Chemicals and reagents
Reagents used for Western blotting – Pierce BCA protein assay kit, SeeBlue™ Plus2 pre-stained standard, NuPAGE™ 4-12% Bis-Tris protein gels, NuPAGE™ MES SDS Running Buffer (20X), NuPAGE™ Transfer Buffer (20X), nitrocellulose 0.1μm membranes and GE Healthcare ECL Amersham™ Hyperfilm™ – were all purchased from ThermoFisher (Waltham, MA, USA). Western Lightning® Plus-ECL was from PerkinElmer (Waltham, MA, USA). Complete™ protease inhibitor cocktail was from Roche (Basel, Switzerland). Primary antibodies targeting human Aβ: anti-Aβ clone W0-2 (MABN10), anti-Aβ40 clone 11A5-B10 (05-799) and anti-Aβ42 clone 12F4 (05-831-I) were from Merck (Kenilworth, MJ, USA). Anti-PS1 (D39D1) and anti-PS2 (D30G3) antibodies were from Cell Signalling (Danvers, MA, USA). Anti-APP-Cter (A8717) and anti-α-tubulin primary antibodies as well as secondary antibodies coupled to horseradish peroxidase (HRP) were obtained from Sigma-Aldrich (St-Louis, MO, USA). Alexa Fluor™ 647 secondary antibody was obtained from ThermoFisher. Thioflavin T (ThT) amyloid stain was obtained from Sigma-Aldrich. Mowiol® 4-88 used for mounting medium was purchased from Merck. Cell culture reagents – Ham’s-F12, DMEM-F12, DMEM and Neurobasal® growth media, penicillin-streptomycin (p-s) cocktail, Lipo2000® transfection reagent, Opti-MEM®, HBSS, glutamine and B-27® – were all purchased from ThermoFisher. Fetal bovine serum (FBS) was from VWR (Radnor, PA, USA). GELFrEE™ 8100 12% Tris-Acetate cartridge kits were purchased from Expedeon (Heidelberg, Germany). ReadyProbes® cell viability assay kit was from ThermoFisher. ELISA strip plates for immuno-Europium assay (F8, high-binding 771261) were from Greiner Bio-One (Frickenhausen, Germany) and reagent diluent-2 10x (DY995) from R&D systems (Minneapolis, MN, USA). Anti-CD9 primary antibody (MAB1880) was from R&D systems, anti-CD81 (TAPA-1, 349502) from BioLegend (San Diego, CA, USA), anti-CD63 (MCA2142) from Serotec Bio-Rad (Kidlington, UK) and anti-GM130 (610823) from BD transduction (Franklin Lakes, NJ, USA). The anti-mouse IgG-biotin (NEF8232001EA), Europium-labeled streptavidin (1244-360), Delfia® wash concentrate 25x (4010-0010), Delfia® assay buffer (1244-111) and Delfia® enhancement solution (1244-105) were all from PerkinElmer.
DNA constructs
The pSVK3-empty (EP), -C42 and -C99 vectors used for expression in rodent cell lines (CHO, MEF) were described previously [30,19]. C42 and C99 are composed of the APP signal peptide fused to the human Aβ42 and βCTF sequences, respectively. For expression in human cell lines (HEK293, SH-SY5Y), the C99 construct in a pCDNA3.1 plasmid was kindly provided by R. Pardossi-Piquard (University of Sophia Antipolis, Nice, France). The pCDNA3.1 plasmid bearing the C99-GVP construct used in reporter gene assays was a gift from H. Karlström (Karolinska Institute, Stockholm, Sweden). The associated Gal4RE-Firefly luciferase reporter gene (pG5E1B-luc) and Renilla luciferase reporter vector (pRL-TK) have been described previously [31,27,32].
Cell lines culture and transfection
Chinese hamster ovary (CHO) cell lines were grown in Ham’s-F12 medium. Human neuroblastoma SH-SY5Y and mouse embryonic fibroblasts (MEF) in DMEM-F12. Human embryonic kidney (HEK293) in DMEM. All media were supplemented with 10% of heat-inactivated FBS and 100units/ml p-s. All cell cultures were maintained at 37°C in a humidified atmosphere and 5% CO2.
