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TrkB phosphorylation in serum extracellular vesicles correlateswith cognitive function enhanced by ergothioneine in humans

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Takahiro Ishimoto1, Reiya Yamashita1, Ruri Matsumoto1, Satoshi Matsumoto2, Yusuke Matsuo1, Shunsuke Nakao1, Yusuke Masuo1,
Makoto Suzuki2 and Yukio Kato 1 ✉Oral administration of the food-derived antioxidant amino acid ergothioneine (ERGO) results in its efﬁcient distribution in the brain
and enhances cognitive function. However, effect of ERGO deﬁciency on cognitive impairment and the underlying mechanisms
remain unknown. We revealed that cognitive function and hippocampal neurogenesis were lower in mice fed an ERGO-free diet thanin those fed the control diet. Furthermore, ERGO supplementation to achieve the control diet ERGO levels reversed these effects andrestored ERGO concentrations in the plasma and hippocampus. The ERGO-induced recovery of cognitive function and hippocampal
neurogenesis was blocked by inhibiting the neurotrophic factor receptor tropomyosin receptor kinase B (TrkB), with a concomitant
reduction in hippocampal phosphorylated TrkB, suggesting the involvement of TrkB in these events in mice. Phosphorylated TrkB
was also detected in extracellular vesicles (EVs) derived from serum of volunteers who had been orally administered placebo or
ERGO-containing tablets. Importantly, the ratio of serum EV-derived phosphorylated TrkB was signiﬁcantly higher in the ERGOtreated group than in the placebo-treated group and was positively correlated with both serum ERGO concentrations and several
cognitive domain scores from Cognitrax. Altogether, TrkB phosphorylation is involved in ERGO-induced cognitive enhancement in
mice, and TrkB phosphorylation levels in serum EVs may quantitatively represent ERGO-induced cognitive enhancement in humans.
npj Science of Food (2024)8:11 ; https://doi.org/10.1038/s41538-024-00250-5

INTRODUCTION
Over 50 million people worldwide have dementia, and thisnumber is rising. However, no satisfactory treatment is currently
available for this disease. Dementia is difﬁcult to treat owing to its
progressive and irreversible pathology in the brain, accompaniedwith mild memory loss in the early stages1,2. Therefore, intervention before the onset of symptoms is desirable2–4. Some clinical
trials have shown that preventive nutritional supplements andphysical exercise lower the incidence of dementia2–4. Particularly,
food-derived compounds that improve cognitive function may be
useful to safely prevent dementia. For example, astaxanthin,epigallocatechin, and resveratrol have prevented cognitive
impairment5–7; however, the detailed underlying mechanisms
remain unclear. Notably, the administration of a naturally
occurring ﬂavone, 7,8-dihydroxyﬂavone, ameliorated cognitive
decline in dementia model mice8,9 possibly by activating thetropomyosin receptor kinase B (TrkB), which is a neurotrophin
receptor and involved in enhancement of neuroplasticity, anessential process for learning and memory10–13.
The food-derived amino acid ergothioneine (ERGO) is notsynthesized in the body but is orally absorbed from the daily dietin mammals. ERGO has a plasma baseline concentration of 1–3 µM
in humans14,15. ERGO levels in the systemic circulation of patientswith dementia are signiﬁcantly lower than those in age-matchedhealthy subjects16,17 and are negatively correlated with neurodegeneration, including hippocampal atrophy18. Therefore, low
ERGO levels may be a risk factor for dementia. Repeated oral
administration of ERGO enhances learning and memory in both
healthy and dementia mice models19,20. Moreover, the daily intake
of food-extract tablets containing ERGO enhances verbal memory
in humans, as demonstrated by the Cognitrax test, which
evaluates cognitive function21. These ﬁndings imply that ERGO

is essential for sustaining memory function, and ERGO supplements may have a beneﬁcial effect on cognitive impairment.
However, the detailed mechanisms of action of ERGO and theeffect of its deﬁciency on cognitive function remain unknown.
Despite its hydrophilicity and low membrane permeability, ERGO
is efﬁciently distributed to the brain following oral administration,
primarily via the carnitine/organic cation transporter (OCTN1/
SLC22A4), which is ubiquitously expressed in organs22–24. In brain
parenchymal cells, OCTN1 is localized in the neurons, neural stem
cells (NSCs), and microglia24–26 but not in astrocytes27. Oraladministration of ERGO increased the number of mature spines
in the hippocampus, and exposure to ERGO promoted synapse
formation in cultured neurons of mice19. ERGO also promoted theneuronal differentiation of NSCs in primary cultures28, implyingthat OCTN1-mediated ERGO uptake may contribute to neural cellhomeostasis. Notably, activation of neurotrophin/TrkB signaling
has been proposed to be involved in these events19,28. Genetic
polymorphisms in TrkB are associated with the onset ofdementia29,30, and TrkB activation is considered a preventive
strategy9. Thus, the enhancement of neurotrophin signaling by
ERGO might promote neurogenesis and/or neuronal maturation,
thereby affecting cognitive function. However, the involvement ofneurotrophin signaling in promoting the beneﬁcial effect of ERGO
on cognitive impairment has not been investigated.
The purpose of the present study was to determine thefundamental role of ERGO in learning and memory by analyzing
the effects of ERGO deﬁciency and supplementation on cognitive
function in mice. Furthermore, we veriﬁed whether TrkB activationand hippocampal neurogenesis are the mechanisms underlying
ERGO-induced cognitive function. It is also necessary to develop adiagnostic marker for the beneﬁcial effect of ERGO to understandthe cognitive enhancement and its mechanism in humans. As

1Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan. 2L·S Corporation Co. Ltd., 3-10-1
Ningyocho-Nihonbashi, Chuo-ku, Tokyo 103-0013, Japan. ✉email: ykato@p.kanazawa-u.ac.jp

