s2fieldsofstudy: Biology Medicine

text:
Differential expression of immune-regulatory proteins C5AR1, CLEC4A and NLRP3 on peripheral blood mononuclear cells in early-stage non-small cell lung cancer patients

Changes in gene expression profiling of peripheral blood mononuclear cells (PBMC) appear to represent the host’s response to the cancer cells via paracrine signaling. We speculated that protein expression on circulating T-lymphocytes represent T-lymphocyte trafficking before infiltration into the tumor microenvironment. The possibility of using protein expression on circulating T-lymphocytes as a biomarker to discriminate early-stage non-small cell lung cancer (NSCLC) was explored. Four independent PBMC gene expression microarray datasets (GSE12771, GSE13255, GSE20189 and GSE3934) were analyzed. We selected C5AR1, CLEC4A and NLRP3 based on their significant protein expression in tumor-infiltrating lymphocytes, but not in normal lymphoid tissue. A validation study using automated flow cytometry was conducted in 141 study participants including 76 treatment-naive early-stage non-small cell lung cancer patients (NSCLC), 12 individuals with non-malignant pulmonary diseases, and 53 healthy individuals. Median ratios of C5AR1, CLEC4A and NLRP3 specific antibody staining to CD3 positive cells in early-stage NSCLC patients compared to healthy controls were 0.014 [0–0.37] vs. 0.01 [0–0.07, p = 0.13], 0.03 [0–0.87] vs. 0.02 [0–0.13, p = 0.10] and 0.19 [0–0.60] vs. 0.09 [0.02–0.31, p < 0.0001], respectively. Median fluorescence intensity (MFI) of CD3+C5AR1+, CD3+CLEC4A+ and CD3+NLRP3+ expression in early-stage NSCLC patients compared to healthy volunteers was 185 [64.2–4801] vs. 107.5 [27–229, p < 0.0001], 91.2 [42.4–2355] vs. 71.25 [46.2–103, p = 0.0005], and 1585 [478–5224] vs. 758.5 [318–1976, p < 0.0001], respectively. NLRP3:CD3 ratio, CD3+C5AR1+, CD3+CLEC4A+ and CD3+NLRP3+ MFI were significantly higher in early-stage NSCLC than healthy volunteers with an area under the ROC curve of 0.69–0.76. The CD3+NLRP3+ MFI provided the most distinguishable expression at 71.5% sensitivity and 70% specificity. Furthermore, CD3+NLRP3+ MFI potentially discriminated between early-stage NSCLC from malignant-mimic inflammation and infection pulmonary disease. Further validation in various pulmonary inflammatory disease might be warranted. Our proof-of-principle findings strengthen the hypothesis that malignancies generate distinctive protein expression fingerprints on circulating T-lymphocytes.

Lung cancer is the most common cancer and the leading cause of cancer-related deaths worldwide with around 2.2 million patients and an estimated 1.8 million deaths in 2020 1 . The majority of lung cancer patients are diagnosed with advanced stage disease in which curative treatment is not suitable. Despite improvement in therapies, the overall 5-year survival rate for all stages is approximately 21% 2 . Thus, there is a major effort for discovery of screening tools for early-stage disease. The National Lung Screening Trial (NLST) using low-dose helical computed tomography (LDCT) demonstrated a 20% reduction in lung cancer mortality among high-risk individuals, but produced a high rate of false-positive results which often led to unneeded invasive procedures 3 .
Exploring complementary noninvasive diagnostic testing may offer better results especially if a blood-based biomarker can be identified. Peripheral blood mononuclear cells (PBMCs) consist of monocytes, T cells, B cells, granulocytes, and natural killer cells and serve as the immune system's first line of defense against malignancy 4 . The changes in gene expression profiling in PBMCs appear to represent the host's response to the cancer cells via paracrine signaling 5 . Several studies have demonstrated that the characterization of gene expression in PBMCs may be useful as a diagnostic test for early detection of various types of cancer 4,6-11 . However, several technical issues limit the use of gene expression on PBMCs in real-life practice. The main challenge is timely sample processing because delayed processing affects the gene expression results in PBMCs 12 . Proteins are less complex and usually correlates to mRNA expression on PBMCs 13 . Given these advantages over gene expression characterization, protein expression on PBMCs may provide a more feasible biomarker that could help determine early cancer status.
In this study, we postulated that the immune-related protein expression of PBMCs from early-stage nonsmall cell lung cancer (NSCLC) patients would differ from that of healthy controls. Discriminative expression of non-malignant pulmonary disease could also enhance the potential possibility of these blood-based biomarkers. Therefore, specific protein expression on PBMCs from three separate groups of early-stage NSCLC patients, non-malignant pulmonary disease patients, and healthy control were conducted.

Results
Demographic characteristics of study participants. We enrolled a total of 141 study participants consisting of 76 treatment-naïve early-stage NSCLC patients, 12 non-malignant pulmonary diseases patients, and 53 healthy individuals. The median age of early-stage NSCLC patients was 67 years [range 41-81]. Fifty percent of patients were female. All patients had ECOG performance of 0-1. Nearly all (98.7%) histology showed adenocarcinoma. Pathological stage I disease accounted for 70%. All patients received curative attempt operations including lobectomy and bilobectomy. The median age for the non-malignant pulmonary diseases and healthy controls were 38 years [range  and 62 years [range 53-64], respectively. Ninety-five percent of non-malignant pulmonary disease patients presented with pulmonary nodules mimicking lung cancer and underwent operative procedures. A majority (70%) of resected lung tissue histology showed infection and inflammation. Details of patient demographics and disease characteristics at study enrollment are presented in Table 1.
Quality control assessment and storage time effect. We explored individual CD3 T-cell positive expression using flow cytometry detection over 5,000 viable PBMCs as the minimal accepted rate for quality control (QC). There were 44 healthy controls (83%), 10 Table S1. Candidate up-and down-regulated gene expressions from these four independent microarray experiments were identified by using CU-DREAM (Connection Up-and Down-Regulation Expression Analysis of Microarrays) 16 . 1885 significant gene expressions with p-values and odd ratios > 1 which indicated a strong association between the two independents studies were retrieved (Table S2). Overlapping candidate gene expressions from each gene expression microarray dataset are illustrated (Fig. S1). Overlapping significant up-regulated genes at least 3 datasets were retrieved for biological functions using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system (available at http:// www. panth erdb. org) 17 (Fig. S2). Seventy-five genes with immune system processes were identified and then mapped with pathology-based protein expression profiling in the Human Protein Atlas (available at https:// www. prote inatl as. org) 18 (Table S3). Three significant up-regulated genes which had the presence of antibodyspecific, immunohistochemistry-based protein expression on tumor-infiltrating lymphocytes (TILs) in tumor specimen, but not in normal lymphoid tissue, were selected including CLEC4A, C5AR1, NLRP3. Details of selected gene ontology in homo sapiens based on the PANTHER are summarized in Table S4.  Table 2). In addition to the number  and adjusted expression (c) of C5AR1, CLEC4A and NLRP3 to CD3 + lymphocytes between healthy controls (n = 44) and early-stage NSCLC patients (n = 63). The ROC curve of C5AR1, CLEC4A and NLRP3 ratio and MFI between healthy controls and early-stage NSCLC patient (c,d). The CD3 + NLRP3 + MFI provided the most distinguishable between early-stage NSCLC and healthy volunteer at 71.5% sensitivity and 70% specificity. Fluorescence intensity of C5AR1, CLEC4A and NLRP3 expression on CD3 positive in early-stage NSCLC patients were significantly higher than healthy controls. These results suggest that either MFI or adjusted C5AR1, CLEC4A and NLRP3 protein expression on the circulating T-lymphocytes exhibit changes in the presence of cancer. The ratio of NLRP3 expression on CD3 lymphocytes was also significantly higher in early-stage NSCLC patients compared to healthy controls. These results show the feasibility of using either the NLRP3 ratio or MFI in CD3 positive cells for distinguishing between the early-stage NSCLC and healthy controls.

