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Relationship: 934

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Inhibition, NADH-ubiquinone oxidoreductase (complex I) leads to Mitochondrial dysfunction

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Inhibition, NADH-ubiquinone oxidoreductase (complex I)

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Mitochondrial dysfunction

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits	adjacent	High	Moderate	Andrea Terron (send email)	Open for citation & comment	WPHA/WNT Endorsed

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Sex Applicability

An indication of the the relevant sex for this KER. More help

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Inhibited CI is unable to pass off its electron to ubiquinone and it cannot translocate protons across the mitochondrial inner membrane. This creates a back-up of NADH within the mitochondrial matrix (Brown and Borutaite, 2004). This leads to an arrest of the citric acid cycle and a failure to build a proton gradient (mitochondrial membrane potential, Δψm) across the inner membrane. This results in impaired ATP production. In addition, the direct transfer of electrons from CI to oxygen is increased. This leads to oxidative stress as ROS (e.g. superoxide, hydrogen peroxide) are produced, which can damage DNA, proteins, lipids and other cell components and function (Sanders et al., 2014).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

The weight of evidence supporting the relationship between inhibition of CI and mitochondrial dysfunction is strong. The mechanisms behind this KER are partially understood and well documented based on in vitro as well as in vivo experiments (e.g., Sanders et al., 2014), complemented by data from human post-mortem PD brain evaluations (Parker et al., 1989; Greenamyre et al., 2001; Sherer et al., 2003; Schapira et al., 1989).

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

The biological plausibility that inhibition of CI activity triggers mitochondrial dysfunction is strong. It is well understood, how the inhibition of CI can lead to mitochondrial dysfunction as measured by: a) decreased oxygen consumption, b) decrease or loss of ATP production, c) decrease of Δψm, d) the loss of mitochondrial protein import and protein biosynthesis, e) reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, f) elevated levels of ROS, g) the loss of mitochondrial motility, causing a failure of mitochondria to re-localize to sites of increased energy demands (such as synapses), h) destruction of the mitochondrial network, i) increased mitochondrial uptake of Ca2+ causing mitochondrial Ca2+ overload (Graier et al., 2007) and opening of mitochondrial PTP, (j) rupture of the mitochondrial inner and outer membranes, leading to release of mitochondrial pro-death factors, including cytochrome c, AIF and endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013). These pathological mechanisms are extremely well studied.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Many studies show that the pathophysiological consequences of a partial or total CI inhibition are linked to mitochondrial dysfunction. In many of these experiments the cellular damage caused by mitochondrial dysfunction is reduced (or entirely prevented) by treatment with antioxidants. Different degrees of CI inhibition by rotenone have been studied in the human osteosarcoma-derived cell line (143B). A quantitative correlation between increasing inhibition of CI and mitochondrial dysfunction (as shown by inhibition of mitochondrial respiration, reduced ATP production, increased ROS release and lipid peroxidation, as well as decreased Δψm) was established (Fig. 1 and Table 1 based on Barrientos and Moraes, 1999). Based on the existing literature it is suggested that rotenone exerts toxicity via oxidative stress, rather than via decrease of ATP synthesis (bioenergetics effects).

A few examples illustrating mitochondrial damage and oxidative stress in animal model of PD and human cells induced by:

Rotenone

Rotenone administered subcutaneously for 5 weeks (2.5 mg/kg/d) caused a selective increase (by ~2 folds) in oxidative damage in the striatum, as compared to the hippocampus and cortex, accompanied by massive degeneration of DA neurons (~80% decrease) in the substantia nigra. Rotenone reduced intracellular ATP levels in the striatum (by >40%), increases malondialdehyde (MDA, indicative of lipid peroxidation, by ~60%), reduced GSH levels (by ~20%), thioredoxin (by ~70%), and manganese superoxide dismutase (SOD, by ~15%) (all parameters significantly changed in the striatum). Antioxidant polydatin (Piceid) treatment significantly prevented the rotenone-induced changes by restoring the above parameters to control levels, confirming that rotenone- induced mitochondrial dysfunction resulted in oxidative stress (Chen et al., 2015).
Rotenone was administered 2.5 mg/kg body weight to male Wistar rats for 4 weeks in the presence or absence of ferulic acid (FA, at the dose of 50 mg/kg) that has antioxidant and anti-inflammatory properties. Rotenone administration caused DA neuronal cell death (~50%), significant reduction in endogenous antioxidants, such as superoxide dismutase (~75%), catalase (~40%), and glutathione (~50%), and induced lipid peroxidation evidenced by increased MDA formation (~2 folds). Treatment with FA rescued DA neurons in substantia nigra pars compacta area and nerve terminals in the striatum, as well as restored antioxidant enzymes, prevented depletion of glutathione, and inhibited lipid peroxidation induced by rotenone (Ojha et al., 2015).

