Pharmacological Blocker of NF-κb and Mitochondrial Ros Restrict NLRP3 In ammasome Activation and Rescue Dopaminergic Neurons in Vitro and in Vivo Parkinson’s Disease

Sahabuddin Ahmed NIPER Guwahati: National Institute of Pharmaceutical Education and Research Guwahati Samir Ranjan Panda NIPER Guwahati: National Institute of Pharmaceutical Education and Research Guwahati Mohit Kwatra NIPER Guwahati: National Institute of Pharmaceutical Education and Research Guwahati Bidya Dhar Sahu NIPER Guwahati: National Institute of Pharmaceutical Education and Research Guwahati VGM Naidu (  vgmnaidu@gmail.com ) NIPER Guwahati: National Institute of Pharmaceutical Education and Research Guwahati https://orcid.org/0000-0003-1520-2177


Introduction
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by degeneration of dopaminergic neurons within the substantia nigra par compacta (SNpc) causing depletion of dopaminergic transmission within the striatum that impact smotor coordination [1]. The cardinal features of the disease include symptoms like tremor at rest, rigidity, muscle stiffness, slowness of movement (bradykinesia) and poverty of movement (hypokinesia) [2]. The prime neuropathological hallmarks of the disease include intracellular inclusions containing aggregates of α-synuclein and formation of Lewy bodies [3]. Mounting evidence states the role of oxidative stress and microglia mediated neuroin ammation as a nearly pathological changes in the progression of neurodegeneration [4].
However, underlying mechanism still requires further investigation and novel potential therapeutics are needed to be explored in halting microglia mediated neurodegeneration.
Microglias are immune cells of brain which principally function as a surveillance system in regulating several signals to maintain brain homeostasis. Neuronal survival directly depends upon the microglia cells function, which includes biochemical homeostasis, neuronal integrity and neuronal remodelling within brain [5]. Microglia can be activated by a variety of stimuli including pathogen-associated molecular pattern (PAMPS) like lipopolysaccharide (LPS) and endogenous damage-associated molecular pattern (DAMPS) which varies from ATP, mitochondrial DNA, potassium e ux, urate crystal and reactive oxygen species (ROS) [6]. Nucleotide-binding oligomerization domain (Nod)-like receptor family pyrin domain-containing 3 (NLRP3) in ammasome is a widely studied in context to microglia activation and plays an essential role in initiating in ammatory cascade in PD brain [7]. Active IL-1β and IL-18 production remains the foremost in ammatory cytokines generates from microglia and plays a crucial role in both acute and chronic neuroin ammation through amplifying innate and adaptive immunity [8]. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) mediated NLRP3 in ammasome regulation and production of active IL-1β and IL-18 are tightly controlled both at transcriptional and posttranslational levels [9]. Generally, a two-step mechanism of NLRP3 activation and generation of active IL-1β and IL-18 is widely studied and accepted. Priming signal (Signal 1) acts via TLR-4 receptors and causes NF-κB translocation to the nucleus and produces biologically inactive intracellular precursors of both pro-IL-1β and pro-IL-18 [10]. The activation signal (Signal 2) involves in ammasome oligomerization in response to endogenous signals from malfunctioned mitochondria and elicit its action by activating the caspase-1 enzyme which then cleaves pro-IL-1β and pro-IL-18 to its matured forms responsible for neuroin ammation and pyroptosis within brain microglia [11,12].
Mitochondrial dysfunction remains as one of the prominent features in several neurodegenerative disorders and is associated with generation of several endogenous insults which causes neuroin ammation within the brain regions [13][14][15]. Similarly, to cross talk, the role of mitochondria in maintaining the glial homeostasis is well documented in PD [16,17,15]. NLRP3 in ammasome can sense any redox changes within its environments allowing its activation through a variety of stimuli including generated reactive oxygen species (ROS). Several studies have proven that many NLRP3 stimulators cause mitochondrial dysfunction but still there is a lacuna for understanding the role ofNLRP3 activators in mitochondrial damage. Recently some studies have shown the role of mitochondrial ROS (mtROS) which act as one of the major endogenous DAMPs (Signal 2) in activating NLRP3 in ammasomes complex within the glia [18,19]. However, the central mechanisms of NLRP3 in ammasomes activation in microglia through free radicals generation is unclear and require further explanation.
Perillyl alcohol (PA), a monoterpene is found in essential oils of plants such as peppermint, sage, mints, cherries, citrus fruits and lemongrass [20]. PA has been reported to exhibit anti-in ammatory [21], antioxidant, anticancer [22] and neuroprotective effects [23]. PA is shown to intensify the memory formation in scopolamine treated rats by restoring dopaminergic neurons and acetyl-cholinesterase activity in brain [24]. PA was found promoting neurogenesis in Neuro-2a cells by inhibiting ubiquinone (CoQ) synthesis [25]. Line of evidences has also suggested its bene cial effects in Alzheimer's disease owing its antioxidant and anti-in ammatory activity [26]. Further, PA was also seen to inhibit neuronal death in unilateral 6-hydroxydopamine (6-OHDA) toxicity by promoting mitochondrial biogenesis [27]. However, the effect of PA in context to microglial mediated neuroin ammation in PD largely remained unexplored. Therefore, the current study is aimed to understand the role of mitochondrial dysfunction and NLRP3 in ammasome activation utilizing complex I inhibitor (MPTP) and exogenous ROS generation (hydrogen peroxide) that mediates in ammation-driven neurodegeneration. Furthermore, the study also involves screening the neuroprotective activity of PA and to understand how targeting mtROS generation resilience in ammasome mediated neurodegeneration in PD mice model. Invitrogen. β-actin (Cat no: 4967S), tyrosine hydroxylase (TH, Cat no: 2792S), phospho-NF-κB p65 (Cat no: 3033), anti-rabbit IgG(Cat no: 7074S), anti-mouse IgG (Cat no: 7076S), anti-rat IgG (Cat no: 7077), normal goat serum (Cat no: 5425)were obtained from cell signaling technology.Anti-caspase-1(p20) (Cat no: AG-20B-0042) was obtained from Adipogen. Mouse IL-1β (Cat no: AF-401-NA) was obtained from R&D system. IL-18 rabbit pAb (Cat no: A1115) was procured from ABclonal, USA. In vitro microglial neuroin ammation model was established by priming LPS and H 2 O 2 at different concentration. For LPS/H 2 O 2 priming treatment, cells were serum-starved for 2 h and priming was done with LPS at a concentration of 1µg/ml for next 4 h. Further, the media was washed with sterile PBS to remove any trace of LPS, and then H 2 O 2 (100 µM) was further added for 2 h. The cell viability was assessed through standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and absorbance were recorded at a wavelength of 570nm. Cell viability was expressed as a percentage of the value in the control cells. Subsequently, neuroin ammatory protein markers was assessed by immunoblotting and ELISA assay.

