Assuntos
Separação Celular/métodos , Retina/embriologia , Células Horizontais da Retina/citologia , Animais , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Centrifugação com Gradiente de Concentração , Embrião de Galinha , Proteínas de Homeodomínio/metabolismo , Células Horizontais da Retina/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
PURPOSE: The apoptosis of retinal neurons plays a critical role in the pathogenesis of diabetic retinopathy (DR), but the molecular mechanisms underlying this phenomenon remain unclear. The purpose of this study was to investigate the cellular localization and the expression of microRNA-29b (miR-29b) and its potential target PKR associated protein X (RAX), an activator of the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway, in the retina of normal and diabetic rats. METHODS: Retinas were obtained from normal and diabetic rats within 35 days after streptozotocin (STZ) injection. In silico analysis indicated that RAX is a potential target of miR-29b. The cellular localization of miR-29b and RAX was assessed by in situ hybridization and immunofluorescence, respectively. The expression levels of miR-29b and RAX mRNA were evaluated by quantitative reverse transcription PCR (qRT-PCR), and the expression of RAX protein was evaluated by western blot. A luciferase reporter assay and inhibition of endogenous RAX were performed to confirm whether RAX is a direct target of miR-29b as predicted by the in silico analysis. RESULTS: We found that miR-29b and RAX are localized in the retinal ganglion cells (RGCs) and the cells of the inner nuclear layer (INL) of the retinas from normal and diabetic rats. Thus, the expression of miR-29b and RAX, as assessed in the retina by quantitative RT-PCR, reflects their expression in the RGCs and the cells of the INL. We also revealed that RAX protein is upregulated (more than twofold) at 3, 6, 16, and 22 days and downregulated (70%) at 35 days, whereas miR-29b is upregulated (more than threefold) at 28 and 35 days after STZ injection. We did not confirm the computational prediction that RAX is a direct target of miR-29b. CONCLUSIONS: Our results suggest that RAX expression may be indirectly regulated by miR-29b, and the upregulation of this miRNA at the early stage of STZ-induced diabetes may have a protective effect against the apoptosis of RGCs and cells of the INL by the pro-apoptotic RNA-dependent protein kinase (PKR) signaling pathway.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Proteínas de Homeodomínio/metabolismo , MicroRNAs/metabolismo , Células Ganglionares da Retina/metabolismo , Células Horizontais da Retina/metabolismo , Transdução de Sinais/genética , eIF-2 Quinase/metabolismo , Animais , Apoptose/genética , Western Blotting , Diabetes Mellitus Experimental/genética , Retinopatia Diabética/genética , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Homeodomínio/genética , Hibridização In Situ , Luciferases/análise , Masculino , MicroRNAs/genética , Ratos , Ratos Wistar , Células Ganglionares da Retina/citologia , Células Horizontais da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , eIF-2 Quinase/genéticaRESUMO
Many animal species make use of ultraviolet (UV) light in a number of behaviors, such as feeding and mating. The goldfish (Carassius auratus) is among those with a UV photoreceptor and pronounced UV sensitivity. Little is known, however, about the retinal processing of this input. We addressed this issue by recording intracellularly from second-order neurons in the adult goldfish retina. In order to test whether cone-driven horizontal cells (HCs) receive UV cone inputs, we performed chromatic adaptation experiments with mono- and biphasic HCs. We found no functional evidence of a projection from the UV-sensitive cones to these neurons in adult animals. This suggests that goldfish UV receptors may contact preferentially triphasic HCs, which is at odds with the hypothesis that all cones contact all cone-driven HC types. However, we did find evidence of direct M-cone input to monophasic HCs, favoring the idea that cone-HC contacts are more promiscuous than originally proposed. Together, our results suggest that either UV cones have a more restricted set of post-synaptic partners than the other three cone types, or that the UV input to mono- and biphasic HCs is not very pronounced in adult animals.