Reagents and chemicals

SLAB51 formulation was provided by Mendes Sa (Lugano, Switzerland). Substrates for assaying the chymotrypsin-like (ChT-L), trypsin-like (T-L), and peptidyl glutamyl-peptide hydrolyzing (PGPH) activities of the proteasomal complex were purchased from Sigma-Aldrich S.r.L. (Milano, Italy). The substrate Z-Gly-Pro-Ala-Leu-Ala-MCA to test the branched chain amino acids preferring (BrAAP) activity was obtained from Biomatik (Cambridge, Ontario). Aminopeptidase N (EC 3.4.11.2) for the coupled assay utilized to detect BrAAP activity36 was purified from pig kidney as reported elsewhere37. Cathepsin B and cathepsin L substrates (Z-Arg-Arg-AMC and Z-Phe-Arg-AFC.trifluoroacetate) were from Sigma-Aldrich S.r.L. (Milano, Italy). Membranes for western blotting analyses were purchased from Millipore (Milano, Italy). Proteins immobilized on films were detected with the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Milano, Italy). p27 antibody was purchased from Calbiochem (EDM Millipore, Billerica, MA). All the other antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany). ELISA Kit for Aβ 1–40 and Aβ 1–42 peptide determination in brain homogenates were purchased from Invitrogen (Camarillo, CA). Proteases inhibitors tosyl phenylalanyl chloromethyl ketone (TPCK) and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF or Pefabloc) were from Sigma-Aldrich S.r.L. (Milano, Italy).

Animals

The triple-transgenic mouse model of AD, B6;129-Psen1 tm1Mpm Tg (APPSwe,tauP301L)1Lfa/J (named 3xTg-AD) and their respective wild types mice were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA). 3xTg-AD mice were previously characterized and represent a reliable model of human AD patients. In this model, Aβ intracellular immunoreactivity can be detected in some brain regions as early as three to four months of age38. Experiments were conducted using 8-week-old male mice (weight 15–25 g) in accordance with the guidelines laid down by the European Communities Council (86/609/ECC) for the care and use of laboratory animals. Mice were housed in plastic (Makrolon) cages (4 animals per cage) in a temperature controlled room (21 ± 5 °C) and 60% humidity on 12 h light/dark inverted cycle (light was switched on at 8:00 P.M.) and maintained on laboratory diet (Mucedola, Italy) with water ad libitum. All appropriate measures were taken to minimize pain and discomfort in experimental animals. Brains, livers, feces and urines were properly stored at −80 °C after sacrifice.

All procedures were in accordance with the guidelines laid down by the European Communities Council (86/609/ECC) for the care and use of laboratory animals under an approved protocol (EUFTP#261473) by Veterinary Health Dept. of the Italian Ministry of Health.

SLAB51 administration

Two groups of mice (n = 32 animals for each group) were treated for four months as follows: a 3xTg-AD group orally treated with vehicle (water), a 3xTg-AD group orally treated with SLAB51, a formulation made of nine live bacterial strains (Streptococcus thermophilus, bifidobacteria (B. longum, B. breve, B. infantis), lactobacilli (L. acidophilus, L plantarum, L. paracasei, L. delbrueckii subsp. bulgaricus, L. brevis)). The dosage (200bn bacteria/Kg/day) was calculated by application of the body surface area principle39. Fresh drinking solution was changed every day. The body weight was measured every 2 weeks before starting the treatment and then once a month to ensure adequate intake of the experimental food.

Behavioral assessments

All behavioural experiments were performed during the animal’s dark phase, with testing performed from 8:00 to 15:00. Animals were handled for 3 days before testing in order to accustom them to the experimenter. The investigators were blinded to the groups’ allocation during the tests.

