TU Darmstadt / ULB / TUbiblio

Modulation of autophagy impacts the fate of senescent skin fibroblasts

Som, Gregory (2021):
Modulation of autophagy impacts the fate of senescent skin fibroblasts. (Publisher's Version)
Darmstadt, Technische Universität Darmstadt,
DOI: 10.26083/tuprints-00017861,
[Ph.D. Thesis]

Abstract

Skin aging is accompanied by an accumulation of senescent cells, which promote a low chronic inflammation and degradation of the extracellular matrix. Autophagy is a conserved biological process involved in cellular homeostasis through the recycling of long-lived or damaged proteins and organelles. Impaired during aging, it has been speculated that autophagy could be involved in senescence. The role of autophagy in the context of cellular senescence remains elusive and is not understood yet, seemingly dependent on the cell type, tissue, and manner in which senescence is induced. Why damaged cells resort to senescence instead of apoptosis remains a key question in the understanding of aging and age-related pathologies.

In this work, 2D-LC-MS/MS labeling-assisted proteomics identified 230 upregulated and 228 downregulated proteins in senescent normal human dermal fibroblasts (NHDF). When upregulated proteins were subjected to a gene set enrichment analysis (GSEA), 27autophagy-related biological processes were found enriched, showing that stress-induced premature senescence (SIPS) leads to an increased autophagic activity in senescent NHDF. This included biological processes such as lysosome biogenesis, mTOR signaling and macroautophagy. Although autophagy is increased in SIPS, whether it is involved in senescence development or a consequence remained to be clarified. In WS1 human fibroblasts, rapamycin-mediated stimulation of autophagic flux prior to SIPS induction delayed the onset of senescence, whereas its inhibition through chloroquine showed no effect. After SIPS induction, stimulation of autophagy delayed the onset of senescence, whereas its inhibition changed the cell fate from senescence to cell death. The latter observation was further shown to be specific to senescent WS1, suggesting the existence of an autophagy-mediated “apoptotic switch”. Although the exact molecular mechanism of this switch remains to be determined, this work implicates mitochondrial abundance and/or mass as well as calcium signaling as part of the process. These data highlighted a dual beneficial/detrimental role of autophagy in skin fibroblasts senescence and thus its modulation is a potential target for age-related pathologies. In this regard, carbohydrates and derivatives have been tested for the ability to modulate autophagy. Whereas raffinose, sucrose, isomaltulose, sorbitol and methyl-α-glucopyranoside could be use as novel mTOR- independent activators of autophagy, trehalose could have an inhibitory effect. Aside from autophagy, GSEA allowed the identification of approximately 40 other biological processes significantly enriched in senescent NHDF, some of which could directly be involved in senescent fibroblasts immortality. Selection of four upregulated candidates involved in (I) alcohol metabolism (APOL2), (II) fatty acid metabolism (CES2), (III) response to hypoxia (MGARP) and (IV) regulation of apoptotic signaling (PTTG1IP) allowed to validate their role in senescent cell immortality, as the siRNA-mediated silencing of these 4 proteins led to cell death.

The understanding of molecular mechanisms involved in cellular senescence, autophagy, and apoptosis becomes increasingly important in aging and cancer research. Sufficient understanding how they regulate each other could provide novel therapeutic opportunities for the specific elimination of senescent skin fibroblasts. Current biological markers used to detect senescent cells in vitro and in vivo are limited and lack specificity. Thus, identification of novels senescent markers would have diagnostic and therapeutic potential.

Item Type: Ph.D. Thesis
Erschienen: 2021
Creators: Som, Gregory
Status: Publisher's Version
Title: Modulation of autophagy impacts the fate of senescent skin fibroblasts
Language: English
Abstract:

Skin aging is accompanied by an accumulation of senescent cells, which promote a low chronic inflammation and degradation of the extracellular matrix. Autophagy is a conserved biological process involved in cellular homeostasis through the recycling of long-lived or damaged proteins and organelles. Impaired during aging, it has been speculated that autophagy could be involved in senescence. The role of autophagy in the context of cellular senescence remains elusive and is not understood yet, seemingly dependent on the cell type, tissue, and manner in which senescence is induced. Why damaged cells resort to senescence instead of apoptosis remains a key question in the understanding of aging and age-related pathologies.

