Mitochondrial dysfunction leads to Degeneration of dopaminergic neurons of the nigrostriatal pathway

Mitochondria are organelles essentials for multiple cellular processes, including production of ATP, maintenance of calcium homeostasis, management of ROS production and apoptosis. Mitochondrial dynamics are also critical for the maintenance of cellular homeostasis, which involve multiple factors controlling mitophagy (Youle et al. 2012). Deregulation of mitochondrial functions may impact any neuronal population; however, SNpc DA neurons are indeed the most vulnerable population in PD. Multiple factors are related to their vulnerability: These include autonomous activity, broad action potentials, low intrinsic calcium buffering capacity, poorly myelinated long highly branched axons and terminal fields, and use of a catecholamine neurotransmitter, often with the catecholamine-derived neuromelanin pigment (Sulzer et al. 2013; Surmeier et al.2010).

The above mentioned factors imply a significantly higher total cell surface and a high energy requirement in order to maintain the re-distribution of ions across the membrane following an action potential. In addition, SNpc DA neurons are characterized by significantly higher numbers of synapses compared with other neuronal types or with DA neurons of different anatomical localizations (Anden et al., 1966; Kawaguchi et al., 1990; Kita et al., 1994; Bevan et al., 1998; Wu et al., 2000; Tepper et al., 2004). In humans, ca. 10 times higher numbers of synapses compared with rats are expected, making human DA neurons particularly vulnerable (Bolam et al., 2012; Matsuda et al., 2009). These extreme bioenergetics demands pose SNpc DA neurons energetically “on the edge”. Any stressor that might perturb energy production would hence lead to conditions under which the energy demand would exceed energy supply, resulting in cell damage and ultimately to cell death.

The mechanistic link between mitochondrial dysfunction and loss of SNpc DA neurons also comes from evidence of mutated proteins related to mitochondrial function in familial PD, resulting in reduced calcium capacity, increased ROS production, increase in mitochondrial membrane permeabilization and increase in cell vulnerability (Koopman et al. 2012; Gandhi et al. 2009). In addition, excessive ROS production can damage mitochondrial DNA and activate the intrinsic pathway of apoptosis (Tait et al. 2010). Additional sources of oxidative stress come from the autoxidation of dopamine and the active generation of ROS by activated glia cells; furthermore, the mitochondrial respiratory chain itself represents a source of constant superoxide formation, even under normal conditions (Moosmann et al., 2002).

Imbalance of mitochondrial dynamics have been also reported in a wide range of experimental models of PD and inhibition of the mitochondrial fission proteins (i.e. Drp1) promote mitochondrial fusion and fission and enhanced the release of dopamine from the nigrostriatal terminals (Tieu et al. 2014).

Additional link between mitochondrial dysfunction and the degeneration of DA neurons of the nigrostriatal pathway comes from studies indicating a reduced activity of mitochondrial complex I in human idiopathic PD cases in the substantia nigra (Keeney et al., 2006; Parker et al., 1989, 2008; Swerdlow et al., 1996). The impairment in complex I activity was directly correlated with an elevated sensitivity of SNpc DA neurons and their demise. Transfer of mitochondria from human platelets collected from idiopathic PD subjects into fibroblasts or neuronal cells resulted in elevated levels of basal oxidative stress, a declined supply with ATP, and an elevated vulnerability towards exogenous stressors such as the complex I inhibitors rotenone or the redox cycler paraquat (Swerdlow et al., 1996; Gu et al., 1998). Systemic application of complex I inhibitors such as rotenone or MPTP lead to a preferential loss of nigrostriatal DA neurons, while other brain areas or peripheral cells are not affected to the same degree (Langston et al., 1983).

The experimental support linking mitochondrial dysfunction with the degeneration of DA neurons of the nigrostriatal system is based on the analysis of mitochondria from PD patients, from genetic mouse models, from in vitro knockdown and overexpression systems, and from in vitro and in vivo toxin models.