The open field (OF) test was used to evaluate the locomotor activity of mice using automated locomotor activity boxes (Med Associates, VT 05478) as previously reported40. Locomotor activity was recorded for 5 mins, starting 1 min after placing the animal in the test cage. Each mouse was automatically recorded by interruptions of orthogonal light beams (3.5 cm above the activity box floor), which were connected to automatic software (Activity Monitor, Med Associates). The behavioral parameters observed were ambulatory (number of horizontal episodes) and stereotype counts (number of grooming movements).

The novel-object recognition (NOR) test is used to evaluate recognition memory and it is based on the spontaneous tendency of rodents to spend more time exploring a novel object than a familiar one. Mice were challenged in the open-field arena explored on the day before during OF. Following a training period, the animal was removed from the environment for a delay period of 3 h and then it was returned to the arena, where one of the two identical objects had been replaced by a new, dissimilar novel object (test phase). The amount of time the rodent spends exploring each object in 10 mins provides a powerful measurement of memory integrity and attention.

Results were expressed as discrimination score (seconds spent with novel object − seconds spent with familiar object)/(total time spent with both objects). Lower score indicates memory impairment in this task. Objects were different for shape, color and texture at each time point41 and maintained throughout the study to obtain reproducible data. Preliminary experiments were done to select novel and familiar object pairs on the basis that each object in the pairs elicited the same amount of spontaneous investigation.

The passive avoidance test is a fear-motivated test used to assess memory function based on the association formed between an aversive stimulus such as a mild foot shock and a specific environmental context. The amygdala plays a pivotal role in passive avoidance learning.

Apparatus and procedures were previously described42. Briefly, during the training test, each mouse received an electric shock when it entered the dark compartment. In the retention test, passive defensive reactions, assessed in terms of the latent period of transfer from the light to the dark compartment, were tested 24 h and 7 days after foot shock. Higher latency value translates to better retention of memory from the foot shock given during the learning phase43.

The elevated plus maze (EPM) is a test used to detect anxiety-related behavior in animals44. The apparatus, a cross-shaped wooden elevated maze, consisted of two opposite open arms 30 cm × 5 cm, and two opposite arms enclosed by 20-cm-high walls with two open arms and two closed arms. The maze was elevated 50 cm from the floor and lit by dim light. The procedure is described in Nasuti et al.45. Changes in the percentage of time spent and number of entries into the open arms indicate changes in anxiety-like behavior. A greater percentage of time spent and number of entries in open arms indicates less anxiety-like condition.

Microbiota analysis

An aliquot of 100 mg (wet weight) of each fecal sample DNA was extracted with a DNA isolation kit (MoBio Power soil, MoBio Laboratoroies, USA) following the manufacturer’s instructions. The V4 region of the 16S rRNA gene was amplified with primers 515 F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806 R (5′-GGACTACVSGGGTATCTAAT-3′) at the MR DNA Laboratory (Shallowater, TX, USA) as previously described46. The Nextera® DNA sample Preparation kit including sequencing adapters and sample specific barcodes was used to prepare a DNA library and sequenced at MR DNA on an Illumina MiSeq instrument.

