Effects of melatonin and its analogues on neural stem cells

Accepted Manuscript Effects of melatonin and its analogues on neural stem cells Jiaqi Chu, Yalin Tu, Jingkao Chen, Dunxian Tan, Xingguo Liu, Rongbiao Pi PII:

S0303-7207(15)30113-1

DOI:

10.1016/j.mce.2015.10.012

Reference:

MCE 9314

To appear in:

Molecular and Cellular Endocrinology

Received Date: 3 May 2015 Revised Date:

27 September 2015

Accepted Date: 18 October 2015

Please cite this article as: Chu, J., Tu, Y., Chen, J., Tan, D., Liu, X., Pi, R., Effects of melatonin and its analogues on neural stem cells, Molecular and Cellular Endocrinology (2015), doi: 10.1016/ j.mce.2015.10.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Effects of melatonin and its analogues on neural stem cells

Department of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510080, China.

2

International Joint Laboratory (SYSU-PolyU HK) of Novel Anti-Dementia Drugs of

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Guangdong, Guangzhou 510006, China.

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Jiaqi Chu1,2,3, Yalin Tu1,2,3, Jingkao Chen1,2,3, Dunxian Tan4, Xingguo Liu5, Rongbiao Pi1,2,3*

National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-Sen University, Guangzhou 510080, China.

4

Department of Cellular and Structural Biology, The University of Texas, Health Science

5

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Center at San Antonio, 7703 Floyd Curl, San Antonio, TX 78229, USA.

School of Chemical Engineering and Light Industry, Guangdong University of Technology,

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Guangzhou, P.R.China

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*To whom correspondence should be addressed; Telephone: +86-20-3994-3122, E-mail: [email protected] .

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Abstract

Neural stem cells (NSCs) are multipotent cells which are capable of self-replication and

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differentiation into neurons, astrocytes or oligodendrocytes in the central nervous system (CNS). NSCs are found in two main regions in the adult brain: the subgranular zone (SGZ) in the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ). The recent discovery

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of NSCs in the adult mammalian brain has fostered a plethora of translational and preclinical studies to investigate novel approaches for the therapy of neurodegenerative diseases.

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Melatonin is the major secretory product synthesized and secreted by the pineal gland and shows both a wide distribution within phylogenetically distant organisms from bacteria to humans and a great functional versatility. Recently, accumulated experimental evidence showed that melatonin plays an important role in NSCs, including its proliferation,

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differentiation and survival, which are modulated by many factors including MAPK/ERK signaling pathway, histone acetylation, neurotrophic factors, transcription factors, and apoptotic genes. The purpose of this review is to summarize the beneficial effects of

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melatonin on NSCs and further to discuss the potential usage of melatonin and its derivatives

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or analogues in the treatment of CNS neurodegenerative diseases.

Keywords

melatonin; neural stem cells; proliferation; differentiation; neurogenesis

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Abbreviations:

Parkinson's disease; AD, Alzheimer's disease; TH, tyroxine hydroxylase ;CVS,cardiovascular system; PVN,

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paraventricular nucleus; Aβ, β-amyloid protein; MSCs, mesenchymal stem cells; GFAP, glial fibrillary acidic protein; MAP2, microtubule associated protein 2; bHLH, helix–loop–helix; MT, melatonin receptors; MAPK, mitogen-activated protein kinase; ERK, Extracellular signal-regulated kinase; BDNF,

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Brain-derived neurotrophic factor; PI3K, phosphoinositide 3-kinase; HD, Huntington's disease; GDNF, Glial cell line-derived neurotrophic factor; Bcl-2, B-cell lymphoma 2; Nrf2, nuclear factor erythroid

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2-related factor 2; ARE, antioxidant response element; Keap1, kelch-like ECH associating protein 1; AChE, acetylcholinesterase; MTLs, multi-targeted ligands.

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1. Introduction

Neural stem cells (NSCs) are multipotent cells present in the developing and adult brain.

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They have the potential to generate both neurons and glia of the developing brain and they also account for the limited regenerative potential in the adult brain. In the adult brain, NSCs

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reside in defined regions (“neurogenic niches”) that sustain their multipotency and regulate the balance between symmetrical self-renewal and fate-committed asymmetric divisions (Sommer and Rao, 2002).