Many studies have shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) functions as a cellular protector against oxidative stress by detoxification of cytotoxic aldehydes. Dopamine is metabolized by monoamine oxidase to yield 3,4-dihydroxyphenylacetaldehyde (DOPAL) then converts to a less toxic acid product by ALDH. The highly toxic and reactive DOPAL has been hypothesized to contribute to the selective neurodegeneration of DA neurons. In this study, rotenone (100 nM, 24 hr) in both SH-SY5Y cells and primary cultured substantia nigra (SN) DA neurons, was shown to reduce DA cell viability (~40%), reduce Δψm (~40%, as shown by TMRM), induce mitochondrial ROS production (~30%, as shown by increase of MitoSox Red), and increased cytosolic protein levels of proteins related to the mitochondrial apoptotic pathway (i.e. Bax, cytochrome c, active caspase-9 and active caspase-3) (~ 2 folds for all proteins).

The neuroprotective mechanism of ALDH2 was observed as overexpression of wild-type ALDH2 gene (but not the enzymatically deficient mutant ALDH2*2 (E504K)) reduced rotenone-induced DA neuronal cell death, prevented rotenone-induced reduction in TMRM signal (95.7±1.6% v.s. 67±3.5%), and prevented rotenone-induced increase in MitoSox Red intensity (103.1±1% v.s. 133.4±0.8%). Additionally, pre-treatment of cells with Alda-1 (activator of ALDH2) (1–10 μM, for 24 hr) prevented rotenone-induced loss of Δψm and ROS production in a dose-dependent manner. These results were confirmed by in vivo studies. Rotenone (50 mg/kg/day, oral administration for 14 days) or MPTP (40 mg/kg/day, i.p. for 14 days) both administered to C57BL/6 mice caused significant SN TH+ DA neuronal cell apoptosis (~50%). Alda-1 attenuated rotenone-induced apoptosis by decreasing ROS accumulation, reversing Δψm depolarization, and inhibiting the activation of proteins related to mitochondrial apoptotic pathway. The present study demonstrates that rotenone or MPP+ induces DA neurotoxicity through oxidative stress. Moreover, Alda-1 is effective in ameliorating mitochondrial dysfunction by inhibiting rotenone or MPP+ induced mitochondria-mediated oxidative stress that leads to apoptosis (Chiu et al., 2015).

Rotenone-induced mitochondrial dysfunction was observed in human neuroblastoma cells exposed to 5 nM rotenone for 1-4 weeks. After 3-4 weeks of treatment, rotenone-treated cells showed evidence of oxidative stress, including loss of GSH (by 5%) and increased oxidative DNA (qualitative, measured by using antibodies to 8-oxo-dG) and protein damage (223 ± 29% of control, as shown by the large increase in protein carbonyls in the insoluble fraction) (Sherer et al. 2002). This chronic rotenone treatment markedly sensitized cells to further oxidative challenge since in response to H2O2 cytochrome c release from mitochondria and caspase-3 activation occurred earlier and to a greater extent in rotenone-treated cells vs Ctr (1.44 ± 0.02% vs 0.38 ± 0.07% apoptosis/hr). This study indicates that chronic, low-level CI inhibition by rotenone induces progressive oxidative damage, and caspase-dependent neuronal cell death (Sherer et al., 2002).
By using anti-oxidant, kaempferol (6 μM, 1 hr prior addition of rotenone) and rotenone (50 nM, max up to 24 hr) on SH-SY5Y cells, kaempferol was found to counteract rotenone-induced ROS production (especially superoxide: with kaempferol, ethidium fluorescence decreased below the control (Ctr) levels), rotenone-induced mitochondrial oxidative dysfunction (protein carbonyls values: 2.5 in Ctr, 6.2 with rotenone, 2.7 with kaempferol + rotenone), rotenone-induced oxygen respiration (values of nmol of atomic oxygen/minute/mg protein: 5.89 Ctr, 0.45 with rotenone, 2.47 with kaempferol + rotenone), rotenone-induced Δѱm decrease (~70% cells of with rotenone only vs ~30% with kaempferol + rotenone) (Filomeni et al., 2012).

