Stimulatory effects of royal jelly on the generation of neuronal and glial cells from cultured neural stem cells
Expectation of protection against certain neurological disorders
Shoei Furukawa, Ph.D.
Labolatory of Molecular Biology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University
Mitahora-higashi 5-6-1, Gifu 502-8585, Japan
Tel, Fax: +81-58-237-8589, furukawa@gifu-pu.ac.jp
1. Introduction
Recently, the number of senior citizens has been increasing all over the world, and there has been a concomitant increase in the number of patients suffering from Alzheimer's or Parkinson's disease. Alzheimer's disease suffer currently affects 600,000 people in Japan, and the prevalence rate is 4-6 % of those 65 years old or older and 15 % of those 80 years old or above. Moreover, the number of patients with Parkinson's disease has shown a tendency to increase in recent years. The etiology of these diseases is still uncertain, and there is no fundamental treatment for therapy. On the other hand, there is also an increase in the number of patients suffering from depression due to various stresses of the lingering economic depression, too much competition in contemporary society, etc. The number of patients displaying mood disorder including depression increased from 440,000 in 1999 to 710,000 in 2002, and to 870,000 in 2005 according to statistical data from the Ministry of Health, Labour, and Welfare of Japan. This situation has caused a very serious social problem, because over 30,000 Japanese have committed suicide each year over a 9-year period until 2006. Needless to say, not only political effort but also the establishment of fundamental prevention against and therapy for brain-related diseases is indispensable; because about 70% of those who commit suicide are thought to be the patients with depression. Although major disorders of the brain such as Alzheimer's disease, Parkinson's disease, and depression are caused by different etiology, they share commonly the failure of a part of higher brain functions including sleep-wake regulation, circadian and feeding rhythms, learning, and memory in health and disease. As Alzheimer's and Parkinson's diseases induce progressive neuronal death of particular brain regions, it can be easily understood that supply with newly generated neuronal cells in the adult brain would help to recover neuronal functions lost by these diseases. Furthermore, it has been clarified in recent years that neuronal generation (neurogenesis) in the dentate dyrus of the hippocampus is associated with the etiology of depression. That is, when the symptoms of depression are improved by anti-depressant treatment, increased neurogenesis is observed in the hippocampus. Therefore, it is thought that neurogenesis in the hippocampus is a key event for the disappearance or reduction in the degree of the symptoms of depression.
In this article, I will describe results demonstrating that royal jelly (RJ) and its components promote the generation of neuronal and glial cells from cultured neural stem cells (NSCs), suggesting their potential as a health food that prevents certain neurological disorders such as Alzheimer's disease, Parkinson's disease, and/or depression .
2. Effect of royal jelly on neural stem cells (NSCs)
2-1. What are NSCs?
The central nervous system （ CNS ） including brain and spinal cord is composed of 3 kinds of cells, i.e., neurons, astrocytes, and oligodendrocytes. NSCs have a self-renewal capacity and multipotent activity to differentiate into neurons, astrocytes, and oligodendrocytes during brain development (Fig. 1click to view). Besides being present in the developing embryonic brain, NSCs also reside in the adult forebrain, 1, ２ ) where they constitutively give rise to proliferating progenitor cells, and differentiate into neurons, suggesting that the injured brain has the capacity for self-repair by activated NSCs. 3, 4) Therefore, NSCs in the mature brain are a promising target for therapy of degenerative neurological disorders including Alzheimer's and/or Parkinson's disease.