2.3Evaluation of mitochondrial membrane potential
Mitochondrial membrane potential (Δψm) was evaluated by utilizing speci c mitochondrial uorescent probe JC-1 dye. Under normal conditions, Δψm JC-1 forms an aggregate with high red uorescence intensity. Loss in the Δψm is indicated with decrease red uorescence and increase green uorescence as the dye shift from aggregate to monomeric form. Thus, the ratio of red/green serves as an indicator of loss of Δψm.

Evaluation of mitochondrial superoxide generation
Mitochondrial ROS was measured using MitoSOX™ Red FM. After priming the cells with LPS/H 2 O 2 and treatment with test compound, cells were stained with 5µM MitoSOX™ Red for 10min at 37 0 C. Cells were washed with warm PBS buffer and observed under a confocal microscope (Leica TCS SP-8) with absorption/emission maxima: ~510/580 nm.

Immunocytochemistry (ICC)
N9 cells were plated on poly D-lysine coated coverslips in a 6 well culture plate at a density of 2x10 6 cells per well. After priming the cells with LPS/H 2 O 2 and treatment with test compound, cells were washed with PBS, xed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100. Cells were blocked with 5%normal goat serum (NGS), washed and incubated overnight with primary antibodies: NLRP3 (1:250 dilution), and pNF-κB 65 (1:800 dilution ) at 4 0 C. Next, cells were washed with PBS and then incubated with secondary antibodies namely Alexa Flour™ 488 goat anti-rabbit IgG(H + L) and Alexa Flour™ 594 goat anti-mouse IgG(H + L). Subsequently, nuclei were stained with Vectashield mounting medium for uorescence with 4′,6-dia-midino-2-phenylindole (DAPI) (Cat no. H-1200, Vector Laboratories, Burlingame, CA). Negative control slides were prepared by the exclusion of the primary antibody. Slides were kept in a cool place until observed under oil emersion at 40x magni cations with a confocal microscope.

2.7Animal studies
Male C57BL/6 mice (10-weeks-old, weighing between 22g to 25g) were obtained from Palamur Biosciences Private Limited, Hyderabad, India, and were acclimatized for two weeks before initiation of the study.
The study and protocol procedures were approved by the Institutional Animal Ethics Committee (IAEC) All the experiments were conducted as per the guidelines laid by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. All the animals were accessed to food and water ad libitum.