Protein expression on PBMCs in early-stage lung cancer patients and healthy controls.
Protein expression on PBMC as a potential candidate to discriminate early-stage non-small cell lung cancer patients from healthy controls. For the clinical use of a potential biomarker to discriminate early-stage NSCLC patients from healthy controls, the ratio and MFI cutoff of significant specific antibody staining expressed on CD3 + cells were calculated. An NLRP3 ratio of more than 0.12 was found to significantly discriminate early-stage NSCLC patients from healthy volunteers with an area under the ROC curve of 0.72 (p < 0.0001). This cutoff provided 60% sensitivity and 75% specificity (Fig. 1c). CD3 + C5AR1 + , CD3 + CLEC4A + and CD3 + NLRP3 + MFI also significantly discriminated between early-stage NSCLC patients and healthy volunteers with an area under the ROC curve of 0.74 (p < 0.0001), 0.69 (p = 0.0006) and 0.76 (p < 0.0001) respectively (Fig. 1d). The CD3 + C5AR1 + MFI of more than 139 units could distinguish early-stage NSCLC patients from healthy controls at 62% sensitivity and 70% specificity. The CD3 + CLEC4A + MFI of more than 81.5 units could distinguish early-stage NSCLC patients from healthy controls at 60% sensitivity and 75% specificity. Lastly, the CD3 + NLRP3 + MFI of more than 1054 units provided the best sensitivity at 71.5% between early-stage NSCLC patients and healthy controls and 70% specificity.

Protein expression on PBMC in early-stage lung cancer and non-malignant pulmonary dis-
ease. An important clinical issue in the discovery of a biomarker is the ability to discriminate non-malignant pulmonary nodules from the early-stage NSCLC patients. We used specific antibodies for staining the candidate proteins on CD3 positive in PBMCs of non-malignant pulmonary diseases compared to early-stage NSCLC patients. The median ratios of C5AR1, CLEC4A and NLRP3 expression in CD3+ of non-malignant pulmonary diseases were 0.01 [range 0-0.02, p = 0. 12 16, p = 0.07]. The NLRP3 MFI on CD3 positive of early-stage NSCLC patients showed higher expression than non-malignant pulmonary disease whereas C5AR1 and CLEC4A expression levels were not different (Fig. 2a-c). Even though limited number of non-malignant www.nature.com/scientificreports/ pulmonary disease patients in our study, higher CD3 + NLRP3 + MFI in early-stage NSCLC patients compared to non-malignant pulmonary disease patients probe the possibility of these PBMCs protein expressions as a biomarker to discriminate between those 2 conditions. Further validation in various pulmonary inflammatory disease might warrant.

Discussion
Previous studies from our group reported paracrine signal-induced PBMC gene upregulation in various cancer types through epigenetic regulation and expression 5,9,19,20 . On this basis, we explored the possibility of using protein expression on circulating T-lymphocytes as a biomarker to discriminate between early-stage NSCLC and healthy individuals by automated flow cytometry analysis. We speculated that protein expression on circulating T-lymphocytes might represent T-lymphocyte trafficking before infiltration into the tumor microenvironment. We chose specific proteins which are related to immune function in either innate and/or adaptive immune processes. NLRP3:CD3 ratio, CD3 + C5AR1 + , CD3 + CLEC4A + and CD3 + NLRP3 + MFI were significantly higher in early-stage NSCLC compared to healthy volunteers with an area under the ROC curve of 0.69-0.76. The CD3 + NLRP3 + MFI provided the highest discriminatory values at 71.5% sensitivity and 70% specificity. Our proof-of-principle findings strengthen the hypothesis that malignancies generate distinctive protein expression fingerprints on circulating T-lymphocytes. Mechanisms of the 3 chosen markers are detailed below. C5AR1 (complement component 5a receptor 1) is a G protein-coupled receptor for C5a. It functions as a complement receptor and modulates inflammatory responses via chemokine and cytokine signaling pathways. C5AR1 signaling also contributes to the promotion of tumor growth by suppressing the adaptive immune response against tumor antigens and recruits myeloid-derived suppressor cells (MDSCs) into tumor cells 21,22 . A study to evaluate C5AR1 expression by immunohistochemistry in neoplastic and non-neoplastic human tissue sample demonstrated that C5AR1 was strongly expressed by different types of immune cells including monocytes, macrophages, eosinophil, neutrophil and mast cell, but negative staining in T and B lymphocyte 23 , consistent with The Human Protein Atlas database (Fig. S3). Furthermore, C5AR1 expression in cancer cells promotes cancer invasion through motility activation and matrix metalloproteinases 24 . Both these data and our present findings support C5AR1 as a primary mechanism in non-small cell lung cancer.
CLEC4A (C-type lectin domain family 4-member a) or dendritic immunoreceptor (DCIR) have been reported as an immune suppressor of dendritic cells which play a crucial role in the adaptive immune response. CLEC4A expresses on various immune cells depending on the stage of maturation 25 . One study described the role of CLEC4A down-regulation via small hairpin RNA inhibiting tumor progression in animal models 26 . In human tumorigenesis, a previous study explored the role of CLECs and their potential association with reshaping the immune system in the development of hepatocellular carcinoma. The result demonstrated that hepatocellular tissues had significantly higher mRNA levels of CLEC4A and CLEC4L compared to normal liver tissues 27 . CLEC4A is believed to be a biomarker involved in carcinogenesis with further research in NSCLC needed.
NLRP3 (NOD-, LRR-and pyrin domain-containing 3) inflammasome complex plays a role in innate immune signaling. Aberrant NLRP3 are involved in various inflammatory conditions, infection immune responses, and tumorigenesis including lung cancer 28,29 . Several previous studies demonstrated that NLRP3 inflammasome regulates lung cancer proliferation and metastasis through a variety of mechanisms [30][31][32][33] including promoting phosphorylation of Akt, ERK1/2, and CREB, enhancing Snail expression, decreasing E-cadherin expression 30 , and increasing inflammatory cytokine secretion such as interleukin-1 β 32-34 . Our results supported this concept. The ratio of NLRP3 protein expression on CD3 positive cells and MFI in PBMCs in early-stage NSCLC patients had significantly higher levels compared to healthy individuals.
To the best of our knowledge, this is the first study to explore specific protein expressions on PBMCs by automated flow cytometry in early-stage NSCLC. Many different methods were used to measure protein expression. Several limitations of immunohistochemistry have been reported such as various levels of expression being affected by dilution of antibody, diverse clonal of specific antibodies, and interpretation inconsistencies by pathologists. Our group also reported the results of using immunofluorescence to detect particular protein expressions on lymphocytes in early breast cancer detection 35 . While immunofluorescence assay requires laborious hands-on effort to complete, flow cytometry can determine the target particles of a whole cell presentation in various graphic features and can measure large numbers of analyzed cells (up to 300,000 cells per second). Flow cytometry is a high throughput assay which measures fluorescence signals through a laser source. Gating techniques allow for the simultaneously measurement of both intracellular expression and surface protein expression at a single cell level 36 . It can determine both the intrinsic size and the internal complexity of a cell including the extrinsic characteristics such as specific protein expression. Standardized quantitative flow cytometry using median fluorescence intensity unit has had great impact in the research field and the clinic 37 . Automate flow cytometry is a promising tool for the profiling of both cell surface proteins and intracellular protein signaling from peripheral blood samples in clinical application. However, our study has some limitations. First, C5AR1, CLEC4A and NLRP3 expression was not explored in other subsets of immune cells which prohibit comparative expression to CD3+ T-lymphocyte. Second, the results of this study could not serve the explanation of pathophysiological relevance of diverse expression of C5AR1, CLEC4A and NLRP3 in CD3+ T-lymphocyte amount cancer patient and healthy individuals.

