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oxidative stress.29 Mutations in LRRK2 gene (PARK 8) cause autosomal dominant PD and have been found with high frequency in sporadic PD cases.30,31

Recently, epigenetic factors have been shown to be involved in the susceptibility of dopaminergic cells to death. Early changes in epigenetic regulation of gene expression may be responsible for epistatic genetic e ects by a ecting several genes or can exert additive e ects on gene mutation in genes associated to PD.32

13.3Signaling Pathways Involved in Selective Dopaminergic Neuronal Death

As previously mentioned, mechanisms involved in selective loss of dopaminergic neurons in the SN in PD include oxidative stress, calcium imbalance, glutamatergic overstimulation and abnormal protein processing. In the next section, it will be discussed how these proposed initiators originate intracellular signals and the pathways involved in the signal transduction that result in selective dopaminergic neuronal death in PD and PD models.

13.3.1Initiators and Signaling Molecules

Proposed mechanisms by which progressive damage and cell death occur in PD may include cell autonomous and non-autonomous factors or the interplay between the two. For instance, genomic mutations result in an abnormal protein structure that creates a toxic intracellular environment, whereas environmental toxicants require entering dopaminergic cells by transporters or membrane receptors in order to exert their toxic e ects. Causative factors act as intracellular or extracellular signals to initiate the transduction of a message using intracellular pathways. The initiation of signal transduction involves highly specific processes such as binding to membrane receptors and inducing molecular modifications that are able to change physiological cell conditions and turn on signaling molecules.

13.3.1.1Response to Oxidative and Nitrosative Stress

Increased production of ROS and reactive nitrogen species (RNS) has been consistently found in PD and in cellular and animal PD models. ROS and RNS are produced as by-products of normal cell respiration, metabolism and aging; however, it has been suggested that dopaminergic neurons in the SN in PD could generate more ROS and RNS as a result of their impaired respiratory chain function.33 Similarly, in PD models, MPP1 has a direct inhibitory e ect on complex I.34 As a result of complex I dysfunction, increased amounts of superoxide anion and hydrogen peroxide leak into the cytosol increasing the production of hydroxyl radical, nitric oxide (NO) and peroxynitrite and turning the redox balance towards oxidative stress.3,4 Additionally, NO is also pro-

duced by the induction of nitric oxide synthase (NOS) activity by glutamate35 as well as MPP1 (Figures 13.2 and 13.3).36,37 Neurotoxins such as Paraquat

and Rotenone also have an inhibitory e ect on complex I inhibition.

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Additionally, Paraquat induces ROS production via reaction with molecular

oxygen38 and Rotenone directly oxidizes endogenous dopamine11 a ecting redox cycling (Figure 13.3). In vitro7,12 and in vivo39 response to exposure to 6-OHDA

involves an early increase in intracellular oxidants through auto-oxidation to aminochrome (Figure 13.3), however, the relevance of 6-OHDA as an endogenous toxic oxidized dopamine intermediate in PD patients remains unknown.

To prevent the damage caused by ROS and RNS, cells have antioxidant systems that protect them from specific reactive species. High levels of superoxide in the cytosol are dismutated to hydrogen peroxide by superoxide dismutases (SOD). Hydrogen peroxide is cleared out by peroxidases or catalases preventing formation of hydroxyl radicals and peroxynitrite.40,41 There are three types of peroxidases: peroxiredoxins (Prxs), catalases and glutathione peroxidases (GPxs). GPxs are involved in the reduction of hydrogen peroxide mediated by glutathione (GSH). GSH is a major antioxidant system in neuronal cells that is involved in both enzymatic reductions as well as non-enzymatic redox regulation. Studies on antioxidant systems in PD are

inconclusive. Peroxidase and catalase levels have not been found altered, whereas superoxide has been found increased and GSH decreased.42,43 Antioxidant

systems in PD models such as MPP1 toxicity have been found to be important toxicity mediators. Transgenic mice over-expressing Cu-Zn SOD (SOD1) have been shown to be resistant to MPTP neurotoxicity.44 It should also be noted that MPP1 redox cycles with the production of oxygen radicals.45

