Exploring mitochondrial function by assessing ROS and antioxidants levels in N. furzeri brains

Details

Supervisors

Page, Melissa ; Debier, Cathy

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

The global demographic landscape is currently undergoing a substantial shift, marked by a notable increase in the aging population. Increased life expectancy due to advances in medical science and improved living conditions has resulted in an increasing proportion of people aged 65 and up. However, this demographic shift has also brought to light the heightened vulnerability of older individuals to chronic diseases. Addressing and managing these health challenges, while promoting a healthy lifestyle, has become a paramount scientific priority. And in the context of aging, mitochondrial failure takes center stage, being an inherent feature of the aging process and a common factor in numerous age-related diseases. Despite its significance, the mechanisms contributing to mitochondrial dysfunction remain elusive. This thesis investigates the mechanisms linked to mitochondrial dysfunction through the study of the African turquoise killifish (Nothobranchius furzeri). Noteworthy for both its display of mitochondrial dysfunction and its unique captive lifespan of only 4 to 6 months, covering all typical biological phases observed in vertebrates. The overall objective was to determine whether oxidative damage accumulates in N. furzeri during aging. Lipid peroxidation levels and protein carbonyl content were compared in the brain of N. furzeri at 6, 14 and 20 weeks of age, representing juvenile, young adult, and adult fish, respectively. We expected to observe an increase in oxidative stress related to aging. However, we did not obtain any results indicating a significant change in these markers of oxidative stress during aging. Western blots were used to measure the levels of SOD1 (superoxide dismutase) and SOD2 in aging brains. The activity of these enzymes was also assessed using a colorimetric assay. Our results showed that SOD1 protein levels remained constant between N. furzeri brains isolated from fish aged 6 and 20 weeks, while SOD2 levels were significantly higher in brains from older fish compared to those from younger fish. This increase in SOD2 suggests a possible intensification of the antioxidant response, potentially linked to a progressive accumulation of oxidative damage at the mitochondrial level. However, this interpretation needs to be confirmed by more in-depth analyses, including directly measuring oxidation products and evaluating the level of other antioxidant systems. Unfortunately, the results concerning SOD activity were unsatisfactory, as we always observed inhibition rates in excess of 80%, even after dilutions. These results suggest that there may be interference with the WST-1 reduction assay. In addition, the control reaction (without SOD) did not conform to expectations, which calls into question the reliability of these measurements, suggesting that the protocol needs to be optimised to obtain more consistent results.These results do not allow us to conclude that oxidative damage accumulates in the brain of N. furzeri with age. These observations may reflect methodological constraints and highlight the importance of conducting further studies using more sensitive techniques to explain more precisely the link between aging and oxidative stress. Thus, it is not possible to draw a definitive conclusion on the role of antioxidant defenses in the aging process in N. furzeri.