*1.3. ERK1/2 Signaling*

The ERK1/2 (extracellular signal–regulated kinases 1 and 2) signaling cascade is a central MAPK pathway that plays a role in the regulation of various cellular processes such as proliferation, differentiation, development, learning, survival, apoptosis etc. [6]. This pathway is activated by growth factors [53], insulin [54], ligands of G protein-coupled receptors [55], or stress factors [56]. All these activators trigger a signal transmission by interacting with specific receptors such as receptors with tyrosine kinase activity (RTKs) or G protein-coupled receptors (GPCRs) [57].

Under a normal condition, the ERK1/2 pathway plays an essential role in the regulation of transcription. It phosphorylates and activates different transcription factors such as Elk1, c-Fos, p53, Ets1/2, c-Myc, and NFAT, which, in turn, activate numerous genes that encode proteins involved in proliferation [58]. Activation of ERK1/2 is crucial for efficient G1/S phase progression in a normal cell cycle. As mentioned above, ERK1/2 directly phosphorylate Elk1, which is involved in the expression of immediate-early genes. In addition, through direct phosphorylation of c-Fos, ERK1/2 promotes its association with c-Jun and the formation of a transcriptionally active AP-1 (activator protein 1) complex. The expression of cyclin D1, which is a protein that interacts with CDKs (cyclin-dependent kinases), permits G1/S transition depending on AP-1 activity [9]. In addition, ERK1/2 extend the MAPK cascade by phosphorylating and activating MAPKAPK (MAPK activated protein kinase) family members including RSKs (ribosomal S6 kinases), MSKs (mitogen and stress activated protein kinases), and MNKs (MAPK interacting protein kinases) [8]. Additionally, ERK1/2 phosphorylate members of the STAT transcription factor family (STAT1, STAT3, STAT4, and STAT5), which are known to mediate many aspects of survival, proliferation, differentiation [59], cytokine dependent inflammation [60], and apoptosis [61]. Genetic studies highlight differences between ERK1 and ERK2 isoforms. Some of them show that ERK1-null mice are neurologically normal with no impairment in the ability to learn, which may suggest that ERK2 compensates for the loss of ERK1 [62]. In contrast, it was shown that ERK2 knock-out mice are embryonically lethal [63].

In an adult mammalian central nervous system, ERK1/2 are expressed at a higher level in post-mitotic neurons. Immunohistochemical studies have demonstrated that ERK2 is localized in the soma and dendritic trees of neurons of the neocortex, the hippocampus, the striatum, and the cerebellum [64].

The ERK1/2 pathway mediates dopaminergic and glutamatergic signaling in the central nervous system and maintains normal activity of striatal neurons. Activation of ERK1/2 is important for associative learning, memory, visual cortical plasticity, etc. [65]. In addition, it is involved in the activation of D1 and D2 dopamine receptors in the striatum and ionotropic or metabotropic glutamatergic receptors in the dentate gyrus [66]. Moreover, it has been shown that the ERK1/2 signaling pathway plays an important role in the maintenance of spatial memory and long-term fear memory [67]. Another study has revealed the importance of ERK1/2 phosphorylation in neuronal development. Transiently repressed ERK1/2 phosphorylation in mice during the neonatal stage by intraperitoneal injection of MEK1/2 inhibitor, SL327, caused apoptosis of brain cells and had an effect on brain functioning: reduced LTP, impaired memory, and deficits in social behavior [68].

ERK1/2 signaling is involved in neuronal death, which is a major phenomenon in all neurodegenerative diseases including Parkinson's disease [69]. Using the oligodendroglial CG4 cell line, it was shown that H2O2-induced cell death is prevented by the application of the ERK1⁄2 pathway inhibitor, PD98059 [70]. Additionally, it has been shown that nitric oxide produced by glial cells induces neuronal degeneration through ERK1⁄2 activation [71] and that this degeneration might be blocked by applying, PD98059 [72]. Application of another inhibitor of this pathway, U0126, also indicated that death of striatal neurons induced by dopamine was associated with ERK1⁄2 activation [73].

