Brain tissues from longitudinally followed, autopsy-confirmed subjects with sporadic PD (n = 17) and age- and postmortem delay-matched clinical and neuropathological controls (n = 10) (Table 1) were obtained from the Sydney Brain Bank following study approval and University of New South Wales human ethics approval (#HC14046). All PD cases met the UK Brain Bank Clinical Criteria for diagnosis and had no other neurodegenerative conditions. The cases analyzed have been previously used to assess lysosomal proteins, lipids, and α-synuclein levels in the affected brain regions .
Protein extraction from frozen brain tissue
Tris-buffered saline (TBS) and SDS-soluble proteins were serially extracted from 250 mg of fresh-frozen brain tissue from the anterior cingulate cortex and occipital cortex as previously described . Briefly, tissue was mechanically homogenized in ten volumes of TBS homogenization buffer (50 mM Tris, 125 mM NaCl, pH 7.4, 5 mM EDTA, 0.02% sodium azide) containing protease inhibitor cocktail (Roche), followed by centrifugation at 100,000×g for 2 h at 4 °C, with supernatant collected as the TBS-soluble fraction containing cytosolic proteins. The pellet was resuspended in SDS solubilization buffer (TBS homogenization buffer containing 5% SDS), sonicated (2 × 10 s bursts) and centrifuged at 100,000×g for 30 min at 25 °C, with supernatant collected as the SDS-soluble fraction containing membrane-associated proteins. Protein concentration of all fractions was measured using a bicinchoninic acid assay (Pierce BCA Protein Assay Kit, Thermo Scientific), according to the manufacturer’s instructions. Samples were stored at −80 °C until use.
Up to 30 μg of protein lysate was heated with sample buffer (2% SDS, 20% glycerol, 2.5% bromophenol blue, 12.5 mM Tris–HCl, pH 6.8, 5% 2-mercaptoethanol) and separated by reducing SDS-PAGE before transfer to nitrocellulose membrane (BioRad). Membranes were fixed in 0.4% paraformaldehyde followed by blocking in 5% skim milk dissolved in 1 × TBS-T (0.87% NaCl, 0.01 M Tris, pH 7.4, with 0.1% Tween20). Membranes were then cut into strips based on molecular weight markers and incubated overnight in primary antibodies prior to protein detection using either horseradish peroxidase-conjugated secondary antibodies (Biorad) with enhanced chemiluminescence (Amersham ECL Plus Western Blot Detection System, GE Healthcare) or Alexa Fluor-conjugated secondary antibodies (Life Technologies). Primary antibodies for immunoblotting were rabbit monoclonal TLR2 (Abcam, 1:500 dilution), rabbit monoclonal TLR1 (Abcam 1:1000 dilution) mouse monoclonal NeuN (Millipore, 1:250 dilution), mouse monoclonal α-synuclein (BD Biosciences, 1:3000 dilution), phospho-α-synuclein (P-129, Abcam, 1:1000 dilution), MYD88, phospho-NFκBp105 (Ser933), phospho-p38 MAPK (Thr180/Tyr182), p62/SQSTM1 and Beclin1 (all Cell Signaling Technology, 1:1000 dilution), HLA-DR (Dako, 1:5000 dilution), LC-3 (Novus, 1:100 dilution) and goat polyclonal Iba1 (Abcam, 1:1000 dilution) with β-actin (Abcam, 1:50,000 dilution) used as a protein loading control. For immunoblotting of Ser-129 phosphorylated α-synuclein, membranes were fixed with 4% PFA and 0.1% glutaraldehyde prior to blocking as described . A Biorad Chemidoc MP system was used to capture images and the relative levels of each protein of interest were analyzed using Image J software (US National Institutes of Health). The intensity of each protein band was quantified and expressed as arbitrary units standardized to β-actin.
