Animals and treatments

Tg2576 transgenic mice carry a transgene coding for the 695-amino acid isoform of human APP derived from a large Swedish family with early-onset AD [25]. These mice express high concentrations of the mutant Aβ, develop significant number of amyloid plaques and display memory deficits. Tg2576 mice and their non-transgenic littermates (001349-W), which served as controls, were purchased from Taconic Europe (Lille Skensved, Denmark). Mice were all genotyped for retinal degeneration.

Six-months-old transgenic females and aged-matched non-transgenic littermates were used. Transgenic and non-transgenic mice (N = 17–27 per treatment group) were treated for 13 months with CHF5074 (125 and 375 ppm in the diet), DAPT (375 ppm/day in the diet) or standard diet (herafter designated as “vehicle”). CHF5074- and DAPT-medicated diets were provided by Mucedola (Settimo Milanese, Italy). The estimated ingested doses of CHF5074 were about 20 and 60 mg/kg/day (behaviourally effective dose when given chronically for 9 months) [21]; the estimated ingested dose of DAPT was about 60 mg/kg/day. The dose of DAPT was selected based on previous studies showing that the drug given orally at doses ranging from 10 to 100 mg/kg dose-dependently inhibited cortical Aβ levels in hAPP transgenic mice [26]. After behavioural testing, mice were sacrificed and their brains were split in the two hemispheres. The left hemisphere was divided into anterior and posterior parts at level −0.70 according to the Paxinos and Franklin atlas of the mouse brain [27]. The anterior part, containing the olfactory bulb, was quickly frozen into liquid N2, while the posterior part, including the hippocampus, was fixed for amyloid plaque and Aβ oligomer analysis. The right hemisphere was divided as above and fixed for immunohistochemistry (anterior part) and Golgi-Cox staining (posterior part, including the hippocampus). Animal care and treatments were in accordance with the EU Directive 2010/63/EU for animal experiments and in conformity with protocols approved by the Ethical Committee of Animal Experimentation, University of Bologna.

Novel object recognition test (NOR)

Long-term memory was evaluated in 18 months-old mice using NOR, measuring recognition memory under spontaneous behavioural conditions. Before NOR, all animals were tested for the papillary reflex and only positive animals were included in the study. Mice were tested in an open-square grey arena (46 x 46 cm), 30 cm high (Ugo Basile, Comerio, Italy). The task started with a habituation trial in which the animals were placed into the empty arena for 10 min. The next day, mice were placed into the same arena containing two identical objects (familiarization phase). In order to evidence side preferences, exploring times spent on left and right familiar objects were recorded separately. The exploratory behaviour was analyzed by calculating the investigation time on both objects. Sniffing and touching the object at a distance not greater than 2 cm were scored as object investigation. Four hours later (test trial) mice were placed in the arena containing one object identical to the one presented during the familiarization phase (familiar object), and a new one (novel object); the time spent exploring the two objects was recorded for 10 min. The videotracking software AnyMaze (Stoelting, Wood Dale, Illinois) was used for analysis. Memory was expressed as recognition index: (seconds on novel – seconds on familiar)/(total time on objects). Animals with no memory impairment spent longer time investigating the novel object, giving a higher discrimination index.

Golgi-Cox staining

FD Rapid GolgiStain™ Kit (FD NeuroTechnologies, Ellicott City, MD, USA) was used for detailed morphological analysis of dendritic spines. Tissue was rapidly frozen, cut on a cryostat and mounted on gelatin-coated microscope slides. Sections were processed with the NDT104 FD Rapid GolgiStain™ Kit. For each mouse, ten fully impregnated hippocampal CA1 neurons were identified under low magnification (20×, 0.5NA). The acceptance criteria for spines were according to Middei et al. [28]. Dendritic spines were measured under high magnification (100×, 1.25NA) from images acquired using a digital camera (Olympus F-View). Measurements were performed on secondary and tertiary branches of CA1 basal dendrites irrespective of their orientation. On each neuron, at least five dendritic spines were selected according to the criteria proposed by Knafo et al. [29]: only considering spines separated throughout their entire length from neighbour spines or dendrites and excluding spine-like protrusions with bifurcated heads or with heads longer than 3.5 μm. Spine length was defined as the distance from the dendritic shaft to the tip of the spine.

