Dynamic Changes in the Levels of Amyloid-β₄₂ Species in the Brain and Periphery of APP/PS1 Mice and Their Significance for Alzheimer’s Disease

Introduction

Alzheimer’s disease (AD) is an age-related, irreversible form of dementia that affects nearly 50 million people worldwide (Collaborators, 2019). A defining pathological feature of AD is the presence of extracellular deposits of aggregated amyloid-β (Aβ) in the form of senile plaques in specific brain tissues and vascular walls (Murphy and LeVine, 2010), the main components of which are the peptide isoforms Aβ₄₀ and Aβ₄₂, of which Aβ₄₂ predominates in neuritic plaques of AD (Gu and Guo, 2013; Hong and Yaqub, 2019). Autopsy and Aβ positron emission tomography (PET) have indicated that Aβ deposition precedes cognitive decline by a decade or more (Driscoll et al., 2006; Sperling et al., 2011; Oxtoby et al., 2018). Aβ, a 4–4.5 kDa peptide containing 39–42 residues, is produced by sequential proteolytic cleavage of amyloid precursor protein (APP), which is expressed in brain cells and peripheral tissues (such as adrenal gland, kidney, heart, liver, spleen, muscles, and blood vessels) (Roher et al., 2009). Considering that skeletal muscle, accounting for 40% of total body weight (Kim et al., 2016), is only one of many peripheral sources of Aβ, peripheral Aβ may account for a large portion of total Aβ. As observed in human and animal models, brain-derived Aβ peptides can be transported from the brain to peripheral blood (Roberts et al., 2014; Xiang et al., 2015). The communication between the periphery organs and the brain allows for active and dynamic Aβ exchange in distinct reservoirs. The brain accumulation of Aβ aggregates is affected by the levels of Aβ₄₂ in the brain and peripheral tissues, but the contribution of peripheral Aβ toward the progression of AD is poorly understood.

Aβ₄₂ monomers (Aβ₄₂Ms), mainly α-helical and random coil structures, easily aggregate to form various soluble oligomers. The transition of monomers to oligomers triggers the aggregation and pathogenic transformation of Aβ peptides (Nag et al., 2011). A high concentration of Aβ₄₂ peptide promotes the formation of its aggregates from soluble monomers into toxic Aβ₄₂ oligomers (Aβ₄₂Os), and eventually forms extracellular neurotoxic plaques. It has been shown that the brain level of Aβ₄₂ increases in the early stage of AD, but decreases with the decline of cognitive ability in the late-stage AD (Naslund et al., 2000). The balance between Aβ production and clearance determines the concentration of Aβ in various reservoirs (Wang et al., 2006). Increasing evidence has implicated that diffusible and soluble Aβ oligomers (Bernstein et al., 2009), rather than insoluble fibrils and small monomers, are the main form of neurotoxicity. These oligomers induce neurotoxic intracellular signaling pathways such as neuronal injury (Wang W. et al., 2019), inflammatory (Salminen et al., 2008), mitochondrial dysfunction, and oxidative stress (Mucke and Selkoe, 2012). The fluctuation of amyloid subtypes in cerebrospinal fluid and blood mainly depends on Aβ₄₂Ms and Aβ₄₂Os, which are used as core biomarkers to reflect the progression of AD (Benilova et al., 2012). Therefore, dynamic measurement of the levels and profile of Aβ₄₂Ms and Aβ₄₂Os in the brain and periphery is crucial for studying their physiological metabolism and their substantial correlation with AD progression.

The reliable detection of Aβ₄₂Ms and Aβ₄₂Os is technically challenging because Aβ₄₂Os are transient, heterogeneous, and in dynamic equilibrium with Aβ₄₂Ms. In this study, we screened two sequence- and conformation-specific antibodies 1F12 and 2C6, that recognize different epitopes of Aβ₄₂. The preferred antibody pairs 1F12/2C6 and 1F12/1F12 were chosen to evaluate the potential significance of various sources and pools of Aβ₄₂ on the overall pathology of AD. The correlation between blood Aβ₄₂ levels and brain Aβ₄₂ levels in APP/PS1 and age-matched C57BL/6J mice at 3 and 9 months was dynamically assessed. Another comprehensive longitudinal study determined the potential effects of intestinal tissue-derived Aβ₄₂ on blood and brain Aβ₄₂ levels in APP/PS1 mice. We expect that our research will promote the understanding of the role of Aβ₄₂Ms and Aβ₄₂Os in the progression of AD.

Materials and Methods

Chemicals and Materials

The Aβ_3–9, Aβ_13–19, Aβ_18–25, Aβ_29–36, Aβ_36–42, Aβ₄₀, and Aβ₄₂ were custom-synthesized as lyophilized powders by Royo Biotech Co., Ltd (Shanghai, China) with a purity of >95%. Anti-Aβ (6E10) antibody was obtained from Invitrogen. The goat anti-mouse IgG (H + L), protein A resin, and protein L resin were ordered from GenScript (Nanjing, China). The mouse monoclonal antibody isotyping kit was purchased from Southern Biotech (Birmingham, AL, United States). The Pierce streptavidin-coupled poly-HRP and protein marker were ordered from Thermo Scientific (Massachusetts, United States). Thioflavin S, BSA, Freund’s complete adjuvant, and Freund’s incomplete adjuvant were obtained from Sigma-Aldrich. Cy3-NHS ester and Biotin-PEG4-NHS ester were provided by Lumiprobe (Hannover, Germany). All other chemicals were purchased from commercial suppliers and used as received.

Oligomeric and Monomeric Aβ Preparations

The Aβ₄₀ monomers (Aβ₄₀Ms) and Aβ₄₂Ms were obtained by dissolving lyophilized Aβ₄₀, Aβ₄₂ peptides in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), followed by incubation overnight at room temperature. HFIP was evaporated with nitrogen gas to form a film, and the Aβ was redissolved in dimethyl sulfoxide. The prepared Aβ₄₀, Aβ₄₂ monomers solution (50 μM) was stored at −20°C as stock solution. The Aβ₄₀ oligomers (Aβ₄₀Os) and Aβ₄₂ oligomers (Aβ₄₂Os) were obtained from 24 h incubation of 50 μM Aβ₄₀ and Aβ₄₂ monomer solutions at 37°C in the dark, respectively.

Cryo-Transmission Electron Microscopy

The patterns of the prepared Aβ₄₂Ms and Aβ₄₂Os were confirmed by cryo-transmission electron microscopy (Cryo-TEM). In brief, 5 μL of each sample was deposited on a copper grid, and the excess liquid was removed using a filter paper, leaving a thin film of the solution on the grid. The Tecnai G20 transmission electron microscope (FEI Ltd., United States) was used to characterize the morphology of the above-mentioned samples.