The raw sequences obtained were analyzed using the software QIIME v.1.8. A total of 5,343,083 were obtained. Sequences were demultiplexed, low quality reads were filtered using default parameters, chimeras removed and sequences were then clustered into operational taxonomic units (OTUs) using an open-reference OTU picking protocol at the 97% sequencing identity level against the Greengenes47 database. For further analysis, each was rarefied to an even sequencing depth of 24,800 sequences to adjust for uneven sequencing depth across all samples. Observed species richness, Chao 1, and Shannon indexes were determined using QIIME. The software PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to make functional gene content predictions based on 16S rRNA gene data generated by all organisms found in the data and represented in the Greengenes phylogenetic tree of 16S rRNA gene sequences. Because most datasets did not meet the assumptions of normal distribution as assessed by the D’Agostino and Pearson normality test, non-parametric statistical tests were used. The Friedman test with Dunn’s post hoc test for repeated measures ANOVA was performed to evaluate changes among all timepoints. The resulting p-values were adjusted for multiple comparisons using the Benjamini & Hochberg’s False Discovery Rate (FDR), and an adjusted p < 0.05 was considered statistically significant. Data were analyzed using Prism software 5.0 (GraphPad Software, San Diego, CA) and JMP software (SAS Institute, Cary, NC, USA). Linear discriminant analysis effect size (LEfSe), freely available online in the Galaxy workflow framework, was used to elucidate taxa and genes associated with treatments at the various time-points. Analysis of beta-diversity was performed using unweighted Unifrac distance metrics. Statistical significance of the resulting distance metric was tested by analysis of similarities (ANOSIM) using the QIIME software. Sequences were deposited in the SRA archive under the accession number: SRP064106. The software PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used to predict the functional gene content in the fecal microbiome based on the 16S rRNA genes found in the data and represented in the Greengenes phylogenetic tree of 16S rRNA gene sequences. PICRUSt was used online in the Galaxy workflow framework. Linear discriminant analysis effect size (LEfSe) was used to elucidate bacterial taxa (16S rRNA genes) and functional genes (PICRUSt) associated with healthy or diseased cats.

Short chain fatty acids (SCFAs) determination

Fecal content of acetic, propionic and butyric acid has been quantified by means of headspace solid-phase microextraction coupled to gas chromatography with flame ionization detection by using a polydimethylsiloxane/carboxen/divinyl benzene coated fiber, following the procedure by Fiorini et al.48. Data are expressed as mean content (mmol/Kg) ± SD and were statistically analyzed using one-way analysis of variance, followed by the Tukey-Kramer method for post-hoc analysis. Different superscript letters (a, b) indicate significant variations at P < 0.05 in the table.

ELISA assay for ghrelin, leptin and GIP, GLP-1

Plasma hormone concentrations were measured through ELISA using plasma treated with protease inhibitors (Pefabloc and TPCK).

Briefly, the Rat/mouse Ghrelin Active ELISA kit is a sandwich ELISA based on the capture of ghrelin molecules (active form) in the plasma by anti-ghrelin IgG and the immobilization of the resulting complex to the wells of a microtiter plate coated by a pre-titered amount of anchor antibodies. After the binding of a second biotinylated antibody to ghrelin and the wash away of unbound materials, followed by conjugation of horseradish peroxidase to the immobilized biotinylated antibodies, the quantification of immobilized antibody-enzyme conjugates is performed by monitoring horseradish peroxidase activities in the presence of the substrate 3,3′,5,5′-tetra-methylbenzidine. The enzyme activity is measured spectrophotometrically by the increased absorbency at 450 nm, corrected from the absorbency at 590 nm, after acidification of formed products. Since the increase in absorbency is directly proportional to the amount of captured rat/mouse ghrelin (active form) in the unknown sample, the concentration of active ghrelin can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of rat/mouse ghrelin.

Leptin and GIP were determined using sandwich ELISA kit based on anti-leptin and anti-GIP monoclonal antibodies respectively.

Similarly, the quantitative determination of mouse glucagon like peptide-1 was performed using a sandwich ELISA kit (CUSABIO Cat #CSB-E08118m). Antibody specific for GLP-1 was pre-coated onto a microplate. Standards and samples are pipetted into the wells and any GLP-1 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for GLP-1 is added to the wells. After washing, avidin-conjugated horseradish peroxidase is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of GLP-1 bound in the initial step. The color development is stopped and the intensity of the color is measured.

Cytokine analyses

The plasma levels of pro- and anti- inflammatory cytokines were measured through ELISA using the Mouse Inflammatory Cytokines & Chemokines Multi-Analyte ELISArray Kit (QIAGEN, Italy). Samples and standards were prepared following the manufacturer’s protocols. Each cytokine level was calculated based on its own standard curve and expressed as mean concentration (pg/ml) ± SE.

