Design of plasmid constructs and viruses:pcDNA3 plasmid expressing Aηα or Aηβ

Aηα and Aηβ constructs were obtained by PCR amplification using APP695 cDNA as template and the forward primer 5ʹ-GATAAGCTTGCCACCATGATTAGTGAACCAAGGATCAGTTAC-3ʹ for both Aηα and Aηβ. This primer contains a Hind III restriction site and a Kozak sequence. We used the reverse primer 5ʹ-GATCTCGAGCTATTTTTGATGATGAACTTCATATCCTGAGTC-3ʹ for Aηα and the reverse primer 5ʹ-GATCTCGAGCTACATCTTCACTTCAGAGATCTCCTCC-3ʹ for Aηβ. Both reverse primers contain the XhoI enzyme restriction site. The amplicon was then digested by Hind III and XhoI enzymes and subcloned in the pcDNA3 vector.

pcDNA3 plasmid expressing ηCTF 

the ηCTF plasmid construction contains the APP signal sequence and two additional residues (Leu-Glu) from APP695 cDNA in frame with the 5ʹ end of the ηCTF sequence. As previously described for SPA4CT [10], SP-C99 and SP-C83 constructs [11], the APP signal sequence is required for a correct membrane insertion of the ηCTF fragment. In a first PCR reaction, the APP signal sequence was amplified using the primers 5ʹ-GATAAGCTTATGCTGCCCGGTTTGGCACT-3ʹ that contains an HindIII restriction site and 5ʹ-TTCACTAATCATCTCCAGCGCCCGAGCC-3ʹ containing the first nucleotides of the 5’ end of ηCTF sequence. In an additional PCR, ηCTF sequence was amplified using the primer 5ʹ-GCGCTGGAGATGATTAGTGAACCAAGGATCAGTTA-3ʹ that contains the last nucleotides of the 3ʹ end of the signal peptide sequence, and the primer 5ʹ-GATCTCGAGCTAGTTCTGCATCTGCTCAAAGAA-3ʹ containing XhoI restriction site. Finally, the amplicons resulting from the first two PCR were incubated together in a third PCR reaction to obtain a final DNA fragment containing the APP signal peptide in frame with ηCTF. This amplicon digested with HindIII and XhoI enzyme was then subcloned in pcDNA3 vector.

AAV-10 plasmid expressing ηCTF under the control of synapsin-1 promoter

For viral plasmid construction, the pcDNA3 plasmid expressing ηCTF described above was used as a template with the primers 5ʹ-GATGCTAGCCCACCATGCTGCCCGGTTTGGCACTGCTCCT-3ʹ and 5ʹ-GATGCTAGCCTAGTTCTGCATCTGCTCAAAGAACTTGTAGGTT-3ʹ that both contain a NheI restriction enzyme site. The PCR product was then digested with NheI and sub-cloned into AAV10 plasmid.

pGEX plasmid for expression of recombinant ηCTF

the DNA sequence encoding the ηCTF was amplified by PCR, using the forward primer 5ʹ-GATGGATCCATGATTAGTGAACCAAGGATCAGTTA-3ʹ, which has a BamH1 restriction site and the reverse primer 5ʹ-GATCTCGAGCTAGTTCTGCATCTGCTCAAAGAA-3ʹ, containing a XhoI restriction site. The PCR product was then sub-cloned between BamH1 and XhoI restriction enzymes sites of pGEX-4T-1 vector.

All above constructs were verified by full sequencing.

Recombinant ηCTF production

Recombinant ηCTF fragment was obtained as previously described for the production of recombinant PrPc fragment N1 [12], with some minor changes. Briefly, pGEX-4T-1 vector containing ηCTF sequence was transformed into BL21 gold strain of Escherichia coli. After induction with Isopropyl 1-thio-d-galactopyranoside (200 μM), the medium was centrifuged. Cells pellets were resuspended with PBS supplemented with a protease inhibitor mixture, DTT (10 mM) and lysozyme (0.2 mg/ml), then proteins were solubilized by the addition of Triton X-100 (10%), MgCl2 (1 M), and DNase (1u/μl). After centrifugation, glutathione-sepharose beads (GE Healthcare) were added to the supernatant, pelleted, and resuspended in PBS (1 ml). Peptides were cleaved with thrombin (5 units/ml; GE Healthcare), and thrombin was removed using Sepharose benzamidine beads (GE Healthcare).