For transient transfection, 40.000cells/cm2 were seeded 24h before transfection. Transfection mixes containing desired DNA and Lipo2000® were prepared in Opti-MEM® and pre-incubated for 15min at room temperature (rt). One day after transfection, medium was changed to fresh FBS-free culture medium and incubated for another 24h. Cell lysates and culture media were harvested 48h after transfection for analysis.
Western blotting
Cells were rinsed and scraped in phosphate-buffered saline (PBS) and centrifuged for 5min at 7.000 x g. Pellets were sonicated in lysis buffer (125mM Tris pH 6.8, 20% glycerol, 4% SDS) with Complete™ protease inhibitor cocktail. Protein concentration was determined using the Pierce BCA protein assay kit. Proteins were heated for 10min at 70°C in loading buffer (lysis buffer supplemented with 50mM dithiothreitol (DTT) and NuPAGE™ LDS sample buffer (ThermoFisher)). Samples were loaded and separated by SDS-PAGE electrophoresis on Nupage™ 4-12% Bis-Tris gels with MES SDS running buffer, using SeeBlue™ Plus2 pre-stained as a standard. Proteins were then transferred for 2h at 30V with NuPAGE™ transfer buffer onto 0.1μm nitrocellulose membranes. After blocking (5% non-fat milk in PBS-Tween®20 0.1%), membranes were incubated overnight at 4°C with the primary antibodies, then washed and incubated with the secondary antibodies coupled to HRP for 1h prior to ECL detection. Primary antibodies were used as follows: anti-human Aβ clone W0-2 (1:1.500), anti-APP-C-ter (1:2.000), anti-PS1 (1:1.000), anti-PS2 (1:1.000), anti-α-tubulin (1:3.000). Secondary antibodies were used as follows: HRP-coupled anti-mouse IgG (1:10.000) or anti-rabbit IgG (1:10.000).
GELFrEE™ isolation of cell-derived hexameric Aβ
CHO cells culture media was collected 48h after transfection with either pSVK3-EP, -C42 or -C99, lyophilized, re-suspended in ultrapure water and pre-cleared with recombinant protein A sepharose (GE Healthcare, Chicago, IL, USA). Immunoprecipitation of Aβ species was performed with the monoclonal anti-human Aβ clone W0-2 antibody. Samples were separated through a gel-eluted liquid fraction entrapment electrophoresis (GELFrEE™ 8100) system to allow the collection of the desired kDa range of proteins directly in liquid fraction. The following method was used for hexameric Aβ collection: step 1: 60min at 50V, step 2: 6min at 70V, step 3: 13min at 85V and step 4: 38min at 85V. Fractions 1, 2 and 3 (Fig.1c) were collected at the end of steps 2, 3 and 4 respectively. All samples were collected in the system running buffer (1X buffer: 1% HEPES, 0.01% EDTA, 0.1% SDS and 0.1% Tris) and kept on ice. Absorbance at 280nm of each fraction was read using a BioPhotometer® D30 (Eppendorf, Hambourg, Germany) and the concentration of the collected hexameric Aβ was calculated using the molar extinction coefficient ε280nm=1490 M-1cm-1.
Dot blotting
5µl of isolated hexameric Aβ (150µM for isoform characterization, 15µM for fractions evaluation prior to intracerebral injection) and 5μl of 50µM synthetic monomeric Aβ (mAβ) with 40 (mAβ40) or 42 residues (mAβ42) were spotted onto 0.1µm nitrocellulose membranes and allowed to dry. Another 5µl of sample were then spotted twice on top and dried. The membranes were boiled twice in PBS for 3min, then blocked with 5% non-fat milk in PBS-Tween®20 0.1%, washed and incubated with primary and secondary antibodies prior to ECL detection, as described above. Primary antibodies dilutions were used as follows: anti-human Aβ clone W0-2 (1:1.500), anti-Aβ40 (1:1.000), anti-Aβ42 (1:1.000). Secondary antibodies were used as described for Western blotting. Synthetic mAβ40 and mAβ42 were prepared as previously described [33].
