Sprague dawley rats were bred and reared in house from stock animals obtained from Charles River UK Ltd (Margate, Kent, UK). Human embryonic kidney 293a cells (HEK293a; an adherent clone of HEK293 cells) were purchased from Quantum Biotechnologies (Canada). N9 microglial cells were a kind gift from Dr Paola Ricciardi Castagnoli (CNR Cellular and Molecular Pharmacology Centre, Milan, Italy). The TOPflash firefly-luciferase reporter construct was purchased from Millipore (Watford, UK). Fetal calf serum, Dulbeccos Modified Eagle Medium (DMEM), Neurobasal medium, B27 supplement and the rat cytokine 10-plex immuno-fluorescent assay panels (LRC0002) were obtained from Invitrogen (Paisley, UK). Papain digestion kits were from Lorne Labs (Berkshire, UK). Recombinant carrier-free mouse Wnt3a, Wnt5a, Wnt5b, dickkopf 1 (DKK1), reelin and sonic hedgehog were from R&D Systems (Abingdon, UK). α-synuclein was from Enzo Life Sciences (Exeter, UK). β-amyloid (1-42) was from Dr David Teplow (UCLA, California, USA). Recombinant insulin, percoll, lipopolysaccharide (LPS), Hoechst 33342, fluoroscein diacetate, mouse anti-β actin and lithium were supplied by Sigma (Dorset, UK). FuGENE6 was from Roche (Burgess Hill, UK). Rabbit anti-Wnt3a and mouse anti-Alix were from Cell Signaling Technology (Hertfordshire, UK). Mouse anti-Rab5 was from Abcam (Cambridge, UK). Goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were from Autogen Bioclear (Wiltshire, UK). Anti-mouse and anti-rabbit IgG conjugated to 10 nm colloidal gold was from British Biocell (Cardiff UK). Enhanced chemiluminescence reagents were from GE Healthcare (Buckinghamshire, UK). C18 PepMap columns were from Dionex (Surrey, UK). Amicon centrifugal devices (3kDa) were from Fisher Scientific (Loughborough, UK). Immobiline Drystrips (pH 3-11) were from GE Healthcare (Buckinghamshire, UK). The ‘Cell Titre 96 AQueous One Solution Proliferation Assay kit’ (MTT assay) and the CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (measures LDH’) and Dual-Glo reagents were obtained from Promega (Southampton, UK). Pioloform and EM grids come from Agar Scientific (Stansted, UK).
Aggregation of β-amyloid and α-synuclein
β-amyloid 1-42 (Aβ) peptide was resuspended in 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP: 1 mM) and homogenized using a Teflon plugged (250 μl) Hamilton syringe. HFIP was removed by evaporation in a SpeedVac and Aβ was resuspended at a concentration of 5 mM in dimethylsulfoxide (DMSO) and sonicated for 10 minutes. For oligomer formation, Aβ (1-42) was diluted to 0.4 mM in phosphate buffered saline (PBS) plus 0.2% sodium dodecyl sulfate and incubated at 37°C for 24 hours then diluted to 100 μM with PBS and incubated at 37°C for 18 hours before use
. α-synuclein was resuspended in PBS at a concentration of 68 μM and incubated for 7 days at 37°C
Primary microglial cell culture and treatment
Primary cultures of microglia were prepared from the brains of P6 Sprague Dawley rats using percoll density gradients as previously described
. In line with the Animals Scientific Procedures Act (1986) UK, the animal procedures undertaken to produce microglial cultures fall under Schedule 1 and were approved by the local ethics committee at the Institute of Psychiatry. All animals used in this study were treated in accordance with standard guidelines for laboratory animal care. Microglia were maintained at 37°C in a humidified atmosphere of 5% CO2 in air and were used after 1 day in vitro. Using this approach microglial cultures were > 98% pure as demonstrated by CD11b reactivity and morphological criteria. Primary microglia were treated with Wnt3a (10 nM), or other agents as stipulated in the text, in serum free medium (SFM) (Dulbeccos modified eagles medium: DMEM) for 8 or 24 hours. Control cultures were incubated in SFM for the corresponding period.
