Mitochondrial Electron Transport Chain and Oxidative Stress in pd

Mitochondria are primary organelles responsible for ATP generation for cellular activities. The mitochondrial generation of energy is regulated by five electron transport chain complexes located in the inner mitochondrial membrane. The redox energy from reduced electron carriers, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2), is transferred to O2 (terminal electron acceptor in the chain) to produce H2O via four respiratory chain complexes. The establishment of a proton gradient across the inner mitochondrial membrane forms the basis of the inner mitochondrial transmembrane potential (AYm). The energy released from the proton gradient is used to drive ATP synthesis via fifth complex in the electron transport chain.

Inner mitochondrial membrane contains five electron transport chain complexes and two mobile carriers, coenzyme Q and cytochrome c (Figure 7). Complex I is NADH dehydrogenase that transfers one electron from NADH to coenzyme Q and two protons to the mitochondrial intermembrane space. Complex II is succinate dehydrogenase that catalyzes oxidation of succinate to fumarate, reducing FAD to FADH2, allowing electron flow from succinate to coenzyme Q. No proton transport occurs in this complex. Complex III is coenzyme Q-cytocrome c reductase that drives proton transport through Q cycle where reduced coenzyme QH2 passes its electrons to cytochrome c. Complex IV is cytochrome c oxidase that accepts electrons from cyt c and directs them to two electron reduction of O2 to form H2O. Complex V is ATP synthase that uses the energy of proton gradient to synthesize ATP from ADP and P; in the matrix (Figure 7).

Deficiencies in mitochondrial electron transport chain underlie defects in energy metabolism and have been implicated in the neurodegenerative process. A reduction in complex I activity in PD is thought to cause bioenergetic dysfunction with subsequent loss of DA-ergic neurons [105]. Experimental evidence also points the involvement of certain genes, such as SNCA, Parkin, DJ-1, PINK1, GSTO1, LRRK2, and HTRA2 that encode corresponding proteins, including a-synuclein, E3 ubiquitin-protein ligase (parkin), transcriptional co-activator DJ-1, phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK-1), glutathione S-transferase omega 1 (GSTO1), leucine-rich repeat kinase 2 (LRRK2), and serine protease (HTRA2), in the pathogenesis of PD (Table 4).

8.1. a-Synuclein a-Synuclein is a small 140 amino acid presynaptic protein especially abundant in the brain. This protein is characterized by repetitive imperfect repeats (KTKEGV) throughout most of the amino-terminal half of the polypeptide, a hydrophobic middle region, and acidic carboxy-terminal region [106]. It is found in both cytosolic and membrane bound forms, but mainly located in the cytoplasm (Figure 7). a-Synuclein is a major constituent of Lewy bodies [73], An important property of K-Synuclein's is that in the presence of specific phospholipids conformational changes occur in this protein [107-108]. Aggregation of a-synuclein is toxic to vulnerable neurons (Table 4), as observed in the brains of patients with PD. When expressed in nM concentration, a-synuclein protects neurons against cellular stress through the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway, whereas its overexpression (^M concentration) exerts cytotoxic effect [109]. These data suggest dual roles of a-synuclein (neuroprotection and neurotoxicity) depending on its expression levels. In MN9D cells (an immortalized dopamine-containing neuronal hybrid cell line) transfected with wild-type or A53T mutant gene, overexpression does not alter TH protein levels but markedly reduces TH activity, suggesting that a-synuclein regulates dopamine biosynthesis [110].

Mutations in a-synuclein gene are associated with mitochondrial abnormalities and cell death due to increased susceptibility to oxidative stress [111-112]. Furthermore, the overexpression of a-synuclein in human embryonic kidney (HEK) cells results in increased mitochondrial susceptibility to rotenone-induced toxicity compared to control HEK cells [113]. A significant reduction in intracellular ATP levels in response to subtoxic concentrations of rotenone observed in human embryonic kidney (HEK) cells overexpressing a-synuclein suggests that at high concentration this protein is translocated from cytoplasm to mitochondria, causing enhanced toxicity. It is associated with an increased rate of neuronal cell death when gene is either in its mutant or highly expressed form. The exact cellular function of a-synuclein remains unclear, but it may regulate synaptic plasticity.

