Animals and antibodies
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed, and all procedures were approved by the Institutional Animal Care and Use Committees at the University of Washington or the VA Puget Sound Health Care Center. Eight week old male CD-1 mice were purchased from Charles River and kept on a 12/12-h light/dark cycle with ad libitum food and water. CD-1 mice are an outbred strain for use for genetics, toxicology, pharmacology, and aging research. In this study, a total of 166 mice were used in six different in vivo experiments: 14 for the clearance of EVs from blood, 30 for the uptake of RBC-EVs by brain, 30 for capillary depletion, 56 to test saturation using unlabeled RBC-EVs, 22 for the effect of WGA on permeability of RBC-EVs and 14 for immunofluorescence staining, respectively.
Mouse monoclonal antibodies against human Alix (2171S, Cell Signaling Technology, Danvers, USA) were used in Western blot (WB); mouse monoclonal antibodies against CD235a (MA5-12484, Thermo Fisher Scientific, Waltham, MA, USA) were used in WB; rabbit polyclonal antibodies against induced nitric oxide synthase (iNOS) (ab15323, Abcam, USA) were used in WB and IF; chicken polyclonal antibodies (ab139590, Abcam, USA) and rabbit polyclonal antibodies (019–19,741, Wako, Japan) against Iba-1 were used in IF; chicken polyclonal antibodies against GFAP (Thermo Fisher Scientific) were used in IF; mouse polyclonal antibodies against GAPDH (HC301, TRANS, China) were used in WB. Horseradish peroxidase (HRP)-conjugated secondary antibodies used in WB and Alexa Fluor 405, 488, 568 or 633 conjugated secondary antibodies used in IF were purchased from Thermo Fisher Scientific.
Isolation of RBC-EVs from PD and normal human erythrocytes
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Approval was granted by the Institutional Review Board at Peking University Health Science Center, and all subjects were fully informed and consented to the study. Human erythrocytes from healthy control subjects (ZenBio, Research Triangle Park, North Carolina), Parkinson’s disease (PD) patients, or age-matched controls (Additional file 1: Table S1), were first subjected to a RPMI-1640 culture medium containing 25 mM HEPES. Cells were maintained at 37 °C in a CO2 incubator for 48 h. RBC-EVs were then isolated from culture medium via serial centrifugations. Culture medium was collected and centrifuged at 1500×g for 10 min, followed by centrifugation at 3000×g for 15 min (two times) to remove the intact cells, cell debris and apoptotic blebs. EVs were concentrated from the supernatants by centrifugation at 150,000×g for 2 h, the resulting pellet washed with PBS, and collected by centrifugation at 200,000×g for 2 h. Sepharose CL-2B (Sigma, St Louis, MO, USA) was found to be suitable for separating EVs from small molecular proteins such as albumin [10, 57]. The EV sample was purified using CL-2B column. Each elution (0.5 mL) was collected to a 1.5 mL tube and the protein concentration of each fraction was measured based on UV absorbance at 280 nm by NanoDrop™ Lite Spectrophotometer (Thermo fisher Scientific). Two different observed peaks (Peak 1 and Peak 2) were further concentrated using Amicon® Ultra centrifugal filter devices (cut-off MW 100 kDa, Millipore Corporation, Billerica, MA, USA).
Nanoparticle tracking analysis (NTA)
The number of particles and size distribution in each peak were analyzed with NTA (NS300; Nanosight, Amesbury, UK). Each fraction was diluted 1000-fold in PBS in order to optimize the number of particles in each fraction. For each fraction, three videos (60 s each) were captured, and then all fractions were analyzed using the same threshold. Analysis was performed by NTA 3.1 software (Nanosight, Amesbury, UK).