Animal models
Transgenic 5xFAD mice (Tg6799) harboring human APP and PSEN1 transgenes were originally obtained from the Jackson Laboratory: B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax (34840-JAX). Colonies of 5xFAD and non-transgenic (wild-type, WT) mice were generated from breeding pairs kindly provided by Pr. Jean-Pierre Brion (ULB, Brussels, Belgium). All mice were kept in the original C57BL/6 background strain. Animals were housed with a 12h light/dark cycle and were given ad libitum access to food and water. All experiments conducted on animals were performed in compliance with protocols approved by the UCLouvain Ethical Committee for Animal Welfare (reference 2018/UCL/MD/011).
Protein extraction from mouse brain tissues
WT and 5xFAD mice of either sex were euthanized by cervical dislocation or using CO2, and brains were quickly removed. The hippocampus and a portion of temporal cortex were immediately dissected on ice. Brain tissues were then homogenized by pipetting up and down with a 1000μl pipette and sonicating in ice-cold lysis buffer (150mM NaCl, 20mM Tris, 1% NP40, 10% glycerol) with Complete™ protease inhibitor cocktail until homogenous. Samples were stored at -80°C until use. Protein concentration was determined using the Pierce BCA protein assay kit prior to analysis.
Cerebrospinal fluid collection
Cerebrospinal fluid (CSF) was collected by lumbar puncture from AD patients and symptomatic controls undergoing diagnostic work-up at the Cliniques Universitaires Saint-Luc (UCL, Brussels, Belgium), both of either sex, following the international guidelines for CSF biomarker research [34]. Collected samples were directly frozen at -80°C until analysis and were always manipulated on ice during Western blotting and ECLIA experiments. Included patients signed an internal regulatory document, stating that residual samples used for diagnostic procedures can be used for retrospective academic studies, without any additional informed consent (ethics committee approval: 2007/10SEP/233). AD patients participated in a specific study referenced UCL-2016-121 (Eudra-CT: 2018-003473-94). In total, CSF samples from eight subjects were retrospectively monitored in this study (see Supplementary Table.S1).
Electro-chemiluminescence immunoassay (ECLIA) for monomeric Aβ quantification
Aβ monomeric peptides were quantified in human CSF or in SH-SY5Y cells media using the human Aβ 6E10 multiplex ECLIA assay (Meso Scale Discovery, Gaithersburg, MD, USA) as previously described [35]. For SH-SY5Y, cells were conditioned in FBS-free medium for 24h. After collection, medium was lyophilized and re-suspended in ultrapure water prior to analysis.
Primary neuronal cultures
Primary cultures of neurons were performed on mouse embryos of either sex at embryonic day 17 (E17), as described previously [36]. Briefly, cortices and hippocampi were isolated by dissection on ice-cold HBSS and meninges were removed. Tissues were then dissociated by pipetting up and down 15 times with a glass pipette. Dissociation was repeated 10 times with a flame-narrowed glass pipette and samples were allowed to sediment for 5min. Supernatants containing isolated neurons were then settled on 4ml FBS and centrifuged at 1.000 x g for 10min. Pellets were resuspended in Neurobasal® medium enriched with 1mM L-glutamine and 2% B-27® supplement medium. 100.000cells/cm2 were plated in 12w plates pre-coated with poly-L-lysine (Sigma-Aldrich). Cultures were maintained at 37°C and 5% CO2 in a humidified atmosphere.
Cell viability assay (ReadyProbes®)
Primary neuronal cultures performed from WT and 5xFAD mouse embryos of either sex were incubated at 7 days in vitro (DIV7) with 1 or 5µM of either cell-derived hexameric Aβ (C42 fraction) or control (EP fraction). At DIV8, 2drops/ml of each reagent of the ReadyProbes® assay were added to cells: NucBlue® Live reagent for the staining of all nuclei and NucGreen® Dead reagent for the nuclei of cells with compromised plasma membrane integrity. Staining were detected with standard DAPI and FITC/GFP filters respectively, at an EVOS® FL Auto fluorescence microscope. Quantification was performed by counting dead vs total cells on ImageJ.