Methods
Study participants. All  www.nature.com/scientificreports/ # TCTR20190508003. Recruited case studies in a prospective manner were classified into three groups as following: (1) 76 patients with early-stage non-small cell lung cancer (NSCLC), (2) 12 patients with non-malignant pulmonary disease, and (3) 53 healthy volunteers with no history of malignant disease and normal chest radiography within 6 months at enrollment. We defined NSCLC staging system based on pathologic results in accordance with either the seventh edition or the eighth edition of the American Joint Committee on Cancer (AJCC) Cancer Staging System at the time of diagnosis 38 . The exclusion criterion were prior systemic therapies including cytotoxic chemotherapy, small molecule targeted therapy, immunotherapy, history of another malignancy within 5 years before enrollment, and receiving corticosteroids or any immunosuppressive agents. All patients were > = 18 years. Demographic characteristics of individual patients are presented in Table 1.
Blood collection and preparation. Whole blood samples were collected in EDTA containers from all participants. Peripheral mononuclear cells (PBMC) were isolated using Ficoll-Paque™ PLUS (GE Healthcare Life Sciences, Canada) as previously described 39 . Briefly, blood samples were centrifuged at 1600×g at 16 °C for 10 min. After plasma removal, the specimen was diluted (1:1) with phosphate buffered saline (PBS) solution. The diluted specimen was then gently layered on top of the ficoll in a new conical tube and centrifuged at 2000 rpm for 30 min. The interface between the diluted specimen and ficoll layers was collected and then washed twice with 10 ml of PBS solution. Specimens were kept in 5% DMSO mixed with culture media at − 196 °C until further analyzed.
Staining process and flow cytometry analysis. The staining protocol was adapted from Junqueira et al. 22  In the final step, these pellets were washed three times and then resuspended in 1% PFA in 1X PBS. The samples were kept at 4 °C until flow cytometry analysis. In cases of the isotype control, PE/ Cyanine7 mouse IgG2a, Alexa Fluor 488 mouse IgG1 and PE rat IgG2a from Biolegend® were used instead of human C5AR1, CLEC4A and NLRP3 antibodies. These results were analyzed by using FlowJo Version 10 for discriminating cell death and using viable PBMCs more than 5,000 CD3 + as the threshold of detection. For the analytical process, all samples were previously gated only CD3+ lymphocyte cells from other immune cells and debris. We used one of the healthy individual samples for setting the compensation pattern. In the compensation setting, unstained and single-stained fluorescence samples were used as the negative and positive controls. After adjusting the positive area of each fluorescence, we set the cut-off boundary values between one fluorescence signal and the others. (Fig. S4). Finally, this setting compensation was applied for all samples. We used the isotype control as the negative control for non-specific binding. The examples of both the isotype control and sample are shown in Fig. S5. Statistical analysis. Statistical parameters used to delineate protein expression on T-lymphocytes were as follows: (1) ratio of specific-antibody positive to CD3 + cells, (2) fluorescence intensity of each protein on CD3 + cells, and (3) adjusted specific protein expression on CD3 + by the ratio of each protein to CD3 multiplied by the fluorescence intensity. Mann-Whitney U test was used to assess differences in non-parametric continuous ratios of expression and adjusted specific proteins to CD3 positive between groups. Receiver operative characteristic (ROC) curve analysis was used to compute sensitivity and specificity of protein expressions as a potential diagnostic marker for early-stage NSCLC compared to healthy controls. The two-sided test p-values < 0.01 were considered significant. All the statistical analyses were performed with the use of GraphPad Prism, version 9.


Probiotic Bifidobacterium breve MCC1274 Mitigates Alzheimer’s Disease-Related Pathologies in Wild-Type Mice

Probiotics improve brain function, including memory and cognition, via the microbiome–gut–brain axis. Oral administration of Bifidobacterium breve MCC1274 (B. breve MCC1274) improves cognitive function in AppNL-G-F mice and mild cognitive impairment (MCI) subjects, and mitigates Alzheimer’s disease (AD)-like pathologies. However, its effects on wild-type (WT) mice have not yet been explored. Thus, the effects of B. breve MCC1274 on AD-like pathologies in two-month-old WT mice were investigated, which were orally administered B. breve MCC1274 for four months. Aβ levels, amyloid precursor protein (APP), APP processing enzymes, phosphorylated tau, synaptic protein levels, glial activity, and cell proliferation in the subgranular zone of the dentate gyrus were evaluated. Data analysis was performed using Student’s t-test, and normality was tested using the Shapiro–Wilk test. Oral administration of B. breve MCC1274 in WT mice decreased soluble hippocampal Aβ42 levels by reducing presenilin1 protein levels, and reduced phosphorylated tau levels. It also activated the protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) pathway, which may be responsible for the reduction in presenilin1 levels and inhibition of tau phosphorylation. B. breve MCC1274 supplementation attenuated microglial activation and elevated synaptic protein levels in the hippocampus. These findings suggest that B. breve MCC1274 may mitigate AD-like pathologies in WT mice by decreasing Aβ42 levels, inhibiting tau phosphorylation, attenuating neuroinflammation, and improving synaptic protein levels.

