27-Hydroxycholesterol contributes to cognitive deficits in APP/PS1 transgenic mice through microbiota dysbiosis and intestinal barrier dysfunction

Specific pathogen-free (SPF) male APP/PS1 transgenic and sex- and age-matched wild-type C57BL/6J mice, 6 months old, weighing 30–35 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China; certification number YXK (Beijing) 2017-0022). The mice were housed in standard individual ventilated cages in a temperature- (22–25 °C) and humidity- (50–60%) controlled room with a 12 h dark-light cycle. Food and water were available ad libitum.

Our previous study has already investigated the effects of intravenous injection of 27-OHC in rats [24]. However, to date, there is no established dosage for subcutaneous injection of 27-OHC in C57BL/6J mice. Here, we conducted the first dose-finding study for subcutaneous administration of 27-OHC to C57BL/6J mice to pave the way for further studies investigating the effects of 27-OHC on the gut microbiota and intestinal barrier in APP/PS1 transgenic mice.

The 27-OHC doses used for the current study were based on the known effective dose in rats (70 μM/day) [24], the physiological levels of 27-OHC in the blood (0.15–0.73 μM) [25], and that taken up by the brain per 5 mg/24 h [26]. The whole blood volume of mice is 7.6 mL/100 g. Thus, a dose of approximately 5.5 mg/g would be effective if assuming direct translatability from rats to mice. Nevertheless, since differences in metabolism can affect the actual effective dose, 27-OHC was tested in six different doses ranging around this value (i.e., 0.275, 0.55, 1.65, 3.3, 5.5, and 8.25 mg/kg). 27-OHC (10 mg, R&D Systems Co., Ltd., USA) was dissolved in 1 mL ethanol and 9 mL 0.9% saline and then diluted to 1 mg/mL before use. The C57BL/6J mice (n = 10/group) were randomly assigned to seven groups (i.e., 0.275, 0.55, 1.65, 3.3, 5.5, and 8.25 mg/kg 27-OHC/day or vehicle [0.9% saline]). After treatment for 3 weeks, behavioral tests were carried out.

Next, the APP/PS1 mice (6 months old, n = 10/group) and matched wild-type C57BL/6J mice (6 months old, n = 10/group) were randomly divided into five groups: group I: WT control (C57BL/6J mice); group II: APP/PS1 control; group III: WT mice treated with 27-OHC (selected effective dose for subcutaneous injection); group IV: APP/PS1 mice treated with 27-OHC (selected effective dose for subcutaneous injection); group V: APP/PS1 mice treated with anastrozole (ANS, an inhibitor of CYP27A1, Enzo Life Sciences, Inc., Switzerland, 0.2 mg/day, subcutaneous injection); group VI: WT mice cotreated with 27-OHC (selected effective dose for subcutaneous injection) plus ANS (0.2 mg/day, subcutaneous injection); and group VII: APP/PS1 mice cotreated with 27-OHC (selected effective dose for subcutaneous injection) plus ANS (0.2 mg/day, subcutaneous injection). The APP/PS1-control and C57BL/6J-control groups received the same volume of 0.9% normal saline alone according to the same schedule.

Spatial learning and memory ability were evaluated by the Morris water maze test, which consists of orientation navigation and spatial probe tests. The test was performed in a circular tank (120 cm in diameter, 50 cm height) divided into four quadrants. A hidden escape platform (9 cm in diameter) was placed 2.0 cm below the water in the center of one quadrant. The water was colored with titanium dioxide for the C57BL/6J and APP/PS1 mice and adjusted to 22 ± 1 °C. The tank was placed in a dimly lit and soundproof room with various visual cues.

The orientation navigation test was performed for five consecutive days, and the mice were released into the water from different starting positions and allowed to swim freely until they reached the hidden platform. This was repeated four times each day with an interval of 30 min. The latency to reach the platform and swimming trajectory were recorded by a computer-controlled video tracking system (Morris water maze video analysis system, Institute of Materia Medica, Chinese Academy of Medical Sciences, China) for a maximum duration of 90 s. Mice that failed to reach the hidden platform within 90 s were placed on it for 15 s. The escape latency was determined as the time from the starting position to the platform. Afterwards, the spatial probe test was carried out. The platform was removed on the sixth day. The mice were released from a position opposite from where the platform used to be and allowed to swim for 90 s in the maze. The time spent in the target quadrant, the time spent crossing where the platform was originally located, swimming speed, and swimming trajectory were recorded.