Luminex immunoassays
RBC-EVs or RBC cell lysates were used to quantify α-syn with an established Luminex protocol as previously described [24]. Briefly, the Luminex assay was performed in 96 well MultiScreen Filter plates (Millipore) in the dark at room temperature. The plate was pre-wetted with a working solution (Na2HPO4 1.175 mg/mL, NaH2PO4 0.228 mg/mL NaCl 8.77 mg/mL, NaN3 0.05 mg/mL 0.2% Tween, 0.2% TrironX-100, pH 7.4). The capturing antibody (anti-α-syn antibody ASY-1, a gift from Dr. Poul H. Jensen, University of Aarhus, Denmark)-coupled beads were mixed thoroughly by vortexing (30 s) and brief sonication (30 s) and were immediately added to the pre-wetted plate. 5 μg of RBC-EVs or RBC cell lysates were added into each well (100 μl/well) and incubated for 3 h at 600 rpm on a plate shaker. After incubation, the sample solution was removed, and then the plate was washed three times by working solution. The detecting antibodies in assay diluent [0.1% Bovine Serum Albumin (BSA) in PBS] [1 ng/ml; biotinylated anti-human α-synuclein antibody (R&D systems, Minneapolis, MN, USA)] were added at 100 μl /well and incubated for 3 h on a plate shaker (600 rpm). After the detecting antibody solution was removed, the plate was washed three times with washing solution. A streptavidin-RPE (1 ng/ml; Prozyme, St. Louis, MO, USA) in assay diluent was added (100 μl /well) and incubated for 30 min on a plate shaker (600 rpm). The plate was then washed four times and 100 μl of washing solution was added to each well. After incubation for 5 min on a plate shaker, the plate was read on a LiquiChip Luminex 200TM Workstation (Qiagen, Valencia, CA, USA). A calibration curve consisting of a series of recombinant full-length human α-synuclein (rPeptide, Athens, GA, USA) standards diluted in assay diluent solution were run in parallel.
Radioactive labeling of RBC-EVs and albumin
RBC-EVs were radioactively labeled with Na125I or Na131I (Perkin Elmer, Waltham, MA, USA) by chloramine-T (Sigma Aldrich) as previously described [11]. Radioactively labeled RBC-EVs were purified on an Illustra NAP5 column (GE Healthcare, Buckinghamshire, UK). Albumin (Sigma Aldrich) was radioactively labeled with 99mTc. Radioactively labeled albumin (99mTc-Alb) was purified on a Sephadex G-10 column (Sigma Aldrich). The radiolabeling efficiency was determined by trichloroacetic acid precipitation method. 1 μl of sample was added into 500 μl of Lactated Ringer solution containing 1% BSA (LR-BSA) and mixed with an equal volume of 30% TCA, and then centrifuged at 5400 × g for 10 min. The levels of radioactivity in the pellet and the supernatant were separately measured in a gamma counter for 3 min (PerkinElmer). The radiolabeling efficiency was calculated as the percentage of pellet radioactivity to total (pellet plus supernatant) radioactivity. The radiolabeling efficiency for RBC-EVs and Albumin was about 80 and 90%, respectively, across different preparations.
To assess the stability of radiolabeled RBC-EVs, samples loaded into mouse serum were incubated for 0, 15 and 60 min. These samples were mixed with 30% TCA and then centrifuged at 5400 × g for 10 min. The levels of radioactivity in the pellet and the supernatant were separately measured in a gamma counter for 3 min (PerkinElmer). The radiolabeling efficiency was calculated as the percentage of pellet radioactivity to total radioactivity. Then, the stability or radiolabeled RBC-EVs were calculated as the percentage of radiolabeling efficiency at 15 min or 60 min to radiolabeling efficiency at 0 min. The stability of radiolabeled RBC-EVs at 15 min and 60 min was about 98 and 90%, respectively.