Introduction
Alzheimer's disease (AD) is a neurodegenerative disease caused by a combination of genetic and environmental factors, and a lifestyle that gradually affects brain function. The brains of patients with AD reveal the presence of extracellular amyloid-β peptide (Aβ) deposition, forming senile plaques and hyperphosphorylated tau protein to form neurofibrillary tangles, which leads to neuronal cell death that causes cognitive decline and memory impairment [1]. The enzymatic cleavage of amyloid-β precursor protein (APP) by βand γ-secretase produces Aβ [2]. Alternatively, APP can be cleaved by α-secretase to prevent Aβ generation. Neuroinflammation is also a common hallmark of AD, and is associated with Aβ and tau pathologies [3,4]. Activated microglia and astrocytes are involved in neuroinflammation in the AD brain, where their activation produces pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1β, subsequently resulting in neuronal cell death [5]. The most common sign of the disease is a decline in cognitive function and motor abilities, which ultimately leads to death [6]. AD cases are increasing worldwide, and the number of cases doubles every anesthesia, and their brains were collected for biochemical analyses and immunostaining. The animal experiment schedule is shown in Figure 1. All studies and protocols were approved by the Nagoya City University Institutional Care and Use of Laboratory Animals Committee, and the experiments were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Figure 1. A schematic figure explaining the timeline of the experiment. Two-month-old wild-type (WT) mice were randomly divided into saline and probiotic groups: the saline group received saline, and the probiotic group (n = 20 per group) was supplemented with B. breve MCC12741 (1 × 10 9 cfu/6.25 mg/200 μL saline/mouse) via oral gavage five times/week for 4 months. At the end of supplementation, mice were injected with BrdU and were then sacrificed, and their brains were collected for immunostaining and biochemical analyses.

Western Blot Analysis
Both cortical and hippocampal tissues were removed quickly, and carefully placed in liquid nitrogen and weighed. They were then homogenized in ten volumes of precooled RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.6) containing protease and phosphatase inhibitor cocktails (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Then, the homogenates were centrifuged at 12,000 rpm for 30 min at 4 °C before collecting the supernatants. Protein concentration was determined using the Pierce TM BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's instructions. The homogenates were mixed with 4 x sample buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, and 40% bromophenol blue), followed by heating for 7 min at 95 °C. Protein lysates (20 μg) were separated using SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). These membranes were blocked with 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature (RT), and incubated overnight at 4 °C with the following primary antibodies  Two-month-old wild-type (WT) mice were randomly divided into saline and probiotic groups: the saline group received saline, and the probiotic group (n = 20 per group) was supplemented with B. breve MCC12741 (1 × 10 9 cfu/6.25 mg/200 µL saline/mouse) via oral gavage five times/week for 4 months. At the end of supplementation, mice were injected with BrdU and were then sacrificed, and their brains were collected for immunostaining and biochemical analyses.

Assessment of the Levels of Soluble Aβ in the Brain by ELISA
Both the cortex and hippocampus (n = 7 per group) were weighed and homogenized in 10× TBS buffer. Cortical and hippocampal homogenates were centrifuged at 100,000 rpm for 20 min at 4 • C. The supernatant was stored at −80 • C until use for detection of soluble Aβ40 and Aβ42 by using sandwich Aβ ELISA kits (Wako Pure Chemical Industries) by following the manufacturer's instructions. In brief, the standard and samples were incubated overnight at 4 • C, followed by washing and incubation for 1 h at 4 • C with HRPconjugated antibody solution, and then incubation for 30 min at RT with TMB solution after extensive washing. The stop solution was then added, and the absorbance of each well was read at 450 nm. The levels of Aβ are presented as pmol of Aβ per gram of protein.
All samples were run in duplicate for each analyte, and exhibited parallel displacement to the standard curve.

Tissue Processing for Immunohistochemistry and BrdU Staining
At the end of the supplementation period, seven mice per group were administered three consecutive daily (twice per day) intraperitoneal injections of 5-Bromo-2-deoxyuridine (BrdU) (Wako). Mice were anesthetized with sevoflurane, and intracardially perfused with phosphate buffer saline (PBS), and then 4% paraformaldehyde (PFA) solution. Brains were post-fixed in 4% PFA for 3 days at 4 • C after removal from the skull, and transferred to 30% sucrose for 2-3 days. Thereafter, the brains were frozen by immersion in dry ice, embedded in optimal cutting temperature compound, cut into 40 µm-thick sections on a vibratome (Leica Microsystems, Wetzlar, Germany), placed in a cryoprotection solution (30% sucrose, 150 mM NaCl, and 250 mM polyvinylpyrrolidone in 0.1 M PB), and stored at −20 • C until used for staining.

Immunofluorescence Staining and Cell Proliferation Analysis
Antigen retrieval for brain slides was achieved by boiling in citrate buffer for 5 min followed by cooling at RT. Brain sections were blocked with 5% goat serum/0.25% Triton X/PBS for 30 min. The sections were incubated overnight at 4 • C with the primary antibodies against Iba1 (1:200, WAKO) and GFAP (1:100, Sigma-Aldrich). After washing, the sections were incubated with goat anti-rabbit Alexa Fluor 568 or goat anti-mouse Alexa Fluor 488 secondary antibody (Thermo Fisher Scientific) for 1 h at RT in the dark. Finally, the cells were mounted with DAPI solution (Vector Laboratories Inc., Burlingame, CA, USA). Images were acquired with a confocal microscope (Olympus, Southall, UK). The number of Iba1-and GFAP-positive cells was quantified in randomly selected two fields (1 × 1 mm 2 ) of the hippocampus and four fields (1 × 1 mm 2 ) of the cortex per mouse using ImageJ. BrdU staining was performed using a BrdU Labeling and Detection kit (Roche, San Francisco CA, USA). Briefly, sections were passed through antigen retrieval, blocking solution, and then incubated with the anti-BrdU antibody for 45 min at 37 • C, followed by incubation with anti-mouse FITC antibody for 30 min at 37 • C. Finally, they were cover-slipped with DAPI solution. BrdU-positive cells were imaged using a confocal microscope (Olympus) and quantified in eight sections per mouse using ImageJ software.

Statistical Analysis
GraphPad Prism software (San Diego, CA, USA) was used for statistical analysis. All data are presented as mean ± SD. Results were analyzed using a two-tailed unpaired Student's t-test (for normally distributed variables). Significance was defined when p < 0.05. Normality was tested using the Shapiro-Wilk test. The results of these tests indicate that the data for both saline and probiotic groups are normally distributed.

B. breve MCC1274 Supplementation Reduces Soluble Aβ42 Level and Presenilin1 (PS1) Protein Level in the Hippocampus
Aβ production in the brain is affected by altered levels of APP and APP-processing enzymes, such as ADAM10, BACE1, and PS1, which are α-secretase, β-secretase, and γ-secretase components, respectively. A previous study demonstrated that oral supplementation with B. breve MCC1274 reduced hippocampal Aβ production in App NL-G-F mice by increasing the ADAM10 levels [27]. Thus, the effect of probiotic supplementation on Aβ levels in WT mice was investigated, and the results showed a significant decrease in soluble Aβ42 levels in the hippocampal extracts of mice supplemented with probiotics compared to those of mice that received saline ( Figure 2A). However, soluble Aβ40 levels in the hippocampus (Figure 2A), as well as soluble Aβ40 and Aβ42 levels in the cortex, were not affected by probiotic supplementation ( Figure 2B). Intra-assay coefficients of variation (CVs) were calculated by dividing the standard deviation by the duplicate mean and multiplying by 100. CVs for soluble Aβ40 and Aβ42 in both the cortex and hippocampus ranged from 0.57 to 6.9%, 1.6 to 7.1%, 0.6 to 7.7%, and 1.5 to 6.7%, respectively. data are presented as mean ± SD. Results were analyzed using a two-tailed unpaired Student's t-test (for normally distributed variables). Significance was defined when p < 0.05. Normality was tested using the Shapiro-Wilk test. The results of these tests indicate that the data for both saline and probiotic groups are normally distributed.

