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

Abstract

The prevention of age-related memory decline and dementia has been becoming a high priority because of the rapid growth in aging populations. Accumulating epidemiological and clinical studies indicate that intake of fermented dairy products rich in β-lactolin improves memory retrieval and executive function and attenuates cognitive decline in the elderly. However, the effects of long-term consumption of β-lactolin on Alzheimer's disease (AD) pathologies have not been investigated. In the present study, we examined the effects of β-lactolin and whey digestion rich in β-lactolin on AD pathology in 5×FAD transgenic mice and PS19 tauopathy mice. Intake of β-lactolin and whey digestion rich in β-lactolin reduced the levels of inflammatory cytokines, suppressed the infiltration of activated microglia, decreased the levels of amyloid-β, ameliorated impaired long-term object memory, and attenuated decreased synaptophysin, dopamine, brain-derived neurotrophic factor, and insulin-like growth factor 1 levels in the cortex in 5×FAD transgenic mice. In addition, intake of β-lactolin and whey digestion rich in β-lactolin improved behavioral abnormality and reduced the ratio of phosphorylated tau to total tau in the cortex in PS19 tauopathy mice. These findings indicate that consumption with β-lactolin and whey digestion rich in β-lactolin suppresses inflammation and attenuates AD pathology and cognitive impairment.

Keywords: Alzheimer’s disease; amyloid-β; cognitive function; inflammation; memory; microglia; peptide; tauopathy; β-lactolin.

Fig.1

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

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.

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.

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.

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.

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.