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serum extracellular vesicles (EVs) contain brain-derived EVs, we
utilized these to investigate the potential effect of ERGO on TrkB
signaling and memory function in humans22. Phosphorylated TrkB
(p-TrkB), the activated form of TrkB, was monitored in serum EVs
derived from a previous clinical study in which the subjects wereorally administered either ERGO-containing or placebo tablets21.
We compared the p-TrkB levels in serum EVs with both exposure
to ERGO in systemic circulation and ERGO-induced cognitiveenhancement to reveal the possible involvement of TrkB in theenhancement of cognitive function in humans. In the present
study, the ratio of p-TrkB to TrkB in serum EVs was proposed as aquantitative diagnostic marker of long-term ERGO-induced
cognitive improvement.
RESULTSERGO deﬁciency suppressed object recognition andlocation memory
To evaluate the effect of ERGO deﬁciency on learning andmemory, ERGO-deﬁcient mice were prepared by feeding an ERGOfree diet from 3 to 8 weeks of age. ERGO concentration in theplasma of the ERGO-deﬁcient mice was below the quantiﬁcation
limit (<0.03 µM), whereas that in control mice fed with a normal
diet was 1.24 ± 0.42 µM (Supplementary Fig. 1a). The hippocampal
ERGO level in ERGO-deﬁcient mice was 0.23 ± 0.03 nmol/g tissue,
which was approximately nine times lower than that in control
mice (2.00 ± 0.11 nmol/g tissue; Supplementary Fig. 1b). Learningand memory abilities were then assessed using the novel object
recognition test (NORT) and spatial recognition test (SRT) at
8 weeks of age. In NORT, the exploration time for a novel object
was signiﬁcantly higher than that for a familiar object in control
mice, whereas no signiﬁcant difference in exploration time wasobserved between the novel and familiar objects in ERGOdeﬁcient mice (Supplementary Fig. 1c). The discrimination index(DI), an indicator of learning and memory ability, was signiﬁcantly
lower in ERGO-deﬁcient mice than in control mice (Supplementary
Fig. 1d). In the SRT, the exploration time for a moved object wassigniﬁcantly higher than that for an unmoved object in control
mice, whereas no signiﬁcant difference in the exploration time
was observed between the novel and familiar objects (Supplementary Fig. 1e). The DI in ERGO-deﬁcient mice tended to belower than that in control mice (Supplementary Fig. 1f).
To further examine the effects of ERGO on memory function,
ERGO or vehicle alone was orally administered (three times per
week for two weeks) to ERGO-deﬁcient and control mice fromnine weeks old to determine whether supplementation with ERGO
recovered the decline in systemic ERGO concentration andmemory function. ERGO-deﬁcient mice were orally administered
ERGO at 0, 2, and 20 mg/kg for two weeks and subjected to NORTand SRT, followed by the measurement of ERGO concentrations in
the plasma and hippocampus. Oral administration of ERGO
increased ERGO concentration in the plasma and hippocampus
in a dose-dependent manner (Fig. 1a, b). We found that
supplementation with 20 mg/kg of ERGO was sufﬁcient to recover
the hippocampal ERGO levels in ERGO-deﬁcient mice to concentrations similar to that of control mice fed a normal diet (Fig. 1a, b).
In the NORT, the exploration time for a novel object wassigniﬁcantly higher than that for a familiar object in ERGOdeﬁcient mice administered 20 mg/kg ERGO (Fig. 1c). Administration of 2 and 20 mg/kg ERGO signiﬁcantly increased the DI in
ERGO-deﬁcient mice (Fig. 1d). In the SRT, the exploration time for a
moved object was signiﬁcantly higher than that for an unmoved
object in ERGO-deﬁcient mice administered 20 mg/kg ERGO
(Fig. 1e). Administration of 20 mg/kg ERGO signiﬁcantly increased
the DI in ERGO-deﬁcient mice (Fig. 1f). Thus, oral supplementation
with ERGO alone reversed the decline in object recognition and
npj Science of Food (2024) 11

spatial memory in ERGO-deﬁcient mice, suggesting that ERGO
plays an essential role in these memory functions.
Effect of ERGO deﬁciency and supplementation onneurogenesis and TrkB activation in the hippocampus
ERGO promotes synapse formation in neurons19 and neuronaldifferentiation of NSCs in primary cultures28; however, no evidence isavailable on its stimulatory effect on neurogenesis in vivo. Therefore,
immunohistochemical analysis was performed in the hippocampal
dentate gyrus following the ﬁnal administration of ERGO to evaluate
its effect on neurogenesis (Fig. 2a, b). The number of the newborn
neuron marker Dcx-positive (Dcx+) cells in ERGO-deﬁcient miceadministered with vehicle alone appeared to be lower than that incontrol mice administered with vehicle alone, and administration of 2
and 20 mg/kg of ERGO increased the number in ERGO-deﬁcient mice
(Fig. 2a). Quantitative analysis revealed that the percentage of thearea of Dcx+ cells in the neuronal nuclei marker NeuN-positive(NeuN+) cells in ERGO-deﬁcient mice was signiﬁcantly lower thanthat in control mice, and 20 mg/kg ERGO administration signiﬁcantly
increased the percentage, while 2 mg/kg of ERGO tended to increaseit (Fig. 2b). Regarding hippocampal neurogenesis, the expression ofphosphorylated TrkB (p-TrkB), an activated form of TrkB, wassigniﬁcantly lower in the dentate gyrus of ERGO-deﬁcient miceadministered with vehicle alone than in the dentate gyrus of control
mice administered with vehicle alone, and ERGO administration
signiﬁcantly increased p-TrkB expression in a dose-dependent
manner in ERGO-deﬁcient mice (Fig. 2c, d). No difference wasobserved in the expression of total TrkB among all groups (Fig. 2c).
Concomitant increase in ERGO-induced hippocampal
neurogenesis and memory functions
Neurogenesis generally involves the proliferation of NSCs andtheir differentiation into neurons, which is a slow process.
Therefore, to understand the possible relevance of ERGOinduced neurogenesis and memory function, we examined theincrease after the start of ERGO administration at different time
points. The percentage of area occupied by Dcx+ cells from thetotal area occupied by NeuN+ increased only two weeks after thestart of ERGO administration, whereas no increase was observed at
4 or 7 days (Fig. 3a, b). In NORT, ERGO administration signiﬁcantly
increased DI only two weeks after the start of ERGO administration, whereas DI did not change at 4 or 7 days (Fig. 3c, e, g;
Supplementary Fig. 2a, c, e). Similarly, the neurogenic effect of
ERGO on DI in SRT was not observed at 4 or 7 days but wasobserved two weeks after the start of ERGO administration(Fig. 3d, f, h; Supplementary Fig. 2b, d, f). In addition, ERGO
signiﬁcantly increased the expression of p-TrkB normalized bytotal TrkB in hippocampal dentate gyrus only 14 days after thestart of administration (Supplementary Fig. 3). Thus, two weeks oftreatment with ERGO seemed to be necessary to induce
enhancement of phosphorylation of TrkB, hippocampal neurogenesis, and cognitive function.
Administration of TrkB inhibitor suppressed ERGO-induced
enhancement of neurogenesis and memory function
To examine the involvement of TrkB in ERGO-induced enhancement of neurogenesis and memory function, ERGO-deﬁcient mice
were orally administered ERGO with or without a TrkB inhibitor,
ANA-12, for 2 weeks. NORT showed that the exploration time forthe novel object was signiﬁcantly longer than that for the familiar
object in the ERGO-treated group, while no difference between
the novel and familiar objects was observed between the ERGOand ANA-12 treated groups (Fig. 4a). ERGO also increased the DI of
NORT, and ANA-12 suppressed this increase in DI induced by
ERGO treatment (Fig. 4b). SRT showed that the exploration time forthe moved object increased in the ERGO-treated group (Fig. 4c).
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T. Ishimoto et al.

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ERGO-freeFig. 1 Recovery of learning and memory by oral ERGO administration in ERGO-deﬁcient mice. Mice fed an ERGO-free diet were orally
administered ERGO (0, 2, and 20 mg/kg) for two weeks, and ERGO concentration in plasma (a) and hippocampus (b) was measured. Mice fed
the control diet were administered solvent alone. Each value represents the mean ± SD (n = 4–8). The statistical signiﬁcance of the difference
was determined by one-way ANOVA, followed by Tukey’s post hoc test (*P < 0.05). c–f Learning and memory ability was assessed via NORT(c and d) and SRT (e and f) after the ERGO treatment for two weeks. c Open and closed symbols represent exploration time for familiar andnovel objects, respectively. e Open and closed symbols represent exploration time for unmoved and moved objects, respectively. d, f An
indicator of learning and memory ability (DI) was calculated from the exploration time. Each value represents the mean ± SD (n = 3-12).
*P < 0.05; one-way (d and f) or two-way (c and e) ANOVA followed by Tukey’s post hoc test.

