Table A.6.

Quantitative evaluation of the KER

KE 2 upstream	KE 4 downstream	Comments	Reference
Rotenone experiments
Mitochondrial membrane potential reduced by 50% upon rotenone treatment. Back to 80% compared to controls in the presence of the flavonoid rutin Intracellular Ca2+ elevated by a factor of 3 by rotenone, reduction to an increase of 1.5 in the presence of rutin ROS increased by a factor of 6.5; increase of ROS by a factor of 2 in the presence of rutin	Rotenone (10 μM) resulted in a reduction of cell viability by 50% In the presence of rutin, cell viability was only reduced by 10% upon rotenone treatment	SH‐SY5Y cells exposed to rotenone (10 μM) for 24 h When applied alone, rutin displayed no toxic effects, up to 100 μM Rutin was added to the cells 30 min prior rotenone at concentrations from 0–10 μM	Park et al. (2014)
Mitochondrial membrane potential reduced by ca. 66% upon rotenone treatment; in the presence of celastrol, reduction by ca. 55% ROS formation increased by a factor of 2 in the presence of rotenone; ROS increase by a factor of 1.5 in the presence of celastrol	Cell viability was reduced by 50% by rotenone; In the presence of the triterpene celastrol, cell viability was only reduced by ca. 10%	SH‐SY5Y + rotenone (10 μM). Celastrol (2.5 nM) was applied 90 min prior to rotenone Cells were incubated with the two compounds for a period of 24 h	Choi et al. (2014)
	TH staining in the SNpc in arbitrary units: Control (25) Rotenone (14) Rotenone + NDI 1(22) TH staining in the striatum Control (70) Rotenone (40) Rotenone + NDI 1 (65) DA levels in the striatum: Control (2.5) Rotenone (1.3) Rotenone + NDI 1 (2.2)	5 month old male Sprague–Dawley rats (ca. 500 g) received intracerebral injection of recombinant adeno‐associated virus with the NADH dehydrogenase NDI 1 gene 45 days after virus injection, rats were treated with rotenone‐loaded microspheres (poly(DL‐lactide‐co‐glycolide) 100 mg rotenone /kg body weight s.c With this method, HPLC analysis of plasma rotenone revealed levels of 2 μM 14 days after microsphere treatment, and 1 μM 60 days after microsphere treatment Behavioural experiments and brain sample collection was conducted 30 days after rotenone treatment	Marella et al. (2008)
MPP + experiments
Decline in mitochondrial transmembrane potential by MPP+; 50% prevention from this decline by rosmarinic acid NADH levels were reduced by ca. 50% in the presence of MPP+; loss of NADH was completely prevented by the presence of rosmarinic acid ROS levels increased by 50% in the presence of MPP+. Rosmarinic acid lead to a reduced increase of ROS by only 20% compared with the untreated control	Cell viability reduced by MPP+ by 30%, complete protection by the presence of the antioxidant rosmarinic acid Striatal DA content reduced by 40% by MPP+ treatment, partially protected by rosmarinic acid back to a value of 25% reduction compared with the untreated control	MES23.5 cells exposed to MPP+ (200 μM) for 24 h Rosmarinic acid (1 nM) was applied 30 min prior to MPP+ treatment	Du et al. (2010)
Reduction in mitochondrial membrane potential by 60% (MPP+), by 50% (rotenone), complete recovery by the co‐incubation with ISB, PHT, PHO	SH‐SY5Y + MPP+: Cell viability reduced by 66%; ISB, PHT, PHO partially protected from cell death with a reduction in cell viability by ca. 20% SH‐SY5Y + rotenone: reduction in cell viability by 60% Partial protection by ISB, PHT, PHO to a reduction in cell viability by 25–50% SH‐SY5Y + BSO: Reduction in cell viability by 80% ISB, PHT, PHO partially protected with a residual decline in cell viability by ca. 20%	SH‐SY5Y + MPP+ (200 μM) or rotenone (150 nM) or BSO (150 μM) for 60 h and 72 h Antioxidants tested: Iminostilbene (ISB) Phenothiazine (PHT) Phenoxazine (PHO) The antioxidants were applied 2 h prior to rotenone, MPP+, or BSO treatment	Hajieva et al. (2009)
Circumvention of endogenous complex I
Wt cells exposed to rotenone: increase in carbonyl content as marker of oxidative stress by 100%; completely prevented in NDI 1 expressing cells In midbrain slice cultures exposed to rotenone: increase in carbonyl content by 20% Rats exposed to rotenone: increase in carbonyl content: 27% in the striatum, increase by 41% in the midbrain	SK‐N‐MC cells: rotenone evoked cell death protected by ca. 90% in NDI 1 expressing cells Rotenone induced cell death prevented by 80% by α‐tocopherol (62.5 μM and 125 μM)	SK‐N‐MC human neuroblastoma cells transfected with the rotenone insensitive NADH dehydrogenase NDI 1; Cells were treated with rotenone (100 nM) for 48 h or with BSO (10 μM) for 24 h When both compound were used in a combined experiment, cells were first treated with BSO (10 μM) for 24 h, then rotenone (10 nM) was added for additional 36 h	Sherer et al. (2003)