Experimental design and drug treatment
Mice were randomly divided into ve groups consisting of 15 animals in each and the treatment was performed as mentioned below: Group 1 (Normal control): Animals were administered orally with corn oil (5ml/kg body weight) for 14 consecutive days and normal saline (1ml/100g body weight) was administered intraperitoneally (i.p.) for 5 consecutive days starting from day 1 to day 5.
Group 2 (MPTP-25mg/kg): Animals were administered i.p. with MPTP-HCl (dissolved in sterile normal saline, 25mg/kg body weight) for 5 consecutive days starting from day 1 to day 5. Corn oil (5ml/kg body weight) as a drug vehicle was administered orally for 14 consecutive days.
Group 3 (MPTP + PA-100mg/kg): Mice were administered i.p. with MPTP-HCl(dissolved in sterile normal saline, 25mg/kg body weight) for 5 consecutive days starting from initial day 1 to day 5 and a suspension of PA (100mg/kg body weight) in Corn oil (5ml/kg body weight) was administered orally for 14 consecutive days. Group 4 (MPTP + PA-200mg/kg): Animals were administered i.p. with MPTP-HCl (dissolved in sterile normal saline, 25mg/kg body weight per day) for 5 consecutive days starting from initial day 1 to day 5 and a suspension of PA( 200mg/kg body weight) in Corn oil (5ml/kg body weight) was administered orally for 14 consecutive days.
Group 5 (MPTP + Mito-TEMPO-1mg/kg): Animals were administered i.p. with MPTP-HCl (dissolved in sterile normal saline, 25mg/kg body weight) for 5 consecutive days starting from initial day 1 to day 5 and Mito-TEMPO(1mg/kg body weight) dissolved in normal saline was administered i.p. for 14 consecutive days.
The dose and duration of treatment of PA and Mito-TEMPO were selected based on previously published report [30]. The dose and duration of MPTP to induce experimental Parkinson-like phenomenon in mice were selected based on our pilot study and earlier reports [31]. MPTP-HCl solution preparations, dosing of animals, and handling of bedding material were done in accordance with the safety guidelines outlined by Przedborski et al [32]. The behavioral parameters were recorded on 0th day, 7th, and 14th day. On the 15th day, animals were euthanized using a high dose of iso urane, and the brains was immediately collected and stored at -80ºC for further analysis (Fig. 1).

Catalepsy test
Catalepsy (bar test) test was performed to assess the behavioral abnormalities in rodents. In this method, front limbs of the mice were suspended on a wooden bar 5 cm high. Length of time in seconds were recorded by the control and treated animals to move the limb back to the surface[33].

Grip strength test
Grip strength of the forelimbs was measured using a digital grip strength meter (Ugo Basile, Gemonio (VA) Italy) [34]. Mice were allowed to grab an iron grid with its forelimbs and were gently pulled back to record its grip strength. The grip strength was expressed in gf (gram x force).

Rota-rod test
Rota-rod experiment was performed to assess the motor coordination and grip performance of all experimental animals. Brie y, mice were placed on the apex of the rota-rod treadmill (Ugo Basile, Gemonio (VA) Italy), and the latency to fall (in seconds) were reordered automatically by individual sensors. Before start of experiment, baseline training was given to all experimental mice. Data was collected on the 0th, 7th, and 14 th day in acceleration mode (4-45 rpm) over 5 min.

Pole test
Pole test is used to measure the degree of bradykinesia, basal ganglia related motor disturbances in experimental animals as described earlier [35]. The mice were allowed to run a vertical pole 50cm length and the time taken to reach the ground was recorded in seconds.

Walking track analysis
Both the limbs of trained mice were dipped in black ink and were allowed to walk on a white paper (4.5 cm wide, 42 cm long) towards a dark cage. Before start of experiment, mice were subjected to training trials to acclimatize the environment. Stride length was measured as the distance between successive paw prints [34]. Data were presented as the average of ve stride lengths for three different animals from each group.

Open eld test
An open eld test (OFT) is used to evaluate spontaneous locomotor activity. The apparatus consists of a wooden box (50×50×38 cm). Mice were placed into the centre square and behavioral activities were videotaped for 5 min. The parameters used for analysis includes the total distance travelled; average speed, and immobility time for a ve-minute period. All the behavior analysis was done by Any-maze video tracking software version-6.1(Stoelting Co, USA).