NSCs have become one of the most intensively studied cell types in neurobiology. NSCs offer a unique and powerful tool for basic research and regenerative medicine in the field of Central Nervous System (CNS). The discovery of NSCs and neurogenesis in the adult

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mammalian CNS has tremendously changed our view of the plasticity and function of the brain. This has prompted excitement for the possible exploitement of intrinsic neurogenic

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activity to cure CNS diseases and rescue brain function after injury or dysfunction. Mobilization of endogenous NSCs has thus emerged as a potential therapeutic approach for neural repair (Honmou, 2015,Tong, Fong and Huang, 2015).

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Melatonin is an endogenous indoleamine present in different tissues, cellular compartments and organelles including mitochondria. In the past decades, among the

proliferation,

migration

and

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numerous functions of melatonin, the effects of the indoleamine on NSCs, including survival, differentiation,

had

been

extensively

researched.

Receptor-dependent and -independent responses to melatonin are suggested to occur in NSCs.

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1.1 NSCs and neurogenesis

NSCs develop into new neurons, astrocytes and oligodendrocytes in successive process of proliferation, migration and differentiation (Wade, McKinney and Phillips, 2014) and

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neurogenesis occurs throughout whole life in the mammalian brain (Wang, Liu, Liu et al.,

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2011). In the early embryo, embryonic stem cells (ESCs) receive the differentiation signal and then the formation of mesoderm, endoderm and ectoderm is guided (Thomson, Itskovitz-Eldor, Shapiro et al., 1998). There exists a type of neurally specified ectoderm cells known as the earliest NSCs within the ectoderm, which are called neuroepithelial cells and the neuroepithelial cells in the telencephalon later develop into radial glial cells in the ventricular zone (VZ) (Fishell and Kriegstein, 2003). At early stages, the cells undergo rapid expansion through symmetrical divisions to generate a stem cell pool (Temple, 2001) and

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then they initiate self-renewal through asymmetrical divisions, generating a committed neuronal precursor cell and a daughter cell identical to the parent. Resembling the

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transit-amplifying cells in many adult lineages, the committed neuronal cells might undergo further symmetrical divisions before differentiation, and are thus called neural precursor cells (NPCs) (Gotz and Huttner, 2005). Throughout the stem cell divisions, radial glial cells remain

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in the region of the VZ while the committed precursors created migrate from the VZ into an overlying secondary proliferative region called the subventricular zone (SVZ), where they can

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undergo a small number of symmetrical divisions before differentiating into neuronal or glial cells (Miyata, Kawaguchi, Saito et al., 2004,Noctor, Martinez-Cerdeno, Ivic et al., 2004,Tabata and Nakajima, 2003). Along with the development, the VZ shrinks, while the SVZ increases in size till adulthood. The SVZ and the subgranular zone (SGZ) in the

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hippocampal dentate gyrus (DG) of the hippocampus are considered as the two neurogenic niches in the adult brain (Zhao, Deng and Gage, 2008).

NSCs or neural precursor cells (NPCs) in SGZ and SVZ continuously participating in

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neurogenesis and gliogenesis under both normal and pathological conditions (Kriegstein and

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Alvarez-Buylla, 2009,Zhao et al., 2008). NPCs-based therapy has been considered as a promising therapeutic approach to protect and to restore the damaged CNS, which were reinforced by discovery of potential benefits of NSCs in several animal models of various neural diseases, such as stroke, Parkinson's disease (PD) and Alzheimer's disease (AD) (Martino, Pluchino, Bonfanti et al., 2011). However, a large percentage of neurons generated by NSCs die during the first two weeks, and only a few of them survive for a relatively long period of time (Kempermann, Gast, Kronenberg et al., 2003), though the continuous

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neurogenesis and gliogenesis. Thus, further investigation of the mechanisms of NSCs proliferation and differentiation are needed.

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1.2 Functions of melatonin

Melatonin is an ancient and highly conserved indoleamine derived from tryptophan and it

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is present in all phyla of muticellular animals. It is recognized as a neurohormone that possesses numerous functions and has a wide range of distribution (Hardeland and Poeggeler,

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2003). In mammals, melatonin is mainly synthesized by the pineal gland in a circadian manner and released into blood and cerebrospinal fluid to exert regulatory roles on seasonal and circadian rhythms (Luchetti, Canonico, Betti et al., 2010), which is critical for the physiological functions of the neuroimmuno-endocrine system such as sleep-wake cycle,

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pubertal development and seasonal adaptation (Reiter, Tan, Manchester et al., 2009,Salti, Galluzzi, Bindi et al., 2000,Shochat, Luboshitzky and Lavie, 1997).

