1. Preparation of the antigens.
Briefly, β-Amyloid and TDP-43 antigens were prepared as previously described [20]. In the case of β-Amyloid antigen, the peptide used was palmitoylated peptide 1–16. For the TDP-43 antigen, the peptide used was non-palmitoylated peptide 311-344.
2. Immunizations, antibody fragments (Fabs) cloning and production.
The immunizations were done by Absolute Antibody (Oxford, UK). To raise antibodies against our peptides, C57BL/6 mice were immunized for 98 days with liposomes presenting the antigen at their surface. Their spleen B-cells were then fusioned with P3-X63-Ag8 myeloma line to obtain Hybridomas. Hybridomas were then sequenced by whole transcriptome shotgun sequencing. After identification of the mature VH and VL regions sequences, they were subcloned in expression vectors to be expressed in Human Embryonic Kidney 293 (HEK293) cells. Cells were transiently transfected with heavy and light chain expression vectors and cultures for a further 6 to 14 days. Cultures were harvested and the Fabs were purified using affinity chromatography and analyzed for purity by SDS-PAGE. The quantity of Fabs were finally quantified by ELISA.
3. Plasmids.
To create pSF-CAG.InsSP-EGFP, the OG4678 vector (OxGene, Oxford, UK), encoding the CAG promoter, was used as parent vector. PCR was performed to append the human insulin signal peptide to EGFP. Restriction and ligation were performed with the EGFP PCR product and OG4678 to create the final construct for expression and secretion of EGFP. pl.DualCAG.Hygro.cAb2789 encodes both chains of an anti-βAmyloid Fab with optimized peptide signals driven by dual CAG promoters. In addition, a His tag is included to simplify the screening of the Fab production. pl.DualCAG.Hygro.cAb2789 was created with pSF-CAG.InsSP-GFP as parent vector. The parent vector was restricted and was subsequently ligated with a restricted DNA fragment corresponding to the ubiquitin promoter, a downstream hygromycin resistance marker for cell selection, and a polyadenylation sequence. PCR was performed to append optimized peptide signals to both chains of the anti-β-Amyloid Fab encoding sequences (CH1-VH and CL-VL). In addition, a 10-His tag encoding sequence was added to the heavy chain encoding sequence. Finally, the fragments were subcloned into the parent vector with CAG promoters for both chains to create the final vector expressing and secreting the anti-βAmyloid Fab. Using the same method, pl.DualCAG.Hygro.cAb2508 was synthetized and encodes both chains of an anti TDP-43 Fab. pSF-CAG.InsSP-GFP, pl.DualCAG.Hygro.cAb2789 and pl.DualCAG.Hygro.cAb2508 were verified with restriction digests and Sanger sequencing. All plasmids were commercially prepared with endotoxin levels confirm to be <100 EU/mg (OxGene, Oxford, UK) and diluted to 2 mg/ml in physiological saline.
4. Cell line.
MAgEC 10.5 cells (murine endothelial progenitor cell line) [Kieda C et al (2011) Human and murine stem-cell lines: models of endothelial cell precursors / Fasc. European Patent EP 2 524 030 B1] were grown as previously described by Collet et al. [19] in Opti-MEM containing 2% FBS (Gibco, ThermoFisher Scientific, Irvine, CA, USA) at 37 °C with 5% CO2.
5. Cell transduction with tdTomato lentiviral expression vectors.
To establish the MAgEC 10.5 cell line displaying tdTomato expression, the third-generation lentiviral system consisting of pMDLg/pRRE, pRSV-Rev, pMD2.G (a gift from Didier Trono (Addgene plasmid # 12251, 12253, 12259)) and expression plasmids pLV[3Exp]-EF1A>{tdTomato}:IRES:Puro (VectorBuilder) were used. Lentiviral vectors were produced using Lenti-X™ 293T cell line (Clontech, USA) according to the protocol described by Rossowska et al. [25]. Stable MAgEC 10.5/tdTomato cell line, renamed MagEC 10.5 RT, was obtained after selection with puromycin (10 μg/mL, from Sigma-Aldrich). Transduction efficacy was analyzed for their fluorescence emission of the tdTomato protein in cells by flow cytometry (FACS Aria, Becton Dickinson).
