Final Report Summary - OLDMITO (Oxidation, Lipids, DNA and Mitochondria)
Many studies carried out with cell cultures and animal models support the fact that reactive oxygen species (ROS), particularly those produced by the mitochondria, play a key role in the aging process. ROS are continuously produced as the result of the normal functioning of the electron transport chain (ETC) and oxidative phosphorylation, and they cause a cumulative and irreversible damage to the tissues, leading to cellular dysfunction with age. It would appear that the mitochondria are not only the main source of ROS, they are also the first target of the oxidative attack. According to the mitochondrial theory of aging, the ROS produced by the mitochondrial ETC under normal physiological conditions cause damage to proteins, lipids and mitochondrial DNA (mtDNA). Some authors believe that the first target of oxidative damage in the mitochondria is the mtDNA and that as more and more mutations accumulate in critical DNA regions, the complexes that make up the ETC become less efficient or are rendered inactive, leading to mitochondrial dysfunction. In recent years, many diseases associated with senescence in mammals and humans have been related to the accumulation of mutations in the mtDNA. The explanation of the high rate of mutations observed in the mtDNA (much higher than for nuclear DNA) may have something to do with the fact that the mtDNA is greatly exposed to ROS: 1) it is located very near the internal mitochondrial membrane where ROS are produced, 2) it is not overly condensed or protected by histones and 3) it has limited repair activity. However, even though all mitochondria molecules are attacked by ROS, it should be pointed out that lipid peroxidation is the most important oxidative process, primarily due to the extreme sensitivity to oxidation of polyunsaturated fatty acids (PUFA), the constituent molecules of cell membrane phospholipids (PL). Lipid peroxidation also gives way to a whole series of highly reactive derivatives that propagate the oxidation reaction throughout the mitochondrial membrane and to other molecules. One recent theory on aging, that of the membrane pacemaker, suggests that the membrane lipids are the first target of ROS, and damage to these molecules causes an alteration in the membrane fluidity, resulting in a mismatch between the ETC and the oxidative phosphorylation, which in turn increases the rate at which ROS are produced. Finally, the cell's repair and defence mechanisms would be overwhelmed and the oxidative damage would spread, particularly to the mtDNA. In cross-sectional studies on different animal species, including mammals, birds and reptiles, it has been observed that species with lipid membranes that are highly unsaturated, and therefore more fluid, have a high level of metabolic activity, due to the greater mobility of the membrane proteins, which are responsible for a large part of the cell’s metabolism. A higher metabolic rate is associated with greater oxidative stress, which according to this theory, would primarily affect the PUFA in the membrane in a vicious circle, which would lead to a high rate of aging and a low life expectancy. Considering that mitochondrial membranes have a distinctive composition of lipid types, PL, glycolipids and cholesterol related to the role of this organelle in energy metabolism and oxygen consumption, it is easy to understand that any change, either in terms of the membrane content of the different lipid types or the fatty acid (FA) composition of the PL, would affect their functions and, ultimately, the functionality of the mitochondria.
The objective of the OLDMITO project was to test these hypotheses in zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss), two fish species with different life-span and interesting for different reasons. Zebrafish is a short-lived species (2-3 years), considered an important vertebrate model organism for scientific research and also proposed as a possible model of ageing. Rainbow trout is a long-lived fish (6-7 years), a well-studied species of interest for aquaculture. The specific aim of the project was to investigate the role of the mitochondrial membrane lipid composition and mtDNA damage as possible regulators of the processes associated with ageing and fish welfare.
The OLDMITO project success is demonstrated by the number of scientific papers (6) published in international peer-reviewed journals. Experiments performed have shown alterations in mitochondrial PL content and composition in fish with age that could be the propagators of the reactions related with ageing. Particularly important were the changes observed in cardiolipin (CL) and sphingomyelin (SM) as these PLs have been proposed as mediators in mitochondrial dysfunction and apoptosis as consequences of situations of high oxidative stress and ageing. Changes in individual PL FA composition with age were also found. With age, mitochondrial PLs became more unsaturated and mitochondrial oxidative stress increased which is in agreement with the membrane pacemaker theory of ageing. Moreover, data from the OLDMITO project showed that dietary lipid composition markedly influences PL contents and FA compositions of individual PL classes of mitochondrial membranes from fish tissues and have a significant impact upon mtDNA gene expression. These effects differed in relation with fish age and pointed to the key role that diet lipid composition can have upon mitochondrial function.
