DOI: 10.5061/dryad.xsj3tx9jt
title: The c-Abl inhibitor IkT-148009 therapeutically suppresses neurodegeneration in models of heritable and sporadic Parkinson’s Disease

description: Parkinson’s Disease (PD) is the second most prevalent neurodegenerative
 disease of the central nervous system, with an estimated 5,000,000 cases
 worldwide. PD pathology is characterized by the accumulation of
 misfolded a-synuclein, which is thought to play a critical role in the
 etiopathogenesis of the disease. Animal models of PD suggest that
 activation of the Abelson Tyrosine Kinase, or c-Abl, plays an essential
 role in the initiation and progression of a-synuclein pathology and
 initiates processes leading to the degeneration of dopaminergic and
 non-dopaminergic neurons. Given the essential role of c-Abl in the
 disease, a proprietary c-Abl inhibitor library was developed to identify
 potent, orally bioavailable c-Abl inhibitors capable of crossing the
 blood-brain barrier based on pre-defined characteristics, leading to the
 discovery of IkT-148009. IkT-148009 is a selective, potent,
 brain-penetrant c-Abl inhibitor with a favorable toxicology profile that
 was analyzed for therapeutic potential in animal models of slowly
 progressive, a-synuclein-dependent disease. In models of both inherited
 and sporadic Parkinson’s disease in the mouse, IkT-148009 suppressed c-Abl
 activation to baseline and substantially protected neurons from
 degeneration when administered therapeutically by once daily oral gavage
 beginning four weeks after disease initiation. Recovery of normal
 behavioral function in diseased mice occurred within 8 weeks of initiating
 treatment and occurred concomitantly with a substantial reduction of
 a-synuclein pathology in the brain. These disease-modifying outcomes in
 mice suggest IkT-148009 has the potential to be a disease-modifying
 therapy in human disease.
description: <strong>Study Design</strong>: The
 <em>in-silico</em> design method known as
 RAMP<sup>TM</sup> was applied to design and develop inhibitors
 of the non-receptor Abelson tyrosine kinases c-Abl1 (c-Abl) and c-Abl2/Arg
 as described in Supplementary Materials. Three candidate molecules emerged
 from RAMP<sup>TM</sup> that were first screened in a
 modification of the pre-exposure prophylaxis MPTP model of Parkinsonism
 and subsequently compared to the commercial inhibitors nilotinib and
 dasatinib in the same model. Following the selection of one candidate
 inhibitor, IkT-148009, a model of slowly progressive inherited disease
 using the clinical mutation A53T of a-synuclein and a model of slowly
 progressive sporadic disease using pre-formed fibrils (PFFs) were utilized
 to evaluate the therapeutic activity of daily oral administration of
 IkT-148009 as a modifier of neurodegeneration, markers of
 neuroinflammation and a-synuclein pathology. The number of animals
 utilized in each sub-study described was not determined by power analysis,
 rather, was determined by minimizing the number of animals used while
 enabling measures of statistical significance as described below. Details
 of the measurements of neuroanatomy and neurological function in both
 progressive disease models are described in Supplementary
 Materials. <strong>RAMP<sup>TM</sup>
 and Initial Screening of Drug Properties</strong>:
 RAMP<sup>TM</sup> begins with a template molecule whose
 properties are aligned with the desired outcome. In this case, a virtual
 library was constructed in which substituted five or six-membered rings
 were attached to the anticancer agent Imatinib using SmiLib. The library
 consisted of five-and six-membered aryl and heterocycle substituents with
 and without branched aliphatic and polar substitutions ortho- or para-to
 the site of attachment. The library was designed and enumerated to capture
 all potential isomers and mono-substitution patterns. When appropriate,
 tautomers and ionized species were also included. The libraries were
 imported into PyRx minimized and prepared as Autodock ligands. Visual
 inspection of the resulting molecules was necessary to catch occasional
 compilation errors. A minimized structure of substituted template
 molecules was docked into the crystal structure of c-Abl (PDB: 1IEP) as
 the initial basis for the search space. 1IEP was prepared for Autodock
 using the tools in PyRx, to include manual editing to retain 4 water
 molecules found in the Imatinib binding site. PyRx was used as the front
 end to run Autodock Vina for the various libraries.