Generation and Purification of 1F12 and 2C6

The antigen was prepared using lyophilized synthetic Aβ₄₂ peptide dissolved in 10 mM NaOH and diluted with PBS to a final concentration of 1 mg/mL. In the first immunization, BALB/c mice were immunized with 50 μg Aβ₄₂ peptide mixed with Freund’s complete adjuvant. In the second and third immunizations, Aβ₄₂ peptides and Freund’s incomplete adjuvant mixtures were used (Zhang et al., 2018). After the third immunizations, 50 μg Aβ₄₂ peptide was used for booster immunization. Three days later, spleen cells were collected from the immunized mice and fused with SP2/0 cells via PEG at 37°C (Wang X. et al., 2019). The fused cells were maintained in HAT medium for 7 days and then cultured in HT medium. The positive hybridomas in each plate were screened by the limited dilution method (Kohler and Milstein, 1975). Three positive hybridoma cell lines were screened from the initial positive wells and cultured to prepare ascites. The immunoglobulins of 1F12, 2C6, and 2E2 were purified using protein A resin, according to the manufacturer’s instructions.

Dot Blot Assay

One microgram of each Aβ₄₂ truncated peptide including Aβ_3–9, Aβ_13–19, Aβ_18–25, Aβ_29–36, and Aβ_36–42 was pipetted onto a PVDF membrane activated by methanol. After the peptide samples were deposited, the PVDF membrane was air-dried and blocked in 5% skimmed milk in phosphate-buffered saline (PBS) with Tween-20 detergent (PBS-T) at 37°C for 1 h. The membranes were then incubated with mAb 1F12 (1:1000) or 2C6 (1:1000) at 37°C for 2 h. After washing three times in PBS-T, the membrane was incubated with the secondary antibody HRP-conjugated goat anti-mouse IgG (H + L) at 37°C for 2 h, and the immunological signals were detected with ECL-substrate (Vazyme, China) using Tanon 5200 Muiti (Shanghai, China).

Biotinylated 1F12 or 2C6 Antibody Preparation

The biotinylated antibodies were generated based on the reaction of amino groups of 1F12 or 2C6 antibody with biotin N-hydroxysuccinimide (NHS) ester. Briefly, 10 nmol of 1F12 or 2C6 antibody was dissolved in PBS, and the solution pH was adjusted to 8.5 with Na₂CO₃ (0.1 M), followed by a reaction with 20 nmol biotin-PEG4-NHS esters in DMSO at room temperature for 2 h. The biotin-modified 1F12 or 2C6 antibody was purified using size exclusion PD-10 columns with PBS as the mobile phase. Indirect ELISA determined the activities and titers of biotinylated 1F12 or 2C6. The indirect ELISA assay was performed using serially diluted biotin-labeled antibodies (from 1:100 to 1:409,600) instead of the primary antibody. After incubation with the Pierce streptavidin-coupled poly-HRP, the immunoreaction was visualized with a soluble TMB substrate solution.

Measurement of the Titer and Binding Affinity of 1F12 or 2C6 Antibody

The 96-well plates were coated with 0.5 μg/well of Aβ₄₂ and blocked with 5% skimmed milk dissolved in a PBS-T buffer for 2 h at room temperature. Subsequently, series of diluted 1F12 or 2C6 ranging from 1:100 to 1:409,600 were added into each well for 2-h incubation at 22°C. The subsequent steps were performed as described in the ELISA section above. The binding affinities of 1F12 or 2C6 with Aβ₄₂ species were determined using serially diluted preparations of 1F12 or 2C6 with serials of concentrations (from 10 μg/mL to 10 ng/mL) instead of different dilutions (from 1:100 to 1:409,600). After incubation with the HRP-conjugated goat anti-mouse IgG (H + L), the immunoreaction was visualized with a soluble TMB substrate solution.

Screening of Preferred Antibody Pairs for Total Aβ₄₂ and Aβ₄₂Os Sandwich ELISA

Preferred antibody pairs of total Aβ₄₂ and Aβ₄₂Os sandwich ELISA were screened from the combination of different antibody pairs. The screening process of antibody pairs for total Aβ₄₂ was as follows: 96-well plate was coated with 1 μg/well of 1F12 or 2C6 as the capture antibody for 2 h at room temperature, and then blocked with 5% skimmed milk. Five ng total Aβ₄₂ containing Aβ₄₂Ms and Aβ₄₂Os or Aβ₄₂Os was loaded into each well and incubated 2 h at room temperature, followed by the addition of biotinylated 2C6 or 1F12 as the detection antibody. Finally, streptavidin-coupled poly-HRP was used to visualize the immunoreaction of each well.

Sandwich ELISA for the Detection of Total Aβ₄₂ and Aβ₄₂Os

The 96-well plates were coated with 1 μg/well of 1F12 in the citrate-buffered saline buffer for 2 h, and then blocked with 5% skimmed milk dissolved in PBS-T for 2 h at room temperature. A standard series of synthetic Aβ peptides (Aβ₄₀Ms, Aβ₄₂Ms, Aβ₄₀Os, and Aβ₄₂Os) and the biological samples (mouse blood and organ homogenization) to be analyzed were added to the plates in triplicates and incubated for 40 min at room temperature. The plate was washed three times with PBS-T and incubated with biotinylated mAb 2C6 for total Aβ₄₂ detection or biotinylated mAb 1F12 for Aβ₄₂Os detection, followed by incubation with streptavidin-coupled poly-HRP for 1 h at room temperature. The immunoreaction in each well was detected using TMB substrate solution.

Quantification of Aβ Peptides in Blood, Brain, and Intestinal Tissue

All procedures involving animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology. Herein, 3-month-old APP/PS1 mice (six mice for blood collection including three female and three male mice, and four mice for tissue collection, including two female and two male mice), 9-month-old APP/PS1 mice (six mice for blood collection including three female and three male mice, n = 4 for tissue collection, including two female and two male mice), and age-matched non-transgenic C57BL/6J mice (six mice for blood collection, including three female and three male mice, and four mice for tissues collection, including two female and two male mice) were anesthetized with 0.4 mL Avertin (25 mg/mL) and intracardially perfused with 0.9% saline solution. The mouse blood was collected, and tissues were organically extracted with antigen extraction tris-buffered saline (TBS, 20 mM Tris and 137 mM NaCl, pH 7.6) at a ratio of 1:50 (w/v) using a tissue grinder. The TBS included a complete protease inhibitor cocktail (Roche). The supernatant of homogenates was centrifuged at 10,000 g for 30 min in Type 60 Ti fixed angle rotor at 4°C to obtain TBS-soluble proteins. The collected mouse blood, prepared brain, and intestinal tissue samples were used for Aβ₄₂ quantification by the above-prepared sandwich ELISA.

