Human brain collection and characterization
Frozen brain samples (parietal cortex) from eight sporadic AD patients as well as two age-matched control individuals were collected from a brain donation program of the NeuroCEB and the CNR-prion brain banks (Additional file 1: Table S1). Consent forms were previously signed by either the patients themselves or their next of kin, in accordance with French bioethics laws. AD patients had a classical evolving form of the pathology, characterized by a disease duration of 5 to 8 years (n = 4) or a more rapidly evolving form characterized by a disease duration of 6 months to 3 years (n = 4). No case of hippocampal sclerosis was reported and all brain samples were PrPSc negative. Samples from AD brains were also negative for α-synuclein and TDP-43. All brain tissues were assessed by immunohistochemistry, as previously described in Lam et al. 2021 [14]. Briefly, 4-μm-thick paraffin sections were cut, deparaffinized in xylene, successively rehydrated in ethanol (100, 90, and 70%) and rinsed under running tap water for 10 min before immunohistological staining. They were then incubated in 99% formic acid for 5 min, quenched for endogenous peroxidase with 3% hydrogen peroxide and 20% methanol, and washed in water. Sections were blocked at room temperature for 30 min in 4% bovine serum albumin (BSA) in 0.05 M tris-buffered saline, with 0.05% Tween 20, pH 8 (TBS-Tween, Sigma). They were then incubated overnight at + 4 °C with the 6F3D anti-Aβ antibody (Dako, 1/200), polyclonal anti-tau antibody (Dako, 1/500), monoclonal anti-alpha-synuclein (LB509, Zymed, 1/250), polyclonal anti-TDP43 (Protein Tech Group, 1/1000) routinely used for Aβ, tau, alpha-synuclein and TDP43 detection, respectively. Sections were further incubated with a biotinylated secondary antibody for 25 min at room temperature, and the presence of the secondary antibody was revealed by a streptavidin–horseradish peroxidase conjugate using diaminobenzidine (Dako, Glostrup, Denmark). Slices were counterstained with Harris hematoxylin.
Brain samples were also evaluated by biochemistry. For tau protein extraction, brain homogenates were sonicated on ice for 5 min, centrifuged for 5 min at 3000 × g at + 4 °C, diluted in 20 mM Tris/2% SDS and sonicated on ice for 5 min. For Aβ, Iba1 and GFAP protein extractions, brain homogenates were sonicated (6 strokes, cycle 0.5, 30% amplitude) in a lysis buffer at a final concentration of 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% Triton-X-100 supplemented with 1X protease inhibitors (Complete™ Mini, EDTA-free Protease Inhibitor Cocktail, Roche) and 1/100 diluted phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2, Sigma-Aldrich). Samples were centrifuged at 20,000 × g for 20 min at + 4 °C and the supernatant was collected for further use. Extracted samples were stored at − 80 °C after evaluation of total protein concentration by a BCA assay (Pierce™). For tau characterization, samples were diluted to 1 μg/μL, diluted in 2X lithium dodecyl sulfate (LDS, Thermo Fisher Scientific) buffer with reducers and heated at + 100 °C for 10 min. 15 μg of samples were loaded on a 12% Bis-TrisCriterion™ gel (Bio-Rad) and migrated in MOPS buffer for 1 h at 165 V on ice. After protein transfer on nitrocellulose sheets, migration and quality of the transfer were checked with a ponceau S staining. The membrane was saturated for 1 h at room temperature, and was then incubated with the AT100 (pT212-pS214, Life technologies MN1060), 2H9 (pS422, 4BioDx 4BDX-1501), tau-Nter (12–21, LB lab-made) or tau-Cter (clone 9F6, LB lab-made) antibodies overnight at + 4 °C. A peroxidase coupled secondary anti-rabbit or anti-mouse antibody was then applied for 45 min at room temperature. Immunoblotting was revealed by ECL. GAPDH (Sigma 9545) was used as a loading control. For Iba1 and GFAP evaluations, extracted samples were denatured at + 90 °C for 5 min in a buffer containing 1X LDS (NuPAGE® LDS sample buffer, Invitrogen) and DTT 1X (NuPAGE® sample reducing agent, Invitrogen). 