  Over 2500 compound/conformation profiles were generated with
 Autodock Vina. The docked poses of the Imatinib derivatives were visually
 inspected energy-minimized and found to be consistent with the crystal
 structure as well as known Structure-Activity Relationships (SAR) around
 drugs in this class. Candidate molecules were synthesized as previously
 described and pharmacology properties were assessed. In particular, the
 kinase inhibition IC<sub>50</sub> values were measured for
 on-target enzymes of the Abelson Tyrosine Kinase family (c-Abl1,
 c-Abl2/Arg, c-Kit, PDGFRa/b) and off-target kinase inhibition was measured
 with the KinaseProfiler<sup>TM</sup> screen (Cerep Eurofins)
 at 500 nM IkT-148009. In addition, the in vitro human liver microsome
 half-life and Caco-2 permeability were measured to assess metabolic
 stability and oral absorption characteristics. Rank-ordered candidates
 were then screened for their ability to be orally absorbed with high
 bioavailability in C57Bl/6 mice, high plasma exposures and then the most
 promising candidates were analyzed in the MPTP Screening Model described
 below to assess c-Abl inhibition in mouse brain in the context of an acute
 neurotoxin. 
 <strong>Pharmacokinetics</strong>: Single dose and
 7-day multiple dose pharmacokinetics were measured at 50, 100, and 200
 mg/kg/day in plasma and whole brain. Briefly, 9-week-old male C57Bl/6 mice
 (n=3 per timepoint) were administered IV IkT-148009 (10 mg/kg in water)
 through the tail vein or orally at 50, 100 or 200 mg/kg/day in water (5
 mL/kg). Plasma sampling was done at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24
 hr. post-dose after IV and at. 0.5, 1, 2, 4, 8, 12, and 24 hr. post-dose
 after oral. Following blood collection, the whole brain was harvested
 immediately, rinsed in cold distilled water to remove blood, and weighed.
 Each whole brain was homogenized using pre-cooled water at the ratio of
 1:4. Dosing solutions were analyzed to measure the accuracy of drug
 delivery IV or oral. Plasma and brain concentrations were determined using
 LC/MS-MS methodology. Plasma concentration versus time data were analyzed
 by non-compartmental approaches using the Phoenix WinNonlin 6.3 software
 program. Values for clearance (CL<sub>p</sub>), volume of
 distribution (Vd<sub>ss</sub>), peak plasma concentration
 (C<sub>max</sub>), the time to maximum plasma concentration
 (T<sub>max</sub>), terminal elimination half-life
 (T<sub>½</sub>), area under the time-concentration curve from
 time zero to the last time point measured
 (AUC<sub>(0-t)</sub>), area under the time-concentration curve
 from time zero to time infinity (AUC<sub>(0-inf)</sub>), mean
 residence time from time zero to the last time point measured (MRT(0-t)),
 mean residence time from time zero to time infinity
 (MRT<sub>(0-inf)</sub>), and bioavailability (%F) were
 determined. Brain concentration versus time data were also analyzed by the
 same non-compartmental approach to derive exposure parameters
 (C<sub>max</sub>, T<sub>max</sub>,
 T<sub>½</sub>, AUC<sub>(0-t)</sub>,
 AUC<sub>(0-inf)</sub>) including brain penetrance.
