Investigating Therapeutic Effects of Indole Derivatives Targeting Inflammation and Oxidative Stress in Neurotoxin-Induced Cell and Mouse Models of Parkinson’s Disease

Neuroinflammation and oxidative stress have been emerging as important pathways contributing to Parkinson’s disease (PD) pathogenesis. In PD brains, the activated microglia release inflammatory factors such as interleukin (IL)-β, IL-6, tumor necrosis factor (TNF)-α, and nitric oxide (NO), which increase oxidative stress and mediate neurodegeneration. Using 1-methyl-4-phenylpyridinium (MPP+)-activated human microglial HMC3 cells and the sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, we found the potential of indole derivative NC009-1 against neuroinflammation, oxidative stress, and neurodegeneration for PD. In vitro, NC009-1 alleviated MPP+-induced cytotoxicity, reduced NO, IL-1β, IL-6, and TNF-α production, and suppressed NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in MPP+-activated HMC3 cells. In vivo, NC009-1 ameliorated motor deficits and non-motor depression, increased dopamine and dopamine transporter levels in the striatum, and reduced oxidative stress as well as microglia and astrocyte reactivity in the ventral midbrain of MPTP-treated mice. These protective effects were achieved by down-regulating NLRP3, CASP1, iNOS, IL-1β, IL-6, and TNF-α, and up-regulating SOD2, NRF2, and NQO1. These results strengthen the involvement of neuroinflammation and oxidative stress in PD pathogenic mechanism, and indicate NC009-1 as a potential drug candidate for PD treatment.


Introduction
Parkinson's disease (PD), characterized by resting tremor, rigidity, bradykinesia, and postural instability, is the second most common neurodegenerative disorder affecting the elderly [1]. The pathological studies find a massive loss of dopaminergic (DAergic) neurons located in the pars compacta of the substantia nigra (SN) in the midbrain and subsequent depletion of dopamine in their projections [2]. The neurodegeneration of PD could be caused by a complex interaction of genetic and environmental factors [3].
Although the disease etiology remains to be clarified, it has been shown that oxidative stress contributes to neurodegeneration of PD [4]. A variety of environmental insults, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), specifically increase oxidative stress, damage DAergic neurons, and produce parkinsonism with symptoms like the main features of PD [5]. 1-Methyl-4-phenylpyridinium (MPP + ), the toxic metabolite of MPTP, inhibits mitochondrial electron transport chain complex I (NADH:ubiquinone oxidoreductase) to compromise mitochondrial oxidative capacity [6]. In MPTP-treated mice, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α mRNA expression levels increase significantly both in the SN and caudate-putamen in comparison with untreated mice [7]. Lines of evidence also demonstrate that microglial activation, release of inflammatory factors, and IgG targeting of neurons may play important roles in the neurodegeneration of PD [8,9], and anti-inflammatory drugs provide a protective effect both in animal models and epidemiological studies [10]. Therefore, strategies or compounds that reduce oxidative stress and neuroinflammation may be beneficial to PD patients.
In the past years, our group has focused on screening novel synthetic compounds to test their therapeutic effects on several neurodegenerative disease models. Indole is an aromatic heterocyclic compound with a wide range of biological activities. Its chemical reactivity makes it a suitable candidate for modification, leading to the development of various novel derivatives with potential as drug candidates for the treatment of various diseases [11]. Previously, indole compound NC009-1 (C 19 H 16 N 2 O 3 ) has been shown to have aggregation-reducing and neuroprotective effects by activating heat shock protein beta 1 (HSPB1) in tauopathy cell model [12] and spinal spinocerebellar ataxia (SCA) type 17 cell and mouse models [13]. In addition, NC009-1 activates apolipoprotein E (APOE) and neurotrophic receptor tyrosine kinase 1 (NTRK1) in amyloid beta (Aβ)-GFP SH-SY5Y cells, and Aβ precursor protein (APP) Swe /presenilin 1 (PS1) M146V /microtubule associated protein tau (Tau) P301L triple transgenic Alzheimer's disease (AD) mouse models [14], and decreases IL-1β-mediated pathway in SCA type 3 SH-SY5Y cell model inflamed with IFN-γ-primed HMC3 conditioned medium [15]. In the present study, we aimed to investigate the neuroprotective potential of NC009-1 and derivative compounds with methyl, phenyl or methyl formate substituent present on the benzene ring in MPP + -activated human microglial HMC3 cells and/or the sub-chronic MPTP-induced mouse model of PD.
The cytotoxicity of NC009 compounds (1-100 µM) in human HMC3 microglial cells was examined by MTT assay after 24 h compound treatment. As shown in Figure 1C, all test compounds had cell viability greater than 80% in compound-treated HMC3 cells. These results demonstrated the low cytotoxicity of the test compounds.
The cytotoxicity of NC009 compounds (1-100 μM) in human HMC3 microglial cells was examined by MTT assay after 24 h compound treatment. As shown in Figure 1C, all test compounds had cell viability greater than 80% in compound-treated HMC3 cells. These results demonstrated the low cytotoxicity of the test compounds.

