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Relationship: 933

Title

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Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I) leads to Inhibition, NADH-ubiquinone oxidoreductase (complex I)

Upstream event

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Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I)

Downstream event

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Inhibition, NADH-ubiquinone oxidoreductase (complex I)

Key Event Relationship Overview

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AOPs Referencing Relationship

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AOP Name Adjacency Weight of Evidence Quantitative Understanding
Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits adjacent High Low

Taxonomic Applicability

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Sex Applicability

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Life Stage Applicability

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Key Event Relationship Description

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It is well documented that binding of an inhibitor to CI inhibits its activity (see MIE). Naturally occurring and synthetic CI inhibitors have been shown to inhibit the catalytic activity of CI, leading to partial or total inhibition of its activity in a dose response manner (Degli Esposti and Ghelli, 1994; Ichimaru et al. 2008; Barrientos and Moraes, 1999; Betarbet et al., 2000). Indeed, binding of inhibitors stops the electron flow from CI to ubiquinone. Therefore, the Fe-S clusters of CI become highly reduced and no further electrons can be transferred from NADH to CI. This leads to the inhibition of the NADH oxido-reductase function, i.e. CI inhibition.

Evidence Supporting this KER

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Biological Plausibility

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The weight of evidence supporting the relationship between binding of an inhibitor to NADH-ubiquinone oxidoreducatse and its inhibition is strong.

Biological Plausibility

There is an extensive understanding of the functional relationship between binding of an inhibitor to NADH-ubiquinone oxidoreductase (CI) and its inhibition. As the first entry complex of mitochondrial respiratory chain, CI oxidizes NADH and transfers electrons via a flavin mononucleotide cofactor and several Fe-S complexes to ubiquinone. The electron flow is coupled to the translocation of protons from the matrix to the intermembrane space. This helps to establish the electrochemical gradient that is used to fuel ATP synthesis (Greenamyre et al., 2001). If an inhibitor binds to CI, the electron transfer is blocked. This compromises ATP synthesis and maintenance of Δψm, leading to mitochondrial dysfunction. As CI exerts a higher control over oxidative phosphorylation in synaptic mitochondria than in non-synaptic mitochondria in the brain (Davey and Clark, 1996), specific functional defects observed in PD may be explained. It is well documented that CI inhibition is one of the main sites at which electron leakage to oxygen occurs. This results in a production of ROS, such as superoxide (Efremov and Sazanow, 2011) and hydrogen peroxide, which are main contributors to oxidative stress (Greenamyre et al., 2001).

Empirical Evidence

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A variety of studies show a significant correlation between binding of an inhibitor to CI and its inhibition, usually measured by the decreased mitochondrial respiration. Different classes of CI inhibitors, such as rotenone, MPP+, piericidin A, acetogenins, pyridaben, and various piperazin derivatives (Ichimaru et al. 2008) have been shown to bind to the ubiquitin site of CI, leading to a partial or total inhibition of oxidoreductase activity in a dose response manner (Grivennikova et al., 1997; Barrientos and Moraes, 1999; Betarbet et al., 2000). The reduction of CI activity is well documented in a variety of studies using isolated mitochondria or cells, as well as in in vivo experiments and in human post mortem PD brains. Usually it is measured by assays described in 2nd Key Event Relationship (KER): Inhibition of complex I leads to mitochondrial dysfunction. It has been shown that binding of rotenone to CI (e.g. Betarbet et al., 2000, Greenamyre et al., 2001) or MPP+ (e.g. Krug et al., 2014; Langston, 1996) can reproduce the anatomical, neurochemical, behavioural and neuropathological features of PD. Therefore, the empirical support for this KER will be mainly based on the experiments performed after exposure to rotenone or MPP+.

