Effects of ageing, calorie restriction and ageing-associated diseases on the mitochondrial proteome

This thesis outlines the effects of ageing, calorie restriction and ageing-associated diseases on the mitochondrial proteome. Several techniques were tested to study the mitochondrial proteome: Techniques for visualisation of proteins after two dimensional (2D) blue native (BN) / sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) were compared: staining of gels with Coomassie Brilliant Blue G-250 (CBBG), silver and the fluorescent dye SYPRO®Ruby as well as labelling of the proteins before gel run with fluorescent dyes from Refraction-2D™ Labeling Kit (differential gel electrophoresis, DIGE). A recommendation was elaborated either for staining with fluorescent dye, when enough amount of protein is available, or for labelling with fluorescent dye, when analysis of low amount of protein is needed. Quantitation of the amount of mitochondrial oxidative phosphorylation (OxPhos) complexes after BN PAGE and subsequent CBBG staining was compared to quantitation after 2D-BN/SDS-PAGE and subsequent SYPRO®Ruby staining, using the rat brain tissue samples for the ageing and calorie restriction analyses (cerebellum, hippocampus and cerebrum). The results showed, that quantitation in CBBG stained BN gels is less accurate than in 2D-BN/SDS gels and subsequent SYPRO®Ruby staining, and therefore not applicable for detailed quantitation studies. The effects of ageing and calorie restriction were studied on the mitochondrial proteome of three different brain tissues of Fischer 344 rats: cerebellum, hippocampus and cerebrum. Animals were fed ad libitum (AL) or calorie restricted (CR, intake about 60 % of ad libitum fed animals, start point: after the age of 6 weeks) and analysed at two ages: 6.5 months (young, Y) and 27 months (old, O). Mitochondria were isolated and the mitochondrial proteins were separated by 2D-BN/SDS PAGE and identified by peptide mass fingerprint (PMF) with MALDI-MS. The protein amount of the assigned subunits was quantified by SYPRO®Ruby staining. BN PAGE of mitochondrial proteins and subsequent in-gel activity tests were applied to analyse the specific activity of the respiratory chain complexes I and IV. The mitochondrial membrane “fluidity” was determined by measurement of the steady-state fluorescence anisotropy of the intercalated probe diphenyl-1,3,5-hexatriene (DPH). In general, it was found that animals of young age under CR (YCR) developed a smaller body weight than young AL ones (YAL). Ageing under AL increased the body weight to the same extent as under CR. Commonly for all three tissues, the results of PMF showed that the migration pattern of subunits of mitochondrial proteins from rat brain tissues in 2D-BN/SDS PAGE are highly comparable, also to other mammalian tissue such as bovine heart. Therefore analyses of the proteome of the different tissues could be compared. The mtDNA encoded protein subunits CYB of OxPhos complex III (CIII) and COX2 of complex IV (CIV) did not show specific changes during ageing neither AL nor CR. Their changes followed the trends of total CIII and total CIV. The highest activities of CI were found in supercomplex I1III2IV3 and of CIV in supercomplex I1III2IV2 for all three brain tissues. However, the changes during ageing and calorie restriction of specific CI and CIV activities were not very pronounced. In cerebellum ageing of AL fed rats reduced the tissue weight and total mitochondrial protein amount as well as the fluidity of the mitochondrial membranes. The amount of OxPhos complexes and the activity was not altered. Already at young age the tissue weight of cerebellum decreased under CR and developed in the same way as in AL fed animals during ageing. The total mitochondrial protein amount in young and in old rats under CR was higher compared to AL fed rats, but it decreased during ageing under CR (YCR vs. OCR), thereof were proteins of cellular stress management, energy metabolism and neuronal composition. The amount of OxPhos complexes was decreased in young animals under CR compared to AL but increased during ageing to the same level of protein amount from animals under AL at old age. In general, the mitochondrial membrane fluidity decreased with ageing, even more in cerebellum of animals under CR than under AL. Already at young age under CR the fluidity was decreased compared to AL. For hippocampus the tissue weight, the total mitochondrial protein amount and CI activity in some (super-)complexes decreased by ageing independently from nutrition. Most of the analysed proteins decreased under AL and in young animals under CR. However, during ageing under CR the amounts of analysed proteins increased to a higher level in aged CR animals than aged AL animals. During ageing under AL the membrane “fluidity” did not change but in young and old animals under CR the membrane fluidity was decreased compared to the AL animals, even though the fluidity increased during age in CR animals. Decreased fluidity could be explained by increased cholesterol amount or increased membrane protein amounts and these could be needed to compensate for the decreased CI activity. In cerebrum the tissue weight decreased during ageing independently from nutrition. The amount of OxPhos complexes (excl. CV) decreased during ageing under AL while ageing under CR increased the protein amount of OxPhos complexes (excl. CV) in old rats. Ageing decreased the mitochondrial membrane “fluidity” in AL animals. Under CR the fluidity did not change, but was still increased in aged animals under CR compared to AL. In conclusion, ageing under AL decreased brain tissue weight, total protein amount and fluidity of mitochondrial membrane. The amount of the OxPhos complexes and their activity remained mainly unchanged. During ageing under CR the total protein amount increased and membrane fluidities decreased which, in terms of remained OxPhos complexes activities, seem to be part of compensation processes. Together with Dr. Koziel (IBA) the effects of ageing (senescence) were studied on a cell culture model, human umbilical vein endothelial cells, in which NADPH oxidase 4 (Nox4), known as producer of reactive oxygen species, was knocked down. The results showed a depleted CI amount and reduced CI activity under the influence of Nox4 which were both increased when Nox4 was knocked down. In collaboration with Dr. Kuter (IF-PAN) an early stage model of the ageing-associated disease Morbus Parkinson was studied. The effects of neuronal degradation in substantia nigra on the mitochondrial proteome were analysed in Wistar rats. For identification of proteins, 2D-BN/SDS PAGE and subsequent PMF were performed. Quantitation was achieved by application of DIGE. In-gel activities of OxPhos complexes I and IV were measured as well as membrane “fluidity”. The evaluation of the obtained results is still in progress. Some of the data were already submitted and are under review and for copyright reasons not described in this thesis.