Discussion
Microglia, the innate immune responders of the central nervous system, are key mediators of neuroinflammation in neurodegenerative diseases [28]. The pronounced activation of microglia in the brains of PD patients up-regulates inflammatory factors, and increases reactive oxygen species (ROS) and neuroinflammation [29,30]. Here we demonstrate that indole derivative NC009-1 alleviates MPP + -induced production of inflammatory mediators in HMC3 microglia. By down-regulating neuroinflammation and up-regulating cellular redox signaling, NC009-1 ameliorates behavioral impairments, increases dopamine and dopamine transporter levels in the striatum, and reduces oxidative stress as well as activation of microglia and astrocytes in the ventral midbrain of the sub-chronic MPTP-induced mouse model. The study results strengthen the roles of oxidative stress and neuroinflammation in PD pathogenesis, suggesting the potential of NC009-1 for the treatment of PD.
Activated microglial infiltrations are observed in the SN of postmortem PD brain [31]. Pronounced microglial activation is also observed in LRRK2 p.G2019S transgenic rats and PLA2G6 knockout mice [32,33], indicating neuroinflammation as a common pathogenesis in both sporadic and genetic-defined PD. Pathological α-synuclein aggregation in PD can induce microglial activation and dysfunction. The extracellular α-synuclein fibrils can bind to toll-like receptor 2 and 4 (TLR2 and TLR4), and thereby activate NLRP3 inflammasome and its downstream CASP1 and IL-1β pathway [34]. The pathological study reveals that NLRP3 is up-regulated and co-localized with microglia in the SN of PD patients [35]. Levels of IL-1β are also elevated in the striatum of PD patients [36,37]. Higher serum levels of IL-1β are observed in PD patients compared to control subjects [38]. Sustained expression of IL-1β in the striatum causes DAergic neuronal death and motor disabilities in rats [39]. Small molecule NLRP3 inhibitor MCC950 decreases inflammasome activation and effectively mitigates motor deficits, nigrostriatal DAergic degeneration, and accumulation of α-synuclein aggregates in 6-hydroxydopamine (6-OHDA)-and α-synuclein fibrils-treated mice [35], indicating NLRP3 as a sustained source of neuroinflammation driving progressive DAergic neuropathology. In this study, the results of NC009-1 to down-regulate NLRP3 and IL-1β in MPP + -treated HMC3 microglia and MPTP-treated mice highlight its potential to reduce PD neuroinflammation and neurodegeneration.
The activation of microglia up-regulated the activity of NADPH oxidase to increase the production of ROS [28], which results in lipid peroxidation, and subsequently, generation of cytotoxic 4-HNE [65,66]. Anti-oxidative responses up-regulate a number of anti-oxidative factors, such as SOD2, NRF2, and NQO1, to reduce these radicals [67]. Increased levels of SOD have been reported in the frontal and motor cortex of PD patients [68,69]. NRF2 inactivation is observed in MPTP-or 6-OHDA-treated SH-SY5Y cells or mice [70,71]. The expressions of NRF2 and NQO1 in DAergic neurons derived from iPSCs carrying a PARKIN mutation are consistently down-regulated [72]. The levels of NRF2 in peripheral leukocytes are elevated in PD patients [73]. Antioxidants may have neuroprotective effects in PD by enhancing activities of SOD or NRF2 pathways. For example, gypenosides mitigate the MPTP-induced reduction of SOD activities in mouse SN [74]. Dimethyl fumarate, a potent NRF2 enhancer in treating multiple sclerosis, demonstrates neuroprotection against MPTPand α-synuclein-induced neurotoxicity in mice through activating NRF2 [75,76]. Indole derivative NC001-8 also protects DAergic neurons derived from SH-SY5Y or iPSCs carrying a PARKIN mutation against MPP + and H 2 O 2 -induced neurotoxicity by up-regulating NRF2 and NQO1 [71]. By up-regulating autophagy and the NRF2 pathway, disaccharides including trehalose, lactulose, and melibiose demonstrate neuroprotective effects against αsynuclein-induced neurotoxicity [77]. Our study demonstrated the anti-oxidative property of NC009-1 to reduce neurodegeneration in PD.

