DOI: 10.5061/dryad.51c59zwf1
title: Enhanced mTORC1 signaling and protein synthesis in pathologic alpha-synuclein cellular and animal models of Parkinson’s disease

description: Pathologic alpha-synuclein plays an important role in the pathogenesis of
 alpha-synucleinopathies such as Parkinson’s disease (PD). Disruption of
 proteostasis is thought to be central to pathologic alpha-synuclein
 (alpha-syn) toxicity; however, the molecular mechanism of this
 deregulation is poorly understood. Here we report that pathologic
 alpha-syn activates the mechanistic target of rapamycin (mTOR) complex 1
 (mTORC1) leading to enhanced mRNA translation via binding tuberous
 sclerosis protein (TSC) 2 and destabilizing the TSC1-TSC2 complex. Genetic
 and pharmacologic inhibition of mTOR and protein synthesis rescue the
 dopamine neuron loss, behavioral deficits, and aberrant biochemical
 signaling in the alpha-syn preformed fibril (PFF) and Drosophila alpha-syn
 transgenic models of pathologic alpha-syn induced degeneration. Our
 findings establish a potential molecular mechanism by which pathologic
 alpha-syn activates mTORC1 leading to enhanced protein synthesis and
 concomitant neurodegeneration in PD.
description: <em><strong>In vitro translation (IVT)
 assay</strong></em> Translation extract was
 prepared from human neuroblastoma (SHSY-5Y) or HEK293 cells grown in 175
 cm<sup>2</sup> flask to achieve ~80% confluence. The growing
 cells were harvested using trypsinization and neutralization with complete
 DMEM media. The cell pellets were washed with 10 mL PBS to remove residual
 media from the cell pellet. After PBS wash, the cell pellets were kept in
 ice and lysed immediately in freshly made translation extract lysis buffer
 (30 mM HEPES-KOH, 100 mM potassium acetate, 2 mM magnesium acetate, 5 mM
 DTT, 1 mg/mL Pefabloc SC). The cell pellet from two 175
 cm<sup>2</sup> flasks was lysed in 500 mL lysis buffer. The
 lysis was performed using mechanical homogenizer and Corning PYREX Tissue
 Grinder in ice. A total of twenty strokes were applied to homogenize the
 cell pellet. The lysates were centrifuged at 12500 rpm for 20 mins at 4°C.
 After centrifugation, the clear cytoplasmic fractions were collected from
 the top of the centrifuge tube. The cytoplasmic fractions are aliquoted
 and stored at -80°C.
 <em><strong>Translation assay
 design</strong></em> Translation reaction
 was assembled as described previously (Gebauer et al., 1999). A 25mL
 translation reaction consisting of 50 ng luciferase reporter, 40% (v/v)
 translation extract and 60% (v/v) translation mix (16 mM HEPES-KOH at pH
 7.4, 100 mM complete amino acid mix, 50 mM potassium phosphate, 2.5 mM
 magnesium acetate, 100 mM spermidine, 250 mg/mL yeast tRNA, 80 mg/mL
 creatine kinase, 20 mM creatine phosphate, 800 mM ATP, 100 mM GTP) were
 incubated at 30°C thermo cycler for 30 minutes. Firefly luciferase
 activity was measured using Promega luciferase assay system. 
 <strong>Drosophila</strong><em><strong>
 S35 assay</strong></em> S35 assay in
 <em>Drosophila</em> was performed as described previously
 (Martin et al., 2014). a-Syn transgenic and age matched control flies (5
 days old) were fed with radioactive S35 (Perkin Elmer) for 10 hours. 10
 mCi S35 in 1 mL PBS (supplemented with 2% sucrose) applied to wet Kim
 wipes in 9 cm empty Drosophila vials (Genesee Scientific, cat no. 32–113).
 In each fly vial 20 flies were housed for S35 feeding. After S35 feeding
 flies were snap frozen in liquid nitrogen and heads were separated from
 body by vortexing and sieving. Equal number of fly heads were lysed in 1X
 RIPA buffer (supplemented with protease inhibitor) using mechanical
 homogenizer. Fly brain homogenates were collected in clean Eppendorf tube
 and protein concentrations were measured using BCA (Thermo) kits. For
 autoradiograph, total 10 mg protein was loaded per sample in 4-20%
 Tris-Glycine grading PAGE (Invitrogen), transferred to PVDF membrane
 (BIO-RAD) overnight, stained for Ponceau (Ponceau S solution, SIGMA), then
 the PVDF membrane was air dried and exposed to hot plate and
 autoradiographed in Typhoon FLA 9500 (GE Healthcare) phospho imager. For
 direct scintillation counts total of 5 mg protein per sample was added to
 scintillation fluid and counts were measured in LS 6500 Scintillation
 Counter (Beckman Coulter).