To model the systemic mitochondrial impairment, rats were exposed to rotenone. A single rotenone dose (10 nM, for 24 hr) induced mtDNA damage in midbrain neurons (>0.4 lesions/10kb vs 0 lesions/10kb in vehicle), but not in cortical neurons; similar results were obtained in vitro in cultured neurons. Importantly, these results indicate that mtDNA damage is detectable prior to any signs of neuronal degeneration and is produced selectively in midbrain neurons. The selective vulnerability of midbrain neurons to mtDNA damage was not due to differential effects of rotenone on CI since rotenone suppressed respiration equally (~60%) in midbrain and cortical neurons compared to vehicle. However, in response to CI inhibition, midbrain neurons produced more mitochondrial H2O2 (5 min of rotenone increased MitoPY1 fluorescence of ~10% in midbrain mitochondria vs vehicle, and progressively for the duration of measurement), than cortical neurons. The selective mtDNA damage in midbrain could serve as a molecular marker of vulnerable nigral neurons in PD. Oxidative damage to cell macromolecules in human PD and the rotenone model have been recently reviewed (Sanders et al., 2014).
Adult male Sprague–Dawley rats were intranigrally infused with rotenone (6 μg in 1 μl) alone or in the presence of L-deprenyl (0.1, 1, 5 and 10 mg/kg; i.p.) at 12 h intervals for 4 days. Rotenone alone (100 μM, 30 min) increased the levels of hydroxyl radials in the mitochondrial P2 fraction 2,3-DHBA (122.90 ± 5.4 pmol/mg protein) and 2,5-DHBA (146.21 ± 6.3 pmol/mg protein). L-deprenyl (100 nM–1 mM) dose-dependently attenuated rotenone-induced ·OH generation in the mitochondrial P2 fraction. L-deprenyl-induced attenuation in the rotenone-mediated 2,3-DHBA generation was from 17 ± 1.1% to 67 ± 4.3%, respectively, for 100 nM–1 mM of the MAO-B inhibitor. Also, rotenone caused about 51 ± 3.3% reduction in GSH levels in the cell body region, SN and 34 ± 1.1% decrease in the nerve terminal region, NCP (nucleus caudatus putamen). L-deprenyl alone did not cause any significant difference in the GSH content in either region. L-deprenyl treatment dose-dependently attenuated the rotenone-induced GSH depletion in SN from 51 ± 3.1% to 44 ± 2.1%, 32 ± 1.7% and 9 ± 1.0%, respectively, for doses of 1, 5 and 10 mg/kg. Additionally, SOD activity was assayed in rotenone-lesioned animals, which were treated with l-deprenyl at different doses (1–10 mg/kg). SN exhibited 2- and 3-fold activity of Cu/Zn-SOD (i.e. cytosolic SOD fraction) and Mn-SOD (i.e. particulate SOD fraction), respectively, compared to the nerve terminal region, NCP. L-deprenyl (5 and 10 mg/kg) in rotenone-lesioned animals caused a significant increase in the cytosolic Cu/Zn SOD activity in SN of both the sides. Intranigral infusion of rotenone alone caused a significant increase in the enzyme activity in SN of the side of infusion as compared to the non-infused side (~20%). L-deprenyl (5 and 10 mg/kg) further increased catalase activity in both ipsilateral SN and striatum, as compared to the contralateral side of infusion. Finally, rotenone caused a 74% reduction in the striatal TH staining intensity, which was partially recovered by L-deprenyl. These results showed that oxidative stress is one of the major causative factors underlying DA neurodegeneration induced by rotenone and they support the view that L-deprenyl is a potent free radical scavenger and an antioxidant (Saravanan et al., 2006). Similar results were obtained after exposure to MPP+ (Wu et al., 1994).

Antioxidant (Piperaceae; PLL) with some anti-inflammatory activities demonstrated in preclinical studies protective effects in PD animal models. Rats treated with rotenone and PLL-derived alkaloids showed decreased ROS, stabilized Δψm, and the opening of the mitochondrial PTP - which is triggered by ROS production - was inhibited. In addition, rotenone-induced apoptosis was abrogated in the presence of these alkaloids (Wang H. et al., 2015).
In SK-N-MC human neuroblastoma cells, rotenone (10 nM - 1µM, 48 hr) caused dose-dependent ATP depletion (~35% reduction by 100 nM rotenone vs Ctr), oxidative damage (100% increase of carbonyls levels upon 100 nM rotenone), and death (100 nM rotenone after 48 hr caused 1.1 AU (arbitrary units) increase of cell death vs untreated – 0.00 AU -). α-Tocopherol pre-treatment (62.5 or 125 μM 24 hr before rotenone (10 nm)) attenuated rotenone toxicity (Sherer et al., 2003).

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) or MPP⁺ (1-methyl-4-phenyl-pyridinium ion)

