Background: The enhancement of learning and memory through food-derived ingredients is of great interest to healthy individuals as well as those with diseases. Ergothioneine (ERGO) is a hydrophilic antioxidant highly contained in edible golden oyster mushrooms (Pleurotus cornucopiae var. citrinopileatus), and systemically absorbed by its specific transporter, carnitine/organic cation transporter OCTN1/SLC22A4. Objective: This study aims to examine the possible enhancement of object recognition memory by oral administration of ERGO in normal mice. Method: Novel object recognition test, spatial recognition test, LC-MS/MS, Golgi staining, neuronal culture, western blotting, immunocytochemistry, and quantitative RT-PCR were utilized. Result: After oral administration of ERGO (at a dose of 1–50 mg/kg) three times per week for two weeks in ICR mice, the novel object recognition test revealed a longer exploration time for the novel object than for the familiar object. Oral administration of ERGO also revealed a longer exploration time for the moved object in the spatial recognition test in mice fed ERGO-free diet. The discrimination index was significantly higher in the ERGO-treated group than the control in both behavioral tests. ERGO administration led to an increase in its concentration in the plasma and hippocampus. The systemic concentration reached was relevant to those found in humans after oral ERGO administration. Golgi staining revealed that ERGO administration increased the number of matured spines in the hippocampus. Exposure of cultured hippocampal neurons to ERGO elevated the expression of the synapse formation marker, synapsin I. This elevation of synapsin I was inhibited by the tropomyosin receptor kinase inhibitor, K252a. Treatment with ERGO also increased the expression of neurotrophin-3 and -5, and phosphorylated mammalian target of rapamycin in hippocampal neurons. Conclusion: Oral intake of ERGO which provides its plasma concentration achievable in humans may enhance object recognition memory, and this enhancement effect could occur, at least in part, through the promotion of neuronal maturation in the hippocampus.

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Hydrophilic antioxidant ergothioneine (ERGO) is not synthesized in mammals, but ingested from daily life in humans. Oral administration of ERGO exhibits several beneficial effects in the brain in experimental animals. ERGO promotes neuronal differentiation of neural progenitor cells in primary culture, and involvement of the activation of mTOR pathway has been proposed (Cell Signal 53, 269, 2018), although its directly interacting proteins have not yet been clarified. The aim of the present study is to perform comprehensive study to clarify the pathways involved in the pharmacological activity of ERGO. After repeated oral dose of ERGO or vehicle alone in normal mice, hippocampal dentate which is important in neurogenesis was isolated, and membrane proteome analysis using LC-MS/MS was conducted. Accordingly, 3,337 proteins were identified, and we found change in expression of proteins associated with mitochondria and synapse formation. Mouse neural stem cells were primarily cultured for 6 days, followed by incubation with ERGO. Gene product of neuronal marker b-III tubulin was increased by ERGO, confirming neuronal differentiation. Effect of ERGO on mitochondrial proteins is now under investigation.

SLC22A4, also known as carnitine/organic cation transporter OCTN1 is ubiquitously expressed in the body. OCTN1 is functionally expressed in neurons, microglia, and neural stem cells in the brain, and may play a protective role in pathophysiological conditions via its in vivo substrate ergothioneine, a food-derived antioxidant. On the other hand, we have recently noticed that pentylenetetrazole-induced seizure was limitedly observed in octn1 gene knockout (octn1 -/-) mice, and this may not be explained by the absence of this antioxidant. To find new OCTN1 substrates in the brain, we here conducted untargeted metabolome analysis using LC-TOF-MS. Hippocampus, cerebral cortex, and plasma of wild-type and octn1 -/mice were subjected to metabolomics, and 2,599, 2,676, and 1,697 ion peaks, respectively, were observed. Among them, five ion peaks with m/z 455, 426, 158, 154, and 144 exhibited at least two times difference between the two strains, only m/z 158 being found to be commonly lower in octn1 -/in all the samples. Time-dependent and saturable transport of chemically synthesized compound corresponding to this peak was observed in HEK293/OCTN1 cells. In addition, systemic elimination of this compound in octn1 -/mice was more rapid compared with wild-type. Thus, we have newly identified OCTN1 substrate in the brain. YIA