Abnormal function of PD-related proteins, such as DJ1, PINK1 and parkin, has been linked to deficient antioxidant systems. DJ1 has been proposed as a redox responsive chaperone that, when oxidized, can translocate from the mitochondrial matrix and inter-membrane space into the outer mitochondrial membrane.46 Also, DJ1 can undergo self-oxidation and act as an atypical mitochondrial ‘‘peroxiredoxin-like peroxidase’’, decreasing levels of hydrogen peroxide.47 Indirect antioxidant e ects of DJ1 include the formation of nuclear complexes with RNA-binding proteins and DNA-binding proteins that regulate gene transcription and stabilization of the antioxidant regulator nuclear factor erythroid 2-related factor (Nrf2)48 (Figure 13.2). This occurs by preventing Nrf2 association to its inhibitor, Keap1, and therefore Nrf2 ubiquitination.49 Nrf2 binding to antioxidant response element (ARE) gene regulatory regions activates transcription of neuroprotective genes such as heme oxygenease (HO-1) and glutamate cysteine ligase (GCL). HO-1 has also been shown involved in cell survival processes and its over-activity is coupled to excessive levels of heme-derived free iron and carbon monoxide.50 GCL is the rate-limiting enzyme in the GSH synthesis; both its mRNA expression and activity have failed to be induced in response to oxidative stress in rat dopaminergic cells.51 By inducing gene expression changes, DJ1 may contribute to cell survival and prevent apoptotic cell death under oxidative stress conditions.52

PINK1 is a ubiquitously expressed protein located in the inner mitochondrial membrane with its C-terminus exposed to the intermembrane space53 (Figure 13.1). PINK1 regulates Ca21 e ux from the mitochondria. PINK1 deficiency or expression of disease-associated mutated PINK1 is associated with

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mitochondrial Ca21 overload, decrease in mitochondrial complex I activity and an increase of levels of ROS production.54 PINK1 deficient cells are more susceptible to die after exposure to MPP1 or Rotenone.55 A collaborative function for PINK1 with parkin and DJ1 in protecting from oxidants has been suggested; this is supported by the susceptibility of PINK1 deficient cells to die, which can be rescued by over-expressing parkin, implicating that PINK1 is upstream on a linear pathway.56 This also indicates that DJ1 stabilizes PINK1 allowing for their binding to have a synergistic action protecting from MPP1 exposure.57

In PD etiology, genetic deficiencies or environmental insults generate toxic cell conditions that a ect the balance between production of reactive species and antioxidant defenses giving rise to oxidative and nitrosative stress. ROS and RNS react rapidly with cell components and are responsible for nitration and oxidation to proteins, lipids and/or nucleic acids. Protein nitration, oxidation and carbonylation are increased in PD.58–60 Specifically, PD-related proteins are oxidatively damaged, including a-synuclein oxidation and nitration and parkin nitrosilation. Additionally, in PD more ubiquitous enzymes are found to be oxidatively modified a ecting central cell pathways. Aconitase, an iron-sulfur enzyme, is inactivated by superoxide-induced loss of the labile Fe21 atom, which a ects the tricarboxylic acid cycle and consequently cellular energy metabolism (Figure 13.1).61 Lipid peroxidation is increased in PD as

high levels of lipid hydroperoxides and 4-hydroxynonenal (HNE) are found in the SN.62,63 HNE is found as part of LBs and also forming adducts

with nucleophilic groups on proteins, such as dopamine transporter.64 Lipid peroxidation in PD has been associated with apoptosis, PARP-induced cleavage, decreased GSH levels and inhibited mitochondrial complexes I and II.62 Lastly, oxidative stress in PD produces several oxidative lesions to genomic and mitochondrial DNA. Mitochondrial DNA (mDNA) is particularly susceptible given its proximity to the mitochondria, an important ROS production site. A higher number of deletions in mDNA are found in PD patients as a result of oxidative damage48 and missense mutations a ect respiratory chain genes65 (Figure 13.1). Additionally, loss of function of PINK1 has resulted in decreased mDNA synthesis.66 Levels of 8-hydroxyguanine and 8-hydroxy-2- deoxyguanosine are increased in PD,67 whereas exposure to 6-OHDA induces an increase of double strand breaks.68 Severely damaged nucleic acids could a ect gene expression and lead to apoptotic cell death in PD. Given that neuronal cells are post-mitotic and DNA does not benefit from turnover, DNA repair systems are critical to defend against oxidative lesions in neuronal cells. The e ciency of DNA repair systems may influence aging in response to oxidative stress.69