10 µg of denatured protein were loaded per well. Samples and molecular weight marker (Bio-Rad Precision Plus Protein™ Dual Color standards) were loaded on 4–20% Criterion™ TGX™ gels (Bio-Rad) and migration was performed in a 1X tris–glycine buffer (Bio-Rad) at 120 V for 1 h. Proteins were then transferred to a nitrocellulose membrane using the Trans-Blot® Turbo™ (Biorad) system. Migration and quality of the transfer were checked with a ponceau S staining. The membrane was then blocked with a TBS/0.1%Tween, 5% milk solution for 1 h at room temperature, and incubated with the primary antibody Iba1 (Wako 1919741, 1/2000), GFAP (Dako Z0334, 1/5000) or actin (Sigma A2066, 1/5000) diluted in saturation buffer overnight at + 4 °C. After washing in TBS/0.1%Tween solution, the membrane was incubated with the appropriate secondary HRP-conjugate antibody diluted to 1/5000 in TBS/0.1%Tween for 1 h at room temperature. The chemiluminescent signal was revealed using the Clarity western ECL (Bio-Rad) kit and the Chemidoc™ MP (Bio-Rad) imaging system. Protein band intensities were quantified on the ImageJ software and normalized by the actin expression level. For Aβ protein quantification, all assay-specific material (pre-coated microtiter plate, buffers, antibodies, standard solutions) was provided in the V-PLEX kit Aβ Peptide Panel 1 (6E10) (MSD®). Human brain homogenates were diluted to 1/5 (Ctrl samples) or 1/10 (AD samples) in the dilution buffer. As described in the manufacturer's protocol, the microtiter plate was blocked for 1 h at room temperature with the appropriate buffer. After washing, 25 µl of detection antibody and 25 µl of diluted sample or standard were added in duplicate to the wells and incubated under continuous agitation for 2 h at room temperature. Wells were washed and 150 µl of reading buffer was added. Plate reading was performed with the MSD Sector Imager 2400 (model 1200) multiplex assay system. Aβ1–38, Aβ1–40 and Aβ1–42 quantifications were performed with the Discovery Workbench 4.0 MSD® software. Tau protein quantifications (total tau and phosphot-tau181) were performed according to the manufacturer’s protocol. Briefly, brain homogenates were diluted to 1/100 and 1/200 in the provided dilution buffer. 50 µl of standards or samples, as well as 50 µl of detection antibody solution were added to wells and incubated for 14 h at + 4 °C. After washing, 100 µl of 1X anti-rabbit IgG HRP solution was added for a 30 min incubation period at room temperature. 100 µl of stabilized chromogen were then added to each well for 30 min at room temperature, in the dark. The reaction was stopped by adding 100 µl of Stop solution and the plate was read at 450 nm within the hour. Data were analyzed with GraphPad Prism 7 using the 4PL method. All samples were tested in duplicates.
Brain extracts preparation
Parietal cortex samples from each patient were individually homogenized at 10% weight/volume (w/v) in a sterile 1X Dulbecco’s phosphate buffer solution in CK14 soft tissue homogenizing tubes at 5000 rpm for 20 s (Precellys®, Bertin technologies). They were then sonicated on ice for 5 s at 40% amplitude and centrifuged at 3000 g for 5 min at + 4 °C. The resulting supernatant was aliquoted in sterile polypropylene tubes and stored at − 80 °C until use. Ten percent individual brain extracts were thawed on ice and combined together according to three groups: (i) Control (n = 2), (ii) AD patients with a disease duration of 5 to 8 years (n = 4, AD1), or (iii) AD patients with a disease duration of 6 months to 3 years (n = 4, AD2). Aβ levels, total tau and phospho-tau181 as well as Iba1 and GFAP protein levels were assessed by biochemistry in each combined brain extract.
