Final Report Summary - MISTRAL (MITOCHONDRIAL STEROIDOGENESIS in the ADRENAL)
Our lab has established several mutant strains of steroidogenesis with the TALEN genome editing approach to study their functional relevance during embryonic development and in adult life. This includes a mutant fdx1b line, which is the zebrafish equivalent of the mammalian fdx1 gene responsible for the synthesis of pregnenolone, a precursor of steroidogenic hormones. We have first studied the effects of fdx1b deletion on steroidogenesis and have shown that mutant embryos are darker due to a failure in their so-called Visual Background Adaptation behavior, a cortisol mediated pigmentation response in teleosts (1). In line with this observation, we detected a massive impact on the stress response and cortisol synthesis in mutant embryos. In addition, the mutants show higher levels of pomca expression, which explains the pigmentation phenotype in these embryos. Interestingly, hyperpigmentation due to cortisol deficiency can also be observed in patients suffering, for example, from Addison disease. The outcomes of our study were published at the beginning of this year in Endocrinology (2) and was highlighted in a News & Views article in Endocrinology (3).
After characterizing the fdx1b mutant line as a model for glucocorticoid deficiency, we next aimed to understand the effects of fdx1b mediated cortisol depletion on a global transcriptome level. This approach aims to pinpoint severe changes in biological pathways that might be linked to pathogenesis. Therefore, we carried out a next-generation sequencing approach. Our results revealed enrichment of genes playing a role for metabolism and metabolic disorders. This was interesting, as patients suffering from adrenal insufficiency reveal metabolic symptoms including weight loss, muscular weakness and hypoglycemia4. However, little is known about the metabolic pathways impaired due to cortisol deficiency and that lead to metabolic alterations. Our transcriptome study showed that several genes involved in the synthesis of biomolecules (e.g. nucleotides, amino acids) and also the generation of energy (e.g. glycolysis, citrate cycle) are altered in the cortisol deficient fdx1b mutants. Interestingly, we also observed an enrichment of oxidative stress genes and of oxidative stress responsive genes in fdx1b mutant embryos, suggesting higher oxidative stress levels in this mutant. Alterations in oxidative stress have been previously linked to complications in diabetes (5) and might explain the observed pathogenesis in patients suffering from adrenal insufficiencies. Importantly, many of these observed gene expression changes were rescued by glucocorticoid replacement, further supporting the important role of glucocorticoids for the correct function of these metabolic pathways. In parallel to our transcriptome approach, we have also carried out metabolic profiling using NMR and Mass spec in order to test whether the observed gene expression changes result in alterations on a metabolite level. Ours studies verified the observations gained with our transcriptomic approach. We overserved changes in glutamine, a precursor metabolite for several metabolic pathways, which were enriched in the fdx1b mutant, and also in the oxidative stress markers glutathione/ glutathione disulfide. The combination of our global transcriptome study and metabolic profiling allowed us to get a detailed understanding of the pathogenic alterations of fdx1b mediated cortisol deficiency on metabolism. These results are summarized in a manuscript (6).
We have also investigated the expression and function of fdx1b in the adult brain. Our own PCR experiments have revealed a strong expression of the fdx1 genes in the adult zebrafish brain, indicating that these genes have an important role. This is particularly interesting, because many aspects of the role of steroids in the adult brain remain elusive. Therefore, we have characterized the spatial expression of several steroidogenic genes (cyp11a1, cyp11a2, cyp11c1, cyp17a1, cyp17a2, fdx1, fdx1b, cyp21a2, hsd3b1, hsd3b2) in the adult zebrafish brain using whole-mount in situ hybridization. We have demonstrated that almost all examined genes are expressed in the zebrafish brain. Interestingly, expression can be also observed in the so-called ventricular zone of the brain, a region with a high regenerative capacity (7). A manuscript summarizing the outcomes of this study is currently in preparation and will be soon submitted for publication (8).
After characterizing the fdx1b mutant line as a model for glucocorticoid deficiency, we next aimed to understand the effects of fdx1b mediated cortisol depletion on a global transcriptome level. This approach aims to pinpoint severe changes in biological pathways that might be linked to pathogenesis. Therefore, we carried out a next-generation sequencing approach. Our results revealed enrichment of genes playing a role for metabolism and metabolic disorders. This was interesting, as patients suffering from adrenal insufficiency reveal metabolic symptoms including weight loss, muscular weakness and hypoglycemia4. However, little is known about the metabolic pathways impaired due to cortisol deficiency and that lead to metabolic alterations. Our transcriptome study showed that several genes involved in the synthesis of biomolecules (e.g. nucleotides, amino acids) and also the generation of energy (e.g. glycolysis, citrate cycle) are altered in the cortisol deficient fdx1b mutants. Interestingly, we also observed an enrichment of oxidative stress genes and of oxidative stress responsive genes in fdx1b mutant embryos, suggesting higher oxidative stress levels in this mutant. Alterations in oxidative stress have been previously linked to complications in diabetes (5) and might explain the observed pathogenesis in patients suffering from adrenal insufficiencies. Importantly, many of these observed gene expression changes were rescued by glucocorticoid replacement, further supporting the important role of glucocorticoids for the correct function of these metabolic pathways. In parallel to our transcriptome approach, we have also carried out metabolic profiling using NMR and Mass spec in order to test whether the observed gene expression changes result in alterations on a metabolite level. Ours studies verified the observations gained with our transcriptomic approach. We overserved changes in glutamine, a precursor metabolite for several metabolic pathways, which were enriched in the fdx1b mutant, and also in the oxidative stress markers glutathione/ glutathione disulfide. The combination of our global transcriptome study and metabolic profiling allowed us to get a detailed understanding of the pathogenic alterations of fdx1b mediated cortisol deficiency on metabolism. These results are summarized in a manuscript (6).
We have also investigated the expression and function of fdx1b in the adult brain. Our own PCR experiments have revealed a strong expression of the fdx1 genes in the adult zebrafish brain, indicating that these genes have an important role. This is particularly interesting, because many aspects of the role of steroids in the adult brain remain elusive. Therefore, we have characterized the spatial expression of several steroidogenic genes (cyp11a1, cyp11a2, cyp11c1, cyp17a1, cyp17a2, fdx1, fdx1b, cyp21a2, hsd3b1, hsd3b2) in the adult zebrafish brain using whole-mount in situ hybridization. We have demonstrated that almost all examined genes are expressed in the zebrafish brain. Interestingly, expression can be also observed in the so-called ventricular zone of the brain, a region with a high regenerative capacity (7). A manuscript summarizing the outcomes of this study is currently in preparation and will be soon submitted for publication (8).