reasoning approach. We show that the embryonic stem cell differentiation protocol is highly reproducible in producing endocrine precursor cells and generates cells that recapitulate many aspects of human embryonic pancreas development, including maturation into functional endocrine cells when transplanted into recipient animals. The availability of whole genome gene and miRNA expression data from the early stages of human pancreatic development will be of great benefit to those in the fields of developmental biology and diabetes research. Our causal reasoning algorithm suggested the involvement of novel gene networks, such as NEUROG3/E2F1/KDM5B and SOCS3/STAT3/IL-6, in endocrine cell development We experimentally investigated the role of the top-ranked prediction by showing that addition of exogenous IL-6 could affect the expression of the endocrine progenitor genes NEUROG3 and NKX2.2.


Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Redes Reguladoras de Genes , Islotes Pancreáticos/metabolismo , Algoritmos , Animales , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Islotes Pancreáticos/embriología , Ratones , Proteínas Nucleares , Factores de Transcripción
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