The capacity of neural stem cells to proliferate and differentiate into neuronal cells in the brain contributes to prevention and recovery from memory loss and other neurodegenerative diseases [1
]. In rodents, neurogenesis has been experimentally demonstrated in two specific regions of the brain: the sub-ventricular zone (SVZ) and dentate gyrus of the hippocampus [2
]. The dentate gyrus is a vital part of the hippocampus with the ability to self-renew in young rodents, although whether this occurs in adults is unclear. Neurogenesis in the dentate gyrus region plays a significant role in hippocampus-dependent learning and memory [4
], and depletion of newly regenerated cells in this region leads to brain damage [6
]. Promotion of neurogenesis in the hippocampal dentate gyrus region by enhancing the self-regenerative ability of neuronal stem cells offers potential for maintaining and improving cognitive function [7
(L.) Wettst. (BM) is a medicinal plant traditionally used as a neural tonic to improve impaired cognitive function and promote longevity [8
]. Recent evidence demonstrates that BM extracts (BME) possess neuroprotective effects against brain dysfunction, such as epilepsy, cognitive deficits in animal models and cerebral ischemia [8
]. In previous studies, we reported that BME made with alcohols, containing 21.8% bacoside A and 11.0% bacopaside I, attenuated transient cerebral ischemia-induced cognitive deficits. Our studies suggested that bacopaside I, a major triterpenoid component of BME, has a significant role in the effect of BME, since it protected neuronal cells from oxygen- and glucose-deprivation-induced damage via protein kinase C (PKC) and phosphatidylinositol-3 kinase (PI3K)/Akt mechanisms in organotypic hippocampal slice cultures [8
]. In addition, we recently reported that BME ameliorates trimethyltin (TMT)-induced cognition dysfunction by protecting hippocampal neurons from TMT-induced lesions and partly by promoting neurogeneration in the hippocampal dentate gyrus region [15
]. These results prompted us to hypothesize that BME may have the potential to improve cognitive function in healthy animals by promoting neurogenesis in the hippocampus. To test this hypothesis, we have examined whether systemic administration of BME improves cognitive function in healthy animals of different ages (adolescent and adult), as the number, density, and proportion of neuronal and non-neuronal cells dramatically changes between adolescent and adult rodents [16
]. We measured changes in gene expression and replication in the hippocampus, and also evaluated the effects of bacopaside I on neurogenesis, using an in-vitro neural stem cell culture system. The results demonstrate that BME enhancement of cognitive performance is accompanied by promotion of neurogenesis in the hippocampal dentate gyrus of adolescent mice, but not adult mice.
The present study investigated the effects of daily BME treatment (50 mg/kg/day) on spatial working memory performance of adolescent 5-week-old and adult 8-week-old mice. The results demonstrated that the administration of BME daily to adolescent mice enhanced spatial cognitive function and correlated with the promotion of endogenous neurogenesis in the hippocampal dentate gyrus region. Furthermore, our in-vitro experiments using an NPC culture system suggested that bacopaside I, a major triterpenoid constituent of BME, contributes to the effect of BME on the hippocampal neurogenesis by enhancing proliferation of NPCs.
In this study, we employed a modified version of the Y-maze test to elucidate the spatial working memory of subject mice because this paradigm can be repeated with the same animal groups, when separated by intervals of no less than one week [10
]. When 5-week-old mice were treated daily with BME for 7–28 days from day 0, significant enhancement of the spatial cognitive function in the modified Y-maze test occurred on day 7 but not on day 28. Notably, these results are consistent with clinical studies showing that supplementation with a combination of BME and multiple micronutrients to school children aged 7–12 years for 60 days but not 121 days significantly improved spatial working memory [19
]. Furthermore, the treatment of neonatal rats with BME caused improvement in spatial memory performance and memory retention at 2–6 weeks after starting the administration [20
]. Mice at the age of 5 and 8 weeks are adolescent and adult, respectively [21
], and development of cognitive and physical function during adolescence is susceptible to training, drugs, nutrition exposure, or other environmental factors [25
]. Together, the present results allow us to hypothesize that the enhancing effect of BME on spatial cognitive function depends on the age of mice used and becomes evident only when the animals are treated during adolescence.
This hypothesis is supported by the results obtained with three different experimental protocols. As shown in Figure 3
, the 5-week-old adolescent mice treated with BME daily for 7 days showed enhanced cognitive performance compared to the same animal group treated with vehicle water. In contrast, the 8-week old adult mice did not responde to the BME treatment and showed no difference in cognitive performance from the vehicle-treated adult animals. Moreover, it is of interest to note that the enhancement of working memory performance by BME administration during adolescence was maintained even 3 weeks after terminating the BME treatment. These results suggest that repeated treatment with BME during adolescence may be essential for long-lasting enhancement of cognitive function in mice.
