Leader: Andrea Graziani (UNIPO); Other collaborator(s): RTDA-Age-it, UniUPO: Valeria Malacarne (RTDA, UniUPO), Elia Angelino (RTDA, UniUPO), Assegnisti di Ricerca Age-it, UniUPO: Giulia Rossino Suvham Barua Ph.D. students: Raluca Minea (DIN Life Course Research, UniTO), Alessia Labate (DIN Life Course Research, UniTO), Lorenza Bodo (UniTO), Sabrina Mula (UniTo)
Upon antigen encounter, naïve and memory T cells undergo a strong proliferative burst exposing their genome to replicative stress and DNA damage. Recent evidence revealed the role of DNA damage-induced T cell senescence and dysfunction in driving aging-associated diseases. We plan to explore the molecular pathways coupling DNA-damage to T cell dysfunction by focusing on DGKa, a lipid kinase induced by DNA damage and in senescent cells, which is a strong negative regulator of T cell activation. We will investigate the role of DGKa in coupling DNA damage/senescence to cell surface expression and function of the TCR and other co-receptors driving T cells activation and cell fate, in human T cells and in POLG-aging murine models.
Brief description of the activities and of the intermediate results: From bioinformatic gene expression analysis in human T cells, DGKA emerged as key node regulating transcriptional networks activated selectively in aged T cells. In primary T cells we showed that upon antigen-acitivation DGKA restricts TCR cell surface expression by promoting CD3zeta lysosomal degradation upon antigen activation. By using Chimeric antigen receptor as a model, we showed that DGKA favours Cbl recuitment to the antigen activated receptor. In addition, in a sarcopenia model (cancer-induced cachexia) we showed through P-CREB1 CHIP expreiments impairmeent of cAMP/CREB signaling, which contirubtes to downregulation of mitocondrial proteins expressing genes and mitochiodnrial dysfunction. Trasncriptome analysis of old rodents suggest that cAMP/CREB1 signaling is also impaired in old muscles.
Main policy/Industry/practice implications
At the moment we do not envisage policy/Industry/practice implications
Bioinformatic gene expression analysis in human T cells indicate that DGKA is a key node regulating transcriptional networks activated selectively in aged T cells, and our preliminary data indicate that DGKA restricts TCR cell surface expression by promoting CD3zeta lysosomal degradation upon antigen activation, likely through Cbl recruitment to the antigen activated receptor. Thus, based on data showing that DGKA is induced upon DNA damage in fibroblasts, we set to investigate the hypothesis that DGKA may contribute to age-associated T cell dysfunction, which is triggered by DNA-damage response.Thus, we have generated and partially characterized an in vitro system to trigger DNA damage response in primary T cells, upon shRNA-silencing of TRF2, a component of shelterin complex. Indeed, TRF2-silenced T cells feature telomer deprotection and activation of a DNA-damage response and senescence as detected by an array of biochemical and cell surface markers. Despite we have not detected any induction of DGKA at mRNA and protein level, TRF2 silencing results in the reduction of the number of TCR molecules at the cell surface. In addition, we extended to human primary myocytes our previous finding that tumor released inflammatory factors, which largely overlap with SASP factors, result in the impairment of cAMP/CREB signaling thereby contributing to downregulation of mitochondrial proteins expressing genes and mitochondrial dysfunction.In addition, we have also showed, both in murine and human models of cachexia, in vitro and in vivo, that deregulation of PDE4D transcript variants in muscle fibers, contribute to cAMP/PKA/CREB1 impairment
In the second part of the year, we have made more robust the preliminary findings that TRF2-silenced primary T cells feature enhanced DNA-Damage Reponse (DDR), and impaired TCR expression at the at the cell surface. In addition, we verified that indeed, TRF2 silencing induced the n° of CD57+ve cells, a senescence cell surface marker. Consistently with our hypothesis, “bona fide” senescence cells feature lower TCR expression at the cell surface. Moreover, we are still optimizing the experimental conditions to functionally evaluate bona fide senescent T cells, i.e. their impaired effector functions upon antigen activation. Finally, we will also characterize in these cells, TCR trafficking at the cell surface, i.e. its internalization, recycling and degradation rates. In addition, we are also optimizing experimental conditions to explore the hypothesis that T cells exposure to pro-inflammatory factors may impair T cell function through the deregulation of T cells trafficking. In addition, we have extended the data demonstrating that tumor released inflammatory factors, which largely overlap with SASP factors, result in the impairment of cAMP/CREB signaling thereby contributing to downregulation of mitochondrial proteins expressing genes and mitochondrial dysfunction. In the second part of the year, we have focus on the experiments in vitro and in vivo requested by the reviewers of our ms. submitted to Nature Metabolism in order to make more robust our original findings.
International Conference Communications
· Cold Spring harbour Conference, “Gene Expression and Signaling in the Immune System”, 16-2074/2024 Andrea GrazianI: Diacylglycerol Kinase alpha (DGKα) couples antigen-induced T cell activation to the negative feedback mechanism leading to TCR and CAR ubiquitination and lysosomal degradation
· Keystone Symposium “CACHEXIA AND WASTING SYNDROME IN CANCER AND CHRONIC DISEASES” | BUCK INSTITUTE FOR RESEARCH ON AGING (CA, USA), 5/5-5/7 2024 Andrea Graziani (Invited speaker): ): 'Impaired cAMP/CREB1 signaling drives mitochondrial dysfunction in skeletal muscle during cancer cachexia'
· Society on Sarcopenia, Cachexia, & Wasting Disorders (SCWD) 17th Conference, Washington DC, USA, 6-8 December 2024. Andrea Graziani (invited speaker): 'Impaired cAMP/CREB1 signaling drives mitochondrial dysfunction in skeletal muscle during cancer cachexia'
· European Association for Cancer Research (EACR) Conference “The Systemic Consequences of Cancer: Cancer as a Whole-body Disease”, virtual event, 25-26/02/2025 Lorenza Bodo: 'Impaired cAMP/CREB1 signaling drives mitochondrial dysfunction in skeletal muscle during cancer cachexia' (oral presentation selected from abstracts)
Manuscripts under revision
· Angelino et al (2024) Impaired cAMP/CREB1 signaling drives mitochondrial dysfunction in skeletal muscle during cancer cachexia, under revision Nature Metabolism