Leader: Marco Sandri (UNIPD); Other collaborator(s):
We plan to study sarcopenia and frailty, namely how imbalanced autophagy-lysosome and ubiquitin-proteasome systems contribute to tissue degeneration and reverberates from one organ to the whole organism. We will use several inducible muscle-specific transgenic mice that show premature senescence and precociuos death and will apply proteomic and single-cell transcriptomic to identify novel biomarkers of unhealthy ageing.
Brief description of the activities and of the intermediate results: We have expanded the C16orf70/MYTHO knockout mice and found a problem of fertility and embryonic/fetal lethality. Few homozygous have been obtained so far. We are checking by EchoMRI whether the knockout mice have problem in skeletal muscle and white adipose tissue. We have also generated a zebrafish C16orf70/MYTHO knockout animals that are under characterization. The fishes in which C16orf70/MYTHO was ablated show already a premature death and fertility issues. In OPA1/MetRS transgenic mice, which show premature ageing, we have set up a protocol to successfully label by click chemistry the new synthetized proteins in skeletal muscle with Biotin. Then we have pulled down the Biotin-labelled proteins and checked by western blots the presence of FGF21, a bona fide positive control that is highly expressed in these mice, to verify that the protocol properly works. We are repeating the experiments in cell culture media in which FDB myofibers from OPA1/MetRS transgenic mice have been cultured.
During this period, we have competed the characterization of C16orf70/MYTHO knockout fish, which showed autophagy impairment, premature lethality, tumour onset (pancreatic cancer, seminomas), sarcopenia, decreased motility and abnormal neuromuscular tests. A manuscript is under preparation. We have expanded C16orf70/MYTHO knockout mice and confirmed the premature death. Histological analyses showed accumulation of protein aggregates in neurons. Functional and morphological analyses on muscles and other organs in ongoing.
We have shown that mitochondrial fragmentation in muscles, which is typical of sedentary sarcopenic people, induces mitophagy and lysosomal overload resulting in activation of TLR9-NFkB pathway and IL6 upregulation. The increase expression of IL6 in muscle and consequent secretion in blood is sufficient to induce a systemic inflammatory response that promotes premature senescence in peripheral tissues contributing to precocious animal death. We are writing the paper on these data.
We have performed proteomic analyses on the labelled proteins from muscles of the OPA1/MetRS transgenic mice, which show premature ageing. The data are currently under bioinformatic screening to check for the presence of secreted proteins.
Franco-Romero A, Morbidoni V, Milan G, Sartori R, Wulff J, Romanello V, Armani A, Salviati L, Conte M, Salvioli S, Franceschi C, Buonomo V, Swoboda CO, Grumati P, Pannone L, Martinelli S, Jefferies HB, Dikic I, van der Laan J, Cabreiro F, Millay DP, Tooze SA, Trevisson E, Sandri M. C16ORF70/Mytho promotes healthy ageing in C. elegans and prevents cellular senescence in mammals. J Clin Invest. 2024 Jun 13:e165814. doi: 10.1172/JCI165814. I.F. 13,3
Franco-Romero A, Leduc-Gaudet JP, Hussain SN, Gouspillou G, Sandri M. PHAF1/MYTHO is a novel autophagy regulator that controls muscle integrity. Autophagy. 2023 Jun 12:1-3. doi: 10.1080/15548627.2023.2224206- IF: 13.3
During this period we have performed life span analyses of C16orf70/MYTHO knockout mice and found that survival was dramatically reduced in males with 100% lethality at 15 months of age. We also showed a newborn lethality, and we are testing the role of this gene during development. We are doing necroscopy to determine the cause of animal death. Preliminary data showed an increase incidence of tumors.
We have finalized the paper of C16orf70/MYTHO knockout fish that is ready to be submitted. During the finalization of the paper we have added data showing that absence of C16orf70/MYTHO led to DNA damage characterized by double strand break. We are investigating the role of this gene in DNA repair systems and analyses in vitro support the role of C16orf70/MYTHO in activation of ATM-p53 pathway.
We are finalizing the paper on mitochondrial fragmentation in muscles, which induces IL6 induction and secretion and cause inflammaging and multiorgan senescence. To further show the role of muscle in triggering systemic inflammation we have generated an inducible muscle specific IL6 knockout mouse line that we are ageing and characterizing.
The proteomic data on OPA1/MetRS transgenic mice, which show premature ageing, identified several contractile proteins but few proteins that could be secreted. To enhance the possibility to identify secreted proteins we have set up an approach to extract extracellular fluids from muscle tissue to perform proteomic and establish the secretome of young and sarcopenic muscles. We have applied this technique to OPA1/MetRS transgenic mice, in which muscle protein can be labelled by synthetic aminoacids. The labelled proteins from extracellular fluids of different muscles will be pulled down and proteomic will be performed. We are collecting samples from different muscles for the pull down experiments.
During this period, we have performed morphological studies on tissues from 12 months old C16ORF70/MYTHO knockout mice. The histology of brain revealed axon degeneration in cerebellum and hypothalamus. In spleen we identified the presence of lymphoma with liver metastasis. In liver we identified signs of cholestasis. Kidney and heart look normal. We also found animals with gastrointestinal cancer. Due to the presence of lymphoma, we are monitoring and characterizing the cell types in the bone marrow, spleen and blood. Due to brain alteration, we are performing behavior tests to check the status of the neuromuscular system.
Because of the presence of cancer and because interactome analyses of C16ORF70/MYTHO identified several enzymes of pyrimidine biosynthesis, we are investigating confirming the role of C16ORF70/MYTHO in nucleotide generation and DNA repair. Coimmunoprecipitation experiments confirmed the interaction with TK1. We are developing split GFP probes to visualize in which are of cytoplasm such interaction happens. We are cloning and performing pull down experiments for the other enzymes of pyrimidine pathway.
We are writing the paper showing that mitochondrial fragmentation during ageing induced the expression in muscles of IL6, which caused inflammaging and multiorgan senescence. To further show the role of muscle in triggering systemic inflammation we have generated an inducible muscle specific IL6 knockout mouse line that we are ageing and characterizing.
The proteomic data on OPA1/MetRS transgenic mice, which show premature ageing, identified several proteins. To better identify the secreted proteins, we have set a methodology that enables us to collect extracellular fluids from muscle tissue. We have collected the extracellular fluids from gastrocnemius, tibialis anterior and quadriceps muscles and ship for proteomic analyses.