Transgenic mice
Mouse experiments used the APPswe/PS1dE9 mouse model of amyloidosis (C57Bl6 background) [8]. Aβ plaques can be detected as early as 4 months of age in these mice and increase in number and total area with age [8]. This model expresses endogenous murine tau protein isoforms and is not transgenic for any human tau. At the time of the inoculation of AD or Ctrl brain extracts, at 2 months of age, these mice did not have Aβ plaques. Animals were studied for four or eight months after intracerebral inoculation of the brain extracts (at 4 and 8 months post-inoculation (mpi) respectively, nCtrl = 11 and 15, nAD1 = 14 and 15, nAD2 = 12 and 20). Wild-type littermates injected with the Ctrl brain sample were used as controls for the behavioral tests (at 4 and 8 mpi respectively, nWT = 6 and 12). A cohort of 21 animals was studied by immunohistochemistry one month post-inoculation (nCtrl = 6, nAD1 = 7, nAD2 = 8). As the Aβ plaques and tau loads were similar between AD1 and AD2 inoculated animals at a given time point (see results), measures from AD1 and AD2 animals were pooled within a single group that we called ADbe-inoculated and brain extracts are referred to as ADbe. All APPswe/PS1dE9 mice were born and bred in our center (Commissariat à l’Energie Atomique, centre de Fontenay-aux-Roses; European Institutions Agreement #B92-032-02). All animals were randomly assigned to the experimental groups using a simple procedure: They were identified using increasing numbers based on their birthdate. Animals with increasing numbers were alternatively assigned to the Ctrl (animal 1, 4, 7…), AD1 (animal 2, 5, 8…) and AD2 groups (animal 3, 6, 9…). Males were exclusively used in this study in order to optimize group homogeneity (Aβ plaque load is known to vary between males and females). Mice were injected during different inoculation sessions and each group was randomly inoculated at each session to avoid an "order of treatment" confounding effect. All experimental procedures were conducted in accordance with the European Community Council Directive 2010/63/UE and approved by local ethics committees (CEtEA-CEA DSV IdF N°44, France) and the French Ministry of Education and Research (A17_083 authorization), and in compliance with the 3R guidelines. Animal care was supervised by a dedicated in-house veterinarian and animal technicians. Human endpoints concerning untreatable continuous suffering signs and prostrations were taken into account and not reached during the study. Animals were housed under standard environmental conditions (12-h light–dark cycle, temperature: 22 ± 1 °C and humidity: 50%) with ad libitum access to food and water. The design and reporting of animal experiments were based on the ARRIVE reporting guidelines [7]. Sample size was based on previous experiments for Aβ induction in APPswe/PS1dE9 mice after inoculation of human brain (n = 8 at 4 mpi estimated with significance level of 5%, a power of 80%, and a two-sided test) [9] and increased to take into account uncertainties for new markers (tau lesion load, memory and synaptic changes). No animals were excluded from the study. SL was aware of initial group allocation, but further analyses (memory evaluations and post-mortem studies) were performed blinded.
Stereotaxic surgery
Ten percent Ctrl, AD1 and AD2 brain extracts were thawed on ice. Extracts were then pooled together according to their group and the three resulting combined samples (Ctrlbe, AD1be, AD2be) were sonicated (70% amplitude, 10 s on/off; Branson SFX 150 cell disruptor sonicator, 3.17 mm microtip probe Emerson, Bron) on ice in a sterile environment, extemporaneously before stereotaxic injection.