Application of the complex I inhibitors: Rotenone Fenazaquin Fenpyroximate Pyridaben Tebufenpyrad Pyridaben	Time and concentration‐dependent cell death with rotenone and a series of other complex I inhibitors NDI 1 expressing cells were resistant towards the different complex I inhibitors	SK‐N‐MC human neuroblastoma cells expressing the rotenone‐insensitive NADH dehydrogenase NDI 1 from Saccharomyces cerevisiae All complex I inhibitors applied were added at the concentrations: 10 nM, 100 nM, 1 μM Pyridaben was applied at 1 μM, 10 μM, 100 μM Viability was assessed after 48 h, ATP was detected after 6 h. Carbonyl content was detected after 24 h	Sherer et al. (2007)
Oxygen consumption rate doubled by MB in the absence of complex I inhibitor Oxygen consumption reduced by 50% by rotenone; completely reversed to control levels by the presence of MB Complex I‐III activity reduced by 95% by rotenone. Reversed to control levels by the presence of MB	HT22 cell viability reduced by 70% by rotenone In the presence of MB, reduction by only 10% of cell viability was observed In rats treated with rotenone, rotarod retention time was reduced by 50% by rotenone. Completely reversed to control levels by the co‐administration of MB In rats, rotenone evoked a reduction of striatal DA by 50%; completely reversed to control levels by MB Complex I‐III activity in the striatum of rats was reduced by 50%, residual inhibition of 10% observed in rats that were additionally treated with MB	The study included: Isolated rat heart mitochondria exposed to rotenone (5 μM) (instant treatment) Hippocampal HT‐22 cells exposed to rotenone (2–8 μM) for 24 h Rats receiving rotenone (5 mg/kg per day via osmotic minipumps for 8 days Test of methylene blue (MB) (10 and 100 ng/mL in isolated mitochondria; 1 and 10 µg/ml in HT 22 cells) to circumvent the complex I/III blockade	Wen et al. (2011)
Cybrid cells with PD mtDNA display a reduction in complex I activity by 20%	Cybrid cells: increase in basal formation of reactive oxygen species by 80% 2‐times higher sensitivity towards MPP+ as stressor	SH‐SY5Y cells devoid of mtDNA; fused with platelets from PD patients for mitochondria transfer: cybrid cells Treatment with MPP+ (40 or 80 μM) for 24 h or 48 h	Swedlow et al. (1996)
Oxidative stress causes mitochondrial dysfunction
Isolated mitochondria: Exposure to DA: loss of ca. 50% membrane potential. Completely protected by GSH or N‐acetyl‐cystein (NAC) Decline of mitochondrial respiration capacity by 90% In the presence of NAC or GSH, only a reduction by 25–30% was observed PC12 cells exposed to DA, then isolation and analysis of mitochondria: inhibition of complex I activity by ca. 50%, prevented by co‐incubation with NAC Inhibition of complex II and III; prevented by NAC Intact PC12 exposed to DA: Mitochondrial transmembrane potential reduced by ca. 50%; prevented by NAC Intracellular ATP reduced by ca. 50%; Cell death increased by DA by ca. 30%, caspase 3 activity increased by a factor of 3; all increases prevented by the presence of NAC	PC12 cells exposed to DA: Increase in intracellular ROS by a factor of 2; completely reversed by NAC Quinoprotein formation increased by a factor of 3; completely prevented by the presence of NAC or GSH Cell death increased from 3% (control) to 37% (DA). Reduced to 10% in the presence of NAC	PC12 cells and isolated rat brain mitochondria exposed to dopamine (100–400 μM) N‐acetyl cysteine or GSH for protection were added at a concentration of 2.5 mM In experiments including isolated mitochondria, NAC and GSH were added 2 h prior to DA. In experiments including PC12 cells, NAC and GSH were added 1 h prior DA Isolated mitochondria were exposed to DA for 2 h; PC12 cells were expose to DA for 24 h	Jana et al. (2011)
Reduction of intracellular GSH by 50% and of intramitochondrial GSH by 60% leads to: Mitochondrial ROS increased by 30% ATP levels reduced by 66% Mitochondrial activity reduced by 66% State 3 respiration reduced by 60% Complex I activity inhibited by 60%	Whole cell ROS increased by 30%	PC12 cells with inducible knockdown of glutamyl cysteine synthetase (inhibition of GSH synthesis) by addition of 25 μg/mL doxycycline Treatment for 24 h with doxycycline resulted in a GSH decline by ca. 50%	Jha et al. (2000)
Reduction of GSH levels by ca. 50% result in: Complex I inhibition by 40%; completely reversed by DTT	No cell toxicity under the applied conditions	N27 cells exposed to BSO (2.5 μM) for 7 days: Total glutathione was declined by ca. 50% by this chronic treatment; absence of cell toxicity under these conditions. DTT for restoration of complex I activity was added at 1 mM	Chinta et al. (2006)