Leader: Antonino Colanzi (CNR); Other collaborator(s):
Secretion of soluble factors is an important contributor to ageing. The secretory pathway plays a key role in this process, but its contribution to senescence is unclear. So, we propose to carry out the following experiments in control and senescent cells: 1) systematically analyze the morphological alterations of the secretory pathway and the proteostasis of glycosylation enzymes implicated in this pathway by high-resolution microscopy (confocal, super-resolution and electron microscopy) techniques. 2) Investigate changes in cellular glycosylation, especially of plasma membrane receptors, by lectin binding and mass-spectrometry analysis. 3) Study the post-translational regulation of the secretory pathway, e.g. by mono-ADP-ribosylation and phosphorylation-based control systems.
Brief description of the activities and of the intermediate results: The aim of the research group affiliated with the Secondary Unit of the IEOS (SU-IEOS) is to investigate the transcriptional and post-translational regulation of the secretory pathway in senescent cells. Recent research at the SU-IEOS has shown that a sophisticated network of signalling components and mechanisms regulates the secretory pathway. Different cargo classes can activate specific regulatory machinery located at the Trans Golgi Network (TGN) to promote their transport and secretion. More recent studies have shown that cargo proteins activate GPRC5A, a TGN-localised orphan G-protein coupled receptor, which trigger a signalling leading to the recruitment and activation of Protein Kinase D at the TGN. Based on this evidence, we tested role of GPRC5A in ageing. The more recent data indicate that the knock-down of GPRC5A induces a strong reduction of cell proliferation and increased spreading and Beta-galactosidase plasma-membrane staining compared to control cells. FACS analysis of cell cycle distribution revealed that GPRC5A knock-down resulted in increased fraction of cells in the G0-G1 phase and decreased fraction of cells in the S phase, suggesting a partial block of the cell cycle at the G1-S transition. This finding is supported by the biochemical characterization of cell cycle markers, which revealed that GPRC5A downregulation resulted in reduced expression of Cyclin A2 and Cyclin B and concomitant upregulation of p21. All the data mentioned above are coherent with a senescent phenotype. In addition, we have investigated the role of GPRC5A in the secretion of APP using neuronal and non-neuronal cell lines, the preliminary results suggest that knock-down of GPRC5A alters APP processing, favouring the non-amylogenic cleavage of the protein. Finally, we have initiated the setting up of imaging-based assays for investigating the intracellular traffic of SASP factors, such as matrix metalloproteinases and interleukins. Cellular senescence induced by different stimuli in human primary cell cultures from subjects of different ages and senescence-associated phenotypes characterisation and modulation.
SASP factors, such as matrix metalloproteinases and interleukins can be secreted via the conventional as well as the un-conventional secretory pathway, depending on the specific protein. This difference must be considered to study the effects of senescence on their secretion, since the secretory phenotypes might be deriving from defects in the specific trafficking machinery. Moreover, secretion defects might derive from altered transport kinetics at different levels in the secretory pathway. To try to answer to these questions, we took advantage of several methods to synchronize protein secretion that allowed us to visualize and monitor a specific soluble protein by both imaging-based assays and biochemical approaches. One of the best and most used synchronization method is the RUSH (retention upon selective hook) approach. We used this approach with different secretory proteins, thus finding out that their secretion kinetics in control HeLa cells is specific for different cargo proteins. Our aim now is to measure the secretion kinetics under different senescence-inducing stimuli. Regarding the role of GPRC5A in the secretion and processing of APP, we managed to produce a GPRC5A CRISP-KO HeLa cell line that stably expresses a synchronizable APP-RUSH protein, that will be now used to further characterize the localization and dynamics of APP cleavage products in control and GPRC5A-KO cells. In addition, we are planning to follow the processing and transport of the endogenous APP in iPS-derived cortical neurons.
In parallel, we are investigating Interleukin-8 (IL-8) in the aging process, considering its role as a key driver of chronic inflammation, often referred to as inflammaging. This phenomenon is closely tied to the accumulation of senescent cells with age, which release various inflammatory molecules as part of the SASP phenotype. Among these molecules, IL-8 stands out for its ability to sustain a persistent, low-grade inflammatory state that contributes to tissue dysfunction and increases the risk of age-related conditions, including cancer. In this context, we have identified the tyrosine phosphatase Shp1 as a key regulator of IL-8 signaling in breast cancer. Specifically, we discovered that Shp1 plays an essential role in promoting IL-8-driven tumor invasiveness by controlling the turnover of its receptor, CXCR2. When Shp1 activity is inhibited, the receptor undergoes irreversible downregulation, which helps suppress CXCR2 function. This, in turn, reduces IL-8 signaling within the tumor microenvironment and limits the effects of excessive inflammation. The mechanisms involved in this process are under investigation.
The Golgi complex undergoes significant structural disassembly during cell division and cellular senescence. While the functional consequences of these alterations are not fully understood, they may impair the secretion of proteins and signaling molecules, potentially disrupting tissue homeostasis and contributing to aging-associated pathologies. Chemotherapy drugs can also induce changes in Golgi architecture, leading to alterations in cellular metabolism and stress responses.
Our investigation into the mechanism of Golgi disassembly during mitosis has revealed that disruptions in Golgi segregation cause notable cell division defects, including spindle multipolarity and tetraploidy. This is particularly relevant because tetraploidy promotes cellular senescence through genomic instability and checkpoint activation, contributing to aging-related cellular dysfunction. These findings have therapeutic potential for targeting senescence mechanisms in both aging and oncology.
Additionally, we have developed a specific approach to restore Golgi integrity using a membrane-permeable peptide derived from the Golgi structural protein GRASP65. This peptide contains a phosphorylation site essential for Golgi disassembly and effectively inhibits Golgi disassembly under various conditions and in different cell lines. In contrast, a control peptide with a non-phosphorylable alanine substitution showed no such effects, confirming the specificity of this tool. Notably, the active peptide reverses Golgi disassembly induced by the chemotherapeutic agent doxorubicin. This peptide could provide valuable insights into the molecular mechanisms of cellular senescence induced by chemoterapics and help identify potential targets for mitigating senescence-related cellular decline.
The progress in the investigation into the interplay between IL8 and Shp1 cascades, are also now exploited for translational applications. Indeed, targeting the CXCR2/Shp1 axis may offer promising avenues for developing new treatments for breast cancer and potentially other age-related diseases where IL-8 and chronic inflammation play a pivotal role.
Continuing the investigation of the role of CXCR2, we further examine the functional consequences of its depletion when Shp1 is inhibited. Our findings indicate that this regulation occurs at the post-translational level, as no significant effects were observed at the mRNA level. Currently, we are investigating the contribution of both the proteasomal and lysosomal pathways to IL8-induced CXCR2 degradation. These ongoing analyses will help clarify the mechanisms through which Shp1 influences CXCR2 stability and may provide insights into how IL8 signaling is fine-tuned in different cellular contexts.
Understanding how IL8-induced CXCR2 degradation is modulated in aging cells could open avenues for targeted interventions, as CXCR2 dysregulation has been linked to aging-related pathologies, including cancer, tissue degeneration, and immune dysfunction.
Regarding the role of the Golgi structure in the development of a senescent phenotype, new and recent data indicate that senescence induced by various types of DNA damage requires Golgi fragmentation as a mediator of the activation of several senescence-associated signaling pathways.