Tmrm Rotenone And Hek293 Cells

Figure 7. Schematic diagram of a mitochondrion showing the electron transport chain and location of selected proteins involved in oxidative stress. IMM, intermitochondrial membrane; OMM, outer mitochondrial membrane; IMS, inner matrix surface; ROS; Suc, succinate; Fum, Fumurate, DOPAL, 3,4-dihydroxyphenyl- acetaldehyde; mito. DNA, mitochondrial DNA; DA, dopamine; MAO, monoamine oxidase. PINK1, PTEN-induced kinase 1; LRKK2, Leucine-rich repeat kinase 2 ; DJ-1, mitochondrial antioxidant protein; HTRA2, high temperature requirement protein A2; GST, glutathione S-transferase; parkin, E3 ubiquitin-protein ligase; a-synuclin, a presynaptic protein. Auto-oxidation of dopamine produces dopamine quinones, and deficiency of glutathione results in cellular toxicity.

Figure 7. Schematic diagram of a mitochondrion showing the electron transport chain and location of selected proteins involved in oxidative stress. IMM, intermitochondrial membrane; OMM, outer mitochondrial membrane; IMS, inner matrix surface; ROS; Suc, succinate; Fum, Fumurate, DOPAL, 3,4-dihydroxyphenyl- acetaldehyde; mito. DNA, mitochondrial DNA; DA, dopamine; MAO, monoamine oxidase. PINK1, PTEN-induced kinase 1; LRKK2, Leucine-rich repeat kinase 2 ; DJ-1, mitochondrial antioxidant protein; HTRA2, high temperature requirement protein A2; GST, glutathione S-transferase; parkin, E3 ubiquitin-protein ligase; a-synuclin, a presynaptic protein. Auto-oxidation of dopamine produces dopamine quinones, and deficiency of glutathione results in cellular toxicity.

8.2. Parkin

Parkin is a 465-amino acid protein that has E3 ubiquitin-protein ligase activity [114]. Parkin is mainly located in the cytoplasm and in the outer mitochondrial membrane (Figure 7). It is involved in the UPS to clear misfolded proteins and other target molecules. In PD, death of nigrostriatal DA-ergic neurons is accompanied by the accumulation of a wide range of poorly degraded proteins and formation of Lewy bodies, implicating that mutation in parkin gene causes impairment in protein clearance (Table 4). A wide variety of mutations, such as exon deletions and duplications as well as point mutations in the parkin gene result in the autosomal recessive early-onset parkinsonism [115-118]. Mutations in genes of two enzymes in the ubiquitin-proteasome system (parkin and ubiquitin C-terminal hydrolase) are associated with neurodegeneration in some familial forms of PD, supporting the notion that failure of the UPS may contribute to neurodegeneration and formation of Lewy bodies in PD [119]. Parkin serves as a neuroprotective role by forming polyubiquitin chains in substrate proteins that target them for proteasomal degradation [120]. Two of its subsrates are glycoslyated form of cr-synuclcin and synphilin-1 (a-synuclein interacting protein), thus parkin may be involved in modulating the turnover and function of these presynaptic proteins.

Table 4. Genes involved in Parkinson Disease (PD)



Gene mutation- linked to diseases



Binding with lipids promotes formation of oligomers, | sensitivity to oxidative stress

An inherited form of PD

[188, 107-108]


E3-ubiquitin ligase controls protein degradation via UPS, & improves mitochondrial dysfunction

Autosomal recessive PD of early onset. Selective neural cell death without forming Lewy bodies



A neuroprotective transcriptional co-activator that blocks oxidative stress, and maintains mitochondrial function

Early onset autosomal recessive form of PD

[127, 134, 137]


Mitochondrial protein kinase has a neuroprotective role in the mitochondria against oxidative stress

