Nrf2 activator via interference of Nrf2-Keap1 interaction has antioxidant and anti-inflammatory properties in Parkinson's disease animal model
Kim S, Indu Viswanath AN, Park JH, Lee HE, Park AY, Choi JW, Kim HJ, Londhe AM, Jang BK, Lee J, Hwang H, Lim SM, Pae AN and Park KD
Parkinson's disease (PD) is a neurodegenerative disorder characterized by abnormal movement, including slowed movements, shuffling gait, lack of balance, and tremor. Oxidative stress has been shown to play a decisive role in dopaminergic neuronal cell death in PD. The nuclear factor E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling pathway provides the main defense system against oxidative stress by inducing the expression of antioxidant enzyme genes. Direct interference in the Keap1-Nrf2 protein-protein interaction (PPI) has emerged as an effective strategy for Nrf2 activation. Therefore, we searched for novel Nrf2 activators that can disrupt Nrf2-Keap1 interaction by using a virtual screening approach and identified a potent Nrf2 activator, KKPA4026. KKPA4026 was confirmed to induce the expression of the Nrf2-dependent antioxidant enzymes heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase regulatory subunit, and NAD(P)H:quinone oxidoreductase 1 in BV-2 cells. Furthermore, KKPA4026 showed anti-inflammatory effects in an Nrf2-dependent manner. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, KKPA4026 effectively attenuated PD-associated behavioral deficits and protected dopaminergic neurons. In summary, we identified KKPA4026 as a novel Nrf2 activator and suggested that Nrf2 activation through interference with the Nrf2-Keap1 interaction may be effective for PD treatment.
Nur77 attenuates inflammatory responses and oxidative stress by inhibiting phosphorylated IκB-α in Parkinson's disease cell model
Yan J, Huang J, Wu J, Fan H, Liu A, Qiao L, Shen M and Lai X
Neuroinflammation and oxidative stress play key roles in the pathological development of Parkinson's disease (PD). Nerve growth factor-induced gene B (Nur77) is closely related to dopamine neurotransmission, and its pathogenesis is unclear. This study aims to investigate the role and mechanism of Nur77 in a cell model of Parkinson's disease. Silencing Nur77 with siRNA can aggravate intracellular LDH release, increase the expression of pro-inflammatory genes (such as tumor necrosis factor α, nuclear factor κB (p65), monocyte chemotactic protein 1, interleukin-6), and decrease cell survival, decrease expression of nuclear factor E2-related factor(Nrf2), heme oxygenase 1, NADPH quinineoxidoreductase-1. Cytosporone B (Nur77 agonist) has the opposite effect to Nur77 silencing. PDTC (NF-κB inhibitor / antioxidant) can also inhibit pro-inflammatory genes to a similar degree as Cytosporone B. Phosphorylated IκB-α can be inhibited by Cytosporone B, while silencing Nur77 can increase the protein expression level of phosphorylated IκB-α. After silencing IκB-α, both Cytosporone B and siNur77 did not affect pro-inflammatory genes and antioxidant stress. These findings reveal the first evidence that Nur77 exerts anti-inflammatory and antioxidant stress effects by inhibiting IκB-α phosphorylation expression in a Parkinson cell model. Nur77 may be a potential therapeutic target for Parkinson's disease.
In silico and in vivo evaluation of oxidative stress inhibitors against Parkinson's disease using the C. elegans model
Hanumanthappa P, Ashok A, Prakash I, Priya CI, Zinzala J, Marigowda VV and Sosalegowda AH
Parkinson's disease ranks second, after Alzheimer's as the major neurodegenerative disorder, for which no cure or disease-modifying therapies exist. Ample evidences indicate that PD manifests as a result of impaired anti-oxidative machinery leading to neuronal death wherein Cullin-3 has ascended as a potential therapeutic target for diseases involving damaged anti-oxidative machinery.
