APPPS1 mice

APPPS1 transgenic mice were maintained at the Ecole Polytechnique Fédérale de Lausanne animal core specific pathogen free facility. There was unlimited access to autoclaved food and water. APPPS1 animals co-express the KM670/671NL Swedish mutation of human amyloid precursor protein (APP) and the L166P mutation of human presenilin 1 (PS1) under the control of the Thy-1 promoter, and show age-dependent accumulation of parenchymal Aβ plaques with minimal vascular Aβ amyloid that is restricted to the pial vessels14. APPPS1 mice were generated on a C57BL/6 background. Both male and female APPPS1 mice as well as age-matched control wild-type (WT) littermates were used. APPPS1 mice and wildtype littermate were housed together in grouped cages (n = 5 mice/cage) until analyzed. Throughout the study these mice are referred to as conventionally-raised (CONVR) APPPS1 and WT mice.

Approval of animal studies

Animal studies were performed according to regulations issued by the Swiss government and approved by the ethic veterinary committee of the cantons of Vaud and Bern, Switzerland.

APPPS1 germ-free mice

For germ-free rederivation, 4–6 week-old APPPS1 transgenic female mice were superovulated by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG, VWR) on day 0, and 5 IU of human chorionic gonadotropin (hCG) on day 2. Females were paired with APPPS1 males on day 2. Two days later, plugged females were euthanized, oviducts collected, and the embryos were flushed out of the oviducts. The fertilized 2-cell embryos were collected and extensively washed in M2 medium (Sigma) containing Penicillin/Streptomycin (Invitrogen). Subsequently these embryos were transferred into pseudo-pregnant germ-free NMRI recipient females under aseptic conditions. Resulting litters were maintained in flexible film isolators at the Clean Mouse Facility at the University of Bern with unlimited access to autoclaved food and water. Both male and female GF-APPPS1 mice as well as age-matched control wild-type littermates (GF-WT) were used. Note that GF-APPPS1 and GF-WT mice were fed with the same autoclaved diet (Kliba-Nafag PN3437) as was used for the CONVR-APPPS1 and CONVR-WT mice (see above). Moreover GF-APPPS1 and GF-WT mice were housed in grouped cages (n = 5 mice/cage), were handled 2 times a week exactly the same as CONVR-APPPS1 and CONVR-WT mice (see above). Due to the complexity of the experimental design (generation of germ free animals), no statistical methods could be used to predetermine sample size. Furthermore the experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Bacteriological status of the GF-APPPS1 model

Mice were regularly checked for germ-free status by aerobic and anaerobic culture, DNA staining (Sytox-green, Invitrogen) and gram staining of cecal content to detect uncultivable contamination. In addition, serological testing for known viruses and pathogens was performed periodically by ELISA, PCR or IFA. The obtained negative results confirm the germ free status of our animals (Tables S1 and S2).

Preparation of transgenic samples for analyses

All mice were prepared at the EPFL, i.e. GF-APPPS1 mice and GF-WT mice raised in Bern were shipped to EPFL in sterile containers 24 hours prior to analysis. On the day of sacrifice, mice were weighed and euthanized by lethal intraperitoneal injection of pentobarbital (150 mg/kg; <200 mg/ml); stomachs, livers, pancreas and brains were collected, weighed and snap frozen in liquid nitrogen for further analysis. Brains were extracted and cut in half; one half was immediately incubated in 4% paraformaldehyde in ice-cold PBS and used for histology (see below); the other hemisphere was snap frozen and used for biochemical analysis (ELISA and western blot; see below). Blood was collected from the veina cava, transferred to Heparin coated tubes, centrifuged 4′500 rpm 4 °C for 15 min. Plasma was transferred to fresh clean tubes and snap frozen for further analysis.