Immunohistochemistry and image analysis

Indirect immunofluorescence was used to determine immunoreactivity associated with intracellular Aβ, cyclin A, NeuN. Brain tissue was immersed in 4% paraformaldehyde for 24 hours and then washed in 5% sucrose in phosphate buffer. Sections (14 μm thick) were cut with a cryostat to include the medial cortex. The following primary antibodies were used: 6E10 anti-Aβ 1–16 monoclonal antibody (Covance, Princeton, NJ) for APP/Aβ (this antibody reacts with the sAPPβ precursor as well as with the processed forms of Aβ) at a 1:1000 dilution; 4G8 anti-Aβ 17–24 monoclonal antibody (Covance) for APP/Aβ (this antibody reacts with the sAPPβ precursor as well as with the processed forms of Aβ) at a 1:800 dilution; rabbit anti Cyclin A (ab7956, AbCam, Cambridge, UK) diluted 1:200; mouse anti neuronal nuclei antibody (NeuN, MAB377 Chemicon) diluted 1:200. Appropriate Rhodamine Red-X-and FITC-conjugated secondary antibodies were used for detection (Jackson ImmunoResearch, Baltimore, PA).

For intraneuronal Aβ immunostaining, about 50 neurons in the anterior cingulated cortex were analyzed for each animal. Stained specimens were analyzed with a Nikon 600 Eclipse microscope equipped with a Nikon DXM1200F digital camera (Nikon Italia, Florence, Italy). The ProPlus software (Media Cybernetics Inc, Bethesda, MD) was used to evaluate optical density in single cells. The expression of cell cycle proteins was analyzed in two sections/animal, in both genotypes. NeuN-positive cells were scored within layers II/III of the frontal cortex, and the percentage of cyclinA immunoreactive, NeuN-positive cells was recorded [30]. For all markers, the mean value/animal was used for statistical analysis. All analyses were performed in a blinded manner.

Brain β-amyloid plaques and activated microglia

Brain samples corresponding to the posterior half of the left hemisphere were used for quantitative analysis of plaques and activated microglia. They were fixed in 10% formalin and then embedded in paraffin according to a standard procedure. Coronal sections (10 μm thick) ranged from bregma −1.46 mm (anterior) to −2.06 mm (posterior) [27]. Aβ plaque immunohistochemistry was performed using the biotinylated 6E10 monoclonal antibody (Signet Laboratories, Dedham, MA) diluted 1.250 as primary antibody. Pretreatments were: incubation in a 3% H 2 O 2 solution in distilled water for 15 minutes to block endogenous peroxidase; incubation in 80% formic acid for 30 minutes for antigen retrieval. After rinsing in TBS for 10 minutes, sections were incubated overnight at 4°C in a humid atmosphere with the primary antibody diluted in TBS containing 0.3% Triton X-100. After rinsing in TBS for 10 min, sections were incubated for 60 min in a humid atmosphere with the streptavidin-peroxidase solution, according to the mouse-on-mouse kit procedure (Dako Cytomation, Glostrup, Denmark) using a peroxidase-based revealing system. Peroxidase activity was detected by treatment with 3,3′-diaminobenzidine (DAB) for 5 minutes. Slides were photographed using a digital Nikon DS microscope colour camera. Digital images were analyzed using NIS-Elements software (Nikon, Tokyo, Japan). Each image was analyzed using the automated target detection mode. Imagesize was 1280 x 960 pixels with a target area size of 68,000 μm2. The software determined the number of plaques, the plaque mean area and the plaque area fraction (immunopositive area/total area used as scan object). Twelve counts were performed for each of the two levels considered. Analyses were performed in analogous areas of the cortex and hippocampus using a 10x objective.

Activated microglia in CA1 region of hippocampus was immunodetected using the Iba1 Rabbit polyclonal antibody (Biocare, Concord, CA). A 20x objective and a target area of 16,900 μm2 was used for this analysis, which involved three counts in homologous areas of CA1 region of hippocampus. After rinsing in distilled water, sections were incubated in a 3% H 2 O 2 solution in distilled water for 15 minutes. After rinsing in TBS for 10 minutes, sections were incubated for 30 minutes with normal goat serum diluted 1:20 with TBS. The sections were then incubated overnight at 4°C in a humid atmosphere with the primary Iba1 antibody diluted 1:250 with TBS. After rinsing in TBS for 10 min, sections were incubated for 60 min in a humid atmosphere with the secondary antibody solution provided by the Goat anti Rabbit Envision System (Dako, Denmark) diluted 1:1 with TBS. Peroxidase activity was detected by treatment with DAB.