  • The binding of rotenone to CI resulted in time- and dose-dependent inhibition of CI activity measured in sub-mitochondrial particles. The kinetics of the active CI inhibition was determined after exposure to rotenone at 20, 30 and 40 nM at different times of exposure (30 sec, 1 min or 2 min) (Grivennikova et al., 1997). This study suggests that two rotenone binding sites exist in CI: one affecting NADH oxidation by ubiquinone and the other one operating in ubiquinol-NAD+ reductase action.
  • Partial inhibition of CI produces a mild, late-onset mitochondrial damage. The threshold effect seen in brain mitochondria (25–50% decrease in activity) may not directly impact ATP levels or Δψm but could have long-term deleterious effects triggered by oxidative stress, as it has been shown that an electron leak upstream of the rotenone binding site in CI leads to ROS production (Greenamyre et al., 2001).
  • Exposure of rats to rotenone (2 days, 2 mg/kg) produced free brain rotenone concentration of 20–30 nM and resulted in 73% inhibition of specific binding to CI of [3H] dihydrorotenone (Betarbet et al., 2000). However, oximetry analysis indicated that in brain mitochondria (but not liver mitochondria) this rotenone concentration (30 nM maximum) was insufficient to inhibit glutamate (CI substrate)-supported respiration (Betarbet et al., 2000) suggesting that this rotenone concentration did not alter mitochondrial oxygen consumption in isolated brain mitochondria.
  • Rotenone has been reported to be a specific and potent mitochondrial CI inhibitor with IC50 values from 0.1 nM to 100 nM depending on the system and methods used (Lambert and Brand, 2004; Ichimaru et al., 2008; Chinopoulos and Vizi, 2001; Beretta et al., 2006).
  • Mesencephalic cultures prepared from C57/BL6 mice and treated with 5, or 10 nM rotenone for 24 h inhibited CI activity by 11% or 33%, respectively (Choi et al., 2008).
  • The inhibition of CI was studied in the human osteosarcoma-derived cell line (143B) after the exposure to rotenone or using a genetic model (40% loss of CI activity in human xenomitochondrial cybrids (HXC) lines). Different degrees of CI inhibition were quantitatively correlated with levels of decreased cellular respiration (Barrientos and Moraes, 1999). Only when CI was inhibited by 35-40% (< 5 nM rotenone), cell respiration decreased linearly until 30% of the normal rate. Increasing concentrations of rotenone produced further but slower decrease in CI activity and cell respiration (Fig. 1). Cells with the complete rotenone-induced CI inhibition still maintain a cell respiration rate of approximately 20% because of an electron flow through complex II. At high concentrations (5–6-fold higher than the concentration necessary for 100% CI inhibition), rotenone showed a secondary, toxic effect at the level of microtubule assembly (Barrientos and Moraes 1999).
  • Bovine sub-mitochondrial particles were used to test rotenone affinity binding at 20 nM. This concentration of rotenone reduced the NADH oxidation rate by approximately 50% (Okun et al., 1999.
  • MPP+ (an active metabolite of MPTP) is an inhibitor of CI (Nicklas et al., 1987; Mizuno et al, 1989; Sayre et al., 1986). Inhibition of the mitochondrial CI by MPP+ supresses aerobic glycolysis and ATP production (Book chapter in Cheville 1994).
  • MPP+ binds loosely to CI and causes reversible inhibition of its activity: approximately 40% inhibition was observed at 10 mM concentration within 15 min of incubation. However, prolonged incubation (> 15min) produces up to 78% of irreversible inhibition of CI (Cleeter et al., 1992).
  • MPP+, interrupts mitochondrial electron transfer at the NADH dehydrogenase-ubiquinone junction, as do the respiratory chain inhibitors rotenone, piericidin A and barbiturates. The 4'-alkyl derivatives of MPP+ inhibit NADH oxidation in submitochondrial particles at much lower concentrations than does MPP+ itself. The MPP+ analogues with short alkyl chains prevent the binding of 14C-labelled piericidin A to the membrane and thus must act at the same site, but analogues with alkyl chains longer than heptyl do not prevent binding of [14C]piericidin (Ramsay et al., 1991).

  • 13 complex I inhibitors  were found to decrease labelling at the PSST site without effect on ND1 labelling.  The results suggest that the common action of MPP(+) and stigmatellin on the functional coupling of the PSST and ND1 subunits is initiated by binding at a semiquinone binding site in complex I (Schuler and Casida, 1991).

Human studies

  • There are many studies that show impaired catalytic activity of CI in multiple PD post-mortem brain tissues. For example (Parker and Swerdlow, 1998), five PD brains were used to measure activities of complexes I, III, IV, and of complexes I/III together (NADH: cytochrome c reductase). These measurements were performed in purified frontal cortex mitochondria and revealed a significant loss of CI activity in these PD samples as compared to controls.
  • Human data indicate that impairment of CI activity may contribute to the pathogenic processes of PD (for example, Greenamyre et al., 2001; Schapira et al., 1989; Shults, 2004).