UHPLC analysis of neurotransmitters
Brie y, the animals were sacri ced and the striatum region was isolated and homogenized in 0.1M perchloric acid (PCA) followed by centrifugation at 13000 rpm at 4 0 C for 10 min. The supernatant was collected and ltered through a 0.22 µm lter and15µl volume was injected in a thermo scienti c-UHPLCultimate 3000 with an electrochemical detector (ECD) using an isocratic elution system. The column used was hyper cell gold-150×4.6mm, ow rate-0.8ml/min, run time-45min, ECD-1 potential-550mv, ECD-2 potential-300mv. A linearity standard curve (1000ng/ml, 500ng/ml, 250ng/ml, 100ng/ml, 50ng/ml, 25ng/ml 5ng/ml) was plotted by using freshly prepared standards of nor-epinephrine (NE), dopamine (DA), serotonin (5-HT) and 5-hydroxy indole acetic acid (5-HIAA). The mobile phase was prepared by using 75 mM sodium dihydrogen phosphate, 1.7 mM 1-octanesulfonic acid, 100 µl/l triethylamine, 25 µM EDTA, 10 % acetonitrile and pH of mobile phase was adjusted to 3 with phosphoric acid. The calibration curve was generated with the Chromeleon-7 software and relative amines concentrations were obtained as an integrated peak area for the individual analyte.
2.12. Cresyl Violet staining of SNpc tissue (Nissl-stained positive neurons) Brain tissues were embedded in Optimal cutting temperature (OCT) medium and sectioned at 20 µm thickness using a cryostat. Sections were mounted on Poly d-lysine coated slides and dried for several hours at room temperature. Slides were stained with cresyl violet stain for 6 to 8 mins in a 60 0 C preheated water bath. Slides were allowed to cool and washed with running tap water to drain excess stain. Slides were then rehydrated with a series of alcohol and cleared in xylene. Sections were mounted with DPx mounting media and observed under a light microscope (Evos FL Auto 2). Nissl-stained positive neurons were quanti ed using ImageJ (Fiji version) software as described by Paul R et al [36]. Low (40x) and high magni cation images (400x) of the SNpc region were acquired by a bright eld microscope.

Immunohistochemistry (IHC)
Animals were anesthetized with ketamine and xylazine combination at a dose of 100mg and 10mg/kg body weight respectively and perfused intracardially with 50 mL of PBS followed by 100 ml of4% paraformaldehyde (PFA).After euthanasia, brain tissue was isolated and further xed in 4% PFA for 24h at 4 ºC. Then brain tissue was immersed in 10%, 20%, and 30% sucrose solutions gradually for cryoprotection until the tissue sink at the bottom. Midbrain was excised out and embedded in an OCT medium and sections approximately 20 µm were cut with a cryostat throughout the midbrain and identi ed the SNpc region. Antigen retrieval step was done in the microwave oven and heating the sections at 95 ºC in 10mM trisodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by dipping sections in 3% hydrogen peroxide. Sections were washed with PBS and blocked with NGS for 1h at room temperature. Sections were incubated with primary antibody (tyrosine hydroxylase (1:200) and Iba1 (1:200) in a humidity chamber and kept at 4ºC overnight. Sections were washed and incubated with biotinylated universal antibody and avidin-biotin complex as per manufacture's protocol (Vecta stain Elite-ABC Universal kit, Vector Labs PK-6200). Sections were visualized with 3,3′-Diaminobenzidine(DAB) until brown color develops. Sections were further dehydrated with graded alcohol, cleared in 100% xylene and mounted in DPx mounting medium. Sections were observed under a bright eld illumination using a trinocularmicroscope (Evos FL Auto 2). The total numbers of immunoreactive neurons and microglia in the entire extent of SNpc were stereologically counted from four mouse brains per group by observer blind to the experiment. Tyrosine hydroxylase and Iba-1 were quanti ed using ImageJ (Fiji version) software as described by Paul R et al [36]. Low (40x) and high magni cation images (200x) of the SNpc region were acquired.
2.14.Isolation of mitochondriaand ATP estimation Mitochondria rich fractions was isolated from the SNpc region using a standard MITOISO1 kit (Sigma, St. Louis, MO) as per manufacture instructions. Protein content in the sample was estimated using the BCA protein estimation kit. The ATP content was measured in the mitochondrial pellet by a colorimetric method using a standard kit. The amount of ATP in the samples was expressed as nanogram per mg of tissue.
2.15 Estimations of pro-in ammatory cytokines (TNF-α and IL-1β) SNpc homogenates were prepared using 50mM PBS, pH 7.4 containing protease inhibitor cocktail (1%). The concentrations of TNF-α and IL-1β were measured using respective mouse-speci c TNF-α and IL-1β commercial ELISA kits. The respective concentrations were obtained by drawing a standard reference curve and expressed as picograms per mg of protein.

Measurement of lipid peroxidation
Lipid peroxidation is the marker for the generated oxidative stress in the tissue. The malondialdehyde (MDA) is the formed end-product of lipid peroxidation. The obtained supernatant of the SNpc was processed separately for MDA level estimation. Brie y, 100µl supernatant was mixed with 200µl of 8.1% SDS, 20% acetic acid (pH 3.4) and thiobarbituric acid (0.8%). The resultant mixture in glass tubes kept for 1 hour in a water bath at 95°C followed by cooling it under running tap water. Finally, absorbance was measured at 532 nm on a multi-plate reader (SpectraMax i3x, Molecular devices). Thereafter, the results were compared among the groups and expressed as nmoles of MDA per mg of protein) (Jangra et al., 2016b).