 <strong>Screening of c-Abl inhibitor
 candidates</strong>: A modification of the MPTP mouse model was used
 for screening studies of drug candidates for c-Abl target engagement in
 the brain in the context of neurodegenerative disease. Drug candidates or
 vehicle were administered orally in 8-9 week old male C57BL/6 mice for
 pretreated three days followed by MPTP
 (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride, CAS #
 23007-85-4) injections on day 4 (20mg/kg every 2 hours x 4). Twenty-four
 hours later mice were euthanized, and brain samples were collected for
 target engagement studies. Mice (n = 5 per group) were divided into
 treatment groups as follows: Control Plus Placebo, MPTP Plus Placebo, MPTP
 Plus IkT-148009 (100 mg/kg/day), MPTP Plus IkT-148032 (100 mg/kg/day) and
 MPTP Plus IkT-1427 (100 mg/kg/day). All drug treatment groups were
 co-administered Elacridar (100mg/kg) a P-glycoprotein inhibitor, to ensure
 passage across the blood-brain barrier. Selected drug
 candidates were also screened against the commercial c-Abl inhibitors
 Nilotinib and Dasatinib to evaluate any potential improvement in brain
 penetration relative to inhibitors that have limited potential to cross
 the blood-brain barrier. Mice (n = 5 per group) were divided into 5
 treatment groups: (1) Saline/placebo, (2) MPTP treatment alone, (3)
 Nilotinib (25mg/kg/day) plus with MPTP treatment, (4) Dasatinib
 (25mg/kg/day) with MPTP treatment and (5) IkT-148009 (100 mg/kg/day) +
 Elacridar (100 mg/kg) with MPTP treatment. Nilotinib and Dasatinib were
 solubilized in a vehicle containing 10% N-Methyl-2-pyrrolidone (NMP) and
 90% polyethylene glycol 300 (PEG 300); candidate inhibitors were dissolved
 in buffered or unbuffered water as appropriate.
 <strong>Pre-exposure prophylaxis study of IkT-148009 in the
 MPTP mouse model</strong>: Selection of IkT-148009 from the MPTP
 screening studies was followed by functional assessment of IkT-148009 in a
 pre-exposure prophylaxis MPTP model in which IkT-148009 treatment daily
 for three days preceded MPTP intra-peritoneal (i.p.) injection (20 mg/kg).
 IkT-148009 treatment resumed after the MPTP dosing day for an additional 7
 days. The mice (n = 10 per group) were divided into 4 treatment groups as
 follows: Control (Saline/Placebo), MPTP (placebo), Saline Plus IkT-148009
 (100 mg/kg/day), and IkT-148009 (100 mg/kg) before and after MPTP
 treatment (see schematic in Fig. 3a). The Saline and MPTP groups were
 pre-treated with placebo or IkT-148009 daily for 3 days by oral gavage. On
 the 4th day, the Saline and IkT-148009 groups received four i.p.
 injections of Saline. The MPTP and IkT-148009 plus MPTP groups received
 four i.p. injections of MPTP (20 mg/kg free base) at 2 hr. intervals.
 Following day 4th, Saline and MPTP groups received only vehicle for an
 additional 7 days. IkT-148009 alone and IkT-148009 plus MPTP groups
 received one week of IkT-148009 daily. Behavioral tests were performed on
 day 10. In each group, five mice were sacrificed on day 11 to collect the
 brain samples for neurochemical, biochemical. Five mice per group were
 perfused brains for immunohistochemistry studies.
 <strong>Conditional hA53T-α-synuclein progressive disease
 model</strong>: The therapeutic effect of IkT-148009 was evaluated
 in the TetP-A53T α-synuclein mice. The treatment groups as follows: (1)
 Vehicle + WT-tTA-Placebo (n=15), (2) Vehicle + tet A53T-tTA (n=14), (3)
 IkT-148009 (100 mg/kg) + WT-tTA-Placebo (n=13), (4) IkT-148009 (100 mg/kg)
 + tet A53T-tTA (n=13), (5) IkT-148009 (50 mg/kg) + Elacridar (100 mg/kg) +
 tet A53T-tTA (n=14), and (6) IkT-148009 (100 mg/kg) + Elacridar (100
 mg/kg) + tet A53T-tTA (n=15). Oral administration (p.o.) of IkT-148009 or
 Vehicle were initiated one month after tTA virus injections and
 continuously administered for five months until mice were euthanized for
 sample collections. Amphetamine-induced stereotypic rotation was performed
 at 3 and 6 months. At 6 months, mice were sacrificed, and brains were
 removed for western blot and monoamine analysis. For TH-stereology and
 immunofluorescence analysis, mice were perfused, and the brain was
 processed for immunohistochemistry.