The passive avoidance test is a fear-motivated test used to evaluate learning and retention memory. It is conducted in an apparatus that consists of two identical compartments (light and dark) 25 × 25 × 25 cm in size with a sliding door and grid-metal floor. The test enables mice to learn to restrain their innate behavior through moving from the illuminated compartment to the dark compartment. During the passive avoidance preacquisition trial, the mice were placed into the light compartment for a 5-min adaptation. The sliding door was automatically closed as soon as the mouse completely entered the dark compartment. Subsequently, the mouse was subjected to an electrical foot-shock (0.5 mA for 3 s) via the floor grid. The latency to enter the dark compartment was detected and analyzed and mice with latencies longer than 300 s were omitted from the study.

The retention memory test trial was tested 48 h after the acquired response had been established by recording their latency to enter the dark compartment. The latency to enter the dark compartment and frequency of entries into the dark compartment were recorded and directly linked to the retention memory. A greater latency and lower frequency of entry indicates better memory.

APP/PS1 and C57BL/6J mice were anesthetized with 5% chloral hydrate (Beijing Solarbio Life Sciences Co., Ltd., China). Fasting blood was drawn via cardiac puncture with a syringe. After being centrifuged at 3000 g for 15 min at 4 °C, serum and ethylenediaminetetraacetic acid↗ (EDTA)-plasma samples were collected and stored at − 80 °C until use. Following blood collection, the mice were perfused transcardially with ice-cold 0.2 M phosphate buffer solution (PBS). Their brains and the entire intestine were removed and fixed in 4% paraformaldehyde at 4 °C followed by processing for paraffin embedding and sectioning for histopathological analysis. Meanwhile, at necropsy, tissue samples from the brain, liver, kidneys, spleen, and intestine that showed gross changes were examined. The organ coefficient is expressed as the ratio to body weight to account for differences in organ weight-related effects and overt signs of toxicity.

A part of the fixed brain as well as the distal ileum and proximal colon tissues were embedded in paraffin, cut into 5 μm sections, stained with hematoxylin and eosin (HE), and examined by light microscopy (n = 6/group). The Aβ load in the brains was assessed using Bielschowsky silver stain (BSS), which was performed according to the previously published protocols [27]. A portion of intestinal (distal ileum specimens and proximal colon) tissue was processed for ultrastructural examination. After being fixed, dehydrated, dried, and stained, the intestinal sections were examined by a JEM-2100 transmission electron microscope↗ (TEM, Tokyo, Japan).

High-performance liquid chromatography-mass spectrometry (Agilent Technologies Inc., USA) was used to measure the plasma and brain levels of 27-OHC (n = 6/group) according to our previously reported protocol [22].

The levels of Aβ1-40 and Aβ1-42 in the plasma and brains were determined by ELISA kits (Thermo Fisher Scientific Co., Ltd., USA) in accordance with the manufacturer’s instructions (n = 6/group). Each experimental sample was run in triplicate. The data were recorded at 450 nm using a microplate reader (Bio-Rad Laboratories Co., Ltd., USA).

Plasma and liver levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). All of the procedures were performed according to the manufacturers’ protocols (n = 6/group).

Levels of IL-1β, IL-10, TNF-α, and IL-17 in the plasma were measured by enzyme-linked immunosorbent assay ELISA kits (Thermo Fisher Scientific Co., Ltd., USA) following the manufacturers' instructions (n = 6/group).

Intestinal permeability (DAO and D-lactate) and tight junction proteins (occludin, claudin 1, claudin 5, and ZO-1) as well as intestinal inflammatory cytokines (IL-1β and IL-10) were measured to assess intestinal barrier dysfunction.

DAO activity and d-lactate in serum were measured by commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). All of the procedures were performed according to the manufacturers’ protocols (n = 6/group).

The mRNA and protein expression of occludin, claudin 1 (CLOD-1), claudin 5 (CLOD-5), and ZO-1 in the ileum and colon were measured by quantitative real-time polymerase chain reaction (qRT-PCR, n = 6/group) and Western blots (n = 6/group). RNA extraction was performed according to the manufacturer’s instructions using the SV Total RNA Isolation System (Promega Corporation Co., Ltd., USA). The RNA concentration was measured and 1 μg of RNA was reverse-transcribed using a RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific Co., Ltd., USA) according to the manufacturer’s instructions. For relative quantification of mRNA levels, qRT-PCR was performed on a Real-time PCR Detection System (Bio-Rad Laboratories Co., Ltd., USA). All samples were measured in triplicate. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as the reference for standardization. Quantitative measurements of target gene levels relative to controls were performed with the 2-ΔΔCt method.