 <em><strong>Direct autoradiography of fly
 heads</strong></em> Transgenic and age
 matched control flies (5 days old) were fed with radioactive s35 (Perkin
 Elmer) for 10 hours. 10 mCi s35 in 1 mL PBS (supplemented with 2% sucrose)
 applied to wet Kim wipes in 9 cm fly vial. Total 20 flies were housed in
 each fly vials for s35 feeding. After feeding flies were snap frozen in
 liquid nitrogen and heads were separated from body by vortexing and
 sieving. The fly heads were placed on clean white paper, three heads were
 placed in each squire and the heads were fixed on the paper surface using
 adhesive plastic wrapper to prevent moving. The fly heads were
 autoradiographed in Typhoon FLA 9500 (GE Healthcare) phospho
 imager.               <strong>SUnSET
 assay</strong> The SUnSET assay was performed as
 previously described with minor changes (Schmidt et al., 2009). Primary
 cortical neurons were prepared from E15 pups and cortices from each pup
 were lysed and plated using neural basal medium. Mature cortical neurons
 (~7 days old) were treated with human a-synuclein Pre-Formed Fibril (a-Syn
 PFF) in 5 mg/mL concentration in 12 wells culture plate for 15 hours.
 After PFF treatment the neurons were treated with Puromycin (Gibco) in 10
 mg/mL concentration for 20 mins. After puromycin pulse the neurons were
 treated with cycloheximide (100 mg/mL) to block new protein synthesis. For
 western blot the PBS washed harvested cells were lysed in RIPA buffer and
 homogenized using mechanical homogenizer using 20 strokes. The clears
 lysates were collocated in Eppendorf and protein concentrations were
 estimated to run equal amount of protein for each sample. For each sample
 total 10 mg protein was loaded for western blot. To quantify new protein
 synthesis, the western blots were developed using anti-puromycin mouse Ab
 (Millipore) and quantifications were normalized by respective actin level.
 Puromycin immunohistochemistry was performed to visualize the
 incorporation of puromycin in the newly synthesized proteins. For
 puromycin histochemistry, primary neurons were grown and cultured on glass
 coverslip in 24 wells plate, a-Syn PFF treatment and puromycin pulse were
 given as above mentioned for puromycin western blot experiment. The
 cycloheximide treated neurons were washed with PBS and fixed with 300 mL
 cold 4% PFA by incubating at room temperature for 15 mins. The neurons on
 the coverslip were washed with 500 mL PBS for three times (5 mins each).
 After PBS wash primary antibodies were added in 1xPBS with 5% normal goat
 serum and 0.3% Triton-X in 250 mL volume for each coverslip. The primary
 antibodies were added in 1:1000 dilution for anti-puromycin (Mouse) and in
 1:500 dilution for anti-NeuN (Rabbit) antibody and incubated overnight at
 4OC shaker incubator. After primary antibody incubation, the coverslips
 were washed with 1xPBS plus 0.3% Triton-X for 30 mins. The secondary
 antibodies (Alexa 488-anti Rb/Ms NeuN (1:1000) + Alexa 555-anti Ms/Rb
 p-a-syn (1:1000) were added and incubated at 37OC for 60 mins. After
 secondary antibody incubation the coverslips were washed for 30 mins and
 mounted on glass slides for confocal microscopy. 
 <em><strong>Biotinylated a-Syn-PFF pulldown from
 mouse striatum</strong></em> Three months
 old C57BL6 male mice were used for striatal injections of biotinylated
 human a-synuclein PFF. Mice were injected with 10 mg a-Syn PFF into both
 hemispheres. To control this experiment, C57BL6 male mice were injected
 with non-biotinylated human a-Syn PFF. After 5 days of injection, both
 mice were sacrificed and striatal tissues were isolated and lysed in 20 mM
 HEPES buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM
 MgCl<sub>2</sub>, 0.1% Triton X-100, 0.1 % NP-40). Striatal
 tissues were homogenized in 800 mL lysis buffer using mechanical
 homogenizer and Corning PYREX Tissue Grinder in ice. Total twenty strokes
 were applied to homogenize the brain tissues. The homogenates were
 centrifuged at 12,000 rpm for 15 mins at 4°C and the clear lysates were
 collected in clean Eppendorf tubes. For streptavidin immunoprecipitation,
 200 mL streptavidin M-280 Dynabeads (ThermoFisher) were washed and
 incubated with brain lysates for 4 hours at 4°C with continuous rotation.
 The immunoprecipitated beads were washed 5 times. Two sets of
 immunoprecipitations were performed. One pair of mice was used for mass
 spectrometry analysis and another pair was used for western blot
 analysis.       <em><strong>In situ
 labelling of a-Syn binding partners using A53T a-Syn-APEX2 assay in HEK293
 cell </strong></em> In situ biotin
 labelling assay was performed as described by Lam et al. (Lam et al.,
 2015) with minor changes. In A53T-a-Syn-APEX2 construct, APEX2 was tagged
 to the C-terminal of A53T a-Syn under the CMV promoter. HEK293 cells were
 grown in complete DMEM and A53T a-Syn-APEX2 plasmid was transfected using
 Xtreme transfection reagent (Thermo). After 24 hours of HEK293
 transfection, 500 mM biotin tyramide dissolved in Optimem was added to the
 transfected cells and incubated for 30 min at 37°C. To activate APEX2
 enzyme activity, H<sub>2</sub>O<sub>2</sub> was
 added at 1 mM final concentration for exactly 1 min at room temperature.