During the daytime, the melatonin secretion level is very low (below 10 pg/mL), yet the

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concentration exhibit higher values (10–15-fold increase, up to 120 pg/mL) during the night

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(Karasek and Winczyk, 2006,Konturek, Konturek, Brzozowska et al., 2007,Sanders, Chaturvedi and Hordinsky, 1999). This circadian rhythm is present in all living organisms. For example, in human beings, the rhythm is developed during the first months of life and reaches the greatest magnitude between the 4th and 7th year of age and then decreases (Escames, Lopez, Garcia et al., 2010). Accordingly, the melatonin level varies during the lifespan. In the fetal period, the fetus uses maternal melatonin that crosses the placenta and the level of melatonin increases from birth to a peak around puberty, and then declines in

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middle-aged and elderly individuals (Sharma, Palacios-Bois, Schwartz et al., 1989,Waddell, Wharfe, Crew et al., 2012). The association between the decline in melatonin’s levels with

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aging and insomnia as well as the development of neurodegenerative diseases such as AD has been reported (Bubenik and Konturek, 2011).

In addition to its multiple physiological functions, much attention has been attracted to the

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antioxidant action of melatonin (Reiter, Paredes, Manchester et al., 2009). Studies indicated that melatonin prevented neuronal cell death induced by toxins, such as 6-hydroxydopamine

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(Sharma, McMillan, Tenn et al., 2006), 1-methyl-4-phenylpyridinium ion (MPP+) (Chetsawang, Govitrapong and Chetsawang, 2007), amphetamine (Klongpanichapak, Phansuwan-Pujito, Ebadi et al., 2007), β-amyloid protein (Aβ) (Gunasingh, Philip, Ashok et al., 2008) and kainic acid (Lee, Chun, Kong et al., 2006) via its free radical-scavenging

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mechanisms. Thus, melatonin has been proposed to be a hormone that could play a role in the development of diseases associated with oxidative damages.

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Amounting evidence shows that melatonin has numerous functions in mesenchymal stem cells (MSCs), a heterogeneous population of multipotent elements resident in tissues such as

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bone marrow, muscle, and adipose tissue, which are primarily involved in developmental and regeneration processes, tissue repair and restoration. The roles of melatonin in MSC lineage commitment and adipogenic differentiation as well as their mechanism are well discussed in a wonderful review published recently (Luchetti, Canonico, Bartolini et al., 2014). Very recently, several reports indicated the potential clinical applications of melatonin on MSCs to treat the kidney injury (Chen, Lin, Wallace et al., 2014), acute interstitial cystitis (Chen, Tan,

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Jing et al., 2014), skin wound (Lee, Jung, Oh et al., 2014), and also cerebral ischemia (Tang, Cai, Yuan et al., 2014). Interestingly, emerging evidence suggests that melatonin receptors are

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expressed in NSCs/NPCs (Niles, Armstrong, Rincon Castro et al., 2004,Ramirez-Rodriguez, Klempin, Babu et al., 2009) and raises the obvious question of whether melatonin also play a key role in the development of NSCs/NPCs. Moreover, it has been documented that

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neurogenesis in the DG occurs under a circadian rhythmic pattern. BrdU-labeled proliferating cells show a dark/light cycle–dependent pattern (Guzman-Marin, Suntsova, Bashir et al.,

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2007). M-phase cells increase during the night (Tamai, Sanada and Fukada, 2008), and constant light exposure decreases neurogenesis in the DG (Fujioka, Fujioka, Tsuruta et al., 2011). Clock genes (Per1, Per2, Cry1, Cry2, Bmal1, and Clock), which are expressed in NPCs in the DG of the hippocampus (Jilg, Lesny, Peruzki et al., 2010), can regulate the differentiation

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transcription factors including NeuroD1, Id2, Hey1, Olig1 (Kimiwada, Sakurai, Ohashi et al., 2009). These data suggest melatonin, which secretes following light/dark rhythms (Zawilska, Skene and Arendt, 2009) and acts as a regulator of circadian rhythms in mammals (Stehle,

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von Gall and Korf, 2003), can play an important role in the development of NSCs. Therefore, a better understanding of the potential interaction between melatonin and NSCs is required to

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shed novel lights on the effects and their mechanisms of melatonin on NSCs and to facilitate the development of new therapies for neurodegenerative diseases.

2. Effects of Melatonin on NSCs

The survival, proliferation and differentiation and also migration of NSCs are very important for their functions in the developing or adult brain as well as in the pathological

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conditions. Amounting evidence demonstrated that melatonin plays important roles in these aspects (summarized in Table 1).

