**1. Introduction**

Parkinson's disease (PD) is a neurodegenerative disorder of the central nervous system (CNS), which affects about 1% of human population over the age of 60 [1] around the world and, for which, up to now, no cure has been developed [2]. Resting tremor, rigidity, hypokinesia, and postural instability are the four cardinal motor symptoms of PD resulting from the loss of dopaminergic neurons in the substantia nigra pars compacta, which is a key regulatory structure of basal ganglia circuitry. As the disease progresses, patients frequently develop cognitive impairment and depression. Most motor symptoms can be attributed to the degeneration of dopaminergic neurons within the substantia nigra pars compacta [3]. Nonetheless, in recent years, it has become increasingly appreciated that several other non-dopaminergic neuronal populations also degenerate (Figure 1). These include various autonomic nuclei and the locus coeruleus as well as glutamatergic neurons throughout the cerebral cortex. PD is characterized by the formation of specific inclusions called Lewy bodies (LBs) in neurons of several brain structures. LBs consist mostly of misfolded proteins such as α-synuclein, tubulin and microtubule associated proteins, ubiquitin, amyloid precursor protein, synaptic vesicle proteins, various enzymes, and chaperons/co-chaperons [4].

**Figure 1.** Principal pathological processes in PD etiology and clinical hallmarks of the disease. SNpc—substantia nigra pars compacta, VTA—ventral tegmental area.

The MAP (mitogen-activated protein) kinase family is one of the oldest and evolutionally conserved family of serine/threonine protein kinases responsible for intracellular signaling in *Eukaryota* [5]. MAPKs (MAP kinases) regulate many physiological processes such as gene expression, mitosis, metabolism, cell differentiation and motility, stress response, survival, or cell death [6]. In mammalian cells, there are four main groups of conventional MAPKs: ERK1/2 (called also MAPK3 and MAPK1, respectively), ERK5, JNKs (JNK1, JNK2, and JNK3 called MAPK8, MAPK9, and MAPK10, respectively) and p38 MAPKs (p38α, p38β, p38γ, and p38δ called also MAPK14, MAPK11, MAPK12, and MAPK13, respectively). All these isoforms share sequence similarities but their cellular targets/substrates differ substantially. In addition, atypical MAPKs including NLK (Nemo-like kinase), ERK3/4, and ERK7/8 classified into a separate group have been described [6]. All these kinases collaborate in transmitting signals from numerous extracellular stimuli and control intracellular processes triggered by them. Thus, in consequence, MAPKs are capable of phosphorylating and altering the activities of countless substrates in different subcellular compartments. MAPK substrates

have been found not only in the cytoplasm but also in mitochondria, the Golgi apparatus, the endoplasmic reticulum, and the nucleus [7,8].

#### *1.1. MAPK Signalling*

The MAPK signalling cascade provides a mechanism for cells to respond to a catalogue of external signals. In fact, the diversity and specificity of cellular responses is facilitated through a linear cascade of events, which is comprised of a sequentially operating set of three evolutionarily conserved groups of protein kinases known as: MAPK, MAPK kinase (MAP2K), and MAPK kinase kinase (MAP3K). MAP3Ks are serine/threonine kinases, which are activated either via phosphorylation and/or due to the interaction with a small GTP-binding protein of the Ras/Rho family in response to extracellular stimulus. MAP3Ks activation results in phosphorylation and activation of MAP2Ks, which consequently stimulate MAPKs activity through dual phosphorylation of threonine and tyrosine residues positioned in the activation loop of kinase subdomain VIII. The activated MAPKs then phosphorylate target substrates specifically on serine or threonine residues followed by a proline residue. MAP2Ks such as MEK3 and MEK6 are activated by a wide range of MAP3Ks (MEKK1–3, MLK2/3, ASK1, Tpl2, TAK1, and TAO1/2), which become activated in response to oxidative stress, UV irradiation, hypoxia, ischemia, and cytokines including IL-1 (interleukin-1) and TNF-α (tumor necrosis factor alpha). Lastly, these events lead to altered gene expression and modulate crucial cellular functions under normal and pathological conditions such as Parkinson's disease [9].

