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Oxidative stress factors in Parkinson’s disease

Parkinson’s disease (PD) is the second most common cause of neurodegeneration. Over the last two decades, various hypotheses have been proposed to explain the etiology of PD. Among these is the oxidant-antioxidant theory, which asserts that local and systemic oxidative damage triggered by reactive oxygen species and other free radicals may promote dopaminergic neuron degeneration. Excessive reactive oxygen species formation, one of the underlying causes of pathology in the course of PD has been evidenced by various studies showing that oxidized macromolecules including lipids, proteins, and nucleic acids accumulate in brain tissues of PD patients. DNA oxidation may produce various lesions in the course of PD. Mutations incurred as a result of DNA oxidation may further enhance reactive oxygen species production in the brains of PD patients, exacerbating neuronal loss due to defects in the mitochondrial electron transport chain, antioxidant depletion, and exposure to toxic oxidized dopamine. The protein products of SNCA , PRKN , PINK1 , DJ1 , and LRRK2 genes are associated with disrupted oxidoreductive homeostasis in PD. SNCA is the first gene linked with familial PD and is currently known to be affected by six mutations correlated with the disorder: A53T, A30P, E46K, G51D, H50Q and A53E. PRKN encodes Parkin, an E3 ubiquitin ligase which mediates the proteasome degradation of redundant and disordered proteins such as glycosylated α-synuclein. Over 100 mutations have been found among the 12 exons of PRKN . PINK1, a mitochondrial kinase highly expressed in the brain, may undergo loss of function mutations which constitute approximately 1–8% of early onset PD cases. More than 50 PD-promoting mutations have been found in PINK1 . Mutations in DJ-1, a neuroprotective protein, are a rare cause of early onset PD and constitute only 1% of cases. Around 20 mutations have been found in DJ1 among PD patients thus far. Mutations in the LRRK2 gene are the most common known cause of familial autosomal dominant PD and sporadic PD. Treatment of PD patients, especially in the advanced stages of the disease, is very difficult. The first step in managing progressive PD is to optimize dopaminergic therapy by increasing the doses of dopamine agonists and L-dopa. The next step is the introduction of advanced therapies, such as deep brain stimulation. Genetic factors may influence the response to L-dopa and deep brain stimulation therapy and the regulation of oxidative stress. Consequently, research into minimally invasive surgical interventions, as well as therapies that target the underlying etiology of PD is warranted.


Introduction
Parkinson's disease (PD) is one of the most common central nervous system (CNS) degenerative diseases and affects almost 2% of the population over the age of 65 and 5% over the age of 85. Although PD was first described over 200 years ago, it still remains an incurable disease and its cause is not fully understood. Given that there are currently no specific diagnostic biomarkers, the diagnosis of PD is based on clinical criteria including the presence of motor disorders (tremor, rigidity, bradykinesia, and postural instability), other motor features (gait disturbance and abnormal posture), and nonmotor symptoms (dementia, depression and disordered sleep) (Braak et al., 2004;Tarakad et al., 2017). It is known that degenerative processes in PD begin years prior to the appearance of clinical symptoms. Currently, several hypotheses exist to expound the mechanisms underlying the etiopathogenesis of PD (Figure 1).
One cause of PD might be the accumulation of pathological proteins in abnormal spatial conformations within specific CNS structures, leading to disorders of nerve cell metabolism and damage to macromolecular compounds (Deas et al., 2016;Draoui et al., 2020). According to another hypothesis, neuronal damage and ultimately death occur due to failure of the ubiquitin-proteasome system in removing pathological protein aggregates. Under physiological conditions, the ubiquitin-proteasome system is involved in recognizing and ubiquitinating abnormal proteins, thereby targeting them for proteasomal degradation. Energy is required for the process of protein removal, at the level of both ubiquitination and degradation. Intelligibly, diminished performance of the ubiquitin-proteasome system in degenerative diseases may stem from a decrease in energy in the form of ATP caused by defects in the mitochondrial electron transfer chain (Bragoszewski et al., 2017). Similarly, exposure to oxidative stress may lead to aberrant cellular bioenergetics and precipitate pathologic processes culminating in clinicallyidentifiable PD (Isobe et al., 2010;Andican et al., 2012;Subramanian et al., 2013;Puspita et al., 2017). Numerous reports also indicate a relationship between PD pathogenesis and the processes of apoptosis and/or autophagy (Banerjee et al., 2010;Lynch-Day et al., 2012).
