Pharmaceutical Potential of Casein-Derived Tripeptide Met-Lys-Pro: Improvement in Cognitive Impairments and Suppression of Inflammation in APP/PS1 Mice

Abstract

Background: Tripeptide Met-Lys-Pro (MKP), a component of casein hydrolysates, has effective angiotensin-converting enzyme (ACE) inhibitory activity. Brain angiotensin II enzyme activates the NADPH oxidase complex via angiotensin II receptor type 1 (AT1) and enhances oxidative stress injury. ACE inhibitors improved cognitive function in Alzheimer's disease (AD) mouse models and previous clinical trials. Thus, although undetermined, MKP may be effective against pathological amyloid-β (Aβ) accumulation-induced cognitive impairment.

Objective: The current study aimed to investigate the potential of MKP as a pharmaceutical against AD by examining MKP's effect on cognitive function and molecular changes in the brain using double transgenic (APP/PS1) mice.

Methods: Experimental procedures were conducted in APP/PS1 mice (n = 38) with a C57BL/6 background. A novel object recognition test was used to evaluate recognition memory. ELISA was used to measure insoluble Aβ40, Aβ42, and TNF-α levels in brain tissue. Immunohistochemical analysis allowed the assessment of glial cell activation in MKP-treated APP/PS1 mice.

Results: The novel object recognition test revealed that MKP-treated APP/PS1 mice showed significant improvement in recognition memory. ELISA of brain tissue showed that MKP significantly reduced insoluble Aβ40, Aβ42, and TNF-α levels. Immunohistochemical analysis indicated the suppression of the marker for microglia and reactive astrocytes in MKP-treated APP/PS1 mice.

Conclusion: Based on these results, we consider that MKP could ameliorate pathological Aβ accumulation-induced cognitive impairment in APP/PS1 mice. Furthermore, our findings suggest that MKP potentially contributes to preventing cognitive decline in AD.

Keywords: APP/PS1 mice; Alzheimer’s disease; MKP; Met-Lys-Pro; dementia; peptide.

Fig. 1

Met-Lys-Pro (MKP) p.o. administration prevents working memory deficit in APP/PS1 mice as measured by the novel object recognition task. A) Both groups spent approximately the same time exploring two identical objects during the training trial, indicating no inherent place preference. B) MKP-treated APP/PS1 mice spent a significantly longer time exploring the novel object compared to control APP/PS1 mice, as indicated by the higher mean recognition index in 10 min measurement. Data are presented as mean±SEM. Data were analyzed with the Student’s t-test or Mann–Whitney U-test. ^*p < 0.05 compared to Control. (Control, n = 19; MKP, n = 19).

Fig. 2

Met-Lys-Pro (MKP) p.o. administration enhances spatial learning in APP/PS1 mice as measured by the Morris water maze test at 14.5 months of age. A) Each mouse performed 5 trials per day for 5 consecutive days to locate and climb onto the hidden platform in training trials. MKP-treated APP/PS1 mice showed significant reductions in latencies to the platform from day 5 to day 6, compared with day 1. Data are presented as mean±SEM. Significance (Tukey’s multiple comparison test after two-way repeated measures ANOVA): ^*p < 0.05, ^**p < 0.01 versus day 1; (Dunnett’s multiple comparison test after one-way repeated measures ANOVA): ^†p < 0.05 versus day 1. (Control, n = 19; MKP, n = 19). B) Probe test 1 day after the last trial. The time spent in the target quadrant in both groups was approximately the same. C) On day 6 of place navigation training, no significant differences in the swim speed were observed between groups. D) The cued navigation test (visible platform test) was conducted. No significant differences were detected between groups. E) On the cued navigation test, no significant differences in the swim speed were observed between groups. Data are presented as mean±SEM. Data were analyzed with the Student’s t-test or Mann–Whitney U-test. ^*p < 0.05 compared to Control. (Control, n = 19; MKP, n = 19).