Immunoprecipitation and Western Blotting

The homogenates of organ tissues (brain, stomach, duodenum, jejunum, ileum, cecum, colon, and intestinal lysates) or blood were incubated for 30 min at room temperature with 40 μg/mL of 1F12 antibody conjugated on the protein A/G magnetic bead, according to the manufacturer’s instructions. The immunoprecipitated proteins were eluted with 0.1 M glycine (pH 3.0) and immediately neutralized to pH 7.4 using a neutralization buffer (1 M Tris-HCl, pH 8.5). The samples were then denatured in loading buffer (Boster Biotech, United States) and boiled for 10 min. Following this, the proteins were run on a 12% reduced tris-tricine SDS-polyacrylamide gel via SDS-PAGE. Proteins were transferred onto a polyvinylidene fluoride membrane at 160 mA for 1 h, at 4°C. Membranes were blocked with 5% skimmed milk dissolved in 1 × PBS-T and incubated with 1F12 or 2C6 for 2 h at 37°C. Subsequently, the membranes were washed in PBS-T followed by 1 h incubation with secondary antibody HRP-conjugated goat anti-mouse IgG (H + L), and the immunological signals were detected using the ECL-substrate (Vazyme, China) on Tanon 5200 Muiti (Shanghai, China).

Immunofluorescence Assays of Tissues

The mice were anesthetized with 0.4 mL Avertin (25 mg/mL), and cardiac perfusion was performed with 4% paraformaldehyde (PFA) for 30 min. The tissues (brain, duodenum, jejunum, ileum, cecum, and colon) from mice were collected, fixed in 4% PFA, and dehydrated in sucrose solution. After embedding in OCT compound (Tissue-Tek; Sakura Finetek, Netherlands, United States), 15 μm coronal frozen sections of tissue samples were serially cut on a Leica CM3050 S cryostat. Each slice was mounted on glass slides and permeabilized with 0.2% Triton X-100 overnight at 4°C. The slice was blocked with 3% BSA for 2 h at room temperature and incubated with thioflavin S or commercially available anti-Aβ antibody 6E10, Cy3-conjugated 1F12 or 2C6 overnight at 4°C. All slides were washed five times with PBS and stained with thioflavin S. The Zeiss LSM710 microscope was used to image slides with red, green, and blue fluorescence filters.

Sequencing Data and Statistical Analyses

Sequencing data from an independent study of the APP/PS1 mice gut microbiota was obtained. Raw 16S sequences were downloaded from SRA accession PRJNA543965 and preprocessed using the ‘dada2’ R package. Reads were truncated at 150 bp, then a maximum expected error rate threshold of 1 was imposed. Taxonomy was assigned using the Silva v138 rRNA database using the default classify algorithm of dada2 (v1.16.0).

Downstream analysis was performed by several R packages including ‘phyloseq,’ ‘MicrobiotaProcess,’ ‘DESeq2,’ and ‘ggplot2.’ Beta diversity was measured by principal coordinate analysis (PCoA) based on weighted UniFrac phylogenetic distance. The significantly differed genus between groups was determined using ‘DESeq2,’ and their significance of difference was assessed by Wilcoxon rank-sum test. The significance cut-off was set at ^∗p < 0.05.

The data, except for sequencing data, are presented as means ± SEM. One-way or two-way analysis of variance (ANOVA) was used for multiple group comparisons. Statistical significance is represented in the figure by ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^****p < 0.0001, and n.s. (indicating no significance). All statistical analyses were performed with GraphPad Prism7.0 software.

Results

Screening and Identification of Aβ₄₂ Sequence- and Conformation-Specific Antibodies

BALB/c mice were immunized with human Aβ₄₂ peptide preparations, and a pool of approximately 1,500 clones was generated via hybridoma technology. In addition, 75 of these clones reacted with Aβ monomers and oligomers in ELISA (Supplementary Figure 1A). Then three positive hybridoma clones 1F12, 2C6, and 2E2 were selected, and their immunoglobulin isotypes were determined. The results showed that the isotype of 1F12 and 2C6 was IgG2a, and the isotype of 2E2 was IgA, and the light chains of these three monoclonal antibodies (mAbs) all belonged to the kappa chain (Figure 1A). Reduced SDS-PAGE identified the purity and molecular weight of these three mAbs (Supplementary Figure 1B). Therefore, 1F12 and 2C6 were chosen to characterize their reactivity by Western blotting brain natural Aβ₄₂ and immunostaining brain slices of APP/PS1 mice. The results displayed strong bands of Aβ₄₂ peptides recognized by 1F12 and 2C6, comparable to those obtained with 6E10 (Supplementary Figure 1C). Immunofluorescence imaging (using thioflavin S or commercially available anti-Aβ antibody 6E10) showed that Aβ plaques were colocalized with Cy3-labeled 1F12 (Figure 1B and Supplementary Figure 2A) or 2C6 (Supplementary Figures 1D, 2B). The titer of mAbs was determined by indirect ELISA and calculated as 1:204800 for 1F12, 1:102400 for 2C6, 1:51200 for 2E2 (Supplementary Figure 1E).

Figure 1. Characterization of sequence and conformation-specific antibodies for Aβ₄₂Ms and Aβ₄₂Os. (A) The isotypes of the prepared monoclonal antibodies. (B) Confocal fluorescence images of murine APP/PS1 brain sections using Cy3-labeled anti-Aβ₄₂ monoclonal antibody 1F12 and commercially available anti-Aβ antibody 6E10. (Scale bar: 200 μm). (C) Linear epitopes of Aβ₄₂ recognized by 1F12 and 2C6. (D) The binding affinities of 1F12 and 2C6 to Aβ₄₂ and Aβ₄₀ were determined by indirect ELISA. (E) The morphologies of Aβ₄₂Ms and Aβ₄₂Os were analyzed by cryo-transmission electron microscopy in parallel. (Scale bar: 500 nm). The purity and molecular weight of freshly prepared Aβ₄₂Ms and Aβ₄₂Os were determined by 12% reduced SDS-PAGE using Coomassie blue staining (F) and Western blotting with 1F12 (G). (H) The K_d value of 1F12-binding Aβ₄₂Ms and Aβ₄₂Os was detected with indirect ELISA. Data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used for multigroup comparisons. Statistical significance is indicated in the figures by ****p < 0.0001.

To identify the linear fragments (epitopes) of Aβ₄₂ recognized by 1F12 and 2C6, we performed epitope mapping experiments using a series of peptides starting from +3 to +42 in the Aβ₄₂ sequence. Based on a series of dot-blot analyses, we observed that 1F12 displayed a linear epitope, amino acids 3–9 located in the N-terminal region of the Aβ₄₂ peptide (Figure 1C, left). In contrast, 2C6 exhibited discrete epitopes, including four distinct Aβ₄₂ fragments (Figure 1C, right). To confirm the binding efficacy of 1F12 and 2C6 to Aβ₄₂ and Aβ₄₀ peptides, indirect ELISAs were performed on separate Aβ₄₂ and Aβ₄₀ coated plates. The 1F12 and 2C6 detection signals of Aβ₄₂ were significantly higher than those of Aβ₄₀ (Figure 1D). Taken together, 1F12 and 2C6 were Aβ₄₂ sequence-specific antibodies, and both showed a preference for the conformational epitope presented by Aβ₄₂ rather than Aβ₄₀.