One may infer that the failure to improve cognitive performance in mice treated with BME for 4 weeks was due to the BME-induced toxicity. However, this possibility is unlikely because our preliminary study showed that BME at doses up to 1.77 g/kg failed to induce any acute toxic symptoms in mice. Moreover, in agreement with other reports [26
], daily treatment of rats with BME at a dose equivalent to the pharmacological effects in mice (50 mg/kg) for 30 days induced no mortality or adverse effects on the biological and morphological indices, including body weight, relative organ weights, hematological, and biochemical parameters, histopathology of liver and kidneys.
To have a better understanding of the mechanism by which the administration of BME during adolescence enhanced the spatial cognitive function, we conducted an immunohistochemical analysis of neurogenesis in the hippocampus. The results revealed that the treatment of adolescent mice with BME daily for 7 days significantly enhanced cell proliferation in the dentate gyrus region without affecting cell death. Besides, a significant increase in the hippocampal neurogenesis persisted even 3 weeks after terminating the BME administration. Evidence indicates that promotion and suppression of hippocampal neurogenesis are associated with improvement and impairment in learning and memory performances, respectively [6
]. Indeed, our previous study using a trimethyltin (TMT)-induced neurotoxicity model of mice demonstrated that the daily administration of BME improved cognitive deficits caused and that the effects of BME were due to not only protection of hippocampal cell from TMT toxicity but also facilitation of hippocampal neurogenesis [15
]. Given these findings, the present results suggest that the daily administration of BME during adolescence is essential to elicit long-term promotion of a neurogenesis process in the dentate gyrus. This enhancement of cognitive function in adolescent animals lasts even after the animals reached adulthood.
The RNAseq analysis demonstrated several noteworthy features of treatment with BME. Those are, first, a significant decrease in the expression of gene coding PP2A, a modulator of the PI3K/Akt pathway that is involved in neuronal signaling and neurodevelopment [27
], and second, changes in the expression of genes coding Wnt, neurogenin, Igf, VEGF, ChAT, and CREB, that are involved in hippocampal neurogenesis and cognitive function in rodents [28
]. For example, the activation of Wnt signaling pathway reportedly enhances the cognitive function of adult mice and rescues memory deficit in APP/PS1-transgenic mice, an Alzheimer’s disease model [35
], whereas blockade of Wnt signaling reduces hippocampal neurogenesis [28
]. Moreover, our previous study demonstrated possible involvement of ChAT and CREB in the mechanism by which BME treatment enhanced cognitive function in OBX mice [10
]. Taken together, the data obtained by the RNAseq analysis provide mechanistic evidence for BME treatment-induced enhancement of cognitive function and neurogenesis in the hippocampus.
Finally, we conducted in vitro experiments using NPCs to elucidate the role of bacopaside I in BME-induced promotion of neurogenesis in adolescent animals. We employed bacopaside I, a major triterpenoid component of BME, because our previous study using a brain ischemia model suggested that bacopaside I (25 μM) has a major role in the neuroprotective effects of BME in this animal model via PKC and PI3K/Akt pathways [8
]. Bacopaside I is also reported to enhance the BDNF signaling pathway in a mouse model of depression, at least in part via activation of pERK/pCREB/BDNF signaling [36
] involved in the neurotrophic activity and neurogenesis [37
]. In agreement with these reports, we found that bacopaside I at 20 μM can promote the proliferation of NPCs probably via ERK1/2 and Akt mechanisms. ERK1/2, a subclass of mitogen-activated protein kinases (MAPKs), are involved in the growth, proliferation, and survival of various types of cells, including NPCs [39
]. The present results showed that bacopaside I dose-dependently enhanced the phosphorylation of ERK1 but not the phosphorylation of ERK2.
It is also of interest that bacopaside I, at 20 μM, induced a significant increase in the expression levels of Akt and p-Akt in NPCs. This finding is supported by the RNAseq analysis, in which we demonstrated that BME enhanced the expression of Akt3 mRNA in the hippocampus. Akt3 plays an important role in cell proliferation [41
]. In addition, the extent of the increase in phospho-Akt was more than the extent of the increase in non-phosphorylated Akt in the bacopaside I-treated NPCs, suggesting that bacopaside I enhances not only translation of Akt protein from its mRNA but also the PI3K/Akt signaling pathway in NPCs. Several lines of evidence indicate that the PI3K/Akt pathway controls the proliferation, differentiation, and migration of endogenous neural stem cells, and plays a crucial role in neurogenesis [42
]. In conclusion, the present study demonstrates that BME enhances working memory performance in adolescent mice by promoting hippocampal neurogenesis and that the effects of BME are due in significant amounts, to bacopaside I.