- In vitro/rotenone: Prevention of ROS formation protects from cell death. The concept of mitochondrial dysfunction as a consequence of defects in complex I has been fueled by observations of impaired complex I activity in the SNpc, muscle, and in platelets of PD patients. Human neuroblastoma SK-N-MC cells, exposed to rotenone, displayed a time- and concentration-dependent decline in viability. Transfection of rotenone-insensitive single subunit NADH dehydrogenase (NDI 1) allowed a replacement of endogenous complex I activity. NDI 1 transfected cells showed no oxidative damage, no declined mitochondrial activity, or cell death. A significant amount of endogenously formed ROS at complex I was identified in SK-N-MC cells and in a chronic midbrain slice culture exposed to rotenone. Antioxidants such as -tocopherol prevented cell death evoked by rotenone, but not the rotenone-induced drop in ATP (Sherer et al. 2003).

- In vitro/rotenone/MPP+: Antioxidants protect from rotenone/MPP+ cell death. Analysis of post mortem nigrostriatal material from PD patients regularly revealed the presence of elevated levels of oxidative modified proteins, lipids, and DNA. These observations indicate an elevated formation of ROS in the cells affected by the disease and triggerd the concept of antioxidants as a potential intervention strategy to slow down the progression of PD. In MES23.5 cells, a reduction in viability, DA content, NADH levels, as well as an increase in ROS formation and elevated nuclear condensation was observed upon treatment with MPP+. Rosmarinic acid is well known for its radical scavenging activities and displayed a complete protection from MPP+-mediated cell death and rescued NADH levels. In addition, it lead to a partial protection from the loss of DA and resulted in a rate of nuclear condensation that was about half of that observed with MPP+ alone (Du et al. 2010). The flavonoid rutin has been demonstrated to protect from oxidative stress in 6-OHDA induced motor deficits in rats as well as to inhibit the formation of nitric oxide and proinflammatory cytokines (Khan et al., 2012). In a model of SH-SY5Y cells, exposure to rotenone lead to a reduction in viability by ca. 50% that was almost completely protected in the presence of rutin. Rotenone-dependent increase of ROS formation and an elevation of intracellular Ca2+ was significantly dampened by the presence of rutin, similar to its rescue from rotenone-dependent decrease in mitochondrial membrane potential (Park et al., 2014). Comparable observations were made with the quinone triterpene celastrol that protected SH-SY5Y cells exposed to rotenone almost completely from cell death, from a rotenone-dependent elevation in ROS levels, and from a rotenone-dependent loss of the mitochondrial membrane potential (Choi et al., 2014).

- In vitro/different complex I inhibitors: Inhibition of complex I triggers oxidant formation and cell death. The majority of experimental PD studies were either conducted using rotenone or MPP+. In order to demonstrate that the concept of complex I inhibition and its ROS-mediated triggering of mitochondrial dysfunction and cell demise can be regarded as a general principle, alternative complex I inhibitors were applied to substantiate previous observations made with rotenone. In human SK-N-MC neuroblastoma cells, rotenone as well as the pesticides fenazaquin, fenpyroximate, pyridaben, tebufenpyrad, pyridaben were tested. In all cases, a time- and concentration-dependent decline in intracellular ATP and cell viability was observed. Expression of the rotenone-insensitive NADH dehydrogenase from Saccharomyces cerevisiae (NDI 1) prevented from the toxicity of the different complex I inhibitors completely. Rotenone- and pyridaben-dependent cell death was prevented by ca. 75 % by the presence of the antioxidant α-tocopherol.(Sherer et al., 2007).

- In vitro/rotenone: Mitochondrial dysfunction-dependent cell death is prevented by antioxidants. In a human neuroblastoma SH-SY5Y model, exposed either to the complex I inhibitors MPP+ or rotenone, the imine antioxidants iminostilbene, phenothiazine, phenoxazine in the low nanomolar concentration range partially protected from MPP+ or rotenone toxicity. A reduction in the membrane potential evoked by MPP⁺ and rotenone was completely prevented by these antioxidants (Hajteva et al., 2009).

- In vitro/rotenone: Circumvention of dysfunctional mitochondria protects from cell death. Assuming a direct causal relationship between complex I inhibition, mitochondrial dysfunction, and the demise of DA neurons, the circumvention of endogenous complex I by expression of the NADH dehydrogenase of Saccharomyces cerevisiae (NDI 1) provided initial evidence for the essential role of complex I inhibition in this sequence of events. As an alternative electron carrier, capable of transferring electrons from NADH to cytochrome c, methylene blue was identified. In hippocampal HT-22 cells, a rotenone-mediated reduction in the oxygen consumption rate was completely reversed by the addition of methylene blue. A rotenone-mediated decline in cell viability by 70 % was almost completely prevented by 0.1 µg/ml methylene blue . In rats, rotenone-mediated decline in striatal DA was entirely prevented by methylene blue, the observed elevation of ROS formation evoked by rotenone was reduced to control levels, and rotarod performance impairments evoked by rotenone were completely avoided by administration of methylene blue. These observations illustrate a causal relationship between dysfunctional mitochondria, the degeneration of nigrostriatal DA neurons, and impaired motor performance (Wen et al. 2011).