Two month-old APPswe/PS1dE9 and wild-type littermates were anesthetized by an intraperitoneal injection of ketamine (1 mg/10 g; Imalgène 1000, Merial) and xylazine (0.1 mg/10 g; 2% Rompun, Bayer Healthcare). Local anesthesia was also performed by a subcutaneous injection of lidocaine at the incision site (1 µl/g; 0.5% Xylovet, Ceva). Mice were placed in the stereotaxic frame (Phymep) and bilateral injections of brain samples were performed in the dorsal hippocampus (AP − 2 mm, DV − 2 mm, L ± 1 mm from bregma). Two µl/site of sample were administered using 34-gauge needles and Hamilton syringes, at a rate of 0.2 µl/min. After the injection, needles were kept in place for 5 more minutes before removal and the incision was sutured. The surgical area was cleaned before and after the procedure using povidone iodine (Vétédine, Vétoquinol). Respiration rate was monitored and body temperature was maintained at 37 ± 0.5 °C with a heating pad during the surgery. Anesthesia was reversed with a subcutaneous injection of atipamezole (0.25 mg/kg; Antisedan, Vetoquinol). Mice were placed in a ventilated heating box (25 °C) and monitored until full recovery from anesthesia. Postoperative anticipatory pain management consisted of paracetamol administration in drinking water (1.45 ml/20 ml of water; Doliprane, Sanofi) during 48 h.
Behavioral evaluations
A novel object recognition task in a V-maze was used to investigate cognition at 4 mpi or 8 mpi on brain-extract inoculated APPswe/PS1dE9 mice. Wild-type littermates injected with the Ctrlbe were used as controls for the tests. Mice were handled for 2 min per day, for 5 days prior to any test to prevent stress effects during tasks. Prior to each test, mice were habituated to the experimental room for 30 min. The experimenter was blind to mouse groups. Performances were recorded using a tracking software (EthoVision XT14, Noldus).
The V-maze arena consisted of two 6 cm-wide, 33.5 cm-long and 15 cm-high black arms forming a V shape and exposed to 50 lx-lighting. The test was divided into three phases, each one separated by 24 h. At the beginning of each session, mice were placed at the center of the arena, i.e. at the intersection of the arms. During the habituation phase (day 1), mice were free to explore the empty arena for 9 min. The distance travelled was automatically recorded as an indicator of their exploratory activity. For the training phase (day 2), two identical objects (bicolor plastic balls) were placed at the end of each arm. Exploratory activity was evaluated as the time spent exploring the objects (manually recorded) and the distance travelled during the 9-min trial. On the test day (day 3), one familiar object (a bicolor plastic ball) was replaced by a novel one of a different shape and material (a transparent glass flask). Recognition was assessed using a discrimination index, calculated as follows:
$$Discrimination\,\, index= \frac{Time\,\, exploring\,\, the \,\,novel \,\,object-Time \,\,exploring\,\, the\,\, familiar \,\,object}{Total\,\, exploration\,\, time}$$
It reflects the time spent exploring each object, and therefore, the ability to discriminate a novel object from a familiar, previously explored one. A low discrimination index score reveals that mice spent less time exploring the new object, i.e. still had marked interest in the familiar object, and suggests that memory was impaired. Between each run, the V-maze was cleaned with 10% ethanol, effectively eliminating any scents from previous visites.
Animal euthanasia and brain preparation for histology
Mice were sacrificed at 4 or 8 mpi, after the behavioral tests, with an intraperitoneal injection of a lethal dose of pentobarbital (100 mg/kg; Exagon, Axience). They were perfused intracardiacally with cold sterile 0.1 M PBS for 4 min, at a rate of 8 ml/min. The brain was extracted and post-fixed in 4% paraformaldehyde for 48 h at + 4 °C, transferred into a 15% sucrose solution for 24 h and in a 30% sucrose solution for 48 h at + 4 °C for cryoprotection. Serial coronal sections of 40 µm were performed with a microtome (SM2400, Leica Microsystem) and stored at − 20 °C in a storing solution (glycerol 30%, ethylene glycol 30%, distilled water 30%, phosphate buffer 10%). Free-floating sections were rinced in a 0.1 M PBS solution (10% Sigma-Aldrich® phosphate buffer, 0.9% Sigma-Aldrich® NaCl, distilled water) before use. Washing and incubation steps were performed on a shaker at room temperature unless indicated otherwise.