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2.1 Melatonin modulates cell survival of NSCs

The fate of NSCs is very important. Consistent with previous studies that melatonin

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protects damaged neurons, it was reported that melatonin regulates the cell viability of NSCs from different regions of brain. Kong X, et al. found that melatonin (0.05-1 nM) increased the

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viability of cultured NSCs obtained from rat midbrain in a dose-dependent manner by MTT assay (Kong, Li, Cai et al., 2008). Ramirez-Rodriguez, G., et al. suggested that melatonin promoted survival of precursor cells in vitro and increased survival of newborn neurons in vivo. By quantifying cells that underwent differentiation after 5 days, melatonin (0.1 µM) was

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found to significantly increase the survival of differentiated cells, which is confirmed with the WST-1 assay. In addition, melatonin increased the survival of newborn neurons in the DG of adult mice after 14 days of treatment by BrdU-labelling. The number of surviving cells in the

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DG in melatonin-treated mice (8 mg/kg) was increased by 63%, comparing to that in vehicle-treated mice (Ramirez-Rodriguez et al., 2009). Studies also showed that melatonin

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protected NSCs against hypoxia and increased the cell viability in vitro. Melatonin (0.001-100 µM) obviously improved the cell viability of NSCs in a dose-dependent manner. Meanwhile, melatonin (100 nM) significantly decreased the death of cells subjected to hypoxia when detected by Hoechst staining (Fu, Zhao, Liu et al., 2011). Most recently, melatonin was reported to protect NSCs against LPS-induced inflammation. Melatonin (100 nM) decreased approximately 20% and 35% cytotoxicity level of the NSCs treated with 100ng/mL and

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1µg/mL LPS respectively by lactate dehydrogenase (LDH) assay, which might be related to attenuating NO production in LPS treated NSCs (Song, Kang, Lee et al., 2015). In addition,

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the protection effects were also found in the human bone marrow derived MSCs (BM-MSCs). Pretreatment of melatonin (0.01-100 µM) successfully reversed the H2O2-induced senescent phenotypes of BM-MSCs (Zhou, Chen, Liu et al., 2015).

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2.2 Melatonin promotes NSCs proliferation

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The ability of proliferation of NSCs affects its function. Melatonin was found to induce cell proliferation in various NSCs. Melatonin (1 nM) significantly increased the total number of neural spheres and the number of neural spheres over 60 µm in diameter in NSCs derived from rat ventral midbrain (Kong et al., 2008). Similarly, melatonin (0.001-10 µM) induced

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SVZ precursor cell proliferation in a concentration-dependent manner. The number of neurosphere generated in melatonin-treated cells in vitro was significantly increased. Furthermore, an increase in neurosphere size was observed in melatonin-treated cells

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(Sotthibundhu, Phansuwan-Pujito and Govitrapong, 2010). Moreover, melatonin (0.1-10 µM) treatment for 5 days increased the number of neurosphere of adult hippocampal NSCs

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(Tocharus, Puriboriboon, Junmanee et al., 2014). By using bromodeoxyuridine labeling and immunostaining, melatonin (100 nM) was shown to promote the proliferation of hypoxic NSCs. The ratio of BrdU/nestin positive cells against total nestin-positive NSCs (>95% of all cells) was significantly increased comparing with the untreated cells at 3 days after hypoxia (Fu et al., 2011). Melatonin (5 or 10 mg/kg) treatment dramatically enhanced cell proliferation of ischemic-stroke NSCs in a time-dependent manner, evaluated by ki67 (a protein strictly

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associated with cell proliferation) staining (Chern, Liao, Wang et al., 2012). In addition, synergistic effect of melatonin (10 mg/kg) and exercising on the proliferation of endogenous

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NSCs/NPCs after Spinal Cord Injury were reported (Lee, Lee, Lee et al., 2014). However, contrary to the above, melatonin (0.01–10 nM, physiological concentrations) failed to affect the proliferation of NSCs derived from the mouse embryo striatum by WST-8 assay.

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Pharmacological concentrations (1–100 µM) of melatonin also did not affect cell proliferation after 1 day incubation, but suppressed cell proliferation on days 4 and 7 in a

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concentration-dependent manner, examined by WST-8 assay and BrdU incorporation analysis (Moriya, Horie, Mitome et al., 2007). These results seem to be contrary to other results demonstrating that melatonin promote the proliferation of NSCs and the reason for the difference in the effects of melatonin is unclear at present. The difference in the source of

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NSCs or the experiment system might be the explanation. Recently, melatonin was found to minimize the inhibitory effects of dexamethasone on adult hippocampal progenitor cell proliferation.