Moreover, oxidative stress may affect both α-synuclein (ASN) aggregation and disease progression, as well as the response to L-dopa pharmacotherapy and deep brain stimulation (DBS) therapy (Dorszewska et al., 2014). In this review, we reviewed the molecular factors involved in the generation of oxidative stress in PD and identified potential targets that may improve the diagnosis and treatment of this degenerative disease in the future.

Search Strategy and Selection Criteria
For this review, literature searches were performed on PubMed, Scopus and other public databases. The following keywords were used: ((Parkinson's disease) AND ((oxidative damage) OR (oxidative stress)) AND (((levodopa) OR (L-DOPA)) OR (therapy))). The articles included in this review were chosen base on relevance to the subject. Subsequent search was performed through articles' references. The last literature sweep was performed in June 2020.

Molecular Parameters and Oxidative Stress Generation
Oxidative stress is one of the main factors behind neurodegeneration in PD. Although increased generation Review of reactive oxygen species (ROS) leads to oxidative stress, physiological levels of these molecules are a normal outcome of metabolic processes and play an important role in cellular defense (Puspita et al., 2017;Saito, 2017). ROS are produced via anterograde and retrograde electron motion along the mitochondrial electron transfer chain, metabolism of DA, and other redox reactions (Figure 1). In healthy dopaminergic neurons, levels of ROS are strictly controlled by various antioxidative mechanisms involving glutathione (GSH), superoxide dismutase (SOD) and DJ-1. These processes, however, tend to fail in patients with PD. Indeed, Bender et al. (2006) found a slightly higher number of mutations, specifically somatic deletions in mitochondrial DNA (mtDNA), in the substantia nigra of PD patients compared to agematched controls corroborating the view that nigral neurons are disproportionately more affected by oxidative damage.
Aerobic respiration takes place in the inner mitochondrial membrane (IMM). The transfer of electrons between protein complexes with increasing reduction potentials allows for the concentration of protons in the intermembrane space, generating a potential difference that fuels ATP synthesis (Puspita et al., 2017). Although electrons are typically transferred sequentially from complex I to complex IV, monoelectron reduction of molecular oxygen, the final electron acceptor, occasionally occurs generating superoxide radical (O 2 -). Electrons may escape from complex I (nicotinamide adenine dinucleotide, NADH) and complex III (cytochrome bc1) to generate ROS (Drose et al., 2008). Under physiological conditions, superoxide is converted into hydrogen peroxide by SOD2 and then detoxified by catalase (Subramaniam et al., 2013). However, with insufficient antioxidants, superoxide and other reactive molecules oxidize mtDNA. mtDNA is not only very sensitive to oxidative damage because of its lack of histones and lower-fidelity DNA polymerase (Kowalska et al., 2020), but also encodes electron transfer chain genes, resulting in a degeneration-promoting positive feedback loop. Accumulation of these abnormalities leads to increased neuronal damage and loss resulting in faster progression of PD (Bender et al., 2006). Apart from aerobic respiration and making certain reactive molecules less harmful, mitochondria play a pivotal role in maintaining proper protein function and structural integrity (Misgeld et al., 2017). These complex mechanisms engaging AAA proteases, ubiquitin-proteasome system, mitochondrial derived vesicles, and mitophagy, are collectively called mitochondrial quality control (MQC) (Amadoro et al., 2014;Misgeld et al., 2017; Figure 1). It has been shown that defects in function of Parkin and PINK1, whose interaction constitutes the first steps in the activation of MQC pathways, facilitate progressive mitochondrial dysfunction (Harper et al., 2018). Under physiological conditions, the TOM20 protein complex transfers PINK1 N-terminus from the OMM to the IMM, where it is cleaved and then proteasomally processed, preventing organelle from accumulation of PINK1 at the OMM, an induction of mitophagy signal. Mitochondrial complex disfunction, proteotoxicity, and membrane depolarization impair this process leading to PINK1 ag gregation, homodimerization, and subsequent autophosphorylation promoting activation of C-terminus kinase domains localized at the OMM. This results in the binding of ubiquitin and Parkin, which is then phosphorylated by PINK1 at Ser65 leading to activation of its E3 ligase functionality (van der Merwe et al., 2015). Activated enzyme facilitates formation of ubiquitin chains that attract more Parkin molecules increasing detection of damaged mitochondria signal, targeting substrates for degradation and even inducing mitophagy (Kalinderi et al., 2016;Harper et al., 2018). Above information suggests that mutations in PINK1 and PRKN genes should affect cell's ability to process dysfunctional organelles. Interestingly, mice with both of the genes knocked-out do not display neurodegeneration, but loss of PINK1 expression results in larger mitochondria with functional impairment (Palacino et al., 2004;Gautier et al., 2008). Moreover, results obtained during cell culture based experiments failed to confirm some of the aforementioned processes (van Laar et al., 2011;Rakovic et al., 2013). Elucidating diverse mechanisms of PINK1/Parkin interactions in MQC systems may bring us closer to understanding etiology of PD.