Congo red staining and immunohistochemistry analysis

Three 3 μm-thick parasagittal sections from each animal (n = 8 per sub-group), at ~0.84, 1.20, and 1.56 mm lateral from the midline49, were prepared. Selected sections were deparaffinized and rehydrated according to standard protocols, and they were used for Congo red staining and for Aβ and FGF9 immunohistochemical detection. Hematoxylin and eosin counterstaining was used to provide morphological details.

In detail, Aβ peptides were immunodetected using a polyclonal antibody with specificity for the Aβ 1–42 C-terminus (Millipore, CA). Briefly, for each time point (8, 12, 18, 24 weeks of age), brain slides from treated and untreated wt and AD mice (n = 8 per subgroup) were fixed in a 50:50 mixture of methanol and acetone for 5 min and incubated with the anti-Aβ 1–42 antibody (1:50). The binding of the antibody was detected with the Elite kit (Vector Laboratories), and the immunoreaction was developed using diaminobenzidine chromogen (DAB, Vector).

For FGF9 detection, brain sections were incubated overnight with anti-FGF9 rabbit polyclonal antibody (aa50–99) IHC-plus™ (LSBio Catalog No. LS-B11953), diluted 1:50, cross reacting with mouse, human, bovine and other animal species. Non-specific binding was blocked by incubation of slides for 10 minutes with a protein-blocking agent (Protein-blocking agent, Dako, Carpinteria, CA, USA) before application of the primary antibody. Slides were incubated overnight in a moist chamber. The immunoreaction with streptavidin–immunoperoxidase (Streptavidin–immunoperoxidase, Black & Decker, Towson, MD, USA) was visualized with 3,3′-diaminobenzidine substrate (3,3′-diaminobenzidine substrate, Vector, Burlingame, UK). Tissues were counterstained with Mayer’s hematoxylin. For negative immunohistochemical controls the primary antibodies were omitted. Sections of human and bovine brain, AD and BSE respectively affected, served as positive control tissues for Aβ 1–42 and FGF9 cell staining. For scoring of Congo red, Aβ 1–42 , and FGF9 positive cells, these cells were quantified in different area of the mouse brain, particularly select compartments of the CNS as hippocampal area. All cellular types were evaluated using a light microscope (Carl Zeiss, Jena, Germany), a 40x objective, a 10x eyepiece, and a square eyepiece graticule (10 × 10 squares, having a total area of 62,500 μm2). Ten appropriate fields were chosen for each compartment and arithmetic means were calculated for each brain region. Results were expressed as IHC positive cells per 62,500 μm2. For all parameters, cells on the margins of the tissue sections were not considered for evaluation to avoid inflation of positive cell numbers. Positive Congo red/Aβ 1–42 /FGF9 cells, amyloid interstitial plaques, and other CNS areas, were quantified by using an image-analysis system consisting of a light microscope (Carl Zeiss, Jena, Germany) attached to a Javelin JE3462 high-resolution camera and a personal computer equipped with a Coreco-Oculus OC-TCX frame grabber and high-resolution monitor. Computerized color-image analysis was performed by using Image-Pro Plus software (Media Cybernetics). The entire cerebral cortex and hippocampus were separately sampled with the counting frame size 250 μm × 250 μm for cortex and 100 μm × 100 μm for hippocampus. The area of each section in all cross brain sections in every mouse was recorded, as was the total number of neurons determined by immunostaining as previously described. For each mouse, the total brain area was calculated as the sum of the areas of all fields in all brain cross sections on one slide. Congo red, Aβ 1–42 , and FGF9 positive cells were counted per section, and stained cell densities were expressed as the number of cells per square millimeter of analyzed section area24. The sum of the area of all amyloid plaques was divided by the total area of cerebral cortex or hippocampus to obtain the amyloid burden. The pathologist performing quantification of amyloid burden was blind to age, treatment type, and genotype of mice. The unbiased stereological based quantification of amyloid burden was performed on the basis of methodology suggested by Liu et al.50.