B. breve MCC1274 Supplementation Reduces Soluble Aβ42 Level and Presenilin1 (PS1) Protein Level in the Hippocampus
Aβ production in the brain is affected by altered levels of APP and APP-processing enzymes, such as ADAM10, BACE1, and PS1, which are α-secretase, β-secretase, and γsecretase components, respectively. A previous study demonstrated that oral supplementation with B. breve MCC1274 reduced hippocampal Aβ production in App NL-G-F mice by increasing the ADAM10 levels [27]. Thus, the effect of probiotic supplementation on Aβ levels in WT mice was investigated, and the results showed a significant decrease in soluble Aβ42 levels in the hippocampal extracts of mice supplemented with probiotics compared to those of mice that received saline ( Figure 2A). However, soluble Aβ40 levels in the hippocampus (Figure 2A), as well as soluble Aβ40 and Aβ42 levels in the cortex, were not affected by probiotic supplementation ( Figure 2B). Intra-assay coefficients of variation (CVs) were calculated by dividing the standard deviation by the duplicate mean and multiplying by 100. CVs for soluble Aβ40 and Aβ42 in both the cortex and hippocampus ranged from 0.57 to 6.9%, 1.6 to 7.1%, 0.6 to 7.7%, and 1.5 to 6.7%, respectively. in WT mice were measured by Sandwich ELISA. Aβ levels were normalized to each protein concentration. Data are represented as the mean ± SD, n = 7 per group, n.s. no significant difference, * p < 0.05 compared with the saline group, data analyzed by Student's t-test.
Next, the protein levels of APP and APP-processing enzymes were evaluated by Western blotting, and the results showed that mice supplemented with B. breve MCC1274 had considerably lower protein levels of PS1 in the hippocampus, but not in the cortex than those in mice that received saline ( Figure 3). In contrast, the protein levels of APP, ADAM10, and BACE1 in both the cortex and hippocampus were not substantially different between the two groups ( Figure 3). These findings indicate that B. breve MCC1274 supplementation reduced soluble Aβ42 levels in the hippocampus of WT mice by decreasing PS1 protein levels. Oral administration of B. breve MCC1274 reduces soluble hippocampal Aβ42 levels in WT mice. Soluble hippocampal Aβ40 and Aβ42 levels (A) and soluble cortical Aβ40 and Aβ42 levels (B) in WT mice were measured by Sandwich ELISA. Aβ levels were normalized to each protein concentration. Data are represented as the mean ± SD, n = 7 per group, n.s. no significant difference, * p < 0.05 compared with the saline group, data analyzed by Student's t-test.
Next, the protein levels of APP and APP-processing enzymes were evaluated by Western blotting, and the results showed that mice supplemented with B. breve MCC1274 had considerably lower protein levels of PS1 in the hippocampus, but not in the cortex than those in mice that received saline ( Figure 3). In contrast, the protein levels of APP, ADAM10, and BACE1 in both the cortex and hippocampus were not substantially different between the two groups ( Figure 3). These findings indicate that B. breve MCC1274 supplementation reduced soluble Aβ42 levels in the hippocampus of WT mice by decreasing PS1 protein levels. . The protein levels were quanti fied by densitometry, normalized to the actin level, and expressed as a relative protein level. Data are represented as mean ± SD; n = 7 per group; n.s. no significant difference; p < 0.05 compared with the saline group, as determined by Student's t-test.

B. breve MCC1274 Supplementation Activates Akt/GSK-3β Pathway in the Hippocampus
Next, the underlying molecular mechanism by which probiotic supplementation de creased PS1 protein levels was investigated. It has been reported that an increase in Ak phosphorylation at S473 causes an increase in the level of phosphorylated (p)-GSK-3β a S9, which is an inactive form of GSK-3β that, in turn, contributes to the reduction in PS levels [28]. Thus, the protein levels of p-Akt (S473) and p-GSK-3β (S9) were assessed usin Western blotting, and the results showed that p-Akt and p-GSK-3β protein levels wer significantly increased in the hippocampus of the probiotic-supplemented group com pared to those in the saline-supplemented group ( Figure 4A). However, the protein level of p-GSK-3α (S21) in the hippocampus ( Figure 4A), as well as those of p-Akt and p-GSK 3α/β in the cortex, did not differ between the two groups ( Figure 4B). It has also bee . The protein levels were quantified by densitometry, normalized to the actin level, and expressed as a relative protein level. Data are represented as mean ± SD; n = 7 per group; n.s. no significant difference; * p < 0.05 compared with the saline group, as determined by Student's t-test.

B. breve MCC1274 Supplementation Activates Akt/GSK-3β Pathway in the Hippocampus
Next, the underlying molecular mechanism by which probiotic supplementation decreased PS1 protein levels was investigated. It has been reported that an increase in Akt phosphorylation at S473 causes an increase in the level of phosphorylated (p)-GSK-3β at S9, which is an inactive form of GSK-3β that, in turn, contributes to the reduction in PS1 levels [28]. Thus, the protein levels of p-Akt (S473) and p-GSK-3β (S9) were assessed using Western blotting, and the results showed that p-Akt and p-GSK-3β protein levels were significantly increased in the hippocampus of the probiotic-supplemented group compared to those in the saline-supplemented group ( Figure 4A). However, the protein levels of p-GSK-3α (S21) in the hippocampus ( Figure 4A), as well as those of p-Akt and p-GSK-3α/β in the cortex, did not differ between the two groups ( Figure 4B). It has also been reported that the inhibition of c-Jun N-terminal kinase (JNK) activity and overexpression of p53 reduce APP processing through suppression of PS1 levels [29,30], and that inhibition of the mammalian target of rapamycin (mTOR) phosphorylation suppresses PS1 levels [31]. Therefore, their protein levels were assessed by Western blotting. Unfortunately, no substantial differences were found in both total and phosphorylated JNK, p53, or mTOR levels between the two groups in both the cortex and hippocampus (Supplementary Figure S1). Taken together, these findings indicate that oral supplementation of B. breve MCC1274 in WT mice reduces hippocampal PS1 protein levels through the Akt/GSK-3β-dependent pathway, leading to a reduction in Aβ42 generation.

2543
7 of 16 of p53 reduce APP processing through suppression of PS1 levels [29,30], and that inhibition of the mammalian target of rapamycin (mTOR) phosphorylation suppresses PS1 levels [31]. Therefore, their protein levels were assessed by Western blotting. Unfortunately, no substantial differences were found in both total and phosphorylated JNK, p53, or mTOR levels between the two groups in both the cortex and hippocampus (Supplementary Figure S1). Taken together, these findings indicate that oral supplementation of B. breve MCC1274 in WT mice reduces hippocampal PS1 protein levels through the Akt/GSK-3β-dependent pathway, leading to a reduction in Aβ42 generation.