In this work, 2D-LC-MS/MS labeling-assisted proteomics identified 230 upregulated and 228 downregulated proteins in senescent normal human dermal fibroblasts (NHDF). When upregulated proteins were subjected to a gene set enrichment analysis (GSEA), 27autophagy-related biological processes were found enriched, showing that stress-induced premature senescence (SIPS) leads to an increased autophagic activity in senescent NHDF. This included biological processes such as lysosome biogenesis, mTOR signaling and macroautophagy. Although autophagy is increased in SIPS, whether it is involved in senescence development or a consequence remained to be clarified. In WS1 human fibroblasts, rapamycin-mediated stimulation of autophagic flux prior to SIPS induction delayed the onset of senescence, whereas its inhibition through chloroquine showed no effect. After SIPS induction, stimulation of autophagy delayed the onset of senescence, whereas its inhibition changed the cell fate from senescence to cell death. The latter observation was further shown to be specific to senescent WS1, suggesting the existence of an autophagy-mediated “apoptotic switch”. Although the exact molecular mechanism of this switch remains to be determined, this work implicates mitochondrial abundance and/or mass as well as calcium signaling as part of the process. These data highlighted a dual beneficial/detrimental role of autophagy in skin fibroblasts senescence and thus its modulation is a potential target for age-related pathologies. In this regard, carbohydrates and derivatives have been tested for the ability to modulate autophagy. Whereas raffinose, sucrose, isomaltulose, sorbitol and methyl-α-glucopyranoside could be use as novel mTOR- independent activators of autophagy, trehalose could have an inhibitory effect. Aside from autophagy, GSEA allowed the identification of approximately 40 other biological processes significantly enriched in senescent NHDF, some of which could directly be involved in senescent fibroblasts immortality. Selection of four upregulated candidates involved in (I) alcohol metabolism (APOL2), (II) fatty acid metabolism (CES2), (III) response to hypoxia (MGARP) and (IV) regulation of apoptotic signaling (PTTG1IP) allowed to validate their role in senescent cell immortality, as the siRNA-mediated silencing of these 4 proteins led to cell death.

The understanding of molecular mechanisms involved in cellular senescence, autophagy, and apoptosis becomes increasingly important in aging and cancer research. Sufficient understanding how they regulate each other could provide novel therapeutic opportunities for the specific elimination of senescent skin fibroblasts. Current biological markers used to detect senescent cells in vitro and in vivo are limited and lack specificity. Thus, identification of novels senescent markers would have diagnostic and therapeutic potential.

Place of Publication: Darmstadt
Collation: 122 Seiten
Divisions: 07 Department of Chemistry
07 Department of Chemistry > Fachgebiet Biochemie
07 Department of Chemistry > Fachgebiet Biochemie > Allgemeine Biochemie
Date Deposited: 07 Apr 2021 08:30
DOI: 10.26083/tuprints-00017861
Official URL: https://tuprints.ulb.tu-darmstadt.de/17861
URN: urn:nbn:de:tuda-tuprints-178616
Referees: Kolmar, Prof. Dr. Harald ; Hagen, Prof. Dr. Jörg von
Refereed / Verteidigung / mdl. Prüfung: 7 September 2020
Alternative Abstract:
Alternative abstract Language

Hautalterung geht mit der Akkumulation von seneszenten Zellen einher, welche eine chronische Entzündung und den Abbau der Extrazellulären Matrix fördern. Autophagie ist ein speziesübergreifend konservierter Prozess, der durch den Abbau von alten oder beschädigten Proteinen und Organellen zur Instandhaltung zellulärer Homöostase beiträgt. Da dieser Prozess im Zuge des Alterns beeinträchtigt ist, wird angenommen, dass Autophagie eine Rolle bei der Entwicklung von Seneszenz spielt. Der Zusammenhang von Autophagie und zellulärer Seneszenz ist derzeit unzureichend verstanden und scheint vom Zelltyp, Gewebetyp und der Art und Weise, wie Seneszenz in Modellsystemen initiiert wird, abzuhängen. Warum beschädigte Zellen Seneszenz anstelle von Apoptose einleiten bleibt eine der Hauptfragestellungen in der Erforschung von altersbedingten Krankheiten.