Production of the ηCTF-Nter antibody

The new ηCTF-Nter antibody is a rabbit polyclonal antibody made following the Covalab’s immunization protocol. As immunogen epitope, we designed peptide corresponding to the first 16 N-terminal residues of ηCTF fragment, the C-terminal end of which was blocked by a cystein residue to preserve a free N-terminal part of the peptide and to obtain antibodies directed towards the N-terminal moiety (sequence: MISEPRISYGNDALM-C). Rabbit’s immunoreactivity and titer were controlled by ELISA then the ηCTF-Nter antibody was purified by antigen-specific affinity with the same peptide used for immunization.

Viral production and mice ICV injection:

Virus production was performed following a protocol previously described [13]. Briefly, HEK293 cells were transfected with the adenovirus helper plasmid (pXX6), the AAV packaging plasmid (rAAV2-rh10), and the AAV10 plasmid empty vector or encoding human ηCTF under control of the synapsin-1 promoter (AAV-empty, and AAV- ηCTF). Viruses were produced, purified and vector titers were determined by real-time PCR and expressed as viral genomes per ml (vg/ml). Four µl of AAV virus (5.5 × 1012vg/ml) were administrated in 1-day-old C57Bl6JRj mice (Janvier Labs, France) through intra-cerebroventricular (ICV) injection as described previously [13] then mice brains were analyzed at 3 months of age by western blot and immunochemistry.

Animals

Pregnant C57Bl6JRj females (Janvier Labs, France) were ordered for new born mice viral injection (see above). In addition wild-type and 3xTg (APPswe; TauP301L; PS1M146V) mice were maintained from breeding pairs provided by Dr F. LaFerla [14]. All mice were kept on the original 129/C57Bl6 background strain, backcrossed every 10 generations and genotyped. Animals were housed with 12 h/12 h light/dark cycle and were given free access to food and water. All experimental procedures were in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU) and approved by the French Ministry of Higher Education and Research (project number APAFIS#9766-201704261624789.v3).

Cell Culture and treatments

Human neuroblastoma (SH-SY5Y, ATTC), human epitheloid cervix carcinoma (HeLa, ATCC) and mouse embryonic fibroblasts either wild type (MEF-APPwt), or naturally devoid of APLP1 and knocked out for APP and APLP2 [15] (referred to as MEF APP KO) were cultured in Dulbecco’s modified Eagle’s medium supplemented with fetal calf serum (10%), penicillin (100 U/ml) and streptomycin (50 μg/ml), and incubated at 37 °C in a 5% CO2 atmosphere. Human SH-SY5Y cells stably expressing wild-type full-length APP (SH-SY5Y-APPWT) were generated as already described [16], and maintained in the presence of G-418 (400 µg/ml). Transient transfections of cells were carried out using Lipofectamine 2000 (Life Technologies) for SH-SY5Y and MEFs and JetPrime (Polyplus transfection) for HeLa cells, according to the manufacturer’s instructions. Twenty-four hours post-transfection, cells were treated with pharmacological agents: Lactacystin (5 µM; Sigma-Aldrich), Epoxomicin (1 µM; Enzo), MG132 (5 µM), Bafilomycin A1 (100 nM), Smer28 (50 µM), or with secretases inhibitors: β-secretase inhibitor, Bi (30 µM, Elan Pharmaceuticals), α-secretase inhibitor, Gi (GI254023X; 10 µM; Sigma-Aldrich) or γ-secretase inhibitor D6 (1 µM; Elan Pharm/Imago). Cells were analyzed 48 h post-transfection.