#### *1.2. JNK Signaling*

JNKs (c-Jun N-terminal kinases) are a family of protein kinases activated in response to cytokines, growth factors, pathogens, and stress. JNK-mediated signaling pathways affect gene expression, neuronal plasticity, regeneration, apoptosis, or cellular senescence [10]. JNKs are activated through a dual phosphorylation of threonine and tyrosine residues within a threonine-proline-tyrosine (Thr-Pro-Tyr) motif by two MAP kinase kinases: MKK4 and MKK7. These two MAP kinase kinases can be inactivated by serine/threonine and tyrosine protein phosphatases [9]. In addition to the regulation by upstream kinases, the JNK signaling pathways are modulated by various scaffolding proteins including JNK-interacting protein 1, 2, and 3 (JIP1-3). The JNK family consists of 10 isoforms derived from three genes: JNK1 (four isoforms), JNK2 (four isoforms), and JNK3 (two isoforms). In mammalian cells, JNK1 and JNK2 are ubiquitously expressed while JNK3 is found mainly in the brain, heart, and testis [11]. In order to understand the biological function of JNKs, gene knockout studies were performed. It was found that mice deficient in JNK1, JNK2, JNK3, and JNK1/JNK3 or JNK2/JNK3 survived normally. Compound mutants lacking genes encoding JNK1 and JNK2 were embryonically lethal and had severe dysregulation of apoptosis of brain cells [12]. Under normal conditions, JNKs phosphorylate a variety of substrates. Examples of these substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62), cell membrane receptors (e.g., BMPR2), mitochondrial proteins (e.g., Mcl1 and Bim), or proteins involved in vesicular transport (e.g., JIP1 and JIP3) [13].

In mammalian brains, JNK transcripts have been detected at levels similar to those in peripheral organs. However, JNK activity is noticeably higher in CNS than in peripheral organs. This activity can be increased by noninvasive environmental stimuli, which underlines the important role of JNKs in the brain [14] under norm and pathology and suggests that it may be implicated in neurodegenerative disorders such as Parkinson's disease [15]. In this respect, it should be stressed that JNKs can be activated by a number of factors implicated in PD such as toxicants [16] and unfolded/misfolded proteins [17]. Some studies have demonstrated that JNKs are significantly activated in several common animal models of PD induced by neurotoxins such as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), 6-OHDA (6-hydroxydopamine) or LPS (lipopolysaccharide) [18–21]. Genetic deletion of JNK2 and JNK3 protects against MPTP-induced neurodegeneration in mice [22]. Moreover, some other studies have indicated that antioxidant and

anti-inflammatory compounds provide neuroprotection in the MPTP and 6-OHDA model of PD, at least in part, through the inhibition of JNK activation [23,24]. In addition, it was found that inhibition of JNKs with the SP-600125 inhibitor protects dopaminergic neurons both from MPP+ (1-methyl-4-phenylpyridinium)-induced neuronal apoptosis in vitro and in MPTP and 6-OHDA models of PD [15]. Another inhibitor of JNKs, SR-3306, was found to reduce the loss of dopaminergic cell bodies in the substantia nigra and their terminals in the striatum [25]. SR-3306 was also shown to have a therapeutic effect in Alzheimer's disease. A marked improvement of cognitive deficits, a significant decrease in the amount of β-amyloid plaques, and a decrease in tau phosphorylation in inflammatory responses were observed in transgenic animals treated for 12 weeks with the JNK inhibitor SP-600125 [26]. Recently, it has been reported that instant activation of JNK phosphorylation following treatment of cells with the HMGB1 (high mobility group box 1) protein cause an increase in the expression of tyrosine hydroxylase. The imbalance of this reduces dopamine synthesis and induces PD [27].