Uncertainties and Inconsistencies

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It is not clear the number of subunits constituting CI in mammals, as according to the existing literature different numbers are cited (between 41-46) (Vogel et al., 2007a; Hassinen, 2007). The majority of data claims that mammalian CI is composed of 46 (Greenamyre et al., 2001; Hassinen, 2007) or 45 subunits (Vogel et al., 2007a). It is not sure whether there may exist tissue-specific subunits of CI isoforms (Fearnley et al., 2001). It is unclear, which subunit(s) bind rotenone or other inhibitors of CI. Additionally, it is not clear whether CI has other uncharacterized functions, taking into consideration its size and complexity (43-46 subunits vs. 11 subunits of complex III or 13 subunits of complex IV) (Greenamyre et al., 2001). There is no strict linear relationship between inhibitor binding and reduced mitochondrial function. Low doses of rotenone that inhibit CI activity partially do not alter mitochondrial oxygen consumption. Therefore, bioenergetic defects can not account alone for rotenone-induced neurodegeneration. Instead, under such conditions, rotenone neurotoxicity may result from oxidative stress (Betarbet et al., 2000). Few studies used human brain cells/human brain mitochondria. Therefore, full quantitative data for humans are not available.

Quantitative Understanding of the Linkage

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The kinetics of binding and CI inhibition by rotenone has been quantitatively evaluated in a dose-dependent manner using the sub-mitochondrial particles (Grivennikova et al., 1997). The consequences of CI inhibition were quantitatively measured by a variety of assays that are used to study mitochondrial dysfunction (see Key Event Relationship (KER): Inhibition of Complex I leads to mitochondrial dysfunction). There are also many in vitro and in vivo studies combining the quantification of CI inhibition and DA cell death (e.g. Choi et al., 2008, Betarbet et al., 2000, see KER Mitochondrial dysfunction induces degeneration of nigrostriatal pathway).

The binding of different classes of inhibitors (e.g., pesticides, drugs and other toxins) to CI has been determined quantitatively and I50, and KI values are available. Potency relative to that of rotenone has been determined under the same conditions in beef mitochondria or submitochondrial particles using the ratio of the KI values, when they were available (Degli Esposti, 1998; Okun et al., 1999). Rotenone I50 value is defined as 20 nM (Okun et al., 1999).


Example of a quantitative evaluation of concentration-dependent CI inhibition by rotenone (Fig. 1 from Barrientos and Moraes, 1999).


KER 1Fig. 1.jpg

Fig. 1. Fig.1. Effect of CI (NADH decylubiquinone reductase) inhibition on endogenous cell respiration. Cells were treated with different concentrations of rotenone for 4 h before measuring cell respiration in whole cells and CI activity in isolated mitochondria. Complete CI inhibition was achieved with 100 nM rotenone. The cell respiration was inhibited also in a dose-dependent manner but showed different inhibition kinetics and a saturation threshold. For comparison, the genetically-altered cell line HXC had an approximately 40% CI reduced activity and an approximately 80% residual cell respiration. HXC, human xenomitochondrial cybrids.

 

Time- and concentration-relationship of NADH oxidase inhibition by rotenone (Fig. 2. from Grivennikova et al., 1997).

KER1 Fig. 2.jpg


Fig. 2. Panel A and B: Time- and concentration-relationship of NADH oxidase inhibition by rotenone. The numbers on the curves indicate the final concentrations of rotenone (0, 20, 30, 40, 1000 nM). In Panel B: vo, zero-order rate of NADH oxidation in the absence of rotenone; vt, the `instant' values of the rates approximated within 10 s time intervals. Panel C: The dependence of first-order inhibition rate constant on the concentration of rotenone (for further description see Fig. 1 in Grivennikova et al., 1997).

 

Quantitative evaluation of the 1st KER: Binding of inhibitor to NADH-ubiquinone oxidoreductase (MIE; KE upstream) leads to its inhibition (KE downstream).

