subunit (IKKg/NEMO).202 Degradation of IkB results in translocation of NFkB to the nucleus, where it induces gene expression. NFkB pathway activation is related to anti-apoptotic and cell survival e ects.203 NFkB pathway is activated during MPP1 toxicity followed by p53 activation and caspase 3 activation as well as increased expression of the antioxidant defenses Mn-SOD147 and nNOS.204 In PD patients, nuclear translocation of NFkB is greater and NFkB pathway has been proposed to be an oxidative stressmediated apoptosis signal as it has been found to be inhibited by HNE.205 Parkin has been shown to interact with and promote degradation-independent ubiquitination of the upstream IKK complex regulators: IKKg/NEMO and tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2).206

13.3.3.4Unfolded Protein Response (UPR)

This multi-dimensional signaling cascade is activated in response to accumulation of misfolded proteins and ER stress. UPR comprises a series of precisely intertwined pathways that cells activate independently,85 beginning with three main pathways activating Ire a/b, PERK and ATF6.207 Ire a/b activation induces caspase 12 and JNK pathway activation.208 PERK activates eIF2a and results in inhibition of translation and activation of ATF4, subsequently causing activation of the encoding of the C/EBP homologous protein-10 (CHOP). CHOP is a nuclear protein that is tightly regulated by stress and forms stable heterodimers with C/EBP family members capable of recognizing novel DNA target sequences.122 CHOP enhances cell death via mechanisms

that include downregulation of Bcl-2 expression.209 Bcl-2 protein is found to be increased in PD patients, involving UPR-induced apoptosis.210,211 The third

UPR pathway activator, ATF6, induces the expression of CHOP, Xbp1, chaperones and proteins involved in protein folding, tra cking and degradation.169 In response to 6-OHDA, the three pathways are activated resulting in the dual over-expression of CHOP by PERK and ATF6. Conversely, during MPP1 exposure, PERK pathway is activated singularly and to a lower magnitude than when activated by 6-OHDA exposure.121

13.3.4Potentially Involved Intracellular Signaling Components

There is little evidence supporting the role of some other intracellular signaling components; however, because they are relevant in the context of PD a brief description will be discussed.

13.3.4.1AMP-Activated Protein Kinase (AMPK)

AMPK is an important regulator in adult cells supporting a high metabolism, such as skeletal muscle and myocardium and in development. AMPK is activated by kinases such as CaMK. AMPK activation is regulated by AMP/ATP ratio, Ca21 concentration and ROS (Figure 13.1). AMPK can potentially be

involved in PD given that in other tissues AMPK is activated in response to energy imbalance and genotoxic stress.74

13.3.4.2Leucine-Rich Repeat Kinase 2 (LRRK2)

LRRK2 is a promising initiator of intracellular signaling pathways. LRRK2 has a kinase domain that may activate cell death pathways and contribute to protein aggregation29 (Figure 13.2). Also, LRRK2 has a Roc (Ras of complex proteins) that is a GTPase domain with high sequence similarity to Ras and other related small GTPases.212 The phosphorylation state of LRRK2 is proportional to its toxic e ects and LRRK2 autophosphorylates its own ROC as well as its kinase domains.213 Moesin, Ezrin and Radixin have been found to be substrates for LRRK2 phosphorylation; however, their relevance in PD remains to be elucidated.214 PARK8 mutations are found in a high proportion of sporadic cases of PD and in some familial cases, which likely supports a connection between genetic and environmental factors. Loss of function of LRRK2 has been implicated in the pathogenesis of PD as a result of decreased expression of its gene in nigral neuronal processes in PD brains.215

13.3.4.3PINK1

PINK1 has a kinase domain that functions in auto-phosphorylation and phosphorylation of other substrates,53 such as HtrA2, a protein that is released from the mitochondria into the cytosol after permeabilization of the mitochondrial membrane and binds to inhibitors of apoptosis109 (Figure 13.1). PINK1 may phosphorylate mitochondrial proteins in response to stress and protect against mitochondrial dysfunction.216 PINK1 phosphorylates the mitochondrial chaperone TRAP1/heat shock protein 75 (Hsp75), which prevents cytochrome c release and apoptosis (Figure 13.1).217

13.3.5E ector Pathways and Final E ects

Dopaminergic neurons in the SN respond to the damage caused by exogenous agents, toxic intracellular environment or the lack of neurotrophic support by activating signaling cascades that lead to apoptosis or cell survival. In the chronic course of PD, such sustained response may show to be defective or successful. Cell death, most likely apoptotic, is the final outcome leading to PD and reflecting a defective response against the damage; indeed, a classification has been suggested for the pathogenic mechanisms leading to cell death in PD according to their likelihood of inducing cell death.209 Nevertheless, no more than 50% of the dopaminergic cell population in the SN die as a result of PD,218 which indicates that, at least temporarily, dopaminergic neuronal cells survive by developing adaptive responses. With regard to pro-apoptotic pathways, it is noteworthy to say that the flow of signaling is not unidirectional as multiple signaling pathways may be activated at the same time and also may have modulator functions on each other. For apoptosis to be inevitable,

however, activation of JNK, p53, Rb, cdk-5 or pro-apoptotic members of the Bcl-2 family is almost always required.