Initially, autophagy was thought to be a response to nutrient restriction that resulted in the degradation of specific organelles (Figure 1). Subsequent studies indicated that it is involved in a wide spectrum of physiological and pathological cellular processes such as development, cellular aging, cell death, and suppression of tumor growth. The organelles and proteins that are broken down in its course are building blocks in key biosynthesis pathways. In PD, autophagy should be a response to Lewy body (LB) accumulation and oxidative stress, however, in neurodegenerative diseases this mechanism does not function properly, depriving the cell of the only presently known means for the degradation of structures that cannot be proteasomally processed (Banerjee et al., 2010;Lynch-Day et al., 2012). Oxidation of the proteasome under conditions of elevated ROS significantly impairs its function and contributes to the aggregation of oxidized or otherwise damaged proteins in the cell (Shamoto-Nagai et al., 2003), highlighting the connection between mitochondrial impairment and LB pathology in PD-ravaged dopaminergic neurons (Braak et al., 2004).
Dopaminergic neurons tend to contain large amounts of ROS, derived from the enzymatic and nonenzymatic metabolism of DA (Saito, 2017). Consequently, they are more sensitive to various stress factors than other neurons (Lotharius et al., 2002;Saito, 2017).
DA may be catabolized by monoamineoxidase (MAO), a molecule bound to the OMM, in a process which generates H 2 O 2 as a byproduct (Lotharius et al., 2002). In addition, DA oxidation may occur spontaneously in the presence of iron generating 6-hydroxydopamine (6-OHDA), which is subsequently transformed to a reactive electrophilic molecule, p-quinon in the presence of oxygen. DA can also be oxidized to DA-quinone (DAQ), a molecule whose toxic effects are derived from its reaction with cysteine sulfhydryl groups (Lavoie et al., 1999) (Figure 1). DAQ has been shown to affect the cell's ability to sequester ROS by reacting with Cys-106 of the neuroprotective protein DJ-1 (van Laar et al., 2009;Girotto et al., 2012). In nigral neurons not affected by PD, DJ-1 is mostly localized to the cytoplasm, however, when exposed to high levels of ROS, it is oxidized to the Cys-SO2H form and translocated to the mitochondria and the nucleus (Kim et al., 2012). DJ-1 removes ROS with lower efficiency than other enzymes involved in oxidoreductive homeostasis (e.g. catalase), suggesting that it may act instead as an oxidative stress sensor by modulating signaling pathways and the expression of specific genes (Kahle et al., 2009). Zhou et al. (2005) demonstrated that increased levels of DJ-1 induce GSH synthesis, protecting DA neurons from the harmful effects of H 2 O 2 and 6-OHDA. 6-OHDA, a substance typically elevated in animal models of PD accumulates in nigral neurons, leading to various types of mitochondrial dysfunction such as inhibition of respiratory chain function or a decrease in membrane potential. Further, it has been shown that elevated levels of ROS cause increased binding of DJ-1 to mitochondrial complex I, enhancing activity of NADH: ubiquinone oxidoreductase and ultimately, the entire respiratory chain (Hayashi et al., 2009).