 

MIE (KE upstream)

Binding of inhibitor to NADH-ubiquinone oxidoreductase (nM)

KE (downstream)

Inhibition of CI (%, approximately)

Comments

(in vivo, in vitro or human studies)

References

Administration of rotenone at 2 mg/kg per day for 2 days

resulted in free rotenone concentration of 20–30 nM in the brain.

75%

 

DA neuronal cell death determined after rotenone administration at 1 to 12 mg/kg per day, Sprague Dawley and Lewis rats infused continuously by jugular vein, 7days up to 5 weeks

Betarbet et al., 2000

 

20 nM rotenone

Direct binding studies using bovine and Musca domestica sub-mitochondrial particles

 

50%

Binding studies that defined the I50 and Kd values for three classes of CI inhibitors (12 chemicals) including rotenone.

Okun et al., 1999

Human skin fibroblasts exposed to 100 nM Rotenone for 72 hr

20%

In the same experiment mitochondria morphology, motility was also evaluated.

Koopman et al., 2007

0-2.5 nM Rotenone

5/10 nM Rotenone

Mesencephalic neurons

were cultured from E14 C57/BL6 mouse embryos for 6 days and then treated with rotenone for 24 hr

No effect

11% and33%, respectively


Treatments with 5 or 10 nM rotenone killed 50% or 75% DA neurons respectively.

Choi et al., 2008

1-2.5-5-7.5-10-20 nM

 

1-10-20-80 nM

 

 

10-20-35-50- 65-80 %

5- 75 %

 

 

 

In this study time course of the active and deactivated enzymes inhibition by rotenone and Piericidin A is study in a dose-dependent manner.

Binding studies in sub-mitochondrial particles prepared from bovine heart after 20 min of exposure to rotenone.

Grivennikova et al., 1997

5-10 nM

20 nM

40 nM

100 nM

143B Cells (human osteo-sarcoma), exposed for 4 hrs to rotenone

55-78 %

80%

87%

100%

In the same study similar experiments were performed using HXC cell line (see Fig. 1 above).

Barrientos and Moraes 1999

Response-response Relationship

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Time-scale

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Known modulating factors

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Known Feedforward/Feedback loops influencing this KER

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Domain of Applicability

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The CI is well-conserved across species from lower organism 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). However, the affinity of various chemicals to cause partial or total inhibition of CI activity across species is not well studied (except rotenone).

References

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Barrientos A, and Moraes CT. 1999. Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology. 274(23)16188–16197.

Beretta S, et al. 2006. Partial mitochondrial complex I inhibition induces oxidative damage and perturbs glutamate transport in primary retinal cultures. Relevance to Leber Hereditary Optic Neuropathy (LHON). Neurobiol Dis. 24:308–317.

Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. 2000. Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat Neurosci 3:1301-1306.

Cheville NF. 1994. Ultrastructural Pathology: The Comparative Cellular Basis of Disease Wiley John Wiley & Sons, 09 dic 2009 - 1000 pagine Chinopoulos C, Adam-Vizi V. 2001. Mitochondria deficient in complex I activity are depolarized by hydrogen peroxide in nerve terminals: Relevance to Parkinson’s disease. J Neurochem. 76:302–306.

Choi WS, Kruse SE, Palmiter R, Xia Z. 2008. Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP, or paraquat. PNAS. 105(39):15136-15141.

Cleeter MW, Cooper JM, Schapira AH. 1992. Irreversible inhibition of mitochondrial complex I by 1-methyl-4-phenylpyridinium: evidence for free radical involvement. J Neurochem. 58(2):786-9.

Davey GP, Clark JB. 1996. Threshold effects and control of oxidative phosphorylation in nonsynaptic rat brain mitochondria, J. Neurochem. 66:1617 24.

Degli Esposti M, Ghelli A. 1994. The mechanism of proton and electron transport in mitochondrial complex I. Biochim Biophys Acta.1187(2):116–120.

Degli Esposti (1998) Inhibitors of NADH-ubiquinone reductase: an overview Biochimica et Biophysica Acta 1364-222-235. Efremov RG, Sazanov LA. Structure of the membrane domain of respiratory complex I. Nature. 2011 Aug 7;476(7361):414-20.

Fearnley IM, Carroll J, Shannon RJ, Runswick MJ, Walker JE, and Hirst J. 2001. GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductas (complex I). J. Biol. Chem. 276(42):38345-8.