13.3.5.1Inflammatory Response

Inflammation could be a final result or a contributing factor in PD. Di erent glia populations may participate in the pathologic process. Reactive gliosis is found in the surrounding tissue in the SN in PD. Activated microglia, found in these sites, releases proinflammatory factors such as interleukin-1b, interleukin- 6 and tumor necrosis factor-a (TNFa), which are involved in generation of oxidative stress and neurotoxicity219 as well as correlated to upregulation of inducible NOS (iNOS).36 Microglial-related inflammatory events have shown to be important in MPTP neurotoxicity, whereas ablation of iNOS attenuates toxicity.220,221 An inverse correlation is described between astrocytic reactivity and dopaminergic cell loss. Astrocytes surrounding surviving dopaminergic neurons in PD express high levels of GPx222 while levels of this enzyme are reduced in homogenates of SN in PD.43 Additionally, PD-related proteins may have a role in inflammation. It has been shown that over-expression of a-synuclein can facilitate cell migration and recruitment of microglia to the site of gliosis,223 whereas extracellular a-synuclein induces glial cells to an inflammatory state by activation of all three MAPK pathways ERK, JNK and p38.224 DJ1 expression is predominant in glial cells in PD brains.225 COX-2, the ratelimiting enzyme in prostaglandin E2 (a pyretic agent) synthesis, is upregulated in dopaminergic cells in PD and in the MPTP model through a JNK/c-Jun- dependent mechanism.226

13.3.5.2Dopamine Metabolism

Dopamine production is increased by the phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, thus enhancing its enzymatic activity.227 A number of studies have suggested that CaMKII, ERK and protein kinase A (PKA) are responsible for the phosphorylation of Ser19, Ser31 and Ser40, respectively.228 ERK, specifically ERK1/2 members, phosphorylates TH at Ser31 while JNK and p38 pathways have no e ect on either glutamate-induced phosphorylation of ERK or TH. After exposure to glutamate, phosphorylation of TH at Ser19 and 31 promptly increases and elicits the release of dopamine not at all changing dopamine amounts intracellularly. The phosphorylation at Ser19 has no direct e ect on TH activity, but can potentiate the phosphorylation at Ser40 and subsequent activation of TH.228 Lindgren et al.229 have suggested that NMDA decreases cyclic AMP production, counteracting the phosphorylation at Ser40 by PKA. The phosphorylation at Ser31 directly results in approximate two-fold activation of TH, but to a lesser extent than that at Ser40.230 In glutamate-stimulated neurons, blocking p38 and JNK pathways and inhibiting TH activity are additive and fully prevent the dopaminergic neurotoxicity.7 TH is also a target for a-synuclein. a-synuclein

has the ability to bind and inhibit TH phosphorylation and activity, resulting in a decrease of dopamine synthesis.231

13.3.5.3Cell Cycle

Re-entry of post-mitotic neurons to the cell cycle has been proposed as the cause of cell death in several acute or chronic neurodegenerative disorders,232 including PD. Cell cycle progression is regulated by cyclins, which bind to and activate specific serine-threonine kinases called cyclin-dependent kinases (cdk). Cyclins are important regulators of the cell cycle and have been shown to inhibit the start of DNA replication. p21 allows cyclin-cdk complexes to phosphorylate their target proteins.233 Importantly, cyclin D1 and p21 are increased and arrest the cell cycle in post-mitotic cells173 and in neuron-like PC12 cells.234 Intracellular redox states can regulate cell cycle progression.235 Both cyclin D1 and p21 have been found to decrease after exposure to intracellular oxidants236–238 although several antioxidants and inhibitors of cell proliferation only partially prevent apoptosis.239

In addition to changes in cyclin and p21, cdk5 and retinoblastoma protein (pRb) have an important role in cell cycle regulation. cdk5 is a cyclin-dependent kinase predominantly associated with post-mitotic neurons. cdk5 expression and activity are found to be increased in PD patients, specifically in LBs.165 pRb is a specific target of cyclin D1-cdk complexes during G1/S transition. Rb hyperphosphorylation (P-pRb) decreases pRb-E2F binding, allowing E2F to trigger transcription of S phase-related genes such as the PCNA, a nuclear protein involved in DNA replication,240 and avoids the repression of apoptosis-related genes silenced by such complexes241 (Figure 13.2). PD patients have increased expression of the P-pRb in the nucleus of dopaminergic neurons of the SN and P-pRb staining also co-localizes with LBs.242 In the MPTP model, cdk5 has been found to be increased whereas the cyclin D1/cdk4/6/Rb pathway is not induced.243

13.3.5.4Autophagy

Autophagy involves degradation of cell components by the lysosomes and can be done in three ways: macroautophagy, chaperone-mediated and microautophagy. Macroautophagy involves degradation of large debris and organelles, such as mitochondria (mitophagy). Chaperone-mediated autophagy involves direct lysosomal targeting and degradation of proteins that have a specific target sequence. Microautophagy is poorly understood and involves uptake of nutrients into lysosomes. Macroautophagy and chaperone-mediated autophagy are related to PD pathogenic e ects.209 Mitophagy is increased in PINK1-deficient cells244 while chaperone-mediated autophagy is involved in a-synuclein altered turn-over.245 In physiological conditions a cell induces autophagy in response to low levels of protein intake. In PD, autophagy has been found to be activated in response to abnormal intracellular protein accumulation and induces cell death. As protein degradation systems are interlinked to autophagy, there is a proposed inter-play between ER stress