Autosomal recessive form of PD



Serine protease maintains mitochondrial integrity

Autosomal recessive form of PD



The increased kinase activity in mutants, but its function not clearly elucidated

Familial and sporadic PD

[151-152, 156]


Post-translational modification of IL-1ß protects against inflammation

Linked to PD


Inactivation of the parkin gene in mice may cause motor and cognitive deficits, inhibition of amphetamine-induced dopamine release and inhibition of glutamate neurotransmission, suggesting that parkin facilitates dopaminergic neurotransmission [121]. This finding is supported by a study reporting that parkin-expressed substantia nigra in mice shows a significant increase in the survival of tyrosine hydroxylase-positive neurons [122]. Intracellular oxidation of dopamine plays a critical role in the degeneration of DA-ergic neurons [123]. The overexpression of parkin results in reduction of DOPAC levels, produced during dopamine oxidation by MAO, further supporting a stimulatory role of parkin for the presynaptic dopamine neurotransmission. Parkin suppresses the expression of monoamine oxidases [124]. Overexpression of parkin has been reported to protect human dopamine neuroblastoma (SH-SY5Y) cells against dopamine-induced apoptosis by decreasing ROS produced during dopamine metabolism [125]. Thus, parkin may limit the production of ROS generated by MAO during dopamine oxidation, suggesting its protective role for the survival of DA-ergic neurons.

DJ-1 is a putative mitochondrial antioxidant protein of 189 amino acids. DJ-1 acts as a H2O2 sensor [126]. It is found in a variety of tissues, including brain, and partially localized to the mitochondrial matrix and intermembrane space (Figure 7). Homodimerization of DJ-1 is critical for its catalytic activity, therefore, absence or inactivation of DJ-1 may result in PD [127-128]. A number of missense mutations in DJ-1, such as L166P, disrupt homodimerization resulting in a poorly folded protein, and other mutations A104T, E163K, and M26I subtly perturb DJ-1 structure and reduce thermal stability, implicating rare forms of familial PD [129].

The downregulation of DJ-1 increases ROS-mediated cell death in neuronal cell lines [130]. DJ-1-deficient mice have normal number of DA-ergic neurons in the substantia nigra, but evoked dopamine overflow in the striatum is significantly reduced primarily due to increased reuptake [131]. Nigral neurons lacking DJ-1 are also less sensitive to the inhibitory response of presynaptic D2 dopamine receptor stimulation. Nigrostriatal DA-ergic dysfunction, motor deficits, and hypersensitivity to MPTP in DJ-1-deficient mice suggest an essential role for DJ-1 in dopaminergic neurotransmission [131-132]. Another independent study showing age-dependent progression of motor deficits and DA-ergic dysfunction in nigrostriatal pathway in DJ-1 null mice supports the importance of DJ-1 in the pathogenesis of PD [133]. In Drosophila melanogaster, inhibition of DJ-1a (a homolog of the human DJ-1) results in cellular accumulation of ROS, hypersensitivity to oxidative stress and degeneration of DAergic neurons [134]. Furthermore, DJ-1A RNAi flies show reduction in Akt phosphorylation causing impairment in PI3K/Akt signaling, implicating that DJ-1 promotes neuronal survival [134]. Double knockout flies for DJ-1a and DJ-ip display selective sensitivity to other toxins, paraquat and rotenone, further confirming its protective role against oxidative stress [135]. Collective findings suggest that DJ-1 plays an important player (Table 4) in cellular defense against oxidative stress and maintains mitochondrial function [127,135-137]. Therefore, loss of DJ-1 may lead to PD by conferring hypersensitivity to dopaminergic insults.

8.4. PINK1

PTEN-induced kinase 1 (PINK1) protein is a serine-threonine kinase, which is localized in mitochondria. It is not yet clear whether PINK1 is selectively localized (Figure 7) within the outer or the inner mitochondrial membrane [138]. PINK1 protects cells from stress-induced mitochondrial dysfunction and is required for long-term survival of DA-ergic neurons [139-141].