Binding Affinities and Selectivity of 1F12 and 2C6 for Different Aβ₄₂ Species

We further evaluated the binding affinity of 1F12 and 2C6 to Aβ₄₂Ms and Aβ₄₂Os with different conformations. Cyo-TEM confirmed the morphology of the prepared Aβ42Ms and Aβ42Os. The results showed that the morphology of Aβ₄₂Ms was α-helical and random coil structures (Figure 1E, up), while Aβ₄₂Os formed a β-sheet and typical fibril three-dimensional structures (Figure 1E, down). 12% reduced SDS-PAGE gel confirmed the purity and molecular weight of the prepared Aβ₄₂Ms and Aβ₄₂Os (Figure 1F), and their components were verified by Western blotting (Figure 1G and Supplementary Figure 1F). Indirect ELISA results showed that the K_d values of 1F12 bound to Aβ₄₂ species were 1.66 ± 0.09 nM for Aβ₄₂Ms and 0.38 ± 0.04 nM for Aβ₄₂Os (Figure 1H), while the K_d values of 2C6 were 3.59 ± 0.27 nM for Aβ₄₂Ms and 0.61 ± 0.03 nM for Aβ₄₂Os (Supplementary Figure 1G). Taken together, 1F12 and 2C6 were Aβ₄₂ sequence- and conformation-specific antibodies and could bind Aβ₄₂ species with different conformations.

Preferred Antibody Pairs for Specific Detection of Total Aβ₄₂ and Aβ₄₂Os

The screening of capture and detecting antibodies for sandwich ELISA is a prerequisite for developing techniques to detect and quantify Aβ₄₂Ms and Aβ₄₂Os. Both 1F12 and 2C6 were biotinylated, and indirect ELISA showed that their bioactivities and titers were high (Supplementary Figures 1H,I). To achieve the specific detection of total Aβ₄₂ and Aβ₄₂Os, a combination of different antibody pairs (1F12/2C6, 1F12/2E2, 2C6/1F12, 2C6/2E2, 2E2/1F12, 2E2/2C6) was screened for preferred antibody pairs by comparing their susceptibility to total Aβ₄₂ and Aβ₄₂Os in sandwich ELISA (Figure 2A). The capture/detection antibody pair 1F12/2C6 had the highest detection signal and specificity for total Aβ₄₂ among the six antibody pairs in ELISA (Figures 2B,C), while the 1F12/1F12 antibody pair was significantly more effective and specificity in detecting Aβ₄₂Os (Figures 2D,E).

Figure 2. Screening of preferred antibody pairs 1F12/2C6 and 1F12/1F12 against Aβ₄₂Ms and Aβ₄₂Os. (A) Schematic representation of 1F12/2C6, 1F12/1F12 ELISA to detect total Aβ₄₂, Aβ₄₂Os, respectively. (B) Preferred antibody pairs were screened for the detection of total Aβ₄₂. (C) The specificity assay of 1F12/2C6 ELISA for total Aβ₄₂ detection. (D) Preferred antibody pairs were screened for the detection of Aβ₄₂Os. (E) The specificity assay of 1F12/1F12 ELISA for Aβ₄₂Os detection. Data are presented as means ± SEM. Two-way analysis of variance (ANOVA) was used for (B,D) and One-way analysis of variance (ANOVA) was used for (C,E). Statistical significance is indicated in the figures by **p < 0.01 and ****p < 0.0001.

Dynamic Monitoring of Changes in Total Aβ₄₂, Aβ₄₂Os, and Aβ₄₂Ms in Blood and Brain

To dynamically monitor the changes of total Aβ₄₂, Aβ₄₂Os, and Aβ₄₂Ms in the brain and periphery blood, 1F12/2C6 and 1F12/12 ELISAs were performed to quantify Aβ₄₂ in peripheral blood and brain extracts of 3 and 9-month-old APP/PS1 mice. The results showed that regardless of age (at 3- or 9-month-old), the total Aβ₄₂ in blood and brain extracts of APP/PS1 mice was significantly higher than that in the tissue extracts of C57BL/6J (Figure 3A). 1F12/2C6 ELISA showed that compared with the 3-month-old APP/PS1, the total Aβ₄₂ levels in the blood of the 9-month-old APP/PS1 did not change significantly (p = 0.1787), but the Aβ₄₂ content in the brain tissue was significantly increased (p = 0.0062, Figure 3A). In comparison, 1F12/1F12 ELISA showed that the Aβ₄₂Os level in the blood (p = 0.005) and brain (p < 0.0001) of APP/PS1 at 9 months old were significantly higher than that of APP/PS1 mice at 3 months old (Figure 3B). The amount of Aβ₄₂Ms was calculated by subtracting the total Aβ₄₂ from Aβ₄₂Os. The level of Aβ₄₂Ms in the blood of APP/PS1 mice at 9 months old was significantly lower than that of APP/PS1 mice at 3 months old (p = 0.0004, Figure 3C). The levels of total Aβ₄₂, Aβ₄₂Os, and Aβ₄₂Ms in C57BL/6J mice between 3 and 9 months old did not change significantly (Figures 3A–C). To further validate the 1F12/2C6 and 1F12/1F12 ELISA results, an immunoprecipitation (IP) assay was performed using blood and brain extracts of 3- and 9-month-old APP/PS1 mice. As shown in Figure 3D, a clear single band was observed in the blood and brain extracts of 3-month-old APP/PS1, and its molecular weight was similar to Aβ₄₂Ms. The results were consistent with the ELISA results as above-mentioned, confirming that the level of Aβ₄₂Ms at the early stage of AD (3-month-old APP/PS1) was high and the level of Aβ₄₂Os in the late stage of AD (9-month-old APP/PS1) were elevated. Western blotting analysis of immunoprecipitated proteins in blood and brain extracts of 9-month-old APP/PS1 mice revealed a prominent Aβ₄₂Os band with a molecular weight of more than 5 kDa and several clear bands of monomers to tetramers (Figure 3E). Taken together, Aβ₄₂ mainly existed as a monomer in the blood at the early stage of AD (e.g., 3-month-old APP/PS1 mice with less Aβ plaque load and Iba 1-positive cells staining, Figures 3F–H), whereas it appears as oligomers in blood and brain extracts at the late state (e.g., 9-month-old APP/PS1 mice with more Aβ plaque load and Iba 1-positive cells staining, Figures 3F–H).