Compounds and Cell Culture
Indole derivatives NC009-1, -2, -3, and -11 were synthesized by iodine-catalyzed C-alkylation of indoles as previously described [78]. The synthesized compounds were examined by nuclear magnetic resonance spectroscopy. The test compounds stayed soluble in cell culture medium at concentrations up to 100 µM. In addition, quercetin was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) as a control for measuring radical scavenging capacity. Human HMC3 microglial cells (ATCC CRL-3304) were routinely maintained in Dulbecco's modified Eagle medium/Nutrient mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured in an incubator at 37 • C (NuAire, Plymouth, MN, USA) with 95% relative humidity and 5% CO 2 .

Antioxidant Assay
1,1-Diphenyl-2-picrylhydrazyl (DPPH) (Sigma-Aldrich), a stable free radical for measuring the antioxidant activity of different samples, was used to measure the free radicalscavenging activity of the studied indole compounds. Briefly, DPPH solution (100 µM) was prepared in ethanol. After adding test compounds (10-160 µM), the mixture was vortexed for 15 s and allowed to stand for 30 min at room temperature. Subsequently, the mixture was measured spectrophotometrically at 517 nm (Multiskan GO microplate spectrophotometer; Thermo Fisher Scientific). The free radical scavenging activity was calculated as the percentage of DPPH discoloration using the formula 1-(absorbance of sample/absorbance of control) × 100%-with EC 50 calculated using the interpolation method.
The test for oxygen radical absorbance capacity [79] was performed using OxiSelect™ kit (Cell Biolabs, San Diego, CA, USA). Briefly, serial dilutions of Trolox standard (2.5-50 µM) and test compounds (4-100 µM) were prepared in 50% acetone. After adding fluorescein to blank (50% acetone), standards or samples, the mixture was mixed and incubated at 37 • C for 30 min. Subsequently, free radical initiator 2,2 -azobis(2-methylpropionamidine) dihydrochloride (AAPH) was added to produce peroxyl radicals (ROO•). The quenching of fluorescent probe over time was recorded for 60 min, with excitation at 480 nm and emission at 520 nm wavelengths (Bio-Tek FLx800). To quantify the oxygen radical absorbance capacity in a sample, the area under the curve (AUC) for blank, standards, and samples were calculated. After subtraction of the blank, the equivalent Trolox concentrations of samples were expressed based on the Trolox calibration curve.

HMC3 Microglia Activation and Inflammatory Mediators Detection
HMC3 cells were plated into 6-well (2 × 10 5 /well) dishes, grown for 20 h, and treated with MPP + (0-10 mM) (Cayman, Ann Arbor, MI, USA) for 20 h. The cell viability was evaluated by MTT assay as described, and the release of nitric oxide (NO) in cell culture medium was evaluated by Griess assay according to manufacturer's protocol (Thermo Fisher Scientific). To examine anti-inflammatory potential of test compounds, HMC3 cells were pre-treated with these compounds (1-10 µM) for 8 h before MPP + (3 mM) addition for 20 h, and cell viability and NO release were examined as described.
The levels of IL-1β, IL-6, and TNF-α in medium pre-treated with 10 µM compound were determined using enzyme-linked immunosorbent assay (ELISA). Specifically, human Instant IL-1β, IL-6, and TNF-α ELISA TM kits (Invitrogen, Carlsbad, CA, USA) were used, according to the experimental procedures supplied by the manufacturer. The OD at 450 nm was detected using Multiskan GO spectrophotometer (Thermo Fisher Scientific).