2-2. Induction of cell differentiation by royal jelly (RJ) 5)
In a previously published study we obtained NSCs from mouse brain at embryonic day 14, and cultured them in medium containing FGF-2, a growth factor that stimulates the proliferation of NSCs. 6) The floating neurospheres that formed were dissociated into individual cells, and these cells were then plated in new culture vessels. The cells stopped proliferating, and began to differentiate into neurons, astrocytes or oligodendrocytes, when FGF-2 was removed from the culture medium. To estimate the effect of RJ on the NSCs, we cultured the cells for several days in the FGF-2-free medium containing RJ, and fixed them with 4 % paraformaldehyde solution. The fixed cells were then analyzed for the expression of particular cell-specific proteins by using an immunohistochemical technique. Nestin, TuJ1 (neuron-specific type III beta-tubulin), GFAP (glial fibrillar acidic protein), and CNPase (2', 3'-cyclic nucleotide 3'-phosphodiesterase) were used as markers of undifferentiated NSCs, neurons, astrocytes, and oligodendrocytes, respectively. The ratio of the nestin-expressing cells to total cells was significantly decreased, whereas the ratios of Tuj1-, GFAP-, and CNPase-positive cells were increased, when the cells were treated with RJ (Fig. 2B click here to view figure). That is, the RJ acted on the NSCs and stimulated the generation of all 3 types of neural cells. 5) However, it was uncertain whether the RJ stimulated the differentiation from the NSCs or promoted the survival of the differentiated cells. It is important to clarify the mechanism underlying the actions of RJ toward NSCs. Therefore, we examined the ratio of nestin-positive cells to total cells after treatment with RJ in the medium supplemented with FGF-2 (Fig. 2A click here to view). The ratio was essentially the same under both circumstances , suggesting that the RJ facilitated cell differentiation rather than promoted cell survival. If the RJ could allow the differentiated neurons, astrocytes or oligodendrocytes to survive more effectively, the treatment with RJ before FGF-2 removal would not facilitate the generation of these differentiated cells. Namely, the RJ was likely to increase the generation of the differentiated cells and to decrease the number of undifferentiated cells such as nestin-positive cells. These results highlight a novel and outstanding property of RJ, namely, that it facilitates the differentiation of all types of brain cells, i.e., neurons, astrocytes, and oligodendrocytes, and suggests that RJ contains plural active components that influence the respective cell lineages differently.
3. Effect of 10-hydroxy-2-decenoic acid (HDEA) on NSCs 5)
In that same study we next examined the effect of HDEA on NSCs, because it is a unique fatty acid of RJ. Exposure of the cells to HDEA after the removal of FGF-2 increased the percentage of Tuj1-positive cells and simultaneously decreased that of GFAP- or CNPase-positive cells (Fig. 3click here to view figure). This reciprocal response between neuronal and glial populations suggests that HDEA affected a neuronal lineage of the neural progenitor cells having the ability to generate both neuronal and glial cells.
Docosahexaenoic acid (DHA), a predominant polyunsaturated fatty acids in the brain having 22 carbons and belonging to the omega-3 fatty acid group, has been reported to facilitate neuronal differentiation of cultured NSCs. DHA is essential for normal brain development, and its chronic oral administration enhances long-term memory in young and aged rats. 7) A reduction in DHA concentration in brain impairs spatial learning tasks regulated by the olfactory bulb, where neurogenesis occurs in adulthood. 8) Thus, DHA is proposed to play crucial roles in brain development and to act to maintain brain function. As suggested by our study, HDEA may have stimulated neurogenesis in the mature brain, and play roles similar to those of DHA. HDEA might have a greater ability to pass through the blood-brain barrier, because HDEA is an unsaturated fatty acid smaller than DHA.
Neurogenesis is promoted by basic helix-loop-helix transcription factors Mash1, Math1 and/or NeuroD but suppressed by another set, Hes1 and Hes5 9, 10, 11) . Our previous report demonstrated that brain-derived neurotrophic factor (BDNF) facilitated an increase in the levels of Mash1, Math1, and NeuroD mRNAs in cultured NSCs, which resulted in substantial neuronal differentiation. 6) Although the nature of the intracellular signals of HDEA that regulate the fate of NSCs is still unknown, HDEA is likely to partly mimic the effects of BDNF. 5)
４． Effect of AMP N 1 -oxide on NSCs 12)
4-1. Induction of neuronal differentiation of PC12 cells 13)
We have reported that an extract of RJ induces neurite outgrowth from cultured PC12 cells, a cell line of rat pheochromocytoma, via adenosine A 2A receptors and identified adenosine monophosphate (AMP) N 1 -oxide as the first active component in RJ that affects the nervous system. 13 ） AMP N 1 -oxide is a unique compound not found in natural products other than RJ; and it suppresses the proliferation of PC12 cells and stimulates the expression of neurofilament M, a specific protein of mature neurons in them, thus demonstrating that AMP N 1 -oxide can induce neuronal differentiation of these cells. 13 ） A recent investigation has added the new idea that the AMP N 1 -oxide-induced neurite outgrowth requires integrin signaling. １ 4 ） These findings prompted us to examine the effects of AMP N 1 -oxide on the CNS, particularly on NSCs.
