#	Pagina
attuale pagina	/open-h2020/projects/207073/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MILEAGE (Microelements in Life Expectancy and Aging)

Teaser

Summary

Cells have different mechanisms to prevent or repair oxidation and in the front line are reducing agents. Zinc (Zn) in cells protects against oxidation of sensitive biochemicals by controlling the production of reducing agents and stabilising molecules that contain oxygen-reactive groups. Copper (Cu) can exist in both oxidised and reduced form. In living cells, Cu performs useful functions, but it can also participate in unwanted redox (reduction-oxidation) reactions which generate harmful free radicals. Damage caused by too much oxidation is difficult for cells to counteract and repair and so the harm is cumulative over time. This process at a while body level is called ageing but at a cellular level is called senescence.
Zn and Cu are found in food and as they pass through the stomach and intestine, they are absorbed through gut cells into the blood. They are used by the body in many ways but some is excreted, through the pancreatic juice to the gut in the case of Zn and from the liver to bile and to the gut in the case of Cu. There are 3 types of cells that act as gatekeepers for maintaining body Zn and Cu homeostasis. The gut cells controling Cu and Zn absorption, the pancreatic cells controlling excretion of Zn and the liver cells that control Cu excretion. In ageing, these cells become senescent through cumulative damage and they are less efficient at maintaining body metal homeostasis. So why is this important? We need Zn and Cu to maintain our health, and imbalance usually leads to disease. Since Zn in particular is used for every bodily function, decreased Zn status accelerates ageing and encourages chronic diseases progression, thus reducing both longevity and quality of life. We hypothesise that ageing decreases body Zn and Cu status through increased oxidation and senescence of the gatekeeper cells. The project aims to study the rate at which Zn and Cu can pass through senescent gatekeeper cells compared to that in healthy cells. We also want to know why oxidation affects the efficiency with which these metals are metabolised. There are complex mechanisms for controlling metal passage into, across and out of these cells involving transport proteins, some of which sit on the membranes and grab the metal atoms from the gut or blood. Other transporters move metals safely through cells and yet others move the metals out of the cells. We wish to discover which transporters are sensitive to oxidation-induced senescence. We are designing and producing drugs that can modify the function of these proteins. We can then look at the consequence of modulating transporter efficiency on the resistance of gatekeeper cells to oxidation and senescence. Finally, we believe that the gut bacteria composition might have a role to play in gut zinc and copper speciation and gut cell senescence.

Work performed

This project has 3 scientific work packages WP2, WP3 and WP4. For WP2, the impact of ageing on Cu and Zn uptake by the gut was measured in WP2 using a model system, namely the fruit fly Drosophila melanogaster. This fly has an average life-span of only 40-50 days, a period well-suited to practical experimentation, and use of large numbers ensures statistical power. The nutritional conditions for fly culture has been developed and mineral content was assessed during ageing. The intestinal Zn level increased and Cu decreased with ageing in this model. Using these conditions, subsequent studies were made on the impact of different nutritional metal status. In WP3, mammalian cell cultures are being utilised to study the rate at which Zn and Cu are transferred across the gatekeeper cells, namely gut, pancreatic and liver cells, which control body metal homeostasis. Stimulators and markers of cell senescence have been evaluated using special culture conditions. Stable isotopes are used to trace the movement of these metals through cells and a novel system of measuring the rate of Zn and Cu passage (kinetics) in real-time is being constructed using dual mass spectrometers. This system is of interest to researchers outside the Consortium. To identify age-related changes in trace mineral transporters and channels ion fluxes have to be analysed in real time in live cells and compared to protein quantity and localisation. The combination of molecular biology techniques with sophisticated multi-parameter live cell imaging will reveal the role of each protein identified in WP2 and 3 in the response to oxidative stress senescence. Under WP4 the technique of live cell imaging of both labile zinc and labile copper has been developed. For the first time, we were able to determine that copper can be present in a free form in the cells and be captured by fluorescent chelator. This allows for quantification of biological response to stress. To determine the respective roles of each to aging genetic interference is being used to manipulate expression. The result achieved confirms that we can change expression of channels, zinc transporters. A bioinformatics was applied further to the selection of small molecules, which allowed identification of a preliminary library for further in screening. In silico protocol included protein modeling using genomic and proteomic databases, analysis of domain\'s architecture and structure as well as phylogenetic trees followed by ligand retrieving and virtual screening.

Final results

There two lines of research progress expanded to high novelty and significance. First, the novel role of Zn ions in ageing mechanisms has been assessed by the beneficiary INSERM in studies of the Zn-containing deacetylase Sirtuin1 regulating lifespan and energy metabolism. Sirtuin1 contains Zn finger domain and thus is sensitive to oxidation. We found that SIRT1 activation is associated with an increase in the expression of genes implicated in mitochondrial function and the augmentation of mitochondrial biogenesis, which should be balanced in ageing. Second, a unique study has been performed by at the European Synchrotron Research Facility (ESRF) in Grenoble, the largest of its kind in Europe. The grant was awarded to use the ID21 beam line in 2018. The results demonstrate changes in sulphur speciations under stress conditions and for the first time apply spectral analysis to cell imaging of high resolution. We are looking forward to enhance the scientific outcome from MILEAGE with the data on elemental maps at nanoscale levels obtained in parallel with redox status analysis of sulfur ligands. The new data will provide evidence on the homeostatic regulatory mechanisms that are targeted by ageing. We expect to delineate the potential mechanistic links between Zn and Cuâ€™s fine control of cell physiology. ble for longevity. The study aims to transfer the methodology into clinical and biotechnological settings. This will improve the effectiveness of known therapies, refinement of treatment, and help prevent the deteriorating effects of ageing.
The MILEAGE project will provide opportunities for career enhancement of young ERs and ESRs. The incorporation of other networks on microelements, and communications with both academic and non-academic ER members will also initiate opportunities. Cultural and language experiences will prepare ESRs for employment in multinational companies. We also anticipate that the MILEAGE network program will be an example of excellence in the research and management training for experienced researchers and administrators.