Dissertation committee:
Γοργούλης Γ. Βασίλειος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ευαγγέλου Κωνσταντίνος, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Χαβάκη Σοφία, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Κοτσίνας Αθανάσιος, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Λαγοπάτη Νεφέλη, Επίκουρη Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Λουγιάκης Νικόλαος, Επίκουρος Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Πίππα Αναστασία-Γεωργία, Επίκουρη Καθηγήτρια Τμήμα Φαρμακευτικής, ΕΚΠΑ
Summary:
Cellular senescence is a response mechanism to stress stimuli, the activation of which aims to maintain homeostasis. Senescent cells are characterized by a generally permanent arrest of the cell cycle, macromolecular damage, disrupted metabolism, and the secretion of a set of molecules termed SASP. Additionally, the senescent cells accumulate a mixture (an aggregate) consisting of by-products of its metabolism, called lipofuscin, constituting the "dark matter" of the senescent cell. Despite extensive research on cellular senescence field, there is a lack of tools/indicators that can accurately identify, living and fixed, senescent cells. Utilizing GL13, a hydrophobic biotinylated analog of Sudan Black-B (SBB), which accurately detects senescent cells by binding to lipofuscin, we developed the innovative reagent “GLF16”. GLF16 is a chemical analog of SBB coupled with fluorochrome, which, due to its structure, enables dissolution in aqueous solvents and opens a new field of study for senescence. The development of GLF16 reagent coincided with the onset of the COVID-19 pandemic caused by the SARS-CoV-2 virus. During viral infection, an acute inflammatory response is triggered mediated by key molecules such as IL-6, also found in the SASP of senescent cells. Furthermore, infected cells exhibit a similar phenotype to that of senescent cells. In this context, we investigated, using the GL13 and GLF16 reagents, to what extent the virus can induce the phenomenon of senescence during infection. In this frame, we investigated the role of senescent cells via SASP in the cytokine storm that characterize the pathogenesis of COVID-19 and their potential role in SARS-CoV-2 mutagenesis. The GLF16 reagent identified with high sensitivity and specificity fixed senescent cells both in vitro and in vivo in FFPE tissues. Moreover, it recognized fixed senescent cells using a multi-index approach with flow cytometry and FACS. The encapsulation of GLF16 into a mycellar nanocarrier allowed for the precise identification and analysis of live senescent cells with FACS, their analysis at the RNA and epigenome levels, as well as their in vivo detection in established mouse senescence models. Exploiting GLF16 reagent parallel to GL13, we identified for the first time the induction of cellular senescence following SARS-CoV-2 infection in vitro and in vivo. Additionally, senescent and infected cells exhibited high levels of IL-6 and IL-1β, indicating the contribution of SASP to the observed hyper-inflammatory response in COVID-19. Furthermore, these cells overexpressed enzymes of the APOBEC family involved in RNA editing. The overexpression of these enzymes, coupled with the prolonged presence of the virus within senescent cells due to their resistance to apoptosis, revealed their role as a reservoir for generating new virus mutations. In summary, the development of the innovative GLF16 reagent allowed for the first time the detection and further analysis of live and fixed senescent cells at the RNA and epigenome levels. GLF16 was simultaneously utilized with GL13 to study cellular aging following SARS-CoV-2 infection. The role of senescent cells was identified in both pathogenesis and virus mutagenesis, rendering them a potential therapeutic target, with senolytic drugs as a complementary treatment alongside existing ones.
Keywords:
Cellular senescence, Sudan Black-B, m-GLF16, COVID-19, SARS-CoV-2