 <strong>Stereotaxic intranigral virus
 injection</strong>: Stereotaxic injection was performed to inject
 AAV-tTA to drive the tetracycline-inducible promotor. Briefly, 8-week-old
 mice of indicated genotypes were anesthetized with a cocktail of ketamine
 and xylazine. Anesthetized mice were gently placed on the mouse
 stereotaxic frame and incisions were made on the scalp to exposure bregma
 point on the skull. The frame installed with Hamilton syringe (2µl, point
 style 3) were guided to respective coordinates to target SNpc
 (anteroposterior, 3.2 mm from bregma; mediolateral, 1.3 mm; dorsoventral,
 4.3 mm) unilaterally stereotaxic injection was performed. The Hamilton
 syringe was loaded with AAV virus particle and mouse was infused with 2µl
 virus particle (at a rate of 0.2 µl/min for 10min duration). The needle
 was placed on the injection site for five minutes for the complete
 absorption of the particles and then gentle retracted needle. Surgery was
 completed by suturing of skin and applying the antibiotic cream for the
 wound healing process. The mice were removed from the stereotaxic frame
 and placed on the heated recovery cage and once the mice completed
 recovered they were then moved to a home cage. The animal was monitored
 and post-care was provided to all the animals. 
 <strong>a-Synuclein Pre-formed Fibril Progressive Disease
 Model</strong>: C57Bl/6 mice were purchased (Jackson Labs) and
 random numbers were generated using GraphPad software (web-based) for
 mouse identification and for the blinded study. Mice were divided into
 four treatment groups (n= 15 to 18 mice/group) as follows., (1) PBS/
 Placebo (2) PBS / IkT-148009 (3) a-Syn-PFF/ Placebo, and (4) a-Syn-PFF/
 IkT-148009. One month after the PBS or PFF injection, mice were treated
 with Placebo or IkT-148009 (100 mg/kg, once a day, five days per week) for
 up to six months of age by oral administration (p.o.). Behavioral studies
 including the Pole test and Grip Strength, as well as bodyweight were
 conducted at 3 and 6 months of age. At six months, mice were euthanized to
 collect the brain samples. Seven to ten mice were perfused, brains were
 sectioned and processed for histological studies. Similarly, eight to ten
 mice were sacrificed for biochemical and neurochemical studies. A blinded
 study protocol was followed during all experiment procedures to acquire
 the data. <strong>α-Syn-PFF preparation and
 use</strong>: Recombinant mouse α-Syn-PFFs were made as described
 previously. Briefly, BL21-competent E. coli were transformed with
 full-length mouse α-Syn grown overnight in the starter culture, and for
 bulk production, the mixture was grown in the Terrific broth with
 overnight shaking at 37°C. The culture was spun down and bacterial pellets
 were resuspended in ice-cold high-salt buffer (1 mM EDTA, 1mM PMSF, and
 protease inhibitors). Pellets were sonicated on ice, boiled at 100°C for
 15min, then the resultant mixture was spun at 6000 x g and the supernatant
 was collected. The supernatant was dialyzed in dialysis buffer (10 mM Tris
 (pH 7.6), 50 mM NaCl, 1 mM EDTA) for buffer exchange for an hour at room
 temperature, then the supernatant was further dialyzed. in fresh dialysis
 buffer overnight at 4 °C. The overnight protein sample was concentrated
 with a centrifuge filter (3.5 kDa cutoff, Amicon) and collected samples
 were filtered using a 0.22 µm syringe filter and loaded into FPLC
 connected to a Superdex 200 SEC column for separation. Relevant fractions
 of α-synuclein were buffer exchanged (10 mM Tris, pH 7.6, 25 mM NaCl, 1 mM
 EDTA) and further purified by anion-exchange chromatography (Hi-Trap Q HP)
 and fractions were collected, analyzed, and exchanged into 50 mM NaCl
 buffer yielding purified α-synuclein stored at -80 °C. Bacterial endotoxin
 was removed using the Toxineraser<sup>TM</sup> endotoxin
 removal kit (Genscript<sup>®</sup>, Cat # L00338). To prepare