Intracerebral stereotaxic surgery
2-month-old WT and 5xFAD mice of either sex were deeply anesthetized by intraperitoneal injection of a mixture of ketamine (Ketamin®) (10mg/kg) and medetomidine (Domitor®) (0.5mg/kg), and placed in a stereotaxic apparatus (Kopf® Instruments, Tujunga, CA, USA). 2μl of 15μM cell-derived hexameric Aβ (C42 fraction) or control (EP fraction) were injected using a 10μl Hamilton syringe and an automated pump (RWD®, Guangdong, China). Coordinates used for intrahippocampal injection were based on the Paxinos atlas: A/P -1.94; L ±2.17; D/V -1.96; mm relative to bregma, considering a bregma-lambda distance of 4.21mm. When the distance differed, coordinates were proportionally adjusted. 30 days after stereotaxic injection, mice were transcardially perfused with PBS and brains were post-fixed in 4% paraformaldehyde for 24h at 4°C.
Immunohistofluorescence
For immunohistological analysis, free-floating coronal sections (50μm) were generated from agarose-embedded fixed brains using a vibrating HM650V microtome (ThermoFisher), and were preserved in PBS-sodium azide 0.02% at 4°C. Prior to immunomarking, sections were washed in PBS and subsequently blocked and permeabilized with PBS-BSA 3%-TritonX100 0.5% for 1h at rt. Sections were then incubated with anti-human Aβ clone W0-2 (1:100) overnight at 4°C as a marker for Aβ-containing species. After three PBS washes and incubation with goat anti-mouse IgG Alexa Fluor™ 647 secondary antibody (1:500) for 1h at rt, slices were finally washed three times with PBS and mounted on SuperFrost® slides. Slides were then incubated with ThT (0.1mg/ml in ethanol 50%) for 15min at rt as a marker for fibrillar deposits. After three washes with ethanol 80% and a final wash with ultrapure water, coverslips were mounted with Mowiol® 4-88-glycerol. W0-2 and ThT staining were detected with standard FITC/Cy5 and GFP filters respectively at an EVOS® FL Auto fluorescence microscope. Counting of double-positive dots was performed on ImageJ.
Generation of SH-SY5Y PS1 and PS2 deficient cells by CRISPR-Cas9
Kits each containing guide RNA vectors that target human PSEN1 or PSEN2 genes, a GFP-puromycin or RFP-blasticidin donor vector respectively, and a scrambled sequence control were obtained from Origene (CAT#: KN216443 and KN202921RB). Target sequences were flanked with specific homology sequences for the stable integration of donor sequences, based on the homology-directed repair technique [37,38]. SH-SY5Y cells were transfected using Lipo2000® and FACS-sorted 48h later for GFP+ (PS1) or RFP+ (PS2) cells, then seeded in 24w plates. Following a few days for cell-growth, a second selection was performed using puromycin (PS1) or blasticidin (PS2) at a concentration of 15μg/ml or 30μg/ml, respectively. Cells were then allowed to grow again and split twice before subcloning in 96w plates. PS1 and PS2 clonal populations were selected for following experiments on account of the highest gene-extinction efficiency, mirrored by the strongest decrease in protein levels. Puromycin (2.5μg/ml) and blasticidin (7.5μg/ml) were used for the maintenance of PS1 and PS2 deficient cells, respectively.
Dual luciferase assay
SH-SY5Y cells were co-transfected with Lipo2000® in a 1:1:1 ratio with pG5E1B-luc, pRL-TK and either a pCDNA3.1-empty plasmid (EP) or the pCDNA3.1-C99-GVP, bearing a tagged C99 to quantify the release of the APP intracellular domain (AICD). The system setup was previously described [32,27]. Cells were rinsed with PBS 48h after transfection and incubated with the reporter lysis buffer (Promega, Madison, WI, USA) for 15min at rt. Firefly and Renilla luciferase activities were measured using the Dual-Glo® luciferase assay system (Promega, Madison, WI, USA) on a Sirius single tube luminometer (Berthold, Bad Wildbad, Germany). Luciferase activity corrected for transfection efficiency was calculated as the Firefly/Renilla ratio.