 Following H<sub>2</sub>O<sub>2</sub> treatment the
 cells were washed twice with PBS containing quenching reagents (5 mM
 Trolox, 10 mM ascorbic acid and 10 mM NaN3) for 30s followed by another
 two washes with PBS. To control the immunoprecipitation, A53T a-Syn-APEX2
 transfected cells were used without
 H<sub>2</sub>O<sub>2</sub> treatment. The cells
 were homogenized in 20 mM HEPES buffer (20 mM HEPES pH 7.4, 150 mM NaCl,
 2.5 mM MgCl<sub>2</sub>, 0.1% Triton X-100, 0.1% NP-40) and
 biotinylated protein complexes were Immunoprecipitated using streptavidin
 M-280 Dynabeads (ThermoFisher). Two sets of immunoprecipitations were
 performed. One set was used for mass spectrometry and another set was used
 for western blot analysis. 
 <em><strong>Sample
 preparation for Mass
 Spectrometry</strong></em>
 Captured biotinylated proteins
 using APEX protocol from HEK293T cells (Lobingier et al., 2017) and Immunoprecipitated samples from mouse brain
 tissue samples were boiled in 4X LDS sample buffer (Life Technologies)
 supplemented with 100 mM dithiothreitol or 200 mM beta-mercaptoethanol.
 These samples were loaded on Novex 4 to 20% Tris-Glycine protein gel
 (Thermo Fisher Scientific). Following the successful electrophoretic
 steps, gels were stained using a dye, Coomassie blue. The size of the gel
 piece containing bands to be cut out from the gel depends on the density
 (Kang et al.,
 2009). Before enzymatic
 digestion of the proteins, in-gel reduction and alkylation reactions are
 carried out to prevent oxidation and disulfide formation. Subsequently,
 trypsin was applied for protein digestion. Following enzymatic digestion,
 the samples are transferred to lobind tubes (Eppendorf) before
 mass-spectrometry analysis as the use of these tubes significantly reduces
 the binding of hydrophobic proteins to the wall of the tubes
 (Kang et al.,
 2009). After enzymatic
 reaction, all samples were desalted using Sep-Pak C18
 cartridges.
 <em><strong>Mass
 Spectrometry</strong></em>
 Peptide fractions were analyzed
 on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher
 Scientific) interfaced with Easy-nLC 1200 nanoflow LC system (Thermo
 Fisher Scientific). The peptides were reconstituted in 0.1% formic acid
 and loaded on an analytical column at a flow rate of 300 nL/min using a
 linear gradient of 10-35% solvent B (0.1% formic acid in 95% acetonitrile)
 over 120 min. Specific
 conditions for mass spectrometry were set as following; MS1 resolution
 (120,000), MS2 resolution (30,000), fragmentation method (HCD), collision
 energy for MS2 (32%). The fifteen most intense precursor ions from a
 survey scan were selected for MS/MS fragmentation using higher energy
 collisional dissociation (HCD) fragmentation with 32% normalized collision
 energy and detected at a mass resolution of 30,000 at 400 m/z. Automatic
 gain control for full MS was set to 1 × 10<sup>6</sup> for MS
 and 5 × 10<sup>4</sup> ions for MS/MS with a maximum ion
 injection time of 100 ms. Dynamic exclusion was set to 30 sec. and singly
 charged ions were rejected (Agarwal et al., 2020).
 <em><strong>Polysome
 profiling</strong></em>: Fly brain polysome profile was
 performed as described by Darnel et al. with minor changes (Darnell et
 al., 2018). Brain tissue homogenates of a-Syn transgenic and age matched
 control flies (5 days old) were examined and profiled for polysome to
 understand the abundance translating mRNAs in the polysome fractions. Fly
 brains were lysed in polysome lysis buffer (10 mM Tris pH 7.5, 150 mM
 NaCl, 5 mM MgCl<sub>2</sub>, 0.5 mM DTT, 100 mg/ml
 cycloheximide, 40 U/ml RNase inhibitor, 0.1% sodium deoxycholate and
 complete protease inhibitor, ROCHE). Approximately ~ 100 fly heads were
 lysed in 1 ml polysome lysis buffer, protein concentrations of the lysates
 were measured and 0.5 mg equivalent polysome lysates were loaded for
 10-45% sucrose gradient. The sucrose gradients with polysome lysates were
 centrifuged at 40,000 rpm for 2 hours at 4°C. All gradients were
 fractionated in polysome Gradient Station (BIOCOMP) under same parameters
 all samples. Polysome profiles of HEK293 cells were performed using same
 buffer and assay conditions. Transfected cells (with 1 mg of DNA per well
 in six well plate) were harvested after 24 hours and lysed in polysome
 lysis buffer. Each polysome assay was performed using 0.5 mg total
 protein.    