 fibrils, monomer protein was thawed quickly and diluted to 5 mg/mL in PBS,
 and the monomer was agitated continually for one week (1,000 rpm, 37 °C)
 to form the fibrils and stored at -80 °C. On the day of striatal surgery,
 α-synuclein-PFFs were thawed at room temperature and diluted to 2.5 mg/mL
 and then sonicated (on ice) at 30% amplitude for 120 seconds (1s on/off)
 with a probe-tip sonicator (Branson Digital Sonifier, Danbury, CT,
 USA). <strong>Stereotactic surgery for
 intrastriatal αSyn-PFF injections</strong>: The male C57BL/6 mice
 (2-3 months old) were anesthetized with a cocktail of ketamine HCL
 (100mg/kg)/xylazine (10mh/kg) i.p. injections, secured with ear bars into
 a stereotactic frame. The skull was exposed via a scalp incision to locate
 bregma and holes in the skull were made at the following coordinates +0.2
 mm (AP), +2 mm (ML), and -2.6 mm (DV). Hamilton syringe (10µl, point style
 3) installed in the stereotaxic frame were loaded with PBS or α-Syn-PFF.
 Probe was gently lowered into the respective injection site. The according
 group mice either received 2 µl of PBS or α-Syn-PFF injections (αSyn-PFF
 at 2.5 µg/µl) into the right striatum. The probe was kept at the injection
 site for 5min before retracting and followed by the closure of the sutures
 and applying antibiotic ointment. Animal were placed in the recovery
 chamber before moving into the home cage.
 <strong>Behavioral studies</strong>:
 <strong>Amphetamine-induced stereotypic
 rotation</strong>: At three- and six-month time-point, to quantify
 the number of amphetamine-induced stereotypic rotation the different
 treatment group mice were injected with amphetamine (AMPH) (5mg/kg, i.p.).
 After AMPH injection, the mice were placed into a glass beaker (5 L) of
 20-cm diameter, and video was recorded for 20 minutes to observe
 AMPH-induced rotation. The video recording was scored by lab personnel
 blinded to the study, animal genotype, and treatments. The major criteria
 in determining the rotation is when the mice complete ipsilateral full
 complete rotations (clockwise). <strong>Grip
 strength test</strong>: The grip strength test was used to measure
 neuromuscular strength in the treatment group mice by using Bioseb grip
 strength test device (BIO-GS3, Pinellas Park, FL) as previously described.
 Mice were gently placed on a metal grid to grab the metal grid by either
 forelimb (forepaw) alone or the forelimb and hindlimbs. The subject is
 placed on the grid, holding tail by the examiner records the forward
 pulling force in the horizontal plane. The grid is connected to a force
 transducer which records the maximal pulling force (in grams).
 <strong>Pole test</strong>: Animals were acclimatized
 in the procedure room for 30 min prior to the experiments. The pole is
 made up 2.5 ft metal rod with 9 mm diameter and wrapped with bandage
 gauze. Briefly, the mice were placed on the top of the pole (3 inch from
 the top of the pole) facing the head-up. The time taken to turn the whole
 body towards the face-down position (Turn time) and the total time taken
 to reach the base (Descending time) of the pole was recorded. Before the
 actual test, the mice were trained for two consecutive days and each
 training session consists of three test trials. The maximum cut-off
 (allowed time) of time to complete the test was 120 sec. Results were
 expressed in total time (in sec).
 <strong>Biochemical Studies</strong>:
 <strong>Tissue lysate preparation</strong>: Brain
 lysates were prepared as described previously with some modifications.
 Soluble fractions were made by homogenization of tissue in brain lysis
 buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40,
 Phosphatase Inhibitor (Roche), and complete protease inhibitor (Roche)).
 Briefly, the brain tissue was homogenated by PTFE pestle with steel shaft
 Tissue grinder homogenizer (1ml) connected with handheld Cordless motor.