Fig. 3

Changes in Aβ levels in the brains of APP/PS1 mice after Met-Lys-Pro (MKP) p.o. administration. One hemisphere of the forebrain was homogenized and sequentially extracted with TBS (TBS fraction) and FA (FA fraction) in APP/PS1 mice. Levels of Aβ₄₀ (A) and Aβ₄₂ (B) in the FA fraction. Levels of Aβ₄₀ (C) and Aβ₄₂ (D) in the TBS fraction. MKP-treated APP/PS1 mice showed significant reductions in Aβ₄₀ and Aβ₄₂ levels in the FA fraction. Data are presented as mean±SEM. Data were analyzed using the Student’s t-test or Mann–Whitney U-test. ^*p < 0.05 compared to Control. (Control, n = 19; MKP, n = 19).

Fig. 4

Aβ₄₂-immunoreactivities in APP/PS1 mice after Met-Lys-Pro (MKP) p.o. administration. Free-floating brain sections in the sagittal plane were immunostained with Abeta (N) antibodies in the hippocampus (A, B, and C) and frontal cortex (D, E, and F). Immunohistochemistry images of control (A) and MKP (B) in the hippocampus. Percentage of immunoreactive areas in each group in the hippocampus (C). Immunohistochemistry images of control (D) and MKP (E) in the frontal cortex. Percentage of immunoreactive areas in each group in the frontal cortex (F). Scale bar: 1 mm. Data represent mean±SEM. Data were analyzed using the Student’s t-test. ^*p < 0.05 compared to Control. (Control, n = 19; MKP, n = 19).

Fig. 5

GFAP-immunoreactivity in APP/PS1 mice after Met-Lys-Pro (MKP) p.o. administration. Free-floating brain sections in the sagittal plane immunostained with GFAP antibodies for activated astrocytes in the hippocampus (A, B, and C) and frontal cortex (D, E, and F). Immunohistochemistry images of control (A) and MKP (B) in the hippocampus. Percentage of immunoreactive areas in each group in the hippocampus (C). Immunohistochemistry images of control (D) and MKP (E) in the frontal cortex. Percentage of the immunoreactive area in each group in the frontal cortex (F). Scale bar: 1 mm. To calculate GFAP-immunoreactive area (C and F), the areas outlined in yellow (A, B, D, and E) were normalized. High-magnification images revealed single astroglial cell morphology in the cortex and hippocampus of the APP/PS1 mice (A, B, D, and E). Scale bars: 20μm (insets). Data represent mean±SEM. Data were analyzed using the Student’s t-test. ^*p < 0.05 compared to Control. (Control, n = 19; MKP, n = 19).

Fig. 6

Iba1-immunoreactivities in APP/PS1 mice after Met-Lys-Pro (MKP) p.o. administration. Free-floating brain sections in the sagittal plane were immunostained with Iba1 antibodies for activated microglia in the hippocampus (A, B, and C) and frontal cortex (D, E, and F). Immunohistochemistry images of control (A) and MKP (B) in the hippocampus. Percentage of the immunoreactive area in each group in the hippocampus (C). Immunohistochemistry images of control (D) and MKP (E) in the frontal cortex. Percentage of the immunoreactive area in each group in the frontal cortex (F). Scale bar: 1 mm. To calculate Iba1-immunoreactive area (C and F), the area outlined in yellow (A, B, D, and E) were normalized. High-magnification images revealed a single microglial cell morphology in the cortex and hippocampus of the APP/PS1 mice (A, B, D, and E). Scale bars: 20μm (insets). Data represent mean±SEM. Data were analyzed using the Student’s t-test. ^*p < 0.05 compared to Control. (Control, n = 19; MKP, n = 19).

Fig. 7

Changes in the levels of cytokines in the brain of APP/PS1 mice after Met-Lys-Pro (MKP) p.o. administration. One hemisphere of the forebrain was homogenized and sequentially extracted with TBS (TBS fraction) in APP/PS1 mice. Levels of IL-1β (A), IL-6 (B), and TNF-α (C) in the TBS fraction. MKP-treated APP/PS1 mice showed a significant decrease in TNF-α in the TBS fraction. Data represent mean±SEM. Data were analyzed using the Student’s t-test or Mann–Whitney U-test. ^*p < 0.05 compared to Control. (Control, n = 19; MKP, n = 19).