6. Cell transfection and cloning.
MagEC 10.5 RT was seeded at 30 000 cells per well in a 12 wells plate, allowed to adhere for 12 hours, and then the medium was exchanged with serum-free OptiMEM. After 6 hours, cells were transfected with Lipofectamine 2000 and Fabs-encoding vectors (pl.DualCAG.Hygro.cAb2789 or pl.DualCAG.Hygro.cAb2508) according to the manufacturer's instructions. Cells were transfected and kept for further 12 hours in a serum-free medium. After 12 hours, the hygromycin selection (125 µg/ml) was made. After 24 hours of hygromycin selection, the medium was removed and replaced with the fresh one. The medium was changed every two days in two weeks. After two weeks, the hygromycin-resistant colonies were observed. To obtain proper clones, the cells were detached and seeded at 96 wells plate (1 cell/per well). Selected clones were checked for Fab secretion using western blotting technique.
7. Western blot His-tagged Fab.
MAgEC 10.5 clones were cultured for 3-5 days Using OPTI MEM with 2% FBS and 125 ug/ml hygromycin. When the cells reached 90-100% confluence, they were detached by Accutase and counted. Cells were centrifuged, washed with PBS and RIPA buffer with protease inhibitors added. Cells lysates were stored at -20 °C. Then, the lysates were centrifuged at 4000×g for 10 mins at 4°C. Protein concentration was quantified using a PierceTM BCA Protein Assay Kit (ThermoFisher Scientific). Cell lysates were solubilized in 4×Laemmli sample buffer and boiled for 5 mins at 100°C. 15-20 µg of protein extracts were separated on 10% SDS-PAGE (First: 10 minutes, 100V, next: about 60 minutes, 180V at 4°C) and transferred onto the nitrocellulose membrane. Following the transfer, the membrane was blocked with blocking buffer (5% nonfat dried milk in TBS/0.1% Tween-20 (TBST)) for 1 hr at RT and incubated overnight at 4°C with primary antibodies. On the following day, the membrane was washed and then incubated with appropriate horseradish peroxidase-labeled secondary antibodies for 1 hr at RT. Finally, target proteins were visualized using Western Blotting Luminol Reagent (Santa Cruz Biotechnology) according to the manufacturer’s protocol. β-actin was used as a loading control. For MAgECs supernatants, the protocol was the same except for estimating the protein concentration.
Primary antibodies
|
Cat. No.
|
Anti-Histag Chimeric
|
Merck, SAB5600096
|
Anti-Mouse IgG (Fab specific)
|
ThermoFisher, 31413
|
Secondary antibodies
|
|
Anti-Histag Chimeric secondary
|
Merck, A1293
|
Rabbit anti-Goat IgG (H+L) secondary
|
ThermoFisher, 81-1620
|
8. Assessment of surface markers using flow cytometry.
To evaluate expression of surface markers, cells from culture were detached with Acutase Solution (Biolegend, USA). Cell suspension was washed with PBS and incubated with TruStain FcX™ PLUS (anti-mouse CD16/32) (#156603 Biolegend, USA) 10 minutes on ice to block non-specific binding of immunoglobulin to the Fc receptors. The reagent was used according to the manufacturer's protocol 0.25 µg per 106 cells in a 100 µl PBS. The cells were then centrifuged, and staining was performed using 105 cells for single marker. Antibodies and isotopic controls used in experiment listed in Table 1; in concentration recommended by manufacturer in 100µl Stain Buffer (FBS) (BD Pharmingen™, USA). Incubation was continued for 30 minutes on ice. After staining cells were washed twice with 300 µl PBS. Finally, 50 µl of cell suspension in Stain Buffer (FBS) was prepared and cells were analyzed by flow cytometry using CYTOFLEX software v.2.3.0.84 (Becton Dickinson, USA). The lower threshold was used to exclude debris and live cells with gating (104 cells), according to forward scatter (FSC) × side scatter (SSC) and only singlets were analyzed. Due to the red color of MAgEC RT cells, auto-fluorescence in different channels was checked. APC and Pacific Blue450 were selected as the most useful (the weakest auto-fluorescence). Data were presented as delta MFI to reduce the impact of auto-fluorescence.