Brain Aβ oligomers

Aβ oligomers were determined in low-detergent (0.1% Triton-X100, 0.01% Nonidet-P40) and high-detergent (3% SDS, 0.5% Triton X-100, 1% deoxycholate) extracts prepared from left hemi-forebrains specimens as described by Lesné et al. [31] with slight modifications. These included cumulative extraction (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 2 mM EDTA, 0.01% NP40, 0.1% Triton X-100, plus 1 mM phenylmethylsulfonyl fluoride) and mixing of the soluble (extracellular-enriched) and cytoplasmic fractions, and the omission of the endogenous immunoglobulin depletion step in order to avoid any artefactual modification of the samples. The residual pellet remaining after low-detergent extraction was further extracted by gentle agitation in 200 μl of a buffer solution containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 3% SDS, 1% deoxycholate and 1 mM PMSF, followed by centrifugation for 90 min at 13,000 rpm. Total protein content of each extract was determined in triplicate with the Bradford Protein Assay reagent (Bio-Rad) using bovine serum albumin as standard; the average of the resulting values (differing by less than 5% of the mean) was used to balance sample input for immunoblot analysis. All extractions were performed in three experimental sessions, carried out at three-days intervals on randomized subsets of the various brain samples, and the resulting extracts (a total of 65) were flash-frozen in dry ice and stored at −80°C. For immunoelectrophoretic analysis, brain extracts (100 μg or 50 μg total protein/each for the low-detergent and the high-detergent extracts, respectively) were heated for 15 min at 70°C in sample buffer and electrophoresed on pre-cast 4-12% Bis-Tris Midi gradient gels (Invitrogen). Gel-fractionated proteins were electro-transferred to 0.2 μm nitrocellulose membranes, which were boiled for 25 sec in PBS, soaked for 10 min in PBS containing 100 mM β-mercaptoethanol and 0.1% SDS, washed three times (10 min each) with PBS and blocked with 5% bovine serum albumin in Tris-buffered saline, prior to antibody addition and incubation using the Snap i.d. blotting system (Millipore). The 6E10 monoclonal antibody (diluted 1:500) was used for immunodetection. Antigen-antibody complexes were revealed with a goat anti-mouse, IrDye 680-labeled secondary antibody (LI-COR; diluted 1:3000), followed by visualization and quantification of immune-reactive bands by near-infrared fluorescence with an Odyssey imager (LI-COR). A total of 14 independent sets of analyses, each comprising 10 sample extracts with at least one vehicle-treated wild-type control, were performed. Non-specific, 6E10 mAb cross-reactive polypeptides, present in both wild-type and Tg2576 brain extracts, were used as loading controls and internal references for data normalization. Synthetic prefibrillar Aβ42(n) prepared according to Lambert et al. [32], with n-values ranging from 1 to 4, was used as size standard for electrophoretic analysis. Results, expressed as NIRF arbitrary units, were analyzed with the SigmaStat™ software and are presented as mean ± standard error of the mean (SEM).

Aβ40 and Aβ42 plasma levels

At the time of sacrifice, blood samples were collected in EDTA-vacuum collection tubes, centrifuged at 4000 g for 10 min and plasma aliquots were stored at −80°C. Simultaneous quantification of β-amyloid 1-40 (Aβ40) and β-amyloid 1-42 (Aβ42) peptides was performed on properly diluted plasma samples using the INNO-BIA plasma Aß forms kit (Innogenetics NV, Gent, Belgium, through Innogenetics srl, Italy), following the manufacturer’s instructions. This kit is a fluorimetric bead-based immunoassay using Luminex xMAP® technology. Briefly, the Aβ40 and Aβ42 peptides are captured selectively by a mix of beads (xMAP® microspheres) coated with three monoclonal antibodies (mAb): 21 F12 for Aβ42, 2G3 for Aβ40 and AT120 for matrix. Following an overnight incubation step at 4°C, the mix is washed and subsequently incubated with the “detection conjugate” solution (phycoerythrin-labeled streptavidin) at room temperature for 1 h. The mix of beads is then washed and read using the Luminex® 100™ IS Total System, which analyzes the microspheres/beads in a flow stream. The fluorescence signals associated with individual beads are converted into intensity units by a digital signal processor and then related to the concentration of the bound antigen through the Luminex IS 2.3 software.

Statistical analysis

Data were analyzed with the appropriate model of analysis of variance (ANOVA) depending on the type of variable. Behavioural data were analyzed with two-way analysis of variance with “genotype” (non-transgenic and Tg2576) and “treatment” (vehicle, CHF5074/DAPT) as fixed factors and mouse as random factor. For balanced design (object recognition task), the ANOVA model also included “genotype by treatment” as fixed factor. To reduce the loss of power due to multiple testing post hoc comparisons were only directed toward the transgenic control group (Tg2576-vehicle) and were carried out with the Holm-Sidak’s test. Two-tailed p values were calculated. Calculations were performed with the statistical software SigmaStat™ (Version 3.5, SPSS, Chicago, IL). Results were generally presented as mean ± standard error of the mean (SEM).