Figure 3. Dynamic detection of the changes of Aβ₄₂Ms and Aβ₄₂Os in blood and brain of APP/PS1 mice by 1F12/2C6 and 1F12/1F12 ELISA. The level of total Aβ₄₂ (A), Aβ₄₂Os (B), and Aβ₄₂Ms (C) in the blood (n = 6) and brain (n = 4) of 3- and 9-month-old APP/PS1 and C57BL/6J mice. Total Aβ₄₂ in the blood and brain of 3 (D) or 9-month-old (E) APP/PS1 mice were enriched with immunoprecipitation (2C6) and visualized via Western blotting (1F12). (F) Confocal fluorescence images of Aβ plaque and Iba 1-positive cells staining in 3 or 9-month-old APP/PS1 mice. Aβ plaque (G) and Iba 1-positive (H) areas in the brains of 3- or 9-month-old APP/PS1 mice. Data are presented as means ± SEM. Two-way analysis of variance (ANOVA) was used in (A–C) for multigroup comparisons. Unpaired t-test was used in (G,H) for two-group comparisons. Statistical significance is indicated in the figures by **p < 0.01, ***p < 0.001, ****p < 0.0001, and n.s. (indicating no significance).

Correlation of Total Aβ₄₂, Aβ₄₂Os, and Aβ₄₂Ms Level in the Gastrointestinal System With AD Progression

Apart from blood and brain, we also paid attention to the distribution of Aβ₄₂ in the intestinal system of APP/PS1 mice, such as duodenum, jejunum, ileum, colon, cecum, and their lysates. Several research groups have reported that AD may begin in the intestine and is closely related to the imbalance of intestinal flora (Hu et al., 2016; Harach et al., 2017; Jiang et al., 2017; Mancuso and Santangelo, 2018). To detect the distribution of total Aβ₄₂, Aβ₄₂Ms, and Aβ₄₂Os in the intestinal systems and explore its potential correlation with the pathogenesis of AD, organs extracted from different parts of intestines (duodenum, jejunum, ileum, colon, and cecum) and their lysates from 3- and 9-month-old APP/PS1 mice were collected for 1F12/2C6 and 1F12/1F12 ELISA. No Aβ₄₂ was detected in the different parts of intestines in 3-month-old APP/PS1 mice, but a certain amount of Aβ₄₂ was observed in jejunum lysate and colonic lysate (Figure 4A). Further analysis revealed Aβ₄₂ subtype in the jejunum and colon lysates. It was found that Aβ₄₂Os were observed in both jejunum and colonic lysate, with higher levels of Aβ₄₂Os in colon lysates (Figure 4B). But for Aβ₄₂Ms, a weak signal was only observed in the colonic lysate (Figure 4C). However, for 9-month-old APP/PS1 mice, total Aβ₄₂ signals were clearly observed in all gastrointestinal organs and their lysates, except for duodenum, duodenum lysates, and colonic lysates (Figure 4A). In a detailed analysis, we found that Aβ₄₂ mainly existed in the form of oligomers and was detected in all gastrointestinal organs except for the duodenum (Figure 4B). For the lysates, the result was opposite to the Aβ₄₂Ms level, and only two oligomers were detected with weak signals in the jejunum and ileum lysates (Figure 4C). For 3 or 9-month-old C57BL/6J mice, no significant differences were observed in the gastrointestinal organs and their lysates (Supplementary Figures 3A,D). Interestingly, the monomeric (Supplementary Figures 3C,F) or oligomeric (Supplementary Figures 3B,E) forms of Aβ₄₂ levels in APP/PS1 mice were significantly higher than those in the C57BL/6J at 3 or 9 months old. Altogether, the ELISA results confirmed that both Aβ₄₂Ms and Aβ₄₂Os exist in the gastrointestinal system, and their distributions are different in APP/PS1 mice, but their levels increase with age.

Figure 4. Dynamic changes in the levels of Aβ₄₂Ms and Aβ₄₂Os in the intestines were detected and quantified via 1F12/2C6 and 1F12/1F12 ELISA. The level of total Aβ₄₂, Aβ₄₂Os, and Aβ₄₂Ms in the stomach, duodenum, jejunum, ileum, colon, and cecum and their lysates from 3-month-old APP/PS1 (n = 4; A–C), 9-month-old APP/PS1 (n = 4; A–C). (D) Representative IP-Western blotting data to analyze the distribution of Aβ₄₂ in the stomach, duodenum, jejunum, ileum, colon, and cecum and their lysates from 3-month-old APP/PS1 (i), 9-month-old APP/PS1 (iii), and C57BL/6J mice (ii,iv) at the same ages. (E,F) The Western blotting results for Aβ₄₂ in APP/PS1 or C57BL/6J mice were quantified. Data are presented as means ± SEM. Two-way analysis of variance (ANOVA) was used for multigroup comparisons. Statistical significance is indicated in the figures by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Meanwhile, IP-Western blotting further confirmed the presence of Aβ₄₂ in the intestine through 1F12/2C6 and 1F12/1F12 ELISA. The Aβ₄₂Os signal was only observed in jejunal lysates and colon lysates of 3-month-old APP/PS1 mice (Figure 4D, i). In comparison, no Aβ₄₂ species were detected in 3-month-old C57BL/6J mice (Figure 4D, ii). Compared with 3-month-old APP/PS1 mice, the distribution of Aβ₄₂Os and Aβ₄₂Ms in 9-month-old APP/PS1 mice was quite different. In 9-month-old APP/PS1 mice, obvious Aβ₄₂Os bands were observed in the stomach, jejunum, ileum, colon, cecum, jejunum lysate, and ileum lysate, while Aβ₄₂Ms levels were detected in low in the jejunum, ileum, and in high in the lysates of stomach, jejunum, and cecum (Figure 4D, iii). However, in age-matched C57BL/6J mice, no bands of Aβ₄₂Ms or/and Aβ₄₂Os were observed (Figure 4D, iv). The light chain of 1F12 was used as an internal reference to ensure that the same amount of immunomagnetic beads was added to each sample. The quantitative data of Western blotting (Figures 4E,F) were consistent with the 1F12/2C6 and 1F12/1F12 ELISA results (Figures 4A–C and Supplementary Figures 3A–F), indicating that both the levels of Aβ₄₂Ms and Aβ₄₂Os and their distributions in the gastrointestinal system are correlated with AD progression.

Based on the results of IP-Western blotting and sandwich ELISA, we further investigated the accumulation of insoluble Aβ plaques in the intestine because Aβ₄₂Ms and Aβ₄₂Os were the core components of amyloid plaques. The immunofluorescence assay (IFA) showed that the weak fluorescent signals of thioflavin S and Cy3-1F12 were colocalized in the duodenum of 3-month-old mice (Figure 5, left). However, in 9-month-old APP/PS1 mice, obvious fluorescent colocalization signals of thioflavin S and Cy3-1F12 were observed in the duodenum, ileum, and cecum, which of their fluorescence signals were significantly stronger than that of 3-month-old APP/PS1 mice (Figure 5, right). In comparison, in 3- or 9-month-old C57BL/6J mice, no fluorescent signals of thioflavin S and Cy3-1F12 were observed (Supplementary Figure 3 and Figure 4). Overall, the results of sandwich ELISA (Figures 4A–C), IP-Western blotting (Figure 4D), and IFA (Figure 5) convincingly demonstrated that Aβ exists in the intestine of APP/PS1 mice, and their levels correlate to AD progression.