Addition

of

dexamethasone

to

hippocampal

progenitor

cells

from

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eight-week-old rats resulted in a decrease in the number of neurospheres, which could be precluded by pretreatment of melatonin (1 µM) (Ekthuwapranee, Sotthibundhu, Tocharus et

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al., 2014).

2.3 Melatonin promotes NSCs differentiation

Some studies demonstrated that melatonin facilitates NSCs differentiation into neurons. The cultured ventral midbrain NSCs with melatonin treatment (1 nM) were found to differentiate into tyroxine hydroxylase (TH), which is a key enzyme of dopamine synthesis

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and is usually regarded as a marker of dopaminergic neurons, positive neurons. By immunostaining, increased percentage of BrdU-positive cells detected were β-III-tubulin (a

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neuronal marker) positive when treated with melatonin (1 nM), while the glial fibrillary acidic protein (GFAP) positive cells decreased (Kong et al., 2008). The nestin (653 bp) and β-III-tubulin (442 bp) mRNA transcripts were readily detected in all samples of C17.2 cells (a

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neural stem cell line derived from the external germinal layer of mouse cerebellum) treated with melatonin (0.05-100 nM) for 24 hr by RT-PCR, in contrast, the mRNA transcript for

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GFAP (592 bp) was not detected in any samples (Sharma, Ottenhof, Rzeczkowska et al., 2008). Fluorescence-based immunocytochemistry showed that 1µM melatonin-treated cells, isolated from the SVZ of adult mice, exhibited significantly increased differentiation into the β-III-tubulin positive cells, but differentiation into GFAP remained unchanged. These cells

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also showed characteristic neuronal morphologies and established contacts between each other (Sotthibundhu et al., 2010). Adult hippocampal NPCs cultured in the presence of melatonin (0.01-10,000 nM) caused an increase in the density of β-III-tubulin positive cells

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and measuring β-III-tubulin levels by western blot confirmed the increase in neuronal differentiation in response to melatonin (Ramirez-Rodriguez et al., 2009). The percentage of

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neurons in relation to the total cell number was decreased when NSCs were exposed to hypoxia, yet melatonin (100 nM) reversed this and increased the percentage of microtubule associated protein 2 (MAP2, a neuronal marker) positive cells in all melatonin treated groups, especially in the 7 days differentiation group, by immunocytochemistry, as corroborated by Western blot analysis. When compared with the control group, hypoxia did not appear to affect the differentiation of NSCs into astrocytes (Fu et al., 2011). Takahiro Moriya, et al.

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reported that treating with physiological concentration of melatonin (0.01–10 nM) during the proliferation period and differentiation period failed to affect the differentiation of the NSCs either

β-III-tubulin+

neurons

or

GFAP+

astrocytes

when

examined

by

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into

immunocytochemistry and ELISA. On the other hand, NSCs which had been treated with higher concentrations of melatonin (1–25 µM) during the proliferation period differentiated more

abundant

neurons

than

those

in

control

medium,

evaluated

by

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into

immunocytochemistry and ELISA. In contrast, melatonin (1–100 µM) decreased the neural

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differentiation of the NSCs into β-III-tubulin+ neurons when cell were treated during the differentiation period in a concentration-dependent manner. However, melatonin failed to affect the differentiation into GFAP+ astrocytes and the number of total viable cells under differentiation-preferring conditions (Moriya et al., 2007). Melatonin might modulate adult

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neurogenesis by different mechanisms under different concentrations. Thus, melatonin exerts its effects at both physiological (0.01-10 nM) and pharmacological concentrations (1-100 µM).

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of NSCs

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3. The mechanisms of melatonin regulating proliferation and differentiation activities

Studies from several sources indicate that melatonin exerts NSCs regulation via different

mechanisms involving melatonin receptors, MAPK/ERK signaling, histone acetylation, neurotrophic factors, basic helix–loop–helix (bHLH) factors, etc. A brief summary of this evidence was described as follows (summarized in Fig. 1).