Culture and treatment of primary cortical neurons
Primary cortical neuronal cultures were prepared from Sprague Dawley rat embryos on embryonic day 18 using papain dissociation as previously described
. In line with the Animals Scientific Procedures Act (1986) UK; pregnant rats were killed by a Schedule 1 method prior to the Schedule 1 killing of embryos. These animal procedures were approved by the local ethics committee at the Institute of Psychiatry. Neurons were cultured in neurobasal medium supplemented with B27 for 7 days at 37°C in a humidified atmosphere of 5% CO2 in air. Neuronal cultures were > 98% pure as demonstrated by morphological criteria and staining for glial fibrillary acidic protein (GFAP) and CD11b; markers of astrocytes and microglia respectively. Following 7 days in vitro, primary neurons were treated with Wnt3a (10 nM) in neurobasal medium (minus supplements) for 8 hours. Control cultures were incubated in neurobasal medium (minus supplements) for the corresponding period.
Growth and maintenance of cell lines
N9 microglia and HEK293a cells were maintained in DMEM supplemented with glutamine (2 mM), foetal bovine serum (10%), penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified atmosphere of 5% CO2 in air. N9 cells were plated on 13 mm glass coverslips (50000 cells/coverslip) and HEK293a cells were plated in 48 well plates (40000 cells/well) the day before use.
Luciferase TOPflash assays
HEK293a cells were transfected with TOPflash (400 ng) using FuGENE6 (1 μl in a volume of 35 μl SFM). Wnt3a (10 nM), DKK1 (30 nM), lithium (5 μM) or Wnt3a (10 nM) plus DKK1 (30 nM) were added to the culture media 18 hours after transfection. After a further 6 hours at 37°C the medium was removed from the cells and the firefly activity was measured using Dual-Glo reagents in a Wallac Trilux 1450 Luminometer. Data for each set of four replica transfections was averaged, the control in each set normalized to 1 and data presented as fold increases over control. All experiments were performed in triplicate. To statistically compare treatments a one-way analysis of variance (ANOVA) was used in combination with a Tukey post-test. P values < 0.05 were considered statistically significant.
Cell culture supernatant was typically harvested from 2.5 million cells (primary microglia or primary cortical neurones), unless otherwise stated, after 8 hours in culture and was centrifuged at 10000xg for 10 minutes to clear large debris. The supernatant was then concentrated using Amicon 3kDa centrifugal devices according to the manufacturer’s instructions. The concentrated supernatant was subjected to centrifugation at 100000xg for 1 hour to isolate exosomes. In some experiments a further 200000xg centrifugation step was performed for 16 hours to extract smaller vesicular particles.
Western blotting and coomassie gels
Cells, exosomes or other vesicular particles were harvested in lysis buffer (10 μl: 20 mM Tris-acetate, 1 mM EDTA, 1 mM EGTA, 10 mM sodium β-glycerophosphate, 1 mM sodium orthovanadate, 5% glycerol, 1% Triton X-100, 0.27 M sucrose, 1 mM benzamidine, 4 μg/ml leupeptin, 0.1% β-mercaptoethanol, pH 7.4) then incubated on ice for 10 minutes. Protein concentrations were determined according to the method of Bradford and confirmed using a NanoDrop2000 at 280 nm. Proteins (exosomal: 500 ng/well, cellular: 20 μg/well) were resolved by SDS-PAGE (10%) then stained with coomassie blue for 2 hours or transferred on to Immobilon P-PVDF membranes for subsequent western blotting. Membranes were probed according to standard protocols. Briefly, membranes were incubated with either mouse anti-β-actin (1:1000), rabbit anti-Wnt3a (1:1000) or mouse anti-Alix (1:1000) for 2 hours at room temperature. Membranes were then incubated for 2 hours at room temperature with goat anti-mouse IgG-HRP (1:1000) or goat anti-rabbit IgG-HRP (1:1000). Proteins of interest were detected using enhanced chemiluminescence reagents. In some instances to ensure equal protein loading membranes were reprobed using mouse anti-β-actin (1:1000). Western blots were performed in triplicate from 3 independent experiments; therefore the blots shown are representative of a single experiment.
Two dimensional gel electrophoresis
Exosomal proteins were isoelectro-focussed on immobilized pH (3-11), dry, gradient strips using an Ettan IPGphor 3 system. Proteins were subsequently separated by SDS-PAGE (10%) using an Ettan DALTtwelve large vertical two-dimensional gel tank in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). Gels were fixed in strong fix (40% ethanol, 10% glacial acetic acid) for 1 hour and then in weak fix (5% ethanol, 5% glacial acetic acid) for 16 hours before proteins were visualized using silver staining. Two-dimensional gels were performed in triplicate from 3 independent experiments; therefore the images shown are representative of a single experiment.