 <strong>Drosophila</strong><em><strong>
 stocks and maintenance:  </strong></em>
 <em>Drosophila</em> stocks were maintained according
 to the standard laboratory protocols. The fly stocks were maintained in
 standard <em>Drosophila</em> diet containing yeast extract,
 agar, cornmeal, sucrose, and dextrose. For routine maintenance flies were
 kept in a 12 h light/dark cycle and all fly stocks and experiential
 crosses were housed at 25°C. UAS a-Syn, UAS A53T-a-Syn and relevant Gal4
 stocks were obtained from Bloomington Drosophila Stock Center (BDSC),
 Indiana, USA. QUAS-a-Syn transgenic fly was a gift from Dr. Mel Feany,
 Harvard Medical School. Human eIF4E-BP1 and TSC2 constructs were generated
 in the lab and subcloned the cDNA sequences of eIF4E-BP1 and TSC2 into
 pQUAST-att plasmid. The verified constructs were used for microinjection
 into w<sup>118</sup> embryos (The BestGene, Inc). For
 experimental control, the relevant Syb-QF2/+ and Gal4/+ (heterozygous)
 flies were used as controls in the experiments.         
 <strong>Drosophila<em> climbing and life span
 assay</em></strong>: Climbing assay was performed as
 previously described (Nichols et al., 2012). Both test and control flies
 were collected immediately after eclosion and maintained in regular fly
 food vial at 25°C fly incubator with recommended humidity and aeration.
 Approximately ~25 flies were housed in each fly vial. Climbing assays were
 performed every 3 days by transferring 25 flies to an empty 9 cm fly vial
 (Genesee Scientific, cat no. 32–113) with a line drawn 6 cm from the
 bottom. To induce and test an innate climbing response, flies were tapped
 to the bottom. The number of flies were counted that crossed the 6 cm mark
 in 15 sec time. For each batch five to ten technical replicates were
 performed to ensure an accurate reading at each time-point and average of
 the independent trials was used to calculate the percentage of flies with
 climbing defects. For fly lifespan assay Syb-QF2&gt;a-Syn flies were
 collected immediately after eclosion and maintained in the fly food vials
 with drugs of interest and their vehicles at 25°C. The flies were aged in
 a batch of 30 per vial and total 120 flies were aged for each drug and
 corresponding vehicles. In every three days flies were transferred to
 fresh fly food with drug and surviving flies were counted every five days.
 Percent fly survival was plotted using Kaplan Meyer survival curve in
 Graph Pad Prism 9 software. 
 <strong><em>Fly brain
 immunostaining</em></strong>: Fly brains were dissected and TH
 immunostained following previously described protocols with minor
 modifications (Wu and Luo, 2006). Adult <em>Drosophila</em>
 brains were dissected in PBS and fixed in 4% paraformaldehyde (PFA) at
 room temperature (RT) for 30 mins. After incubation with PFA, the brains
 were washed for 30 mins in 1X PBS with 0.3% Triton X-100 (wash buffer),
 blocked for 30 mins in blocking buffer (1X PBS, 0.3% Triton X-100 and 5%
 normal goat serum) at RT. Following blocking step, the brains were
 incubated with primary antibody in blocking buffer for 48 hours at 4°C.
 The brains were washed three times, 10 mins each, at RT. Secondary
 antibody in blocking buffer was added to the brains and incubated for 24
 hours at 4°C. After five 10 mins washes, brains were carefully mounted on
 glass slides using ProLong Gold antifade mountant (Life Technologies). The
 TH (anti-Tyrosine Hydroxylase) primary antibody was used in 1:500 dilution
 (Immunostar, cat no. 22941), Secondary antibody goat anti-mouse was used
 at 1:1000 (Thermo Fisher, cat no. A-11032). 
 <em><strong>Immunostaining of primary cortical
 neurons</strong></em>: Primary cortical neurons on glass
 coverslip in 12-well plate were treated with 5 mg/mL a-Syn PFF in neural
 basal media for 7 days. The cells on the coverslip were washed with 1X PBS
 to remove residual media and fixed with 4% paraformaldehyde (PFA) for 15
 mins and incubated at room temperature (RT). Following PFA treatment cells
 were washed with 1X PBS for three 10 mins washes and blocked in 1X PBS
 with 5% normal goat serum and 0.3% Triton X-100 (blocking buffer) at RT.
 Primary antibodies were added in blocking buffer for 24 hours at 4°C. The
 coverslips were washed in 1X PBS with 0.3% Triton X-100 (Wash buffer) for
 three 10 mins washes at RT. Secondary antibodies were added to the
 coverslips in blocking buffer and incubated for 1 hour at room temperature
 followed by the 5 ten mins washes. The coverslips were mounted on glass
 slides with mounting solutions. The rabbit anti-a-Syn (phospho S129)
 antibody (Abcam) and mouse anti-MAP2 (Millipore) primary antibodies were
 used in 1:500 dilution (Millipore. 22941), Secondary antibody goat
 anti-mouse and anti-rabbit antibodies were used at 1:1000 (Thermo Fisher,
 cat no. A-11032).     Toxicity test in primary cortical
 neuron: Mature primary cortical neurons were treated with 5 mg/mL a-Syn
 PFF in neural basal media for 14 days. Cell death was measured by treating
 the neurons with 7 mM Hoechst 33342 and 2 mM propidium iodide (PI)
 (Invitrogen). Images were captured by a Zeiss microscope and percent cell
 death was counted using ImageJ software.  