The aforementioned high level of iron in DA neurons can also induce ROS generation in a different way. In recent years, there has been growing evidence of interaction between metal ions and ASN second to which decreased GSH levels, increased lipid peroxidation, resulting in membrane structural damage, and elevated formation of H 2 O 2 and hydroxyl radicals may occur (Deas et al., 2016). Other effects of these abnormalities include decreased ATP levels and nucleic acid damage. 8-Oxo-2′-deoxyguanosine (8-oxo2dG) is the main oxidation product of hydroxyl radicals and guanine residues in DNA (He et al., 2018). Its levels have been shown to be higher in cerebrospinal fluid, serum and urine of people with PD as compared to controls (Isobe et al., 2010). This deoxyguanosine derivative can base pair not only with cytosine as it should, but also with adenosine, leading to a possible mutation (Hirano, 2008). Interestingly, Dorszewska et al. (2011) showed that the levels of 8-oxo2dG and corresponding DNA alterations, may be caused by treatment with L-dopa depending on its duration.

L-dopa Therapy and Oxidative Stress Parameters in Parkinson's Disease
The most effective up-to-date treatment of PD symptoms is based on replenishing or mimicking DA in the dopaminergic system, to enhance dopaminergic transmission. Since DA does not cross blood-brain barrier (BBB), replenishing DA requires admission of its precursor, L-dopa (Kostrzewa et al., 2005). The use of L-dopa in the treatment of PD is considered a "gold standard" and L-dopa preparations have been successfully used as pharmacotherapy in PD since the 1960s (Fahn, 2015). Unfortunately, the L-dopa clinical outcome is weakened by motor and nonmotor side effects, including neurotoxic effects (Gesi et al., 2001), subsequent to long-term treatment (Thanvi et al., 2004). In recent years, more and more studies indicate that they may cause enhanced ROS generation. The mechanisms underlying the said side effects of L-dopa treatment likely include oxidative DA metabolism, an indirect outcome of which is the dysregulation of biothiol levels (e.g. homocysteine, Hcy) and induction of inflammatory, apoptotic, and autophagic processes (Dorszewska et al., 2011;Andican et al., 2012;Lynch-Day et al., 2012). Elevated Hcy levels following L-dopa treatment can result in vascular diseases, cognitive impairment, dementia, depression, neurodegeneration, amounting to a poor prognosis for PD patients . In addition, it has been shown that increased Hcy levels are accompanied by reduced levels of cysteine, and in turn reduced synthesis of GSH, changes that leave the cell more susceptible to oxidative stress. The key substance associated with increased production of Hcy and reduced level of GSH in PD might be the hydrogen sulfide as its serum level is decreased in this neurodegenerative process (Yin et al., 2017). Furthermore, the hydrogen sulfide has been shown to play a role in the regulation of GSH synthesis and Hcy turnover (Kimura et al., 2004).
Subsequently, it has been shown that side-synthesis of dihydroxyphenylacetic acid (DOPAC) and H 2 O 2 during the metabolism of DA in the stratum of PD patients by monoamine oxidase (MAO) may be another mechanism underlying elevated ROS formation in DA neurons (Gesi et al., 2001). This data is confirmed by in vitro studies showing that incubation with L-dopa may increase intracellular ROS and lead to cell death. This effect was attenuated by inhibition of MAO by pargyline in studied RN46A-B14 serotoninergic cell line, which were shown to be able to produce dopamine after incubation with L-dopa (Stansley and Yamamoto, 2013). This property was reduced by co-incubation with the AADC inhibitor, NSD-1015, what further reduced ROS production and improved cell viability (Stansley et al., 2013). These data suggest that substantial doses of L-dopa may be toxic not only to dopaminergic neurons, but may also affect serotoninergic cells, leading to psychiatric symptoms of PD, like depression or anxiety (Eskow Jaunarajs et al., 2011).
Moreover, in PD, the brain deposition of heavy metals ions, such as iron ions (Fe 2+ ), may lead to non-enzymatic Fenton reaction that produces ROS, like OH• and may stimulate ASN Review aggregation (Figure 1) (Weinreb et al., 2013). Fenton reaction produces ROS, like OH· (Weinreb et al., 2013). Subsequently, cortical neurons growing in the presence of L-dopa showed significant buildup of intracellular Fe 2+ , followed by reduced cell viability. This effect was partially reduced for cells coincubated with L-dopa in astrocyte conditioned medium, suggesting the significant role of molecular environment to L-dopa toxicity (Du et al., 2009). This observation is in line with the study by Billings et al. (2019) who investigated the effect of L-dopa in transgenic hA53T mice, expressing mutated ASN with or without presence of excessive Fe 2+ . The authors demonstrated that in vivo L-dopa may act as a mild Fe 2+ chelator, comparable to Clioquinol. In the brains of wildtype mice treated with multiple doses of L-dopa, Billings et al. (2019) found no evidence of increased oxidative stress.