Figure 5. Immunofluorescence assay for insoluble Aβ plaques in different parts of the intestines of APP/PS1 mice. The levels of insoluble Aβ plaques in the duodenum, jejunum, ileum, cecum, and colon of 3-month-old (left) and 9-month-old (right) APP/PS1 mice were evaluated by double-staining with thioflavin S and Cy3-IF12 (Scale bar: 100 μm).

Dynamic Changes in Microbiome Composition of APP/PS1 Mice With Age

To investigate the changes in microbiome composition of APP/PS1 mice with age, we compared relative microbial abundance at genus levels among 3-month-old APP/PS1 mice, 9-month-old APP/PS1 mice, and age-matched C57BL/6J mice. Among all three groups, the abundance of Lachnospiraceae fluctuated with age (Figure 6A). To further characterize the microbiome composition, a beta diversity analysis was performed based on PCoA and weighted UniFrac distance in 3 and 9-month-old APP/PS1 mice (Figure 6B). The PERMANOVA results revealed that the microbiome structure was reshaped during the development of AD (Figure 6C). Then, representative bacterial taxa that differed significantly across two APP/PS1 groups were determined by ‘DESeq2’ R package based on the negative binomial distribution. Taxa were sorted by fold change between the two groups at the genus level (Figure 6D). In addition, the content of Turicibacter, Atopostipes, and Lachnospiraceae in 9-month-old APP/PS1 mice was significantly higher than that in 3-month-old mice (Figure 6E). Meanwhile, the Prevotellaceae was less abundant in aged APP/PS1 mice (Figure 6E). Collectively, these results illustrated that in the early stages of AD, the composition of the gut microbiota shows a significant difference.

Figure 6. Comparison of microbiome composition of APP/PS1 mice at 3 and 9 months old. (A) The relative abundance of top microorganisms in the feces of 3 and 9-month-old APP/PS1 mice and age-matched C57/BL/6 mice. (B) Principal coordinate analysis (PCoA) based on weighted UniFrac phylogenetic distance to compare 3 and 9-month-old APP/PS1 mice. (C) Comparison of beta diversity between 3 and 9-month-old APP/PS1 mice based on weighted UniFrac phylogenetic distance. The data was calculated using permutational multivariate analysis of variance (PERMANOVA) by R package ‘vegan.’ **p < 0.01. (D) Significant difference in the bacterial genus with relative abundance ≥ 0.01% between 3 and 9-month-old APP/PS1 mice. (E) The main genus of APP/PS1 mice with significant differences at 3 and 9 months. Comparisons of the relative abundance of gut microbiota between groups at the genus level were performed using two-tailed Wilcoxon rank-sum tests. Statistical significance is indicated in the figures by *p < 0.05, **p < 0.01, and ****p < 0.0001.

Discussion

In this study, we screened Aβ₄₂ sequence- and conformation-specific antibodies 1F12 and 2C6 and selected preferred antibody pairs 1F12/2C6 and 1F12/1F12 for sandwich ELISA to accurately detect Aβ₄₂Ms and Aβ₄₂Os in blood and organs. Of note, in the results of identifying the epitopes of 1F12 and 2C6, we find that the 2C6 exhibited discrete epitopes. Presently, there are about five epitopes on the surface of Aβ₄₂ peptides, mainly including Aβ_3–9, Aβ_13–19, Aβ_18–25, Aβ_29–36, and Aβ_36–42 and several antibodies have been designed for the corresponding epitopes (De et al., 2019). Interestingly, our prepared 2C6 can recognize the Aβ_3–9, Aβ_13–19, Aβ_18–25, Aβ_29–36, but not for Aβ_36–42. In the detailed analysis of the immune response of Aβ_36–42, we used the keyhole limpet hemocyanin-coupled Aβ_36–42 peptides to immunize BALB/c mice, but only one of three mice showed a weak immune response with a low titer. The inadequate immune response may be due to the Aβ_36–42 peptides (VGGVVIA) containing many hydrophobic amino acids, such as V, I, and A, making it difficult for animals to absorb and followed to produce antibodies. Therefore, the explanation of why 2C6 exhibits discrete epitopes is that there are more hydrophilic or neutral amino acids instead of hydrophobic amino acids in the region of Aβ_1–36, making it easier to be exposed and recognized by antibodies. Besides, the successful preparation of mAb A8978 (against the intermediate epitope Aβ_13–28) and mAb 78 (against Aβ_7–11, Aβ_18–24, and Aβ_26–32) indicate that it is possible to generate multiple epitope antibodies in the Aβ_1–36 region (Hatami et al., 2014; Nabers et al., 2018), and all these results also further confirm our explanation. Studies describing the different roles of Aβ₄₂ sequence region in the toxicity mechanisms have been reported by several groups. In general, the C-terminal area of Aβ₄₂ plays a significant role in inducing bilayer permeability (De et al., 2019), while the middle and N-terminal parts of Aβ₄₂ are effective in causing microglial inflammation and TLR signaling (Colvin et al., 2017; De et al., 2019). Therefore, antibodies targeting the C-terminal region of Aβ₄₂ are more effective in reducing membrane permeability induced by Aβ₄₂ aggregates, whereas antibodies targeting the middle and N-terminal areas of Aβ₄₂ are more potent in reducing the inflammatory response induced by Aβ₄₂ aggregates. These differences may be due to the accessibility of solvent-exposed N-terminal fragments in larger aggregates formed in the later stages of Aβ₄₂ aggregation. Altogether, the functional effect of antibodies with more epitopes may be far greater than that of single-epitope antibodies, especially in terms of immunotherapy.

The 1F12/2C6 and 1F12/1F12 ELISAs provided sufficient sensitivity to detect the levels of Aβ₄₂Ms and Aβ₄₂Os in blood and tissue extracts of APP/PS1 mice, and the results were not affected by the peripheral expression of APP or Aβ₄₀ because the prepared 1F12 and 2C6 showed more preference for Aβ₄₂ species. The results of ELISA and immunoprecipitation (IP) performed in blood and brain extracts of 3- and 9-month-old APP/PS1 mice well confirmed that no APP protein brand was observed or detected, except for Aβ₄₂Ms and several small Aβ₄₂Os (Figures 1D, 2C,E, 3D,E). Longitudinal studies of blood Aβ₄₂Ms and Aβ₄₂Os levels showed extensive temporal variation within and among APP/PS1 mice that participated in our research. Based on the 1F12/2C6 and 1F12/1F12 ELISA tests, it was found that the decrease in the levels of total Aβ₄₂ in blood was accompanied by its increase in the brain, but total Aβ₄₂ levels in blood were not correlated with AD progression (p = 0.2146, Figure 3A). The results are unexpectedly consistent with the literature on Aβ₄₂ detection because the Aβ₄₂ level in previous reports did not show a significant change (Hoglund et al., 2005) using a pair of antibodies that recognize different Aβ₄₂ epitopes, which is similar to 1F12/2C6 ELISA. In contrast, soluble Aβ₄₂Os, whose levels are elevated in AD patients, are easily mis-detected in the measurement of Aβ₄₂, resulting in underestimation of Aβ₄₂Ms level and poor performance in assessing the progression of AD (Yang et al., 2013; Liu et al., 2017). However, based on our method, we found obvious decrease in the level of Aβ₄₂Ms and significantly increased level of Aβ₄₂Os in blood, and prominently elevated level of Aβ₄₂Os in the brains of 3 and 9-month-old APP/PS1 mice (Figures 3B,C). Therefore, the levels of Aβ₄₂Ms and Aβ₄₂Os in blood and brain are closely associated with AD progression, which is helpful for us to further understanding of the pathogenesis of AD.