 <em><strong>Rapamycin, S6K Inhibitor and Anisomycin
 treatment for
 </strong></em><strong>Drosophila</strong>: The
 drugs were given to the experimental flies in standard fly food. The flies
 were treated in batches of 20-25 per fly tube containing 0.5 mM rapamycin
 (LC laboratories), 10 mM S6K inhibitor (PF-4708671; Pfizer), and 10 mM
 anisomycin (Sigma) respectively. The drugs were added, in a 200 mL volume,
 to the dry surface of fly media and allowed to absorb and air dry at least
 6 hours prior to the experimental treatment. For both climbing and
 survival assays the flies were treated for 3 days in a tube and then
 transferred to tubes containing fresh food and drugs.         
 <em><strong>Tissue homogenization and Western
 blotting</strong></em>: Brain tissues, both human post mortem
 PD brains as well as mouse brains, were homogenized in 1X RIPA buffer (50
 mM Tris HCl, 150 mM NaCl, 1.0% (v/v) NP-40, 0.5% (w/v) Sodium
 Deoxycholate, 1.0 mM EDTA, 0.1% (w/v) SDS and 0.01% (w/v) sodium azide at
 a pH of 7.4.) with additional phosphatase inhibitor mixture I and II
 (Sigma-Aldrich, St. Louis, MO), and complete protease inhibitor mixture
 (Roche, Indianapolis, IN.). Following homogenization, the samples were
 freeze-thawed three times using dry ice, centrifuged 15000 x g for 15 min
 and collected clear supernatants. Protein concentrations were measured
 using the BCA assay (Pierce, Rockford, IL), SDS-PAGE was used to separate
 proteins and transfer to nitrocellulose membranes for immunoblot analysis.
 For blocking, membranes were incubated in 5% non-fat milk in TBS-T (Tris-
 buffered saline with 0.1% Tween-20) at least 1 h at room temperature (RT)
 and subsequently probed using primary antibodies overnight at 4°C with
 continuous shacking.  After overnight primary antibody. The membranes were
 washed 30 mins ---appropriate HRP-conjugated secondary antibodies (Cell
 signaling, Danvers, MA). The bands were visualized by ECL
 substrate. <em><strong>Isolation of A53T
 a-Syn aggregates from transgenic mice brain
 tissues</strong></em>: Freshly dissected brain stem tissues
 (~30 mg) from late symptomatic (13 months old) A53T a-Syn transgenic and
 age matched control mice were homogenized in lysis buffer (10 mM Tris pH
 7.5, 150 mM NaCl, 5 mM MgCl<sub>2</sub>, 0.5 mM DTT, 100 mg/ml
 cycloheximide, 40 U/ml RNase inhibitor, 0.1% sodium deoxycholate and
 complete protease inhibitor from ROCHE). Tissue lysate volume was adjusted
 to 600mL for ultracentrifugation at 70K rpm in Optima Max Ultracentrifuge
 (Bechman Coulter). For ultracentrifugation 45% sucrose solution was
 prepared in the same lysis buffer. The tissue homogenates were
 ultracentrifuged in Polycarbonate Thick Walled 13 X 56 mm centrifuge tube
 (Beckman Coulter), containing 0.9 ml 45% sucrose solution and 0.6 ml
 tissue homogenate on top of sucrose solution. Centrifugation was performed
 for 2 hours at 4°C. After centrifugation supernatant was decanted and
 hard-shiny pellets were resuspended in 100 mL lysis buffer. The preps were
 dialyzed using MINI Dialysis Device, 10K MWCO (Thermo) in 1L PBS
 overnight. After overnight dialysis the dialysis device was transferred to
 fresh 1L PBS to repeat the process overnight. After 48 hours of dialysis
 the preps were filter sterilized and stored at -80°C.  
 <strong><em>Transmission Electron Microscopy (TEM) of
 </em>Drosophila<em> brains</em></strong>:
 <em>Drosophila</em> heads were fixed with 4% paraformaldehyde
 (freshly prepared from EM grade prill) 2% glutaraldehyde 100 mM Sorenson’s
 phosphate buffer with 5 mM magnesium chloride pH 7.2, overnight at 4° C. 
 Following buffer rinses, samples were microwave fixed twice in 2% osmium
 tetroxide reduced with 1.5% potassium ferrocyanide, in the same buffer.
 Sample temperatures did not exceed 9°C.  Following microwave processing
 samples were rocked in osmium on ice for 2 hours in the dark.  Tissue was
 then rinsed in 100 mM maleate buffer with pH 6.2, then en-bloc stained for
 1 hour with filtered 2% uranyl acetate in maleate buffer, pH 6.2. 
 Following en-bloc staining samples were dehydrated through a graded series
 of ethanol to 100%, transferred through propylene oxide, embedded in
 Eponate 12 (Pella), and cured at 60°C for two days. Sections were cut on a
 Riechert Ultracut E microtome with a Diatome Diamond knife (45 degree). 60
 nm sections were picked up on formvar coated 1 x 2 mm copper slot grids
 and stained with methanolic uranyl acetate, followed by lead citrate. 
 Grids were viewed on a Hitachi 7600 TEM operating at 80 kV and digital
 images captured with an XR80- 8-megapixel CCD by AMT.