In dieser Arbeit wurden mittels 2D-LC-MS/MS labeling-assisted proteomics 230 hochregulierte Proteine und 228 herunterregulierte Proteine in seneszenten normalen humanen dermalen Fibroblasten (NHDF) identifiziert. Unter den hochregulierten Proteinen wurden mittels gene set enrichment analysis (GSEA) 27 biologische Prozesse identifiziert, die möglicherweise im direkten Zusammenhang mit Autophagie stehen. Unter anderem waren die Lysosomen Biogenese, mTOR Signalwege und Makroautophagie erhöht. Dies zeigt, dass Stress-induzierte Seneszenz (SIPS) zu einer Erhöhung des Autophagie Flux in NHDF führt. Zur Untersuchung der Frage, ob die Erhöhung des Autophagie Flux eine Rolle in der Entwicklung von Seneszenz spielt oder eine Folge dieser ist, wurden mehrere Experimente durchgeführt. Die Stimulierung des Autophagie-Flux in humanen WS1 Fibroblasten mittels Rapamycin vor der Induktion von SIPS konnte diese verzögern, wohingegen die Blockierung des Autophagie Flux keinen Effekt zeigte. Die Erhöhung des Autophagie-Flux nach der Induktion von SIPS verzögerte ihre Ausbildung, wobei die Blockierung zum Absterben der Zellen führte. Es wurde gezeigt, dass dies spezifisch seneszente Zellen betrifft, was auf eine Autophagie-bedingte Reprogrammierung zur Apoptose hindeutet. Erste Daten belegen, dass die Anzahl an Mitochondrien und oder Calcium Signalwege eine Rolle spielen. Der genaue molekulare Mechanismus muss allerdings noch aufgeklärt werden. Die in dieser Arbeit erhobenen Daten weisen auf eine duale gegenläufige Rolle der Autophagie bei der Entwicklung von Seneszenz in Hautfibroblasten hin. Siezeigt, dass die Modulation von Autophagie einen potenziellen Angriffspunkt zur Bekämpfung von altersbedingten Krankheiten ist. In dieser Arbeit wurden außerdem mehrere Kohlenhydrate und deren Derivate auf die Fähigkeit getestet Autophagie zu beeinflussen. Raffinose, Sucrose, Isomaltulose, Sorbitol und Methyl-a-glucopyranosid wurden als neue mTOR-unabhängige Aktivatoren des Autophagie-Flux identifiziert, wohingegen Trehalose einen inhibierenden Effekt zeigte. Neben Autophagie wurden mittels GSEA ca. 40 weitere biologische Prozesse identifiziert, die in seneszenten NHDF signifikant verstärkt reguliert werden. siRNA vermitteltes Silencing von vier hochregulierten Proteinen führte zum Zelltod. Damit konnte gezeigt werden, dass (I) der Alkohol Metabolismus (APOL2), (II) der

Fettsäuremetabolismus (CES2), (III) Sauerstoffmangel (MGARP) und die Regulierung des Apoptose Signalwegs (PTTG1IP) eine direkte Rolle bei der Entwicklung von Seneszenz spielen.

Das Verständnis der molekularen Zusammenhänge von Seneszenz, Autophagie und Apoptose werden immer wichtiger in der Alters- und Krebsforschung. Ein Verständnis, wie diese Prozesse sich gegenseitig regulieren, könnte dazu beitragen neue Therapien zur spezifischen Eliminierung von seneszenten Hautfibroblasten zu entwickeln. Derzeit gibt es keine robusten spezifischen biologischen Marker zur Identifizierung von seneszenten Zellen in vitro und in vivo. Die Identifizierung von neuen Seneszenzmarkern hätte großes diagnostisches und therapeutisches Potential.

German
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