Ferulic Acid Ameliorates MPP/MPTP-Induced Oxidative Stress via ERK1/2-Dependent Nrf2 Activation: Translational Implications for Parkinson Disease Treatment
Li X, Zhang J, Rong H, Zhang X and Dong M
Parkinson's disease (PD) is a neurodegenerative disorder closely associated with oxidative stress. The biochemical and cellular alterations that occur after cell and mouse treatment with the parkinsonism-inducing neurotoxin MPP/MPTP are remarkably similar to those observed in idiopathic PD. Previously, we showed that ferulic acid (FA) has antioxidant properties and the ability to activate nuclear factor E2-related factor 2 (Nrf2). The present study tested the hypothesis that FA attenuates MPP/MPTP-induced oxidative stress by regulating crosstalk between sirtuin 2 (SIRT2) and Nrf2 pathways. To test this hypothesis, we performed in vitro and in vivo studies using MPP/MPTP-challenged SH-SY5Y cells or mice treated with or not with FA. FA marginally inhibited SIRT2 in parallel with α-synuclein at levels of transcription and translation in SH-SY5Y cells challenged with MPP. Moreover, FA attenuated MPP-induced oxidative stress, as indicated by reactive oxygen species, lipid hydroperoxides, GSH/GSSG ratio, and NAD/NADH ratio. Mechanistically, FA strongly upregulated the glutamate cysteine ligase catalytic subunit and heme oxygenase-1 expression at the levels of transcription and translation. Interestingly, FA-mediated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation contributed to nuclear accumulation of Nrf2 via de novo synthesis, which was validated by the use of dominant negative ERK2. Surprisingly, activation of the ERK1/2 and inhibition of SIRT2 by FA are mediated by independent mechanisms. Furthermore, FA ameliorated motor deficits and oxidative stress in the ventral midbrain in MPTP-treated (25 mg/kg, i.p., daily for 5 days) wild-type mice and α-synuclein knockout mice, but not in Nrf2 knockout mice. Collectively, FA exerts antioxidant effects through ERK1/2-mediated activation of the Nrf2 pathway, and these results may have important translational value for the treatment of PD.
The transcription factor REST up-regulates tyrosine hydroxylase and antiapoptotic genes and protects dopaminergic neurons against manganese toxicity
Pajarillo E, Rizor A, Son DS, Aschner M and Lee E
Dopaminergic functions are important for various biological activities, and their impairment leads to neurodegeneration, a hallmark of Parkinson's disease (PD). Chronic manganese (Mn) exposure causes the neurological disorder manganism, presenting symptoms similar to those of PD. Emerging evidence has linked the transcription factor RE1-silencing transcription factor (REST) to PD and also Alzheimer's disease. But REST's role in dopaminergic neurons is unclear. Here, we investigated whether REST protects dopaminergic neurons against Mn-induced toxicity and enhances expression of the dopamine-synthesizing enzyme tyrosine hydroxylase (TH). We report that REST binds to RE1 consensus sites in the TH gene promoter, stimulates TH transcription, and increases TH mRNA and protein levels in dopaminergic cells. REST binding to the TH promoter recruited the epigenetic modifier cAMP-response element-binding protein-binding protein/p300 and thereby up-regulated TH expression. REST relieved Mn-induced repression of TH promoter activity, mRNA, and protein levels and also reduced Mn-induced oxidative stress, inflammation, and apoptosis in dopaminergic neurons. REST reduced Mn-induced proinflammatory cytokines, including tumor necrosis factor α, interleukin 1β (IL-1β), IL-6, and interferon γ. Moreover, REST inhibited the Mn-induced proapoptotic proteins Bcl-2-associated X protein (Bax) and death-associated protein 6 (Daxx) and attenuated an Mn-induced decrease in the antiapoptotic proteins Bcl-2 and Bcl-xL. REST also enhanced the expression of antioxidant proteins, including catalase, NF-E2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1). Our findings indicate that REST activates TH expression and thereby protects neurons against Mn-induced toxicity and neurological disorders associated with dopaminergic neurodegeneration.