Histology and immunohistochemistry

After 2 days fixation in 4% paraformaldehyde (PFA), the half brains were incubated for 48 h in 30% sucrose. Brains were then frozen in 2-propanol (Merck) and subsequently sectioned on a freezing-sliding microtome to collect 25 μm coronal sections. For histological staining, brain sections were stained with Thioflavin T dye according to standard protocol (ThT 1% in 50% Ethanol; Sigma Aldrich, St. Louis, MO). Sections were immunostained to visualize Aβ deposits using mouse monoclonal antibody (6E10, 1:500; COVANCE). Microglial reaction was assessed using a rabbit polyclonal antibody to Iba-1 (1:1000; Wako, 019–19141). Vectastain Elite ABC Kits were used and revelation obtained with Vector SG Blue (Vector Laboratories).

Quantification of Thioflavin T-positive amyloid load

Hemi-brain sections encompassing the cortex and hippocampus, and stained for Thioflavin T (ThT), were quantified for compact amyloid load. To acquire images used for quantification, ThT-stained brain sections were imaged with a 10x objective using a Zeiss Axiovert 200 M/ApoTome microscope (Zeiss, Germany) coupled with a Zeiss Axiocam HR camera (Zeiss, Germany). For each of the animals involved in this study, 10 ± 2 stained coronal brain secions with a thickness of 25 μm and spaced from each other by 24 slices were available for imaging. Among these, four sections were selected based on the following criteria: one section encompassing the cortex (between position AP 0.74 mm and AP 0.38 mm from Bregma), one section with the striatum (between the position AP - 0.46 mm and AP - 0.70 mm from Bregma), one section showing the dorsal hippocampus (between position AP - 1.82 mm and AP - 2.18 mm from Bregma) and one section encompassing the ventral hippocampus (between position AP - 2.70 mm and AP - 3.08 mm from Bregma). For each selected section, 1300 × 1030 contiguous images (with less than 5% overlapping) were captured for the entire cortex and hippocampus region (when present). The acquired images were analyzed with the public domain software Image J. Analyzed areas were adjusted manually so that each measurement was accurately measuring only the region of interest and adjusted to exclude brains regions other than the cortex and hippocampus. For ThT-load analysis, the images were inverted and a threshold of approximately 180 was applied (with a 5% variance to best adjust tissue staining variations among sections). ThT-stained brain sections were analyzed with parameters of larger than 5 × 5 pixels and with a circularity of 0.15.

Quantification of Iba-1-positive immunoreactivity

Neocortical microglia immunoreactivy was quantified in hemi-brain sections immunostained for activated microglia (with the Iba-1 antibody). Acquisition of images from Iba-1-stained sections was similar to that described for ThT quantification. Subsequently, a threshold of 110 with a variance of 10% was applied to each image using Image J. This threshold was selected in order to best display the microglia while minimizing the background noise. After visual inspection, ROI were selected such that staining artefacts on the section was manually removed by cropping. The microglia were quantified using the analyze particles tool embedded in ImageJ. In order to consider both the single microglia and the microglial loads, the pixel size was not specified and the circularity parameter was set to 0.0–1.0.

Statistical analysis (Histology)

Data represent the means ± standard errors of the means. Statistical analysis was performed using ANOVA followed by Hom-Sidak’s test for multiple comparisons and unpaired t test followed by Welch’s correction.

Western blot and biochemistry

Brains were extracted and homogenized in RIPA buffer (150 mM sodium chloride, 1.0% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0) (10% weight/volume). Brain homogenates were centrifuged at 14’000 rpm for 15 min at 4 °C. The purified protein fractions were stored at −80 °C until western blotting. Total protein concentration of each sample was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). For Western blot analysis, 25 μg of protein was loaded into a precast 15-well NuPAGE Novex 12% Bis-Tris gel (Invitrogen, Waltham, MA) for separation by electrophoresis and then transferred to a polyvinylidenedifluoride (PVDF) membrane as indicated in the manufacturer’s instructions (GE Healthcare, UK). As primary antibodies, mouse monoclonal anti Aβ antibody (6E10, Covance), mouse monoclonal anti-ß tubulin antibody (cell signaling) were incubated at 4 °C overnight, followed by secondary horseradish peroxidase-anti-mouse antibody (Jackson Labs, Baltimore) incubation. The blots were visualized with BM chemiluminescence Western blot kit (Roche, Basel).

