**1. Introduction**

316 Etiology and Pathophysiology of Parkinson's Disease

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#### **1.1 Clinical characteristics of Parkinson's disease**

Parkinson's disease (PD) is an old-age neurodegenerative disease with a small but significant genetic risk. The prevalence of PD is of 0.3% in the whole population, affecting more than 1% of the humans over 60 years of age (de Lau & Breteler, 2006). Parkinson´s disease is characterized by the progressive loss of dopamine due to degeneration of dopaminergic neurons in the *substancia nigra, striatum* body and brain cortex. In addition, αsynuclein-positive Lewy bodies in brainstem and neocortex are consistently found at autopsy (Forno, 1996; Jellinger & Mizuno, 2003). Therefore, in patients with PD, movements, sleep, autonomic functions and cognition become progressively impaired.

Complex factors contribute to the appearance of PD but with a constant mitochondrial involvement and a decreased capacity to produce energy (ATP) in the affected brain areas (Shapira, 1998; Shapira, 2008). Mitochondrial dysfunction in the human frontal cortex is to be considered a factor contributing to impaired cognition in PD.

#### **2. Environmental aspects and experimental models**

Both environmental chemicals and genetic susceptibility are thought to contribute to the etiology of sporadic PD (Nagatsu, 2002). Despite of familial PD was correlated with a series of genes mutations, the etiology of idiopathic PD, which accounts for more than 90% of PD, is still not fully understood. It is well documented that there is an epidemiological link between PD and individuals who lives and works in rural areas and who has been exposed to various herbicides and insecticides (Gorell et al. 1998; Ayala et al., 2007; Gomez et al., 2007).

Although the etiopathogenesis of PD is still elusive, *post mortem* studies support the involvement of oxidative stress in neurons with an increased production of superoxide

Brain Mitochondrial Dysfunction and Complex I Syndrome in Parkinson´s Disease 319

Physiological, clinical and genetic studies support the relationship between PD and energy metabolism in neurons, including mitochondrial electron transport carriers and cytosolic glucose utilization. *In vivo* and *ex vivo* experimental results have shown that PD is primarily associated to two interdependent situations of brain mitochondria: (a) mitochondrial dysfunction; and (b) mitochondrial oxidative damage. In addition, defective oxidative phosphorylation was reported in muscle, and increased level of 8-hydroxydeoxyguanosine

**4.1 Mitochondrial complex I and physiological production of superoxide, nitric oxide** 

Mitochondrial complex I (NADH-UQ reductase) catalyzes electron transfer from NADH to ubiquinone and it is the main molecular pathway to link the tricarboxylic acid cycle, the coenzyme NADH and the mitochondrial respiratory chain. Complex I is a supra-molecular protein complex composed of about 40 polypeptide subunits and contains FMN and ironsulphur centers (Walker, 1992; Walker et al., 1992). Two complex I-linked UQ-pools have been detected (Raha & Robinson, 2000). Non-covalent hydrophobic bonds are essential in keeping together the whole structure of complex I; low concentrations of detergents, natural and synthetic steroids (Boveris & Stoppani, 1970) and hydrophobic pesticides, such as rotenone and pyridaben (Gomez et al., 2007), are effective in disrupting intra-complex I polypeptide hydrophobic bonds and in inhibiting complex I electron transfer activity. Complex I produces significant amounts of O2- in physiological conditions (0.80-0.90 nmol

/min.mg protein) through the auto-oxidation reaction of flavin-semiquinone (FMNH•

contribution, in complex I, is negligible (Boveris & Cadenas, 2000; Turrens & Boveris, 1980). Superoxide anion production yields an O2- steady state concentration of 0.1-0.2 nM in the mitochondrial matrix (Boveris & Cadenas, 2000; Boveris et al., 2006; Valdez et al., 2006). The O2- production rate by complex I is increased by inhibition of electron transfer with rotenone (Boveris & Chance, 1973) or by complex I dysfunction (Hensley et al., 2000; Navarro et al.,

of complex I. Mitochondrial NO production is carried out by the mitochondrial nitric oxide synthase (mtNOS), an isoenzyme of the NOS family located in mitochondrial inner membrane (Tatoyan & Giulivi, 1998; Giulivi et al., 1998). Nitric oxide is produced at a rate of 1.0-1.4 nmol NO/min.mg protein and kept at a steady state level of 200-350 nM in the mitochondrial matrix (Boveris et al., 2006; Valdez et al., 2006). Peroxynitrite is generated in the mitochondrial matrix through the diffusion controlled reaction (k = 1.9 × 1010 M-1 s-1) between two free radicals: O2- and NO. This reaction contributes with 0.38 μM ONOO-

in the mitochondrial matrix or 0.92 nmol/min. mg protein (Valdez et al., 2000). In this approximation the contribution of cytosolic NO has not been considered. Peroxynitrite is normally reduced by the mitochondrial reductants NADH, UQH2 and GSH and kept at intramitochondrial steady state level of 2-5 nM (Valdez et al., 2000). When the steady state concentration of ONOO- is enhanced up to 25-40 nM, tyrosine nitration, protein oxidation and damage to iron sulfur centers might takes place, leading to a sustained complex I

with molecular oxygen. It is understood that the ubisemiquinone (UQH•

)