BRUP-1, an intracellular bilirubin modulator, exerts neuroprotective activity in a cellular Parkinson's disease model
Kataura T, Saiki S, Ishikawa KI, Akamatsu W, Sasazawa Y, Hattori N and Imoto M
Bilirubin, the end product of heme redox metabolism, has cytoprotective properties and is an essential metabolite associated with cardiovascular disease, inflammatory bowel disease, type 2 diabetes, and neurodegenerative diseases including Parkinson's disease (PD). PD is characterized by progressive degeneration of nigral dopaminergic neurons and is associated with elevated oxidative stress due to mitochondrial dysfunction. In this study, using a ratiometric bilirubin probe, we revealed that the mitochondrial inhibitor, rotenone, which is widely used to create a PD model, significantly decreased intracellular bilirubin levels in HepG2 cells. Chemical screening showed that BRUP-1 was a top hit that restored cellular bilirubin levels that were lowered by rotenone. We found that BRUP-1 up-regulated the expression level of heme oxygenase-1 (HO-1), one of the rate-limiting enzyme of bilirubin production via nuclear factor erythroid 2-related factor 2 (Nrf2) activation. In addition, we demonstrated that this Nrf2 activation was due to a direct inhibition of the interaction between Nrf2 and Kelch-like ECH-associated protein 1 (Keap1) by BRUP-1. Both HO-1 up-regulation and bilirubin restoration by BRUP-1 treatment were significantly abrogated by Nrf2 silencing. In neuronal PC12D cells, BRUP-1 also activated the Nrf2-HO-1 axis and increased bilirubin production, resulted in the suppression of neurotoxin-induced cell death, reactive oxygen species production, and protein aggregation, which are hallmarks of PD. Furthermore, BRUP-1 showed neuroprotective activity against rotenone-treated neurons derived from induced pluripotent stem cells. These findings provide a new member of Keap1-Nrf2 direct inhibitors and suggest that chemical modulation of heme metabolism using BRUP-1 may be beneficial for PD treatment.
DJ-1-binding compound B enhances Nrf2 activity through the PI3-kinase-Akt pathway by DJ-1-dependent inactivation of PTEN
Niki T, Endo J, Takahashi-Niki K, Yasuda T, Okamoto A, Saito Y, Ariga H and Iguchi-Ariga SMM
DJ-1 was identified as an oncogene and also as a causative gene for a familial form of Parkinson disease (PD). DJ-1 plays various roles in anti-oxidative stress response. Superfluous oxidation of DJ-1 at cysteine residue 106 (C106), an inactive form of DJ-1, was observed in PD patients. DJ-1-binding compound B, which specifically bound to the C106 region of DJ-1, has been isolated and it has been shown to prevent oxidative stress-induced cell death through maintaining active forms of DJ-1 by inhibiting its superfluous oxidation. The molecular mechanism of the action of compound B, however, has not been fully elucidated. In this study, we found that compound B stimulated transcriptional activity of Nrf2 in HO-treated SH-SY5Y cells by inhibiting its degradation through the ubiquitin-proteasome system. Although Keap 1 is a major negative regulator of Nrf2, compound B strongly increased Nrf2 activity in Keap1-mutant A549 cells but not in PTEN-null PC3 and PTEN-knockout SH-SY5Y cells. Furthermore, treatment of cells with inhibitors of the PI3-kinase/Akt pathway inhibited the effect of compound B, and compound B increased the binding of PTEN to DJ-1 and decreased lipid phosphatase activity of PTEN concomitantly with increased oxidation of PTEN, an inactive form of PTEN. These results suggest that compound B enhances transcriptional activity of Nrf2 under an oxidative stress condition in a Keap1-independent manner and that its activity is elicited by activation of the PI3Kinase/Akt pathway with DJ-1-dependent inactivation of PTEN, leading to protection of oxidative stress-induced cell death.
Systemic activation of Nrf2 pathway in Parkinson's disease
Petrillo S, Schirinzi T, Di Lazzaro G, D'Amico J, Colona VL, Bertini E, Pierantozzi M, Mari L, Mercuri NB, Piemonte F and Pisani A
Preclinical studies underlined the relevance of Nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor pathway in the pathogenesis of Parkinson's disease (PD).