Liquid chromatography-tandem mass spectrometry (LC/MS/MS)
Exosomal proteins were separated by SDS-PAGE (10%) and visualised with coomassie blue. In-gel reduction, alkylation and digestion (with trypsin) was subsequently performed. Peptides were extracted from the gel by a series of acetonitrile and aqueous washes and lyophilised. Each sample was then resuspended in ammonium bicarbonate (50 mM) and analysed by LC/MS/MS. Chromatographic separations were performed using an Ultimate LC system (Dionex, UK). Peptides were resolved by reversed phase chromatography on a 75 μm C18 PepMap column using a three step linear gradient of acetonitrile in formic acid (0.05%). The gradient was delivered to elute the peptides at a flow rate of 200 nL/minute over 60 minutes. The eluate was ionised by electrospray ionisation using a Z-spray source fitted to a QTof-micro operating under MassLynx v4.0. The instrument was run in automated data-dependent switching mode, selecting precursor ions based on their intensity for sequencing by collision-induced fragmentation. The MS/MS analyses were conducted using collision energy profiles that were chosen based on the mass-to-charge ratio (m/z) and the charge state of the peptide. Database searching was carried out using MASCOT 2.2. All peptide assignments were manually validated.
Assessment of cytokine secretion using a rat cytokine 10-plex immuno-fluorescent assay
Microglia (1 million cells per treatment) were treated with either Wnt3a (10 nM) or LPS (10 ng/ml) for 8 or 24 hours in SFM at 37°C. Control cultures were left in SFM for the corresponding time points. Cell culture supernatants were then collected and analysed simultaneously for the presence of cytokines (GM-CSF, IFN-γ, IL1α. IL1β, IL2, IL4, IL6, IL10, IL12 and TNFα) according to the manufacturer’s instructions and fluorescence was quantified against standards using a Luminex200 instrument. All experiments were performed in triplicate. To statistically compare treatments an ANOVA was used in combination with a Tukey post-test. P values < 0.05 were considered statistically significant.
Cell titre 96 AQueous one solution proliferation assay (MTT assay)
Primary microglia (300000 cells per treatment) were treated with Wnt3a (10 nM), LPS (10 ng/ml), oligomeric Aβ (3 μM) or oligomeric α-synuclein (500 nM) for 24 hours and the medium (SFM) was collected. Control medium was collected from untreated microglia. This medium was then added to cortical neuron cultures (500000 cells per treatment) for a further 24 hours before the assay was performed. Some neuronal cultures were exposed to conditioned medium collected from microglia treated with LPS (10 ng/ml), Aβ (3 μM) or α-synuclein (500 nM) in the presence of medium collected from Wnt3a treated microglia. The assay principle is that MTS [3-(4,5-dimethylthiazol-z-yl)-S-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-2H-tetrazolium)], a tetrazolium compound, is bioreduced by cellular NADP/NADPH to a coloured formazan end-product, which is soluble in tissue culture medium. Following treatment with conditioned medium, neurons (in 250 μl of medium) were incubated with the Cell Titre 96 AQueous One Solution reagent (50 μl), containing MTS, for 4 hours and the absorbance was subsequently read at 492 nm using a 96-well Anthos HTll microplate absorption photometer. Neurons were lysed with triton- X100 (9% v/v) as a positive control (100% death) and neuronal death was calculated as a percentage of this total. All experiments were performed in triplicate. To statistically compare treatments an ANOVA was used in combination with a Tukey post-test. P values < 0.05 were considered statistically significant.
Cytotox 96® non-radioactive cytotoxicity assay kit (LDH assay)
Cells were treated as described for the performance of the MTT assay above. In addition, LPS (10 ng/ml), oligomeric Aβ (3 μM) or oligomeric α-synuclein (500 nM) were directly added to neuronal cultures in the presence of conditioned medium collected from control microglia to account for any direct neurotoxic effects of the microglial activators. The assay measures lactate dehydrogenase (LDH), a cytoplasmic enzyme, released from the cell upon death. The assay is based on the formation of NADH from NAD+ and lactate, which in turn reduces a tetrazolium compound into a coloured formazan end-product. In brief, medium was collected from neurons (50 μl) and added to assay substrate mix (50 μl) and incubated in that dark for 30 minutes at room temperature. Subsequently, stop solution (50 μl) was added and the absorbance was read at 490nm. Neurons were lysed with triton- X100 (9% v/v) as a positive control (100% death) and neuronal death was calculated as a percentage of this total. All experiments were performed in triplicate. To statistically compare treatments an ANOVA was used in combination with a Tukey post-test. P values < 0.05 were considered statistically significant.