JNKs are not only implicated in the survival of dopaminergic neurons but also in dopamine transmission, which is, among the pathways, most impaired during the course of Parkinson's disease [28]. Dopamine plays a central role in motor and cognitive functions as well as in reward processing by regulating glutamatergic inputs in the striatum. Release of dopamine rapidly exerts its influence on synaptic transmission and regulates both AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (*N*-methyl-D-aspartate) receptors [29]. JNKs were found to be downstream targets of postsynaptic NMDA receptors and, moreover, NMDA activity is linked to the presence of a JNK scaffolding protein, JIP1. It was shown that NMDA-evoked glutamate release is controlled by presynaptic JNK-JIP1 interaction. Using JNK2 knock-out mice, it was proven that this kinase is essential in mediating glutamate release [30]. Activation of the glutamatergic pathway together with the dopaminergic one is responsible for synaptic plasticity, long-term potentiation (LTP), and long-term depression (LTD), which underlie motor learning. Accordingly, it has been found that LTP and LTD are altered in animal models of PD [31]. In addition, it has been found that the JNK1-Rac1 signaling pathway mediates phosphorylation of serine 295 in the PSD-95 (postsynaptic density protein 95) protein and, thus, enhances its synaptic accumulation and capability to recruit surface AMPA receptors and, lastly, potentiates excitatory postsynaptic currents [32]. It is worth mentioning that AMPA receptors, which are extremely relevant for synaptic plasticity, are physiological substrates of JNKs [33]. Lately, it has been found that, in a mouse model of PD, the JNK pathway is required for dopamine D1 receptor (D1R)-dependent modulation of corticostriatal synaptic plasticity. Pharmacological activation of D1R evokes a large increase in JNK phosphorylation. Electrophysiological experiments on brain slices from PD mice show that inhibition of JNK signaling in the pathway of striatal projection neurons prevents the increase in synaptic strength caused by activation of D1Rs [28].

It should be stressed that JNKs are implicated in other processes essential for neuronal homeostasis that seem to be severely dysregulated during Parkinson's disease. For example, JNKs seem to play a role in protein transport in the brain. A study on *Caenorhabditis elegans* provides evidence that components of the JNK pathway are necessary for normal protein transport [34] and JNKs have been shown to modulate the interaction of kinesin with microtubules [35]. Therefore, inadequate JNK activity may be at the root of the impairment in the axonal transport frequently observed in PD and a number of many other neurodegenerative disorders [36,37].

JNK signaling is also linked to the apoptosis in neurons [38]. There is a study showing that, in cultured neurons, c-Jun activation is required for NGF (nerve growth factor) withdrawal-induced apoptosis and inhibition of c-Jun protects neurons from induced cell death. For instance, NGF deprivation-induced apoptosis is associated with the activation of the GTPase Cdc42 and JNKs in primary superior cervical ganglion sympathetic neurons. In addition, overexpression of the MAP3K apoptosis signal-regulated kinase 1 (ASK1), has been found to activate JNKs and to induce apoptosis in NGF-differentiated pheochromocytoma PC12 cells and primary rat sympathetic neurons [39]. On the

other hand, inhibition of JNK signaling has been shown to reduce apoptosis of many other cells [40–43]. Therefore, JNKs may be critical for pathological cell death observed in Parkinson's disease [44].

Numerous studies have implicated JNKs in oxidative stress, which is known to play an important role in Parkinson's disease and other neurodegenerative disorders [45–47]. Research on *Drosophila melanogaster* showed that flies with mutations that accelerate JNK signaling accumulate less oxidative damage and live longer than wild-type flies [48]. JNKs activation has been also linked to stress evoked by misfolded proteins. Neuropathogenic forms of the huntingtin receptor and the androgen receptor were shown to inhibit axonal transport [49] and subsequent studies showed that this inhibition is mediated by JNK [37].

It should be noted that several other studies provide new insights into the role of JNK-mediated pathways in the control of the balance of autophagy in response to genotoxic stress i.e., the process that plays an important role in neurodegeneration including Parkinson's disease [50–52].