ERGO also increased the DI of SRT; however, this increase was notobserved when ANA-12 was co-administered (Fig. 4d). ERGO
increased the ratio of p-TrkB to TrkB (p-TrkB/TrkB) in the dentate
gyrus, while ANA-12 administration suppressed this increase(Fig. 5a, b). Thus, administration of ANA-12 was sufﬁcient toinhibit ERGO-induced TrkB phosphorylation. The effect of TrkB
inhibition on hippocampal neurogenesis was examined using
immunohistochemical analysis. Administration of ERGO increased
the percentage of area occupied by Dcx+ cells from the total area
occupied by NeuN+, whereas the effect of ERGO on neurogenesis
was not observed when ANA-12 was co-administered (Fig. 5c, d).
Oral administration of ERGO-containing tablets increased
serum concentrations of ERGO and the ratio of p-TrkB to TrkB
in serum EVs in humans
In our previous clinical study, 5 mg ERGO-containing tablets
prepared from food-extract or placebo tablets were orally
administered healthy volunteers and subjects with mild cognitive
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impairment (MCI) once per day for 12 weeks, and ERGO
concentrations in blood and Cognitrax memory functions were
examined at weeks 0, 4, 8, and 1221. In the present study, serum
concentrations of ERGO and its putative metabolites, S-methyl
ERGO and hercynine, were measured in the same samples. ERGO
and S-methyl ERGO concentrations at weeks 4, 8, and 12 weresigniﬁcantly higher than those at week 0 in the ERGO-treatedgroup, whereas serum concentrations of S-methyl ERGO andhercynine were much lower than those of ERGO in both the ERGOtreated and placebo groups (Supplementary Fig. 4a–c). BloodERGO concentration at week 12 in the ERGO-treated group(114 ± 54 µg/mL) was signiﬁcantly higher than that in the placebogroup (72.1 ± 42.1 µg/mL). These blood ERGO concentrations were~100 times higher than the serum concentration (Supplementary
Fig. 4).
To determine whether ERGO stimulates TrkB phosphorylation inhumans, EVs were isolated from human serum samples, as they
contain brain-derived EVs31. The average diameter of the EVs
isolated from the control serum was 104 nm (Fig. 6a). The EVs
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4aControl

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ERGO-freeFig. 2 Recovery of hippocampal neurogenesis and TrkB phosphorylation by oral ERGO administration in ERGO-deﬁcient mice. Mice fed
an ERGO-free diet were orally administered ERGO (0, 2, and 20 mg/kg) for two weeks, and immunostaining and western blotting were
performed for the hippocampus. Mice fed the control diet were administered solvent alone. a Images represent the expression of DCX (red)and NeuN (green) in the hippocampal dentate gyrus. Scale bar is 100 μm. b Percentage of the area occupied by Dcx+ cells from the total area
occupied by NeuN+ cells was quantiﬁed using ImageJ. Each value represents the mean ± SD (n = 6-7). *P < 0.05; one way ANOVA followed by
Tukey’s post hoc test. c p-TrkB, TrkB, and β-actin were detected in the hippocampal dentate gyrus via western blotting. Typical samples wereshown. d The protein levels were measured using ImageJ software. Intensity of each band of p-TrkB was normalized by that of TrkB. Each value
represents the mean ± SD (n = 4). *P < 0.05; one way ANOVA followed by Tukey’s post hoc test.

expression of TrkB, NT-5 (a TrkB ligand), or SNAP25 normalized by
the expression of CD63 (TrkB/CD63, NT-5/CD63, or SNAP25/CD63,
respectively) (Fig. 7c–e).

obtained were then immunoprecipitated with antibodies against
SNAP25, a marker for neuron-derived EVs, and p-TrkB and TrkB
were detected in the immunoprecipitated EVs (Fig. 6b). We also
conﬁrmed that TrkB-expressing EVs were secreted from neuronalcells. EVs were isolated from the medium of Neuro2a cells
transfected with TrkB-3xFlag, and both CD63, a marker for EVs,
and TrkB-3xFlag were detected in the EVs of culture medium in
TrkB-3xFlag transfected cells but not in cells transfected with
tdTomato (Fig. 6c). EVs were isolated from all serum samples
obtained from previous clinical studies21, and western blot
analysis detected CD63 and SNAP25 (Fig. 7a), conﬁrming thatbrain-derived EVs were included in the isolated EVs. The p-TrkB/
TrkB ratio in the ERGO-treated group was signiﬁcantly higher thanthat in the placebo group at week 12 (Fig. 7b). Furthermore, in the
ERGO-treated group, p-TrkB/TrkB at weeks 8 and 12 wassigniﬁcantly higher than that at week 0 (Fig. 7b). In contrast, theadministration of ERGO-containing tablets did not affect theCorrelation of p-TrkB/TrkB in serum EVs with systemic ERGO
concentration and cognitive function in humans
Correlations between protein expression in serum EVs (p-TrkB/
TrkB, TrkB/CD63, NT-5/CD63, and SNAP25/CD63) and serum ERGO
concentration were ﬁrst assessed using Pearson’s correlation
analysis (Fig. 8a–d). Serum ERGO concentrations were signiﬁcantly
correlated with the p-TrkB/TrkB ratio (Fig. 8a) but not with TrkB/
CD63, NT-5/CD63, or SNAP25/CD63 (Fig. 8b–d) when all serum EVs
samples from the ERGO-treated and placebo groups were
included. A signiﬁcant correlation was also observed between
the blood ERGO concentration and p-TrkB/TrkB (Supplementary
Fig. 5a) but not between TrkB/CD63, NT-5/CD63, or SNAP25/CD63

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Fig. 3 Time-dependent hippocampal neurogenesis and increase in cognitive function during oral administration of ERGO. Mice fed an
ERGO-free diet were orally administered ERGO (50 mg/kg) or vehicle alone for 4, 7, or 14 days, followed by immunohistochemical analysis andbehavior tests. a Images represent the expression of DCX (red) and NeuN (green) in the hippocampal dentate gyrus. Scale bar is 100 μm.
b Percentage of the area occupied by Dcx+ cells from the total area occupied by NeuN+ was quantiﬁed using ImageJ. Each value represents
the mean ± SD (n = 3–5). *P < 0.05; two-way repeated measures ANOVA followed by Tukey’s post hoc test. Learning and memory ability were
evaluated via NORT (c, e, and g) and SRT (d, f, and h) at day 4 (c and d), 7 (e and f), and 14 (g and h). An indicator of learning and memory
ability (DI) was calculated from the exploration time shown in Supplementary Fig. 2. Each value represents the mean ± SD (n = 3–5). *P < 0.05;
Student’s t test.