MPTP converted into MPP⁺ inhibits mitochondrial CI activity, resulting in excessive intracellular ROS production followed by further mitochondrial dysfunction leading to mitochondrial-dependent apoptosis. Lutein, a carotenoid of xanthophyll family (antioxidant) reversed MPTP-induced mitochondrial dysfunction, oxidative stress, apoptotic cell death and motor abnormalities. These results revealed that antioxidant protected DA neurons and diminished mitochondrial dysfunction and apoptotic death (Nataraj et al., 2015).
Antioxidant (salidroside; Sal) pre-treatment protected DA neurons against MPTP/MPP⁺ induced toxicity in a dose-dependent manner by: (1) reducing the production of ROS, (2) regulating the ratio of Bcl-2/Bax, (3) decreasing cytochrome-c and Smac release, and inhibiting caspase-3, caspase-6, and caspase-9 activation, which are known to trigger apoptosis following mitochondrial dysfunction. Sal acted as an effective neuroprotective agent through modulation of the ROS-induced mitochondrial dysfunction in vitro and in vivo (Wang S. et al., 2015).
In an in vitro study, MPP⁺ (1 mM, 24 hr) was found to elicit production of ROS (by 2 fold vs Ctr) and reduce by 50% SOD (by about 50%) and catalase (by about 65%) activity in SH-SY5Y human neuroblastoma cells. Pre-treatment with the antioxidant astaxanthin (AST; 50 μM, 24 hr) inhibited MPP⁺- induced production of ROS and attenuated both SOD and catalase activity decrease. Furthermore, MPP⁺ (1 mM, 48 hr) increased caspase-3 activity to 243% of the Ctr and also increased cleaved caspase-3 in the cells (qualitative). Addition of 50 μM AST attenuated MPP⁺-induced caspase-3 activation (57% suppression). MPP⁺ induced also a 70% reduction of Δψm and cytochrome c release (qualitative), while AST prevented both these effects. The protective effects of AST on MPP⁺ induced mitochondrial dysfunction was due to its anti-oxidative properties and anti-apoptotic activity via induction of expression of SOD and catalase (as shown above) and regulating the expression of Bcl-2 and Bax (Bax/Bcl-2 ratio increased to 1.6-fold vs Ctr upon treatment with MPP+, while AST prevented the MPP⁺-induced increase of the Bax/Bcl-2 ratio). These results were confirmed by in vivo studies (Lee et al., 2011).

DA neurons in primary mesencephalic cultures treated with MPP⁺ (100 μМ, for 48 hr) underwent reduction of cell viability (~55% MTT reduction), LDH release (~90%), about 60% reduction of TH+ cells, disruption of Δψm (~45% decline) and ROS production (~60% increase), upregulation of Nox2 (~45%) and Nox4 (~60%), while promoting a decrease of both SOD (~45%) and GSH activity (~85%). Additionally, MPP induced apoptosis via mitochondrial dysfunction, as shown by induction of cytochrome c (~55%), cleaved-caspase-3 (~75%), upregulation of Bax expression (~55%), and downregulation of Bcl2 (~60%). Liuwei dihuang (LWDH), a widely used traditional Chinese medicine (TCM), has antioxidant characteristics. LWDH-WH, derivative of LWDH (0.01-10 μg/ml, added 1 hr prior to MPP⁺addition) reduced oxidative damage via increasing antioxidant defence (SOD, GSH), decreasing ROS production, and down-regulating NADPH oxidases (Nox2 and Nox4). LWDH-WH also inhibited neuronal apoptosis by increasing anti-apoptotic protein Bcl-2 expression, and down-regulating apoptotic signalling (Bax, cytochrome c, cleaved-caspase-3) in MPP⁺-treated neurons. All these protective effects were induced in a dose-dependent manner (Tseng et al., 2014).

PC12 cells treated with MPP⁺ (500 μM, for 24 hr) underwent reduction of viability (~55% MTT reduction), oxidative stress (~160% increase in ROS production) and downregulation of heme oxygenase-1 expression (~ 2 folds). Pre-treatment with edaravone, a novel free radical scavenger, (25, 50, 75, 100 μM, for 1 h prior MPP+ treatment) protected PC12 cells against MPP+-cytoxicity via inhibiting oxidative stress and up-regulating heme oxygenase-1 expression in a dose-dependent manner (Cheng et al., 2014).

The protective effects of antioxidant, apigenin (AP), naturally occurring plant flavonoids were observed on the MPP+ induced cytotoxicity in cultured rat adrenal pheochromocytoma cells (PC12 cells). The PC12 cells were pre-treated with various concentrations of the test compound for 4 h, followed by the challenge with 1,000 µM MPP⁺ for 48 h. Pre-treatment with AP (3 - 6 - 12 µM) before MPP⁺ significantly reduced the level of intracellular ROS and elevated Δψm in the MPP⁺ treated PC12 cells. In addition, AP markedly suppressed the increased rate of apoptosis and the reduced Bcl 2/Bax ratio induced by MPP⁺in the PC12 cells. The findings demonstrated that AP exerts neuroprotective effects against MPP⁺induced neurotoxicity in PC12 cells, at least in part, through the inhibition of oxidative damage and the suppression of apoptosis through the mitochondrial pathway (Liu et al., 2015).