 The samples were homogenized with 1:10 (wt./vol) soluble lysis buffer on
 ice for about 20 stoke with homogenizer. The homogenate was collected and
 centrifuged for 30 minutes at 4 °C, 15000 x g, and the resulting
 supernatant (soluble) fractions were carefully transferred to
 microcentrifuge tube. The pellet was washed with soluble lysis buffer spin
 down max at cold and the supernatant was discarded. To the resulting
 pellet insoluble lysis buffer (contains 10 mM Tris-HCl, pH 7.4, 150 mM
 NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 2% SDS and 0.5% sodium deoxycholate,
 Phosphatase Inhibitor (Roche), and complete protease inhibitor (Roche))
 were added and resuspended and then sonicated (30% amplitude 5sec ON, 3sec
 OFF for 3 cycle) on ice. The homogenate was centrifuged at max for 30 min,
 and the resulting supernatant was collected. Protein levels in tissue
 lysates were quantified using a Pierce BCA protein assay kit (Thermo
 Scientific). <strong>Western blot
 analysis</strong>: The detergent-soluble, or detergent-insoluble
 samples were normalized based on the protein concentration, boiled in 2x
 Laemmle buffer, and loaded. The samples were electrophoresed on 4-20%
 SDS-PAGE and transferred to nitrocellulose membranes. Membranes were
 blocked with 5% BSA or 5% nonfat dry milk (wt./vol) in TBS-T and incubated
 with primary antibodies overnight at 4°C. On day two, washed blots were
 incubated with secondary antibody conjugated with HRP and the signal was
 detected using chemiluminescent substrates. For detecting using Odyssey
 CLx, blots were probed with secondary antibodies conjugated with infrared
 dyes (680nm and 800nm) and scanned using an Odyssey CLx (LiCor).
 <strong>Immunohistochemistry</strong>: Anesthetized
 mice were perfused with ice-cold phosphate-buffered saline (PBS, pH 7.4).
 Brains were removed and post fixed for 2 days in 4% paraformaldehyde/PBS
 solution. Brains were embedded with 30% sucrose/PBS (pH 7.4) for 2 days,
 then frozen brain blocks were prepared on dry ice. The blocks were
 sectioned with a temperature-controlled cryostat (Leica CM 3050s) and
 serial coronal sections of 50 μm sections were collected. Free-floating
 sections were incubated with 3% H2O2 washed and blocked with 4% normal
 goat serum/PBS plus 0.2% Triton X-100 and incubated with an antibody
 against TH (NB300-109, 1:2000, rabbit polyclonal; Novus Biologicals)
 followed by incubation with biotin-conjugated anti-rabbit antibody
 (anti-rabbit polyclonal; Vector Labs), VECTASTAIN Elite ABC-HRP kit
 (Vector Laboratories, PK-6101, RRID: AB_2336820) and SigmaFast DAB
 Peroxidase Substrate (Sigma-Aldrich, Cat # D4293, CAS no. 91-95-2). A
 standard staining protocol was used for counterstaining with Nissl (0.09%
 thionin). <strong>Stereology analysis and fiber
 density</strong>: Stereo Investigator (Micro Bright-Field,
 RRID:SCR_002526) software was to use to count the number of TH-positive
 and Nissl positive neurons. By using optical fractionators workflow, we
 draw the contour area around the SNpc region using 2.5x and counted the
 neurons at 100x (oil) magnification. This is an unbiased method for cell
 counting was carried out by using a computer-assisted image analysis
 system consisting of an Axioplan 2 imaging microscope (Zeiss), motorized
 stage (Ludl Electronics), and a color camera (Optronics). Serial striatal
 sections were processed for TH staining following the same procedure as
 above except no Nissl staining was carried out. Images were acquired using
 the Axiophot 2 Imaging brightfield microscope (Zeiss). Fiber density in
 the striatum was quantified by optical density (OD). ImageJ
 (RRID:SCR_003070) software (NIH) was used to analyze the OD as previously
 described. <strong>Immunofluorescence
 analysis</strong>: Immunofluorescence staining for phosphorylated
 a-synuclein (S129 and Y39) was performed in the regions of SNpc and cortex