Immunofluorescent labeling using human tissue
Immunohistochemistry was performed with 10 μm formalin-fixed paraffin-embedded tissue sections from the substantia nigra and anterior cingulate cortex of 50% of the cases used for immunoblotting (9 PD and 5 matched control cases randomly chosen). Briefly, sections were deparaffinized in xylene and rehydrated in graded ethanols. Antigen retrieval was performed using 99% formic acid for α-synuclein immunohistochemistry, and boiling with citrate buffer (pH 6.0) for all other antibodies, after which sections were cooled to room temperature. Throughout the protocol, all washes were three times with 0.1 M Tris buffer. For immunofluorescent double labeling, sections were blocked with 10% horse serum in TBS buffer containing 0.5% triton-X-100 and incubated overnight with primary antibody pairs: anti-α-synuclein (1:200 dilution) and Abcam anti-TLR2 (1:100 dilution) or anti-Iba1 (1:400 dilution) and anti-TLR2 or anti-NeuN (1:200 dilution) and anti-TLR2 or anti-tyrosine hydroxylase (Sigma, 1:400 dilution) and anti-TLR2. Alexa fluor-labeled secondary antibodies (Life Technologies) diluted 1:500 in 0.1 M Tris buffer were then used. Auto-fluorescence was reduced by incubating in autofluorescence eliminator buffer (Millipore) following the manufacturer’s instructions. Slides were then stained with DAPI (Sigma) and coverslipped using Vectashield mounting medium (Vector Laboratories). Images were visualized using a Nikon Confocal Microscope ECLIPSE 90i and captured using EZ C3.80 software. At least ten images from each slide of the anterior cingulate cortex were used to quantify (1) Iba1-immunoreactive microglia expressing TLR2 immunoreactivity, (2) all DAPI-labeled neuronal nuclei expressing TLR2 immunoreactivity, and (3) α-synuclein-immunopositive Lewy bodies colocalizing TLR2 immunoreactivity. Neurons were identified by their nucleus being wider and fainter with a bright nucleolus compared with the nuclei of surrounding glial cells in the DAPI stain. Quantitation was performed using 590 microglia (average of 42 ± 12 per case), 1483 neurons (average of 106 ± 70 per case), and 117 Lewy bodies (average 23 ± 6 per case that contained Lewy bodies in the cingulate cortex). To assess interrater variation in the cell counts, 16 ± 6 images from each combination of double labeling were used. There was <5% variation in counts made by two researchers, with a correlation between counts of 0.96.
Immunoperoxidase staining using human tissue
For immunoperoxidase staining, sections were incubated with either rabbit monoclonal anti-TLR2 from abcam, or goat polyclonal anti-TLR2 antibody from R&D systems, as outlined in the supplementary methods. Absorption controls were also performed for both TLR2 antibodies to demonstrate the antibody binding specificity. The antigen to antibody mixture was made at a working dilution of 10:1 (molar ratio) and was pre-incubated overnight at 4 °C. The pre-absorbed antibodies were then be incubated with tissue sections in place of the primary antibodies with the same immunoperoxidase staining protocols for the respective TLR2 antibody.
Tissue culture experiments with SHSY5Y cells
Human neuroblastoma SHSY5Y cells were cultured in Dulbecco’s modified Eagle medium/Hams F12 supplemented with 10% low endotoxin fetal bovine serum and 1 × penicillin/streptomycin solution (all from Gibco, Life Technologies). Cells were differentiated for 7 days in the same media except with 1% fetal bovine serum and 10 μM retinoic acid (Sigma). Cells were treated with TLR agonists (all purchased from Invivogen) dissolved in endotoxin-free water and/or small molecule inhibitors (Calbiochem, Cayman Chemicals or Sigma) dissolved in DMSO at indicated concentrations for indicated time points. For cytokine ELISA assays, tissue culture media were removed and snap-frozen in liquid nitrogen and stored at −80 °C until analysis. For gene expression analysis, cells were lysed according to the manufacturer’s instructions (ReliaPrep, miniprep systems, Promega) and stored at −80 °C. For immunoblot analysis, cells were lysed either in buffer containing 50 mM Tris HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.27 M sucrose, 1 mM benzamadine, 1 mM phenylmethylsulfonyl fluoride and 1% (v/v) Triton X-100, or straight into 1 × LDS sample buffer (Life Technologies). For the former, lysates were clarified by centrifugation at 13,000×g for 20 min and protein concentrations were measured by bicinchoninic acid assay as mentioned above. Reactive oxygen species were measured using a 2′,7′-dichlorofluorescin diacetate (DCFDA) assay (Abcam) following the manufacturer’s instructions. Briefly, 25,000 cells were seeded into 96 well clear bottom microplates and differentiated for 7 d before treatment for 4 h with 1 μg/ml PAM3CSK4. The included 55 mM tert butyl hydrogen peroxide was used as a positive control. Median fluorescence intensity was measured using a fluorescence microplate reader (Polarstar Omega, BMG). Apoptosis and cell viability were assessed by Annexin V/propidium iodide staining following the manufacturer’s instructions (Annexin V-FITC kit from Miltenyi Biotech). Median fluorescence intensity was measured using a FACS Canto cell analyzer (BD Biosciences) with at least 20,000 events captured. Flow cytometry data were acquired with FACS Diva software (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Coverslips with adherent cells were washed in 1 × PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Coverslips were washed again in 1 × PBS after fixing and permeabilized with 0.3% Triton X-100 for 15 min. Cells were then blocked in 3% BSA for 1 h and incubated overnight at 4 °C in primary antibody at a dilution of 1:200 in 3% BSA. After incubation, cells were washed 3 × 5 min in 1 × PBS and incubated with Alexa Fluor secondary antibodies (Abcam, 1:300 dilution) in 3% BSA for 1 h at room temperature in the dark. Cells were washed again for 3 × 5 min in 1 × PBS with DAPI added to the last wash at a concentration of 1:10,000. Coverslips were mounted face down using fluorescent mounting medium (Dako) and allowed to dry in the dark for 1 h before visualizing. Images were captured on a confocal microscope (Nikon) using NIS elements AR software. Images were obtained in each channel at 40× magnification for analysis. Intensity settings were kept constant for all images, which were analyzed using Image J 1.49v (NIH). The threshold tool on Image J was used to highlight all stained areas within the cells and the intensity of each highlighted particle was obtained using the analyze particles tool. Once selected, the threshold was kept constant for all images. The cell counter plugin was used to count the number of DAPI positive cells in the image and a total of 400–500 cells were counted for each condition. Cells falling on the edges of the image were excluded from the analysis.