In vivo evaluation of RBC-EV permeability
Mice were weighed and given an intraperitoneal (IP) injection of LPS (3 mg/kg dissolved in sterile normal saline) from Salmonella typhimurium (Sigma Aldrich) or control saline three times (at 0, 6 and 24 h). Mice underwent the experiment 28 h after the first injection of LPS or control saline. Multiple-time regression analysis [8, 34] was used to calculate blood-to-tissue (brain, liver, spleen and kidney) uptake of 125I-EVs and Tc99m-Alb. In vivo evaluation of RBC-EVs was carried out according to the methods described previously [4, 6]. CD-1 mice were anesthetized with 40% urethane and a 200 μL injection of Lactated Ringer solution containing 1% BSA (LR-BSA), 300,000 cpm of 125I-RBC-EVs and Tc99m-Alb each was injected into the jugular vein (IV injection). Between 1 to 15 min after the IV injection, the arterial blood was collected from the carotid artery. The collected whole blood was centrifuged at 3000×g for 15 min, and 50 μL of serum transferred into a new glass tube. Levels of radioactivity in 50 μL of serum were measured in a gamma counter for 3 min. The tissue was removed after blood collection at each time-point and weighed. The levels of radioactivity in the tissue were determined in a gamma counter. The results were expressed as tissue (brain, liver, kidney and spleen)/serum ratios in units of μL/g and plotted against exposure time (expt) in units of minutes. The slope of the line for the linear portion of the relation between tissue/serum ratios and expt measures the unidirectional influx rate (Ki) in units of μL/g-min and the intercept of that line measures the initial volume of distribution in tissue (Vi) in units of μL/g. The half-time clearance and initial volume of distribution in the body (Vd) were calculated according to the method as previously described [5].
For self-inhibition studies, 300,000 cpm of 125I-RBC-EVs with or without 1 or 30 μg of unlabeled RBC-EVs was injected via jugular vein and mice were decapitated 15 min after IV injection and brain and serum collected.
To examine the effect of wheat germ agglutinin (WGA) (Sigma Aldrich) on the permeability of RBC-EVs, 131I-RBC-EVs with or without 10 μg per mouse of WGA was injected via jugular vein and mice were decapitated 15 min after IV injection and collect brain and serum.
Capillary depletion was carried out according to the methods described previously [6]. Mice were decapitated 15 min after IV injection and brain and serum collected. The collected mouse brain was weighed and homogenized with 0.8 mL of physiological buffer (10 mM HEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, NaH2PO4 and 10 mM d-glucose adjusted pH 7.4) at 4 °C. 1.6 mL of 26% dextran solution in physiological buffer added to the brain homogenate. The pellet containing the brain capillary was carefully separated from the supernatant containing the brain parenchyma after centrifuge at 5400×g for 15 min at 4 °C. The levels of radioactivity in capillary or brain parenchyma were determined in a gamma counter. The results were expressed as capillary/serum ratios and brain parenchyma/serum in units of μL/g.
Culture of primary BMECs
Primary BMECs were isolated from 8-week-old CD-1 mice as previously described [2]. Briefly, isolated BMECs were cultured using BMEC medium, which contained Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 20% plasma-derived fetal bovine serum (Animal Technologies), 1% GlutaMAX (Thermo fisher Scientific), basic fibroblast growth factor (bFGF, 1 ng/ml; Roche Life Sciences), heparin (100 μg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), selenium (5 ng/ml) (Thermo fisher Scientific), and gentamicin (50 μg/ml) (Sigma Aldrich). Puromycin (4 μg/ml) (Sigma Aldrich) for the first 48 h after plating. Cells were maintained at 37 °C in a CO2 incubator for 24 h. Culture medium was changed at 24 h after plating to remove non-adhering cells, RBCs, and debris. At 48 h after plating, the medium was changed to BMEC medium in the absence of puromycin. The purified primary BMECs were used to construct in vitro models when reached 80% confluency.
Construction of in vitro BBB models
In vitro BBB models were constructed as previously described [2]. Briefly, BMECs (4 × 104 cells/ well) were seeded onto fibronectin and collagen type IV pre-coated (0.1 mg/ml, each) Transwell inserts (0.33 cm2, 0.4-μm pore size, Corning). For the negative controls, no BMECs were seeded to the collagen type IV plus fibronectin pre-coated Transwell insert. BMEC medium containing additional hydrocortisone (500 nM) was used to reinforce tight junctions of in vitro BBB models [22]. An EVOM volt ohmmeter connecting with a STX-2 electrode (World Precision Instruments; Sarasota, FL) was used to analyze of transendothelial electrical resistance (TEER, in ohms per square centimeter) prior to treatment. The TEER value of cell-free Transwell inserts (negative controls) was subtracted from TEER value of BMECs containing Transwell inserts. BMEC monolayers were established within 7 days after seeding in Transwell inserts.