- In vivo/rotenone: Circumvention of dysfunctional mitochondria prevents from nigrostriatal cell degeneration. Circumvention of a dysfunctional complex I by the rotenone-insensitive NADH dehydrogenase NDI 1 in vivo and its influence on nigrostriatal DA neuron integrity was demonstrated in a rat model with an unilateral injection of a recombinant adeno-associated virus, carrying the NDI 1 gene into close special vicinity to the SNpc. The animals were treated with rotenone after the unilateral expression of NDI 1. NDI 1 almost completely prevented from the rotenone-mediated loss of TH staining in the SNpc and the striatum. Striatal DA levels that were reduced by ca. 50 % by rotenone, in the presence of NDI 1, DA levels were also almost identical to the values of untreated controls. These observations highlight a causal relationship between the inhibition of complex I and the degeneration of nigrostriatal DA neurons (Marella et al. 2008).

- In vitro/DA: Exogenously added oxidants lead to mitochondrial dysfunction and cell death. Next to an elevated formation of reactive oxygen species evoked by endogenous defects in complex I or in response to pharmacological inhibitors of complex I, nigrostriatal DA neurons are characterized by the neurotransmitter dopamine and its tendency to undergo autoxidation when exposed to physiological pH and oxygen tension conditions. To assess the role of DA-mediated oxidative stress as a cause of mitochondrial dysfunction and its influence on cell viability, PC12 cells were exposed to DA. The observed increase in intracellular ROS was completely reversed by the presence of the antioxidant N-acetly-cysteine (NAC). The amount of oxidative modified protein increased by DA treatment, its rise was completely prevented by the presence of NAC, and partially prevented by the presence of exogenously added GSH. DA-dependent PC12 cell death, decline in the transmembrane potential and in intracellular ATP, and decline in complex II/III activities were observed and were all completely prevented by the presence of NAC. (Jana et al., 2011). - In vitro/ GSH depletion: Oxidative stress causes mitochondrial dysfunction and neurodegeneration. Several reports indicated a declined activity of complex I in the brain, but also in muscle and platelets of PD patients. In order to investigate the mutual interaction between pro-oxidative conditions and complex I activity, a PC12 subclone was generated, allowing the inducible downregulation of -glutamyl-cystein synthetase involved in the synthesis of glutathione (GSH). This system allows a controlled decrease of intracellular GSH by ca. 50 % and a decrease in mitochondrial GSH by ca. 40 %. Under these conditions, intracellular and intramitochondrial ROS increased by ca. one third, mitochondrial complex I activity and ATP levels were reduced by ca. two thirds. The observed inhibition of complex I was completely reversed by DTT. These observations indicate that an impairment of complex I activity as a key event in the initiation of mitochondrial dysfunction and ultimately cell death, can be evoked by elevated levels of oxidants, respectively by a declined cellular antioxidant capacity (Jha et al., 2000).