Increasing evidence indicates that the intestine or intestinal flora is associated with the progression of AD. However, to date, almost no literature directly reports the presence of Aβ₄₂ in the intestinal tissues and further elaborates the relationship between Aβ₄₂ in the intestine and AD progression. Our study showed that significantly increased levels of Aβ₄₂ were observed in the gastrointestinal organs (including stomach, jejunum, ileum, colon, cecum, jejunum lysate, ileal lysate, and cecum lysate) of 9-month-old APP/PS1 mice but not in 3-month-old APP/PS1 mice or age-matched C57BL/6J mice (Figures 4, 5). The level of Aβ₄₂Ms in the blood and brain of 3-month-old APP/PS1 mice was significantly higher than that in the intestine, indicating that the human Aβ produced by APP/PS1 mice is mainly distributed in the blood and brain. While the increasing Aβ₄₂ levels in the gastrointestinal organs still needed more evidence to confirm whether it originates from the brain or/and blood. Presently, several potential mechanisms of how brain Aβ is released into peripheral tissues have been mentioned. Some convincing evidence suggests that brain-derived soluble Aβ₄₂Ms and Aβ₄₂Os can be absorbed by neurons and enter the intestine along the vagus nerve (Foley et al., 2020). In contrast, the administration of Aβ₄₂ into the gastrointestinal tract may induce amyloidosis in the central nervous system (CNS) and AD-related pathologies, such as dementia (Sun et al., 2020). Besides, a study provided by Cintron et al. (2015) demonstrated the ability of peripheral monocytes to transport Aβ₄₂ aggregates from the abdominal cavity to the brain, spleen, and liver. Several other groups claimed that Aβ seeds could be transported via axons (Clavaguera et al., 2014; Guo and Lee, 2014; Boluda et al., 2015; Ye et al., 2015). In addition, the levels of Aβ₄₂ in the peripheral blood neuronal-derived exosomes in AD patients were higher than those in aMCI and healthy people, indicating that exosomes could also act as transport vehicles for Aβ₄₂ (Jia et al., 2019; Lakshmi et al., 2020). Altogether, neurons, axons, neuronal-derived exosomes, and peripheral monocytes can be used as potential carriers of Aβ₄₂ delivery, and there is currently evidence supporting axonal transport as a critical mode of disease propagation within the nervous system. In addition to the brain, other non-neural tissues, including the pancreas, kidney, spleen, heart, liver, testis, aorta, lung, skin, adrenal glands, and thyroid, also express amyloid-β protein precursor (APP). It is worth noting that the peripheral expression of APP will have the opportunity to be hydrolyzed to produce Aβ₄₂ when APP is transferred to its resident site tans-Golgi network (TGN), where β- and γ-secretase are distributed (Roher et al., 2009; Querfurth and LaFerla, 2010; Sakono and Zako, 2010; Hodson, 2018). The produced Aβ₄₂ is recycled to the cell membrane surface, which may increase the level of Aβ₄₂ in peripheral tissues. However, to date, it is difficult to evaluate the impact of APP produced by peripheral tissues on Aβ levels in the brain and blood. The current evidence supports the brain as the primary source of Aβ pools because Aβ levels are the highest. Whether the source of APP or/and Aβ in peripheral tissues contributes to the Aβ pool of brain, blood or gastrointestinal organs and its influence on the detection of Aβ₄₂ levels in the brain and blood need to be further confirmed to clarify.

In a detailed analysis of the intestinal origin of Aβ₄₂, we found that Aβ₄₂Ms and Aβ₄₂Os were first detected in colonic lysates of 3-month-old APP/PS1 (Figures 4B–D, i). Interestingly, this phenomenon is partly consistent with the study reported by Hui et al. (2012) that the colon is considered the first segment of the gastrointestinal tract where Aβ deposits occur. This phenomenon may be explained that in the early stage of AD, the Aβ₄₂, especially Aβ₄₂ monomers or small oligomers from the brain and blood pools, can be easily transmitted through the enteric nervous system (ENS) to the digestive tract of the stomach and intestines. According to reports, the colon contains the most neurons (Gershon, 1998; Rao and Gershon, 2016; Brierley et al., 2018; Borgmann et al., 2021), and the monomers that gradually accumulate in the colon can be aggregated into small molecular weight oligomers. It is worth noting that the colon undergoes vital processes to ensure our health. These processes are coordinated by transmitting sensory signals from the periphery to the central nervous system, allowing communication from the intestine to the brain via the “gut-brain axis” (Brierley et al., 2018). Foley et al. (2020) have confirmed that soluble Aβ₄₂Ms and Aβ₄₂Os could be absorbed by neurons and enter the intestine along the vagus nerve. Therefore, there is a strong correlation between the colon and degenerative neurological disease. In general, our results and previous evidence may well explain the observation of Aβ₄₂ in colon lysates, but the precise correlation of Aβ₄₂ and the intestine still needs to be studied. Besides, the intestine and enteric nervous systems play an important role in neurological disorders and deserve our attention.

Compared to 3-month-old APP/PS1, we did not observe an increase in Aβ₄₂ peptides in colonic lysates, although the levels of Aβ₄₂Ms and Aβ₄₂Os increased sharply in 9-month-old APP/PS1 (Figures 4B–D, iii). The significant difference in Aβ₄₂ levels in colonic lysates of 3- and 9-month-old APP/PS1 mice may be due to the migration of Aβ₄₂ in the colon from the colon to other gastrointestinal organs. This migration process has been confirmed by multiple groups (Cintron et al., 2015; Sun et al., 2020). In addition, high Aβ₄₂ levels were detected in the cecum, ileum, jejunum, and even stomach of 9-month-old APP/PS1 mice. Among them, the Aβ₄₂ level in the ileum was more significant and higher than that in the colon or colon lysate. These results further confirmed the ability of Aβ₄₂ peptides to spread in the colon as seeds. Thus, the disappeared Aβ₄₂ peptide in the colonic lysate of 9-month-old APP/PS1 may have migrated to other gastrointestinal organs such as the cecum, ileum, jejunum, and stomach because their Aβ₄₂ levels were significantly higher than colon lysates. In summary, our study, together with previous studies, confirms the ability of Aβ₄₂ peptides to spread as seeds, which leads to extensive changes in Aβ₄₂ levels within and between different APP/PS1 mice.

