Leader: Luca Rampoldi (UNISR); Other collaborator(s):
To ensure in-depth mechanistic dissection and validate targets, we will employ a wide array of dedicated genetically modified mouse models characterized by age-related damage, either systemic or organ-specific (including brain, hematopoiesis, skeletal muscle, heart, kidney, liver). We will explore several age-associated mechanisms, including clonal hematopoiesis, neuroinflammation, SASP, HMGB1, endosomal trafficking, (dys)metabolism, autophagy, hypoxia and protein homeostasis. Selected mechanisms will be modelled in zebrafish. Additional insight will be granted by ongoing exclusive studies on Chiroptera (bats), unique mammals characterized by exceptional longevity and exemption from age-related diseases.
Brief description of the activities and of the intermediate results
Here we aim at mechanistic dissection and target validation in a wide array of dedicated genetically modified mouse models characterized by age-related damage, either systemic or organ-specific. We established a model of CKD by administering low dose of aristolochic acid (AA) to mice with different Umod dosage (TgUmodwt/wt, Umod+/+, Umod-/-). Interestingly, AA-treated Umod-/- mice were protected against kidney function decline, damage, inflammation and fibrosis. These results, consistently with the genetic effect described in human cohorts, reflect a protective effect of reduced/absent uromodulin expression. We highlighted that a pharmacological activation of HIF-1alpha in adult skeletal muscle stem cells (satellite cells), isolated from 18-month-old mice, determines: a) an increase in stemness through an increase in the number of PAX7+ satellite cells; b) a decrease in intracellular accumulation of p16, reflecting a reduction in the senescent phenotype. We: i) established an Institutional biobank containing now >400 primary bat-derived cell coltures; ii) performed metabolomics analyses highlighting novel mechanisms in bats bioenergetics; iii) performed transcriptomic and proteomic characterizations of primary bat derived cells, in search for adaptations in autophagic, DNA damage repair and proliferative pathways in bats versus murine counterparts. We demonstrated that SDH deficiency commits cells to rely on mitochondrial glutamate-pyruvate transaminase (GPT2) activity for proliferation. By driving reductive glutamine carboxylation, GPT2 activity fuels a metabolic circuit maintaining a favorable intracellular NAD+ pool to enable glycolysis, thus meeting the energetic demands of SDH-deficient cells. We have observed that loss of ACOD1, the enzyme responsible for itaconate biosynthesis, in mice stimulates spontaneous formation of metastases following subcutaneous transplantation of syngeneic mouse LLC1 lung cancer cells. Moreover, physiologically relevant concentrations of itaconate trigger invasive phenotype of LLC1 cell spheroids in vitro.
To assess the contribution of HMGB1 to aging, we analyzed total body (tKO) and muscle-specific (mKO) HMGB1 knockout mice during aging, finding that HMGB1 deficiency causes motor deficits tied to muscle homeostasis and cognitive impairments without worsening age-related decline. Additionally, HMGB1 loss reduces fat mass and body weight, implicating it in lipid metabolism, with further ex vivo investigations ongoing to explore these effects.
By using analytical chemistry approaches, we have observed that the loss of SDH activity generates dysfunctions in the metabolism of nucleotides in cancer cells. Ongoing experiments aim to explore how such changes might be targeted in SDH-deficient cells xenografted in immunocompromised mice. We have observed that the loss of itaconate production in mice stimulates the formation of metastases following the subcutaneous transplantation of syngeneic cancer cells. We are now exploring the metabolic mechanisms by which itaconate produced in the tumor immune microenvironment suppresses ferroptosis in cancer to support metastasis formation.
LC-MS analyses of satellite cells isolated from mice at 2 and 18 months of age, treated with FG-4592, and then stimulated to differentiate, revealed that glycolytic metabolism increases during myogenesis and counteracts muscle atrophy.
We generated the kidney proteome and metabolome profile of mice expressing different Umod gene dosage. The analysis of such data is currently ongoing and will allow to better dissect the well-established role of uromodulin as a main risk factor of chronic kidney disease in the aging population.
In an aged mouse model of stroke, we are investigating how physical training can modulate inflammation and outcome. Also, we are exploting a model of juvenile Parkinson’s disease to investigate mechanisms of accelerated aging and neurodegeneration.
We quantified and characterized constitutive autophagy as a putative mechanism curbing proliferative senescence and conferring superior stress-adaptive capacity in bat cells as compared to murine and human counterparts.
By using stable isotopic labelling approaches, we have found that loss of SDH activity impairs the de novo synthesis of purine nucleotides and engage their salvage pathway in cancer cells. By measuring metabolite exchange rates between cells and their media, we also observed that SDH loss increases consumption of nucleosides and nucleobases. Work is in progress to determine the extent to which SDH loss affects the individual contribution of purine salvage pathway genes in supporting proliferation of cancer cells in vitro.
Beyond its role in mitigating skeletal muscle atrophy during aging, preliminary findings suggest that FG-4592 stimulates satellite cells to release extracellular vesicles, which, in turn, inhibit cardiac fibroblast activation and reduce alpha-SMA production—the primary marker of cardiac fibrosis, a condition commonly associated with aging and sarcopenia.
We are currently validating the most interesting candidates from kidney proteome from mice expressing different Umod gene dosage, with main focus on mice overexpressing uromodulin, a model of the well-established role of uromodulin as a main risk factor of chronic kidney disease in the aging population.
The Training in Aged Stroke project is progressing with the analysis of neuroprotective mechanisms induced by physical exercise in experimental models of stroke in aging.
We further inquired into the lower activation of replicative senescence in bats by testing the weight of their upregulated autophagic activity in determining such adaptation and designed a novel imaging-based experiment to assess the rate of bats' uniquely evolved constitutive autophagic activity, which could play a crucial role in the evolution of extended lifespan within this taxonomic unit.
We characterized age-associated endosomal defects in neurons, and we are currently investigating how these alterations induce changes in mRNA trafficking.
Coming soon