Sub-Chronic MPTP Mouse Model
The animal experiments were conducted in accordance with the guidelines and were approved by the National Taiwan Normal University (NTNU) Research Committee. Male C57BL/6 mice (8 weeks old, 18-22 g) were purchased from the National Laboratory Animal Center (Tainan City, Taiwan). The mice were housed in individually ventilated cages under controlled temperature (25 ± 2 • C), relative humidity (50%), and 12 h on/off light cycle with ad libitum access to food and water at the Animal House Facility of NTNU.
After one-week habituation, mice were randomly divided into 4 groups (n = 7). NC009-1 (40 mg/kg) or vehicle (DMSO:Cremophor EL:0.9% saline = 1:2:7) was intraperitoneally (i.p.) administrated 5 times per week for 6 weeks. Experimental parkinsonism was established by i.p. injections of 20 total doses of MPTP (25 mg/kg in 0.9% saline; Toronto Research Chemicals, Toronto, Ontario, Canada) along with probenecid (250 mg/kg in 0.1 M NaOH; Sigma-Aldrich), while control group received injections of saline. Probenecid was administered 1 h prior to MPTP administration as it decreases the clearance of MPTP and intensifies its neurotoxicity [80]. The dosage regimen was administered over 4 weeks with 5 doses per week (once daily for five consecutive days). Appropriate guidelines were abided in handling MPTP.

Behavioral Tests
To assess the gait performance, the CatWalkXT automatic quantitative gait analysis system (Noldus, Wageningen, The Netherlands) was utilized. The animals were placed in a walkway of 4 cm width with a glass bottom and recorded by a high-speed digital camera from below. The footprints were analyzed using the Catwalk XT 9.1 software. In addition, tail suspension test was performed for screening antidepressant-like activity in mice [24], as depression is one of the non-motor symptoms in PD [81]. This test relies on immobility as a measure of "behavioral despair" once the mouse perceives that escape from the apparatus is impossible. As described [82], each mouse was individually suspended to the edge of a table, 50 cm above the floor, by adhesive tape placed approximately 1 cm from the tip of the tail. During the test, each mouse was acoustically and visually isolated from other mice. The trials were conducted for 6 min, during which the duration of immobility was recorded. Mice were considered immobile when they hung passively and motionless.

HPLC Analysis of Dopamine
Levels of dopamine in striatum were determined by high performance liquid chromatography (HPLC) analysis. Briefly, the isolated brain striatum was homogenized in 500 µL of PRO-PREP TM protein extraction solution (iNtRON Biotechnology Inc., Gyeonggido, Republic of Korea). Samples were centrifuged at 10,000 × g for 30 min and then filtered through a 0.45 µm syringe membrane. Dopamine from the supernatant was analyzed by the HPLC system using a C18 column with a UV detector at 254 nm. The sample was passed through the HPLC system using a mobile phase of 87.5% 90 mM of sodium phosphate, 40 mM of citric acid, 10 mM of 1-octanesulfonic acid, 3 mM of ethylenediaminetetraacetic acid (EDTA), and 12.5% acetonitrile (pH3.0) at a flow rate of 1.0 mL/min.

Statistical Analysis
For each data set, three independent experiments were performed and data were expressed as the means ± standard deviation (SD). Differences between groups in the cell experiments were evaluated by one-way analysis of variance (ANOVA) with a post hoc Bonferroni test. In the animal experiments, the Kruskal-Wallis test (nonparametric test to compare unmatched groups) with a post hoc Dunn's test was applied to compare the differences (n = 5-7). Nonparametric Spearman's correlations were applied to evaluate the relationship between the 4-HNE/TH ratio in immunohistochemistry and parameters of behavioral tests. All p values were two-tailed, with values lower than 0.05 considered being statistically significant.

Conclusions
In the present study, we show that NC009-1 exerts neuroprotective effects by modulating inflammatory and anti-oxidative pathways ( Figure 6). It is of note that NC009-1 demonstrates good bioavailability and BBB penetration potential [13], further enhancing its potential for translation to clinical practice. Future studies in other animal models of PD will be necessary to validate its potential for treating PD before moving toward to clinical trials.