 <em><strong>Antibodies for SiMPull
 Assay</strong></em> All antibodies used for
 SiMPull were obtained from commercial sources as follows: biotinylated
 anti-RFP from Abcam (ab34771), anti-YFP and anti-Flag from Rockland
 Antibodies and Assays and anti-myc from Sigma-Aldrich. Primary antibodies
 for TSC1 and TSC2 were obtained from Cell Signaling Technology. Alexa-647
 tagged goat anti-rabbit IgG was purchased from ThermoFisher Scientific.
 Neutravidin and BSA were procured from Thermo-Fisher and New England
 Biolabs respectively.  <strong><em>Cell
 Culture and Transfection for SiMPull
 Assay</em></strong> Assembly of functional
 mTORC2 was achieved by co-expression of mTOR, Rictor, mSin and mLST8,
 while mTORC1 and TSC complexes were generated via co-expression of mTOR,
 Raptor and TSC1, TSC2 respectively. HEK293 cells were grown in DMEM
 containing 10% (vol/vol) FBS and 2 mM L-glutamate at 37°C with 5%
 (vol/vol) CO<sub>2</sub>. Transfection of plasmids was carried
 out using Lipofectamine 2000 (Thermo Fisher Scientific) following the
 manufacturer’s protocol when the cells reached 60-70% confluence in 6-cm
 plates. A day after transfection, cells were lysed in 300 mL of lysis
 buffer containing 40 mM HEPES, pH 7.5, 120 mM NaCl, 10 mM sodium
 pyrophosphate, 10 mM b-glycerophosphate, 1X protease inhibitor mixture and
 0.3% CHAPS. PKA complexes were obtained from HEK cells, transiently
 transfected with R-Flag-mCherry and C-HA-YFP constructs. PKA-RIIb and Ca
 isoforms were used as the regulatory and catalytic subunits respectively.
 After 24 h expression, cells were lysed using a buffer containing 10mM
 Tris pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM benzamidine, 10 mg/ml
 leupeptin, 1mM NaF, 1mM
 Na<sub>3</sub>VO<sub>4</sub>. The lysate thus
 obtained was centrifuged at 14,000 X g for 20 min and subsequently used
 for SiMPull (Jain et al., 2011). Pre-determined
 concentrations of monomers and PFFs of a-synuclein were added to the
 cell-lysates and incubated at 4°C for 3h. The cell lysates were diluted
 50-150 fold in T50-BSA buffer (10 mM Tris, pH 8, 50 mM NaCl and 0.2 mg/mL
 BSA) to obtain a surface density optimal for single-molecule analysis (~
 600 molecules in 5,000 mm<sup>2</sup> imaging area) (Aggarwal
 and Ha, 2014; Jain et al., 2011). 
 <em><strong>Single-Molecule Imaging and
 Analysis</strong></em> Single-molecule
 experiments were performed on a prism-type TIRF microscope equipped with
 an electron-multiplying CCD camera (EM-CCD) (Roy et al., 2008). For
 single-molecule pull-down experiments quartz slides and glass cover slips
 were passivated with 5000 MW methoxy poly-(ethylene glycol) (mPEG, Laysan
 Bio) doped with 2-5% 5000 MW biotinylated PEG (Laysan Bio). Each
 passivated slide and cover slip was assembled into flow chambers. The cell
 lysates were pulled down with biotinylated antibodies against Flag, myc,
 YFP, or RFP, already immobilized on the surface via neutravidin-biotin
 linkage. TSC1 and TSC2 were visualized using Alexa-647 tagged anti-rabbit
 secondary antibodies. YFP-, m-Cherry- and Alexa-647 tagged proteins were
 excited at 488 nm, 568 nm, and 640 nm respectively and the emitted
 fluorescence signal was collected via band pass filters (HQ 535/30, Chroma
 Technology for YFP, BL 607/36, Semrock for mCherry and 665LP from Semrock
 for Cy5). 15 frames were recorded from each of 20 different imaging areas
 (5,000 mm<sup>2</sup>) and isolated single-molecule peaks were
 identified by fitting a Gaussian profile to the average intensity from the
 first ten frames. Mean spot-count per image for YFP and mCherry was
 obtained by averaging 20 imaging areas using MATLAB scripts. All
 experiments were carried out at room-temperature (22-25°C).
 The complexes identified were next subjected to photobleaching
 step analysis to determine the stoichiometry of mTOR and Raptor/Rictor in
 mTORC1 and mTORC2. A single photobleaching step can be characterized by an
 abrupt drop in fluorescence intensity. Single-molecule fluorescent time
 traces from individual YFP or mCherry spots were manually scored for the
 number of photobleaching steps and the stoichiometry of the molecules was
 assessed subsequently (Aggarwal and Ha, 2014; Jain et al., 2011). All
 images were collected at a time resolution of 100 ms. Each molecule was
 arrayed based on the number of photobleaching steps (typically 1-3) or was
 discarded if no distinct photobleaching step could be identified. All
 spots with no fluorescent signal from either of the fluorophores were
 rejected. A minimum of 500 molecules acquired from at least four different
 imaging areas were analyzed for each experimental condition.
 <strong><em>Rapamycin treatment of a-Syn PFF injected
 mice</em></strong> This experiment was
 performed in compliance with the regulations of the Animal Ethical
 Committee of the Johns Hopkins University Animal Care and Use Committee.