There are several processes that link ERK1/2 and Parkinson's disease. In particular, these include: oxidative stress and mitochondria dysfunction, cell survival and apoptosis, neuroprotection, and inflammation. Regarding mitochondria dysfunction, it was found that, in the substantia nigra of PD patients, there is a mild deficiency in mitochondrial complex I [74]. Moreover, using confocal microscopy, it was established that phosphorylated ERK1/2 (p-ERK1/2) immune-reactivity was associated with mitochondrial proteins called MsSOPs and that some vesicular-appearing p-ERK1/2 granules enveloped enlarged mitochondria. In addition, p-ERK1/2 were found within the mitochondria of degenerating neurons derived from Parkinson's disease patients and patients with Lewy body dementia [75]. There are also some other studies that support an idea that ERK1/2 inhibition activates both apoptotic and necrotic cell death-inducing pathways [76]. ERK1/2 directly phosphorylates mitochondrial transcription factor A (TFAM) on serine 117, which affects TFAM-DNA binding and, in consequence, leads to mitochondrial dysfunction. In addition, it was found that TFAM, which is downregulated by ERK1/2 in cells chronically treated with a complex 1 inhibitor, MPP+, regulates mitochondrial biogenesis [77]. Regarding oxidative stress, Wang et al. [78] have shown that the DJ-1 transcription factor interacted with ERK1/2 and was required for the nuclear translocation of ERK1/2. This translocation was suppressed in DJ-1 knock-down cells and DJ-1 null mice treated with an oxidative insult. Additionally, endoplasmic reticulum (ER) stress seems to play a critical role in the progression of Parkinson's disease. Results obtained by Cai et al. [79] indicate that ER stress-induced apoptosis in PD might be inhibited by a basic fibroblast growth factor (bFGF). Administration of bFGF improved motor function recovery, increased tyrosine hydroxylase positive neuron survival, and upregulated the levels of neurotransmitters in the brain of a rat model of Parkinson's disease. Another study has shown that a redox protein, thioredoxin-1, protects neurons from injuries and attenuates symptoms of Parkinson's disease [80]. Additionally, it has been shown that the ERK1/2 and JNK1/2-c-Jun systems are linked with *L*-DOPA-induced neurotoxicity of dopaminergic neurons in a cellular model of PD [81] and that PI3K/Akt and ERK1/2 signaling pathways are involved in the protection of dopaminergic neurons against MPTP/MPP+-induced neurotoxicity [82]. In addition to that, it was found that, in LPS-induced PD models in vivo and in vitro, a flavonoid known as licochalcone A (Lico.A) significantly inhibited the production of pro-inflammatory mediators and microglial activation by blocking phosphorylation of ERK1/2 [83].

Lastly, it should be mentioned that some of the functions attributed to ERK1/2 in neuronal survival might be carried out by ERK5 since PD98059 and U0126 known as MEK1/2 inhibitors might inhibit the ERK5 pathway as well [84,85]. There is also a study that sheds light on the distinct roles of ERK1/2 and ERK5 in the survival of dopaminergic neurons under physiological conditions and acute oxidative stress. The latter condition is extensively linked to the molecular pathogenesis of Parkinson's disease. The interaction between ERK5 and ERK1/2 pathways was found to promote basal survival of dopaminergic neurons when exposed to oxidative stress. When both pathways were inhibited, the decline in basal survival of MN9D dopaminergic cells after exposure to a toxic agent, 6-OHDA, was observed. In addition, it was found that ERK5 and ERK1/2 have different roles in neuronal metabolism. Activation of ERK5 promoted the survival of MN9D cells but had no influence on the toxic effect of 6-OHDA on these cells [86].

#### *1.4. p38 MAPK Signaling*

The p38 MAPKs are strongly activated by extracellular stimuli such as UV light, heat shock, osmotic shock, inflammatory cytokines (e.g., TNF-α, IL-1β), or growth factors (e.g., CSF-1). Thus, these kinases are also known as stress-activated ones [87]. There are four isoforms of p38 MAPKs known as α, β, γ, and δ. All of them share up to 60% sequence similarities and 40% to 45% with other MAP kinase family members [88]. p38 MAPK isoforms have a different expression pattern. p38α MAPK is ubiquitously expressed in most cell types. p38β MAPK is mainly expressed in the brain while p38γ MAPK—in skeletal muscle and p38δ MAPK—is expressed in endocrine glands [89].

Regarding the role of p38α MAPK, it was found that the knockout of the gene encoding this protein is lethal [90] while mice lacking the *p38β* gene were viable and exhibited no apparent health problems. When embryonic fibroblasts from *p38β*−*/*<sup>−</sup> mice were analyzed, expression and activation of p38α MAPK, ERK1/2, and JNKs in response to cellular stress remained unchanged, which suggests that the α isoform of p38 MAPK is the main one responsible for controlling all of the detrimental consequences of the p38 MAPK activation such as microglia activation, neuro-inflammation, oxidative stress due to reactive oxygen species (ROS) accumulation, nitric oxide activity, and neuronal apoptosis [91–93].