For the Western blots, intestinal tissues were lysed in freshly prepared RIPA buffer (50 mM Tris buffer saline, 0.5% deoxysodium cholate, 1 mM EDTA, 150 mM NaCl, 1% NP-40, and 1 mM PMSF). The concentrations of the protein extracts were determined by a BCA assay kit (Beyotime Biotechnology Co., Ltd., China). Proteins (50 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel by electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Merck Millipore Co., Ltd., Germany). The membranes were blocked with 5% bovine serum albumin prepared in TBS (Tris-buffered saline, pH 7.5 containing 0.1% Tween-20) for 1 h at room temperature, then incubated with the primary antibodies overnight at 4 °C, followed by the HRP-conjugated secondary antibodies (1:5,000. Abcam Company Co., Ltd., USA). All primary antibodies were as follows: anti-ZO-1 (1:5000; Abcam Company Co., Ltd., USA), anti-Occludin (1:50,000; Abcam Company Co., Ltd., USA), anti-Cldn-1 (1:5000; Abcam Company Co., Ltd., USA), anti-Cldn-5 (1:5000; Abcam Company Co., Ltd., USA), and β-actin (1:5000; Abcam Company Co., Ltd., USA). Protein bands were visualized by an enhanced chemiluminescence kit (Pierce Protein Biology Co., Ltd., USA). The density of each band was measured using Image System Fusion FX (Vilber Lourmat Co., Ltd., France) and normalized to β-actin levels. The experiments were repeated three times. The plots for Western blot were individual samples representative for each group.

Levels of IL-1β, IL-10, TNF-α, and IL-17 in the distal ileum specimens and proximal colon were also measured by ELISA kits (Thermo Fisher Scientific Co., Ltd., USA) following the manufacturers’ instructions (n = 6/group).

At the end of the treatment, fecal samples were collected (n = 6/group) and subjected to DNA isolation using a Bacterial DNA Kit (Omega, Shanghai, China). The DNA concentration was determined using a Nanodrop 1000 (Thermo Fisher Scientific, Wilmington, DE, USA). The 16S rDNA gene library preparation was conducted by using PCR amplification of the V3–V4 region. All libraries were sequenced using an Illumina HiSeq2500 platform (Illumina, San Diego, California, USA) at Biomarker Technologies, Beijing, China. Bioinformatic analyses were performed to compare the differences in the gut microbiota between the indicated experimental groups.

Fecal samples (50 mg) were mixed with an aliquot of 1000 μL of methanol/water (v/v = 1:1) mixture and then vortexed for 1 h and centrifuged at 132,000 r/min for 10 min. Then, 50 μL supernatant was transferred into a 1.5 mL centrifuge tube and mixed with 50 μL of propionic acid-d2 (1 μg/mL), which acts as an internal standard. Samples were then derivatized with 3NPH and centrifuged at 132,000 r/min for 10 min, and the supernatant was collected. An aliquot (5 μL) of the resulting derivatized material was injected into an ultra-performance liquid chromatograph (Waters UPLC I-Class) coupled to a tandem quadrupole mass spectrometer detector (Waters XEVO TQ-S Micro). Standard stock solutions of acetate, propionate, isobutyrate, butyrate, valerate, isovalerate, caproate, and heptanoic acid (ca. 1 mg/mL) were prepared by dissolving specific amounts of authentic standards in chloroform-methanol (1:1, v/v). The working standard solutions of the short-chain fatty acids (SCFAs) were serially diluted with chloroform-methanol (3:1, v/v) to obtain the concentrations needed for calibration curve standards (2, 10, 50, 200, 500, 1000 ng/mL). The estimate of the relative concentrations was based on the ratio of the area under the curve of the internal standard for each SCFA (n = 6/group).