Induced pluripotent stem cells
Human fibroblasts from a neurologically normal subject were obtained from the NINDS genetic repository at Coriell (#ND38530). Briefly, primary fibroblasts at passage 4 were reprogrammed to induced pluripotent stem cells (IPSCs) using the EPi5 reprogramming kit (Life Technologies) as per the manufacturer’s instructions. Electroporation of fibroblasts was performed with the Neon transfection system (Life technologies) and colonies with the morphology of IPSCs were selected ~30 days post-electroporation. IPSCs were maintained on geltrex (Life Technologies)-coated dishes and stained for both pluripotency (Pluripotent stem cell marker immunocytochemistry kit, Life Technologies) and tri-lineage differentiation potential (Human pluripotent stem cell functional identification kit, R&D Systems) as per the manufacturer’s instructions. Neural progenitor cells were then derived from IPSCs using the PSC neural induction kit (Life Technologies) as per the manufacturer’s instructions. Differentiation of neural progenitor cells was confirmed by immunocytochemistry for nestin and paired box protein 6 (PAX6). Only cell lines confirmed >95% positive for both markers were used. Neural stem cells were maintained in neural expansion medium comprising 50% RPMI media (Life Technologies) and 50% neurobasal medium (Life Technologies), supplemented with neural induction supplement at 1/50 dilution and penicillin–streptomycin at 1/100 dilution. Media were changed every second day and neural stem cells were passaged in the presence of ROCK inhibitor Y27632 (Cayman Chemicals) at a final concentration of 5 µM. For all experiments described, neural stem cells were used at a passage <8. Images were captured on a confocal microscope (Nikon) using NIS Elements software (Nikon). For the directed differentiation to neurons, neural progenitor cells were plated at a density of 2.5 × 104 cells per ml on coverslips coated with poly-l-ornithine (Sigma) and laminin (10 μg/ml, Life Technologies). Cells were cultured in neurobasal media containing 2% B27 supplement and 2 mM glutamax (all Life Technologies). For the first 3 days, media were changed daily. After this, half of the media were replaced every second day and it was supplemented with 1 μg/ml PAM3CSK4. In total, cells were differentiated for 10 days with 7 days of PAM3CSK4 treatment. At this stage, neurons no longer expressed nestin, but now expressed the neuronal markers MAP2 and TUJ1.
qRT-PCR and ELISA
RNA was extracted using the ReliaPrep miniprep system (Promega) following the manufacturer’s instructions. RNA was reverse transcribed to cDNA using the iScript cDNA synthesis kit (Biorad). qRT-PCR was performed on a realplex thermocycler (Eppendorf) using sybr green-based chemistry (Biorad). All primer sequences were obtained from qPrimer depot  and synthesized by Sigma. Gene expression was quantified using the comparative Ct method  with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. Melt-curve analysis was performed to ensure that a single product was amplified. Cytokine ELISA assays were performed using Bioplex xMAP assays (Biorad) following the manufacturer’s protocol with plates read on a Magpix instrument (Luminex).
All statistical analyses for human tissue were performed using SPSS Statistics software (IBM, Chicago, IL, USA) and statistical significance set at p < 0.05. Group demographic statistics have been previously reported  with cases well matched for age, sex and postmortem delay (Table 1), and univariate statistical analysis covarying for neuron loss (NeuN levels) shows that the relative levels of SDS-soluble α-synuclein protein increased in PD . There was also a positive Spearman’s correlation between the levels of SDS-soluble α-synuclein protein and Braak Lewy body stage of Parkinson’s disease for these cases .
To determine TLR2 levels and location changes in PD, two multivariate analyses were performed, the first on the quantitated immunoblot data to identify any changes in TLR2 levels in association with α-synuclein levels (covarying for age, postmortem delay and levels of NeuN, Iba1 and HLA-DR), and the second on the quantitated immunohistochemistry data to identify any changes in the numbers of TLR2-immunopositive microglia, neurons and Lewy bodies (covarying for age, postmortem delay and Braak neuritic stage). Non-parametric Spearman’s correlations were used to identify related variables.
Statistical analysis of data obtained from tissue culture experiments was performed using Graphpad software (Prism). Students t test was used for comparisons between two groups and one-way ANOVA with Tukey’s post hoc test was used for comparisons between multiple groups. Significance was accepted at p < 0.05 and data are presented as mean ± standard error of the mean.