conventional PKC have been activated in MPP1 toxicity and upon glutamatergic stimulation (Figure 13.3).144 The activation of PKC requires the function of phospholipase C (PLC), which is also activated by Ca21. PLC is a mem- brane-bound enzyme that produces DAG from hydrolysis of phosphatidyl inositol-bisphosphate (PIP2). PIP2, Ca21 and DAG activate PKC and induce its recruitment to the plasma membrane. Importantly, PKC can be activated depending on its spatial and temporal localization, as the binding partners needed for activation are also spatial and temporal specific.145

13.3.2.2Small GTPases

Typically, small GTPases such as Ras and Rap transduce an extracellular signal into intracellular pathway activation. These small GTPases are activated by guanine nucleotide exchange factors (GEF), which put on guanosine triphosphate (GTP) and take away a guanosine diphosphate (GDP). GEF are directly activated by the second messenger cAMP, which is in turn provided by the activation of G proteins. The compartmentalization and controlled activation of small GTPases are crucial and controlled by ubiquitination. Ras and Rap activation determines downstream MAPK cascades. Ras activation induces Raf activation and the PI3K pathway activation. Blocking of Ras activation protects nigral cells from MPTP-induced death.146 Raf protein has been found to be caspase-3 degraded as a result of apoptotic induction mediated by MPTP.147 Described first as an oncongene, DJ1 was shown to act cooperatively with Ras and c-myc inducing tumoral cell transformation in response to growth factors,148 but the significance of these findings to PD is uncertain.

13.3.3Intracellular Signaling Cascades

As was mentioned earlier, di erent initiators activate intracellular signaling

pathways involved in both cell toxicity and protection in dopaminergic neuronal cells.149,150

13.3.3.1Mitogen Activated Protein Kinases (MAPK) Pathway

The MAPK pathway is activated by a protein kinase cascade. A first upstream kinase, MAPKKK, is phosphorylated by small GTPases or PKC. Raf and MEKK1-5 are specific MAPKKKs involved in PD (Figures 13.2 and 13.3).151 MAPKKKs in turn phosphorylate the MAPKKs MEK 1/2, MKK4/7 and MKK3/6, followed by phosphorylation of the last kinases, MAPKs: the extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38. Activation of these three MAPKs determines di erent intracellular

signaling pathways and has been implicated in selective induction of dopaminergic neuronal apoptosis in PD.152,153 Two of these pathways are ROS-

dependent whereas the third one (p38) is not. It has been proposed that induction of the three pro-apoptotic pathways is required in order to drive the

cells to cell death. In the 6OHDA model, ERK, JNK and p38 activation is necessary for inducing apoptosis because blocking either of them is su cient to keep the cells alive. Additionally, the three cascade activator kinases are found phosphorylated after exposure of mesencephalic cultures to glutamate.126 In contrast, dopaminergic neurons also activate two intracellular pathways that

compete with cell death pathways including ERK154 and phosphoinositide 3-kinase (PI3K)/Akt,155,156 thus promoting cell protection and neuronal

survival.157

The role of ERK pathway has been found to be highly dependent on the

experimental paradigm tested. In some cases its activation has a protective role,158,159 while in other cases it mediates neurotoxicity.160–162 ERK pathway

has been found to be activated in response to neurotrophic factors stimulating the activity and/or expression of anti-apoptotic proteins, including BCL2,163 and the transcription factor cyclic AMP responsive element-binding protein (CREB, Figures 13.2 and 13.3).164 In support of a neuroprotective role for ERK pathway activation in PD, ERK phosphorylation is reduced in SH-SY5Y cells after treatment with MPP1.147 In contrast, a neurotoxic e ect is also supported by the findings that ERK and also p38 pathway are found to be highly activated after exposure of mesencephalic cultures to glutamate126 and that ERK phosphorylation status is required for regulating the splicing of a-synuclein14 (Figure 13.2). Moreover, some studies have shown no relation of

ERK pathway activation with PD.165

JNK is one of the molecules reportedly activated by oxidative stress,166,167 and it is considered an essential molecule in neurodegeneration.168 JNK and downstream c-jun are activated by Ire a/b in response to ER stress resulting in ER-specific apoptosis.169 The JNK activator MKK7 (Erk kinase kinase-1/ MAPK kinase 4 and MAPK kinase 7) is phosphorylated by the intracellular serine/threonine kinase MLK3 (ASK1 and mixed-lineage kinase 3), and is therefore considered to be a mitogen-activated protein kinase kinase kinase (MAPKKK).170 JNK1 and JNK2 isoforms have a broad tissue distribution, while JNK3 is predominantly found in brain and is specifically related to

neuronal death.171 JNK, and particularly JNK3, have shown to increase and activate its target c-jun after 6-OHDA treatment.172,173 MPP1 neurotoxicity is

dependent upon JNK and c-jun activation.173 JNK activation is involved in apoptosis induced in vitro by several oxidants.79,175–177 Also, JNK is involved in

dopamine-induced neuronal death in the striatum166 and hydrogen peroxideinduced toxicity of primary cortical neurons.178 JNK is found to be activated after oxidative stress in in vivo experimental models179 and in PD patients.180 c-jun activates AP-1 causing elevated genetic expression of Fas ligand (FasL)181 and cycloxigenase 2 (COX2), which are proposed as final mediators in activation of JNK by MPTP.174 In the 6-OHDA model, JNK activates the NFkB cascade182 and JNK2 traslocates to the mitochondia and phosphorylates 14-3-3 protein, which facilitates translocation of Bax to the mitochondria where

it generates cytochrome c release79 (Figure 13.1). JNK is also phosphorylated, as well as BH3-only members of the Bcl-2 family, via P75NTR in an alternate

pro-apoptotic function of neurotrophic factors.183