RESUMO
The circadian clock is a highly conserved 24 h biological oscillation mechanism and is affected by environmental stimuli such as light, food and temperature. Disruption of the circadian clock results in disorders of diverse biological processes, including the sleep-wake cycle and metabolism. Although we previously identified several components of the circadian clock in zebrafish, our understanding of the relationship between light-inducible clock genes and metabolism remains incomplete. To investigate how light-inducible clock genes regulate metabolism, we performed transcriptomic and metabolomic analyses of the light-inducible clock genes zPer2, zCry1a, and zCry2a in zebrafish. Transcriptomic analysis of zPer2/zCry1a double knockout (DKO) and zPer2/zCry1a/zCry2a triple knockout (TKO) mutants showed that their gene expression profiles differed from that of wild type (WT) zebrafish. In particular, mRNA levels of zKeap1a, which encodes an oxidative stress sensor, were increased in DKO and TKO mutants. Metabolomic analysis showed genotype-dependent alteration of metabolomic profiles. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) showed the alteration of cysteine/methionine metabolism and glutathione metabolism. Specifically, cysteine and glutathione were decreased but methionine sulfoxide was increased in TKO zebrafish. These results indicate that the light-inducible genes zPer2, zCry1a, and zCry2a are involved in regulating the oxidative status of zebrafish.
Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Criptocromos/genética , Proteínas de Ligação a DNA/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica , Estresse Oxidativo/genética , Proteínas Circadianas Period/genética , Proteínas de Peixe-Zebra/genética , Animais , Cisteína/metabolismo , Perfilação da Expressão Gênica , Glutationa/metabolismo , Luz , Metionina/metabolismo , Modelos Animais , Oxirredução , Análise de Componente Principal , RNA Mensageiro/metabolismo , Transcriptoma , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Autophagy is a dynamic process that degrades subcellular constituents, and its activity is measured by autophagic flux. The tandem proteins RFP-GFP-LC3 and GFP-LC3-RFP-LC3ΔG, which enable the visualization of autophagic vacuoles of different stages by differences in their fluorescent color, are useful tools to monitor autophagic flux, but they require plasmid transfection. In this study, we hence aimed to develop a new method to monitor autophagic flux using small cell-permeable fluorescent probes. We previously developed two green-fluorescent probes, DALGreen and DAPGreen, which detect autolysosomes and multistep autophagic vacuoles, respectively. We here developed a red-fluorescent autophagic probe, named DAPRed, which recognizes various autophagic vacuoles. By the combinatorial use of these green- and red-fluorescent probes, we were able to readily detect autophagic flux. Furthermore, these probes were useful not only for the visualization of canonical autophagy but also for alternative autophagy. DAPRed was also applicable for the detection of autophagy in living organisms.
RESUMO
Patients suffering from neuropsychiatric disorders such as substance use and addiction disorders show impaired decision-making, which may be associated with their psychiatric disorders. Previously, using a gambling test for rodents, we demonstrated that methamphetamine-dependent rats showed alterations in their decision-making strategy. In this study, we investigated the effect of nicotine on impaired decision-making strategy in rats which have been treated repeatedly with methamphetamine. Nicotine has previously been shown to have therapeutic effects on attentional and cognitive abnormalities in psychosis. Rats were administered methamphetamine subcutaneously (sc) at 4â¯mg/kg once a day, for 30 days, and their decision-making was then assessed with a rodent gambling task. We found that methamphetamine-treated rats preferred the high-risk/high-return actions, which is consistent with our previous findings. Methamphetamine-induced impairment of decision-making was reversed by daily nicotine treatment (0.3â¯mg/kg, sc). This effect was associated with the reduction of lose-shift behavior after negative reward prediction error. Repeated treatment with nicotine had no effects on arm-choice behavior in naïve rats. Varenicline, an α4ß2-nicotinic acetylcholine receptor partial agonist, also ameliorated the altered decision-making in methamphetamine-treated rats. Our findings suggest that nicotine treatment is useful for ameliorating the altered decision-making caused by methamphetamine treatment, and that the α4ß2-nicotinic acetylcholine receptor is a therapeutic target for poor decision-making.