Brain mitochondria isolated from ventral midbrain of mitochondrial matrix protein cyclophilin D (CYPD) knockout mice were significantly less sensitive to acute MPP⁺ (20 µM) -induced effects. CYPD ablation attenuated in vitro Ca2+-induced mitochondrial dysfunction and ROS generation upon Ca2+ loading, both in the absence and in the presence of MPP⁺, compared to wild-type mice. CYPD ablation conferred a protection to mitochondrial functions upon in vivo treatment with MPTP.

Ventral midbrain mitochondria (that constitutes < 5% of SNpc DA neurons) isolated from brains of wild type (wt) mice acutely treated with MPTP (single MPTP 20 mg/kg injection, analysis done after 4 hr), as compared with saline-treated mice, showed a reduction of CI (by 53%), a reduced rate of phosphorylating respiration (by 38%), a reduced respiratory control index (by 37%), and a decreased ADP/O ratio (by 18%). Ventral midbrain mitochondria isolated from brains of CYPD knockout mice acutely treated with MPTP, as compared with MPTP-treated wt mice, exhibited higher activity of CI (~80%, vs 53% wt), higher rate of phosphorylating respiration (~82%, vs 62% wt), a better respiratory control index (~79%, vs 63% wt), and a higher ADP/O ratio (~90% vs 82% wt) (Thomas et al., 2012). CYP plays as a regulatory component of a calcium-dependent permeability transition pores (PTP), and the data suggest that PTP is involved in MPP⁺-induced mitochondrial damage. Under oxidative stress, the prolonged opening of the PTP results in calcium overload and with time mitochondrial dysfunction as they get de-energized, depolarized, triggering apoptotic or necrotic cell death (Bernardi, 1999).

There are many other studies showing that MPP⁺ induces NADH-dependent SOD formation and enhances NADH-dependent lipid peroxidation in submitochondrial particles, confirming that oxidative stress is induced by MPP+ (e.g. Takeshige, 1994; Ramsay and Singer, 1992).

Based on the human post mortem studies of PD brains it is well established that oxidative stress and mitochondrial dysfunction accompany the pathophysiology of PD (e.g. Dias et al., 2013; Zhu and Chu, 2010; Hartman et al., 2004; Fujita et al., 2014).

Examples of human data confirming the presence oxidative stress and mitochondrial dysfunction in PD post mortem brains:

A significant decrease in CI activity has been identified in a large study of post-mortem PD brains, specifically in substantia nigra compared with age matched controls. In idiopathic PD all 10 patients studied had significant reductions of CI activity (Parker et al., 1989). It is hypothesize that the CI dysfunction may have an etiological role in the pathogenesis of PD (Greenamyre et al., 2001; Sherer et al., 2003, Schapira et al., 1989).
The structure and function of mitochondrial respiratory-chain enzyme proteins were studied post-mortem in the substantia nigra of nine patients with PD and nine matched controls. Total protein and mitochondrial mass were similar in the two groups. CI and NADH cytochrome c reductase activities were significantly reduced, whereas succinate cytochrome c reductase activity was normal. These results indicated a specific defect of CI activity in the substantia nigra of patients with PD (Schapira et al., 1990).
Post mortem human studies show that CI deficiency in PD is anatomically specific for the substantia nigra, and they are not present in another neurodegenerative disorder involving the substantia nigra. These results suggest that CI deficiency may be the underlying cause of DA cell death in PD (Schapira et al., 1990; Schapira, 1994).
The mitochondrial respiratory chain function was studied in various brain regions as well as in skeletal muscle and in blood platelets from patients with idiopathic PD and from matched controls. The evidence suggests that the CI deficiency in PD is limited to the brain and that this defect is specific for the substantia nigra (Mann et al., 1992).
Immunoblotting studies on mitochondria prepared from the striata of patients who died of PD were performed using specific antisera against Complexes I, III and IV. In 4 out of 5 patients with PD, the 30-, 25- and 24-kDa subunits of CI were moderately to markedly decreased. No clear difference was noted in immunoblotting studies on subunits of Complexes III and IV between the control and PD. The authors claim that deficiencies in CI subunits seem to be one of the most important clues to elucidate pathogenesis of PD (Mizuno et al., 1989).
Redox markers have been found unchanged in PD patient-derived vs Ctr-derived fibroblasts at baseline. Basal mitochondrial respiration and glycolytic capacity resulted similar at baseline between PD and Ctr fibroblasts, while rotenone-sensitive respiration (analysed by using 0.5 μM rotenone) resulted lower in PD fibroblasts vs Ctr (174.74 ± 48.71 vs 264.68 ± 114.84) (Ambrosi et al., 2014).
Augmented oxidative metabolism has been detected in PD brains by magnetic resonance studies, in conjunction with energy unbalance. Decreased glucose consumption (22% mean reduction), likely reflecting a decrease in neuronal activity, has been reported in the nigrostriatal system of PD patients (Piert et al., 1996). These symptoms were hypothesized to be indicative of mitochondrial dysfunction as early markers, present in the brain of patients with PD even in the absence of overt clinical manifestations (Rango et al., 2006). In particular, by using high temporal and spatial resolution 31P magnetic resonance spectroscopy (31P MRS) technique authors studied mitochondrial function by observing high-energy phosphates (HEPs) and intracellular pH in the visual cortex of 20 PD patients and 20 normal subjects at rest, during, and after visual activation. In normal subjects, HEPs remained unchanged during activation, but rose significantly (by 16%) during recovery, and pH increased during visual activation with a slow return to rest values. In PD patients, HEPs were within the normal range at rest and did not change during activation, but fell significantly (by 36%) in the recovery period; pH did not reveal a homogeneous pattern with a wide spread of values. Energy unbalance under increased oxidative metabolism requirements, that is, the post-activation phase, discloses a mitochondrial dysfunction that is present in the brain of patients with PD even in the absence of overt clinical manifestations,(Rango et al., 2006).