(Supplementary Fig. 5b-d). The ΔAUC of serum ERGO, which
represents an increase in systemic exposure to ERGO by oral
administration of ERGO-containing tablets, was also signiﬁcantly
correlated with composite memory, verbal memory, and processing speed in the ERGO-treated group (Table 1), implying thatERGO may be associated with the enhancement of these cognitive
functions.
Correlations between the protein expression in serum EVs andeach domain score in Cognitrax were assessed (Table 1). Amongthe domains of Cognitrax, composite memory, verbal memory,
processing speed, visual memory, cognitive ﬂexibility, excessivefunction, and working memory were positively correlated with pTrkB/TrkB, whereas reaction time and complex attention were
negatively correlated with p-TrkB/TrkB in the ERGO-treated group
Published in partnership with Beijing Technology and Business University(Table 1), suggesting an association between p-TrkB/TrkB andcognitive function. In contrast, NT-5/CD63 and SNAP25/CD63 were
not correlated with any Cognitrax domains (Table 1).
The correlation with serum EV proteins was also examined using
the Mini Mental State Examination (MMSE) score, a test to evaluate
cognitive function, before ERGO intervention. p-TrkB/TrkB was notcorrelated with MMSE score and was almost comparable between
healthy controls (HC) and MCI before ERGO intervention(Supplementary Fig. 6a), suggesting that p-TrkB/TrkB may not
reﬂect cognitive function without intervention. In contrast, bothNT-5/CD63 and SNAP25/CD63 were signiﬁcantly correlated with
the MMSE score and were signiﬁcantly higher in the HC groupthan in the MCI group (Supplementary Fig. 6c, d). This wasconsistent with a previous report that the expression of SNAP25 in
npj Science of Food (2024) 11

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Fig. 4 TrkB inhibitor suppressed ERGO-induced enhancement of memory function. a, d Mice fed an ERGO-free diet were orally
administered ERGO (50 mg/kg) or vehicle alone with or without a TrkB inhibitor ANA-12. Learning and memory ability were assessed via NORT(a and b) and SRT (c and d) after the ERGO treatment for two weeks. a Open and closed symbols represent exploration time for familiar andnovel objects, respectively. c Open and closed symbols represent exploration time for unmoved and moved objects, respectively. b, d An
indicator of learning and memory ability (DI) was calculated from the exploration time. Each value represents the mean ± SD (n = 6–8).
*P < 0.05; one-way (b and d) or two-way (a and c) ANOVA followed by Tukey’s post hoc test.

DISCUSSION
The present study demonstrated that an ERGO-free diet causes
impairments in both hippocampal neurogenesis and cognition,accompanied by decreases in plasma ERGO levels and phosphorylation of TrkB in the hippocampus (Supplementary Fig. 1 and Fig.
2). Moreover, oral supplementation of ERGO alone recovered
these impairments, with the concomitant restoration of ERGO
levels in the plasma and hippocampus in mice (Fig. 1). Thissuggests that ERGO levels in the body may play an essential role insustaining cognitive function, and supplementation with ERGO
would be beneﬁcial as a preventive therapy for reduced cognitive
function, at least in rodents. In humans, blood ERGO levels insubjects with dementia were reported to be lower than those innormal subjects of the same age16,17, whereas ERGO-containing
tablets enhanced cognitive function in healthy participants21.
Moreover, a positive correlation between plasma ERGO levels andhippocampal volume has been reported in patients18. ERGO is notsynthesized in mammals but is ingested from the daily diet, orally
absorbed, and taken up by neuronal cells via its speciﬁctransporter OCTN1/SLC22A422,23, implying its importance in brain
function.
The present study also detected p-TrkB in serum EVs of humans,revealing that the p-TrkB/TrkB ratio in serum EVs in the group
administered ERGO-containing tablets was signiﬁcantly higher
than that in the placebo group (Fig. 7b), and that p-TrkB/TrkB ratio
in the serum EVs was positively correlated with both the systemiclevels of ERGO and cognitive enhancement after the administration ERGO-containing tablets in humans (Table 1). These ﬁndings
indicate that TrkB phosphorylation is associated with ERGOinduced enhancement of cognitive function in humans. The

present study further focused on TrkB phosphorylation as apossible mechanism underlying ERGO-induced enhancement ofcognitive function; ERGO-free diet decreased both TrkB phosphorylation and neurogenesis in the hippocampus, whereas oral
supplementation with ERGO reversed these effects (Fig. 2a–d).
This is consistent with a previous report that ERGO promotes
neuronal differentiation of NSCs in primary culture via S6K1/NT-5/
TrkB signaling28. It took two weeks for ERGO-induced TrkB
phosphorylation, hippocampal neurogenesis, and cognitiveenhancement (Fig. 3 and Supplementary Fig. 3), which may be
due to the time-consuming process of brain distribution of ERGO
and/or neurogenesis. Furthermore, the effects of ERGO were
blocked by administering a TrkB inhibitor (Figs. 4 and 5). In
addition, it has been reported that antioxidant activity of ERGO
may not be involved in neuronal differentiation of NSCs25. Theseﬁndings may suggest a pivotal role of TrkB phosphorylation in
ERGO-induced enhancement of cognitive function and hippocampal neurogenesis. Activation of TrkB is crucial for neuroplasticity, such as neurogenesis and neuronal maturation11,12, which
are essential for cognitive function12,13. Single nucleotide polymorphisms of TrkB were reported to be signiﬁcantly associatedwith the onset of cognitive impairment in AD29,30. Additionally,
TrkB activation ameliorates impairments in memory performance8,9,32,33, neurogenesis, and neuronal maturation in animalmodels of dementia34,35. All these previous reports suggest therole of TrkB as a target molecule for cognitive function
improvement by ERGO.
In humans, the p-TrkB/TrkB ratio in serum EVs was signiﬁcantly
correlated with serum and blood ERGO levels (Fig. 8a andSupplementary Fig. 5a), and both composite and verbal memory
scores were assessed in Cognitrax (Table 1). These results
suggested that the p-TrkB/TrkB ratio in serum EVs quantitatively
represents the cognitive efﬁcacy of ERGO. Although a large
number of biomarker candidates reﬂecting the pathology ofneurodegenerative disorders have been proposed, only a few

npj Science of Food (2024) 11

Published in partnership with Beijing Technology and Business University

circulating EVs discriminates cognitive function among healthy,
mild cognitive impairment (MCI), and Alzheimer’s disease (AD)31,
whereas no report is available for NT-5/CD63.

T. Ishimoto et al.

7

ANA-12

+
-

+

-

+

-

++ ++

+

+
150
(kDa)

p-TrkB

150

TrkB

50

β-actin

*

1.5

*

*

*

*

*

1.0
0.5
0

ERGO

-

+

+

-

+

-

ANA-12

-

-

++

++

+

+

ERGO

ERGO
+ ANA-12

ANA-126*

2.0

Control

Dcx+/NeuN+(Ratio to control)dP-TrkB/TrkB ratio(Ratio to vehicle)

-

ERGO

c

*

b

a

*

*

4
2
0
ERGO
ANA-12

-

+
-

+
+

+

Fig. 5 TrkB inhibitor suppressed ERGO-induced neurogenesis. a, b Western blot analysis was performed for the lysates of the hippocampal
dentate gyrus in mice fed an ERGO-free diet, which were orally administered ERGO (50 mg/kg) or vehicle alone with or without a TrkB inhibitor
ANA-12. a p-TrkB, TrkB, and β-actin in the typical samples are shown. b Those protein levels were measured using ImageJ software. Intensity ofeach band of p-TrkB was normalized by that of TrkB. Each value represents the mean ± SD (n = 3-4). *P < 0.05; one way ANOVA followed by
Tukey’s post hoc test. c, d Immunohistochemical analysis was performed for the hippocampus of mice fed ERGO-free diet, which were orally
administered ERGO or vehicle alone with or without ANA-12. c Images represent expression of DCX (red) and NeuN (green) in the
hippocampal dentate gyrus. Scale bar is 100 μm. d Percentage of area occupied by Dcx+ cells from the total area occupied by NeuN+ wasquantiﬁed using ImageJ. Each value represents the mean ± SD (n = 7–12). *P < 0.05; one way ANOVA followed by Tukey’s post hoc test. −, notreatment; +, 6 times per two weeks on days 0, 2, 4, 7, 9, and 11; ++, daily treatments.