Immunohistochemistry for Aβ, tau, microglia and astroglia
Aβ deposits were evaluated using a 4G8 labelling. Tau was evaluated using labelling with AT8 directed against hyperphosphorylated tau and AT100 that binds to a conformational epitope including phosphorylated Thr212 and Ser214. Microglia were evaluated using Iba1 and CD68 antibodies. Astrocytes were stained with the GFAP antibody.
4G8 labelling was performed after pretreating brain sections with 70% formic acid (VWR®) for 20 min at room temperature. AT8 and AT100 labellings were performed after a pre-treatment with EDTA 1X citrate (Diagnostic BioSystems®) for 30 min at 95 °C. All tissues were then incubated in 30% hydrogen peroxide (Sigma-Aldrich®) diluted 1/100 for 20 min to inhibit endogenous peroxidases. Blocking of non-specific antigenic sites was achieved over 1 h using a 0.2% Triton X-100/0.1 M PBS (Sigma-Aldrich®) (PBST) solution containing 4.5% normal goat serum or 5% bovine serum albumin. Sections were then incubated at + 4 °C with the 4G8 (Biolegend 800706, 1/500), Iba1 (Wako 1919741, 1/1000), CD68 (Serotec—Biorad MCA 1957, 1/800) or GFAP (Dako Z0334, 1/10000) antibody diluted in a 3%NGS/PBST solution for 48 h, or with the AT8 (Thermo MN1020B, 1/500) or AT100 (Thermo MN1060, 1/500) antibody diluted in a 3%NGS/PBST solution for 96 h. After rinsing, an incubation with the appropriate biotinylated secondary antibody diluted to 1/1000 in PBST was performed for 1 h at room temperature, followed by a 1 h incubation at room temperature with a 1:250 dilution of an avidin–biotin complex solution (ABC Vectastain kit, Vector Laboratories®). Revelation was performed using the DAB Peroxidase Substrate Kit (DAB SK4100 kit, Vector Laboratories®). Sections were mounted on Superfrost Plus slides (Thermo-Scientific®). For the AT8 and AT100 labellings, a cresyl violet counterstain was performed. All sections were then dehydrated in successive baths of ethanol at 50°, 70°, 96° and 100° and in xylene. Slides were mounted with the Eukitt® mounting medium (Chem-Lab®).
Stained sections were scanned using an Axio Scan.Z1 (Zeiss®—Z-stack images acquired at 20× (z-stacks with 16 planes, 1 µm steps with extended depth of focus)). Each slice was extracted individually in the .czi format using the Zen 2.0 (Zeiss®) software. Image processing and analysis were performed with the ImageJ software. Macros were developed for each staining in order to attain a reproducible semi-automated quantification. Images were imported with a 50% reduction in resolution (0.44 µm/pixel), converted to the RGB format and compressed in .tif format. For the 4G8, Iba1 and CD68 immunostainings, segmentation was performed through an automatic local thresholding using the Phansalkar method (radius = 15). Aβ load was evaluated after quantification of the 4G8-labelled particles between 8 and 2000 µm2, and normalization to the surface area of each ROI. Microglial status was evaluated as a percentage of Iba1- or CD68-positive surface area in each ROI. For the AT8 and AT100 stainings, the blue component of each image was extracted to remove the cresyl violet counter-staining from the analysis. AT8-positive or AT100-positive tau loads were assessed following an automatic local thresholding of the staining with the Phansalkar method followed by an evaluation of the percentage of AT8-positive or AT100-positive surface area in each ROI.