There are many other studies providing evidence that oxidative stress and mitochondrial dysfunction play an important role in PD pathophysiology (see indirect KER Mitochondrial dysfunction induced DA neuronal cell death of nigrastriatal pathway).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

Some studies suggest that rotenone may have effects other than CI inhibition, and it has been claimed that rotenone induces microtubule disruption, rather than ETC CI inhibition (Feng, 2006; Ren et al., 2005). Some studies suggested that there was no evidence for significant change in mitochondrial CI function in PD patients' brains (Jenner et al., 1992). It is still unclear whether the site of superoxide production in CI inhibited mitochondria is CI itself or not (Singer and Ramsay, 1994).

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

Based on the available data, the threshold effect seen in brain mitochondria indicates that modest CI inhibition (~ 25-50% decrease in activity) may not directly impact ATP levels or Δψm. Indeed, low levels of CI inhibition produces an oxidative stress without any significant changes in mitochondrial respiration (Betarbet et al., 2000; Greenamyre et al., 2001) or causes not significant changes in ATP levels (Sherer et al., 2003). In particular, in rotenone-infused animals (2.0 mg/kg per day for 2 days), [3H] dihydrorotenone binding to CI in brain was reduced by about 73%. Based on this degree of binding inhibition, the rotenone concentration in brain was estimated to be between 20−30 nM. Complexes II and IV were unchanged by rotenone infusion (Betarbet et al., 2000).

However, such defects have long-term deleterious effects. It is well documented that that there is a site of electron leak upstream of the rotenone binding site in CI (i.e., on the ‘NADH side’ of the complex) (Hensley et al., 1998) leading to the superoxide (O2-) and followed up by H2O2 production by CI (Greenamyre et al., 2001). The relative role of each ETC complex in forming superoxide differs by tissue; however CI is a major source of O2- in the brain (Halliwell, 2007).

Thus, a low inhibition of CI activity that is insufficient to affect cell respiration may lead to mitochondrial damage and chronic up-regulation of ROS production. Therefore, it is suggested that rotenone that binds to CI with an affinity of 10-20 nM induces toxicity not by bioenergetics effects but rather via accumulative oxidative stress. Sustained oxidative stress leads to decrease levels of reduced glutathione; activation of superoxide dismutase (SOD) (scavenger of O2-), catalase and indeed, treatments with antioxidants reduce the oxidative stress-induced damage. Such data are abounded in the existing literature based both on in vivo and in vitro studies and a few examples are described in the Empirical support for linkage.

The selective CI defects (other complexes were unaffected) (Schapira et al., 1990a) and induced mitochondrial damage followed by oxidative stress is also described in PD patients brains as documented by: (a) reduced glutathione levels (Jenner at al., 1992); (b) increased content of 8-oxo-deoxyguanine, a marker of oxidatively damaged nucleic acids (Alam et al., 1997; Mecocci et al., 1993); (c) increased level of malondialdehyde (marker of lipid peroxidation) (Navarro et al., 2009); (d) increased cholesterol lipid hydroperoxide (Dexter et al., 1994); (e) increased protein oxidation measured e.g. by elevated levels of methionine sulfoxide formation or protein carbonyl content (Alam et al., 1997). These studies in human brain present a semiquantitataive evaluation of the oxidative stress, as there is no data showing KER between the various degrees of CI inhibition and mitochondrial damage (ROS production) and the parameters described above. However, these studies clearly confirmed that oxidative stress in PD patient brain is increased as shown by the measured biomarkers (Sanders and Greenamyre, 2013).

In in vitro and in in vivo animal studies there are some data showing the quantitative relationship between the oxidative stress produced by inhibition of CI and mitochondrial damage measured by the same assays, as described in human studies, and a few examples of such experiments are discussed below. The quantitative evaluation of the causative relationship between the CI inhibition (KE up) induced by rotenone (4 hr exposure) and mitochondrial dysfunction (KE down) measured in human-chimpanzee isolated mitochondria (xenomitochondrial cybrids; HXC) by a decreased cell respiration and Δψm, increased ROS production and lipid peroxidation showed linear, time- and concentration-dependent effects (below Fig.1 from Barrientos and Moraes, 1999).

