Adaptation within mitochondrial oxidative phosphorylation supercomplexes and membrane viscosity during degeneration of dopaminergic neurons in an animal model of early Parkinson's disease.

In Parkinson's disease (PD) motor symptoms are not observed until loss of 70% of dopaminergic neurons in substantia nigra (SN), preventing early diagnosis. Mitochondrial dysfunction was indicated in neuropathological process already at early PD stages. Aging and oxidative stress, the main factors in PD pathogenesis, cause membrane stiffening, which could influence functioning of membrane-bound oxidative phosphorylation (OxPhos) complexes (Cxs) in mitochondria. In 6-OHDA rat model, medium-sized dopaminergic lesion was used to study mitochondrial membrane viscosity and changes at the level of OxPhos Cxs and their higher assembled states-supercomplexes (SCxs), during the early degeneration processes and after it. We observed loss of dopaminergic phenotype in SN and decreased dopamine level in striatum (STR) before actual death of neurons in SN. Behavioural deficits induced by lesion were reversed despite progressing neurodegeneration. Along with degeneration process in STR, mitochondrial Cx I performance and amount decreased in almost all forms of SCxs. Also, progressing decrease of Cx IV performance in SCxs (I1III2IV3-1, I1IV2-1) in STR was observed during degeneration. In SN, SCxs containing Cx I increased protein amount and a shifted individual Cx I1 into superassembled states. Importantly, mitochondrial membrane viscosity changed in parallel with altered SCxs performance. We show for the first time changes at the level of mitochondrial membrane viscosity influencing SCxs function after dopaminergic system degeneration. It implicates that altered mitochondrial membrane viscosity could play an important role in regulation of mitochondria functioning and pathomechanisms of PD. The data obtained are also discussed in relation to compensatory processes observed.

Modulation of oxidative phosphorylation machinery signifies a prime mode of anti-ageing mechanism of calorie restriction in male rat liver mitochondria.

Mitochondria being the major source and target of reactive oxygen species (ROS) play a crucial role during ageing. We analyzed ageing and calorie restriction (CR)-induced changes in abundance of rat liver mitochondrial proteins to understand key aspects behind the age-retarding mechanism of CR. The combination of blue-native (BN) gel system with fluorescence Difference Gel Electrophoresis (DIGE) facilitated an efficient analysis of soluble and membrane proteins, existing as monomers or multi-protein assemblies. Changes in abundance of specific key subunits of respiratory chain complexes I, IV and V, critical for activity and/or assembly of the complexes were identified. CR lowered complex I assembly and complex IV activity, which is discussed as a molecular mechanism to minimize ROS production at mitochondria. Notably, the antioxidant system was found to be least affected. The GSH:GSSG couple could be depicted as a rapid mean to handle the fluctuations in ROS levels led by reversible metabolic shifts. We evaluated the relative significance of ROS generation against quenching. We also observed parallel and unidirectional changes as effect of ageing and CR, in subunits of ATP synthase, cytochrome P450 and glutathione S-transferase. This is the first report on such 'putatively hormetic' ageing-analogous effects of CR, besides the age-retarding ones.

Proteome alterations in rat mitochondria caused by aging.

Analysis of the protein profile of mitochondria and its age-dependent variation is a promising approach to unravel mechanisms involved in aging and age-related diseases. Our studies focus on the mammalian mitochondrial membrane proteome, especially of the inner mitochondrial membrane with the respiratory chain complexes and other proteins possibly involved in life-span control and aging. Variations of the mitochondrial proteome during aging, with the emphasis on the abundance, composition, structure, and activity of membrane proteins, are examined in various rat tissues by native polyacrylamide gel electrophoresis techniques in combination with MALDI-TOF mass spectrometry. In rat brain, age-modulated differences in the abundance of various mitochondrial and nonmitochondrial proteins, such as Na,K-ATPase, HSP60, mitochondrial aconitase-2, V-type ATPase, MF(o)F(1) ATP synthase, and the OXPHOS complexes I-IV are detected. During aging, a decrease in the amount of intact MF(o)F(1) ATP synthase occurs in the cortex. As analytical technique, native PAGE separates not only individual proteins but also multi-subunit (membrane) proteins, (membrane) protein supercomplexes as well as interacting proteins in their native state. It reveals the occurrence and architecture of supramolecular assemblies of proteins. The age-related alterations in the oligomerization of the MF(o)F(1) ATP synthase observed by us in rat cortex might be one clue for understanding the link between respiration and longevity. Also, the abundance of OXPHOS supercomplexes, that is, the natural assemblies of the respiratory complexes I, III, and IV into supramolecular stoichiometric entities, such as I(1)III(2)IV(0-4), can differ between young and aged cortex tissue. Age-related changes in the supramolecular architecture of OXPHOS complexes might explain alterations in ROS production during aging.

Unraveling age-dependent variation of the mitochondrial proteome.

Blue-native and colorless-native gel electrophoresis combined with subsequent 2D-SDS-PAGE and MALDI mass spectrometry are successfully applied for understanding the role of mitochondria in cellular dysfunction, aging, and cellular death. The partial mitochondrial proteome maps of various tissues (liver, brain, kidney, heart, and skeletal muscle) obtained from rat serve now as a database for the elucidation of age-dependent changes, including alterations in protein-protein interactions as well as in posttranslational modifications.

Native DIGE proteomic analysis of mitochondria from substantia nigra and striatum during neuronal degeneration and its compensation in an animal model of early Parkinson's disease.

Cause of Parkinson's disease (PD) is still not understood. Motor symptoms are not observed at early stages of disease due to compensatory processes. Dysfunction of mitochondria was indicated already at preclinical PD. Selective toxin 6-OHDA was applied to kill dopaminergic neurons in substantia nigra and disturb neuronal transmission in striatum. Early phase of active degeneration and later stage, when surviving cells adapted to function normally, were analysed. 2D BN/SDS difference gel electrophoresis (DIGE) of mitochondrial proteome enabled to point out crucial processes involved at both time-points in dopaminergic structures. Marker proteins such as DPYSL2, HSP60, ATP1A3, EAAT2 indicated structural remodelling, cytoskeleton rearrangement, organelle trafficking, axon outgrowth and regeneration. Adaptations in dopaminergic and glutamatergic neurotransmission, recycling of synaptic vesicles, along with enlargement of mitochondria mass were proposed as causative for compensation. Changed expression of carbohydrates metabolism and oxidative phosphorylation proteins were described, including their protein-protein interactions and supercomplex assembly.

Upregulation of cytochrome c oxidase subunit 6b1 (Cox6b1) and formation of mitochondrial supercomplexes: implication of Cox6b1 in the effect of calorie restriction.

Calorie restriction (CR), a non-genetic intervention that promotes longevity in animals, may exert anti-aging effects by modulating mitochondrial function. Based on our prior mitochondrial proteome analysis, we focused on the potential roles of cytochrome c oxidase (Cox or Complex IV) subunit 6b1 on formation of mitochondrial supercomplexes comprised of Complex I, III, and IV. Blue native polyacrylamide gel electrophoresis followed by immunoblotting showed that the amount of Cox6b1 and the proportion of high molecular weight supercomplexes (SCs) comprised of Complexes I, III, and IV were increased in the liver of mice subjected to 30 % CR, compared with the liver of mice fed ad libitum. In in vitro experiments, in Cox6b1-overexpressing NIH3T3 (Cox6b1-3T3) cells, Cox6b1 was increased in the SC, III2IV1, and III2IV2 complexes and Cox was concomitantly recruited abundantly into the SC, compared with control (Con)-3T3 cells. The proportions of III2IV1, and III2IV2, relative to IV monomer were also increased in Cox6b1-3T3 cells. Cox6b1-3T3 cells showed increased oxygen consumption rates, Cox activity, and intracellular ATP concentrations, indicating enhanced mitochondrial respiration, compared with Con-3T3 cells. Despite the increased basal level of mitochondrial reactive oxygen species (ROS), cell viability after inducing oxidative stress was greater in Cox6b1-3T3 cells than in Con-3T3 cells, probably because of prompt activation of protective mechanisms, such as nuclear translocation of nuclear factor E2-related factor-2. These in vivo and in vitro studies show that Cox6b1 is involved in regulation of mitochondrial function by promoting the formation of SC, suggesting that Cox6b1 contributes to the anti-aging effects of CR.

Ageing alters the supramolecular architecture of OxPhos complexes in rat brain cortex.

Activity and stability of life-supporting proteins are determined not only by their abundance and by post-translational modifications, but also by specific protein-protein interactions. This holds true both for signal-transduction and energy-converting cascades. For vital processes such as life-span control and senescence, to date predominantly age-dependent alterations in abundance and to lesser extent in post-translational modifications of proteins are examined to elucidate the cause of ageing at the molecular level. In mitochondria of rat cortex, we quantified profound changes in the proportion of supramolecular assemblies (supercomplexes) of the respiratory chain complexes I, III(2), IV as well as of the MF(o)F(1) ATP synthase (complex V) by 2D-native/SDS electrophoresis and fluorescent staining. Complex I was present solely in supercomplexes and those lacking complex IV were least stable in aged animals (2.4-fold decline). The ATP synthase was confirmed as a prominent target of age-associated degradation by an overall decline in abundance of 1.5-fold for the monomer and an 2.8-fold increase of unbound F(1). Oligomerisation of the ATP synthase increases during ageing and might modulate the cristae architecture. These data could explain the link between ageing and respiratory control as well as ROS generation.

Defining the mitochondrial proteomes from five rat organs in a physiologically significant context using 2D blue-native/SDS-PAGE.

In accordance with their manifold tasks, various dysfunctions of mitochondria are critically involved in a large number of diseases and the aging process. This has inspired considerable efforts to identify all the mitochondrial proteins by denaturing approaches, notably, the standard gel-based method employing isoelectric focusing. Because a significant part of the mitochondrial proteome is membrane-associated and/or functions as homo- or heterooligomeric protein complexes, there is an urgent need to detect and identify mitochondrial proteins, both membranous and soluble ones, under conditions preserving protein-protein interactions. Here, we investigated mitochondria of five different rat organs (kidney, liver, heart, skeletal muscle, and brain) solubilized with digitonin, enabling the quantitative extraction of the five oxidative phosphorylation (OXPHOS) complexes. The analysis by blue-native (BN)-PAGE recovered the OXPHOS complexes to a large extent as supercomplexes and separated many other protein complexes and individual proteins which were resolved by subsequent 2D SDS-PAGE revealing the tissue-diverse mitochondrial proteomes. Using MS peptide mass fingerprinting, we identified in all five organs 92 nonredundant soluble and membrane-embedded non-OXPHOS proteins, among them, many as constituents of known mitochondrial protein complexes as well as novel ones such as the putative "stomatin-like protein 2 complex" with an apparent mass of ca. 1800 kDa. Interestingly, the identification list included 36 proteins known or presumed to be localized to nonmitochondrial compartments, for example, glycolytic enzymes, clathrin heavy chain, valosin-containing protein/p97, VoV1-ATPase, and Na,K-ATPase. We expect that more than 200 distinct non-OXPHOS proteins of digitonin-solubilized rat mitochondria separated by 2D BN/SDS-PAGE, representing a partial "protein interactome" map, can be identified.