Damage to macromolecules due to oxidative and nitrosative stress results in cell components dysfunction. Two of the most a ected organelles in PD are mitochondria and endoplasmic reticulum (ER). Mitochondrial dysfunction in PD is likely the result of complex I inhibition, increased ROS production and Ca21 overload.70 Under these conditions, mitochondria lose their control over Ca21 levels, mitochondrial membrane potential (MMP) and generation of NADH and ATP. Mitochondrial Ca21 overload stimulates mitochondrial

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permeability transition pore (mPTP) opening, which results in mitochondrial membrane depolarization and mitochondrial swelling. As a result, there is loss of GSH and NAD(P)H from the mitochondria and release of cytochrome c to the cytosol.71 Under physiological conditions, mitochondrial Ca21 stimulates tricarboxylic acid cycle and oxidative phosphorylation, however, it is uncertain how this stimulation changes under Ca21 overload conditions.71 Decreased MMP a ects normal mitochondrial processes such as fusion and fission72 and mitochondrial tra cking.73 By nature, the main function of mitochondria is to supply energy to maintain physiological cell processes, and impaired ATP production directly a ects the function of ATP-dependent processes such as maintenance of plasma membrane potential, glucose transport into the mitochondria, ubiquitination and the proteasome function. Additionally, master cell energy regulators, such as AMP-activated protein kinase (AMPK), respond to changes in AMP/ATP ratio, Ca21 levels and ROS initiating the activation of an intracellular signaling pathway that prevents cell death.74

PD-related proteins that are localized in the mitochondria, such as DJ1, PINK1 and parkin, can show di erences in their functions under mitochondrial dysfunction.75 Parkin redistribution from the mitochondria into the cytosol occurs in response to inhibitors of respiratory chain activity and cell cycle blockers.48 Oxidized DJ1 changes its distribution from being di used throughout the cytoplasm to the proximity of the mitochondria. Additionally, mitochondrial dysfunction could induce ER stress due to their adjacency in the cytoplasm. ER stress is found in PD and it is accompanied by the unfolded protein response (UPR) and protein aggregation (discussed in the next section). It has been proposed that a cross-talk between the mitochondria and ER could initiate a signaling pathway resulting in apoptosis in dopaminergic cells.76

Although believed to be only damaging, a new role for ROS in normal intracellular signaling has emerged. Such function in signaling has especially been applied to hydrogen peroxide since its half-life and specificity allows it to fit criteria for being a second messenger.77 Thiols in proteins can be oxidized to sulfenic acid residues, glutathionylated residues or to the formation of intramolecular disulfide bonds in the presence of hydrogen peroxide. These protein modifications determine the origin of cell messages. In the 6-OHDA model, oxidation of thioredoxins (Trx), such as Trx1, allows apoptosis signalregulated kinase1 (ASK1) to dimerize and activate the apoptosis-inducing pathways, p38 and JNK intracellular pathway.78,79 Using a similar mechanism, in cells treated with hydrogen peroxide, oxidized DJ1 converts Cys-106 to cysteine sulfinic acid (Cys-SO2H), and binds to ASK1 inducing cytoprotection.80 The activation of another cell survival intracellular signaling cascade, the Akt pathway, has been found to be facilitated by DJ1.81 Additionally, the ‘‘floodgate hypothesis’’ proposes that, under oxidative stress conditions, hydrogen peroxide oxidizes peroxydases, thus making them inactive.77 Peroxidase inactivation, specifically Prx1, has been described to induce cell-cycle arrest mediated by the activation of p38 and caspase-3.82 Some promising examples of hydrogen peroxide-mediated signaling include oxidative modifications in AP-183 and PTEN.84