 Rapamycin was administered to α-syn PFF injected mice model in two
 different ways. First, to examine α-syn pathology in one month of α-syn
 PFF injected mouse model, both PBS and α-syn PFF mice were injected
 intraperitonially with Rapamycin (6mg/Kg; #R-5000, LC laboratories) or
 vehicle (10% PEG400, 10% Tween 80 in water) for 30 days. Rapamycin
 initiated at day 1 after PFF injection and continued for 30 days and three
 times per week. Second, to examine toxicity, α-syn PFF injected mice were
 fed with rapamycin in mouse chow (5LG6-JL) for six months. Mouse feeding
 experiment was performed as described previously by Harrison et al.
 (Harrison et al., 2009). Microencapsulated rapamycin (eRAPA) was
 incorporated into mouse chow and provided by Rapamycin Holdings (San
 Antonio, TX 78249). Briefly, Rapamycin (LC Labs) was microencapsulated by
 Southwest Research Institute (San Antonio, Texas) using enteric coating
 material Eudragit S100 (Ro ̈hm Pharma). Microencapsulation protects
 rapamycin from digestion in the stomach and the encapsulated rapamycin was
 administered at 14 mg per kg food (2.24 mg of rapamycin per kg body weight
 per day). The diet containing only the coating material (Eudragit) used as
 control food in the experiment. 
 <strong><em>Stereotactic Injection of α-Syn PFF
 </em></strong> Mice were anesthetized with
 ketamine (100 mg/kg) and xylazine (10 mg/kg). PBS or α-syn PFF (10
 μg/mouse) was injected into the striatum (anteroposterior (AP) = +0.2 mm,
 mediolateral (ML) = + 2.0 mm, dorsoventral (DV) = +2.6 mm, relative to
 bregma). A 2 μl syringe was used for injections at a rate of 0.4 μl/min,
 and post-surgical care was provided after surgery.
 <strong><em>Immunohistochemistry </em></strong> Immunohistochemistry was performed on 40 mm thick serial brain sections. For α-syn phosphorylation detection, sections were blocked with PBS containing 10% normal goat serum and 0.2% Triton X-100 for 1 hour, and then incubated with primary antibodies to p-α-syn, and MAP2 or Tyrosine hydroxylase (TH) overnight at 4°C, followed by incubations with appropriate fluorescent secondary antibodies conjugated to Alexa-fluor 488, 594 or/and 647. P-α-syn pathology was displayed by tracing all visible immunoreactive inclusions/cells and neurites using Keyence Microscope at 10 × magnification. Fluorescent images with higher magnification (20 × or 40 ×) were acquired by confocal scanning microscopy (LSM880, Carl Zeiss). Images were processed using Zen software (Carl Zeiss), and the signal intensity was measured using ImageJ. For histological studies, free-floating sections were rinsed in Tris-buffered saline (TBS, pH 7.4) and incubated with 0.5% H<sub>2</sub>O<sub>2</sub> in TBS to inhibit endogenous peroxidase. After blocking in TBS containing 10% goat serum and 0.2% Triton X-100, sections were incubated with Rabbit anti-TH antibody overnight at 4°C, followed by incubation with biotin-conjugated anti-rabbit antibody. After three washes, ABC reagent (Vector laboratories) was added, and the sections were developed using DAB peroxidase substrate (Sigma). Sections were counterstained with Nissl (0.09% thionin). For the quantification, both TH and Nissl-positive DA neurons from the substantia nigra pars compacta (SNpc) region were counted using a computer-assisted image analysis system consisting of an Axiophot photomicroscope (Carl Zeiss) equipped with a computer controlled motorized stage (Ludl Electronics), a Hitachi HV C20 camera, and Stereo Investigator software (MicroBright-Field).  <strong>Behavior Tests</strong> <strong><em>Grip Strength</em></strong> Neuromuscular strength was measured by maximum holding force generated by the mice (Biosed). Animals were allowed to grasp a metal grid with either by their fore and/or hind limbs or both. The tail was gently pulled, and the maximum holding force recorded by the force transducer when the mice released their grasp on the grid. The peak holding strength was digitally recorded and displayed as force in grams. <strong><em>Pole Test</em></strong> The pole test was used to measure bradykinesia (Karl et al., 2003). A metal rod (2.5 foot long with a 9 mm diameter) wrapped with bandage gauze was used as the pole. The pole test protocol was performed as described previously (Kam et al., 2018): two consecutive days of training consisted of three test trials, followed by the actual test. Each animal was placed directly under the top of the pole (3 inch from the top of the pole) with the head held upwards. Results were expressed in turn time and total time. The maximal cutoff of time to stop the test was 60 s.