B. breve MCC1274 Supplementation Inhibits Tau Hyperphosphorylation in the Hippocampus
Tau hyperphosphorylation is believed to be crucial in the pathogenesis of AD because it promotes neurofibrillary tangle formation in the brains of patients with AD, and causes neuronal cell death [32,33]. In addition, Akt activation inhibits GSK-3β activation, which is known to decrease tau phosphorylation at T231, S202, and T202 [34]. Therefore, the effect of B. breve MCC1274 supplementation on both total and p-tau (T231 and S202/T205) protein levels was investigated using Western blotting. The results showed lower levels of phosphorylated tau at T231, S202, and T205 in the hippocampus of the probiotic-supplemented group than in the saline-supplemented group, whereas total tau . Oral supplementation of B. breve MCC1274 increases Akt and p-GSK-3β phosphorylation levels, but not GSK-3α phosphorylation levels, in the hippocampus of WT mice. Western blot analysis of phosphorylated (p-) Akt, total (t-) Akt, p-GSK-3α/β, t-GSK-3α/β, and actin in the hippocampus (A) and cortex (B). Quantification of phosphorylated (p-) protein levels normalized to total (t-) protein levels and expressed as values relative to the control. Data are represented as mean ± SD, n = 7 per group; n.s. no significant difference; * p < 0.05 compared with the saline group, as determined by Student's t-test.

B. breve MCC1274 Supplementation Inhibits Tau Hyperphosphorylation in the Hippocampus
Tau hyperphosphorylation is believed to be crucial in the pathogenesis of AD because it promotes neurofibrillary tangle formation in the brains of patients with AD, and causes neuronal cell death [32,33]. In addition, Akt activation inhibits GSK-3β activation, which is known to decrease tau phosphorylation at T231, S202, and T202 [34]. Therefore, the effect of B. breve MCC1274 supplementation on both total and p-tau (T231 and S202/T205) protein levels was investigated using Western blotting. The results showed lower levels of phosphorylated tau at T231, S202, and T205 in the hippocampus of the probiotic-supplemented group than in the saline-supplemented group, whereas total tau protein levels did not differ between the two groups in the hippocampus ( Figure 5A). However, there was no change in total and phosphorylated tau protein levels in the cortex ( Figure 5B). These results indicate that oral supplementation with B. breve MCC1274 inhibits hippocampal tau hyperphosphorylation in WT mice through the Akt/GSK-3β-dependent pathway.

B. breve MCC1274 Supplementation Enhances Synaptic Plasticity in the Hippocampus
Because of the essential role of synaptic proteins in the early development of neurons and regulation of neurotransmitter release, the protein levels of the scaffolding postsynaptic protein, PSD-95, and three different presynaptic proteins, synaptotagmin (SYT), syntaxin, and synaptophysin (SYP), in both the hippocampus and cortex of WT mice, were assessed using Western blotting. Supplementation with B. breve MCC1274 significantly increased the protein levels of SYT and syntaxin, and showed a tendency to increase the protein levels of SYP and PSD-95 in hippocampal extracts ( Figure 6A). In contrast to the hippocampus, these protein levels did not change in the cortical extracts between the two groups ( Figure 6B). These findings indicate that B. breve MCC1274 supplementation increases the expression of synaptic proteins in WT mice, which is consistent with previous findings that B. breve MCC1274 supplementation increases these synaptic protein levels in App NL-G-F mice [27]. . Quantification of phosphorylated (p-) protein levels normalized to total (t-) protein levels and expressed as values relative to the control. Data are represented as mean ± SD; n = 7 per group; n.s. no significant difference; * p < 0.05 compared with the saline group, as determined by Student's t-test.

B. breve MCC1274 Supplementation Enhances Synaptic Plasticity in the Hippocampus
Because of the essential role of synaptic proteins in the early development of neurons and regulation of neurotransmitter release, the protein levels of the scaffolding postsynaptic protein, PSD-95, and three different presynaptic proteins, synaptotagmin (SYT), syntaxin, and synaptophysin (SYP), in both the hippocampus and cortex of WT mice, were assessed using Western blotting. Supplementation with B. breve MCC1274 significantly increased the protein levels of SYT and syntaxin, and showed a tendency to increase the protein levels of SYP and PSD-95 in hippocampal extracts ( Figure 6A). In contrast to the hippocampus, these protein levels did not change in the cortical extracts between the two groups ( Figure 6B). These findings indicate that B. breve MCC1274 supplementation increases the expression of synaptic proteins in WT mice, which is consistent with previous findings that B. breve MCC1274 supplementation increases these synaptic protein levels in App NL-G-F mice [27]. . The protein levels were quantified by densitometry, normalized to the actin level, and expressed as a relative protein level. Data are represented as mean ± SD; n = 7 per group; n.s. no significant difference; ** p < 0.01 compared with the saline group, as determined by Student's t-test.

B. breve MCC1274 Supplementation Attenuated Microglial Activation in the Hippocampus
As activated microglia and astrocytes are involved in AD-like pathologies, we also investigated whether B. breve MCC1274 supplementation alters their levels in the brains of WT mice. Brain sections were stained with anti-Iba1 (a marker for activated microglia) and anti-GFAP (a marker for reactive astrocytes) antibodies, and the numbers of Iba1 + and GFAP + cells were quantified. In the hippocampus of mice, supplementation with B. breve MCC1274 remarkably decreased the number of Iba1 + cells compared with those in vehicle treatment ( Figure 7A). However, the number of GFAP + cells in the hippocampus showed no difference between the two groups ( Figure 8A). Although both Iba1 + and GFAP + cells were rarely detected in the cortex of WT mice, Iba1 + and GFAP + cells around the brain surface were observed. No differences were found in the number of Iba1 + cells in the cortex ( Figure 7B) or GFAP + cells in the hippocampus and cortex (Figure 8) between the two groups. Furthermore, the protein levels of Iba1 and GFAP were investigated using Western blotting, and obtained similar results to those of immunostaining analysis. That is, the Figure 6. Oral supplementation of B. breve MCC1274 upregulates synaptic protein levels in the hippocampus of WT mice. Western blot analysis of SYT, syntaxin, SYP, PSD95, and actin in the hippocampus (A) and cortex (B). The protein levels were quantified by densitometry, normalized to the actin level, and expressed as a relative protein level. Data are represented as mean ± SD; n = 7 per group; n.s. no significant difference; ** p < 0.01 compared with the saline group, as determined by Student's t-test.