The distribution of the variables was first assessed by the Kolmogorov-Smirnov (K-S) test. If the data followed a normal distribution, data are presented as the mean ± standard deviation and parametric tests (one-way ANOVA for three or more groups) were performed; for ANOVA, least significance difference (LSD) testing was carried out for post hoc analysis. If the data were not normally distributed, the data are presented as the median (interquartile range) and nonparametric tests (Kruskal-Wallis test with Dunn’s post hoc analysis for three or more groups) were used by applying SPSS 19.0 software (Chicago, IL, USA) and Prism 6.0 (GraphPad Software, Inc., USA). Repeated measures analysis of variance (ANOVA) were performed for the water maze data as appropriate. Correlations were analyzed by Spearman’s test. P < 0.05 was considered statistically significant.

For 16S rDNA sequencing, sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs). Representative sequences for each OTU were screened for further annotation. For each representative sequence, the Silva Database was used based on the mothur algorithm to annotate the taxonomic information. The following downstream data analyses were conducted in R software. Alpha diversity was applied for analyzing the complexity of species diversity for a sample through three indices, including Chao1, Shannon, and Ace. ANOVA was performed to evaluate α-diversity among the different groups. Beta diversity analysis was used to evaluate differences of the samples in species complexity. The linear discriminant analysis (LDA) effect size (LEfSe) method was performed to detect differentially abundant features with statistical significance and biological relevance among different groups and an LDA score of > 4 was used as the threshold. Metastats analysis was used to compare bacterial relative abundance between different groups.

In the passive avoidance test, the latency to enter the dark area (Fig. 4g) was significantly shortened and the frequency of entries into the dark compartment (Fig. 4h) was significantly increased in the mice receiving doses of 27-OHC higher than 1.65 mg/kg.

In summary, higher 27-OHC concentrations caused brain and intestine cell loss, Aβ deposits, neuroinflammation, and evident neurobehavioral impairment in mice. Only the 5.5 mg/kg dose of 27-OHC significantly affected all of the parameters measured in both the neurobehavioral tests and the neuropathologic examinations. Thus, the 5.5 mg/kg dosage can be used in further studies of 27-OHC in mice.

Simultaneously, Bielschowsky silver staining of Aβ in brain sections also showed that 27-OHC-treated APP/PS1 mice exhibited marked Aβ plaques deposition (Fig. 8e). What is more, APP/PS1 control mice exhibited significantly increased intestine and brain levels of 27-OHC, liver TC, TG, and LDL-C as well as brain and plasma levels of Aβ load than WT mice, suggesting pre-existing neuropathological changes in APP/PS1 mice.

We found that 27-OHC administration to APP/PS1 and WT mice significantly increased their latency to locate the platform (P < 0.05). Interestingly, the ANS-treated APP/PS1 group preferentially performed a selective search for the platform and had a shorter distance to the platform than the 27-OHC-treated APP/PS1 group (Fig. 9b). In addition, no significant difference was detected between ANS-treated APP/PS1 mice and 27-OHC + ANS-treated APP/PS1 mice (P > 0.05).

These findings were supported by representative images of the path of travel during the orientation navigation, which demonstrated a longer path length of APP/PS1 or WT mice treated with 27-OHC than the APP/PS1 or WT control mice, or the ANS alone treated mice (Fig. 9c).

In the spatial probe test, compared to the WT mice, the APP/PS1 group mice swam more randomly throughout the tank, suggesting poor memory retention of the location of the platform. The 27-OHC-treated APP/PS1 or WT group showed an obvious decrease in the number of target crossings (Fig. 9d) and target-quadrant abidance (Fig. 9e) compared to the control groups (P < 0.05), while the ANS-treated APP/PS1 group completely reversed this trend. 27-OHC + ANS-treated mice showed no significant differences in the number of target crossings or the target-quadrant abidance in comparison to the control mice (P > 0.05). For swimming speed, the ANS-treated APP/PS1 group had a significantly increased speed relative to both the control groups and the 27-OHC-treated groups (Fig. 9f, P < 0.05).

Next, we applied transmission electron microscopy for ultrastructural analysis of tight junctions and microvilli in the ileum and colon. Figure 15b shows selected micrographs of the ultrastructural analysis of each experimental group. Qualitative and quantitative analyses revealed that APP/PS1 and WT control mice showed a relatively intact apical intercellular ultrastructure, whereas 27-OHC-treated mice were morphologically indistinguishable from controls, as indicated by dilated (larger) intercellular spaces in the tight junctions (Fig. 15c) and shorter intestine microvilli with less density (Fig. 15d). However, the tight junctions and microvilli of the ANS-treated and 27-OHC + ANS-treated groups appeared to be better preserved, which suggests that the tight junctions and microvilli are maintained to some extent in response to ANS.