β-Lactolin,a Whey-Derived Lacto-Tetrapeptide,Prevents Alzheimer’s Disease Pathologies and Cognitive Decline

Materials

GTWY peptide, β-lactolin, (purity: 98%; Fig. 1) was purchased from Bachem (Bubendorf, Switzerland). Whey digestion (containing 1.6 mg of β-lactolin per 1 g of whey digestion) was prepared by Kirin Holdings Company (Tokyo, Japan).

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Fig.1

Chemical structure of β-lactolin. The chemical structure of β-lactolin, glycine-thereonine-tryptophan-tyrosine (GTWY) lactotetrapeptide.

Animals

AD model mice, B6SJL-Tg mice (APPSwFlLon, PSEN1*M146L*L286V, http://jaxmice.jax.org/strain/006554.html, [18]), hereafter referred to as 5×FAD transgenic mice, were purchased from Jackson Laboratory (Sacramento, CA, USA) and were maintained by crossing hemizygous transgenic mice with B6SJLF1/J mice in the experimental facility at the University of Tokyo. 5×FAD transgenic mice overexpress mutant human APP (695) with Swedish (K670N, M671L), Florida (I716V), and London (V717I) Familial Alzheimer’s Disease (FAD) mutations, along with human PS1 harboring 2 FAD mutations, M146L and L286V. Tauopathy model mice (B6;C3-Tg mice (Prnp-MAPT*P301S)PS19Vle/J, https://www.jax.org/strain/008169, [19]), hereafter referred to as PS19 mice, were also used in the present study. PS19 mice overexpress the T34 isoform of microtubule-associated protein tau with one N-terminal insert and four microtubule-binding repeats encoding the human P301S mutation. Nontransgenic wild-type littermates were used as controls in this study.

All experiments were approved by the Animal Care and Use Committee of the Graduate School of Agricultural and Life Sciences, the University of Tokyo, and they were conducted from May 2017 to June 2018 in strict accordance with their guidelines (Approval No; P15-042). All efforts were made to minimize animal suffering. Mice were maintained at room temperature (23°C±1°C) under constant 12-h light/dark cycles (light on from 8:00 am to 8:00 pm). Mice aged < 3 months were fed a standard purified rodent growth diet (AIN-93G; Oriental Yeast, Tokyo, Japan), and those aged≥3 months were fed a maintenance diet (AIN-93M; Oriental Yeast).

To evaluate the effects of β-lactolin and whey digestion rich in β-lactolin on Alzheimer-like disease, we fed 2.5-month-old transgenic 5×FAD and wild-type male mice with a diet that either contained or did not contain 0.05% w/w β-lactolin or 5% w/w whey digestion for 3.5 months (wild-type mice, n = 10; transgenic control mice, n = 10; transgenic mice with β-lactolin, n = 11; transgenic mice with whey digestion, n = 10). In addition, we also fed 3-month-old PS19 and wild-type male mice with a diet that either contained or did not contain 0.05% w/w β-lactolin or 5% w/w whey digestion for 6 months (wild-type mice, n = 12; transgenic control mice, n = 11; transgenic mice with β-lactolin, n = 11; transgenic mice with whey digestion, n = 11). The daily dosage of β-lactolin was 1.75 mg in the group fed with 0.05% w/w β-lactolin and 0.28 mg in the group fed with 5% w/w whey digestion when the consumption of food was 3.5 g. There were no significant differences in body weight and food consumption among different mouse groups (data not shown). After behavioral evaluations, mice were euthanized, and the brains were removed, as described in the following sections.

Quantification of cytokine, synaptophysin, Aβ, and tau by enzyme-linked immunosorbent assay (ELISA)

Homogenate samples of the left hippocampus and the cortex were prepared as described in a previous study [20]. The hippocampus or cortex was homogenized in tris-buffer solution (TBS) containing a protease inhibitor cocktail (BioVision, CA, USA) using a multi-beads shocker (Yasui Kikai, Osaka, Japan). The supernatant (first) was collected after centrifugation at 50,000×g for 20 min, and the pellet was homogenized again in TBS containing 1% Triton X-100 (Wako, Osaka Japan), and the supernatant (second) was collected after centrifugation at 50,000×g for 20 min. The total protein concentration of each supernatant was measured using the BCA Protein Assay Kit (Thermo-Scientific, Yokohama, Japan). The first supernatant was used to quantify soluble Aβ_1-42 (Wako), phosphorylated tau (pS199, ThermoFisher Scientific, Yokohama, Japan), total tau (ThermoFisher Scientific), synaptophysin (LSBio, Seattle, WA, USA), BDNF (Promega, Madison, WI, USA), and IGF-1 (R&D Systems, Minneapolis, MN, USA) by ELISA and cytokines and chemokines by a Bio-Plex assay system (Bio-Rad, Hercules, CA, USA). The second supernatant was used to quantify insoluble Aβ_1-42 (Wako) by ELISA.