Western-blot analysis

Cells were lysed in RIPA buffer [Tris (50 mM); pH 7,4; NaCl (150 mM); EDTA (1 mM); Triton X100 (1%); deoxycholate (0.5%); SDS (0.1%)] supplemented with a cocktail of protease inhibitors (Roche diagnostics). Cell homogenate proteins (50 µg) were separated on 16% tris-tricine gels, and sEVs proteins were separated on 16% tris-tricine gels or Bio-Rad 12% stain-free TM TGX FastCastTM acrylamide gels. It should be noted that in few cases, when protein of interests and loading control proteins have similar molecular weights, to achieve accurate normalizations, gels were run separately in strictly identical conditions. All full gels are provided as indicated in the legends of figures showing gels. Tris-tricine gels were directly transferred to nitrocellulose membranes using a conventional transfer system and boiled in PBS before saturation. Bio-Rad gels were photoactivated for the visualization of proteins before being electrophoretically transferred to nitrocellulose membranes using the Bio-Rad Trans-Blot® TurboTM Transfer System. Membranes were saturated with skimmed milk, and incubated overnight with the following primary antibodies: APPCter rabbit polyclonal (1:5000, gift from Paul Fraser); WO2 mouse monoclonal (1:5000, Sigma–Aldrich); ηCTF-Nter rabbit polyconal (1:1000, homemade with Covalab, see above); α-HSC70 mouse monoclonal (1:1000, Santa Cruz) or α-GAPDH mouse monoclonal (1:5000, EMD Millipore). After probing with primary antibodies, immunological complexes were revealed with anti-mouse or anti-rabbit HRP-conjugated antibodies (1:5000, Jackson ImmunoResearch) followed by electrochemiluminescence (Westernbright™ Sirius™ and Quantum™ chemiluminescent HRP substrate, Advansta, France). Peak height of signal intensities from protein bands were quantified with MultiGauge software.

For western-blot analysis of mice brains, following intracardiac perfusion with PBS, hemibrains were extracted then enzymatically and mechanically lysed using “the adult brain dissociation kit” and a GentleMACS Dissociator (Miltenyi Biotec). PBS was added to the cell suspension, which was then filtered through a 70 μm Smartstrainer and centrifuged at 300×g for 10 min. The cell pellets were lysed in RIPA buffer supplemented with protease inhibitors and then loaded on Bio-Rad 12% stain-freeTM TGX FastCastTM acrylamide gels.

Sandwich ELISA analysis

MEF APP KO cells were transiently transfected with pcDNA.3 or ηCTF constructs using Lipofectamine 2000 and treated or not with secretases inhibitors Bi, Gi and D6. The concentrations of human secreted Aβ40 were measured in the culture medium using the ELISA kit (Invitrogen) following the manufacturer’s instructions.

Aηα immunoprecipitation

MEF APP KO cells were plated on 6 wells plates, transiently transfected with pcDNA.3 or ηCTF constructs using Lipofectamine 2000 and treated or not with secretases inhibitors Bi, Gi and D6, as described above. Culture media were collected, centrifuged at 14,000×g for 5 min to remove cell debris then supernatants were supplemented with RIPA buffer 5X and precleared with 10 μl of Protein A Sepharose (GE Healthcare) for 1 h at 4 °C. Resulting supernatants were incubated for 1 h with WO2 mouse monoclonal antibody (1 μl) then 20 μl of Protein A Sepharose beads were added and left for overnight incubation. After three washes with RIPA buffer (and 1 time in PBS), the beads were resuspended with 20 μl of loading buffer, loaded on a tris-tricine gel (16%) and subjected to western-blot analysis as described above.

Cells immunostaining

Hela cells grown on coverslips were fixed in paraformaldehyde 4% solution for 10 min, permeabilized with Triton-X 100 (0.1%) for 10 min, saturated in BSA (5%)/Tween20 (0.1%), and probed for 1 h with appropriate primary antibodies: APPCter rabbit polyclonal (1:5000), APPCter, mouse monoclonal (1:5000, Biolegend), WO2 mouse monoclonal (1:5000), or ηCTF-Nter rabbit polyclonal (1:1000) for ηCTF detection, α-Lamp2 mouse monoclonal (1:1000, Santa Cruz), α-EEA1 rabbit monoclonal (1:200, Cell Signaling Technology), α-CD63 mouse monoclonal (1:1000, Santa Cruz) and α-TGN46 sheep polyclonal (Serotec, AHP500G, 1:1000) for lysosomes, early and late endosomes and golgi specific markers, respectively. After washes, coverslips were incubated for 1 h with Alexa Fluor-488 and Alexa Fluor-590 conjugated antibodies (Molecular Probes, 1:1000) and DAPI (1:20,000, Roche) staining. Finally, the sections were washed with PBS, then mounted onto glass slides and cover-slipped. The stained slices were kept at 4◦C before analysis with confocal microscopy (Zeiss LSM 780 with 63 × Objective).

