Untersuchungen zur Lokalisation und Expression des respiratorischen Proteins Neuroglobin bei Säugetieren

In der vorliegenden Arbeit wurde Neuroglobin (Ngb), ein evolutiv altes und in Metazoen konserviertes respiratorisches Protein, funktionell untersucht. Mittels des induzierbaren Tet on / Tet off Systems wurde Ngb ektopisch in der murinen Leber und im Gehirn überexprimiert. Die Transkriptome von Leber und Gehirnregionen Ngb-transgener Mäuse wurden mittels Microarrays und RNA-Seq im Vergleich zum Wildtyp analysiert, um Auswirkungen der Ngb-Überexpression zu ermitteln. Die Transkriptom-Analyse in Leber und Gehirn zeigte eine nur geringe Anzahl differenziell regulierter Gene und Stoffwechselwege nach Ngb-Überexpression. Ngb transgene Mäuse wurden CCl4-induziertem ROS-Stress ausgesetzt und die Leberfunktion untersucht. Zudem wurden primäre Hepatozyten-Kulturen etabliert und in diesen in vitro die extrinsische Apoptose induziert. Die Stressversuche zeigten: (i) Die Ngb-Überexpression hat keine protektive Wirkung in der Leber in vivo. (ii) In Leberzellen in vitro hingegen verminderte eine Ngb-Überexpression effizient die Aktivierung der apoptotischen Kaskade. Eine protektive Wirkung von Ngb ist vermutlich von betrachtetem Gewebe und dem verwendeten Stressor abhängig und keine generelle, selektierte Funktion des Proteins.rnWeiterhin wurde eine Ngb-KnockOut-Mauslinie mit einem LacZ-KnockIn-Genotyp etabliert. Hierbei zeigten die KO-Mäuse keinen offensichtlichen Phänotyp in ihrer Entwicklung, Fortpflanzung und Retina-Funktion. Unter Verwendung des LacZ-Knockin-Konstrukts konnten kontrovers diskutierte Ngb-Expressionsorte im adulten Mausgehirn (Hippocampus, Cortex und Cerebellum) sowie in Testes experimentell bestätigt werden. Parallel wurden öffentlich verfügbare RNA-Seq Datensätze ausgewertet, um die regionale Ngb-Expression systematisch ohne Antikörper-assoziierte Spezifitätsprobleme zu charakterisieren. Eine basale Ngb-Expression (RPKM: ~1-5) wurde im Hippocampus, Cortex und Cerebellum, sowie in Retina und Testes ermittelt. Eine 20-40fach höhere, starke Expression (RPKM: ~160) wurde im Hypothalamus bzw. im Hirnstamm nachgewiesen. Die „digitale“ Expressionsuntersuchung wurde mittels qRT-PCR und Western Blot bestätigt. Dieses Expressionsprofil von Ngb in der Maus weist auf eine besondere funktionelle Bedeutung von Ngb im Hypothalamus hin. Eine Funktion von Ngb in der Sauerstoffversorgung der Retina und eine generelle Funktion von Ngb in der Protektion von Neuronen sind mit dem beobachteten Expressionsspektrum weniger gut vereinbar.In this thesis the function of the metazoan, evolutionary old respiratory protein neuroglobin (Ngb) was analyzed. Using the inducible Tet-on / Tet-off System Ngb was ectopically expressed in murine liver and brain tissues. Ngb-overexpressing transcriptomes of neuronal and hepatic tissues were analyzed to describe the effect of Ngb-overexpression on these tissues compared to wild type. The analysis revealed no formerly in association with Ngb discussed pathways as dysregulated. Ngb-transgenic mice were stressed in vivo with the hepatotoxic CCl4 and the liver-function was assessed. Primary hepatocyte-cultures of Ngb-overexpressing tissues were established and the extrinsic apoptotic pathway was induced in vitro to assess the protective effects of Ngb on liver tissue. The stress regimes showed no protection of the Ngb-expression in vivo from CCl4-induced ROS-damage but an efficient protection through Ngb from activating the extrinsic apoptotic pathway compared to wild type hepatocytes. The reported protective effect of Ngb probably depends on the used stress-regimes and tissues and is not an evolutionary conserved and selected function of Ngb. Furthermore an Ngb-KnockOut and LacZ-KnockIn mouse lines based on the EUCOMM-KnockOut-First-Vector were established. The development, reproduction and retinal function in Ngb-KO mice were analyzed and showed no obvious phenotype. Utilizing the LacZ-reporter mice controversially discussed Ngb expression-sites in the murine brain (hippocampus, cortex and cerebellum) were verified. Analyzing publicly available data the Ngb-expression was re-evaluated using RNA-Seq technique. Here hypothalamus was identified as the main Ngb expression-site in the murine CNS, with a 100x higher expression compared to other regions like cortex, hippocampus, cerebellum and retina a published high Ngb expression-site. This “digital” Ngb-expression was verified using wet lab methods like qRT-PCR and western blot. This new expression profile of Ngb with quantitative differences in the CNS argues with an evolutionary conserved protective function of Ngb and instead hints on different functions of Ngb in low expressing and high expressing cells