Measurement of the cortex

For the thickness measurement of the cortex wall, serial sections, in the coronal plane, from rostral to caudal of each mouse cerebrum were made. Briefly, the brains were removed, placed in 10% buffered formalin, covered with aluminum foil, and refrigerated. Two-mm-thick sections were cut 4, 6, and 8 mm from the frontal pole, and sections were photographed within 24 h. To prevent distortion, brain slices were kept flat in the Petri dish overnight. After the sections had been photographed, they were routinely processed and paraffin embedded, then newly 3 µm sectioned and stained with hematoxylin and eosin (H&E) and newly photographed. The serial sections were sub-divided into frontal, parietal, temporal, and occipital regions of the brain in rostral to caudal direction. Qualitative and quantitative analyses of the wall of the cerebral hemisphere were carried out on every 20th section. For each sub-group, 5 sections were analyzed. In each of these coronal sections, brains were stained with H&E and the cortex thickness was measured. For the assessment of regional cortical atrophy the thicknesses of the wall and laminae was determined by thickness measurements on H&E-stained sections. The different zones were measured using a calibrated integrating graticule (0.01mm) in a single eyepiece. The method of point counting was used to determine the relative volume proportion of the cerebral wall, meninges and ventricles. The same stratified selected sections were projected onto a screen using a Leitz demonstration microscope (x2.5). A grid with 300 points was superimposed on the image. The points falling on the cerebral wall, meninges, and ventricle were counted field-by-field to cover the entire left and right cerebral hemispheres.

Ventricular sizes evaluation in the brain sections

Equivalent sections of 3xTg-AD, and wild-type mice brains were chosen on the basis of common morphological landmarks49. Images of stained tissues were converted into TIFF format using Adobe Photoshop Elements 2.0. The areas of the brain substance and ventricles were measured using NIH ImageJ 1.39 u. Two images from each individual mouse were considered.

TUNEL analysis

In brain sections, apoptotic index was highlighted through a TUNEL colorimetric staining (DeadEnd, Promega®) according to the manufacturer’s instructions. For evaluation of the apoptotic rate, ten random fields of any chamber were examined under a dry- 40x objective. TUNEL-positive cells are characterized by a brownish-black nuclear stain. Lower-power digitized images were acquired with a light microscope (Carl Zeiss, Jena, Germany) attached to a Javelin JE3462 high-resolution camera and a personal computer equipped with a Coreco-Oculus OC-TCX frame grabber and high-resolution monitor, and cells count and quantification were performed as previously reported.

Preparation of brain extracts

Brain extracts were homogenized (1:5 weight/volume of buffer) in 50 mM Tris buffer, 150 mM KCl, 2 mM EDTA, pH 7.5. Homogenates were immediately centrifuged at 13.000 × g for 20 min at 4 °C and the supernatant was collected for enzymes activity assays and western blotting. A small part of this supernatant fraction was immediately supplemented with protease inhibitors for hormones determination by ELISA as described below. Protein content was determined by the Bradford method51 using bovine serum albumin (BSA) as standard.

Proteasome activity assays

Proteasome peptidase activities in brain homogenates (supernatant fraction) were determined using synthetic fluorogenic peptides: Suc-Leu-Leu-Val-Tyr-AMC was used for ChT-L activity, Z-Leu-Ser-Thr-Arg-AMC for T-L activity, Z-Leu-Leu-Glu-AMC for PGPH activity, and Z-Gly-Pro-Ala-Phe-Gly-pAB for BrAAP activity52. The incubation mixture contained brain homogenates (15 μg total proteins), the proper substrate (5 μM final concentration) and 50 mM Tris–HCl pH 8.0, up to a final volume of 100 μL. Incubation was performed at 37 °C for 60 min and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin (AMC) and 4-aminobenzoic acid (pAB) was detected (AMC, λ exc = 365 nm, λ em = 449 nm; pAB, λ exc = 304 nm, λ em = 664 nm) on a SpectraMax Gemini XPS microplate reader. The 26S proteasome ChT-L activity was tested including in the reaction mix 10 mM MgCl 2 , 1 mM dithiothreitol, and 2 mM ATP.