4-2. Effect on cell differentiation of NSCs by AMP N 1 -oxide and its intracellular signaling 15)
Primary NSCs were cultured in the FGF-2-free medium containing or lacking AMP N 1 -oxide for 5 days. AMP N 1 -oxide significantly increased the percentage of GFAP-positive cells (astrocytes) to total cells in a dose-dependent manner, but did not cause any change in that of Tuj1-positive cells (neurons) or CNPase -positive cells (oligodendrocytes, Fig. 4click here to view figure). However, AMP induced no changes in the expression of any differentiation markers. These results demonstrate that AMP N 1 -oxide, but not AMP, facilitated specifically the generation of astrocytes from NSCs, and suggest that AMP N 1 -oxide is one of the components of RJ that influence cell lineages .
It is known that transcription factors STAT3 and Smad1, which are activated by leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2), respectively, form a complex with the coactivator p300 and that this complex synergistically generates astrocytes from NSCs. 16, 17) Furthermore, a recent report indicated that LIF induces the expression of BMP2 via STAT3 activation and leads to the consequent activation of Smad1 to promote generation of astrocytes from NSCs, 18) suggesting that activation of STAT3 is a crucial step for astrogenesis by NSCs. Therefore, we estimated the phosphorylation status of STAT3 in AMP- or AMP N 1 -oxide-treated NSCs. AMP N 1 -oxide , but not AMP, conspicuously induced phosphorylation of STAT3, demonstrating that the induction of astrogenesis by AMP N 1 -oxide was correlated with the activation of STAT3.
These results suggest that AMP N 1 -oxide serves as a tool for protection against and therapy for certain brain injuries, because astrocytes play important roles in brain development and in the response to neural injury. 19)
5. RJ for brain health
5-1. Depression and neurogenesis in the hippocampus
The NSCs exist in the subgranular zone in the dentate gyrus of the hippocampus and the subventricular zone lining the lateral ventricles in mature brain of mammals including humans, and it has been proven that they generate functional neurons. In rodents, about 9000 neurons are generate in a day 20) , and their half-life is about 28 days. 21) The neurogenesis in the hippocampus is less robust in aged animals than younger ones in mammals including monkeys. 22) It is a recent finding that reduced hippocampal neurogenesis may be associated with depression. Stress induces glucocorticoid production from the adrenal gland via stimulation by ACTH from the pituitary gland, and long term-exposure to glucocorticoid attenuates neurogenesis and causes shrinkage of the hippocampus. 23) Depression is a disease in which drug therapy succeeds, and the improvement of the symptoms is usually seen after the administration of anti-depressant drugs, although it usually takes a long time. The improvement of symptoms is well correlated with the recovery of neurogenesis in the hippocampus. The most frequently used anti-depressant drug is the selective serotonin reuptake inhibitor (SSRI), which inhibits degradation of serotonin and increases serotonin usage. Serotonin is considered to act via 5-HT1A receptors, 24) but the details have not yet been clarified.
5-2. RJ as a stimulant of neurogenesis or astrogenesis
Unique components of RJ, the medium-fatty acid HDEA and the AMP analogue AMP- N 1oxide, affect the cell lineage of cultured NSCs in a different manner. HDEA stimulated the generation of neurons, whereas AMP- N 1oxide promoted that of astrocytes, from the cultured NSCs (Figs. 3 click to view, 4 click to view). Although actual proof by the animal experiment is necessary, the HDEA can be expected to pass the blood-brain barrier by presuming from its chemical constitution, and may promote the generation of hippocampal neurons in the mature brain. In addition, whole RJ has the outstanding activity that all 3 kinds of brain cells are stimulated by it to be generated from the NSCs (Fig 2 click to view,). The results to date suggest possible action mechanisms of RJ and its components HDEA or AMP- N 1oxide for the differentiation of NSCs (Fig. 5click to view). RJ may accelerate the differentiation of both neuronal progenitors and glial progenitors from NSCs while inhibiting their cell proliferation, because RJ could facilitate the production of all 3 types of brain cells. It seems clear that HDEA facilitates the neuronal choice of NSCs while suppressing their glial choice. AMP- N 1oxide may enhance the differentiation of astrocytes without affecting a choice between astrocytes and oligodendrocytes, because AMP-N oxide activated transcription factor STAT3, which leads to astrocytic differentiation.