The More, the Merrier? Multiple Myoglobin Genes in Fish Species, Especially in Gray Bichir (Polypterus senegalus) and Reedfish (Erpetoichthys calabaricus)

The members of the globin superfamily are a classical model system to investigate gene evolution and their fates as well as the diversity of protein function. One of the best-known globins is myoglobin (Mb), which is mainly expressed in heart muscle and transports oxygen from the sarcolemma to the mitochondria. Most vertebrates harbor a single copy of the myoglobin gene, but some fish species have multiple myoglobin genes. Phylogenetic analyses indicate an independent emergence of multiple myoglobin genes, whereby the origin is mostly the last common ancestor of each order. By analyzing different transcriptome data sets, we found at least 15 multiple myoglobin genes in the polypterid gray bichir (Polypterus senegalus) and reedfish (Erpetoichthys calabaricus). In reedfish, the myoglobin genes are expressed in a broad range of tissues but show very different expression values. In contrast, the Mb genes of the gray bichir show a rather scattered expression pattern; only a few Mb genes were found expressed in the analyzed tissues. Both, gray bichir and reedfish possess lungs which enable them to inhabit shallow and swampy waters throughout tropical Africa with frequently fluctuating and low oxygen concentrations. The myoglobin repertoire probably reflects the molecular adaptation to these conditions. The sequence divergence, the substitution rate, and the different expression pattern of multiple myoglobin genes in gray bichir and reedfish imply different functions, probably through sub- and neofunctionalization during evolution

Transcriptome analysis reveals a high aerobic capacity in the whale brain

The brain of diving mammals is repeatedly exposed to low oxygen conditions (hypoxia) that would have caused severe damage to most terrestrial mammals. Some whales may dive for > 2 h with their brain remaining active. Many of the physiological adaptations of whales to diving have been investigated, but little is known about the molecular mechanisms that enable their brain to survive sometimes prolonged periods of hypoxia. Here, we have used an RNA-Seq approach to compare the mRNA levels in the brains of whales with those of cattle, which serves as a terrestrial relative. We sequenced the transcriptomes of the brains from cattle (Bos taurus), killer whale (Orcinus orca), and long-finned pilot whale (Globicephala melas). Further, the brain transcriptomes of cattle, minke whale (Balaenoptera acutorostrata) and bowhead whale (Balaena mysticetus), which were available in the databases, were included. We found a high expression of genes related to oxidative phosphorylation and the respiratory electron chain in the whale brains. In the visual cortex of whales, transcripts related to the detox- ification of reactive oxygen species were more highly expressed than in the visual cortex of cattle. These findings indicate a high oxidative capacity in the whale brain that might help to maintain aerobic metabolism in periods of reduced oxygen availability during dives

When the brain goes diving: transcriptome analysis reveals a reduced aerobic energy metabolism and increased stress proteins in the seal brain