Measurement of reduced glutathione level
The reduced glutathione is the indicator of the glutathione-based antioxidant system in the tissue. Supernatant of the SNpc homogenate was mixed in equal ratio with 20% (w/v) trichloroacetic acid and centrifuged at 1000xg for 10 min at 4 0 C.The obtained supernatant was diluted with 0.3 M disodium hydrogen phosphate buffer and250µl of 1mM freshly prepared DTNB [5, 50-dithiobis (2-nitro benzoic acid) solution dissolved in 1 % w/v sodium citrate and incubated for 20 min in dark. Final absorbance was taken at 412 nm on a multi-plate reader (SpectraMax i3x, Molecular devices). The results were calculated as nmoles of GSH/mg of protein [37,38].

Determination of superoxide dismutase (SOD) and catalase (CAT) activity
The superoxide dismutase (SOD) and catalase (CAT) activity were quanti ed using the SOD and CAT assay kits, respectively. The assays were performed as per manufacturer's protocol. Final absorbance of SOD and CAT activity was measured at 440 nm and 520 nm, respectively using a multi-plate reader (SpectraMax i3x, Molecular devices). SOD activity was represented as U/mg of protein while CAT activity was expressed as µmol of H 2 O 2 consumed/min/mg of protein.

Data Analysis
All the results were indicated as the mean ± standard deviation (SD).For comparison between only control and treatment group, data were analysed by one-way analysis of variance (ANOVA) followed by Dunnett analysis. Two-way ANOVA was used for comparing multiple groups and was used for behavior studies. For comparing between different treatment groups, one-way ANOVA followed by Tukey's post hoc test was applied.
Statistical analysis was performed using Graph Pad Prism 8.01 software (GraphPad Software Inc., La Jolla, CA, USA). A value of p < 0.05 was considered statistically signi cant.

Combined LPS and H 2 O 2 treatment causes mitochondrial damage and activates NLRP3 in ammasomes expression in N9 microglia
NLRP3 in ammasome complex is widely studied in correlation to microglial activation butthe exact mechanism for NLRP3 in ammasome activation is still debatable. Our prime focus in this study was to establish a link between LPS and hydrogen peroxide (H 2 O 2 ) to mimic the exposure of in ammation and oxidative stress as a double-hit model in NLRP3 in ammasome activation. To test the hypothesis whether exogenous H 2 O 2 treatment could act as a potent free radical in activating NLRP3 in ammasome in LPS primed microglia, we initially primed the microglia cells with a xed dose of LPS i.e. 1000ng/ml (Signal 1) for 4h and then treated with different concentrations of H 2 O 2 ranging from 50µM to 200µM for next 2h (Fig. 2B). Further data from kinetic studies using the combination of LPS + H 2 O 2 treatment at different time interval showed a robust release of H 2 O 2 at time points from 1hr, 1.5 hr with highest release at 2hr incubation (Fig. 2C). Thus, from the above preliminary studies, exposure of a combination of LPS and H 2 O 2 at a dose of 1000 ng/ml and 100 µM for 4 hr and 2hr respectively was su cient to release ROS without causing signi cant cell death. Next sets of experiments involve in addressing the question whether the combination of LPS and H 2 O 2 was su cient to activate NLRP3 in ammasome complex and its downstream target. Immunoblot data suggested that selected concentrations of LPS and H 2 O 2 were su cient enough to activate NLRP3 in ammasome, together with increased protein expression of active caspase-1(p20), IL-1β and IL-18 (Fig. 2D, 2E, 2F, 2G and 2H). These results were further supported by cytokine analysis from the cell supernatant showing a signi cant increase in the IL-1β level in LPS primed H 2 O 2 treated microglia compared to LPS primed microglia and H 2 O 2 treatment alone (Fig. 2I). However, we fail to detect any active processing of caspase-1(p20) mature IL-1β or mature IL-18 form neither in LPS alone primed microglia nor in H 2 O 2 treatment alone (Fig. 2F, 2G and 2H). Thus, from our data, it can be assumed that H 2 O 2 can itself act as a DAMP (Signal 2) in augmenting the NLRP3 in ammasome complex together with cleavage of caspase-1 and release of active IL-1β and IL-18 in LPS primed microglia. Moreover, cytokine analysis of microglia supernatant treated with LPS and H 2 O 2 and H 2 O 2 alone showed a signi cant increase in TNF-α level as compared to unprimed cells (Fig. 2J). This indicates that TNF-α does not require a secondary signal in the form of DAMP for its activation. Treatments with PA and Mito-TEMPO was seen to rescue Δψm damage and causes a decrease mitochondrial superoxide anion levels compared to untreated microglial cells. Further treatment with PA and Mito-TEMPO was also found to inhibit the release free H 2 O 2 generation as seen from the Amplex red assay (Fig. 3E). From these ndings, we believe that H 2 O 2 treatment in LPS primed microglia augment the ROS generation which cause damage to mitochondrial membrane leading to metabolic crises within microglia.

