The aim of this study is to compare APPPS1/swe to APP CSF1R-/- to understand the role of CSF1R in Alzheimer’s disease.
Animals:
We used only male for this study.
Conditional CSF1R knock out mice CSF1R-lox/CX3CR1-Cre/ER (called, cKO) B6.Cg-CSF1R tm1jwp/J mice (JaxMice; stock number 02212) were crossed with the B6.129-Cx3cr1tm2.1 (CreER)Jung/Orl mice (EMMA mouse respiratory; EM:06350). The resulting mouse has a tamoxifen-inducible CRE activity specifically in microglial cells, leading to a non-functional CSF1R protein. Mice were injected with tamoxifen at 10-week-old.
RosaredTm-CSF1R-lox/CX3CR1-Cre/ER mice: We crossed B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze (JaxMice cat# 007914) mice with CSF1R-lox/CX3CR1-Cre/ER to confirm the deletion of CSF1R. Mice were injected with tamoxifen at 10 weeks-old for 4 consecutive days.
APPSwe/PS1 male transgenic mice bearing a chimeric human/mouse β-amyloid precursor protein (APPSwe) gene and the human presenelin 1 gene (A246E variant). These mice were purchased from The Jackson Laboratory [Strain: B6C3-Tg (APP695)3Dbo Tg(PSEN1)5Dbo/J] and maintained on a C57BL/6J background.
cKO mice and APPSwe/PS1 mice were crossed to obtain an APPSwe/PS1-CSF1R-lox; CX3CR1-Cre/ER (called, APP cKO). The resulting mouse has a tamoxifen-inducible CRE activity specifically in microglial cells, leading to a non-functional CSF1R protein. Mice were injected with tamoxifen at 3-month-old.
All animals were acclimated to standard laboratory conditions (14 h light, 10 h dark cycle; lights on at 06:00 and off at 20:00 h) with free access to rodent chow and water. Protocols were conducted according to the Canadian Council on Animal Care guidelines, administered by the Laval University Animal Welfare Committee.
Tamoxifen preparation and administration:
Tamoxifen was dissolved in corn oil (Sigma-Aldrich cat#C8267) and Ethanol 100% for 1 h at 37 degrees Celsius, vortexed every 15 min. We used ∼75 mg tamoxifen/kg body weight and 100 μl tamoxifen/corn oil solution was administered via intraperitoneal injection for 4 consecutive days.
Sacrifices:
All mice were deeply anesthetized with ketamine/xylazine (90:10) and sacrificed via intracardiac perfusion with 0.9% saline. Brains were retrieved and post-fixed 24 h in 4% PFA pH 7.4 and transferred in 4% PFA pH 7.4 + 20% sucrose for a minimum of 15 h. Brains were sliced in coronal sections of 20-μm thickness with a freezing microtome (Leica Microsystems), serially collected in anti-freeze solution and kept at −20 C until usage.
Western blot:
Brain protein lysate was extracted and quantified as previously described (63). Proteins were loaded in 8-16% agarose precast gels (BioRad) and electroblotted onto 0.45µm Immibilon PVDF membranes. Membranes were incubated with primary antibodies anti-b Catenin (Cell signaling rabbit 1:1000 cat#9562), TREM2 (R&D sheep 1:1000 cat#AF-1729), BDNF (Millipore rabbit 1:1000 cat#AB-1534), Syndecan-1 (Abcam mouse 1:1000 cat#AB-34164), IL-34 (R&D sheep 1:1000 cat#AF-5195), PSD95 (Millipore mouse 1:1000 cat#MAB-1596), BACE1 (Cerderlane rabbit 1:1000 AB-108394), APC (Novus biological rabbit 1:1000 cat#NBP2-15422), ABCB1 (Novus biological rabbit 1:1000 cat#NB-100-80870), followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and revealed by clarity (ECL) substrate (BioRad). Quantification was done by determining integrative density of bands using Thermo Scientific Pierce by Image Analysis software v2.0. Optical values were normalized over actin.
Immunofluorescent staining:
Brain sections were washed (4 x 5 minutes) in KPBS then blocked in KPBS containing 1% BSA and 1% Triton X-100 (Sigma-Aldrich cat#T8787). Tissues were incubated overnight 4 degrees Celsius with primary antibodies Iba1 (Wako rabbit 1:1000 cat#09-19741), 6E10 (Biolegend mouse 1:1000 cat#SIG-39320), CD31 (Ebioscience rat 1:1000 cat#11-0311-81). After washing sections in KPBS (4 x 5 minutes), tissues were incubated with the appropriate secondary antibodies for 2 hours at room temperature. Followed by washes in KPBS (4 x 5 minutes), tissues were incubated 10 minutes with DAPI (Molecular Probes 1:10000 cat#D3571). After washes in KPBS (4 x 5 minutes) sections were mounted onto MicroSlides Superforst® (Fisherbrand cat#22-037-246) and cover slipped (Globe scientific cat#1419-10) with Fluoromount-G (Electron microscopy sciences cat#17984-25).