- In vitro/ GSH depletion: Oxidative stress causes mitochondrial dysfunction and neurodegeneration. PD is characterized by the depletion of glutathione (GSH) in the SNpc. Declined cellular levels of GSH were reported to be associated with morphological changes of mitochondria (Perry et al., 1982; Jain et al., 1991). To investigate the influence of declined GSH levels, N27 cells were exposed to buthionine-S-sulfoximine (BSO), an inhibitor of glutamate cysteine ligase and hence of de novo GSH synthesis. The BSO concentration chosen allowed a reduction in intracellular GSH levels by 50 % in the absence of cell death. Chronic GSH depletion resulted in the S-nitrosation of complex I and its inhibition. Both effects were completely reversed by the addition of DTT (Chinta et al., 2006). - Isolated mitochondria: Exogenous oxidants cause mitochondrial dysfunction. In order to further address the aspect on how DA autoxidation contributes to mitochondrial dysfunction and DA neurodegeneration, isolated rat brain mitochondria were exposed to DA, resulting in an inhibition of complex I by ca. 30 % and in an inhibition of complex IV by ca. 50 %. Both activities of complex I and complex IV were completely protected from DA-dependent inactivation by the presence of GSH. These observations point to a direct inhibitory action of endogenous DA and its autoxidation derivatives on the activity of the mitochondrial respiratory chain. (Khan et al., 2005) - In vitro/cybrid cells: Sensitization of neuronal cells for degeneration by transfer of dysfunctional mitochondria. In a subclone of human neuroblastoma cells (SH-SY5Y), devoid of mitochondrial DNA, mitochondria from platelets of PD patients were transplanted. Analysis after 5-6 weeks in culture after transplantation of mitochondria indicated a 20 % reduction in complex I activity, a 2-fold increase in the basal formation of reactive oxygen species, and a ca. 2-fold higher sensitivity towards the mitochondrial PD toxin MPP+ (Swerdlow et al., 1996).

- In vitro/cybrid cells: Sensitization of neuronal cells for degeneration by transfer of dysfunctional mitochondria. In a subclone of the human A549 cell line, devoid of mitochondrial DNA, mitochondria of platelets from PD patients were transplanted. Complex I activity in platelets of PD patients displayed a reduction of 25 % compared with age-matched controls. After transplantation into the A549 cells, complex I activity was reduced by 25% in its activity (Gu et al., 1998).

- In vivo: Induction of mitochondrial dysfunction by Drp1 deletion leads to neuronal cell loss. Maintenance of functional mitochondria in a cell is regulated by fission/fusion processes that allow the elimination of damaged mitochondria and the spreading of intact mitochondria. Deletion of the central fission protein dynamin related protein 1 (Drp1) leads to an elimination in DA neuron terminals in the caudate putamen and to a loss of DA neuron cell bodies in the midbrain. In Drp1 deficient mice, mitochondrial mass decreases, particularly in axons (Berthet et al., 2014) - In vivo: Induction of mitochondrial dysfunction by Tfam knockdown leads to neuronal cell loss. Mitochondrial transcription factor A (Tfam) is a key regulator of mitochondrial biogenesis. Conditional knockout mice with a selective disruption of the gene for mitochondrial Tfam in DA neurons indicated a reduction in mtDNA levels and deficiencies in the respiratory chain in midbrain DA neurons that progressed to DA cell death. The demise of DA neurons in the SNpc was associated with the onset of PD symptoms such as a reduction in locomotor activity of these mice by ca. 30 %. The decrease in locomotor activity was reversed by L-DOPA treatment (Ekstrand et al., 2007).

- In vivo: MPTP dependent mitochondrial dysfunction and cell death is protected by PGC-1 overexpression. Peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) is a key regulator of mitochondrial biogenesis and metabolism. Transgenic mice overexpressing PGC-1 show protection against MPTP intoxication (50 %). The SNpc in these mice is characterized by elevated levels of SOD2, Trx2. Resveratrol is a known activator of SIRT1, leading to enhanced PGC-1 gene transcription. In MPTP mice, resveratrol protected TH-positve neurons by 80% from cell loss (Mudo et al., 2012).

- In vivo: Prevention of mitochondrial dysfunction protects from nigrostriatal cell loss. In order to demonstrate the causative connection between complex I-dependent mitochondrial dysfunction and the degeneration of DA neurons, a series of in vivo experiments were conducted that indicated partial restoration by antioxidants or by compounds supporting a dysfunctional mitochondrial ATP generation. In MPTP challenged mice that additionally received Q10 treatment, a 37 % higher striatal DA level compared with the MPTP group was detected. TH positive staining in the striatum dropped by ca. 65 % after MPTP. In the MPTP + Q10 group, the loss in striatal TH staining was reduced to ca. 40 % compared with the untreated controls. (Beal et al., 1998). In MPTP challenged marmosets, TH positive cell body numbers were reduced by ca. 60 %, co-administration with ebselen resulted in a reduction of TH staining of only ca. 25 % (Moussaoui et al., 2000). In MPTP challenged mice, a reduction of striatal DA by ca. 70 % was detected. Co-treatment with creatine resulted in a reduction of DA levels of only 42 %. In the same setup, TH positive neuron number in the SNpc was reduced by 70 % in response to MPTP, in the presence of creatine, a drop of only 4 % was observed (Matthews et al., 1999).