quantitative biomarkers have been established to monitor theeffect of therapeutic agents on cognitive function inhumans31,36,37. For example, the expression of synaptic proteins,
such as SNAP25, GAP43, and neurogranin, in the plasma-derived
EVs of patients with preclinical AD is lower than that in healthy
controls31, and the expression of SNAP25 in serum-derived EVs is
positively correlated with age- and education-corrected MMSE
scores36. These results suggest the positive correlation between
SNAP25/CD63 in serum EVs and MMSE scores in the participants
before the intervention and are consistent with the present study(Supplementary Fig. 6d). We also focused on the neurotrophic
factor NT-5, which is a TrkB ligand activating TrkB, because
exposure of cultured NSCs to ERGO is reported to increase
expression of NT-5, followed by promotion of neuronal differentiation28. Importantly, the NT-5/CD63 ratio in the serum EVs ofpatients with mild cognitive impairment was signiﬁcantly lower
than that in healthy volunteers, and the ratio was positively
correlated with the MMSE score (Supplementary Fig. 6c). Thus, NT5/CD63 in serum EVs might be useful for the diagnosis of mild
cognitive impairment, as in the case of SNAP25.
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In the present study, human serum EVs were immunoprecipitated using an antibody against the synaptic protein SNAP25, andboth p-TrkB and TrkB were detected in the precipitated EVs(Fig. 6b), suggesting that TrkB-expressing EVs are at least partiallyderived from the central nervous system. The expression of TrkB in
the brain is much higher than that in the peripheral tissues of bothrodents and humans38,39. Therefore, it can be speculated that TrkB
detected in human serum EVs may reﬂect TrkB expressed in thecentral nervous system. The p-TrkB/TrkB in mouse serum EVs
would support the speculation, but has not yet been detected in
our preliminary studies probably because of too little volume ofserum samples. On the other hand, the speculation was further
supported by the detection of Flag-tagged TrkB, TrkB, and p-TrkB
in the culture medium of neuronal cells Neuro2a exogenouslytransfected with TrkB-3xFlag (Fig. 6c). However, the physiologicalroles of TrkB-loaded EVs in the circulation are still unknown. EVsderived from brain parenchymal cells may be washed out, at least
partially, by cerebrospinal ﬂuid in the systemic circulation andthereby cleared by peripheral organs. Serum EVs have been
proposed to play physiological roles in the transfer of proteins,
nucleic acids, and nutrients40–42. TrkB in adipose tissue has a
npj Science of Food (2024) 11

T. Ishimoto et al.

8administered ERGO (50 mg/kg) on days 0, 2, 4, 7, 9, and 11, andthe mice were administered intraperitoneally with ANA-12(0.5 mg/kg) or vehicle (1% DMSO) daily or 30 min before eachadministration of ERGO.

Concentration(x108 particles/mL)

a
1.5
1.0
0.5
0
0

50
100
150 200
Particle diameter (nm)

b

c
N
:S
IP

In
pu
t

A

P2

5

pAAV-TrkB-3xFLAG(kDa)

p-TrkB

180
135
100

TrkB

180
135
100

SNAP2525

Flag
p-TrkB
TrkB

250

EVs

-

+

Cells

-

+

(kDa)
135
100
135
100
135
100

CD63
β actin

63
48

Fig. 6 Characterization of EVs isolated from human serum. a The
particle size distribution of serum EVs was analyzed using qNano.
Each value represents the mean ± SD (n = 4). b Serum EVs were
immunoprecipitated using anti-SNAP25 antibody, followed bywestern blotting with antibodies for p-TrkB, TrkB, and SNAP25.
Serum EVs without immunoprecipitation was also subjected towestern blotting and shown as input. c Neuro2A cells weretransfected with pAAV-TrkB-3xFlag (+) or pAAV-tdTomato (−), andboth EVs isolated from the culture medium and cell lysates were
subjected to western blotting with antibodies for ﬂag, p-TrkB, TrkB,
CD63, and β-actin.

substantial inﬂuence on feeding behavior and obesity in female
mice43, and the activation of endothelial TrkB induces therelaxation of resistance arteries44. Further studies are required toinvestigate the potential roles of TrkB-loaded EVs derived from thebrain in peripheral organ functions.
Altogether, oral supplementation with ERGO recovered impairments in hippocampal neurogenesis and cognition in mice fed an
ERGO-free diet, possibly via phosphorylation of TrkB. This suggests
a fundamental role of ERGO in brain function. Additionally, TrkB
phosphorylation was detected in human serum EVs and wasconsidered to be associated with ERGO-induced cognitiveenhancement in humans.

Behavioral tests
NORT and SRT were performed to assess learning and memory
abilities according to the method described by Nakamichi et al.19,
with minor modiﬁcations. Fourteen days after the ﬁrst administration of ERGO, NORT was performed. Each mouse was placed inan acrylic chamber (45 cm ×45 cm ×45 cm) without any objects
and allowed to explore for 10 min. The following day, the mouse
was placed in the same chamber with two identical objects
located on a diagonal line and allowed to explore the chamber for
ﬁve minutes. The time spent exploring each object was thenrecorded. Eight hours later, one of the objects was replaced with anovel object of a different shape at the same location in thechamber. Each mouse was allowed to explore the chamber for ﬁve
minutes. The exploration time for each object was recorded. The
DI, an indicator of learning and memory ability, was calculated as:[(novel object exploration time/total exploration time) − (familiar
object exploration time/total exploration time)] × 100.
The SRT was conducted on the day following NORT. Eachmouse was placed in an acrylic chamber (45 cm ×45 cm ×45 cm)
without any objects and allowed to explore for 10 min. The nextday, the mouse was placed in the same chamber with two
identical objects located on a diagonal line and was allowed to
explore the chamber for nine minutes. The time spent exploringeach object was then recorded. One hour later, one of the objects
was moved, and each mouse was allowed to explore the chamber
for ﬁve minutes. The exploration time for each object wasrecorded. The DI was calculated as: [(moved object explorationtime/total exploration time) − (unmoved object exploration time/
total exploration time)] × 100.
In the experiment evaluating the effect of ANA-12, 8 h ofretention time in the NORT was increased to 24 h, and 9 min of
exploration time in the SRT was reduced to 5 min, without any
change in other experimental conditions.