Immunohistochemistry

The right-brain hemispheres (n = 10-11 in each group) were fixed in 10% formalin solution (Wako), paraffin-embedded, and cut into 5-μm serial sections to evaluate Aβ deposition and infiltration of activated microglia and astrocytes. The studied brain regions included the hippocampus and cerebral cortex (bregma 2.30 mm posterior). After dewaxed and rehydrated, the sections for Aβ assay were treated in 98% formic acids, and those for ionized calcium-binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) measurements were autoclaved at 121°C for 10 min in 0.2% citrate buffer (pH 6.0) for antigen retrieval. The sections were then incubated with a blocking solution (8% w/v skimmed milk) for 30 min after inactivation of endogenous peroxidase with 3% H₂O₂ (Wako) in methanol for 5 min. Subsequently, the sections were incubated overnight at room temperature with primary antibodies, including monoclonal anti-human Aβ_x-42 antibodies (12F4, Millipore, Billerica, MA, USA), polyclonal anti-Iba-1 antibodies (Wako), or polyclonal anti-GFAP antibodies (Dako, Glostrup, Denmark). After the sections were incubated with horseradish peroxidase coupled goat anti-mouse or rabbit IgG antibodies (4μg/ml, Nichirei, Tokyo, Japan) for 1 h at room temperature, they were visualized with 3,3’-diaminobenzidine (Wako) and counterstained in hematoxylin. The size of the positive region per area was measured using the Image J image analysis software (NIH, Bethesda, MD, USA).

Assessment of microglial characteristics using a flow cytometer

For microglial activity evaluation, the right-brain hemispheres (n = 5 in each group) were removed, and the complement receptor (CD)11b-positive microglia were isolated and were used for flow cytometry analysis, as described in previous studies [12, 21]. Brain cells were obtained by papain treatment using a Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, MA, USA). The cells were treated with 2μg/ml of anti-CD11b antibody conjugated with microbeads (Miltenyi Biotec), and CD11b-positive cells were isolated by magnetic cell sorting.

To measure the expression of cell surface markers, isolated microglia with more than 90% purity were stained with anti-CD11b-APC-Cy7 (M1/70, BD Pharmingen) and anti-CD86-PE (GL-1, eBioscience) and were then analyzed using a FACS Canto II flow cytometer (BD Bioscience).

To measure intracellular cytokine production, isolated microglia were plated in a 96-well plate (BD Biosciences, MA, USA) at 50,000 per well and were cultured in DMEM/F-12 (Gibco, CA, USA) medium supplemented with 10% fetal calf serum (Gibco) and 100 U/ml of penicillium/streptomycin (Sigma-Aldrich, MO, USA). Microglia were treated with a leukocyte activation cocktail using BD GolgiPlug (BD Biosciences) for 12 hours and were then fixed with a BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences), and subsequently, they were stained with following antibodies: anti-interleukin 1 beta (IL-1β)-FITC (NJTEN3, eBiosciences), anti-tumor necrosis factor (TNF)-α-APC (MP6-XT22, BD Pharmingen, CA, USA), and anti-CD11b-APC-Cy7 (M1/70, BD Pharmingen). The cells were analyzed using a flow cytometer.

Dopamine analysis

To evaluate the level of dopamine in the brain, the tissue was homogenized in 0.2 M perchloric acid (PCA, Wako) containing 100μM EDTA•2Na (Sigma-Aldrich). After centrifugation, the supernatant was analyzed by HPLC using an EICOMPAK SC-5ODS column and a PREPAK column (Eicom, Kyoto, Japan) with an electrochemical detection (ECD) unit. The mobile phase consisted of 83% 0.1 M acetic acid in a citric acid buffer (pH 3.5), 17% methanol (Wako), 190 mg/ml of sodium 1-octane sulfonate sodium (Wako), and 5 mg/ml EDTA•2Na. For ECD, the applied voltage was 750 mV via an Ag/AgCl reference electrode.