Greenamyre TJ, Sherer TB, Betarbet R, and Panov AV. 2001. Complex I and Parkinson’s Disease. Critical Review.IUBMB Life, 52: 135–141.

Grivennikova VG, Maklashina EO, Gavrikova EV, Vinogradov AD. 1997. Interaction of the mitochondrial NADH-ubiquinone reductase with rotenone as related to the enzyme active/inactive transition. Biochim et Biophys. Acta. 1319:223–232.

Hassinen I. 2007. Regulation of Mitochondrial Respiration in Heart Muscle. In Mitochondria – The Dynamic Organelle Edited by Schaffer & Suleiman. Springer ISBN-13: 978-0-387-69944-8.

Ichimaru N, Murai M, Kakutani N, Kako J, Ishihara A, Nakagawa Y, Miyoshi H. 2008.. Synthesis and Characterization of New Piperazine-Type Inhibitors for Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I). Biochemistry. 47(40)10816–10826.

Koopman W, Hink M, Verkaart S, Visch H, Smeitink J, Willems P. 2007. Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy. Biochimica et Biophysica Acta 1767:940-947. Krug AK, Gutbier S, Zhao L, Pöltl D, Kullmann C, Ivanova V, Förster S, Jagtap S, Meiser J, Leparc G, Schildknecht S, Adam M, Hiller K, Farhan H, Brunner T, Hartung T, Sachinidis A, Leist M (2014) Transcriptional and metabolic adaptation of human neurons to the mitochondrial toxicant MPP(+). Cell Death Dis. 8(5):e1222. doi: 10.1038/cddis.2014.166. Lambert AJ, Brand MD. Inhibitors of the quinone-binding site allow rapid superoxide production from mitochondrial NADH:ubiquinone oxidoreductase (complex I). J Biol Chem. 2004 Sep 17;279(38):39414-20.

Langston JW. 1996. The etiology of Parkinson’s disease with emphasis on the MPTP story. Neurology. 47, S153–160.

Mizuno Y, Ohta S, Tanaka M, Takamiya S, Suzuki K, Sato T, Oya H, Ozawa T, Kagawa Y. 1989. Deficiencies in complex I subunits of the respiratory chain in Parkinson's disease. Biochem Biophys Res Commun. 29;163(3):1450-5.

Nicklas WJ, Yougster SK, Kindt MV, Heikkila RE. 1987. MPTP, MPP+ and mitochondrial function. Life Sci. 40:721-729.

Okun JG, Lummen PL, Brandt U. 1999. Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase) 274(5)2625–2630.

Parker Jr WD, Swerdlow RH. 1998. Mitochondrial dysfunction in idiopathic Parkinson disease. Am J Hum Genet 62:758 –762.

Ramsay RR, Krueger MJ, Youngster SK, Singer TP. Evidence that the inhibition sites of the neurotoxic amine 1-methyl-4-phenylpyridinium (MPP+) and of the respiratory chain inhibitor piericidin A are the same. Biochem J. 1991 Jan 15;273(Pt 2):481-4.

Sayre LM, Arora PK, Feke SC, Urbach FL. 1986. Mechanism of induction of Parkinson’s disease by I-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP). Chemical and electrochemical characterization of a geminal-dimethyl-blocked analogue of a postulated toxic metabolite. J Am Chem Sot. 108:2464-2466.

Schapira AH, Cooper JM, Dexter D, Jenner P, Clark JB, and Marsden CD. 1989. Mitochondrial complex I deficiency in Parkinson’s disease. Lancet. 1,1269.

Schuler F, Casida JE.The insecticide target in the PSST subunit of complex I. Pest Manag Sci. 2001 Oct;57(10):932-40.

Shults CW. 2004. Mitochondrial dysfunction and possible treatments in Parkinson’s disease–a review. Mitochondrion 4:641– 648.

Vogel RO, van den Brand MA, Rodenburg RJ, van den Heuvel LP, Tsuneoka M, Smeitink JA, Nijtmans LG. (2007a). Investigation of the complex I assembly chaperones B17.2L and NDUFAF1 in a cohort of CI deficient patients. Mol. Genet. Metab. 91:176–182.