Tau lesions occur in the form of neuropil threads, neurofibrillary tangles (NFTs), and neuritic plaques i.e. tau aggregates within neurites surrounding Aβ deposits. In addition for the AT8 immunostaining, a quantification of neuritic plaques and NFTs was performed by manual counting. The AT8-positive area stained within neuritic plaques was evaluated by drawing circular regions of interest (with a constant area of 6 µm2), and by quantifying the percentage of tau-positive regions within each ROI, using the same thresholding method as previously described. A semi-quantitative analysis of neuropil threads was also performed by assigning a severity score based on the intensity and extent of AT8-positive staining in each ROI. All quantifications were performed on adjacent slices between − 0.34 and − 4.36 mm from bregma. Ten adjacent slices were analyzed for the 4G8 staining, and 5 for Iba1, CD68, AT8, and AT100 stainings. All ROIs were manually segmented using the Paxinos and Franklin neuro-anatomical atlas of mouse brain [20].
Gallyas silver staining
Free-floating sections were mounted on Superfrost Plus (Thermo-Scientific®) slides and dried overnight prior to Gallyas staining. Section were permeabilized by successive incubations in toluene (2 × 5 min) followed by ethanol at 100°, 90° and 70° (2 min per solution). Slides were then incubated in a 0.25% potassium permanganate solution for 15 min, in 2% oxalic acid for 2 min then in a lanthanum nitrate solution (0.04 g/l lanthanum nitrate, 0.2 g/l sodium acetate, 10% H2O2 30%) for 1 h to reduce non-specific background. Several rinses with distilled water were performed followed by an incubation in an alkaline silver iodide solution (3.5% AgNO3 1%, 40 g/l NaOH, 100 g/l KI) for 2 min. The reaction was neutralized with 0.5% glacial acetic acid baths (3 × 1 min) and sections were incubated for 20 min in a developing solution (2 g/l NH4NO3, 2 g/l AgNO3, 10 g/l tungstosilicilic acid, 0.76% formaldehyde 37%, 50 g/l anhydrous Na2CO3). Several rinses with 0.5% acetic acid (3 × 1 min) followed by an incubation in 1% gold chloride solution for 5 min were then carried out. Sections were rinsed with distilled water and the staining was fixed with a 1% sodium thiosulfate solution. All sections were then rinsed with distilled water and dehydrated for 1 to 5 min in successive baths of ethanol at 50°, 70°, 96° and 100° and in xylene. Slides were mounted with the Eukitt® mounting medium (Chem-Lab®). All steps were performed at room temperature.
Co-stainings of microglia and Aβ plaques
In order to evaluate microglial load surrounding Aβ plaques, the co-staining of microglia and Aβ plaques was performed. Free-floating sections were permeabilized in a 0.2% Triton X-100/0.1 M PBS (Sigma-Aldrich®) solution for 3 × 10 min. Slices were stained by MXO4 dye (Tocris #4920, 1/300) for 30 min at room temperature, and then washed in a 0.1 M PBS solution. Sections were blocked in a 4.5%NGS/PBST solution for 1 h at room temperature before being incubated with the Iba1 antibody (Wako 1919741, 1/1000). 24 h later, sections were rinsed in 0.1 M PBS and incubated for 1 h at room temperature with the appropriate secondary antibody diluted to 1/1000 in PBST (anti-rabbit AlexaFluor 633). Sections were rinsed and mounted on Superfrost Plus (Thermo-Scientific®) slides with the Vectashield® mounting medium with a refractive index of 1.45. Images of stained sections were acquired using a Leica DMI6000 confocal optical microscope (TCS SPE) with a 40 × oil-immersion objective (refractive index 1.518) and the Leica Las X software. A confocal zoom of 3 and a pinhole aperture fixed at 1 Airy were applied. Acquisition was performed in sequential mode with a sampling rate of 1024 × 1024 and a scanning speed of 700 Hz. Image resolution was 60 nm/pixel and the optical section was 0.896 µm. Twelve separate planes with a 0.1 µm step were acquired. The excitation wavelengths were 633 nm (for Iba1) or 350 nm (for Aβ). Image acquisition was performed on 2 slices located between − 3.28 and − 4.24 mm from the bregma, with 3 images per slice for the CA1 region and for the perirhinal/entorhinal cortex. 3D deconvolution of the images was performed using the AutoQuant X3 software. The deconvoluted 8-bit images were analyzed using the ImageJ software. Quantification of microglial load around plaques was based on a thresholding procedure applied across all images to segment microglial cells. MXO4-positive surfaces were dilated as circular regions of interest (with a diameter of 40 µm) drawn around the Aβ plaque to define a dilated plaque area. Microglial staining within the dilated surface, e.g. within the plaque area, was included in the analysis.