description: # Enhanced mTORC1 signaling and Protein Synthesis in Pathologic
 alpha-Synuclein Cellular and Animal models of Parkinson’s Disease
 [https://doi.org/10.5061/dryad.51c59zwf1](https://doi.org/10.5061/dryad.51c59zwf1) In this study, we examined the pathological effect of a-synuclein in enhanced mRNA translation and mTORC1 activity in Parkinson’s disease models. We used complementary proteomic approaches and mouse primary neuron cellular and animal models of PD to identify and characterize the pathologic a-synuclein interactome. We demonstrated that the highest biological processes that interact with pathologic a-synuclein include RNA processing and translation initiation. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein (TSC) 2, resulting in the destabilization of the TSC1-TSC2 complex. This destabilization ultimately triggered the activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). The activated mTORC1 complex plays a pivotal role in augmenting mRNA translation and protein synthesis, consequently contributing to neurodegenerative processes. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescued the dopamine neuron loss, behavioral deficits and aberrant biochemical signaling in the a-synuclein  preformed fibril (PFF) mouse model and *Drosophila* a-synuclein  transgenic models of pathologic a-synuclein  induced degeneration. ## Description of the data and file structure Details for each dataset are provided in the README file titled "README\_Khan\_et\_al-STM-add0499v2". Files are organized by Figure.   1.     Pathogenic a-synuclein biochemically interacts with the critical components of translation and mTOR pathways in vitro and in mice · A53Ta-synuclein-APEX mass spectrometry data revealed interactome of A53Ta-synuclein in HEK293 transfected cells · Striatal injection of biotinylated a-synuclein PFF followed by immunoprecipitation and mass spectrometry showed the interactions with the protein complexes. · Validation of interaction by immunoprecipitation of A53Ta-synuclein-APEX confirmed the binding with the partners   · Validation of interaction by immunoprecipitation of biotinylated a-synuclein PFF from the PFF injected mice tissue confirmed the interactions with the protein of interest      2.     Pathogenic a-synuclein enhances mRNA translation in vitro and in in a-synuclein flies · In Vitro Translation (IVT) assay showed pathologic a-synuclein enhanced translation of cap-dependent mRNA in concentration dependent manner.   · Assessment of new protein synthesis using S35 incorporation assay in UAS-a-synuclein transgenic flies showed enhanced new protein synthesis · Polysome profile analysis of fly brain lysates showed increased association of mRNA with polyribosomes in a-synuclein transgenic flies  · Syb-QF2&gt;a-synuclein flies showed enhanced new protein synthesis in S35 incorporation assay. · SUnSET assay in a-synuclein PFF treated mouse primary cortical neuron showed increase in new protein synthesis.  · Isolated a-synuclein aggregates from A53T transgenic mice showed increase in new protein synthesis in primary cortical neuron   3.     Pathogenic a-synuclein mediated increase in protein synthesis depends on mTORC1 kinase activity in a-synuclein flies · New protein synthesis was assessed in Syb-QF2&gt;a-synuclein and control flies by S35 pulse chase assay and rapamycin treatment showed significant reduction in new protein synthesis. · Immunoblot analysis showed reduction of P-S129 a-synuclein and d4E-BP phosphorylation by rapamycin treatment. · TH immunostaining of whole fly brain showed rescue of TH positive cells. · Rapamycin treatment rescued climbing defects in Syb-QF2&gt;a-synuclein flies and enhanced survival of the transgenic flies.         4.     Rapamycin is neuroprotective in the a-synuclein PFF mouse model · In a-synuclein PFF mouse model rapamycin rescued the loss of TH positive cells and reduced a-synuclein pathology in different brain region · Whole brain coronal sections showed reduction of P-S129 a-synuclein in rapamycin treated mice · P-S6K (P-T389) immunostaining showed significant reduction in phosphorylation in a-synuclein PFF mice treated with rapamycin. · Behavioral assessment showed  rapamycin treatment rescued PD pathology which includes locomotion defect assessed by pole test.   5.     Anisomycin rescues a-synuclein pathology and neurotoxicity in a-synuclein flies. · In vivo Syb-QF2&gt;a-synuclein flies showed significant increase in S35 incorporation and that effect is subsided the anisomycin treatment. · Anisomycin treatment reduced a-synuclein and d4E-BP phosphorylation in Syb-QF2&gt;a-synuclein flies · Anisomycin treatment rescued TH and dopamine neuron loss in Syb-QF2&gt;a-synuclein flies · Anisomycin treatment enhanced survival of  Syb-QF2&gt;a-synuclein flies · Cell death in a-synuclein PFF treated primary cortical was rescued by anisomycin treatment.   6.     Pathogenic a-synuclein destabilizes the TSC1-TSC2 complex · Single Molecule Pulldown (SiMPull) assay showed pathologic a-synuclein disassembled TSC1-TSC2 complex in concentration dependent manner · Single Molecule Pulldown (SiMPull) assay showed pathologic a-synuclein has no effect of the mTORC1 complex · Effect of pathologic a-synuclein on TSC1-TSC2 complex is specific since other toxic amyloid proteins (Tau and Ab) didn’t show any effect   7.     Constitutive overexpression of TSC2 rescues motor deficits and PD pathology in a-synuclein fly model · a-synuclein: TSC2 double transgenic fly model showed rescue in climbing defect and a-synuclein pathology · Immunoblot analysis showed rescue in TH loss in a-synuclein: TSC2 fly · Confocal images showed rescue in dopamine neuron loss in the same fly · Immunoblot analysis of human post-mortem PD brains showed the a-synuclein pathology and loss of TSC2 protein ##