METHODS
Animals
Three-week-old male ICR mice were purchased from Sankyo Labo
Service Co. (Toyama, Japan). Mice were fed an ERGO-free diet
(Basal Diet 5755, PMI Nutrition International) to prepare ERGOdeﬁcient mice, following a previously reported protocol19,
whereas mice fed a normal diet (PicoLab® Rodent Diet 5053,
PMI Nutrition International) were used as controls. Mice were kept
in a room under pathogen-free conditions at a controlledtemperature (21–25 °C) with a 12 h dark/light cycle and fed foodand water ad libitum. To evaluate the effect of ERGO on cognitive
decline, ERGO-deﬁcient and normal mice at the age of 9 weeks
were orally administered ERGO dissolved in sterile distilled waterat 0, 2, or 20 mg/kg on days 0, 2, 4, 7, 9, and 11, followed bybehavioral testing. To examine the effect of ANA-12, an inhibitorof TrkB, ERGO-deﬁcient mice at the age of 6 weeks were orally

Immunohistochemistry
After the behavioral tests, each mouse was anesthetized with
isoﬂurane and subjected to transcardial perfusion with 5 mL PBS
and 20 mL 4% paraformaldehyde in phosphate buffer. Each brain
sample was isolated and incubated with 4% paraformaldehyde inphosphate buffer at 4 °C for 24 h. After washing with PBS, eachbrain was sectioned with a Neo-LinearSlicer (DOSAKA EM, Kyoto,
Japan) at a thickness of 100 µm. The sections were incubated for
30 min in 0.1% Tween 20 in PBS (PBST) containing 3% bovine
serum albumin (BSA) at room temperature. They were incubatedwith PBST containing goat anti-doublecortin (Dcx) antibody (1:500
dilution; sc-8066, goat, Santa Cruz; Dallas, TX, USA) and mouseanti-NeuN antibody (1:500 dilution; MAB377, mouse, Merck
Millipore; Burlington, MA) overnight at room temperature,
followed by washing with PBST and probing with anti-goat
Alexa555 (1:500 dilution; A21432, donkey, Sigma-Aldrich; St. Louis,MO) and anti-mouse Alexa488 (1:500 dilution; A21202, donkey,
Thermo Fisher Scientiﬁc; Waltham, MA) overnight at room
temperature. The sections were rinsed again with PBS, treatedwith a mounting medium containing 2-(4-aminophenyl)-1Hindole-6-carboxamidine, and observed under an LSM710 confocal
laser-scanning microscope (Carl Zeiss, Jena, Germany). The newborn neuron marker Dcx and the neuronal nuclei marker NeuN
were colored red and green, respectively. Quantiﬁcation was
performed by measuring the immunoreactive area of Dcx or NeuNin double immunocytochemical analysis.

npj Science of Food (2024) 11

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T. Ishimoto et al.

9

Placebo

a

0

4

8

ERGO
12

0

4

8

12 (Weeks)p-TrkB- 135 (kDa)

TrkB

- 135

NT-5

- 25

SNAP25

- 25

CD63

- 48

b
*

*

*

1.5
1.0
0.5
0

0

4
8
Weeks

2
1
0

12

3

SNAP25/CD63e4NT-5/CD63

d
3
2
1
0

0

4
8
Weeks

12

: ERGO

3
TrkB/CD63

p-TrkB/TrkB

2.0: Placebo

c

0

4
8
Weeks

12

0

4
8
Weeks

12

2
1
0

Fig. 7 TrkB phosphorylation in serum EVs of humans after oral administration of ERGO-containing tablets was higher than that in theplacebo group. a P-TrkB, TrkB, CD63, NT-5, and SNAP25 in the serum EVs of all participants were detected using western blotting. Typicalsamples are shown. b–e Protein levels were measured using ImageJ software. The intensity of each band of p-TrkB was normalized by that of
TrkB (b), and the intensity of each band of TrkB (c), NT-5 (d), and SNAP25 (e) was normalized by that of CD63, followed by a comparison
between the placebo (open symbol, n = 23) and ERGO-treated (closed symbol, n = 25) groups. Each value represents the mean ± SD. *P < 0.05;
two-way repeated measures ANOVA followed by Tukey’s post hoc test.

Clinical study
A randomized, placebo-controlled, double-blind, parallel-groupclinical study (UMIN000034386; placebo group, n = 23; grouptreated with ERGO-containing tablets, n = 25), including healthy
volunteers and subjects with MCI, was conducted for 12 weeks, as
previously described (Supplementary Fig. 7)21. Subjects weredivided into two groups based on their age and MMSE scores.
Participants in the ERGO-treated group were orally administered
four food extract tablets containing 5 mg ERGO daily with water,whereas those in the placebo group were administered ERGO-free
tablets in the same manner as the ERGO-treated group. Neither
tablet could be distinguished by its appearance or smell. All
participants were instructed to refrain from eating foods containing ERGO, such as mushrooms, during the study period. Serumcollection and cognitive function tests (Cognitrax) were performed
at weeks 0, 4, 8, and 12.
Measurement of ERGO and its metabolites
Hippocampal samples were weighed and homogenized using a
Precellys homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) in 12 volumes of methanol containing L(+)-ergothioneine-d9 (ERGO-d9; Toronto Research Chemicals
Inc., North York, ON) as an internal standard, whereas plasma
Published in partnership with Beijing Technology and Business University

and serum samples were mixed with nine volumes of methanol
containing ERGO-d9. The mixtures were centrifuged (21,500 × g,
10 min, 4 °C) twice and subjected to liquid chromatography-mass
spectrometry/mass spectrometry (LCMS-8040; Shimadzu, Kyoto,
Japan) equipped with a Luna 3.0 µm HILIC column (200 Å,
150 × 2.0 mm; Phenomenex, Torrance, CA) to quantify ERGO, Smethyl-ERGO, and hercynine. Chromatography was performed
using step-gradient elution (ﬂow rate, 0.4 mL/min) as follows: The
mobile phases were (A) 10 mM ammonium acetate/0.1% formic
acid/80% water/20% acetonitrile and (B) 10 mM ammonium
acetate/0.1% formic acid/5% water/95% acetonitrile. 0–0.5 min:
1% A/ 99% B; 0.5–1.9 min: 1% A/ 99% B to 20% A/ 80% B;
1.9–4.5 min: 20% A/ 80% B; 4.5–5.5 min: 20% A/ 80% B to 60% A/
40% B; 5.5–6.5 min: 60% A/ 40% B; 6.5–6.7 min: 60% A/ 40% B to
1% A/ 99% B; 6.7–7.2 min: 1% A/ 99% B. Each compound wasmeasured in the ESI positive mode (ERGO: 230.3 > 127.0, S-methylERGO: 244.1 > 140.95, hercynine: 198.0 > 95.05, ERGO-d9:
239.2 > 127.0). The area under the serum concentration curve(AUC) of ERGO subtracted from the baseline level of ERGO at week
0 (ΔAUC) was calculated using the following Eq. (1):
ΔAUC ¼

3
X

2ðy n1 þ y n 2y 0 Þ

(1)

n¼1

npj Science of Food (2024) 11

T. Ishimoto et al.

10

Placebo_0w
ERGO_0w

Placebo_4w
ERGO_4waPlacebo_8w
ERGO_8w

b
3

1.5
1.0
r = 0.177*

0.5

TrkB/CD63

p-TrkB/TrkB

2.0

r = -0.006
2
1
0

0.0

0

0
2
4
Serum ERGO (μg/mL)

2
4
Serum ERGO (μg/mL)

d

4

2.5

3
r = 0.0512
1
0SNAP25/CD63

NT-5/CD63

c

Placebo_12w
ERGO_12w

2.0

r = 0.117

1.5
1.0
0.5
0.0

0

2
4
Serum ERGO (μg/mL)0
2
4
Serum ERGO (μg/mL)

Fig. 8 Scatter plots showing the relationship between serum ERGO concentration and ratio of each protein expression level in humanserum EVs. a p-TrkB/TrkB, b TrkB/CD63, c NT-5/CD63, and d SNAP25/CD63. The plots included all data of human serum samples, serum EVs,
and a cognitive domain score of Cognitrax obtained at weeks 0, 4, 8, and 12 after the start of oral administration of ERGO-containing andplacebo tablets (placebo, n = 23; ERGO, n = 25). *P < 0.05; correlations were assessed via Pearson’s correlation coefﬁcient. r, correlation
coefﬁcient.