Immunohistochemistry:
Brain sections were washed (4 x 5 minutes) in KPBS then blocked in KPBS containing 1% BSA and 1% Triton X-100 (Sigma-Aldrich cat#T8787). Tissues were incubated overnight 4 degrees Celsius with primary antibodies CSF1R (R&D sheep 1:750 cat#AF-3818). After washing sections in KPBS (4 x 5 minutes), tissues were incubated with the appropriate secondary antibodies for 2 hours at room temperature. Followed by washes in KPBS (4 x 5 minutes), tissues were incubated with ABC mix (Vector Laboratories Vectasin® Elite ABC-HPR Kit, Peroxidase (Standard) 2,25 µl/ml sol A and 2,5 µl/ml sol B cat#PK-6100) for 1 hour at room temperature. After incubation sections were washed in KPBS (4 x 5 minutes) and incubated up to 15 minutes with a DAB solution (Sigma-Aldrich H2O2 0.003% cat#H1009 and Sigma-Aldrich DAB 0.05% cat#D7304-1SET), followed by washed in KPBS (4 x 5 minutes). Sections were mounted onto MicroSlides Superforst® (Fisherbrand cat#22-037-246) and cover slipped (Globe scientific cat#1419-10) after dehydration with DPX (Electron microscopy sciences cat#13510).
Behavioral analyses:
Novel object recognition task protocol (34) consists of 3 phases: habituation, familiarization and test phase. The experimenter who observed and recorded the behavior was not aware of treatment and genotype of the tested animals (WT+vehicle, n=10, APPSwe/PS1 + vehicle, n=10 and APP + tamoxifen, n = 10). Baseline data were obtained at 3 months of age, whereas the effects of the treatment were determined at 6 or 8 months. Tests were made at 15 lux.
Nesting protocol (64), mice were placed 1 per cage at 3 month-old. The experimenter who observed and recorded the behavior was not aware of treatment and genotype of the tested animals (WT+vehicle, n=10, APPSwe/PS1 + vehicle, n=10 and APP + tamoxifen, n = 10). After each NOR task, cages were cleaned a new pad was placed inside. After 24h, the nests were analysed and scored. The scores vary from 1 (untouched pad) to 5 (perfect nest).
ELISA:
Total brain homogenates were used for all the ELISA. For the Aβ 1–40 (Millipore cat#EZHS40) we analyzed 50 μg per well following the protocol provided.
Unbiased Stereological count:
Brains were serially sectioned as previously described and were stained with Iba1, 6E10 and DAPI as previously described. We counted microglia and plaques at 20x magnification using an Axio Observer microscope equipped with Axiocam 503 monochrome camera and processed using with ZEN Imaging Software (Carl Zeiss Canada, Toronto, ON, Canada). Sampling characteristics for cortex sections: Sampling interval 12, total number of sections 7, section sampling interval 3 and starting selection 1. For hippocampal sections: Sampling interval 12, total number of sections 7, section sampling interval 2 and starting selection 1. After counting plaques using unbiased dissector, the mean plaque volume (MPV) was estimated using the rotator method. The estimated MPV was based on the length of line crossing each plaque using randomly oriented line. 4 locations for each hippocampus and 3 for each cortex were sampled in 7 sections Plaques and microglia were counted according to Gundersen unbiased counting rules, with optical fractionator method and sampling continued to a coefficient of error of 10% or less (65).
Image Acquisition:
Image acquisition of Fluorescent staining images was performed using a Zeiss LSM800 confocal microscope supported by the Zen software (2.3 system) using the 10×, 20×, and 40× lenses. Confocal images were then processed using Fiji (ImageJ Version 2.0.0-rc-43/1.51n). For analyses and bright field image acquisition of staining, Iba-1 and CSF1R, 8-bit grayscale TIFF images of the regions of interest were taken in a single sitting for whole protocols with a Qimaging camera (Qcapture program, version 2.9.10) attached to Nikon microscope (C-80) with the same gain/exposure settings for every image. To evaluate the level of Iba-1+ immune response in the regions of interest, the images were imported into ImageJ (1.37) and the percentage of area occupied by the staining was measured using the threshold parameter. Cell count was assed manually using ImageJ (1.37). Analysis was performed in double blinded to avoid bias of analysis. Fluorescent staining of images was performed using a Zeiss LSM800 confocal microscope supported by the Zen software (2.6 system). Confocal images were then processed using Fiji (ImageJ Version 2.0.0-rc-43/1.51n).
Statistical analyses and Figure preparation:
Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were carried with the Prism software (version 8.0, GraphPad Software Inc.), comparing control groups vs. tested groups. Values were considered statistically significant if p < 0.05. All panels were assembled using Adobe Photoshop CC 2018 (version 19.1.0) and Adobe Illustrator CC 2018 (version 23.0.1).