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1.
J Biol Chem ; 298(9): 102293, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35868558

RESUMEN

MicroRNA-124a (miR-124a) is one of the most abundantly expressed microRNAs in the central nervous system and is encoded in mammals by the three genomic loci miR-124a-1/2/3; however, its in vivo roles in neuronal development and function remain ambiguous. In the present study, we investigated the effect of miR-124a loss on neuronal differentiation in mice and in embryonic stem (ES) cells. Since miR-124a-3 exhibits only background expression levels in the brain and we were unable to obtain miR-124a-1/2/3 triple knockout (TKO) mice by mating, we generated and analyzed miR-124a-1/2 double knockout (DKO) mice. We found that these DKO mice exhibit perinatal lethality. RNA-seq analysis demonstrated that the expression levels of proneural and neuronal marker genes were almost unchanged between the control and miR-124a-1/2 DKO brains; however, genes related to neuronal synaptic formation and function were enriched among downregulated genes in the miR-124a-1/2 DKO brain. In addition, we found the transcription regulator Tardbp/TDP-43, loss of which leads to defects in neuronal maturation and function, was inactivated in the miR-124a-1/2 DKO brain. Furthermore, Tardbp knockdown suppressed neurite extension in cultured neuronal cells. We also generated miR-124a-1/2/3 TKO ES cells using CRISPR-Cas9 as an alternative to TKO mice. Phase-contrast microscopic, immunocytochemical, and gene expression analyses showed that miR-124a-1/2/3 TKO ES cell lines were able to differentiate into neurons. Collectively, these results suggest that miR-124a plays a role in neuronal maturation rather than neurogenesis in vivo and advance our understanding of the functional roles of microRNAs in central nervous system development.


Asunto(s)
Proteínas de Unión al ADN , MicroARNs , Neurogénesis , Neuronas , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ratones , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Embrionarias de Ratones , Neurogénesis/genética , Neuronas/citología , Neuronas/metabolismo
2.
Exp Eye Res ; 212: 108770, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34562437

RESUMEN

PURPOSE: Cancer-associated retinal ON bipolar cell dysfunction (CARBD), which includes melanoma-associated retinopathy (MAR), has been reported to be caused by autoantibodies against the molecules expressed in ON bipolar cells, including TRPM1. The purpose of this study was to determine the antigenic regions of the autoantibodies against TRPM1 in the sera of CARBD patients, in whom we previously detected anti-TRPM1 autoantibodies. METHODS: The antigenic regions against TRPM1 in the sera of eight CARBD patients were examined by Western blots using HEK293T cells transfected with the plasmids expressing FLAG-tagged TRPM1 fragments. The clinical course of these patients was also documented. RESULTS: The clinical course differed among the patients. The electroretinograms (ERGs) and symptoms were improved in three patients, deteriorated in one patient, remained unchanged for a long time in one patient, and were not followable in three patients. Seven of the eight sera possessed multiple antigenic regions: two sera contained at least four antigen recognition regions, and three sera had at least three regions. The antigen regions were spread over the entire TRPM1 protein: five sera in the N-terminal intracellular domain, six sera in the transmembrane-containing region, and six sera in the C-terminal intracellular domain. No significant relationship was observed between the location of the antigen epitope and the patients' clinical course. CONCLUSIONS: The antigenic regions of anti-TRPM1 autoantibodies in CARBD patients were present not only in the N-terminal intracellular domain, which was reported in an earlier report, but also in the transmembrane-containing region and in the C-terminal intracellular domain. In addition, the antigenic regions for TRPM1 were found to vary among the CARBD patients examined, and most of the sera had multiple antigenic regions.


Asunto(s)
Autoanticuerpos/sangre , Síndromes Paraneoplásicos Oculares/inmunología , Células Bipolares de la Retina/metabolismo , Canales Catiónicos TRPM/inmunología , Anciano , Western Blotting , Electrorretinografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Síndromes Paraneoplásicos Oculares/metabolismo , Síndromes Paraneoplásicos Oculares/patología , Células Bipolares de la Retina/patología , Estudios Retrospectivos , Células Tumorales Cultivadas
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