Nanoparticle analysis
The concentration and size distribution of EVs were measured
with tunable resistive pulse sensing by qNano (Izon Science, UK)
using an NP100 nanopore (Izon Science, UK), according to themethod described by Lane et al.45 with minor modiﬁcations. All
measurements were calibrated with 100 nm polystyrene beads(CPC100; Izon Science, UK). Data analysis was carried out using
Izon Control Suite software v3.1 (Izon Science, UK).

equilibration with PBS, 1 mL of serum was loaded onto thecolumn and eluted with PBS. The eluate was collected into
25 sequential fractions (0.5 mL). The number and size of particles
were determined using qNano for each fraction, and particles
were detected in fractions 6–12. These fractions (3.5 mL) werecombined and concentrated to approximately 200 µL using
Amicon Ultra-15 (Merck Millipore, Burlington, MA, USA). To isolate
brain-derived EVs, an antibody against the mature neuronalmarker SNAP25 (1:25 dilution; sc-390644, mouse, Santa Cruz;
Dallas, TX, USA) was ﬁrst incubated with Protein G Sepharose 4
Fast Flow (Cytiva, Marlborough, MA, USA) in PBS containing 0.01%
Triton-X 100 and phosphatase inhibitors (10 mM sodium ﬂuoride,
10 mM β-glycerophosphate disodium salt hydrate, 10 mM sodium
pyrophosphate decahydrate, and 1 mM sodium orthovanadate) at
4 °C for 2 h, followed by blocking with PBS containing 10% BSA
and phosphatase inhibitors. The beads and EV samples were thenmixed and incubated under rotation at 4 °C for 2 h. The samples
were centrifuged at 15,000 × g for 2 min at 4 °C, and thesupernatant was discarded. The pellets were washed twice with
PBS containing 0.01% Triton-X 100 and phosphatase inhibitors,
followed by western blotting as described below.

Isolation of brain-derived EVs via immunoprecipitation
Serum EVs were isolated via size exclusion chromatography
according to the method described by Böing et al.46, with minor
modiﬁcations. Brieﬂy, the tip of a 10 mL plastic syringe (Terumo,
Tokyo, Japan) was stuffed with nylon stocking (DAISO, Tokyo,
Japan), and the syringe was stacked with 5 mL of washed
Sepharose CL-2B (GE Healthcare, Uppsala, Sweden). After

Western blotting
Western blotting was performed according to the method describedby Ishimoto et al.28, with minor modiﬁcations. Brieﬂy, isolated
hippocampal dentate gyrus and EVs were homogenized using a tip
sonicator (TOMY SEIKO Co. Ltd., Tokyo, Japan) with 20 mM Tris–HClbuffer (pH 7.5) containing 1 mM EDTA, 1 mM EGTA, proteaseinhibitors (0.1 mM 4-(2-aminoethyl) benzenesulfonyl ﬂuoride

npj Science of Food (2024) 11

Published in partnership with Beijing Technology and Business University

where y 0 , y 1 , y 2 , and y 3 represent the serum ERGO concentrations
at weeks 0, 4, 8, and 12, respectively.
Isolation of EVs from serum
One milliliter of serum was ﬁrst centrifuged at 1200 × g for 20 min,and the supernatant was then centrifuged at 17,000 × g for
20 min. The collected supernatant was passed through a 0.22 µm
ﬁlter, followed by ultracentrifugation at 100,000 × g for 90 min(TLS-55 Rotor, Optima MAX-TL, Beckman Coulter, Brea CA). The
pellets were resuspended in 50 µL of phosphate-buffered saline(PBS) and used for each experiment.

T. Ishimoto et al.

11
Table 1. Correlation between each cognitive domain score of Cognitrax and the ratio of each protein expression level in serum EVs or ERGO ΔAUC in
the group treated with ERGO-containing tabletsa.
p-TrkB/TrkB