Neuroprotective potential of chrysin in Parkinson's disease: Molecular mechanisms and clinical implications
Angelopoulou E, Pyrgelis ES and Piperi C
Parkinson's disease (PD) is the most common neurodegenerative movement disorder, with current treatment being mainly symptomatic and often accompanied by serious side effects. In search of novel and safe therapeutic agents for PD, natural flavonoids have been shown to exert significant neuroprotective effects. Among them, chrysin (5,7-dihydroxyflavone) has been demonstrated to exhibit anti-oxidative effects to dopaminergic neurons mainly by increasing the expression of Nuclear Factor Erythroid 2 -related factor 2 (NRF2) which reduces intracellular nitric oxide (NO) levels and regulates anti-oxidant pathways. Moreover, chrysin activates Myocyte Enhancer factor 2D (MEF2D), a critical transcription factor involved in dopaminergic survival. It suppresses the MPP-induced upregulation of c-caspase and Bax as well as the downregulation of anti-apoptotic protein Bcl 2. Chrysin also enhances the production of neurotrophic factors, contributing to neuronal survival. Of interest, the combination of chrysin with protocatechuic acid (PCA) has been demonstrated to inhibit neuronal loss in PD animal models. Along with anti-inflammatory properties, chrysin has also been shown to increase dopamine levels in the striatum via monoamino-oxidase B (MAO-B) inhibition while it restores the behavioral deficits in PD animal models. In this review, we discuss the molecular mechanisms that underlie the possible neuroprotective effects of chrysin in PD pathogenesis along with its therapeutic potential.
Crosstalk between Nrf2 signaling and mitochondrial function in Parkinson's disease
Ammal Kaidery N, Ahuja M and Thomas B
Search for a definitive cure for neurodegenerative disorders like Parkinson's disease (PD) has met with little success. Mitochondrial dysfunction and elevated oxidative stress precede characteristic loss of dopamine-producing neurons from the midbrain in PD. The majority of PD cases are classified as sporadic (sPD) with an unknown etiology, whereas mutations in a handful of genes cause monogenic form called familial (fPD). Both sPD and fPD is characterized by proteinopathy and mitochondrial dysfunction leading to increased oxidative stress. These pathophysiological mechanisms create a vicious cycle feeding into each other, ultimately tipping the neurons to its demise. Effect of iron accumulation and dopamine oxidation adds an additional dimension to mitochondrial oxidative stress and apoptotic pathways affected. Nrf2 is a redox-sensitive transcription factor which regulates basal as well as inducible expression of antioxidant enzymes and proteins involved in xenobiotic detoxification. Recent advances, however, shows a multifaceted role for Nrf2 in the regulation of genes connected with inflammatory response, metabolic pathways, protein homeostasis, iron management, and mitochondrial bioenergetics. Here we review the role of mitochondria and oxidative stress in the PD etiology and the potential crosstalk between Nrf2 signaling and mitochondrial function in PD. We also make a case for the development of therapeutics that safely activates Nrf2 pathway in halting the progression of neurodegeneration in PD patients.
Neuroprotective effects of protocatechuic aldehyde through PLK2/p-GSK3β/Nrf2 signaling pathway in both and models of Parkinson's disease
Guo C, Zhu J, Wang J, Duan J, Ma S, Yin Y, Quan W, Zhang W, Guan Y, Ding Y, Wen A and Zhang Y
Mitochondrial dysfunction and oxidative damage are closely related to the pathogenesis of Parkinson's disease (PD). The pharmacological mechanism of protocatechuic aldehyde (PCA) for PD treatment have retained unclear. The purposes of the present study were to clarify the neuroprotective effects of post-treatment of PCA for PD treatment by mitigating mitochondrial dysfunction and oxidative damage, and to further determine whether its effects were mediated by the polo-like kinase 2/phosphorylated glycogen synthase kinase 3 β/nuclear factor erythroid-2-related factor 2 (PLK2/p-GSK3β/Nrf2) pathways. We found that PCA improved 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced behavioral deficits and dopaminergic cell loss. Moreover, PCA increased the expressions of PLK2, p-GSK3β and Nrf2, following the decrease of α-synuclein (α-Syn) in MPTP-intoxicated mice. Cell viability was increased and the apoptosis rate was reduced by PCA in 1-methyl-4-phenylpyridinium iodide (MPP)-incubated cells. Mitochondrial membrane potential (MMP), mitochondrial complex I activity and reactive oxygen species (ROS) levels in MPP-incubated cells were also ameliorated by treatment with PCA. The neuroprotective effects of PCA were abolished by inhibition or knockdown of PLK2, whereas overexpression of PLK2 strengthened the protection of PCA. Furthermore, GSK3β and Nrf2 were involved in PCA-induced protection. These results indicated that PCA has therapeutic effects on PD by the PLK2/p-GSK3β/Nrf2 pathway.
