Nsun5 in mammalian healthy lifespan

Unsere Forschungsgruppe konnte bereits zeigen, dass der Verlust von Nsun5 die Lebensspanne von einfachen Modellorganismen wie Fliegen, Würmern und Hefe erhöhen kann und diese Lebewesen stressresistenter macht. Die Folgen eines Nsun5-Knockouts auf die Alterung von Säugetieren wurden bisher jedoch noch nicht erforscht. In der vorliegenden Arbeit untersuchten wir die Gesundheitsspanne von Nsun5-Knockout-Mäusen und konzentrierten uns auf verschiedene Organsysteme und Körperfunktionen, die potenziell von der Alterung betroffen sind.Wir fanden heraus, dass Nsun5-Knockout-Mäuse eine verminderte Gebrechlichkeit („Frailty“) aufweisen, was darauf hindeutet, dass sie weniger anfällig für gesundheitliche Beeinträchtigungen im Alter sind. Ungeachtet dessen, waren die Lymphozyten und weißen Blutkörperchen bei Nsun5-Knockout-Mäusen reduziert. Allerdings zeigte keiner der anderen Phänotypisierungstests einen signifikanten Unterschied zwischen Wildtyp- und Knockout-Mäusen. In Übereinstimmung mit früheren Ergebnissen, stellten wir bei Nsun5-Knockout-Mäusen mittleren Alters ein vermindertes Körpergewicht fest. In vivo wurde Nsun5 in proliferierenden Zellen im Vergleich zu nicht-proliferierenden Zellen stark exprimiert, was die Bedeutung dieses Enzyms für die Proliferation unterstreicht. Auf Gewebe-Ebene stellten wir eine altersbedingte Abnahme der Nsun5-mRNA in Wildtyp-Mäusen fest, während der Methylierungszustand des entsprechenden Cytosins auf der 28S rRNA unverändert blieb. Diese Ergebnisse werden zur Charakterisierung von Nsun5 in Säugetieren beitragen und verdeutlichen die Auswirkungen einer einzelnen rRNA-Modifikation auf Gesundheit und Alterung.Im Rahmen eines Nebenprojekts wurde die Raman-Mikrospektroskopie in unserer Forschungsgruppe etabliert und auf zwei verschiedene wissenschaftliche Fragestellungen angewandt. Eine umfassende Literaturrecherche zu den aktuellen Anwendungen dieser spektroskopischen Technik in der Alterungsforschung wurde publiziert.Our research group previously showed that loss of Nsun5 can increase the lifespan of simple model organisms, like flies, worms, and yeast and that lack of this rRNA methyltransferase renders them more stress-resistant. However, the consequences of Nsun5 knockout on mammalian aging have not been investigated so far. In the present study, we assessed the healthspan of Nsun5 knockout mice, focusing on several organ systems and physical functions that are potentially affected by aging.We found that Nsun5 knockout mice show reduced frailty, suggesting to be less prone to adverse health outcomes at old age. Despite this, lymphocytes and white blood cells were reduced in Nsun5 knockout mice, but none of the other phenotyping assays revealed a significant difference between wildtype and knockout mice. In line with previous findings, we found decreased body weight in middle-aged Nsun5 knockout mice. In vivo, Nsun5 was highly expressed in proliferating cells compared to non-proliferating cells, underlining the importance of this enzyme for proliferation. On the tissue level, we characterized the expression and activity of Nsun5 in wildtype mice, revealing an age-related reduction of Nsun5 mRNA, while the methylation state of the respective cytosine on 28S rRNA remained unaffected. These findings will contribute to the characterization of Nsun5 in mammalian organisms, emphasizing the impact of a single rRNA modification on health and aging.In the course of another project, Raman microspectroscopy was established in our research group and applied to two different scientific questions. Comprehensive literature research on the current applications of this spectroscopic technique in aging research resulted in a publication.submitted by Lisa LiendlDissertation Universität für Bodenkultur Wien 2022Mit deutscher Zusammenfassun