What is more, L-dopa improved motor functions in mice with overload of Fe 2+ . This protective effect was attenuated in hA53T transgenic murine model of PD in the presence of Fe 2+ , suggesting that L-dopa effects may depend on Fe 2+ brain content and genetic mutations that affect PD proteins, such as ASN.
While Fe 2+ ions are catalyst of DA and L-dopa oxidation, these processes can occur spontaneously in the brain, as DA and L-dopa may undergo auto-oxidation, thus generating H 2 O 2 and the more stable DA-quinone (Sulzer et al., 1999;Pattison et al., 2002). There are reports showing that DA-quinone further disturbs DA turnover, e.g. by halting the functions of TH enzyme and DA transporter (DAT), followed by mitochondrial damage resulting in ATP deficiency (Berman et al., 1999;Kuhn et al., 1999;Khan et al., 2001).
The toxic DA-quinone is further metabolized to aminochrome, a metabolite that was shown to induce formation of ASN aggregates (Muñoz et al., 2012). In the healthy brain, there are numerous mechanisms protecting against aminochrome toxicity. First, as DA oxidation is mostly dependent on pH and Fe 2+ content, thus DA present in neuronal cytoplasm is quickly absorbed by synaptic vesicles by abovementioned VMAT2. Second, DA-quinone, aminochrome, and other oxidation product, 5,6-indolequinone undergo quick polymerization to neuromelanin that stains dopaminergic neurons black, and renders the toxins inert. Third, toxic aminochrome is reduced in enzymatic reaction catalyzed by DT-diaphorase. The excessive aminochrome is conjugated with GSH by glutathione S-transferase M2-2 (GSTM2) (Segura-Aguilar et al., 2014). On the other hand, it has been hypothesized that GSHaminochrome conjugate, a building block of neuromelanin may be able to accumulate heavy metal ions such as prooxidative Fe 2+ (Zucca et al., 2017). It seems that, neuromelanin, a DA autoxidation endpoint metabolite may also exert neuroprotective and antioxidative effect by eliminating free Fe 2+ . Subsequently, the neuromelinin damage in the course of PD may cause premature ageing due to accumulation of ASN and excessive ROS, followed by neuroinflammation and reduction of neurogenesis (Nekrasov et al., 2018). Thus it is suggested that disturbances in protective mechanisms and accumulation of L-dopa and DA oxidation products may play a role in the development of PD (Herrera et al., 2017).

SNCA
SNCA encodes ASN, a protein involved in synaptic vesicle trafficking, regulation of DA biosynthesis, neuronal differentiation, chaperone activity, antioxidative processes, and suppression of apoptosis. The accumulation of ASN in nerve cells of the substantia nigra, pons, and medulla leading to cell death is a hallmark of PD (Emamzadeh, 2016). Moreover, ASN may interact with other proteins e.g. Parkin and disrupt their function. Mutations in SNCA contribute to ASN aggregation and oxidative stress ( Table 1) and the declined capacity to scavenge ROS can further exacerbate ASN aggregation (Scudamore et al., 2018). Both point mutation and duplication or triplication of SNCA lead to the aggregation of ASN, however, triplications are responsible for the more severe phenotype typified by early onset PD and dementia (Fuchs et al., 2007). Ko et al. (2000) demonstrated that overexpression of mutant SNCA increases the sensitivity of transfected human cells to oxidative injury. Moreover, oxidation of ASN decreases its capacity for forming either αor β-type secondary structures integral to the construction of fibrils.

PRKN and PINK1
PRKN encodes Parkin, an E3 ubiquitin ligase, which mediates the proteasome-dependent degradation of redundant and disordered proteins, e.g. glycosylated ASN. It is necessary for the survival of DA neurons as it protects against ASN toxicity and oxidative stress (Feany et al., 2003). PRKN mutations impair the ability of protein to bind ASN and result in the aggregation of the latter (Shimura et al., 2001) in addition to slower mutant protein turnover and increased levels of oxidative stress markers such as protein carbonyls, lipid peroxides, and nitrated proteins (Hyun et al., 2002). Loss of function mutations in PRKN causes recessively inherited PD and contributes to nearly half of the cases of familial early onset PD (EOPD) and one fifth of sporadic EOPD cases. The clinical symptoms of PD patients bearing PRKN mutations resemble idiopathic PD, with good response to L-dopa, slower disease progression and more symmetric onset (Lücking et al., 2000). Furthermore, patients with PRKN mutations more commonly suffer from depression without an impact on executive function as compared to individuals with EOPD caused by other factors (Song et al., 2020). PRKN mutations can also be identified in late onset PD (LOPD) ( Table 1).