Novel object recognition test

The novel object recognition test was performed during the light period in a polyvinyl chloride box (40×40×40 cm³) without a roof to evaluate the object memory. In the acquisition trial, a pair of wooden triangle poles (4.5×4.5×4.5 cm³) or wooden pyramids (4.5×4.5×4.5 cm³) was used; in the retention trial, a pair of poles or pyramids and a golf ball (4.5-cm diameter) were used. Mice were moved into the sound isolated room 16 h before the test started, and behavioral evaluations were carried out during the light phase. In all trials, the objects were placed 7.5 cm apart from the corner of the box. In the acquisition trial, each mouse was allowed to explore the box with the 2 objects for 10 min. Twenty-four hours after the acquisition trial, the mouse was allowed to explore the box with the novel and familiar objects for 5 min. The discrimination index was calculated by dividing the difference in the time for exploring the novel object and for the familiar object by the total time spent exploring both objects [(novel object exploration time – familiar object exploration time) / (total exploration time)]. A discrimination index of 0 indicated equal exploration of both objects.

Open field test

To evaluate the activity in the novel place, the mice were subjected to the open field test in an open field box (40 cm×40 cm×40 cm, gray polyvinyl chloride) without a roof for 5 min. The mouse activity was monitored using the SMART video tracking software (PanLab Harvard Apparatus, MA, USA).

Statistical analysis

Data are presented as mean±SEM. Data were analyzed using one-way ANOVA followed by the Tukey–Kramer, Dunnett, or Student’s t-tests, as described in figure legends. All statistical analyses were performed using the Ekuseru-Toukei 2012 software program (Social Survey Research Information, Tokyo, Japan).

Effects of β-lactolin on inflammation and microglial activation in 5×FAD mice

The levels of cytokines and chemokines in the cortex and hippocampus were measured to evaluate the effects of β-lactolin and whey peptides rich in β-lactolin on inflammation. The levels of IL-1β, macrophage inflammatory protein (MIP)-1α, MIP-1β, TNF-α, and IL-12p70 were reduced significantly in the cortex and hippocampus in the 5×FAD mice fed with β-lactolin compared with control 5×FAD mice (Table 1). In addition, the levels of IL-1α, IL-1β, MIP-1α, and IL-12p70 were reduced significantly in the cortex and hippocampus in 5×FAD mice fed with whey digestion compared with control 5×FAD mice (Table 1).

Microglia in the brain were characterized using a flow cytometer to evaluate the effects of β-lactolin and whey digestion rich in β-lactolin on microglial activation. The ratios of TNF-α and IL-1β-producing cells to CD11b-positive cells (Fig. 2A) were higher in control 5×FAD mice than in wild-type mice (Fig. 2A) but were lower in 5×FAD mice fed with β-lactolin and whey digestion than in control 5×FAD mice (Fig. 2B, C, respectively). The expressions of CD86 in CD11b-positive microglia were significantly higher in control 5×FAD mice than in wild-type mice but were significantly lower in 5×FAD mice fed with β-lactolin and whey digestion than in control 5×FAD mice (Fig. 2D).

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Fig.2

Effects of β-lactolin on inflammation and microglial activation in 5×FAD mice. Transgenic 5×FAD and wild-type male mice aged 2.5 months were fed a diet with or without 0.05% w/w β-lactolin or 5% w/w β-lactolin-rich whey enzymatic digestion (BL-W) for 3.5 months. A) Characterization of CD11b-positive microglia producing TNF-α and IL-1β by flow cytometry. B, C) The percentage of TNF-α or IL-1β-producing cells to CD11b-positive cells. D) Expressions of CD86 in CD11b-positive cells. M.F.I. is the mean fluorescent intensity. Data are presented as mean±SEM values (5 mice per group). p-values shown in the graph were calculated by one-way ANOVA followed by the Tukey–Kramer test. *p < 0.05, **p < 0.01.

The distribution of activated microglia in the brain was observed using immunohistochemistry (Fig. 3A-D). The levels of Iba-1-positive microglia in the cortex were reduced significantly in 5×FAD mice fed with β-lactolin compared with control 5×FAD mice (Fig. 3E). The distribution of activated astrocytes in the brain was also observed using immunohistochemistry (Supplementary Figure 1A-D). The levels of GFAP-positive astrocytes in the cortex were reduced in 5×FAD mice fed with β-lactolin compared with control 5×FAD mice (Supplementary Figure 1E).