PA suppresses the NF-κB mediated NLRP3 activation in LPS primed H 2 O 2 treated microglial cells
The mechanism of NLRP3 in ammasome activation is a two-step process that involves priming signal (Signal 1 by LPS) and activation signal (Signal 2 by H 2 O 2 mediated ROS generation). LPS is widely known as an in ammatory cytokine inducer which acts through activation of the NF-κB signaling. Further ROS produced from H 2 O 2 and generated H 2 O 2 itselfare believed to be the main activators of NLRP3 in ammasome complex and its downstream targets in LPS primed microglia. Hence, controlling the NF-κB signaling and the ROS generation could be a potential strategy in inhibiting microglia activation. The ndings of our study demonstrated that combined LPS and H 2 O 2 treatment was su cient to activate NLRP3 in ammasomes and its downstream targets i.e. generation of active caspase-1, IL-1β and IL-18 ( Fig. 4A), whereas PA treatment attenuates the NLRP3 in ammasome activation in a dose-dependent manner (Fig. 4B,4C,4D, and 4E).Treatment with PA and Mito-TEMPO further inhibit the increase expression of Iba-1(microglia activation marker) and decreases the extracellular release of IL-1β level in LPS primed H 2 O 2 treated microglia (Fig. 4F, 4G).The results were further con rmed by immunocytochemistry experiments where PA inhibits the nuclear co-localization of P-NF-κBp65 (Fig. 4H) and inhibits the expression of NLRP3 in ammasome protein in microglia cells (Fig. 4I). In our study, treatment with Mito-TEMPO (a standard mitochondria superoxide scavenger) exhibited similar activity alike PA. Thus, results of our study demonstrated that PA by virtue of its antioxidant property scavenges free radicals and inhibits the NF-κB mediated NLRP3 in ammasome activation in microglia cells.

PA ameliorates behavioral anomalies and restored the dopamine level in SNpc in MPTP-administered mice
All mice were trained before the initiation of experiments, and mice failed to pass the baseline behavioural performance were excluded from the experimental study. Five doses of MPTP (25mg/kg body weight) were injected i.p. to produce PD-like symptoms in mice. MPTP treated mice showed a decrease in neuromuscular tone and poor grasping strength with a shorter force to pull the bar in grip strength meter test (Fig. 5A). Similarly, in the rota-rod test, the latency to fall of time in seconds was reduced in the MPTP treated mice on the 7th and 14th day of experiment when compared with the vehicle control group which represents severe motor in-coordination and disease progression (Fig. 5B). Mice treated with MPTP showed a marked increase in the cataleptic response on Day 7 and Day 14 when compared with vehicle control group (Fig. 5C). Pole climbing and OFT were performed to assess the bradykinesia (slow movement) in the MPTP-induced mice. The time taken by the mice to descend from the pole was recorded and compared with vehicle control to assess the disease severity in the MPTPadministered mice (Fig. 5D). Total distance travelled, average speed and immobility time was recorded in the OFT to correlate the motor behaviour in vehicle control, MPTP group and treatment groups (Fig. 5F).
Abnormal gait and the stride length were measured in mice on Day 14 to evaluate motor in-coordination as well as walking pattern behavior (through foot print analysis) (Fig. 5G). 4.5 PA restored the antioxidant enzyme activities and inhibited theNLRP3 in ammasomes activation in brain MPTP, as a complex-I inhibitor of mitochondrial ETC, causes robust generation of ROS with a decrease in several antioxidant enzyme levels along with mitochondria bioenergetic crises. The generated superoxide and peroxide ions were capable of activating NLRP3 in ammasomes together with its downstream targets as seen from the immunoblot data (Fig. 6A, 6B, 6C, 6D, and 6E). Further MPTP causes NF-κB activation which remains one of the major signaling mechanisms for the generation of in ammatory cytokines together with NLRP3 expression (Fig. 6F). The upregulated expression in different proteins were su cient to activate microglia as seen from immunoblot data of Iba-1 in the SNpc region of the brain (Fig. 6G) along with reduced tyrosine hydroxylase (TH) positive stained neurons in the SNpc region of mice brain (Fig. 6H). PA treatment caused a signi cant increase in GSH level, SOD and Catalase activity with lowered MDA levels were seen in the treatment groups thereby exhibiting its strong antioxidant property (Fig. 6K, 6L, 6M and 6N). To understand the microglia activation, we further con rmed the protein expression of Iba-1 in SNpc by employing immunohistochemistry (IHC). The protein expression of Iba-1 was signi cantly decreased in SNpc region of MPTP induced mice when compared with vehicle control group. Treatment with both PA and Mito-TEMPO signi cantly reduced the protein expression of Iba-1, and prevented the microglial activation when compared with MPTP-administered mice (Panel C, Fig. 7).The above data was further supported by estimation of neurotransmitter levels in the striatum region of the mouse brain. The concentrations of NE, 5-HT, 5-HIAA and DA were estimated in the striatum region of the brain tissue. We observed a signi cant decrease in the concentrations of NE; 5-HT and DA in brain of MPTP-administered mice when compared to vehicle control group. Whereas, the concentration of 5-HIAA was signi cantly increased in brain tissue of MPTP-administered mice. Depleted neurotransmitter levels and loss of dopaminergic neurons can be correlated with behavioural data where there was decreased motor function in MPTP-administered mice (Fig. 7F). Treatment with both PA and Mito-TEMPO signi cantly restored the concentrations of NE, 5-HT and DA, and decreased concentration of 5-HIAA in brain tissue when compared to MPTP-administered mice. The aforementioned ndings concluded the neuroprotective effects of PA were comparable to Mito-TEMPO and this ensures PA exhibit strong antioxidant and anti-in ammatory properties. Thus, tightly controlling signals that govern NLRP3 in ammasome activation could be a potential treatment regime in controlling in ammation-induced neurodegeneration in PD.