Neprilysin (NPE) and Insulin degrading enzymes (IDE) activity: NPE and IDE activity have been measured, according to the manufacturer instructions with fluorometric SensoLyte® 520 Neprilysin Activity Assay Kit (AS-72223, ANASPEC) and SensoLyte® 520 IDE Activity Assay Kit (AS-72231, ANASPEC) respectively.

Statistical analysis (Western Blots)

Graphpad Prism 6 software was used to perform statistical analysis. Data represent the means ± standard errors of the means. Statistical analysis was performed using ANOVA followed by Hom-Sidak’s test for multiple comparisons and unpaired t test followed by Welch’s correction.

ELISA

Brain protein extracts and plasma were diluted to the fourth into sampling medium provided by the manufacturer and a final of 25 μl volume was loaded into a 96 well plates. Aβ38, Aβ40 and Aβ42 measurements were performed according to the manufacturer’s instructions (Peptide Panel 1 (6E10) Kit (1 Plate) V-PLEX™K15200E-1 Mesoscale Discovery, Gaithersburg). All levels of Aβ38, Aβ40 and Aβ42 were normalized against total protein amount. For cytokine measurements, total brain homogenates were centrifuged at 14’000 rpm, 30 min, 4 °C and supernatants were analyzed using the mouse pro-inflammatory panel 1 V-plex plate (Mesoscale Discovery) according to the manufacturer’s instructions and normalized against total protein content.

Statistical analysis (ELISA)

Graphpad Prism 6 software was used to perform statistical analysis. Data represent the means ± standard errors of the means. Statistical analysis was performed using ANOVA followed by Hom-Sidak’s test for multiple comparisons.

Colonization study

Cecal contents were pooled from amyloid-depositing (12 month-old) CONVR-APPPS1 mice or CONVR-WT mice. Cecal contents were homogenized in sterile PBS (2 ml per cecum) and a volume of 0.2 ml was immediately administered by oral gavage to 4 month-old littermate GF-APPPS1 mice, as previously described38. Oral gavage was given at day 1 and day 4. The resulting transplanted mice (termed as COLOAD-APPPS1 and COLOWT-APPPS1) were housed in a conventional environment for 8 weeks under the same conditions and fed with the same diet as their CONVR-APPPS1 counterparts. Control colonization study was performed using cecal contents pooled from control wild-type (12 month-old) mice and given intraorally to GF-APPPS1 mice (COLOWT-APPPS1). During feces collection, mice were put in clean litter-free individual cages until stool collection. Fresh feces were collected from each mouse at day 1 and 4 and at week 2, 4 and 6 for bacterial 16S rRNA gene sequencing. Mice were analysed 8 weeks after the beginning of the colonization.

DNA extraction

One frozen faecal pellet/mouse was thawed on ice and DNA was extracted using the QIA amp DNA Stool Mini Kit (Qiagen). The protocol from the manufacturer was followed with an addition of a bead beating step. Sterile glass beads (1 mm) were added together with stool lysis buffer to the samples and cell disruption was performed for 2 × 2 minutes at 25 Hz using a TissueLyser (Qiagen), followed by a heating step at 95 °C for 5 minutes. After lysis, DNA-damaging substances and PCR inhibitors were removed using InhibitEX tablet (provided with the kit) and the DNA was purified on QIAamp Mini spin columns.

PCR amplification of the V3–4 region of bacterial 16 S rRNA genes

16S rRNA genes were amplified by PCR with forward and reverse primers containing Illumina adapter sequences and unique dual indexes used to tag each PCR product39, according to the 16S-protocol provided by Illumina. Briefly, PCR reactions were carried out in 25-μL reactions with 0.2 μM forward and reverse primers, with 12.5 ng template DNA and 12.5 μl of 2 × KAPA HiFi HotStart Ready Mix kit (KAPA Biosystems). Thermal cycling consisted of initial denaturation at 95 °C for 3 min followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, followed by a final step of 72 °C for 5 min. The amplicons (464 bp product) were purified with Agencourt AMPureXP Kit (Beckman Coulter). A second PCR was thereafter performed to attach Illumina adapters and unique dual indexes to each sample, followed by a clean-up step with AmPureXP Kit (Beckman Coulter) (Table S3). PCR amplicons were visualized using 0.1% agarose gel electrophoresis. Negative extraction controls did not produce visible bands.