Fig.1. A dose- and time-dependent effect of CI inhibition by rotenone on (A) reactive oxygen species production (ROS), (B) Lipid peroxidation and (C) mitochondrial membrane potential (Δψm) studied in the human osteosarcoma-derived cell line (143B) or using a genetic model (40% CI inhibited in HXC lines) (for further information see Barrientos and Moraes, 1999).

The endogenous respiration was inhibited in a dose-dependent manner but showed different inhibition kinetics. Only when CI was inhibited by 35-40% (< 5 nM rotenone), cell respiration started decreasing (a threshold for inhibition for cell respiration triggered by rotenone). Between 40 and 60% of CI inhibition (5-10 nM), 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. 100% CI inhibition was achieved with 100 nM rotenone but the cells still maintained a cell respiration rate (through complex II), approximately 20% and the rate of ROS production increased by a maximum of 20-25% (4 hr treatment). ROS production was saturated at 100 nM rotenone but an initial effect was observed already at 1-5 nM (Barrientos and Moraes, 1999). Inhibition of CI activity triggered decrease of cell respiration by different concentrations of rotenone and resulted in mitochondrial damage measured not only by ROS production, but also by lipid peroxidation and decreased Δψm. Inhibition of CI by 25, 50, 75 and 100 % decreased cell respiration by 5, 20, 53, 81 %, increased ROS production by 48, 81, 157, 216%, increased lipid peroxidation by 8, 27, 45, 55 % and decreased Δψm by 6, 13, 20, and 37% respectively (approximately).

Similar studies were also performed using different types of neuronal cells.

Hoglinger and colleagues, by using DA neurones derived from the rat (embryonic day 15.5) ventral mesencephalon, showed that CI inhibition by rotenone at 30 nM, (or MPP+ 3 µM) for 24 hr decreased ATP levels (by > 80%) within the first 6 hr, and neuronal cell death within 24 hr. When residual ATP levels remained above 20%, there was no or little neuronal loss, suggesting that 20% of normal ATP level was the minimum compatible with neuronal survival. Rotenone (and MPP+) increased ROS (≥ 40% over control levels) already at low concentrations that were subtoxic or only moderately toxic (i.e., 10-30 nM for rotenone, 10-30 µM for MPP+) (Fig. 2) (Hoglinger et al., 2003).

Fig. 2. ATP levels, ROS production and neuronal surviving cells in mesencephalic cultures treated with CI inhibitors (rotenone and MPP+) (from Hoglinger et al., 2003, Fig. 4a-b)

Shamoto-Nagai and colleagues showed that 25 or 50 nM rotenone decreased ATP levels over time. In particular, the intracellular ATP level was reduced to 18.0% and 19.6% of control after 44 hr of treatment with 25 and 50 nM of rotenone, respectively, and thereafter the decreased level was sustained (Fig. 3, left) (Shamoto-Nagai et al., 2003). Also, The production of ROS-RNS increased 6 hr after the rotenone treatment, and the increase was about 1.5-fold of the basal value. With treatment with the higher (50 nM) concentration of rotenone, DCF production level was restored to the basal level after 48 hr, whereas, at the lower concentration (25 nM), DCF production increased again at 48 hr and then declined to the basal value after 90 hr (Fig. 3, right) (Shamoto-Nagai et al., 2003).

Fig. 3. Effect of rotenone on ATP level (left) and on ROS and RNS production (right) in SH-SY5Y cells. SH-SY5Y cells were treated with 25 nM (circles) or 50 nM (triangles) of rotenone. * indicates significant difference from control (P < .05) (from Shamoto-Nagai et al., 2003, Figs. 2, 3)

Human neuroblastoma cell line (SK-N-MC) exposed to 5 nM rotenone chronically, for 4 weeks caused reduction in GSH by 44%, GSSG by 40%. These effects were not observed after two weeks of exposure. Total cellular GSH levels were reduced after 4 weeks of exposure by 50% (Sherer et al., 2002). Similarly, in the same study, 1-2 weeks of treatment did not alter protein carbonyl levels (oxidative protein damage) but exposure for 3-4 weeks caused a large increase in carbonyls in the insoluble fraction by approximately 223% of control. Systemic in vivo rotenone infusion (up to 5 weeks, 3.0 mg/kg/day) modestly elevated soluble protein carbonyls in the rat cortex by approximately 19%, in the striatum by 27% and the largest elevation occurred in the DA neurons of midbrain, around 41% (no effect in cerebellum or hippocampus) (Sherer et al., 2003).

