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

Dementia affects millions globally, and effective treatments remain elusive. Preventive strategies, including nutritional supplements, show promise. Ergothioneine (ERGO), a food-derived antioxidant, has demonstrated cognitive-enhancing effects in both animal models and human studies. However, the precise mechanisms underlying ERGO's action and the impact of ERGO deficiency on cognitive function remain poorly understood. Previous research suggests that ERGO's effects may involve the activation of tropomyosin receptor kinase B (TrkB), a neurotrophin receptor critical for neuroplasticity and cognitive function. This study aimed to investigate the role of ERGO in learning and memory by examining the effects of ERGO deficiency and supplementation in mice and to determine if TrkB activation is a key mechanism underlying ERGO-induced cognitive enhancement. Furthermore, the study explored the potential of using serum extracellular vesicles (EVs) as a biomarker to reflect the effects of ERGO on TrkB signaling in humans. Despite ERGO's hydrophilicity and low membrane permeability, it efficiently reaches the brain via OCTN1/SLC22A4 transporter. Previous studies have shown ERGO to increase hippocampal spine numbers, promoting synapse formation in neurons and NSC differentiation, potentially through neurotrophin/TrkB signaling. Given the link between TrkB genetic polymorphisms and dementia, and TrkB activation's role in preventing dementia, this study aimed to test the hypothesis that ERGO's beneficial effects on cognition are mediated, at least in part, through TrkB activation. The identification of a reliable biomarker to quantify ERGO's cognitive-enhancing effects in humans is also crucial for clinical translation.

Numerous studies have explored the potential of various food-derived compounds, including astaxanthin, epigallocatechin, and resveratrol, to prevent or alleviate cognitive impairment. However, the underlying mechanisms often remain unclear. 7,8-dihydroxyflavone, a naturally occurring flavone, has shown promise in ameliorating cognitive decline by activating TrkB, highlighting the importance of TrkB signaling in cognitive function. Previous research has indicated a correlation between lower ERGO levels and increased risk of dementia, along with a negative correlation between ERGO levels and neurodegeneration. Supplementation with ERGO has been shown to improve memory in both healthy and dementia mouse models and to enhance verbal memory in humans, supporting the potential of ERGO as a cognitive enhancer. However, more research is needed to elucidate the specific mechanisms underlying these effects and to identify suitable biomarkers for clinical use.

The study used a two-pronged approach involving both in vivo experiments using mice and in vitro analysis of human serum samples. For in vivo studies, mice were fed an ERGO-free diet to induce ERGO deficiency. Their cognitive function was assessed using the novel object recognition test (NORT) and spatial recognition test (SRT). ERGO was then administered to ERGO-deficient mice, and the effects on cognition, hippocampal neurogenesis (using immunohistochemical analysis of DCX+ and NeuN+ cells), and TrkB phosphorylation (via Western blotting) were measured. To examine the role of TrkB, a TrkB inhibitor (ANA-12) was co-administered with ERGO. Time-course experiments were also performed to investigate the timeline of ERGO's effects. For human studies, serum samples from a previous clinical trial where participants received either ERGO-containing tablets or placebo were analyzed. Extracellular vesicles (EVs) were isolated from serum using size-exclusion chromatography. Brain-derived EVs were isolated via immunoprecipitation with antibodies against SNAP25. Western blotting was used to quantify p-TrkB, TrkB, NT-5, and SNAP25 levels in the EVs. Serum ERGO and its metabolites (S-methyl ERGO and hercynine) were also quantified using LC-MS/MS. Finally, correlations between p-TrkB/TrkB ratios in serum EVs, serum ERGO concentrations, and cognitive domain scores from the Cognitrax test were analyzed using Pearson's correlation coefficient. Statistical analyses were performed using appropriate tests such as Student's t-test, one-way or repeated measures ANOVA, and Tukey's post hoc test. The study employed a variety of techniques, including behavioral tests (NORT and SRT), immunohistochemistry (for neurogenesis analysis), Western blotting (for protein quantification), LC-MS/MS (for metabolite analysis), and nanoparticle analysis (for EV characterization), ensuring comprehensive assessment of ERGO's impact on cognition and TrkB signaling.