B. breve MCC1274 Supplementation Attenuated Microglial Activation in the Hippocampus
As activated microglia and astrocytes are involved in AD-like pathologies, we also investigated whether B. breve MCC1274 supplementation alters their levels in the brains of WT mice. Brain sections were stained with anti-Iba1 (a marker for activated microglia) and anti-GFAP (a marker for reactive astrocytes) antibodies, and the numbers of Iba1 + and GFAP + cells were quantified. In the hippocampus of mice, supplementation with B. breve MCC1274 remarkably decreased the number of Iba1 + cells compared with those in vehicle treatment ( Figure 7A). However, the number of GFAP + cells in the hippocampus showed no difference between the two groups ( Figure 8A). Although both Iba1 + and GFAP + cells were rarely detected in the cortex of WT mice, Iba1 + and GFAP + cells around the brain surface were observed. No differences were found in the number of Iba1 + cells in the cortex ( Figure 7B) or GFAP + cells in the hippocampus and cortex (Figure 8) between the two groups. Furthermore, the protein levels of Iba1 and GFAP were investigated using Western blotting, and obtained similar results to those of immunostaining analysis. That is, the Iba1 protein level was reduced in the hippocampus of the probiotic-supplemented group compared to that in the saline-supplemented group, whereas Iba1 protein level in the cortex, as well as GFAP protein levels in both the cortex and hippocampus, were not substantially different between the two groups ( Figure 9A,B). As microglial activation is associated with the production of pro-inflammatory cytokines [35,36], the mRNA levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in both hippocampal and cortical extracts were assessed using qRT-PCR analysis. Unfortunately, their mRNA levels were undetectable, even in the saline-supplemented group (data not shown), possibly because neuroinflammation had not occurred because the mice were still young. These results suggest that B. breve MCC1274 supplementation in WT mice attenuates microglial activation only in the hippocampus, and does not affect reactive astrocytes, which is consistent with previous findings in App NL-G-F mice [27]. Iba1 protein level was reduced in the hippocampus of the probiotic-supplemented group compared to that in the saline-supplemented group, whereas Iba1 protein level in the cortex, as well as GFAP protein levels in both the cortex and hippocampus, were not substantially different between the two groups ( Figure 9A,B). As microglial activation is associated with the production of pro-inflammatory cytokines [35,36], the mRNA levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in both hippocampal and cortical extracts were assessed using qRT-PCR analysis. Unfortunately, their mRNA levels were undetectable, even in the saline-supplemented group (data not shown), possibly because neuroinflammation had not occurred because the mice were still young. These results suggest that B. breve MCC1274 supplementation in WT mice attenuates microglial activation only in the hippocampus, and does not affect reactive astrocytes, which is consistent with previous findings in App NL-G-F mice [27].   Iba1 protein level was reduced in the hippocampus of the probiotic-supplemented group compared to that in the saline-supplemented group, whereas Iba1 protein level in the cortex, as well as GFAP protein levels in both the cortex and hippocampus, were not substantially different between the two groups ( Figure 9A,B). As microglial activation is associated with the production of pro-inflammatory cytokines [35,36], the mRNA levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) in both hippocampal and cortical extracts were assessed using qRT-PCR analysis. Unfortunately, their mRNA levels were undetectable, even in the saline-supplemented group (data not shown), possibly because neuroinflammation had not occurred because the mice were still young. These results suggest that B. breve MCC1274 supplementation in WT mice attenuates microglial activation only in the hippocampus, and does not affect reactive astrocytes, which is consistent with previous findings in App NL-G-F mice [27].    Photomicrographs of brain sections throughout the hippocampal dentate gyrus (DG). Immunofluorescence staining with BrdU (green) and DAPI nuclear counterstain (blue) (left panel). BrdU (50 mg/kg, intraperitoneal injection) was injected twice/day for 3 days. The BrdU + cell numbers in the SVZ of the DG area were quantified (right panel). Data are represented as mean ± SD, n = 7 per group; n.s. no significant difference; * p < 0.05 compared with the saline group, as determined by Student's t-test.

B. breve MCC1274 Supplementation does Not Alter Cell Proliferation in the Hippocampus
To assess cell proliferation in the subgranular zone of the dentate gyrus after oral supplementation with B. breve MCC1274, brain sections were labeled with BrdU. The number of BrdU + cells did not change in the probiotic-supplemented group compared to that in the saline-supplemented group ( Figure 9C). These results suggest that oral supplementation with B. breve MCC1274 does not alter cell proliferation in the hippocampus of WT mice, which is consistent with the previous finding in App NL-G-F mice [27].

Discussion
In recent decades, several clinical and experimental studies have explored the role of microbiota in modulating the gut-brain axis because the enteric microbiota plays a vital role in the gut-brain axis not only through the intrinsic nervous system, but also through hormonal and metabolic pathways [37][38][39][40][41]. A previous study demonstrated that oral supplementation with B. breve MCC1274 suppressed hippocampal neuroinflammation in Aβinjected mice, which led to the amelioration of cognitive dysfunction [24]. Furthermore, MCI subjects who received this probiotic for 16 weeks showed improved memory function [25]. In addition, recently, it has been found that AD-like pathologies in App NL-G-F mice supplemented with B. breve MCC1274 were mitigated through enhancement of the nonamyloidogenic pathway, as well as attenuation of neuroinflammation, which finally prevented memory deficits [27]. However, the effects of this probiotic on AD-like pathologies in WT mice have remained unclear. In this study, the effect of B. breve MCC1274 on ADlike pathologies in WT mice was explored. A similar beneficial effect was also exerted in these mice by orally-supplemented B. breve MCC1274, as demonstrated by a reduction in soluble Aβ42 levels, attenuation of neuroinflammation, and improvement of synaptic plasticity in the hippocampus.
Aggregation of Aβ in the brain is the most remarkable characteristic of AD, which is generated through the cleavage of APP by β-secretase and then by γ-secretase. PS1 is a key enzyme in the generation of Aβ because it harbors the catalytic site of the γ-secretase Photomicrographs of brain sections throughout the hippocampal dentate gyrus (DG). Immunofluorescence staining with BrdU (green) and DAPI nuclear counterstain (blue) (left panel). BrdU (50 mg/kg, intraperitoneal injection) was injected twice/day for 3 days. The BrdU + cell numbers in the SVZ of the DG area were quantified (right panel). Data are represented as mean ± SD, n = 7 per group; n.s. no significant difference; * p < 0.05 compared with the saline group, as determined by Student's t-test.

B. breve MCC1274 Supplementation Does Not Alter Cell Proliferation in the Hippocampus
To assess cell proliferation in the subgranular zone of the dentate gyrus after oral supplementation with B. breve MCC1274, brain sections were labeled with BrdU. The number of BrdU + cells did not change in the probiotic-supplemented group compared to that in the saline-supplemented group ( Figure 9C). These results suggest that oral supplementation with B. breve MCC1274 does not alter cell proliferation in the hippocampus of WT mice, which is consistent with the previous finding in App NL-G-F mice [27].