Transendothelial permeability assay
A permeability assay for RBC-EVs was conducted according to the method described previously [2]. A 600 μL volume of DMEM/F12 containing 1% BSA (an assay buffer) was added to the wells of a new 24-well plate, and the inserts were put on in these wells. 125I-RBC-EVs (3 × 106 cpm/mL) in the assay buffer (100 μL) were added to the luminal chamber (loading chamber). Samples (400 uL) were mixed with pipetting and collected from the abluminal chamber (collecting chamber) to a new glass tube at 15, 30, 45, 60 min, and added an equal volume of fresh DMEM/F12 containing 1% BSA to the abluminal chamber after sample collection at each time point. 400 uL of 30% trichloroacetic acid (TCA) added to all samples and centrifuged at 5400×g for 15 min at 4 °C. After removal of the supernatant, the radioactivity in the TCA precipitate was determined in a gamma counter. The permeability coefficient of TCA-precipitable 125I-RBC-EVs (cm/min) was calculated from the clearance of TCA-precipitable 125I-RBC-EVs (μL) according to the method described previously [2]. Clearance was expressed as microliters of radioactive tracer diffusing from the luminal chamber to the abluminal chamber and was calculated from the initial level of radioactivity in the luminal chamber (loading chamber) and final level of radioactivity in the abluminal chamber (collecting chamber):
$$ \mathrm{Clearance}\ \left(\upmu \mathrm{L}\right)={\left[\mathrm{C}\right]}_{\mathrm{C}}\times {V}_{\mathrm{C}}/{\left[\mathrm{C}\right]}_{\mathrm{L}}, $$
where [C]L is the initial radioactivity in a microliter of loading chamber (in cpm/μL), [C]C is the radioactivity in a microliter of collecting chamber (in cpm/μL), and V
C is the volume of collecting chamber (in μL). During a 60-min period of the experiment, the clearance volume increased linearly with time. The volume cleared was plotted versus time, and the slope was estimated by linear regression analysis. The slope of clearance curves for the BMEC monolayer plus Transwell® membrane was denoted by PS
app, where PS is the permeability × surface area product (in μL/min). The slope of the clearance curve with a Transwell® membrane without BMECs was denoted by PS
membrane. The real PS value for the BMEC monolayer (PS
e) was calculated from 1/PS
app = 1/PS
membrane + 1/PS
e. The PS
e values were divided by the surface area of the Transwell® inserts (0.33 cm2) to generate the endothelial permeability coefficient (P
e, in cm/min).
N9 Microglia culture and stimulation by RBC-derived EVs
Mouse N9 cell line (N9 microglia), a retroviral-immortalized cell line (kindly provided by Department of Pathology, Peking University Health Science Centers), were plated in 6-well plate at a density of 5 × 105 cells/well with F12/DMEM containing 10% FBS and incubated at 37 °C in a CO2 incubator overnight, then the medium was replaced with F12/DMEM free of FBS. Simultaneously, 100 μl dissolved RBC-EVs derived from PD patients or control subjects was added and cells were stimulated for 30 min.