- In vivo/rotenone: Antioxidants prevent from rotenone-dependent nigrostriatal cell death. Rotenone administered subcutaneously for 5 weeks (2.5 mg/kg/d) caused a selective increase in oxidative damage in the striatum as compared to the hippocampus and cortex, accompanied by massive degeneration of dopaminergic neurons in the substantia nigra. Antioxidant polydatin (Piceid) treatment significantly prevented the rotenone-induced changes in the levels of glutathione, thioredoxin, ATP, malondialdehyde and the manganese superoxide dismutase (SOD) in the striatum, confirming that rotenone- induced mitochondrial dysfunction resulted in oxidative stress (Chen et al., 2015).

- In vivo/rotenone: Degeneration of DA neurons depends on oxidative stress evoked by mitochondrial dysfunction. 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 dopamine (DA) neurons. In this study, 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. Application of a potent activator of ALDH2, Alda-1, was effective in protecting against rotenone-induced (100 nM, 24 hr exposure) apoptotic cell death in both SH-SY5Y cells and primary cultured substantia nigra (SN) DA neurons. These results were confirmed by in vivo studies. Intraperitoneal administration of Alda-1 to C57BL/6 mice treated with rotenone (50 mg/kg/day, oral administration for 14days) or MPTP (40 mg/kg/day, i.p. for 14 days) significantly reduced death of SN tyrosine hydroxylase-positive dopaminergic neurons. The attenuation of rotenone-induced apoptosis by Alda-1 resulted from decreasing ROS accumulation, reversal of mitochondrial membrane potential depolarization, and inhibition of 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).

Human studies: PD patients were found to show striatal oxidative stress directly relating to the progression of disease severity (Ikawa et al., 2011), suggesting that oxidative stress may cause synaptic dysfunction. Indeed, interruption of the activity-driven local ATP synthesis by synaptic mitochondria (an auto-regulated mechanism) can impair synaptic function (Rangaraju et al., 2014).

Additional empirical support added after the external review:

• Humans + MPTP. Neuropathological examination reveals severe depletion of neurons of the SNpc, no Lewy bodies, clustering of glia; indications for self-perpetuating neurodegeneration following initiation by MPTP. Patients display Parkinsonian motor deficits; responsive to L-Dopa (Ann Neurol 1999, 46(4), 588-605). Nigrostriatal DA neurodegeneration confirmed in live patients by PET analysis (Ann Neurol 1994, 36(5), 765-770).
• Mice/rats + MPTP. Intrastriatal infusion of MPP⁺. EC₅₀ for DA neurotoxicity was 0.4 mM (mice) and 4.3 mM (rats). Depletion of striatal DA reflected this ca. 10-fold difference between mice and rats (JPET 1994, 270(3), 1008-1014).
• Rats/intranasal infusion of MPTP (0.1 mg/nostril). Reduction in DA neurons in the olfactory bulb and the substantia nigra by ca. 50 %. Reduction in DA by ca. 50 %. Correlation of nigra-striatal DA neuron loss with the onset of impaired motor functions (Exp Neurol 2006, 202(2), 391-403).
• Rats + intranasal MPTP. Decrease in TH staining in the olfactory bulb and SNpc by ca. 30 %. Reduction of DA by ca. 50% in olfactory bulb and 25 % in striatum. Correlated with olfactory, cognitive, and motor impairments (Ann NY Acad Sci 2009, 1170, 629-636).
• Mice + MPTP. Strain differences in the response towards MPTP. Comparison of C57/bl vs. CD 1 mice. C57/bl mice displayed 90 % reduction in striatal DA, while CD1 mice exhibited a reduction of only 30 %. TH expression levels reduced by 45 % in C57/bl mice vs. 27 % reduction in CD1 mice (Exp Neurol 1994, 126(2), 195-204).
• Mice + MPTP. Mice lacking DAT are resistant towards MPTP. Loss of TH-positive neurons in the nigrostriatal system by 34 % in wt (+/+) mice; loss of 22.5% in DAT (+/-) mice; loss of 0 % in DAT (-/-) mice (Exp Neurol 1999, 155, 268-279).
• Rats + rotenone. Loss of striatal fibers (54 %), loss of nigral neurons (28.5 %). TH staining intensity reduced by 50 % in the striatum, reduced by 70 % in the substantia nigra (J. Neurochem 2003, 84, 491-502).
• Rats + rotenone (low 1.5 mg/kg/day vs. high 2.5 mg/kg/day) Dose-dependent loss of striatal DA, loss of TH immunoreactivity in the striatum; dose-dependent catalepsy (Behav Brain Res 2002, 136, 317-324).
• C57/bl 6 mice + rotenone. Oral administration of rotenone for 28 days leads to nigra-striatal DA neurodegeneration, correlation with the onset of motor deficits (J. Neurochem 2007, 101, 1491-1504).