The prolonged treatment with rotenone (3-4 weeks, not 1-2 weeks) caused also a marked increase in 8-oxo-dG immune-reactivity (i.e., oxidative DNA damage) and redistribution of cytochrome c (Sherer et al., 2002).

The same group showed that exposure of SK-N-MC cells for 6-8 hr to low concentrations of rotenone (100 pM, 1 nM, 10 nM and 100 nM) produced a concentration-dependent decrease in ATP levels by 0, 2.5, 10, and 32.2 % respectively (Sherer et al., 2003).

The oxidative stress (mitochondrial damage) induced by rotenone exposure was confirmed in ex-vivo studies using brain sections at the level of the substantia nigra that were treated with 50 nM rotenone over 1 week. A significant increase of protein carbonyls (indicative of oxidative damage to proteins; biomarkers of oxidative stress) was observed (~ 25%) when compared to the untreated slices. Exposure to 100 μM α-tocopherol, antioxidant (vitamin E) significantly protected the neurons from the oxidative damage induced by 50 nM rotenone over 1 week (~ 25%), as shown by lower protein carbonyl levels (~ 3%), with very similar effects observed with 20 nM rotenone over 2 weeks (Testa et al., 2005).

The same assays for mitochondrial dysfunction evaluation after exposure to rotenone, MPTP or other chemicals were used through a range of different studies (Sherer et al., 2003, Betarbet et al., 2000) and the role of CI inhibition in PD is discussed in many published reviews (Sanders and Greenamyre, 2013, Greenamyre et al., 2001, Schapira et al., 1990a and 1990b).

Summing up, it is well documented in human PD brain studies as well as in vivo and in vitro existing data that CI inhibition induces mitochondrial dysfunction as shown by measuring the decreased cellular respiration and induced oxidative damage to protein, lipids and nucleic acids, as well as compromised function of anitioxidant defense mechanisms (e.g. decreased levels of reduced glutathione). As discussed above, oxidative damage is largely reversed by antioxidants treatments. These data are largely semi quantitative only, as the full dose- and time response curves are available. They indicate that low levels of CI inhibition for long periods of time (4-5 weeks) mostly increase ROS production, having negative effects on DA neurons in SNpc, which seem to be affected more than other neuronal cell types in other brain structures (reviews e.g. by Sanders and Greenamyre, 2013; Greenamyre et al., 2001, Schapira et al., 1990a and 1990b etc.).

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Time-scale

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Mitochondrial CI in eukaryotes has highly conserved subunit composition based on protein databases (Cardol, 2011). The characterization of induced mitochondrial dysfunction phenotypes in zebrafish was studied in the presence of CI and CII inhibitors (Pinho et al., 2013). Exposure of Caenorhabditis elegans (C. elegans) to rotenone, reduced bioluminescence (an assay for mitochondrial dysfunction) after both relatively short (2 hr) and longer exposures (24 hr) to a range of concentrations. A sharp decline in bioluminescence (maximal inhibition) relative to controls occurred at the lowest rotenone concentration of 2.5 μM. This decline in bioluminescence was consistent with reduced cellular ATP (Lagido et al., 2015). The results obtained from C. elegans exposed to rotenone suggested that chronic exposure to low concentration (2 or 4 μM) caused mitochondrial damage through persistent suppression of mitochondrial biogenesis and mitochondrial gene expression leading to mitochondrial dysfunction that contributed to DA neuron degeneration (Zhou et al., 2013).

Drosophila melanogaster has been proven suitable to study signaling pathways implicated in the regulation of mitochondrial function and integrity, such as the PINK1/parkin pathway (controlling mitochondrial integrity and maintenance), DJ-1 and Omi/HtrA2 genes (associated with the regulation of mitochondrial functionality). Notably, PINK1, PARKIN, and DJ-1 genes are associated with recessive forms of PD (Guo, 2012). Drosophila flies lacking DJ-1 result to be viable, but show an increased sensitivity to oxidative stress induced upon rotenone or Paraquat (an herbicide inducer of CI-dependent ROS) feeding (Menzies et al. 2005; Meulener et al. 2005; Meulener et al. 2006). Moreover, it has been reported in Drosophila that inhibition of CI by mean of sublethal chronic exposure to rotenone (<750 μM) via the feeding medium caused a selective loss of DA neurons in all of the brain regions and locomotor impairments, while L-dopa (3,4-dihydroxy-L-phenylalanine) rescued the behavioral deficits (but not neuronal death) (Coulom and Birman, 2004). MPTP causes Parkinsonism in primates including humans. However, rodents (rats) are much less susceptible to MPTP+ but are fully susceptible to MPP+ (due to the differences in toxicokineticks). In all species, CI inhibition leads to mitochondrial dysfunction. Mitochondrial dysfunction is an universal event occurring in cells of any species (Farooqui and Farooqui, 2012).

References

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