Hoechst 33342 staining for nuclear morphology
Primary microglial cells (50000 cells per treatment) were treated with and without Wnt3a (10 nM) for 24 hours. Apoptosis was subsequently assessed using the fluorescent dye 2′[epoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazol (Hoechst 33342)
. Microglia on 13 mm coverslips (50000 cell per treatment) were fixed in formaldehyde (4%) in phosphate buffered saline at 4°C, then incubated with Hoechst 33342 (17.8 μM) for 10 minutes at 20°C. Immunofluorescence was visualised and captured using a Zeiss Axioscope microscope and nuclear morphology was appraised at 365 nm with emission greater than 490 nm collected. Apoptotic cells possess brightly stained pyknotic nuclei, whereas, non apoptotic cells possess large weakly stained nuclei. Apoptotic cells were counted on three coverslips per treatment on three independent occasions (each coverslip comprised 10 fields of view). Apoptotic cells were expressed as a percentage of the total number of cells counted per field.
Fluoroscein diacetate staining for cell membrane integrity
Primary microglial cells (50000 cells per treatment) were treated with and without Wnt3a (10 nM) for 24 hours. Cell membrane integrity was subsequently assessed using fluoroscein diacetate
. Primary microglial cells on coverslips were incubated with fluoroscein diacetate (35 μM) for 10 minutes at 37°C in the dark. Cell viability was then assessed at 380 nm with emission greater than 505 nm collected. Fluoroscein diacetate is metabolised in healthy cells to form a fluorescent product, which emits at 530 nm, staining live cells green. Fluorescent cells were counted on three coverslips per treatment on three independent occasions (each coverslip comprised 10 fields of view) and cells were expressed as a percentage of the total number of cells counted per field.
Exosomes suspended in phosphate buffered saline (PBS: 10 μl) were fixed by the addition of 16% formaldehyde (1 μl). The exosomal solution (3 μl) was then pipetted on to Pioloform coated electron microscopy grids. Excess fixative was quenched by exposing the grids to quenching buffer (PBS, 50 mM glycine, 0.01% triton). Non-specific binding was minimised by blocking the grids in blocking buffer (PBS, 0.01% triton 0.1% acetylated BSA). The grids were then incubated with mouse anti-β-actin (1:1000) or rabbit anti-Wnt3a (1:1000) for 1 hour at room temperature in blocking buffer and washed in 6 changes of blocking buffer. The grids were subsequently incubated with anti-mouse or anti-rabbit IgG conjugated to 10 nm colloidal gold (1:100) for one hour at room temperature before being washed with 4 changes of PBS. The exosomes were then fixed in glutaraldehyde (2%) and washed in 3 changes of distilled water before being contrasted with a mixture consisting of 9 parts uranyl acetate (0.3%) to 1 part methyl cellulose (2%). In other experiments, exosomes were fixed in glutaraldehye (2%) then washed in 3 changes of distilled water before being stained with osmium tetroxide (1%). Exosomes were visualised using a FEI Tecnai T12 BioTWIN transmission electron microscope fitted with an AMT camera. Exosomes were stained in triplicate from 3 independent experiments; therefore images are representative of a single experiment.
N9 microglia (50000 cells per treatment/coverslip on the day of plating) were treated with or without Wnt3a (10 nM) for 2 hours then fixed with methanol at 4°C for 5 minutes. Cells were washed in three changes of PBS and then stained according to standard protocols. Briefly, cells were incubated with rabbit anti-Wnt3a (1:500) and/or mouse anti-Rab5 (1:500) before being incubated with the appropriate fluorescent secondary antibody (1:500). Nuclei were counter-stained with Hoescht 33342. Immunofluorescence was visualized and captured using a Zeiss Axioscope microscope. All experiments were performed in triplicate. Figures shown are representative of a single experiment.