Table 1 List of antibodies:
Antibody
|
Catalog number
|
Details
|
CD34
|
152207 (Biolegend)
|
Rat IgG2a Brilliant Violet 421™
|
CD45
|
103111(Biolegend)
|
Rat IgG2b APC
|
UEA-1
|
DL-1069 (Vector Labratories)
|
DyLight649 labeling
|
Isotype Control Antibody
|
400511 (Biolegend)
|
Rat IgG2a APC
|
Isotype Control Antibody
|
400611(Biolegend)
|
Rat IgG2b APC
|
9. Solubilization capacity of β-amyloid or TDP-43 aggregates.
Reaction tubes containing 30 µg of β-Amyloid 1–42 protein (Bachem, Bubendorf, Switzerland) or 30µg of TDP-43 protein (LS-Bio, Seattle, Washington, USA) in 10µl of PBS, pH 7.4 (Gibco), were incubated for 1 week at 37°C. Aggregation was measured by the thioflavin T (ThT)-binding assay, in which the dye’s fluorescence emission intensity reflects the degree of fibrillar aggregation. Disaggregation was followed after addition of purified antibodies, Fab or supernatants from Fab producing-clones to the preformed fibers (10 µl each). The purified IgG, Fab and an irrelevant control antibody (mouse IgG) were used at a final concentration of 1,5 mg/ml. The reaction incubated for 2 days at 37°C. Fluorescence (excitation:450 nm; emission: 482 nm) was measured after addition of 1 ml of ThT (3 µM in 50 mM sodium phosphate buffer, pH 6.0) on Fluoromax4C fluorometer (Horiba, Kyoto, Japan).
10. Hypoxia sensitivity of murine brain-derived endothelial cells (MBrMECs) recognition by MAgEC 10.5 RT cell line by adhesion experiment.
The adhesion experiment was performed as previously described [26]. A proportion 1:1 for MAgECs/MBrMECs was used. For hypoxia effect assessment, MBrMECs cells were pre-cultured for 48 hours in a cell incubator (19,5% O2) or hypoxia chamber (Biospherix X3 Vivo System, USA) set for a 1% O2 atmosphere for 72 h to reach confluence. After 72 hours, MAgEC 10.5 RT cells (control and selected clones) were counted and added in suspension at a ratio of 1:1 to MBrMECs monolayers and were incubated for 20 min at RT with gentle rocking. Un-attached cells were washed off with warm medium. The remaining cells were detached with Accutase and analyzed by flow cytometry. The ratio of MAgEC 10.5 RT to MBrMECs was counted based on the number of events detected in PerCP-positive (MAgECs) to PerCP-negative (MBrMECs) gates.
11. Animal experiments and immunofluorescence.
All mice were housed and treated in accordance with widely accepted standards, and the protocols were approved by the institutional care and the Regional Animal Health and Food Control Station of Csongrád-Csanád County (permit number: XVI./764/2018).
8–14-week-old BALB/c mice were anesthetized via inhaled isoflurane 4% (v/v) in synthetic air for induction and 1–2% (v/v) for maintenance, using an isoflurane vapourizer (Stoelting). Depth of anesthesia was monitored by toe pinch tests. Endothelial precursor cells (EPC) - 4.105 MAgEC 10.5 RT cells labeled with CellTracker Red (C34552, Thermo Fisher) according to the manufacturer’s instructions, MAgEC 10.5 RT cells or MAgEC 10.5 RT anti-TDP-43 cells - were injected into the common carotid artery while the external carotid artery was ligated. 4 hours to 7 days later the animals were sacrificed and perfused transcardially with 4 w/w % formaldehyde. Overnight post-fixation with the same fixative was followed by vibratome sectioning to 30 micrometer thickness. The sections were immunolabeled using antibodies Fab (1:200, A1293 Sigma-Aldrich), PECAM-1 (1:100, NB100-2284, Novus Biologicals), claudin 5 (1:200, 35-2500, Invitrogen, Thermo Fisher), aquaporin-4 (1:100, sc-390488, Santa Cruz) and secondary antibodies donkey-α-goat-Alexa+647, donkey-α-rabbit-Alexa+488 donkey-α-mouse-Alexa+488 (1:500, A32849, A32790, A32766, Invitrogen, Thermo Fisher)). Syto-13 (1:10000, S7575, Invitrogen) was used as a nuclear counterstain. Immunofluorescence was recorded using a fluorescence microscope (Axiovert Z1, Zeiss) equipped for super-resolution capable laser scanning confocal microscopy (Stedycon, Abberior Instruments).
12. Statistical analysis.
The results were analyzed using GraphPad Prism 9.0 software. Data are expressed as mean ± standard deviation of the mean (±SD). Statistical differences were considered relevant at p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).