In this study, we monitored the dynamic changes of Aβ₄₂Ms and Aβ₄₂Os levels in the intestines and studied their significance for AD. Indeed, our results convincingly demonstrated that Aβ exists in the intestines of APP/PS1 mice, and their levels are correlated with AD progression. However, a current controversy is whether the gastrointestinal organs can directly produce Aβ₄₂. Several studies have reported that microorganisms including Bacillus subtilis, Shigella, Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Mycobacterium tuberculosis can produce functional extracellular amyloid proteins but not for Aβ₄₂ peptides (Hufnagel et al., 2013; Schwartz and Boles, 2013; Friedland, 2015; Pistollato et al., 2016). In fact, it seems that there is insufficient evidence to show that Aβ₄₂ peptides could be directly produced in the organs of the gastrointestinal tract. In addition, the practical and accurate distinction between locally produced and transported Aβ₄₂ peptides is crucial for helping to understand the pathology of AD. Distinguishing the local production and transportation of Aβ₄₂ peptides is our next research plan.

In addition to finding elevated levels of soluble Aβ₄₂, several insoluble Aβ₄₂ plaques were observed in the intestines of 3-month-old APP/PS1 mice. However, it is in the early stages of AD, and there were only a few plaques in the brain (Figure 5, left). With the progression of AD, more plaques were observed in the intestines of 9-month-old APP/PS1 mice (Figure 5, right). Interestingly, changes in microbiome composition were observed in 9-month-old APP/PS1 mice. The effect of increased levels of Aβ₄₂ in the intestine on the changes in its microbial composition remains obscure. There is no doubt that the observed toxicity of Aβ₄₂, especially small oligomers, is the most effective in inducing inflammation of microglia or macrophages, and it causes more damage in inducing bilayer permeability (Flagmeier et al., 2017; Fusco et al., 2017; De et al., 2019). It is certain that in the current investigation, the abundance of Lachnospiraceae fluctuated with age (Figures 6C,D). The content of Turicibacter, Atopostipes, and Lachnospiraceae in aged APP/PS1 mice was significantly higher than in young APP/PS1 mice, but the abundance of Prevotellaceae in old APP/PS1 mice was lower (Figure 6). Overall, these results indicated that the significant difference in gut microbiota composition is accompanied by the sharply increased soluble Aβ₄₂ and insoluble Aβ plaques during AD progression, but whether this change affects or is affected by the change in gut-derived Aβ₄₂ needs to be further explored.

In summary, based on our screened antibody pairs 1F12/2C6 and 1F12/1F12 in sandwich ELISA for specific detection of Aβ₄₂Ms and Aβ₄₂Os, we observed apparent fluctuation in the levels of Aβ₄₂Ms and Aβ₄₂Os in blood and intestines of APP/PS1 mice during the progression of AD. The identification of Aβ₄₂ abnormalities in the gastrointestinal tract could provide new ideas for AD therapeutic interventions that are only used to evaluate Aβ₄₂ levels in peripheral blood and the brain. Furthermore, studying the relationship between the levels of Aβ₄₂Ms and Aβ₄₂Os in peripheral tissues and the progression of AD may help to understand the causes of AD, and may provide new treatment strategies for improving AD or other Aβ-related dementias.

Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article.

Ethics Statement

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology.

Author Contributions

HL, QL, and ZZ coordinated the writing of the manuscript and provided writing guidance and manuscript revision. LZ completed all experiments and contributed to writing the first draft. YL and SN participated in most experiments. XL was involved in APP/PS1 mice breeding and schematic figure preparations. CY performed the bioinformatics data analysis. All authors reviewed and approved the final manuscript.

Funding

This study was financially supported by grants from the Major Research Plan of the National Natural Science Foundation of China (Grant No. 91749209), the National Natural Science Foundation of China (Grant No. 81971025), and the Startup Fund of Huazhong University of Science and Technology.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We thank the Optical Bioimaging Core Facility and the Center for Nanoscale Characterization & Devices (CNCD) of WNLO-HUST for support with data acquisition, the Analytical and Testing Center of HUST for performing spectral measurements, and the Research Core Facilities for Life Science (HUST) for using cryo-transmission electron microscopy.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2021.723317/full#supplementary-material

Supplementary Figure 1 | Characterization of sequence- and conformation-specific antibodies 1F12 and 2C6 against Aβ₄₂Ms and Aβ₄₂Os. (A) The preferred hybridoma colonies were screened via indirect ELISA. (B) The purity and molecular weight of purified 1F12, 2C6, and 2E2 were confirmed via 12% reduced SDS-PAGE with Coomassie blue staining. (C) The bioactivities of 1F12, 2C6, and commercial antibody 6E10 toward natural Aβ₄₂ peptides extracted from murine APP/PS1 brain were evaluated via Western blotting. (D) Confocal fluorescence images of murine APP/PS1 brain sections using Cy3-labeled anti-Aβ₄₂ monoclonal antibody 2C6 and commercially available anti-Aβ antibody 6E10 (E). (Scale bar: 200 μm). (E) Titers of 1F12, 2C6, and 2E2. (F) The purity and molecular weight of freshly prepared Aβ₄₂Ms and Aβ₄₂Os were determined by Western blotting with 2C6. (G) Binding affinities of 2C6 toward Aβ₄₂Ms and Aβ₄₂Os were evaluated via indirect ELISA. The activities (H) and titers (I) of the biotinylated 1F12 or 2C6 antibody.

Supplementary Figure 2 | Confocal fluorescence images of whole murine APP/PS1 brain sections using Cy3-labeled anti-Aβ₄₂ monoclonal antibody 1F12 (A) or 2C6 (B) and thioflavin S.

Supplementary Figure 3 | Comparison of the dynamic distribution of Aβ₄₂ in APP/PS1 and C57BL/6J. The levels of total Aβ₄₂ (A,D), Aβ₄₂Os (B,E), and Aβ₄₂Ms (C,F) in the stomach, duodenum, jejunum, ileum, colon, and cecum and their lysates from APP/PS1 and C57BL/6J at 3-month-old (n = 4) or 9-month-old APP/PS1 (n = 4). Data are presented as means ± SEM. Two-way analysis of variance (ANOVA) was used for multigroup comparisons. Statistical significance is indicated in the figures by **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Supplementary Figure 4 | Representative confocal fluorescence images of the duodenum, jejunum, ileum, cecum, and colon of 3-month-old C57BL/6J mice double-stained with thioflavin S and Cy3-1F12 (Scale bar: 100 μm).

Supplementary Figure 5 | Representative confocal fluorescence images of the duodenum, jejunum, ileum, cecum, and colon of 9-month-old C57BL/6J mice double-stained with thioflavin S and Cy3-1F12 (Scale bar: 100 μm).

References