The rapid progress of OLDMITO activities allowed the fellow to participate in further studies with similar aims and objectives. A study was performed on selenium (Se) antioxidative protection in zebrafish subjected to dietary oxidative stress showing that Se supplementation reduces oxidation in zebrafish, although its effects seemed not to be mediated by transcriptional changes. Several experiments were also carried out on species of genus Nothobranchius, a genus characterized by having an extremely short life span (3-18 months, depending on the species) and, as such, species within this group have been well studied and show a progressive deterioration in several ageing markers. They have been recently proposed as candidates to fill the gap between the animals traditionally used as models to study ageing, the mouse (with short phylogenetic distance with humans but with a long life span) and the worm Caenorhabditis elegans (short life span but very long phylogenetic distance with humans). In Nothobranchius species, similar results to those from zebrafish were found. Taking into account that Nothobranchius presents explosive growth and that most of the changes observed occurred after the growth phase was completed, results in species of this genus pointed to the fact that a high growth rate and rapid attainment of adult size is associated with numerous negative effects, among them a reduction in life expectancy. Data from Nothobranchius species also showed cognitive degeneration as they age, with deterioration of fish biological rhythms and, among them, the rest-activity rhythm, which is determined by the circadian system.
Overall, data from OLDMITO pointed to mitochondria as one of the first responders to various stressors in fish and to mitochondrial membrane lipids deterioration, particularly of CL, and accumulation of mtDNA mutations to be involved in the organelle bioenergetics function impairment and increased oxidative stress.
The objective of the OLDMITO project was to test these hypotheses in zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss), two fish species with different life-span and interesting for different reasons. Zebrafish is a short-lived species (2-3 years), considered an important vertebrate model organism for scientific research and also proposed as a possible model of ageing. Rainbow trout is a long-lived fish (6-7 years), a well-studied species of interest for aquaculture. The specific aim of the project was to investigate the role of the mitochondrial membrane lipid composition and mtDNA damage as possible regulators of the processes associated with ageing and fish welfare.
The OLDMITO project success is demonstrated by the number of scientific papers (6) published in international peer-reviewed journals. Experiments performed have shown alterations in mitochondrial PL content and composition in fish with age that could be the propagators of the reactions related with ageing. Particularly important were the changes observed in cardiolipin (CL) and sphingomyelin (SM) as these PLs have been proposed as mediators in mitochondrial dysfunction and apoptosis as consequences of situations of high oxidative stress and ageing. Changes in individual PL FA composition with age were also found. With age, mitochondrial PLs became more unsaturated and mitochondrial oxidative stress increased which is in agreement with the membrane pacemaker theory of ageing. Moreover, data from the OLDMITO project showed that dietary lipid composition markedly influences PL contents and FA compositions of individual PL classes of mitochondrial membranes from fish tissues and have a significant impact upon mtDNA gene expression. These effects differed in relation with fish age and pointed to the key role that diet lipid composition can have upon mitochondrial function.
The rapid progress of OLDMITO activities allowed the fellow to participate in further studies with similar aims and objectives. A study was performed on selenium (Se) antioxidative protection in zebrafish subjected to dietary oxidative stress showing that Se supplementation reduces oxidation in zebrafish, although its effects seemed not to be mediated by transcriptional changes. Several experiments were also carried out on species of genus Nothobranchius, a genus characterized by having an extremely short life span (3-18 months, depending on the species) and, as such, species within this group have been well studied and show a progressive deterioration in several ageing markers. They have been recently proposed as candidates to fill the gap between the animals traditionally used as models to study ageing, the mouse (with short phylogenetic distance with humans but with a long life span) and the worm Caenorhabditis elegans (short life span but very long phylogenetic distance with humans). In Nothobranchius species, similar results to those from zebrafish were found. Taking into account that Nothobranchius presents explosive growth and that most of the changes observed occurred after the growth phase was completed, results in species of this genus pointed to the fact that a high growth rate and rapid attainment of adult size is associated with numerous negative effects, among them a reduction in life expectancy. Data from Nothobranchius species also showed cognitive degeneration as they age, with deterioration of fish biological rhythms and, among them, the rest-activity rhythm, which is determined by the circadian system.
Overall, data from OLDMITO pointed to mitochondria as one of the first responders to various stressors in fish and to mitochondrial membrane lipids deterioration, particularly of CL, and accumulation of mtDNA mutations to be involved in the organelle bioenergetics function impairment and increased oxidative stress.