PINK1 encodes the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), which as a mitochondrial kinase is highly expressed in the brain, heart, and muscles (Barodia et al., 2017). PINK1 loss of function mutations can cause recessive EOPD and stand for approximately 1-8% of EOPD cases (Kawajiri et al., 2011). The overexpression of PINK1 has neuroprotective properties, and it protects against oxidative stress-induced cell death by inhibiting the release of cytochrome c from mitochondria what depends on phosphorylation of a mitochondrial chaperone TRAP1 by PINK1 (Pridgeon et al., 2007). PINK1 sometimes is called a guard of mitochondrial quality control and its deficiency was linked to changes in mitochondrial morphology associated with loss of mitochondrial enzyme activity, especially complex I. The knockdown of PINK1 results in decreased mtDNA synthesis followed by decreased ATP production and it induces mitochondrial fragmentation and autophagy what promote oxidative stress (Dagda et al., 2009;Dias et al., 2013).
PINK1 together with Parkin is involved in the removal of impaired mitochondria by autophagy. The accumulation of PINK1 on the surface of dysfunctional mitochondria signals Parkin to commence degradation of the damaged organelle (Narendra et al., 2008). PINK1 or Parkin deficiency inhibits autophagy of dysfunctional mitochondria, which can lead to increased ROS and subsequent nigral cell loss. Further, impaired DA release and synaptic plasticity and defects in mitochondrial respiration and ATP-generation were found in pink1 knockout mice. Moreover, reduced fission and increased aggregation of mitochondria follow from conditions of increased oxidative stress (Gispert et al., 2009).
PINK1 can also interact with DJ-1. Overexpression of these proteins synergistically protects cells against MPTP-induced cell death, an effect that was abolished in the presence of PINK1 or DJ1 mutations (Tang et al., 2006). It is proposed that DJ-1 acts either downstream to PINK1 or parallel to the PINK1/Parkin pathway (Thomas et al., 2011), however, the exact mechanism of the interaction between PINK1 and DJ-1 is still unknown.

DJ1
DJ-1 is another neuroprotective protein which regulates numerous processes e.g. anti-oxidative, anti-apoptotic and anti-inflammatory pathways. Mutations in DJ1 gene are a rare cause of EOPD and constitute only 1% of cases. DJ-1 protects substantia nigra DA neurons from oxidative stress. Waragai et al. (2006) found elevated levels of DJ-1 in the cerebrospinal fluid of sporadic PD patients, especially in the early stages of the disease (I-II HY scale), and suggested that the protein plays a protective role against oxidative stress during PD onset. Interestingly, rapid relocalization of DJ-1 to mitochondria has been shown to occur under oxidative stress conditions (Junn et al., 2009) and may be implicated in inhibiting autophagy (Thomas et al., 2011). Cytologic analyses of fibroblasts or lymphoblasts isolated from PD patients with mutations in DJ1 gene showed mitochondrial fragmentation, impaired bioenergetics, and increased sensitivity to ROS and toxins (Yan et al., 2013). Kim et al. (2005) found that dj1 knockout mice had increased levels of mitochondrial oxidative stress and were more sensitive to MPTP toxicity as compared to wild-type animals. Interestingly, however, the dj1 knockout animals did not exhibit any significant motor impairment.

LRRK2
Leucine-rich repeat kinase-2 (LRRK2), also called dardarin, is responsible for numerous processes, including cytoskeletal dynamics, vesicular trafficking, calcium signaling, and mitochondrial function (Weindel et al., 2020). LRRK2 controls mitochondrial network dynamics and mitophagy through interactions with the mitochondrial fusion proteins and mitochondrial outer membrane proteins Hsieh et al., 2016). LRRK2 located within the cytoplasm is necessary for phosphorylation of protein, e.g, ASN and tau (Zimprich et al., 2004). Moreover, overexpression of LRRK2 or synergism with ASN may intensify destructive processes  Review during PD (Schapira, 2006).