In mice fed an ERGO-free diet, object recognition and location memory were significantly impaired, associated with lower hippocampal ERGO levels and reduced TrkB phosphorylation. ERGO supplementation reversed these effects in a dose-dependent manner, restoring ERGO levels and improving cognitive function. Importantly, the ERGO-induced improvement in neurogenesis and memory was blocked by the TrkB inhibitor ANA-12, confirming the crucial role of TrkB in ERGO's mechanism of action. The positive correlation between TrkB phosphorylation and neurogenesis was observed only after 2 weeks of ERGO administration. In humans, oral administration of ERGO-containing tablets significantly increased serum ERGO levels and the p-TrkB/TrkB ratio in serum EVs, especially at weeks 8 and 12. This increase in p-TrkB/TrkB in serum EVs showed a strong positive correlation with both serum ERGO concentrations and several cognitive domain scores from Cognitrax (including composite memory, verbal memory, and processing speed). In contrast, other EV proteins like TrkB/CD63, NT-5/CD63, and SNAP25/CD63 showed no significant correlations with serum ERGO levels or Cognitrax scores. However, the NT-5/CD63 ratio showed a positive correlation with the MMSE score before ERGO intervention, suggesting potential in diagnosis of mild cognitive impairment. Furthermore, the analysis of EVs showed that they contain at least some brain-derived EVs, as evidenced by the detection of SNAP25 (a neuronal marker). Finally, it was shown that TrkB-expressing EVs can be secreted by neuronal cells, as confirmed in vitro using Neuro2a cells transfected with TrkB.

This study demonstrates a critical role for ERGO in maintaining cognitive function and hippocampal neurogenesis, highlighting the importance of adequate ERGO intake. The findings strongly support a mechanism where ERGO enhances cognitive function, at least partly, through TrkB activation, as evidenced by the reversal of cognitive deficits and neurogenesis impairments in mice upon ERGO supplementation, and the blocking of these effects by a TrkB inhibitor. The positive correlation between serum EV-derived p-TrkB/TrkB and both systemic ERGO levels and cognitive performance in humans further solidifies the importance of TrkB signaling in ERGO's action. The identification of p-TrkB/TrkB in serum EVs as a potential biomarker holds promise for non-invasive monitoring of ERGO's efficacy in enhancing cognitive function. Although the specific mechanism by which ERGO acts on TrkB remains to be fully clarified, this research suggests that ERGO may affect the expression of NT-5 (a TrkB ligand), potentially leading to TrkB activation. Furthermore, the role of EVs as carriers of TrkB and their involvement in the systemic effects of ERGO warrant further investigation. This comprehensive study provides valuable insights into the biological mechanisms and clinical applications of ERGO in improving cognitive function.

This study demonstrates that ERGO deficiency leads to impaired cognitive function and hippocampal neurogenesis in mice, which can be reversed by ERGO supplementation, mediated through TrkB phosphorylation. In humans, the p-TrkB/TrkB ratio in serum EVs correlates with serum ERGO levels and cognitive enhancement. This suggests that p-TrkB/TrkB may serve as a quantitative biomarker for assessing ERGO's cognitive effects. Future research should focus on exploring the detailed mechanisms underlying ERGO's action on TrkB and investigating the potential therapeutic applications of ERGO for preventing or treating cognitive decline. Further studies could also explore the precise role of EVs in mediating the effects of ERGO.

The study primarily utilized a mouse model, which may not fully recapitulate the complex pathophysiology of human cognitive decline. While the human study used samples from a previous clinical trial, a larger, specifically designed clinical study with longer follow-up is needed to confirm the findings and establish the p-TrkB/TrkB ratio as a robust biomarker. The precise mechanism by which ERGO activates TrkB signaling requires further investigation. Additionally, while the results suggest brain-derived EVs, definitive proof of central nervous system origin for all EVs analyzed remains to be provided.