Mutations in PINK1 gene (Table 4) cause autosomal recessive early-onset in PD [142]. Some missense mutations in PINK1 are associated with down-regulation of the protein serine/threonine kinase activity [143-144]. Drosophila bearing null mutation for dPINK1 show fragmented cristae, loss of outer membrane, and ATP depletion accompanied with DA-ergic neuronal degeneration, supporting that PINK1 plays functional and structural roles in mitochondria [145-146]. PINK1 knockout mice have been reported to show impairment in evoked dopamine release without any alteration in number of DA-ergic neurons, DA-ergic receptors or dopamine synthesis [147]. Direct activation of appropriate postsynaptic DA-ergic receptors compensates for a presynaptic defect in dopamine release, suggesting that impaired presynaptic release of dopamine may be a common pathophysiological mechanism in PD [147]. In contrast to PINK1 knockout fly, mitochndria in PINK1 knockout mice appear to be structurally intact and preserved in total number, however, mitochondrial key function is impaired [138]. There is a possibility that mitochondrial functional defect in PINK1 knockout mice may be a causal and early pathogenic event of PD. Loss-of-function mutations, including degeneration of DA-ergic neuronal, locomotor defects and mitochondrial defects, in Drosophila PINK1 model system reproduce some aspects of PD. Collectively, PINK1 is a neuroprotective protein and therefore loss of PINK1 function causes PD.

8.5. LRRK2

Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein kinase with autophosphorylation activity. It is an extraordinarily large complex of proteins that contains multiple domains, including a leucine-rich repeat (LRR), serine-threonine kinase mitogen-activated protein kinase kinase kinase (MAPKKK), Ras of complex proteins (ROC) that may act as GTPase (ROC-GTPase), and WD40 domain [148-149]. Each domain of this complex is targeted by pathogenic mutations in familial PD. Although, most of LRRK2 is localized in the cytosol, but ~ 10% is associated with the mitochondrial outer membrane (Figure 7). ROC domain dimer may act as a GTPase to regulate its LRRK2 kinase activity [150]. GTPase can be activated independently, but LRRK2 kinase activity strictly requires activation of GTPase [151]. PD-associated LRRK2 is a dimer that undergoes intramolecular autophosphorylation, and its intact C terminus is required for autophosphorylation activity [152].

Dominant missense mutations in LRRK2 gene are the most common genetic cause of PD, but the mechanisms by which these mutations disrupt neuronal function causing loss of DA-ergic neurons remain poorly understood. It is the most common cause of familial autosomal dominant and also sporadic forms of PD [153-155]. Mutations in LRKK2

(autophosphorylation and the phosphorylation of a generic substrate) augment kinase activity [156]. Thus, mutations increase in kinase activity (Table 4) that may be associated with apoptotic cell death in dopaminergic cell lines and primary neurons [151].

In primary neuronal culture, mutated LRRK2--mediated neurodegeneration is prevented by the functional inhibition of Fas-associated protein with death domain (FADD) or depletion of caspase-8, two key elements of the extrinsic cell death pathway. This finding suggests that mutated LRRK2 may induce death signaling by interacting with FADD and caspase-8, which establishes a direct link between a mutant LRRK2 gene and programmed cell death [157]. Furthermore, heat shock protein 90 (Hsp90) has been reported to interact with LRRK2. This interaction is critical for maintaining the stability of LRRK2. Therefore, any disruption in the association between Hsp90 and LRK2 results in proteasomal degradation of LRRK2 [158]. Destabilization of mutated LRRK2 (by blocking Hsp90-mediated chaperone activity) may be a novel way to limit its detrimental effects. These new findings of LRRK2 research warrant further investigation.

8.6. HTRA2

High temperature requirement protein A2, (HTRA2), is a mitochondrial serine protease that is released from mitochondria to the cytoplasm and inhibits the function of X chromosome-linked inhibitor of apoptosis (XIAP) [159]. A monomeric structure of HTRA2 consists a trimerization motif, a C-terminal PDZ domain, and a central serine protease domain that contains the His-198, Asp 228, and Ser-306 in its active site HTRA2 protein contains a central serine protease domain and a C-terminal PDZ domain [160-162]. HTRA2 is localized in intermembrane space within the mitochondria (Figure 7). HTRA2 is released into the cytosol in response to apoptotic stimuli, where it can induce apoptosis in a caspase dependent or independent manner [163-164].