Background: During long dives, the brain of whales and seals experiences a reduced supply of oxygen (hypoxia). The brain neurons of the hooded seal (Cystophora cristata) are more tolerant towards low-oxygen conditions than those of mice, and also better survive other hypoxia-related stress conditions like a reduction in glucose supply and high concentrations of lactate. Little is known about the molecular mechanisms that support the hypoxia tolerance of the diving brain. Results: Here we employed RNA-seq to approach the molecular basis of the unusual stress tolerance of the seal brain. An Illumina-generated transcriptome of the visual cortex of the hooded seal was compared with that of the ferret (Mustela putorius furo), which served as a terrestrial relative. Gene ontology analyses showed a significant enrichment of transcripts related to translation and aerobic energy production in the ferret but not in the seal brain. Clusterin, an extracellular chaperone, is the most highly expressed gene in the seal brain and fourfold higher than in the ferret or any other mammalian brain transcriptome. The largest difference was found for S100B, a calcium-binding stress protein with pleiotropic function, which was 38-fold enriched in the seal brain. Notably, significant enrichment of S100B mRNA was also found in the transcriptomes of whale brains, but not in the brains of terrestrial mammals. Conclusion: Comparative transcriptomics indicates a lower aerobic capacity of the seal brain, which may be interpreted as a general energy saving strategy. Elevated expression of stress-related genes, such as clusterin and S100B, possibly contributes to the remarkable hypoxia tolerance of the brain of the hooded seal. Moreover, high levels of S100B that possibly protect the brain appear to be the result of the convergent adaptation of diving mammals

Genetic and functional diversity of the multiple lungfish myoglobins

It is known that the West African lungfish (Protopterus annectens) harbours multiple myoglobin (Mb) genes that are differentially expressed in various tissues and that the Mbs differ in their abilities to confer tolerance towards hypoxia. Here, we show that other lungfish species (Protopterus dolloi, Protopterus aethiopicus and Lepidosiren paradoxa) display a similar diversity of Mb genes and have orthologous Mb genes. To investigate the functional diversification of these genes, we studied the structures, O2 binding properties and nitrite reductase enzymatic activities of recombinantly expressed P. annectens Mbs (PanMbs). CD spectroscopy and small‐angle X‐ray scattering revealed the typical globin‐fold in all investigated recombinant Mbs, indicating a conserved structure. The highest O2 affinity was measured for PanMb2 (P50 = 0.88 Torr at 20 °C), which is mainly expressed in the brain, whereas the muscle‐specific PanMb1 has the lowest O2 affinity (P50 = 3.78 Torr at 20 °C), suggesting that tissue‐specific O2 requirements have resulted in the emergence of distinct Mb types. Two of the mainly neuronally expressed Mbs (PanMb3 and PanMb4b) have the highest nitrite reductase rates. These data show different O2 binding and enzymatic properties of lungfish Mbs, reflecting multiple subfunctionalisation and neofunctionalisation events that occurred early in the evolution of lungfish. Some Mbs may have also taken over the functions of neuroglobin and cytoglobin, which are widely expressed in vertebrates but appear to be missing in lungfish

Additional file 4: Table S3. of When the brain goes diving: transcriptome analysis reveals a reduced aerobic energy metabolism and increased stress proteins in the seal brain

Genes overrepresented in the seal brain. Ontology analysis of genes that are at least twofold higher expressed in the visual cortex of the hooded seal compared to the ferret visual cortex. A. The GO terms for the domains “molecular function” and “biological process”, and the “protein class” are given. B and C. PANTHER Overrepresentation Test of the domains “molecular function”, “biological process”, and “protein class” using the complete (B) and PANTHER GO-slim terms. (PDF 24 kb

Additional file 5: Table S4. of When the brain goes diving: transcriptome analysis reveals a reduced aerobic energy metabolism and increased stress proteins in the seal brain

Genes overrepresented in the ferret brain. Ontology analysis of genes that are at least twofold higher expressed in the visual cortex of the ferret compared to the hooded seal visual cortex. A. The GO terms for the domains “molecular function” and “biological process”, and the “protein class” are given. B and C. PANTHER Overrepresentation Test of the domains “molecular function”, “biological process”, and “protein class” using the complete (B) and PANTHER GO-slim terms. (PDF 74 kb

Lilium speciosum Thunb.

原著和名: カノコユリ科名: ユリ科 = Liliaceae採集地: 熊本県 八代郡 坂本村 (肥後 八代郡 坂本村)採集日: 1973/8/5採集者: 萩庭丈壽整理番号: JH014852国立科学博物館整理番号: TNS-VS-96485

Additional file 3: Spreadsheet S1. of When the brain goes diving: transcriptome analysis reveals a reduced aerobic energy metabolism and increased stress proteins in the seal brain

Comparative gene expression in the seal and ferret brain. Gene expression (RPKM values) in the visual cortex of the hooded seal and the ferret. The cut-off was set to RPKM >5 in both species. (XLSX 406 kb