Discussion
In recent decades, several clinical and experimental studies have explored the role of microbiota in modulating the gut-brain axis because the enteric microbiota plays a vital role in the gut-brain axis not only through the intrinsic nervous system, but also through hormonal and metabolic pathways [37][38][39][40][41]. A previous study demonstrated that oral supplementation with B. breve MCC1274 suppressed hippocampal neuroinflammation in Aβ-injected mice, which led to the amelioration of cognitive dysfunction [24]. Furthermore, MCI subjects who received this probiotic for 16 weeks showed improved memory function [25]. In addition, recently, it has been found that AD-like pathologies in App NL-G-F mice supplemented with B. breve MCC1274 were mitigated through enhancement of the non-amyloidogenic pathway, as well as attenuation of neuroinflammation, which finally prevented memory deficits [27]. However, the effects of this probiotic on AD-like pathologies in WT mice have remained unclear. In this study, the effect of B. breve MCC1274 on AD-like pathologies in WT mice was explored. A similar beneficial effect was also exerted in these mice by orally-supplemented B. breve MCC1274, as demonstrated by a reduction in soluble Aβ42 levels, attenuation of neuroinflammation, and improvement of synaptic plasticity in the hippocampus.
Aggregation of Aβ in the brain is the most remarkable characteristic of AD, which is generated through the cleavage of APP by β-secretase and then by γ-secretase. PS1 is a key enzyme in the generation of Aβ because it harbors the catalytic site of the γ-secretase complex [42]. Furthermore, an in vivo study demonstrated that the overexpression of PS1 is sufficient to elevate the production and aggregation of Aβ in the brain, and mutant PS1 produces greater deposition of Aβ in the brain, as it cleaves APP more efficiently at the Aβ42 site than at the Aβ40 site [43]. These findings showed that B. breve MCC1274 supplementation was able to downregulate hippocampal soluble Aβ42 production in WT mice by decreasing the protein levels of the γ-secretase component of PS1. A previous study demonstrated that B. breve MCC1274 supplementation decreased Aβ production and deposition in App NL-G-F mice through upregulation of ADAM10 protein levels [27]. In the current study, B. breve MCC1274 supplementation altered the protein levels of PS1, but not ADAM10. This difference between WT and App NL-G-F mice suggests that there are different regulatory mechanisms underlying the B. breve MCC1274-reduced Aβ42 generation.
Brain dysfunction may be linked to the downregulation of Akt and elevation of the inactive form of GSK-3β [44]. Furthermore, Akt activation may be considered a potential preventive therapy for neurodegenerative diseases [45,46]. It has also been reported that Akt activation with an elevation of the inactive form of GSK-3β contributes to the downregulation of PS1 protein levels [28]. In this study, the results showed that B. breve MCC1274 supplementation increased pAkt (S473) and the inactive form of pGSK-3β (S9), without significant changes in pGSK-3α (S21) in the hippocampi of WT mice. According to a previous study, the inactivation of GSK-3β results from the increased phosphorylation at the S9 site [47]. Another study demonstrated that increasing the inactive form of pGSK-3β resulted in the attenuation of PS1/γ-secretase activity [48]. Taken together, these findings suggest that B. breve MCC1274 supplementation in WT mice reduces PS1 protein levels through the Akt/GSK-3β signaling pathway.
Recently, abnormally hyperphosphorylated tau has attracted considerable attention as a targeted drug for AD [49,50]. In this study, the results showed that oral supplementation with B. breve MCC1274 downregulated the levels of some tau phosphorylation sites (T231, S202, and T205) in the hippocampus of WT mice, which is different from the previous findings in App NL-G-F mice because tau phosphorylation could not be altered by B. breve MCC1274 supplementation in these mice [27]. In this study, the increased phosphorylation levels of both Akt and GSK-3β, without changes in the total protein levels of these two kinases, participate in the inhibition of tau hyperphosphorylation in the hippocampus. Therefore, B. breve MCC1274 supplementation appears to be a potential therapeutic approach for the prevention of tau hyperphosphorylation in WT mice.
It has been reported that impaired synaptic plasticity is found in several neurodegenerative diseases, including AD, and is also an early event in the disease [51]. Synaptic dysfunction is hypothesized to be not only the main hallmark, but also a primary cause of AD [52]. Indeed, synaptic dysfunction is caused by the aggregation of soluble Aβ oligomers that bind to both presynaptic and postsynaptic proteins and impair their function [53]. The levels of four synaptic proteins, SYT, syntaxin, SYP, and PSD95, were determined in both the cortex and hippocampus. The results indicated that supplementation with B. breve MCC1274 substantially increased these protein levels in the hippocampus of WT mice. Furthermore, previous findings demonstrated that this probiotic recovered both pre-and postsynaptic protein levels in App NL-G-F mice [27]. Thus, the current and previous findings suggest that B. breve MCC1274 may be used as a targeted therapy to prevent synaptic dysfunction by increasing both pre-and postsynaptic protein expression in both AD-like models and WT mice.
Neuroinflammation caused by activated astrocytes and microglia contributes to the progression of neurodegenerative diseases [3,4,54,55]. A previous study demonstrated that B. breve MCC1274 supplementation has an anti-inflammatory effect in the brains of App NL-G-F mice, which might be achieved by shifting microglia from the M1 to M2 phenotype, resulting in the elevation of anti-inflammatory cytokines and the reduction of pro-inflammatory cytokines [27]. In the present study, the possible anti-inflammatory effects of this probiotic were demonstrated in WT mice. The results of immunofluorescence staining and Western blotting indicated a substantial downregulation of Iba1 + cell number and protein level in the hippocampus of WT mice, whereas there was no change in GFAP + cell number or protein levels. These results consistently suggest that B. breve MCC1274 has a neuroprotective effect in the hippocampus of WT mice through its anti-inflammatory effect, which may be achieved by reducing activated microglia. Several studies have shown that the dysregulation of the PI3K/Akt signaling pathway leads to microglial activation, which promotes the production of pro-inflammatory cytokines, such as IL-6 and TNFα [56,57]. Therefore, enhanced Akt activation in the brains of mice supplemented with B. breve MCC1274 may play an important role in the attenuation of microglial activation. However, the underlying mechanism by which B. breve MCC1274 supplementation activates Akt is still unclear. Several studies have demonstrated that some probiotics elevate Akt phosphorylation [58,59]. However, further investigations are required to understand the mechanism of B. breve MCC1274-mediated activation of the Akt-GSK-3β pathway.
Interactions between microglia and synapses have been observed in most neurodegenerative diseases. It has been reported that increased microglial activation, along with a reduction in both the number and function of synapses, occurs in the early development of AD [60,61]. Activated microglia secrete pro-inflammatory cytokines that negatively affect synaptic transmission and activity, resulting in impaired cognitive function [62][63][64]. Therefore, this study suggests that the attenuation of microglial activation in the hippocampus of WT mice by B. breve MCC1274 supplementation might play an essential role in the improvement of synaptic protein expression.
As neurogenesis plays a vital role in improving cognitive function and mental health [65], one of the objectives of this study was to investigate the effect of B. breve MCC1274 supplementation on neurogenesis in the brains of WT mice. After four months of probiotic supplementation, no remarkable differences were observed in the number of BrdU + cells in the SVZ of the DG area in these mice, which is consistent with previous findings in App NL-G-F mice [27]. Thus, these results suggest that B. breve MCC1274 supplementation does not affect hippocampal neurogenesis in WT or App NL-G-F mice.

Conclusions
Previous findings showed that oral administration of B. breve MCC1274 improved cognitive function and mitigated AD-like pathologies. However, its effect on WT mice is unknown. In this study, the results showed that B. breve MCC1274 supplementation collectively decreased soluble Aβ42 and PS1 protein levels through the Akt/GSK-3β signaling pathway, attenuated microglial activation, and inhibited tau phosphorylation, which might improve synaptic function. B. breve MCC1274 supplementation had similar potential therapeutic effects on both WT and App NL-G-F mice, except for the difference in the underlying regulatory mechanism of B. breve MCC1274 supplementation-reduced Aβ generation and the impact on tau hyperphosphorylation. Overall, this study and the previous studies support the idea that oral supplementation with B. breve MCC1274 has a beneficial effect on multiple AD therapeutic targets.

Institutional Review Board Statement:
All experiments were carried out in conformity with the National Institute of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Nagoya City University Institutional Care and Use of Laboratory Animals Committee (21-029).