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Fig.3

Effects of β-lactolin on microglial infiltration in 5×FAD mice. Transgenic 5×FAD and wild-type male mice aged 2.5 months were fed a diet with or without 0.05% w/w β-lactolin or 5% w/w β-lactolin-rich whey enzymatic digestion (BL-W) for 3.5 months. A-D) Representative immunohistochemistry images for Iba-1 in wild-type mice, transgenic control mice (Ctrl), and transgenic mice fed a diet containing β-lactolin or BL-W. Scale bars, 400μm. E) Percentage of the Iba-1-positive area detected by immunohistochemistry in the cortex in transgenic control mice (Ctrl) and transgenic mice fed a diet containing β-lactolin or BL-W. Data are presented as means±SEM (sample size: wild-type mice, 10; control transgenic mice, 10; transgenic mice fed with β-lactolin, 11; or transgenic mice fed with BL-W, 10). p-values shown in the graph were calculated by one-way ANOVA followed by the Tukey–Kramer test. *p < 0.05.

These findings indicate that consumption of β-lactolin and whey digestion rich in β-lactolin suppresses inflammation and microglial activation in 5×FAD mice.

Effects of β-lactolin on Aβ deposition in 5×FAD mice

5×FAD mice were fed with β-lactolin and whey digestion, and Aβ levels were measured using immunohistochemistry and ELISA to assess the effects of β-lactolin and whey digestion rich in β-lactolin on Aβ deposition in the brain. Immunohistochemical measurements (Fig. 4A-D) showed that Aβ_1-42 levels in the somatosensory/visual cortex were significantly lower in 5×FAD mice fed with β-lactolin and whey digestion than in control 5×FAD mice (Fig. 4E). Aβ_1-42 levels in the hippocampus were significantly lower in 5×FAD mice fed with β-lactolin, but not in those with whey digestion, than in control 5×FAD mice (Fig. 4F).

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Fig.4

Effects of β-lactolin on Aβ deposition in 5×FAD mice. Transgenic 5×FAD and wild-type male mice aged 2.5 months were fed a diet with or without 0.05% w/w β-lactolin or 5% w/w β-lactolin-rich whey enzymatic digestion (BL-W) for 3.5 months. A-D) Representative immunohistochemistry images for Aβ_1-42 in wild-type mice and transgenic mice with or without β-lactolin or β-lactolin-rich whey enzymatic digestion. Scale bars, 400μm. E, F) Percentage of the Aβ_1-42-positive area detected by immunohistochemistry in the cortex and hippocampus in transgenic control mice (Ctrl) and transgenic mice fed a diet containing β-lactolin or BL-W. G, H) The levels of TBS-soluble or TBS-insoluble/TBS-T soluble Aβ_1-42 in the frontal cortex. Data are presented as means±SEM (sample size: wild-type mice, 10; control transgenic mice, 10; transgenic mice fed with β-lactolin, 11; or transgenic mice fed with BL-W, 10). p-values shown in the graph were calculated by one-way ANOVA followed by the Tukey–Kramer test. *p < 0.05.

Quantification by ELISA revealed that the levels of TBS-soluble Aβ_1-42 and TBS-insoluble and TBS-T soluble Aβ_1-42 were significantly lower in the cortex in 5×FAD mice fed with β-lactolin and whey digestion than in control 5×FAD mice (Fig. 4G, H). These results indicate that the consumption of β-lactolin and whey peptides rich in β-lactolin reduces the deposition of Aβ_1-42, especially in the cortex.

Effects of β-lactolin on memory impairment in 5×FAD mice

5×FAD mice underwent a novel object recognition test to evaluate object memory retrieval. The time approaching the novel object was significantly longer than that approaching the familiar object in wild-type mice and 5×FAD mice fed with β-lactolin and whey digestion, but there was no difference in the time approaching two objects in control 5×FAD mice (Fig. 5A). In addition, the discrimination indexes were reduced significantly in control 5×FAD mice compared with wild-type mice but were increased significantly in 5×FAD mice fed with β-lactolin and whey digestion compared with control 5×FAD mice (Fig. 5B). These results indicate that consumption of β-lactolin and whey digestion ameliorates long-term object memory impairment in 5×FAD mice.

The levels of synaptophysin in the frontal cortex were measured to evaluate the effects of β-lactolin and whey peptides rich in β-lactolin on synapse. The levels of synaptophysin were reduced significantly in 5×FAD mice compared with wild-type mice (Fig. 5C) but were significantly increased in 5×FAD mice fed with β-lactolin (Fig. 5C) compared with control 5×FAD mice. In addition, synaptophysin levels also increased in 5×FAD mice fed with whey digestion compared with control 5×FAD mice (Fig. 5C), although the difference was not significant. The results indicate that β-lactolin and whey peptides rich in β-lactolin suppress synapse loss in 5×FAD mice.