Cathepsin B and L

Cathepsin B and L proteolytic activities were measured following the protocol described by Tchoupè et al.53 using the fluorogenic peptides Z-Arg-Arg-AMC and Z-Phe-Arg-AFC, respectively, at a final concentration of 5 μM. The mixture for cathepsin B, containing 7 μg of protein lysate, was pre-incubated in 100 mM phosphate buffer pH 6.0, 1 mM EDTA and 2 mM dithiothreitol for 5 min at 30 °C. Upon the addition of the substrate, the mixture was incubated for 15 min at 30 °C. The mixture for cathepsin L, containing 7 μg of protein lysate, was incubated in 100 mM sodium acetate buffer pH 5.5, 1 mM EDTA and 2 mM dithiothreitol for 5 min at 30 °C and, upon the addition of the substrate, the mixture was incubated for 15 min at 30 °C. The fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin (AMC, λexc = 365 nm, λem = 449 nm) and 7-amino-4-trifluoromethylcoumarin (AFC, λexc = 397 nm, λem = 500 nm) was detected on a SpectraMax Gemini X PS microplate reader.

Western blotting analyses

Brain homogenates (supernatant fraction) were analyzed through western blotting assays with the aim to measure the following intracellular protein levels: amyloid oligomers, ubiquitinated proteins, p53, p27 and the autophagy related proteins Beclin-1, p62 and LC3-II. In detail, for each time point brain homogenates (20 μg total protein) were loaded on 12% SDS-PAGE (15% for LC3; 10% for ubiquitinated proteins and amyloid oligomers) and electroblotted onto PVDF membranes. Successively, upon incubation with specific antibodies, the immunoblot detections were carried out with Enhanced ChemiLuminescence western blotting analysis system (Amersham Pharmacia-Biotech). Molecular weight markers (6.5 to 205 kDa) were included in each gel. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to check equal protein loading. The bands were quantified by using a densitometric algorithm. Each Western Blot was scanned (16 bits greyscale) and the obtained digital data were processed through Image J (NIH)54 to calculate the background mean value and its standard deviation. The background‐free image was then obtained subtracting the background intensity mean value from the original digital data. The integrated densitometric value associated to each band was then calculated as the sum of the density values over all the pixels belonging to the considered band having a density value higher than the background standard deviation. The band densitometric value was then normalized to the relative GAPDH signal intensity. The ratios of band intensities were calculated within the same Western Blot. All the calculations were carried out using the Matlab environment (The MathWorks Inc., MA, USA)55.

ELISA assay for Aβ levels determination

Brain homogenates (supernatant fraction) promptly supplemented with protease inhibitors (Pefabloc and TPCK) were used to measure Aβ 1–40 and Aβ 1–42 levels using enzyme-linked immunosorbent assay NOVEX® ELISA kits (Invitrogen,). Based on preliminary tests, samples were diluted at 1:5 with diluent buffer provided by the kit. Assays were performed according to the manufacturer’s directions.

Statistical analysis

Results of behavioral tests were expressed as mean ± S.E. In particular, the EPM was analyzed by mean of Student’s t test. For the OP, NOR and passive avoidance, a two-way ANOVA with one factor within (time) and one factor between (treatment) was employed and appropriate post-hoc analysis was carried out using the Newman-Keuls test. Biochemical and IHC data are expressed as mean values ± S.E. Statistical analysis was performed with one way ANOVA, followed by the Bonferroni test using Sigma-stat 3.1 software (SPSS, Chicago, IL, USA). P-Values p < 0.05 were considered to be significant.