Amplicons quantitation, pooling, and sequencing

Mouse fecal amplicon DNA concentrations were quantified using the Qubit3.0 Fluorometer (Life Technologies, Stockholm, Sweden). Amplicons were combined in equimolar ratios into a single tube with a final concentration of 4 pM. As an internal control, 5% of PhiX was added to the amplicon pool. Paired-end sequencing with a read length of 2 × 300 bp was carried out on a Miseq Instrument (Illumina) using a Miseq reagent kit v3 (Illumina Inc., San Diego, USA).

Sequence analysis

Sequences were analysed with the free software package Quantitative Insights into Microbial Ecology (QIIME), which allows analysis of high-throughput community sequencing data. Default values were used for each step, except where otherwise specified40. Sequences were removed if lengths were <200 nt, contained ambiguous bases, primer mismatches or homo polymer runs in excess of six bases. Forward and reverse reads were joined using Fastqjoin. After quality filtering, a total of 6,571,748 sequence reads were generated for the 8 month-old mice (APPPS1 n = 6, WT n = 7), 5,713,081 reads for the 3.5 month-old mice (APPPS1 n = 7, WT n = 8) and 3′688′179 reads for the 1 month-old mice (APPPS1 n = 6, WT n = 8) with an average number of 505,519 reads per sample for the 8 month-old mice, 361,568 reads/sample for the 3.5 month-old and 263,411 reads/sample for the 1 month-old mice. For the colonized mice, a total of 5,550,818 sequence reads was generated after quality filtering with an average of 79,297 reads per sample. Similar sequences were binned into operational taxonomic units (OTUs) using UCLUST41, with a minimum pairwise identity of 97%, generating 1369 OTUs for the CONVR-APPPS1 and WT mice, and 1,837 OTUs for the COLO-mice. The most abundant sequence in each OTU was chosen to represent its OTU. Representative sequences from each OTU were aligned using PyNAST (a python-based implementation of NAST in QIIME)39 and taxonomy was assigned using the Greengenes database (v. 13_8)42 and the RDP classifier43.

Statistical analysis (metagenomics)

Graphpad Prism 6 software was used to identify significant differences in bacterial relative abundances between transgenic CONVR-APPPS1 and WT mice using multiple t-tests and the Holm-Sidak method of correction for multiple comparisons, at phylum and genus level. To find correlations between brain Aβ42 levels and bacterial genera in the aged and COLO-APPPS1 mice, the operational taxonomic units (OTU) tables were rarefied at 291,712 and 22,609 randomly selected sequences/sample, respectively, for the entire data set after which orthogonal partial least squares (OPLS) scatter plot analysis was performed using SIMCA14 (Umetrics, Umeå, Sweden). Pearson’s correlation was calculated for each pairwise combination of brain Aβ42 and bacterial genera using Graphpad Prism 6 and the p-values were then corrected by Benjamin-Hochberg procedure for multiple comparisons44. Further, α- and β-diversities were analyzed in QIIME on rarefied data, using 291,712 sequences/sample for 8 month-old mice, 178,620 for 3.5 month-old mice, 136,829 for 1 month-old mice, and 22,609 for mice colonized with APPPS1 and WT microbiota. Differences in α-diversity were calculated in QIIME using a non-parametric t-test and FDR correction for multiple comparisons and β-diversity differences were analyzed with the ANOSIM and Adonis non-parametric statistical tests in QIIME. Linear discriminant analysis effect size (LEfSE, www.huttenhower.sph.harvard.edu/galaxy/) was applied on the OTU table according to ref. 45.