*3.6. WY Peptide Improved Age-Related Memory Impairment*

Our results showed that the WY dipeptide restored pharmacologically-induced memory impairment in mice. Therefore, we further examined whether the WY dipeptide would have a similar effect on memory impairment in aged mice. Aged (22 months) and young (7 months) C57BL/6J mice were orally administered WY dipeptide, and their performance in the spontaneous alternation test was evaluated. The proportion of spontaneous alternation was reduced in aged mice, compared with young mice, indicating age-related memory impairment. The administration of the WY dipeptide increased the proportion of spontaneous alteration in aged mice (Figure 6), indicating that WY dipeptide also restored memory impairment in aged mice.

**Figure 6.** The effects of the WY peptide on aged mice: 7- and 22-month-old C57BL/6 mice were orally administered a daily dose 1 mg/kg of WY dipeptide for 14 days. At 1 h after oral administration of the peptide, mice were allowed to explore the Y-maze for 8 min. Data represent the mean ± SEM of 8–10 mice per group. The *p* values were calculated using one-way ANOVA followed by the Tukey–Kramer test. \* *p* < 0.05 and \*\* *p* < 0.01.

#### **4. Discussion**

Epidemiological studies have reported that the consumption of fermented dairy products is beneficial for the prevention of cognitive decline in the elderly [1]. Our previous study demonstrated that WY-related peptides derived from enzymatic whey protein digests, such as the GTWY peptide β-lactolin, improve memory impairment in mice with pharmacologically-induced amnesia [7]. However, the underlying mechanism of the effects of the WY dipeptide core included in WY-related peptides on memory improvement is not well-understood. In the present study, we demonstrated that dipeptides with an N-terminal tryptophan, such as WY, WM, WV, WL, and WF improved memory impairment in scopolamine-induced amnesic mice. Especially, the WY dipeptide increased the dopamine levels in the hippocampus and frontal cortex, and the WY-induced memory improvement was attenuated by the blockade of the dopamine D1 receptor. Moreover, the WY dipeptide improved spatial memory impairment in aged mice. These results suggested that orally administered WY dipeptide improved spatial memory impairments in pharmacologically-induced amnestic mice and aged mice via modulating the dopamine system.

We showed that the WY dipeptide inhibited MAO-B activity and increased dopamine levels in the hippocampus and frontal cortex and improved spatial memory impairment in pharmacologically-induced amnestic mice and aged mice. Previous reports have indicated that dopamine is involved in hippocampus-dependent memory functions [12,13]. The dopamine neuronal network is related to an age-related decline in cognitive performance and executive function, and the dopamine precursor levodopa has been shown to improve the task-based learning rate and task performance in the elderly [14]. It has also been reported that MAO-B inhibitors improve cognitive function in rodents [15–17]. MAO-B is an enzyme for dopamine metabolism and thus decreases dopamine levels in the brain [18]. Therefore, MAO-B inhibitors have been used as drugs to induce dopamine levels [19]. It is suggested that some MAO-B inhibitors have a potential as therapeutic or preventive treatment for dementia including Alzheimer's disease [20,21]. We also showed that the administration of WY dipeptide increased total dopamine levels in the hippocampus and frontal cortex. These results suggest that WY dipeptide increase dopamine levels in the brain by inhibiting MAO-B activity. Those findings suggest that the WY dipeptide exerts its beneficial effect on the prevention of age-related cognitive decline by increasing dopaminergic activity in the brain.

In addition, we showed that dopamine D1 receptor is involved in the spatial memory improvement induced by the WY dipeptide. Dopamine exerts its functions via multiple receptor subtypes, D1-like (D1 and D5) and D2-like (D2, D3, and D4) receptors. In the present study, the treatment with D1-like receptor antagonist, SCH-23390 attenuated the improvement caused by the WY dipeptide in scopolamine-induced amnesic mice. It has been reported that activation of dopamine D1-like receptors enhances hippocampus-dependent memory functions [22–25]. Because the hippocampus is crucial for spatial memory as measured by the Y-maze test [26], we examined the involvement of the dopamine D1 receptor in the hippocampus in WY-dipeptide-induced memory improvement. Dopamine D1-like receptors are expressed in many brain areas including the hippocampus, but due to the lack of selective pharmacological drugs, most studies failed to discriminate dopamine receptor subtypes. Recently, the brain-region-specific knockdown of the dopamine D1 receptor subtype was achieved by injecting AAV vectors that express recombinant miRNA targeting this dopamine receptor subtype [9]. This technique made it possible to further examine a potential link between the spatial memory improvement by the WY dipeptide and the dopamine D1 receptors in the hippocampus. Dopamine D1 receptor knockdown in the hippocampus attenuated the improvement by the WY peptide, but it was a partial reduction. These data suggested that the dopamine D1 receptor in the hippocampus is involved in the WY-dipeptide-induced memory improvement for spatial information measured in the Y-maze test at least in part. The reason why the effect of the D1 receptor knockdown was partial remains unclear. It could be due to the partial knockdown of the dopamine D1 receptor in the hippocampus. This finding is consistent with previous reports suggesting that D1 receptor stimulation in the hippocampus augments spatial memory. Alternatively, dopamine in the frontal cortex could be involved in the WY-dipeptide-induced memory improvement, since the WY dipeptide increased the dopamine levels in frontal cortex as well.

In the present study, we compared the effects of several dipeptides containing tryptophan and tryptophan as a single amino acid. We found that the administration of a single dose of dipeptides with an N-terminal tryptophan, WY, WM, WV, WL, and WF improved spatial memory in mice, whereas YW and MW dipeptides did not have this effect. These findings suggest that the dipeptide conformation with an N-terminal tryptophan is crucial for effectively improving the memory impairment after oral administration. It has been shown that dipeptides with an N-terminal tryptophan have a high affinity to peptide transporters, and among the dipeptide library, the WY dipeptide is one of the dipeptides with the highest affinities [27]. Thus, the conformation of dipeptides with an N-terminal tryptophan could be necessary for transporter-mediated absorption from the gut and delivery to the brain. On the other hand, based on our finding that the WY dipeptide, but not tryptophan as a single amino acid, inhibited MAO-B activity in vitro, the dipeptide conformation with an N-terminal tryptophan could be crucial for enhancing dopaminergic activity. Note that these two possibilities are not mutually exclusive but rather synergetic for the memory improving effect of the WY dipeptide.