Extracellular vesicles isolation
Culture medium was collected and underwent several centrifugation steps (all at 4°C): 300 x g for 10min for the elimination of living cells, 1.000 x g for 10min to discard dead cells, 10.000 x g for 30min for the removal of cellular debris, and finally 100.000 x g for 1h to collect extracellular vesicles (EVs) as a pellet and soluble proteins as supernatant. Soluble proteins were precipitated by incubation with 10% trichloroacetic acid (TCA) for 30min on ice. Both EVs and soluble proteins fractions were resuspended in 500µl of PBS for nanoparticle tracking analysis (NTA) and plate-based Europium-immunoassay. For Western blotting, a saved portion of both fractions was sonicated in lysis buffer (125mM Tris pH 6.8, 20% glycerol, 4% SDS) with Complete™ protease inhibitor cocktail. Protein concentration was determined using the Pierce BCA protein assay kit.
Nanoparticle tracking analysis (NTA)
EVs were counted in each fraction by the ZetaView® (ParticleMetrix GmbH, Inning am Ammersee, Germany), which captures Brownian motion through a laser scattering microscope combined with a video camera to obtain size distribution (50-1000nm) and concentration. Samples were diluted 1:50 (v:v) in PBS to reach 50-200particles/frame, corresponding to ~2 x 107-1 x 108particles/ml. Sensitivity was set to 65 and camera shutter to 100 in order to detect less than 3 particles/frame when PBS alone was injected, to assess background signal. Measurements were averaged from particles counted in 11 different positions for 2 repeated cycles with camera at medium resolution mode.
Plate-based Europium-immunoassay
50µl of EVs and soluble proteins fractions were bound to protein-binding ELISA plates. After overnight incubation at 4°C, the rest of the experiment was performed at rt by shaking on a tilting shaker at 30rpm. The plate was washed with Delfia® buffer (diluted to 1x in PBS: Delfia®-W), then blocked with reagent diluent-2 (diluted to 1% BSA in PBS) for 90min. The bound material was labeled with primary antibodies against CD9, CD81, CD63 and GM130 (1µg/ml in reagent diluent-2) for 90min. After three Delfia®-W washes, goat anti-mouse biotinylated antibody (1:2.500 in reagent diluent-2) was added for 60min. After another three Delfia®-W washes, Europium-conjugated streptavidin (diluted to 1:1.000 in Delfia® buffer) was added for 45min. After six final Delfia®-W washes, Delfia® enhancement solution was incubated for 15min before measurement using time-resolved fluorometry with excitation/emission: 340/615nm, flash energy/light exposure: high/medium and integration lag/counting time: 400/400µs (VICTOR® X4 multilabel plate reader, PerkinElmer).
Statistical analyses
The number of experiments (N) and the number of samples per condition in each experiment (n) are indicated in figure legends. All statistical analyses were performed using the GraphPad Prism 8 software (GraphPad Software, La Jolla, CA, USA). All datasets were assessed for gaussian distribution using the Shapiro-Wilk test. A parametric test was applied if the data followed normal distribution. Otherwise, non-parametric tests were used. Statistical analysis performed in each case is indicated in the corresponding figure legend. Briefly, when tested groups were expressed as a fold-change of their corresponding control, the value of the control was set as the hypothetical value for the use of parametric one-sample t test or non-parametric one-sample Wilcoxon single-ranked test. When a correlation between two variables was assessed, Pearson’s R correlation coefficient was calculated. When two groups were compared, parametric t or non-parametric Mann-Whitney tests were used. When more than two groups were compared, parametric ANOVA with indicated post hoc tests or non-parametric Kruskal-Wallis were used. Significance is indicated as: ns=non-significant, *=p<0.05, **=p<0.01, ***=p<0.001. Actual p-values of each test are indicated in the corresponding figure legend.