Astragaloside IV ameliorates motor deficits and dopaminergic neuron degeneration via inhibiting neuroinflammation and oxidative stress in a Parkinson's disease mouse model
Yang C, Mo Y, Xu E, Wen H, Wei R, Li S, Zheng J, Li W, Le B, Chen Y, Pan H, Huang S, Wang S and Wang Q
Oxidative stress and neuroinflammation are the key and early events during the pathological process of Parkinson's disease (PD). Thus, therapeutic intervention to regulate oxidative stress and neuroinflammation would be an effective strategy to alleviate the progression of PD. Astragaloside IV, the main active component isolated from Astragalus membranaceus, has been shown to possess anti-inflammatory and anti-oxidant properties in neurodegeneration diseases, however, the molecular mechanisms of Astragaloside IV in the pathology of PD are still unclear. In this study, we explored the mechanisms of Astragaloside IV of PD on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mice model and lipopolysaccharide (LPS)-induced BV2 microglia cells. Our results showed Astragaloside IV significantly alleviated behavioral impairments and dopaminergic neuron degeneration induced by MPTP. Also, Astragaloside IV inhibited microglia activation and reduced the oxidative stress of MPTP mouse model. In addition, Astragaloside IV significantly inhibited NFκB mediated NLRP3 inflammasome activation and activated Nrf2 both in vivo and in vitro. Furthermore, Astragaloside IV lessened reactive oxygen species (ROS) generation in LPS-induced BV2 microglia cells remarkably. These findings demonstrate that Astragaloside IV protects dopaminergic neuron from neuroinflammation and oxidative stress which are largely dependent upon activation of the Nrf2 pathways and suppression of NFκB/NLRP3 inflammasome signaling pathway. Therefore, Astragaloside IV is a promising neuroprotective agent that should be further developed for neurodegeneration diseases.
Shaping the Nrf2-ARE-related pathways in Alzheimer's and Parkinson's diseases
Fão L, Mota SI and Rego AC
A failure in redox homeostasis is a common hallmark of Alzheimer's Disease (AD) and Parkinson's Disease (PD), two age-dependent neurodegenerative disorders (NDD), causing increased oxidative stress, oxidized/damaged biomolecules, altered neuronal function and consequent cell death. Activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a redox-regulated transcription factor, results in upregulation of cytoprotective and antioxidant enzymes/proteins, protecting against oxidative stress. Nrf2 regulation is achieved by various proteins and pathways, at both cytoplasmatic and nuclear level; however, the elaborate network of mechanisms involved in Nrf2 regulation may restrain Nrf2 pathway normal activity. Indeed, altered Nrf2 activity is involved in aging and NDD, such as AD and PD. Therefore, understanding the diversity of Nrf2 control mechanisms and regulatory proteins is of high interest, since more effective NDD therapeutics can be identified. In this review, we first introduce Keap1-Nrf2-ARE structure, function and regulation, with a special focus on the several pathways involved in Nrf2 positive and negative modulation, namely p62, PKC, PI3K/Akt/GSK-3β, NF-kB and p38 MAPK. We then briefly describe the evidences for oxidative stress and Nrf2 pathway deregulation in different stages of NDDs. Finally, we discuss the potential of Nrf2-related pathways as potential therapeutic targets to possibly prevent or slowdown NDD progression.