Our present results may be a clue to understand a variety of biological activities of RJ toward CNS at the molecular level, and may provide a novel strategy for nutritional and/or clinical applications. It is urgent to examine the neurogenic activity of HDEA and RJ in vivo; if effective, they may serve as a tool for protection against and therapy for some particular neurological disorders.
References:
1) Gage FH, Science , 287, 1433-1438, 2000
2) Nedergaard M, Ransom B, Goldman S.A.,Trends Neurosci , 26, 523-530, 2003
3)Lois C, Alvarez-Buylla A, Science, 264, 1145-1148, 1994
4) Luskin MB, FASEB J , 8, 722-730, 1994.
5) Hattori N , Nomoto H, Fukumitsu H, Mishima S, Furukawa S, Biomed Res, 28, 261-266, 2007
6) Ito H, Nakajima A, Nomoto H, Furukawa S, J Neurosci Res , 71, 648-658, 2002
7) Gamoh S, Hashimoto M, Sugioka K, Hossain MS, Hata N, Misawa Y, Masumura Ｓ , Neuroscience , 93, 237-241 , 1999
8) Greiner RS, Moriguchi T, Hulton A, Slotnick BM, Salem N,Lipids, 34 Suppl, S239-243, 1999
9) Kageyama R, Nakanishi S Curr Opin Genet Dev, 7, 659-665, 1997
10) Lee JE, Curr Opin Neurobiol, 7, 13-20 , 1997
11) Massari ME, Murre C, Mol Cell Biol, 20, 429-440, 2000
12) Hattori N, Nomoto H, Fukumitsu H, Mishima S, Furukawa S, Biomed Res , 28, 295-299, 2008
13) Hattori N, Nomoto H, Mishima S, Inagaki S, Goto M, Sako M, Furukawa S, Biosci Biotechnol Biochem , 70, 897-906, 2006
14) Hattori N, Nomoto H, Fukumitsu H, Mishima S, Furukawa S, Biomed Res , 28, 139-146, 2007
15) Hattori N, Nomoto H, Fukumitsu H, Mishima S, Furukawa S,, Biomed Res, 28 (6):295-299, 2008
16) Gu F, Hata R, Ma YJ, Tanaka J, Mitsuda N, Kumon Y, Hanakawa Y, Hashimoto K, Nakajima K, Sakanaka M, J Neurosci Res , 81, 163-171, 2005
17) Nakashima K, Yanagisawa M, Arakawa H, Kimura N, Hisatsune T, Kawabata M, Miyazono Y, Taga T, Science , 284, 479-482, 1999
18) Fukuda S, Abematsu M, Mori H, Yanagisawa M, Kagawa T, Nakashima K, Yoshimura A, Taga T, Mol Cell Biol , Epub Apr 23, 2007
19) Nedergaard M, Ransom B, Goldman SA , Trends Neurosci , 26, 523-530, 2003
20) Cameron HA, McKay RDG, J Comp Neurol , 435, 406-417, 2001
21) McEwen BS, Ann NY Acad Sci , 1032, part 1, pp. 1–7, 2004
22) Gould E, Reeves AJ, Fallah M, Tanapat P, Gross CG, Fuchs E, Proc Nat Acad Sci USA , 96, 5263–5267, 1999
23) Duman RS, Monteggia LM, Biological Psychiatry , 59, 1116–1127, 2006
24) Lacivita E, Leopoldo M, Berardi F, Perrone R, Curr Top Med Chem 8, 1024-1034, 2008