TrkB/CD63

NT-5/CD63

SNAP25/CD63

ΔAUC for Serum
ERGOb

rc

Pd

rc

Pd

rc

Pd

rc

Pd

rc

Pd

Composite memory

0.286

<0.01

−0.054

0.591

0.055

0.590

−0.023

0.823

0.244

<0.05

Verbal memorye

0.215

<0.05

−0.032

0.752

0.133

0.186

−0.015

0.884

0.240

<0.05

Processing speede

0.204

<0.05

0.003

0.979

0.023

0.823

−0.045

0.660

0.240

<0.05

Visual memorye
Psychomotor speede

0.235
0.113

<0.05
0.264

−0.053
0.073

0.599
0.469

−0.040−0.067

0.695
0.509

−0.021−0.080

0.838
0.428

0.187
0.105

0.108
0.371
0.183

e

Reaction timee−0.284<0.01−0.120

0.234

0.057

0.572

−0.112

0.266

0.156

Complex attentione−0.265

<0.01

0.117

0.246

0.011

0.914

0.111

0.272

−0.042

0.721

Cognitive ﬂexibilitye

0.332

<0.01

−0.0870.388−0.0030.980−0.054

0.596

0.064

0.586

Executive functione

0.335

<0.01

−0.0860.397−0.0030.975−0.051

0.616

0.060

0.607

Working memorye

0.256

<0.05

−0.026

0.797

0.011

0.914

−0.013

0.897

0.167

0.152

Sustained attentione

0.176

0.080

−0.0660.516−0.1180.243−0.115

0.255

0.098

0.403

Simple attentione
Motor speede

0.118−0.006

0.243
0.951

−0.084
0.107

0.406
0.287

0.007
−0.099

0.945
0.330

−0.095−0.063

0.349
0.533

−0.043−0.046

0.713
0.694

a

Correlations were assessed using Pearson’s correlation coefﬁcient.
The area under the serum concentration curve (AUC) for ERGO subtracted from that of the baseline obtained at week 0.
cCorrelation coefﬁcient.
dStatistics p-values.
eCognitive domain scores of Cognitrax. The higher score of all Cognitrax domains, except reaction time and complex attention, indicated a higher score foreach function.
bhydrochloride, 1 μg/mL leupeptin, 1 μg/mL antipain, and 0.5 mM
benzamidine hydrochloride hydrate), and phosphatase inhibitors(10 mM sodium ﬂuoride, 10 mM β-glycerophosphate disodium salt
hydrate, 10 mM sodium pyrophosphate decahydrate, and 1 mM
sodium orthovanadate), followed by protein concentration measurements with a PierceTM BCA Protein Assay Kit (ThermoFisher Scientiﬁc,Waltham, MA). The homogenates were added at a volume ratio of
1:4 to 10 mM Tris–HCl buffer (pH 6.8) containing 10% glycerol, 2%
sodium dodecyl sulfate, 0.01% bromophenol blue, and 5% 2mercaptoethanol, and the mixtures were incubated on a shaker atroom temperature for 1 h, followed by incubation at 95 °C for 5 min.
Each aliquot of 10 µg of protein from the hippocampal dentate gyrus
was loaded onto a 10% polyacrylamide gel for electrophoresis at aconstant current of 40 mA/plate for 90 min at room temperatureusing a PAGE system (Sima Biotech, Chiba, Japan). For EV samples,
each aliquot of 20 µg of protein was loaded on a 12.5%
polyacrylamide gel for electrophoresis at a constant current of
21 mA/plate for 30 min at room temperature using a compact-slab
size PAGE system (ATTO, Tokyo, Japan), followed by blotting to a
polyvinylidene ﬂuoride membrane pretreated with 100% methanol.
For blocking, BSA or skim milk dissolved in 20 mM Tris–HCl buffer(pH 7.5) containing 137 mM NaCl and 0.1% Tween 20 were used
depending on the antibodies (2% BSA: p-TrkB, TrkB, NT-5, Flag, andβ-actin; 1% skim milk: SNAP25; 3% skim milk: CD63). The membranes
were probed with antibodies against TrkB (1:1,000 dilution; 4603 S,
rabbit, Cell Signaling Technology; Danvers, MA), p-TrkB at Tyr816(1:100 dilution; ABN1381, rabbit, Merck Millipore; Burlington, MA),
NT-5 (1:250 dilution; AB1781SP, rabbit, Merck Millipore; Burlington,MA), SNAP25 (1:50 dilution; sc-390644, mouse, Santa Cruz; Dallas, TX),
CD63 (1:100 dilution; sc-15363, rabbit, Santa Cruz; Dallas, TX), Flag
(1:1,000 dilution; 14793S, rabbit, Cell Signaling Technology; Danvers,
MA), and β-actin (1:10,000 dilution; A5441-.2 ML, mouse, SigmaAldrich; St. Louis, MO) diluted with Can Get Signal Solution 1(TOYOBO, Osaka, Japan), followed by probing with a secondary antirabbit IgG antibody conjugated with peroxidase (1:2,000 dilution;
7074, goat, Cell Signaling Technology; Danvers, MA) or anti-mouse
IgG antibody conjugated with peroxidase (1:10,000 dilution; A9917Published in partnership with Beijing Technology and Business University

1ML, goat, Sigma-Aldrich; St. Louis, MO) diluted with Can Get Signal
Solution 2. Proteins that reacted with these antibodies were detectedwith ECL™ detection reagents using a lumino image analyzer (LAS4000; FUJIFILM, Tokyo, Japan). Densitometric analysis of the western
blots was performed using ImageJ software.
Plasmid construction
pAAV-CMV-ZsGreen1 was purchased from Takara Bio. TdTomato wasampliﬁed from the pCSCMV:tdTomato plasmid (Addgene plasmid#30530; http://n2t.net/addgene:30530; RRID: Addgene_30530)47. Murine TrkB-3xFlag was ampliﬁed from the murine hippocampus using
the DNA polymerase KOD FX NEO with the following sense andantisense primers: 5’-tgtaatcgatgtcatgatctttataatcaccgtcatggtctttgtagtccatCGCGCCTAGGATATCCAG-3’, 5’-tcacagggatgccacccgtggatccTCACTTGTCATCGTCATCCTTGTAATCGATGTCATGATCTTTATA-3’.
The
sequence encoding tdTomato in pAAV-CMV-tdTomato was replacedwith TrkB-3xFlag using a NEBuilder HiFi DNA assembly kit (NewEngland Biolabs).
Cell culture
Neuro2a cells were cultured according to the method describedby Nakamichi et al.24, with minor modiﬁcations. Brieﬂy, Neuro2acells were seeded at a density of 1.2 × 106 cells/dish on φ10 cm
dishes in DMEM supplemented with 100 units/mL penicillin,
100 µg/mL streptomycin, and 10% EV-depleted FBS, which was
the supernatant after ultracentrifugation at 100,000 × g for 16 h at
4 °C. After 24 h, the cells were transiently transfected with pAAVCMV-TrkB-3xFlag or pAAV-CMV-tdTomato using PEI MAX (Polysciences, Warrington, PA, USA), according to the manufacturer’s
instructions, and the medium was changed to DMEM supplemented with 2% EV-depleted FBS and 20 µM retinoic acid forneuronal differentiation. At 48 h after seeding, the medium waschanged to DMEM containing 100 nM of the TrkB agonist 7,8dihydroxyﬂavone or vehicle. At 72 h after seeding, the cells andmedium were collected. The cultures were maintained in ahumidiﬁed atmosphere of 5% CO2/ 95% air at 37 °C.
npj Science of Food (2024) 11

T. Ishimoto et al.

12
Statistics
Data were expressed as means ± standard deviation (SD). The
statistical signiﬁcance of the differences was determined using theStudent’s t test or one-way or repeated measures ANOVA, followed
by Tukey’s post hoc test. Correlations were determined using
Pearson’s correlation coefﬁcient after the Kolmogorov–Smirnov
test. Statistical analyses were performed using Prism 7 (GraphPad
Software, San Diego, CA, USA) and IBM-SPSS version 25.

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npj Science of Food (2024) 11

Published in partnership with Beijing Technology and Business University

Study approval
Animal studies were approved by the Committee on the Ethics ofAnimal Experiments of the University of Kanazawa (permit
number: AP-183968) to minimize animal suffering and loss of life.
The clinical studies were conducted following the Declaration of
Helsinki and approved by the Institutional Review Boards ofKanazawa University and the Japan Food Evidence Association. All
participants provided written informed consent for the use of theirsamples and medical information for research purposes.
Reporting summary
Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
DATA AVAILABILITY
The data supporting the ﬁndings reported herein are available on request from thecorresponding author.

Received: 24 February 2023; Accepted: 18 January 2024;

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COMPETING INTERESTS
The authors declare no competing interests.

ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41538-024-00250-5.
Correspondence and requests for materials should be addressed to Yukio Kato.
Reprints and permission information is available at http://www.nature.com/
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AUTHOR CONTRIBUTIONSOpen Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
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material in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted by statutory
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creativecommons.org/licenses/by/4.0/.

IT, YR, and KY designed this study. IT, YR, MR, MY, and NS conducted the experiments
and acquired data. IT, YR, MR, MY, NS, MY, and KY contributed to data analysis. IT, YR,
and KY wrote the manuscript, and all authors reviewed and approved the manuscript.

© The Author(s) 2024

ACKNOWLEDGEMENTS
We thank Naotaka Hashiya for conducting the clinical study, and Taisuke Fukaya for
managing the clinical study. This work was partially supported by Grants-in-Aid for
Scientiﬁc Research to TI (Nos. 19K23797 and 20K15991) and YK (Nos. 22H02781 and
18H02584) from the Ministry of Education, Culture, Sports, Science, and Technologyof Japan.

Published in partnership with Beijing Technology and Business University

npj Science of Food (2024) 11