It is worthy to note that several lines of evidence suggest that p38 MAPKs play a role in neuronal apoptosis, which is linked to Parkinson's disease [94,95]. For instance, it has been reported that they induce apoptosis by phosphorylating Bcl-2 (B-cell lymphoma 2) family members [96]. Interestingly, phosphorylation of one such member, BimEL, on serine 65 may be a common regulatory point for cell death induced by both p38 MAPK and JNK pathways [97]. In addition, oxidative stress in dopaminergic neurons has been shown to trigger the p38 MAPK pathway which, in consequence, may lead to uncontrolled activation of apoptosis in cellular and animal models of Parkinson's disease [98–100]. Together these data suggest that both oxidative stress and p38 MAPK operate to balance the pro-apoptotic and anti-apoptotic phenotypes of dopaminergic neurons. Some other studies show the link between the generation of ROS, initiation of the p38 MAPK/JNK signaling, and apoptosis of neuronal cells in different models of Parkinson's disease [101–105]. Interestingly, it has been shown that the exacerbating effects of deletion of *Park2* gene (encoding parkin protein) on ethanol-induced ROS generation, mitophagy, mitochondrial dysfunction, and cell death were reduced by p38 MAPK inhibitor, SB203580, in vitro and in vivo. In the case of dopaminergic neurons it has been shown that deletion of this gene exacerbates ethanol-induced damage through p38 MAPK dependent inhibition of autophagy and mitochondrial function [106]. Similar data revealed that the p38 MAPK-parkin signaling pathway regulates mitochondrial homeostasis and neuronal degeneration in the A53T α-synuclein mutant model of Parkinson's disease [107].

Attention to p38 MAPKs in terms of neurodegeneration is driven by the fact that these kinases are involved in dopaminergic signaling, which is a pathway known to be disrupted during Parkinson's disease [93]. There is a study by Wu et al. [108] showing that degeneration of nigral dopaminergic neurons was accompanied by an increase in the level of p38 MAPKs and their phosphorylated forms. In agreement are the results published by Yoon et al. [109] showing that phosphorylation of p38 MAPKs by the LRRK2-ASK1 pathway regulated neuronal toxicity and apoptosis. Pharmacological inhibition of this kinase with SB203580 blunted MPTP neurotoxin induced cell apoptosis [110]. Similarly, neuronal protection was observed by applying another p38 MAPK inhibitor, SB239063 [111], or celastrol in rotenone-evoked neuroblastoma SH-SY5Y cellular model of Parkinson's disease [112].

An important issue in Parkinson's disease is neuro-inflammation that can be associated with alterations in glial cells including astrocytes and microglia. The response of neurons to activation of microglia promotes oxidative stress, inflammation, and cytokine-receptor-mediated apoptosis, which eventually contribute to the death of dopaminergic neurons and to the progression of the disease [113]. Rotenone, which is an inhibitor of the mitochondrial complex I, can directly activate microglial cells through the p38 MAPK pathway and initiate dopaminergic neuronal damage in substantia nigra, which ultimately results in parkinsonism. Unfortunately, the exact mechanism behind the selective degeneration of nigral dopaminergic neurons is not fully understood [103]. Moreover, it

has been reported that montelukas, which is a cysteinyl leukotriene receptor antagonist, exerted neuroprotective effects in the rotenone-induced PD animal model through the attenuation of microglial cell activation and p38 MAPK expression [114]. It has been suggested that degeneration of nigral dopaminergic neurons was followed by an increase in the expression of p38 MAPKs, p53, and Bax (Bcl-2-associated X protein). Neurotoxins exhibited a similar effect on the level of these proteins in cultured pheochromocytoma PC12 cells, which shows that this phenomenon occurs both in vitro and in vivo. When activated, Bax is exported into the mitochondrial membrane where it oligomerizes and triggers mitochondrial apoptotic signaling. This observation strongly indicates that p38 MAPK/p53 stimulation of Bax can certainly contribute to rotenone's neurotoxicity in models of Parkinson's disease [108]. p38 MAPK also plays a role in neurotoxicity induced by MPTP [115].

Inflammation and autophagy are highly interdependent cellular processes. Autophagy plays an anti-inflammatory role and suppresses pro-inflammatory process by regulating innate immune signalling pathways and inflammasome activity [116]. Inflammatory signals also function to reciprocally control autophagy [117]. However, the mechanism of mutual regulation of both processes is not yet explained. A recent study has shown that the α isoform of p38 MAPK plays a direct and essential role in relieving autophagic control in response to an inflammatory signal by direct phosphorylation of UNC51-like kinase-1, which is the serine/threonine kinase involved in the autophagic cascade in microglia. Moreover, phosphorylation of UNC51-like kinase-1 by p38α MAPK inhibited activity of this kinase, disrupted its interaction with autophagy-related protein 13, ATG13, and, thus, reduced the level of autophagy [118]. Because autophagy disorders are more commonly associated with neurodegenerative diseases, the role of p38 MAPKs in autophagy was studied in a human neuroblastoma SK-N-SH cellular model of Parkinson's disease. The studies revealed that microRNA (miR)-181a regulated apoptosis and autophagy by inhibiting the p38 MAPK/JNK pathway [119].