DAB and immunofluorescence staining of tissue

For immunohistochemistry, mice were anesthetized by an intraperitoneal injection of a combination of ketamine (150 mg/kg), xylazine (10 mg/kg) and acepromezine (10 mg/kg) then perfused intra-cardiacally with PBS followed by paraformaldehyde (4%) before collecting the brains. Paraformaldehyde-fixed brains were embedded in paraffin and cut on a microtome in 8 μm thick sections (Thermoscientific, France). Brain sections were deparaffined in xylen bath and rehydrated by successive 5 min baths of EtOH (100%, twice), 90%, and then 70%. Antigens were unmasked in a 90% formic acid bath for 5 min and then in citric acid solution at 90–100 °C for 20 min (Pressure cooker, Vector Laboratories). For DAB staining, sections were treated with H2O2 3% for 15 min. Non-specific binding sites were blocked for 1 h in BSA (5%)/Tween20 (0.1%) and brain sections were then incubated at 4 °C overnight with primary antibodies ηCTF-Nter rabbit polyclonal (1: 800) or WO2 mouse monoclonal (1:1000). Sections were incubated for 1 h with Alexa Fluor-488 and Alexa Fluor-594 conjugated antibodies (1:1000) and DAPI (1:20,000) for immunofluorescence, or with horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoResearch, 1:1000) then revealed with the DAB-ImmPACT system (Vector Laboratories) for DAB-staining. Images were taken with a confocal Leica TCS SP5 microscope (Immunofluorescence) or with DMD108 Leica microsystem (DAB) and processed using ImageJ software. For ηCTF-Nter staining, signal was amplified with Avidin Biotin Complex (ABC) method (VECTASTAIN Original ABC Kit, Vector Laboratories).

Purification of exosomes (sEVs)

SH-APPWT cells were cultivated in DMEM for 24 h, rinsed twice with PBS, and treated or not with the β-secretase inhibitor Bi or Bafilomycin A1 as described above, for another 24 h in OptiMEM to allow vesicle secretion. When overexpressing ηCTF, cells were transiently transfected 1 day before secretion in OptiMEM. sEVs were purified by ultracentrifugation as previously described [17]. Briefly, media were first harvested and centrifuged at 2000×g for 20 min, then filtered through a 0.22 μm filter. The supernatant was then sequentially centrifuged at 10,000×g for 30 min and 100,000×g for 125 min. The obtained pellet containing sEVs and contaminating proteins was washed in ice-cold PBS and centrifuged for another 120 min. The final sEVs pellet was resuspended in RIPA buffer and sonicated in an ultrasonic bath for 15 min before western-blot analysis. sEVs isolated from the extracellular space of AAV-ηCTF mice brain were purified according to the protocol previously described [17]. Briefly, intracardiac perfusion with PBS was performed then hemibrains were extracted and enzymatically and mechanically lysed using “the adult brain dissociation kit” and a GentleMACS Dissociator (Miltenyi Biotec). PBS was added to the homogenate suspension, which was then filtered through a 70 μm Smartstrainer and centrifuged at 300×g for 10 min. The supernatant was used for sEVs purification and the cell pellet was kept for further analysis by western blot as described above. For sEVs purification, the supernatant was sequentially centrifuged several times as previously described [16], and the last pellet was loaded on a sucrose gradient and centrifuged at 200,000×g for 16 h. The fractions containing the sEVs were collected, diluted with PBS and centrifuged at 100,000×g for 90 min. The final sEVs pellet was resuspended in PBS, lysed in RIPA buffer complemented with protease inhibitors and ultrasonicated before western-blot analysis. Before loading, nanoparticle analysis was performed on each fraction using the ZetaView instrument (Particle-Metrix) to determine particle size distribution and concentration.

Membrane fraction preparation

Dissected hippocampi of 3xTgAD (AD) and wild-type (WT) mice were homogenized in hypotonic buffer [HEPES (5 mM), pH 7.4, EDTA (1 mM), sucrose (0.25 M) containing a proteases inhibitor cocktail]. The homogenate was centrifuged at 1000×g for 5 min at 4 °C and the supernatant was centrifuged at 100,000×g for 1 h. Membrane pellets were then solubilized in RIPA buffer complemented with protease inhibitor, and centrifuged at 100,000×g for 20 min. Supernatants were recovered as the soluble membrane fractions and loaded on 16% tris-tricine gels.

Statistical analyses

Statistical analyses were performed using Prism software (GraphPad Prism 7). Quantitative data are represented as means ± SEM and subjected to non-parametric tests such as the Mann–Whitney test for single comparisons and the Tukey one-way ANOVA test for multiple comparisons. Statistical significance code is: ****p < 0.0001 and *p < 0.05.