Mutations in the LRRK2 gene are the most commonly known cause of familial autosomal dominant PD and sporadic PD. For the first time they have been described in the Basque population. Most of the LRRK2 mutations are localized in catalytic core domains of the protein, especially Roc-GTPase and kinase domains, and alter the enzyme activity. The results of LRRK2 mutations are abnormalities in mitochondrial fusion and fission dynamics, mitophagy, mitochondrial DNA (mtDNA) damage (Mancini et al., 2020). They contribute to elevated ROS production, inhibition of peroxidase activity, and in consequence to increased oxidative stress (Angeles et al., 2014). The most common mutation in the LRRK2 gene, G2019S, is present with a frequency of 10-18% in the Ashkenazi Jewish population, and with 3-13% in the European population (Lesage et al., 2005;Ozelius et al., 2006;Clark et al., 2006). The penetrance of the G2019S mutation has an age-dependent effect and varies from 28% at the age of 59 years, 51% at the age of 69 years, and 74% at the age of 79 years (Healy et al., 2008).

Other Treatments in Parkinson's Disease and Oxidative Stress
In progressive PD, optimization of dopaminergic therapy is recommended in the first place (increasing the doses of dopamine agonists and L-dopa), and then advanced therapy, including DBS, subcutaneous apomorphine, and enteral L-dopa. DBS is the leading surgical intervention for PD (Lozano et al., 2017). Still, studies have suggested that DBS is overindicated, highlighting the need for careful assessment of patient eligibility (Bronstein et al., 2011).
Following its clinical implementation, DBS quickly became the standard of care for medication-refractory motor disorders, and principally PD (Loznao et al., 2019), however, it was not until 2006 that a randomized-pairs trail evidenced that subthalamic nucleus (STN) DBS combined with medical treatment was superior to medical treatment alone in alleviating L-dopa-related motor dysfunction (Desuchl et al., 2013). A randomized controlled trial published a few years later demonstrated that DBS was superior to medication alone when stratified by patient age (Weaver et al., 2009). Nonetheless, both trials reported more serious adverse health risks associated with DBS as compared to medication alone. Current literature indicates that the STN, GPi, and the ventral intermediate thalamic nucleus (VIM), are the only foci routinely targeted using DBS in clinical practice (Deuschl et al., 2013), however, stimulation of the pedunculopontine nucleus has been shown to improve gait disturbances, and stimulation of the nucleus basalis has been investigated for the treatment of dementia (Gratwicke et al., 2018). Whether an ideal target for DBS in the treatment of PD exists remains contested. A 2010 study failed to observe any significant changes between patients having undergone pallidal or subthalamic DBS 24 months post-operatively, as measured using the Unified Parkinson's Disease Rating Scale 3 (UPDRS3; the segment dedicated to assessing rigidity, tremor, bradykinesia, and postural instability). Further, STN DBS allowed for L-dopa dose reduction, but it was correlated with worsened visuomotor processing and depression. Nonetheless, the STN is the most common target for DBS (Deuschl et al., 2013;Lozano et al., 2019) and researchers have sought to identify optimal regions for neurostimulation within it. This task, however, has been burdened by the STN's shape and anatomic position, in addition to limitations in visualization. Still, the dorsolateral STN, zona in certa and Forel-H2 field, or boundaries contacting both of these regions have been purported to yield maximally efficacious results. Recently, a high angular resolution diffusion imaging analysis evidenced that DBS applied to the central superolateral STN best ameliorated PD tremor and neurostimulation within the posteromedial superolateral STN was most effective for treating rigidity and bradykinesia (Akram et al., 2017).
