Inhibition, NADH-ubiquinone oxidoreductase (complex I) leads to N/A, Mitochondrial dysfunction 1

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).

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.).

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).