The Therapeutic Implications of Tea Polyphenols Against Dopamine (DA) Neuron Degeneration in Parkinson's Disease (PD)
Zhou ZD, Xie SP, Saw WT, Ho PGH, Wang H, Lei Z, Yi Z and Tan EK
Accumulative evidence indicated that the pathologically accumulated metal ions (iron species and Mn) and abnormally up-regulated monoamine oxidase B (MAOB) activity induced oxidation of endogenous dopamine (DA) can lead to mitochondria impairment, lysosome dysfunction, proteasome inhibition, and selective DA neuron vulnerability, which is implicated in the pathogenesis of Parkinson's disease (PD). The DA oxidation can generate deleterious reactive oxygen species (ROS) and highly reactive DA quinones (DAQ) to induce DA-related toxicity, which can be alleviated by DA oxidation suppressors, ROS scavengers, DAQ quenchers, and MAOB inhibitors. On the other hand, the nuclear factor erythroid 2-related factor 2 (Nrf2)-Keap1 and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) anti-oxidative and proliferative signaling pathways play roles in anti-oxidative cell defense and mitochondria biogenesis, which is implicated in DA neuron protections. Therefore, agents with capabilities to suppress DA-related toxicity including inhibition of DA oxidation, scavenge of ROS, detoxification of DAQ, inhibition of MAOB, and modulations of anti-oxidative signaling pathways can be protective to DA neurons. Accumulative evidence shows that tea or coffee consumptions and smoking are related to deceased PD prevalence with unknown mechanisms. In this study, we investigate the protective capabilities of tea polyphenols and other PD relevant agents to inhibit DA-related toxicity and protect against environmental or genetic factors induced DA neuron degeneration in vitro and in vivo. We find that tea polyphenols can significantly suppress DA-related toxicity to protect DA neurons. The tea polyphenols can protect DA neurons via inhibition of DA oxidation, conjugation with DAQ, scavenge of ROS, inhibition of MAOB, and modulations of Nrf2-Keap1 and PGC-1α anti-oxidative signaling pathways. The tea polyphenols with more phenolic hydroxyl groups and ring structures have stronger protective functions. The protective capabilities of tea polyphenols is further strengthened by evidence that phenolic hydroxyl groups can directly conjugate with DAQ. However, GSH and other sulfhydyl groups containing agents have weaker capabilities to abrogate DA oxidation, detoxify ROS and DAQ and inhibit MAOB; whereas nicotine (NICO) and caffeine (CAF) can only modulate Nrf2-Keap1 and PGC-1α pathways to protect DA neurons weakly. The tea polyphenols are identified to protect against overexpression of mutant A30P α-synuclein (α-syn) induced DA neuron degeneration and PD-like symptoms in transgenic Drosophila. Based on achievements from current studies, the excellent and versatile protective capabilities of tea polyphenols are highlighted, which will contribute and benefit to future anti-PD therapy.
Neuroprotection of Indole-Derivative Compound NC001-8 by the Regulation of the NRF2 Pathway in Parkinson's Disease Cell Models
Wei PC, Lee-Chen GJ, Chen CM, Wu YR, Chen YJ, Lin JL, Lo YS, Yao CF and Chang KH
Parkinson's disease (PD) is a common neurodegenerative disease accompanied by a loss of dopaminergic (DAergic) neurons. The development of therapies to prevent disease progression is the main goal of drug discovery. There is increasing evidence that oxidative stress and antioxidants may contribute to the pathogenesis and treatment of PD, respectively. In the present study, we investigated the antioxidative protective effects of the indole-derivative compound NC001-8 in DAergic neurons derived from SH-SY5Y cells and PD-specific induced pluripotent stem cells (PD-iPSCs) carrying a ex5del mutation. In SH-SY5Y-differentiated DAergic neurons under 1-methyl-4-phenylpyridinium (MPP) treatment, NC001-8 remarkably reduced the levels of reactive oxygen species (ROS) and cleaved caspase 3; upregulated nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H dehydrogenase, quinone 1 (NQO1); and promoted neuronal viability. In contrast, knockdown abolished the effect of NC001-8 on the reduction of ROS and improvement of neuronal viability. In HO-treated DAergic neurons differentiated from PD-iPSCs, NC001-8 rescued the aberrant increase in ROS and cleaved caspase 3 by upregulating NRF2 and NQO1. Our results demonstrated the protective effect of NC001-8 in DAergic neurons via promoting the NRF2 antioxidative pathway and reducing ROS levels. We anticipate that our present assays may be a starting point for more sophisticated models or clinical trials that evaluate the potential of NC001-8 as a disease modifier for PD.