/sec

) auto-oxidation

) have been proposed as direct inhibitors

**4. Pathophysiological aspects** 

**and peroxynitrite** 

2009; Navarro et al., 2011).

Both, nitric oxide (NO) and peroxynitrite (ONOO-

inhibition and increased generation of O2- by complex I.

O2-

was found in PD patients plasma (Henchcliffe & Beal, 2008).

radical (O2 - ) and hydrogen peroxide (H2O2) and of mitochondrial dysfunction, especially of complex I of mitochondrial respiratory chain (Shapira et al., 1989; Shapira et al., 1990a, 1990b; Gomez et al., 2007; Navarro & Boveris, 2009; Navarro et al., 2009).

The early hints about the central role of mitochondria in the pathogenesis of PD resulted from the observation that human exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a contaminant in synthetic opiates, triggered an acute and permanent parkinsonism with death of dopamine neurons (Langston et al., 1983). It was found that the MPTP active metabolite is the 1-methyl-4-phenilpyridinium ion (MPP+). This compound is accumulated in mitochondria and produces their toxicity by inhibiting mitochondrial complex I, the proton pumping NADH:ubiquinone oxidoreductase.

As was mentioned above, epidemiological research indicates that exposure to pesticides and welding elevates the risk of PD (Chade et al., 2006; Dhillon et al., 2008). Most of pesticides are inhibitors of mitochondrial complex I, which is the first and the most vulnerable complex in the series of membrane H+ pumps of the mitochondrial respiratory chain (Wallace et al., 1997). The pesticide rotenone ((2R,6aS,12aS)-1,2,6,6a,12,12a-hexahydro-2 isopropenyl-8,9-dimethoxychromeno [3,4-b]furo(2,3-h)chromen-6-one) is a powerful inhibitor of mitochondrial complex I: in isolated beef heart and liver mitochondria, rotenone median inhibitory concentration (IC50) is 0.05 nmol/mg protein with a Ki of 4 nM (Degli, 1998). When neuron cultures are exposed to rotenone, the cells increase the O2 - production rate leading them to death (Ahmadi et al., 2003; Moon et al., 2005). Furthermore, dopaminergic neuronal cells exposed to rotenone reproduce many of the features of PD including α-synuclein inclusions bodies in rats (Betarber et al., 2000; Sherer et al., 2003).

The above mentioned inhibitors of complex I, rotenone and MPTP, are typically used in the experimental model of PD in laboratory animals.

#### **3. Genetic aspects**

Although most PD cases are sporadic, the discovery of genes linked to familial form of disease due to mutations in the SNCA (α-synuclein), PARK2, DJ-1, PINK1, and LRRK2 genes has provided important clues about the disease progress (Henchcliffe & Beal., 2008; Zheng et al., 2010). In the sporadic disease, α-synuclein and degenerating mitochondria are the major components of Lewy bodies, the hall mark cytoplasmic inclusions found in PD brains. Biochemical complex I deficiency is found in PD patients not only in *substancia nigra*  but also in platelets (Henchcliffe & Beal, 2008).

Recently, Zheng and coworkers (2010) reported that decreases in expression of 10 gene sets are associated with PD, even in probable subclinical disease and in tissues, outside *substancia nigra*. These 10 gene sets encode proteins responsible for interconnected cellular processes: nuclear-encoded mitochondrial electron transfer, mitochondrial biogenesis, glucose oxidation, and glucose sensing (Zheng et al., 2010). The authors showed that bioenergetics genes responsive to the master regulation of PGC-1α, including genes for nuclear-encoded electron transfer carriers are under expressed in patients with PD and in incipient Lewy body diseases. Furthermore, co-activation by PGC-1α up-regulates nuclear subunits of mitochondrial respiratory chain complexes I, II, III, IV, and V and blocks dopamine neuron loss in cellular models of PD-linked α-synocleinopathy and rotenone toxicity. Moreover, genetic ablation of PGC-1α in mice markedly enhanced MPTP-induced dopamine neuron loss in the *substancia nigra* (St-Pierre et al., 2006).