 of mouse brain sections. Coronal brain sections were initially treated
 with 1x IHC Antigen retrieval solution (high pH, Invitrogen) at 85°C for
 15min, then sections were washed with PBS. The sections were blocked for
 30 min with 10% goat serum (Sigma-Aldrich, Cat no. S26-M) plus 0.3% Triton
 X-100 in PBS and then sections were incubated with indicated antibodies
 for overnight at 4°C. On day 2, the sections were brief washed 3 times
 with PBS-T and incubated with corresponding secondary antibodies
 conjugated with fluorescent dyes (Alexa Fluor 555–conjugated goat antibody
 to mouse IgG and Alexa Fluor 488–conjugated goat antibody to rabbit IgG)
 and counter stained with DAPI for nucleus. Sections washed and covered by
 a coverslip with using Shandon IMMU-MOUNT media (Thermo Fisher Scientific,
 Cat no. 9990402). Mounted slides were dried, and Images were obtained
 using a confocal microscope (Zeiss Confocal LSM 880) and image analysis
 was performed using ZEN Digital Imaging for Light Microscopy
 (RRID:SCR_013672). <strong>Neurochemical
 Studies</strong>:
 <strong>HPLC</strong>: Striatal biogenic amines were
 measured using high-performance liquid chromatography (HPLC) with
 electrochemical detection (ECD). As in previous work, striatal tissue was
 thawed from -80 °C storage and sonicated in ice-cold 0.01 mM perchloric
 acid with 0.01% EDTA (wt./vol). To control for volume errors, DHBA was
 included as the internal standard. Sonicated samples are centrifuged at
 15,000 x g for 30 minutes at 4 °C. Supernatant was collected and filtered
 with a 0.2 µm filter, then 20 µL per sample was separated (mobile phase)
 with an HPLC column (Atlantis T3 3 μm reverse phase column). Eluted
 compounds were analyzed using a dual-channel Coulochem III electrochemical
 detector (Model 5300, ESA Inc, Chelmsford, MA, USA). Pellets were
 homogenized and total protein was quantified to estimate protein to
 normalize for tissue mass. 
 <strong>MPP<sup>+</sup>
 analysis</strong>: For MPP<sup>+</sup> measurements,
 mice were injected with four doses of saline or MPTP (20 mg/kg, 2h
 interval) and mice were euthanized 2 h after the final injection. As
 previously described MPP<sup>+</sup> levels in the striatum
 were detected using HPLC-UV. Striatal tissues were sonicated in 1:10
 volumes of 5% trichloroacetic acid and the samples were centrifuged at
 14,000 rpm for 20 min and the supernatant was injected onto a
 cation-exchange Ultracyl- CS column (Beckman). The mobile phase consisted
 of 90% of a solution of 0.1M acetic acid and 75 mM triethylamine·HCl
 (pH 2.35 adjusted with formic acid) and 10% acetonitrile. The LC pump
 (Antec leyden model LC110) flow rate was 1.0 mL and UV detector (Kanuer
 model S2550) were set at 295 nm. Results were presented as μg/mg
 protein. <strong>Statistical
 analysis</strong>: The animals used in the study were randomized,
 and random numbers were generated using the GraphPad software. The study
 design and treatment were blinded to personnel feeding the placebo or
 active pharmaceutical ingredient. Lab personnel were blinded to the
 disease state and treatment given throughout each sub-study. Western Blot
 band intensities were quantified using ImageJ software (National Institute
 of Health, USA), and the normalized to b-actin. Statistical analysis was
 performed with GraphPad Prism (Version 8) software. Bar graphs were
 generated using GraphPad Prism and expressed with mean ± standard error.
 Statistical significance was determined by performing a one-way analysis
 of variance (ANOVA) or two-way ANOVA followed by Tukey, Bonferroni posthoc
 analysis to enable multiple comparisons with respective to disease or
 treatment groups. Respective p values are denoted in the figures, and p
 values lower than 0.05 were considered statistically significant.
 Quantitative data are expressed as the mean ± standard error from the
 mean.
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