Evaluation of synaptic density
Synaptic density was evaluated in the hippocampus (CA1) and the perirhinal/entorhinal cortex of all inoculated mice using a double immunolabelling of presynaptic (Bassoon) and postsynaptic (Homer1) markers. Free-floating sections were permeabilized in a 0.5% Triton X-100/0.1 M PBS (Sigma-Aldrich®) solution for 15 min. Slices were incubated with Bassoon (Abcam Ab82958, 1/200) and Homer1 (Synaptic systems 160003, 1/400) antibodies diluted in 3% BSA/PBST solution for 24 h at + 4 °C. Incubation with secondary antibodies coupled to a fluorochrome (Alexa Fluor) diluted in a 3% BSA/0.1 M PBS solution was then performed for 1 h at room temperature. Sections were rinsed and mounted on Superfrost Plus (Thermo-Scientific®) slides with the Vectashield® mounting medium with a refractive index of 1.45. Images of stained sections were acquired using a Leica DMI6000 confocal optical microscope (TCS SPE) with a 63 × oil-immersion objective (refractive index 1.518) and the Leica Las X software. A confocal zoom of 3 and a pinhole aperture fixed at 1 Airy were applied. Acquisition was performed in sequential mode with a sampling rate of 1024 × 1024 and a scanning speed of 700 Hz. Image resolution was 60 nm/pixel and the optical section was 0.896 µm. 26 separate planes with a 0.2 µm step were acquired. The excitation wavelengths were 594 nm or 633 nm. Image acquisition in the CA1 region was performed on 4 adjacent slices located between − 1.82 and − 3.28 mm from the bregma, with 2 images per slice. For the perirhinal/entorhinal cortex, 3 adjacent slices located between − 3.28 and − 4.24 mm from the bregma were analyzed, with 2 images acquired per slice. 3D deconvolution of the images was performed using the AutoQuant X3 software. The deconvoluted 8-bit images were analyzed using the ImageJ software, as described in Gilles et al. [10]. Briefly, automated 3D segmentation of the presynaptic (Bassoon) and postsynaptic (Homer1) stained deconvoluted images was performed using "3D spots segmentation" from ImageJ (with "gaussian fit", "block" and "no watershed" options; https://imagej.net/plugins/3d-segmentation). Co-localization of overlapping objects was evaluated using "3D MultiColoc" from imageJ (https://imagejdocu.list.lu/plugin/analysis/details_about_multi-colocalisation_analysis/start). The number of colocalized objects was quantified as an index of synaptic density.
Statistical analysis
Statistical analysis was performed using the GraphPad Prism software 8. For the behavioral tasks analysis, Kruskal–Wallis tests with Dunn’s multiple comparisons were perfomed except when repeated measures were acquired, in which case, a two-way repeated measures ANOVA with the Geisser-Greenhouse correction and Dunnett’s multiple comparisons was carried out. For the post-mortem analysis, Mann–Whitney tests were performed in order to compare differences between ADbe- and Ctrlbe-inoculated mice. For correlation studies, Spearman correlation test was performed. The significance level was set at p < 0.05. Data are shown on scattered dot plots with mean ± standard error of the mean (s.e.m).