Discussion
Microglia are the resident immune cells within the brain that plays a fundamental role in maintenance of neuronal survival by phagocytising and eliminating cytotoxic insults [39]. Substantial evidences concluded chronic and persistent microglia activation function as a key regulator in initiating neurodegeneration in several neurological diseases including PD [40][41][42]15]. This statement was further proven by the post-mortem brain analysis of patients suffering from PD was found with activated microglia in SNpc region where dopaminergic neurons are mostly concentrated [43]. NLRP3 in ammasome activation is commonly associated with microglial activation and release in ammatory mediators that cause neuronal damage and further progresses process of neurodegeneration in PD brain [44][45][46]. NLRP3 in ammasome activation is triggered by a variety of stimuli including microbial invasion, misfolded protein aggregates, and other DAMPs including ATP, ROS generation that triggers in ammation induced cell death (pyroptosis) [47,48]. However, the regulatory mechanism underpinning NLRP3 activation in microglia through free radical generation is still remains controversial and need great attention [49]. Hence the present study focused the role of NLRP3 in ammasomes in microglial activation by adding a ROS inducer, H 2 O 2 after priming step of LPS induced activation in N9 microglial cells. The study further involves elucidating the underlying neuroprotective mechanisms of PA and Mito-TEMPO by employing both in vitro N9 microglial cells and in vivo MPTP-administered PD model in mice.
Generally NLRP3 in ammasome activation is a two-step signalling process and represents major players in immune cell activation [50]. Signal 1or pattern recognition induced priming signal (mainly by LPS) through TLR-4 receptors, induces transcription of NLRP3, pro-IL-1β, and pro-IL-18 by NF-κB dependent pathway [51]. Signal 2 activation occurs by endogenous insults such as ATP, urate crystals, mitochondrial dysfunction, potassium e ux that activate NLRP3in ammasome and drives active caspase-1 processing which in turn synthesis of active IL-1β, and IL-18 [7,52]. Earlier reports have depicted the role of LPS + ATP combination in activating the NLRP3 in ammasomes complex, but these studies do not imitate exact condition of mitochondria mediated ROS generation in NLRP3 in ammasome activation [53,10,54].
Hence, within this study we tried to establish a cellular model whether changing the redox microenvironment regulates NLRP3 in ammasome activation through oxidative stress in LPS primed microglia. In this study, H 2 O 2 was selected as an exogenous stimulant for mimicking the condition of oxidative stress in LPS-primed microglia. Remarkably, H 2 O 2 -treatment at a dose of 100 µM causes a robust increase in NLRP3 activation along with cleavage of caspase-1 and release of mature IL-1β level and IL-18 level in LPS primed microglia which remains as potent pro-in ammatory cytokines for further exacerbating neuroin ammatory cascade following neurodegeneration. Similar studies in microglia and macrophages have shown the NLRP3 in ammasome activation with LPS priming following extracellular ATP treatment but not with LPS or ATP alone [55,56]. Contrary to this a few studies states the in ammasome activation occurs only by LPS priming but not its downstream cascade proteins (IL-1β, IL-18) [57,58]. Furthermore, studies also provide overview with H 2 O 2 alone may capable to stimulate NLRP3 in ammasome in human placenta and human primary macrophages [59,60]. This provides the overview that NLRP3 in ammasome activation depends on nature, dose and duration of different signals exposure. Our study has utilized the bidirectional approach (Signal 1: LPS; Signal 2: H 2 O 2 ) of employing the exact cellular environment where two signals are necessary for NLRP3 in ammasome activation while H 2 O 2 alone fails to activate the NLRP3 downstream targets.
Oxidative stress mediated NLRP3 in ammasome activation has been recognised as the main perpetrator for in ammation in several neurological disorders [61,13]. Mitochondria are highly sensitive organelle which can respond even to mild redox imbalance via generating H 2 O 2 which is a potent inducer of oxidative related cellular damage [62,63]. Our study showed that treatment with LPS and H 2 O 2 combination showed a robust production of both cytosolic and mitochondrial superoxide anion levels and H 2 O 2 release in microglial cells due to mitochondrial membrane depolarisation. Thus, inhibiting or treatment with Mito-TEMPO was also found inhibiting NLRP3 in ammasome complex and its downstream targets. Hence, we conclude that mtROS plays a major role in activating NLRP3 in ammasome complex and compounds which alleviate ROS originated oxidative stress not only maintain mitochondrial integrity but also inhibiting NLRP3 in ammasome activation.
Data obtained from in vitro experiments were well correlated with in vivo studies; where administering MPTP, to activate NLRP3 in ammasome in the SNpc region of mouse brain which was well corroborated to earlier reports [7,66]. MPTP being a complex-I inhibitor of mitochondrial electron transport chain and possibly via conversion to its active metabolite MPP + in the microglia results in robust production of mtROS and accelerate NLRP3 activation in vivo [67,12]. Further like in vitro model LPS priming it not possible in animal models as MPTP itself can act as priming signal. Our results showed that MPTPadministered mice exhibited severe motor de cits as seen from grip strength, rota-rod, catalepsy test and pole climbing. The results were further supported from open eld test and measuring the distance between two successive paws where severe bradykinesia with reduction in gait velocity were noticed in MPTP-administered mice. Treatment with both PA and Mito-TEMPO mitigated several behavioral abnormities including motor-impairment, tremor and shu ed gait after MPTP-administration.
To further elucidate the neuroprotective mechanism of PA and Mito-TEMPO in vivo models, SNpc region of mice brains were excised out from MPTP-challenged, PA and Mito-TEMPO treated mice. The major mechanism through which MPTP exerts its toxicity is through induction of oxidative stress and mitochondrial damage via generation of ROS such as O 2 •− and H 2 O 2 . The generated ROS hampers the anti-oxidant defence mechanism and therefore compromised anti-oxidants levels along with depleted ATP level that exacerbate bioenergetic crises. Further the generated free radicals were found activating brain microglia through NLRP3 in ammasome complex oligomerization which triggers cleavage of caspase-1withgenerationof active IL-1β and IL-18 in MPTP-administered mice. These results further indicate that NLRP3 driven microglia activation may show signi cant involvement in progression of neurodegeneration with altered neurotransmitter levels evaluated in the striatum region together with decreased tyrosine hydroxylase expression level in the SNpc region of MPTP-intoxicated mice brain.
Treatment with PA and mtROS scavenger (Mito-TEMPO) was seen protecting the cellular antioxidant (reduced GSH) reserves and their activity (SOD and catalase) defence systems and ATP levels when compared with MPTP-administered mice. Overall treatment with PA and Mito-TEMPO has shown inhibition of microglia activation via regulating NLRP3 in ammasome activation and its downstream targets which remains one of major main executor for in ammation induces neuronal cell death in PD (Fig. 8).