Nsun5 in mammalian healthy lifespan

Unsere Forschungsgruppe konnte bereits zeigen, dass der Verlust von Nsun5 die Lebensspanne von einfachen Modellorganismen wie Fliegen, Würmern und Hefe erhöhen kann und diese Lebewesen stressresistenter macht. Die Folgen eines Nsun5-Knockouts auf die Alterung von Säugetieren wurden bisher jedoch noch nicht erforscht. In der vorliegenden Arbeit untersuchten wir die Gesundheitsspanne von Nsun5-Knockout-Mäusen und konzentrierten uns auf verschiedene Organsysteme und Körperfunktionen, die potenziell von der Alterung betroffen sind.Wir fanden heraus, dass Nsun5-Knockout-Mäuse eine verminderte Gebrechlichkeit („Frailty“) aufweisen, was darauf hindeutet, dass sie weniger anfällig für gesundheitliche Beeinträchtigungen im Alter sind. Ungeachtet dessen, waren die Lymphozyten und weißen Blutkörperchen bei Nsun5-Knockout-Mäusen reduziert. Allerdings zeigte keiner der anderen Phänotypisierungstests einen signifikanten Unterschied zwischen Wildtyp- und Knockout-Mäusen. In Übereinstimmung mit früheren Ergebnissen, stellten wir bei Nsun5-Knockout-Mäusen mittleren Alters ein vermindertes Körpergewicht fest. In vivo wurde Nsun5 in proliferierenden Zellen im Vergleich zu nicht-proliferierenden Zellen stark exprimiert, was die Bedeutung dieses Enzyms für die Proliferation unterstreicht. Auf Gewebe-Ebene stellten wir eine altersbedingte Abnahme der Nsun5-mRNA in Wildtyp-Mäusen fest, während der Methylierungszustand des entsprechenden Cytosins auf der 28S rRNA unverändert blieb. Diese Ergebnisse werden zur Charakterisierung von Nsun5 in Säugetieren beitragen und verdeutlichen die Auswirkungen einer einzelnen rRNA-Modifikation auf Gesundheit und Alterung.Im Rahmen eines Nebenprojekts wurde die Raman-Mikrospektroskopie in unserer Forschungsgruppe etabliert und auf zwei verschiedene wissenschaftliche Fragestellungen angewandt. Eine umfassende Literaturrecherche zu den aktuellen Anwendungen dieser spektroskopischen Technik in der Alterungsforschung wurde publiziert.Our research group previously showed that loss of Nsun5 can increase the lifespan of simple model organisms, like flies, worms, and yeast and that lack of this rRNA methyltransferase renders them more stress-resistant. However, the consequences of Nsun5 knockout on mammalian aging have not been investigated so far. In the present study, we assessed the healthspan of Nsun5 knockout mice, focusing on several organ systems and physical functions that are potentially affected by aging.We found that Nsun5 knockout mice show reduced frailty, suggesting to be less prone to adverse health outcomes at old age. Despite this, lymphocytes and white blood cells were reduced in Nsun5 knockout mice, but none of the other phenotyping assays revealed a significant difference between wildtype and knockout mice. In line with previous findings, we found decreased body weight in middle-aged Nsun5 knockout mice. In vivo, Nsun5 was highly expressed in proliferating cells compared to non-proliferating cells, underlining the importance of this enzyme for proliferation. On the tissue level, we characterized the expression and activity of Nsun5 in wildtype mice, revealing an age-related reduction of Nsun5 mRNA, while the methylation state of the respective cytosine on 28S rRNA remained unaffected. These findings will contribute to the characterization of Nsun5 in mammalian organisms, emphasizing the impact of a single rRNA modification on health and aging.In the course of another project, Raman microspectroscopy was established in our research group and applied to two different scientific questions. Comprehensive literature research on the current applications of this spectroscopic technique in aging research resulted in a publication.submitted by Lisa LiendlDissertation Universität für Bodenkultur Wien 2022Mit deutscher Zusammenfassun

Organotypic human skin culture models constructed with senescent fibroblasts show hallmarks of skin aging

AbstractSkin aging is driven by intrinsic and extrinsic factors impacting on skin functionality with progressive age. One factor of this multifaceted process is cellular senescence, as it has recently been identified to contribute to a declining tissue functionality in old age. In the skin, senescent cells have been found to markedly accumulate with age, and thus might impact directly on skin characteristics. Especially the switch from young, extracellular matrix-building fibroblasts to a senescence-associated secretory phenotype (SASP) could alter the microenvironment in the skin drastically and therefore promote skin aging. In order to study the influence of senescence in human skin, 3D organotypic cultures are a well-suited model system. However, only few “aged” skin- equivalent (SE) models are available, requiring complex and long-term experimental setups. Here, we adapted a previously published full-thickness SE model by seeding increasing ratios of stress-induced premature senescent versus normal fibroblasts into the collagen matrix, terming these SE “senoskin”. Immunohistochemistry stainings revealed a shift in the balance between proliferation (Ki67) and differentiation (Keratin 10 and Filaggrin) of keratinocytes within our senoskin equivalents, as well as partial impairment of skin barrier function and changed surface properties. Monitoring of cytokine levels of known SASP factors confirmedly showed an upregulation in 2D cultures of senescent cells and at the time of seeding into the skin equivalent. Surprisingly, we find a blunted response of cytokines in the senoskin equivalent over time during 3D differentiation.</jats:p

Cyclosporin A toxicity on endothelial cells differentiated from induced pluripotent stem cells:Assembling an adverse outcome pathway

Cyclosporin A (CSA) is a potent immunosuppressive agent in pharmacologic studies. However, there is evidence for side effects, specifically in regard to vascular dysfunction. Its mode of action inducing endothelial cell toxicity is partially unclear, and a connection with an adverse outcome pathway (AOP) is not established yet. Therefore, we designed this study to get deeper insights into the mechanistic toxicology of CSA on angiogenesis. Stem cells, especially induced pluripotent stem cells (iPSCs) with the ability of differentiation to all organs of the body, are considered a promising in vitro model to reduce animal experimentation. In this study, we differentiated iPSCs to endothelial cells (ECs) as one cell type that in other studies would allow to generate cells or organoids from single donors. Flow cytometry and immunostaining confirmed our scalable differentiation protocol. Then dose and time course experiments assessing CSA cytotoxicity on iPS derived endothelial cells were performed. Transcriptomic data suggested CDA dependent induction of reactive oxygen species (ROS) and mitochondrial dysfunction, which was confirmed by in vitro experiments. Additionally, CSA impaired angiogenesis via ROS induction. Finally, we combined this information into an AOP, was developed based on here observed and literature based evidence for CSA-mediated endothelial cell toxicity. This AOP will help to design in vitro test batteries, model events observed in human toxicity studies, as well for predictive toxicology.</p

Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology