Key Event Overview
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AOPs Including This Key Event
|AOP Name||Event Type||Essentiality|
|Inhibition of the mitochondrial complex I of nigra-striatal neurons leads to parkinsonian motor deficits||KE||Strong|
|Rattus sp.||Rattus sp.||Strong||NCBI|
Level of Biological Organization
How this Key Event works
Under physiological conditions complex I (CI) couples the oxidation of NADH to NAD+ by reducing flavin mononucleotide (FMN) to FMNH2. FMNH2 is then oxidized through a semiquinone intermediate. Each electron moves from the FMNH2 to Fe-S clusters, and from the Fe-S clusters to ubiquinone (Q). Transfer of the first electron results in the formation of the free-radical (semiquinone) form of Q, and transfer of the second electron reduces the semiquinone form to the ubiquinol form (CoQH2). Altogether, four protons are translocated from the mitochondrial matrix to the inter-membrane space for each molecule of NADH oxidized at CI. This leads to the establishment of the electrochemical potential difference (proton-motive force) that may be used to produce ATP (Garrett and Grisham, 2010). Binding of an inhibitor attenuates or completely blocks the activity of CI, i.e. the oxidation of NADH is impaired and protons are not moved. This causes two major consequences: first, electrons are channelled toward oxygen instead Q. This impairs normal oxygen reduction into water at complex IV and leads to the formation of the ROS superoxide at other sites of the respiratory chain. Superoxide may cause damage of proteins, lipid and DNA of the cell, or damage components of the mitochondria after transformation into e.g. hydrogen peroxide. These processes result in mitochondrial dysfunction (Voet and Voet., 2008). The second consequence is the increase of the NADH/NAD+ ratio in mitochondria. This affects the function of key dehydrogenase enzymes in the citric acid cycle and can lead to its block, resulting in an inhibition of mitochondrial ATP production and mitochondrial respiration. The functional consequences of CI inhibition have been titrated in a time- and dose-dependent manner (Barrientos and Moraes, 1999), with mitochondrial dysfunction measured by a range of different assays (Barrientos and Moraes, 1999; Greenamyre et al., 2001). These included quantification of ROS derived from mitochondria, and of cellular respiration (see KE2: Mitochondrial dysfunction).
How it is Measured or Detected
Methods that have been previously reviewed and approved by a recognized authority should be included in the Overview section above. All other methods, including those well established in the published literature, should be described here. Consider the following criteria when describing each method: 1. Is the assay fit for purpose? 2. Is the assay directly or indirectly (i.e. a surrogate) related to a key event relevant to the final adverse effect in question? 3. Is the assay repeatable? 4. Is the assay reproducible?
As CI has an enzymatic function as such, but also contributes to the overall function of oxidative phosphorylation, there are two fundamental approaches to assess CI inhibition. The first approach measures the enzymatic activity of the complex itself; the second one assesses the overall activity of oxidative phosphorylation of entire mitochondria, and indirectly infers from this a potential dysfunction of CI.
I. Direct detection of complex I activity This type of assay is always performed in homogenates of cells or tissues, and requires at least a partial purification of mitochondria or respiratory chain components. In order to focus on CI activity, the activities of Complexes III (e.g. antimycin A) and complex IV (e.g. cyanide) need to be blocked by pharmacological inhibitors in these setups.
1. Forward Electron Transfer Submitochondrial particles or intact isolated mitochondria are incubated with NADH as electron donor and with an electron acceptor to measure the flow of electrons from NADH, through CI to the acceptor. As readout, either the consumption of NADH, or the reduction of the electron acceptor is followed photometrically or fluorometrically (Lenaz et al. 2004; Spinazzi et al. 2012; Long et al. 2009; Kirby et al. 2007). The physiological electron acceptor of CI is Coenzyme Q10 (CoQ10). Due to its hydrophobicity, it is not suitable for use in an experimental in vitro setup. Short-chain analogs of CoQ10, such as CoQ1 or decylubiquinone (DB) with a 10 carbon-atom linear saturated side chain are hence applied as alternatives. With these non-physiological electron acceptors, it is important to consider that the activity of CI can easily be underestimated. As water-soluble electron acceptors, either ferricyanide or 2,6-dichlorophenolidophenol (DCIP) are used. However the reduction of such compounds is not strictly coupled to the transduction of energy. To identify the portion of rotenone-inhibitable CI activity, all samples investigated are assayed in parallel following treatment with rotenone. In contrast to the autoradiography assays, direct CI activity detection allows the identification also of CI inhibitors that bind to sites of CI different from the rotenone binding site.
2. Reverse Electron Transfer An alternative setup for the direct measurement of CI activity with minimal interference by the activities of complex III and complex IV make use of the observation of a general reversibility of oxidative phosphorylation and electron flow across the mitochondrial respiratory chain (Ernster et al. 1967). With this method, electrons enter the respiratory chain via complex II. Based on the reverse flux, this method allows the complete circumvention of complexes III and IV. As electron donor, succinate is applied, together with NAD+ as electron acceptor. Formation of NADH from NAD+ can be determined photometrically. The succinate-linked NAD+ reduction can be performed either with intact isolated mitochondria or with submitochondrial particles. For the direct assessment of CI activity, submitochondrial particles are used. For assays with intact mitochondria, the succinate-linked reduction of NAD+ is performed in the presence of ATP as energy source. Potassium cyanide (KCN) is added for inhibition of forward electron transport towards complex IV.
3. Complex I activity dipstick assay To assess CI activity and its inhibition in cell or tissue homogenates without interference by other components of the respiratory chain, CI-selective antibodies attached to a matrix (e.g. multiwell plates) are used (Willis et al., 2009). Homogenized tissue can directly be added for capturing of CI, the unbound supernatant is washed away and leaves a complex of the antibody and mitochondrial CI. For activity determination, NADH as electron donor and nitroblue tetrazolium (NBT) as acceptor are added. Reduced NBT forms a colored precipitate, its signal intensity is proportional to the amount of CI bound to the antibody. CI inhibitors can directly be added for an assessment of their inhibitory potential. This method, when applied in e.g. 96-well or 384-well plates, allows screening of large sets of potential CI inhibitors without any interference by other elements of the mitochondrial respiratory chain.
II. Indirect measurements of complex I activity. Such assays mostly require / allow the use of live cells.
1. Oxygen consumption Electrons, fed into the mitochondrial respiratory chain either by CI or complex II, ultimately reduce molecular oxygen to water at complex IV. In a closed system, this consumption of oxygen leads to a drop of the overall O2 concentration, and this can serve as parameter for mitochondrial respiratory activity. Measurements are traditionally done with a Clark electrode, or with more sophisticated optical methods. At the cathode of a Clark electrode, oxygen is electrolytically reduced, which initiates a current in the electrode, causing a potential difference that is ultimately recorded. Clark electrodes however have the disadvantage that oxygen is consumed. Furthermore, interferences with nitrogen oxides, ozone, or chlorine are observed (Stetter et al., 2008). To circumvent these limitations, optical sensors have been developed that have the advantage that no oxygen is consumed, combined with a high accuracy and reversibility. Optical oxygen sensors work according to the principle of dynamic fluorescence quenching. The response of the respective fluorescence dye is proportional to the amount of oxygen in the sample investigated (Wang and Wolfbeis, 2014). In a model of isolated mitochondria in the absence of complex II substrates, oxygen consumption can serve as surrogate readout for the assessment of the degree of CI inhibition. It is however essential to realize that also complex III and complex IV activities are involved and their inhibition also results in a decline in O2 consumption. In addition to that, CI inhibitors can lead to a one-electron reduction of molecular oxygen at the site of CI to yield superoxide. The amount of superoxide formed hence contributes to the consumption of oxygen, but this must not be interpreted as oxygen consumption as a result of controlled and coupled electron flux through the complexes of the mitochondrial respiratory chain. A modern convenient method to measure oxygen consumption is provided by the Seahorse technology of extracellular flux (XF) analysis, in which cells are kept in a very small volume, so that changes of oxygen levels can be detected very sensitively by an oxygen sensor. To allow manipulation of the mitochondria in cells, the cell membrane can be permeabilized with saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), to allow addition of specific substrates to measure activity of different respiratory chain complexes, including CI. (Salabei et al., 2014).
2. Intracellular ATP levels Intracellular ATP levels originate both from mitochondria and from glycolysis. If glycolytic ATP production is impaired or inhibited, the cellular production of ATP is a measure of mitochondrial function. If it is assumed that the ATP consumption remains constant, then the steady state ATP levels can serve as indirect readout for mitochondrial activity, and the latter depends on the functioning of CI. Inhibitors of CI reduce cellular ATP levels, but it has to be remembered that intracellular ATP levels are also affected by inhibitors of other parts of the respiratory chain, of the citric acid cycle or of the transport of energy substrates. For a proper interpretation of assay results, it has to be ascertained in each particular test system, that ATP production from other sources is excluded and that the cellular ATP consumption remains constant. ATP levels can be easily measured from lysates of in vitro cell cultures or from tissues by a luminometric luciferase/luciferin assay. The amount of light emitted is proportional to the amount of ATP in the sample (Nguyen et al. 1988) (Leist, 1997).
3. Other approaches As mitochondrial activity is coupled to many cellular functions, there is a multitude of other indirect assays that are sensitive to inhibitors of CI. Some of these tests may indeed be very sensitive, while they have a low specificity. Thus, their application requires usually a good control of the experimental system and care with the interpretation of the data. One exemplary approach is the measurement of NADH/NAD+ ratios in mitochondria by imaging methods. This provides resolution on the level of individual mitochondria within a living cell (van Vliet et al., 2014)
Evidence Supporting Taxonomic Applicability
The CI is well-conserved across species from lower organisms to mammals. The central subunits of CI harboring the bioenergetic core functions are conserved from bacteria to humans. CI from bacteria and from mitochondria of Yarrowia lipolytica, a yeast genetic model for the study of eukaryotic CI (Kerscher et al., 2002) was analyzed by x-ray crystallography (Zickermann et al., 2015, Hofhaus et al., 1991; Baradaran et al., 2013). The CI of the mitochondria of eukaryotes and in the plasma membranes of purple photosynthetic bacteria are closely related to respiratory bacteria and the close homology of sequences, function, and prosthetic groups shows a common ancestry (Friedrich et al., 1995).
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