Leader: Luca Scorrano (UNIPD); Other collaborator(s):
We plan to characterize how ameliorated mitochondrial dynamics is linked to healthy aging at the cellular and systemic level. We will perform a genome-wide scree in Opa1-/- cells to identify factors whose ablation restores mitochondrial elongation, characterize the top hits and verify if their inhibition counteracts Fgf21 production and systemic ageing in vivo; we will explore if a novel Epac1-Rap1 signalling pathway identified by unbiased RNAseq across multiple models of reduced Opa1 levels is linked to Fgf21 production and systemic ageing in vivo.
Brief description of the activities and of the intermediate results: Senescence is a complex stress response state of the cell, mainly characterized by a stable cell cycle arrest in G1/G2 phase and a relevant secretome release (a.k.a. Senescence Associated Secretory Phenotype). Mitochondria are dynamic intracellular organelles and key players in many aspects of cell biology. This functional versatility is tightly coupled to constant reshaping of the mitochondrial network by fusion and fission (division) events. Mitochondria are also key players in cellular senescence: in senescent cells, the highly interconnected and elongated mitochondria because of low expression of the fission proteins Drp1 and Fis1 display low ATP production. Additionally, downregulation of Fis1 leads to extensive mitochondrial elongation and senescence. Altogether these data represent a proof of concept of a direct correlation between sustained mitochondrial elongation and senescence induction, but the underlying mechanism is unknown. To address this point, we decided to perform a genome-wide RNAi screening based on high content imaging, to identify genes essential for senescence induced by loss of Fis1. We established the screening pipeline using HUVECs cells where genetic silencing of Fis1 induces accumulation of the beta galactosidase marker of senescence that can be readily imaged. Our screening is now under way and expected to highlight the connection between mitochondrial dynamics and the establishment of senescence.
During these 4 months, the screening is ongoing. This screening is performed at the HITS@UniPD facility in an automated fashion, with the operator being blinded to the pooled siRNA being tested. Moreover, once all the repeats are completed, the analysis pipeline will be implemented to rank and identify the results. As this is an unbiased, unbiased approach, we cannot provide any update on hits because they will be unveiled after the three rounds of imaging-based screening and the corresponding analysis have been completed.
Zambello, Ludovica, and Luca Scorrano. “A RalA between high-fat diet and mitochondrial shape.” Nature metabolism, 10.1038/s42255-023-00969-7. 29 Jan. 2024, doi:10.1038/s42255-023-00969-7