Western blot analysis
RBC-EVs and RBC cell lysates were mixed with an equal volume of 2 × Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA). Equivalent amounts of protein from each sample were electrophoretically separated on 4-15% Criterion™ TGX Stain-Free™ Protein Gel (Bio-Rad Laboratories) and then transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories). Membranes were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan). The membrane was probed with corresponding primary antibodies overnight at 4 °C. After washing, membranes were then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The immunoreactive bands were visualized using ECL reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Proteins were extracted from N9 microglia by cell lysis buffer (RIPA cell lysis buffer, PPLYGEN, C1053) and the protein concentration was determined by BCA Protein Assay Kit according to the manufacturer’s instruction. The sample was boiled in 5 × SDS loading buffer for 5 min and loaded onto a 10% SDS-polyacrylamide gel. Following electrophoresis, the proteins were transferred to a PVDF membrane (Merck Millipore). The membranes were blocked for 1 h at RT in 5% BSA (amresco) in TBST buffer (Axygen). Immunoblotting was performed by incubating the membrane in 5% BSA-TBST with corresponding primary antibodies overnight at 4 °C. The membranes were washed three times with TBST, followed by incubation with appropriate HRP-conjugated secondary antibodies, positive bands were detected using enhanced chemiluminescence reagents (Millipore) and quantified using densitometric analyses by Photoshop.
Immunofluorescence staining
The RBC-EVs were labeled with Vybrant™ DiI cell-labeling solution in accordance with the manufacturer’s instructions (Thermo Fisher Scientific). Briefly, RBC-EVs were resuspended in 500 μL of PBS. DiI solution (10−3 μmol) was then added into resuspended RBC-EVs in PBS, followed by 20 min incubation at room temperature. To remove the excess DiI dye, DiI-labeled RBC-EVs were further concentrated using Amicon® Ultra centrifugal filter devices (cut-off MW 100 kDa), and then re-suspended with PBS three times and finally resuspended in 50 μL of PBS.
Mice were anesthetized with i.p. injection of 0.15 ml of 40% urethane (Sigma Aldrich) [52]. DiI-labeled RBC-EVs dissolved in PBS (50 μg per mice) or control PBS were intravenously injected via the jugular vein to LPS (3 mg/kg of mice) or control saline pre-injected (i.p. injection) mice. After 3 h, the mice were perfused. The descending aorta was clamped and both jugular veins were severed. A 23-gauge butterfly needle was injected into the left ventricle of the heart and then PBS infused at a rate of 2 ml/min for 5 min, followed by perfusion of 4% paraformaldehyde solution at a rate of 2 ml/min for 5 min. Brains were removed and immersed in 4% paraformaldehyde solution at 4 °C overnight. After dehydration in 20% sucrose, sagittal brain sections (20 μm) were prepared with a sliding microtome (Leica, Wetzlar, Germany).
Brain slices were washed with PBS and treated with blocking solution (1% BSA, 0.4% Triton X-100 and 4% goat serum in PBS) for 2 h. Next, brain slices were incubated overnight at 4 °C with primary antibodies diluted in blocking solution. Brain slices were then washed with washing buffer (0.1% Tween in PBS) and incubated with corresponding secondary antibodies diluted in PBS containing 0.3% of Triton X-100 for 2 h. After washing with PBS or washing buffer and PBS (when indicated), brain slides were embedded in Vectashield medium or Vectashield medium with DAPI (when indicated).
Immunofluorescence images were captured at room temperature using a Nikon Eclipse Ti (Nikon Instruments Inc., Melville, NY, USA) instrument under 20× or 40× magnification. Z-series images were acquired from randomly selected presence or absence DiI-labeled RBC-EVs fields, following with deconvolution. Interbrain including thalamus and hypothalamus were analyzed. Midbrain including Substantia nigra was analyzed. Isocortex of cerebral cortex was analyzed. Note that DiI signal in the brain may signify that EV membrane is present but cannot inform on the number of EVs or their cargo.
Statistical analysis
The results are shown as means ± S.E.M. The statistical significance of differences between two groups was assessed by the Student’s t-test, one-way or two-way analysis of variance (ANOVA), followed by Tukey-Kramer’s post-hoc test for multiple comparisons and Kruskal-Wallis test, followed by Dunn’s post-hoc test for multiple comparisons (Graph Pad Prism 5.0 (GraphPad, San Diego, CA)). *p < 0.05; **p < 0.01; ***p < 0.001.