Mutational analysis reveals that the phenylalanine 149 in HTRA2 at the homotrimerization motif is crucial for the formation of homotrimeric assembly of HTRA2 [165]. In the absence of this assembly, HTRA2 monomeric form abolishes its autoproteolytic activity and proteolytic activity against XIAP, a mammalian inhibitor of apoptosis protein having caspase inhibitory activity, suggesting that the homotrimeric structure of protein is required for executing its serine protease activity. The missense mutation of the HTRA2 protease domain (substitution of cysteine for serine at residue 276) in mice exhibits progressive loss of striatal neurons, leading to motor dysfunction [166]. Another missense mutation (substitution of serine for glycine at residue 399) is rare in individuals with PD, but absent in healthy humans [167]. HTRA2 phosphorylation decreases in brain tissues of parkinsonian patients carrying PINK1 mutations, suggesting that HTRA2 protease activity is regulated by PD-associated PINK1 [168]. Similarly, HTRA2 mutants share some phenotypic similarities with Parkin and PINK1 mutants in fruit fly [169]. Taken together, HTRA2 plays an important role in maintaining the mitochondrial homeostasis.

The glutathione-S-transferases (GSTs) gene family encodes cytosolic proteins (Figure 7), which are involved in detoxification of toxic chemical compounds, drugs, oxidized metabolites of catecholamines, and environmental pollutants, through conjugation of reduced glutathione. Human cytosolic GST genes (GSTO1, GSTM1, GSTM3, GSTP1, GSTT1, and GSTZ1) are polymorphic. The encoded proteins in GST family may vary in their substrate specificity and tissue distribution, and to some extent may even show some overlap. GSTs are a family of inducible phase II enzymes and possess detoxification and antioxidative functions. Various mechanisms have been implicated in the pathogenesis of PD. Among them, oxidative toxicity (excessive formation ROS and/or absence of detoxification of ROS) is suggested to play an important role. In the DA-ergic system, oxidation of dopamine to o-quinone, its subsequent product, aminochrome (2,3dihydroindole-5,6-dione), may elicit redox cycling, toxicity and apoptosis [170-171].

Omega class of cytosolic GST possesses dehydroascorbate reductase and thioltransferase activities and also catalyzes the reduction of monomethylarsonate, an intermediate in the pathway of arsenic biotransformation. There are two functional Omega class glutathione transferase genes in humans: GSTO1 and GSTO2. GSTO1 encodes glutathione S-transferase omega 1 protein family (GSTO1). GSTO1 modulates ryanodine receptors (a class of intracellular calcium channels in the endoplasmic reticulum of various cells, including neurons), and interacts with cytokine release inhibitory drugs [172-173]. GSTO1 modifies the age-at-onset of PD [173]. It is involved in the post-translational modification of the inflammatory cytokine IL-1P, a proinflammatory cytokine that is overexpressed in the brains of parkinsonian patients (Table 4), suggesting its protective role against inflammation [173174]. GSTO1 uses glutathione in the process of the biotransformation of drugs, xenobiotics and oxidative stress, therefore mutation in GSTO1 is linked to PD. Biological function of GSTO2 is not known, but it is suggested to protect against oxidative stress by recycling ascorbate [175], implicating its role in influencing the age-at-onset of PD. Association of GSTM1 null polymorphism with PD is reported to be strongest in the earlier age range [176]. Under physiological conditions, this enzyme catalyzes the conjugation reaction between glutathione and quinones [177-178]. The deficiency/absence of GSTM1 influences an individual's risk to PD.

Collectively, GSTs protect DA-ergic neurons in PD through their direct antioxidant activity, and facilitate the elimination of endogenous toxins from the cell. In general, they have cytoprotective properties and are hypothesized to protect against neurodegeneration.

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