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Fig. 5

Effects of β-lactolin on memory function in 5×FAD mice. Transgenic 5×FAD and wild-type male mice aged 2.5 months were fed a diet with or without 0.05% w/w β-lactolin or 5% w/w β-lactolin-rich whey enzymatic digestion (BL-W) for 3.5 months. Mice aged 6 months were subjected to the novel object recognition test to evaluate object recognition memory. A, B) The time spent exploring novel and familiar objects during 5 min of re-exploration (A) and the discrimination index [(time spent with object A – time spent with object B) / total time exploring both objects] (B) were measured. C) The levels of synaptophysin in the frontal cortex. Data are presented as means±SEM (sample size: wild-type mice, 10; control transgenic mice, 10; transgenic mice fed with β-lactolin, 11; or transgenic mice fed with BL-W, 10). p-values shown in the graph were calculated by student t-test or one-way ANOVA followed by the Tukey–Kramer test. *p < 0.05, **p < 0.01.

Effects of β-lactolin on dopamine, brain-derived neurotrophic factor (BDNF), and insulin-like growth factor 1 (IGF-1) levels

To evaluate the effects of β-lactolin and whey peptides rich in β-lactolin on neuroprotection, the levels of dopamine, BDNF, and IGF-1 in the cortex were measured. The levels of dopamine, BDNF, and IGF-1 were reduced significantly in 5×FAD mice compared with wild-type mice (Supplementary Figure 2A-C). The levels of dopamine and BDNF were significantly increased in 5×FAD mice fed with β-lactolin compared with control 5×FAD mice (Supplementary Figure 2A, B). In addition, IGF-1 levels in 5×FAD mice fed with β-lactolin or whey digestion and BDNF levels in 5×FAD mice fed with whey digestion also increased compared with those in control 5×FAD mice, although the differences were not significant (Supplementary Figure 2A-C). These results indicate that β-lactolin and whey peptides rich in β-lactolin increase BDNF and IGF-1 levels in 5×FAD mice.

Effects of β-lactolin on behavioral abnormalities and tau levels in PS19 mice

PS19 mice underwent the open field test to evaluate activity and anxiety. The total distances in the open field box were significantly increased in control PS19 mice compared with wild-type mice but were significantly reduced in PS19 mice fed with β-lactolin and whey digestion compared with control PS19 mice (Fig. 6A). The distances in the center zone of the open field box were significantly increased in control PS19 mice compared with wild-type mice but were reduced in PS19 mice fed with β-lactolin and whey digestion compared with control PS19 mice (Fig. 6B).

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Fig.6

Effects of β-lactolin on memory function and tau accumulation in PS19 mice. Transgenic PS19 and wild-type male mice aged 3 months were fed a diet with or without 0.05% w/w β-lactolin or 5% w/w β-lactolin-rich whey enzymatic digestion (BL-W) for 6 months. Mice aged 9 months were subjected to the open field test to evaluate behavioral abnormality. A, B) The total distances in the open field (A) and in the center of the open field (B). C, D) The levels of phosphorylated tau (pTau, C) and total tau (D) in the cortex. E) The ratio of pTau to total tau. Data are presented as means±SEM (sample size: wild-type mice, 12; control transgenic mice, 11; transgenic mice fed with β-lactolin, 11; or transgenic mice fed with BL-W, 11). p-values shown in the graph were calculated by one-way ANOVA followed by the Tukey–Kramer test. *p < 0.05.

The levels of pTau and total tau in the cortex were measured to evaluate the effects of β-lactolin and whey peptides rich in β-lactolin on tau pathologies. The levels of TBS-soluble pTau were reduced significantly in PS19 mice fed with β-lactolin compared with control PS19 mice (Fig. 6C), but the levels of total tau were not changed (Fig. 6D). The ratio of pTau to total tau was significantly reduced in the PS19 mice fed with β-lactolin compared with control PS19 mice (Fig. 6E). In addition, synaptophysin and IGF-1 levels in PS19 mice fed with β-lactolin and BDNF levels in PS19 mice fed with whey digestion increased compared with those in control PS19 mice (Supplementary Figure 5A-C). The results indicate that β-lactolin and whey peptides rich in β-lactolin suppress cognitive impairment and tau accumulation in PS19 mice.