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.

In vitro translation (IVT) assay

Translation extract was prepared from human neuroblastoma (SHSY-5Y) or HEK293 cells grown in 175 cm² 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 cm² 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.

Translation assay design

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. 

Drosophila S35 assay

S35 assay in Drosophila 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).

Direct autoradiography of fly heads

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.              

SUnSET assay

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. 

Biotinylated a-Syn-PFF pulldown from mouse striatum

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 MgCl₂, 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.      

In situ labelling of a-Syn binding partners using A53T a-Syn-APEX2 assay in HEK293 cell

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, H₂O₂ was added at 1 mM final concentration for exactly 1 min at room temperature. Following H₂O₂ 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 H₂O₂ treatment. The cells were homogenized in 20 mM HEPES buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM MgCl₂, 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. 

Sample preparation for Mass Spectrometry

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.

Mass Spectrometry

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 × 10⁶ for MS and 5 × 10⁴ 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).

Polysome profiling: 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 MgCl₂, 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.    

Drosophila stocks and maintenance: 

Drosophila stocks were maintained according to the standard laboratory protocols. The fly stocks were maintained in standard Drosophila 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 w¹¹⁸ embryos (The BestGene, Inc). For experimental control, the relevant Syb-QF2/+ and Gal4/+ (heterozygous) flies were used as controls in the experiments.         

Drosophila climbing and life span assay: 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>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. 

Fly brain immunostaining: Fly brains were dissected and TH immunostained following previously described protocols with minor modifications (Wu and Luo, 2006). Adult Drosophila 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). 

Immunostaining of primary cortical neurons: 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.  

Rapamycin, S6K Inhibitor and Anisomycin treatment for Drosophila: 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.         

Tissue homogenization and Western blotting: 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.

Isolation of A53T a-Syn aggregates from transgenic mice brain tissues: 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 MgCl₂, 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.  

Transmission Electron Microscopy (TEM) of Drosophila brains: Drosophila 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.

Antibodies for SiMPull Assay

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. 

Cell Culture and Transfection for SiMPull Assay

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) CO₂. 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 Na₃VO₄. 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 mm² imaging area) (Aggarwal and Ha, 2014; Jain et al., 2011). 

Single-Molecule Imaging and Analysis

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 mm²) 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.

Rapamycin treatment of a-Syn PFF injected mice

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. 

Stereotactic Injection of α-Syn PFF

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.

Immunohistochemistry 

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% H₂O₂ 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). 

Behavior Tests

Grip Strength

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.

Pole Test

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.