DBS is contraindicated for atypical parkinsonism and affords those patients who are generally responsive to L-dopa, however, nonetheless experience medication-related side effects (e.g. off-periods and dyskinesia) an optimal response (Bronstein et al., 2011). Multiple predictors of DBS outcome exist. Anatomic variations both due to and independent of PD-related regional brain atrophy can influence targeting accuracy and the efficacy of stimulation (Younce et al., 2019). Specifically, mesencephalic surface area (Bonneville et al., 2005), paracentral region thickness (Muthuraman et al., 2017), and PD-related atrophy of the hippocampus (Aybeck et al., 2009) and sensorimotor cortex (Hwang et al., 2013) affect the response to DBS. In a recent MRI analysis, smaller thalamic volumes and larger ventricular volumes were correlated with poorer DBS outcomes, as measured using the UPDRS3 (Younce et al., 2019). Genetic factors also influence response to DBS (Artusi et al., 2019;de Oliveria et al., 2019;Kuusimäki et al., 2019). While carriers of the common p.G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) and PINK1 mutations were generally found to experience positive outcomes following DBS, those with the rarer p.R1441G LRRK2 mutation do not face such a good prognosis. Moreover, improvements in motor function of patients carrying SNCA, GBA, and LRRK2 p.T2031S mutations appear to co-occur with worsened nonmotor symptoms of PD following DBS (Kuusimäki et al., 2019). Interestingly, dysregulation of calcium homeostasis and ensuing oxidative stress underlies many of the mutations for which a correlation with DBS outcome exists. LRRK2 has been shown to upregulate the expression of the mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uptake 1 (MICU1) protein, without altering expression of the mitochondrial Na + /Ca 2+ /Li + NCLX antiporter (Verma et al., 2017). These changes promote mitochondrial calcium overload, which has been implicated in PD pathophysiology (Kowalska et al., 2020). Further, the decreased sequestration of synaptic vesicles (Piccoli et al., 2014) and increased activation of presynaptic voltage-gated calcium channels (Bedford et al., 2016) seen with certain pathologic LRRK2 variants explain the heightened cortical glutamatergic signaling that occurs due to these mutations (Verma et al., 2017). Enhanced excitatory transmission in the cortex might in turn disturb corticostriatal dynamics (e.g. overactivate the indirect pathway). DBS has been shown to induce calciumdependent astrocytic release of glutamate (Tawfik et al., 2010). In contrast to LRRK2, PINK1, a mitochondrial kinase, does regulate NLCX, however, its deficiency in PD also ultimately promotes mitochondrial calcium overload and ROS generation (Ghandi et al., 2009). Lastly, Schöndorf et al. (2014) demonstrated that induced pluripotent stem cellderived neurons from PD patients bearing mutations in the β-glucocerebrosidase (GBA1) gene featured aberrant calcium homeostasis and lysosomal functioning.
Technological improvements including increasingly efficient implantable programmable generators (IPGs), MRI compatible hardware (alleviating the need for stereotactic ventriculography), and more recently, directional leads, have allowed DBS to maintain its ascendancy among treatments for advanced PD (Lozano et al., 2019). Moreover, there is ongoing research into developing and implementing closedloop stimulation systems, wherein feedback between the stimulating electrode and another which detects biomarkers (e.g. action or local field potentials, electrocorticograms, and electroencephalograms) allows for continually adjustable modifications to pulse amplitude, frequency, and duty cycle (the ratio of pulse width to signal period). In this manner, the need for a physician to routinely perform adjustments to the neurostimulation regimen according to a largely trial-and-error approach would be nullified. Nonetheless, closed-loop systems are considerably more energy-demanding, necessitating the use of larger batteries or wireless means of energy transfer (Ghasemi et al., 2018). Further, notwithstanding the foreseeable transition to adaptive systems in the coming years, DBS is an invasive procedure, and as such carries a risk of infection and hemorrhage (Lozano et al., 2019). Seminally, Grossman et al. (2017) showed that the interference of two electric fields oscillating at minimally offset frequencies, each individually too high to stimulate neurons due to their intrinsic low-pass filter, can be used to target subcortical structures from areas outside of the brain. However, although the potential for non-invasive, temporal interference DBS has been demonstrated in mice, the technology has not yet been applied clinically. Lastly, while PD and other motor disorders typically feature complex aberrations to neural circuits, DBS is limited in its ability to modulate a single anatomic target.

Conclusion
Generating oxidative stress in PD may occur in various mechanisms, is associated with the failure of complex I of the respiratory chain and the presence of certain genetic variants of the PARK. At the same time, the level of oxidative stress may be responsible for both the effectiveness of antiparkinsonian pharmacotherapy and DBS therapy. It seems that finding genetic and environmental factors associated with the generation of oxidative stress may be of importance in the future for improving the diagnostic processes and prognostic disorders of the extrapyramidal system and may contribute to the introduction of more effective pharmacologic and nonpharmacologic treatments.