- Several in vitro studies applying rotenone to evoke mitochondrial dysfunction came to the conclusion that rotenone-dependent ROS formation, and not the rotenone-evoked drop in ATP is the primary cause for cell degeneration. These observations are largely based on experimental systems employing the rotenone insensitive NADH dehydrogenase NDI 1. Expression of NDI 1 protected rotenone exposed cells from degeneration. The presence of NDI 1 however results in a substitution of ATP. Endogenously expressed complex I is still present in these models and it can be assumed that rotenone exposure would still lead to a complex I-dependent formation of ROS that precludes the modeling of a precise cause-consequence relationship between either ATP depletion or elevated ROS levels with the demise of DA neurons.

- Several studies indicate a dominant role of ROS in the degeneration of DA neurons, based on models in which rotenone/MPP+ mediated mitochondrial dysfunction and cell degeneration was protected by the presence of exogenously added antioxidants. Maintenance of the endogenous redox potential however is a highly ATP-dependent process. Clear-cut separations between the respective contribution of ROS or the role of an inhibited mitochondrial ATP synthesis on the degeneration of DA neurons is hence difficult to postulate.

- Studies with chronic partial GSH depletions indicated that an experimental reduction of GSH/GSSG by ca. 50 % has no influence on cell viability. Reports involving rotenone and MPP+ however regularly observe degeneration of DA neurons under conditions of GSH depletion around 50 %. These observations indicate a more prominent role of the intracellular drop of ATP evoked by the complex I inhibitors in the process of cell degeneration.

- Studies in which oxidative stress is generated e.g. by the application of DA or 6-OHDA not only observed a challenge of the cellular redox potential, but also reversible and irreversible inhibitory mechanisms of mitochondrial respiratory chain complexes (nitration, S-nitrosation) that are accompanied by an inhibition of the respiratory chain in the absence of pharmacological complex I inhibitors. These observations illustrate the close mutual interaction between oxidative stress and the inhibition of mitochondrial respiration and point to a profound role of direct mitochondrial inhibition also under oxidative stress conditions.

- Mitochondrial dysfunction is generally associated with conditions of oxidative stress. Dysfunctional mitochondria can act as potent source of superoxide. Oxidative stress associated with PD however not only originates from mitochondrial ROS, but also from DA autoxidation and the Fenton reaction, as well as from inflammatory activated adjacent glia. Interpretations on the role of oxidative stress in DA neurons and its role in DA neurodegeneration is hence hampered by the fact that the respective origin of the reactive oxygen species formed (mitochondria, DA autoxidation, inflammation of glia cells) is rather difficult to identify and often shows overlappings (Murphy et al., 2009; Starkov et al., 2008, Cebrian et al., 2015).

- In PD patients, a reduction in complex I activity in the SNpc, but also in peripheral tissue and cells such as platelets, was reported. Studies with isolated mitochondria indicated that for efficient inhibition of mitochondrial ATP formation, an inhibition of complex I by ca. 70 % is necessary (Davey et al., 1996). Reports on the reduction of complex I activity in PD patients however repeatedly indicated an inhibition of only 25-30 % (Schapira et al., 1989; Schapira et al., 1990; Janetzky et al., 1994).

- Data available on the respective inhibition of the components of the respiratory chain are highly dependent on the experimental setup used. Analysis of mitochondrial respiratory chain complex activities in mitochondrial homogenates provide results different from data obtained with intact, isolated mitochondria. These aspects need to be considered in the interpretation of such data (Mann et al., 1992; Parker et al., 2008; Mizuno et al., 1989; Schapira et al., 1990; Cardellach et al., 1993)