Conclusion
Thus, from both in vitro and in vivo experiments we can conclude that NLRP3 in ammasomes activation remains as major pathway for microglia induction in both mouse microglia (N9 cells) and in SNpc brain region of MPTP-administered mice. Both PA and Mito-TEMPO treatment was found exhibiting potential neuroprotective effects. Hence, alleviates motor defects, neuroin ammation and neuronal damage associated with PD by maintaining cellular redox homeostasis, limit microglia activation and simultaneously support neuronal survival via targeting NLRP3 in ammasome pathway.  Ionized calcium binding adaptor molecule 1; IL-1β: Interleukin 1 beta; IL-18: Interleukin-18;  JC-1: 5,5′,6,6′-Tetrachloro-1  and TNF-α levels in the supernatant of LPS primed microglia treated with H2O2 for 4h and 2h respectively by standard ELISA kit (n=6). Data's were expressed as mean±SD. Statistical signi cance was determined by one-way ANOVA followed by Dunnett analysis where all the groups were compared with normal control group with statistical signi cance de ned as *p < 0.05, **p < 0.01, ***p < 0.001 respectively. were expressed as mean±SD. Statistical signi cance was determined by one-way ANOVA followed by Tukey's post hoc analysis where #p < 0.05, ##p < 0.01, ###p < 0.001 represents control vs. LPS+H2O2 group and*p < 0.05, **p < 0.01, ***p < 0.001 represents LPS+H2O2 vs. different treatment group. Walking track analysis with graph representing distance (cm) between two successive paws. All behavioral data were conducted using (n=8) mice per group. Data's were expressed as mean±SD.
The activated microglia releases neurotoxic factors which ultimately causes neuroin ammation and neuronal loss. Perillyl alcohol inhibits NF-κB mediated NLRP3 in ammasomes activation and subsequently rescues dopaminergic neuronal damage.