3.1 Melatonin receptors

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Till now, four types of melatonin receptors have been identified, membrane receptors (MT1, MT2), cytosolic binding sites (MT3 and calmodulin), and nuclear receptors of the RZR/ROR

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family (Cutando, Aneiros-Fernandez, Lopez-Valverde et al., 2011). Numerous biological functions of melatonin in mammals are mediated via activation of the G-protein-coupled high-affinity melatonin receptors 1 and 2 (MT1 and MT2). Both MT1 and MT2 receptors are

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expressed in various organs, the MT1 subtype is found in retina, ovary, testis, mammary gland, coronary arteries, gall bladder, aorta, liver, kidney, skin and brain, high density of melatonin

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receptors has been shown to be in the SCNs, particularly in the adenohypophysis, suprachiasmatic nucleus (SCN), paraventricular nucleus (PVN) and area postremaand thus should be related to the function of melatonin in SCNs. The MT2 subtype is found in the retina, SCN, hippocampus, substantia nigra, ventral tegmental area (Luchetti et al.,

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2010,Singh and Jadhav, 2014).

MT1 is a 350- amino protein, presenting a sequence homology of 60% with the 362-amino acid MT2 and has higher homology than MT2 with rhodopsin (Reppert, Godson, Mahle et al.,

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1995). MT1 and MT2 are typical the G-protein-coupled receptor (GPCR) with

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7-transmembrane (TM) α-helices and 4 intracellular and 4 extracellular domains. The receptors couple to G proteins (Gai2/3, Gaq, and Gbg) during activation by binding sites of the MT isoforms, which lie in the same part of the receptor: in the immediate vicinity of TM5 and in the extracellular half of the transmembrane domain (Mazna, Grycova, Balik et al., 2008,Rivara, Lorenzi, Mor et al., 2005).

The effects of melatonin have been found to be linked with the activation of functional

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melatonin receptors. The transcripts of melatonin receptors are detected in precursor cells and the increased proportion of neurons induced by melatonin was almost entirely blocked (84%)

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by luzindole, a melatonin receptor antagonist. The antagonist alone, however, did not induce changes in neuronal differentiation (Ramirez-Rodriguez et al., 2009). Luzindole partially suppressed the melatonin-induced proliferation of precursor cells isolated from SVZ of adult

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mouse in a concentration-dependent manner. Double labeling showed that MT1 was expressed in cells during both proliferation and differentiation periods (Sotthibundhu et al.,

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2010). Chern CM, et al. found that MT2 melatonin receptor is involved in ameliorating neural function in ischemic-stroke mice (Chern et al., 2012). The protective effect of melatonin was largely reversed by pretreating with either luzindole or 4P-PDOT, two MT antagonists with different chemical structures. The latter is a much more selective MT2 antagonist than

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luzindole (Dubocovich, Masana, Iacob et al., 1997).

3.2 MAPK/ERK signaling

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The mitogen-activated protein kinase (MAPK) signal pathway is evolutionarily conserved among eukaryotes and plays important roles in numerous biological functions, including cell

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proliferation and cell death. Extracellular signal-regulated kinase (ERK) is one of the main subgroups of the MAPKs. ERK1 and ERK2 are key transducers of proliferation, differentiation and survival signals. It was originally found to be phosphorylated on Tyr and Thr residues, and almost expressed in all tissues, mainly affecting the growth of cells by G1-to S-phase progression. Generally, growth factors are the most well-known activators, which then cause the activation of upstream kinases MEK1/2 MAPK kinases and Raf MAPK

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kinases (Lei, Wang, Mei et al., 2014). ERK1/2 is reported to be activated by phosphorylation in response to melatonin in neuronal cell cultures (Roy and Belsham, 2002). Studies found

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that the MAPK/ERK pathway, which has been linked to histone acetylation (Cohen-Armon, Visochek, Rozensal et al., 2007,Lee, McCool, Murdoch et al., 2006), is involved in melatonin-induced proliferation and differentiation of NSCs. Data showed that melatonin

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significantly increased the phosphorylation of the main MAPK (ERK1/2) and c-Raf , which are protein kinases involved in the MAPK signaling pathway, and the phosphorylation was

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significantly reduced when melatonin receptor antagonist was pretreated (Fu et al., 2011,Niles, Pan, Kang et al., 2013,Tocharus et al., 2014). However, whether melatonin will exert its effect when ERK is blocked was not discussed.

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3.3 Neurotrophic factors

Neurotrophic factors are secreted proteins that play important roles in the development and maintenance of the nervous system, influencing the development of distinct neuronal

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populations, and maintaining the survival and neuritic arbors of mature neurons into adulthood (Hegarty, O'Keeffe and Sullivan, 2014). Brain-derived neurotrophic factor (BDNF)

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can maintain neuronal survival and regulate synaptic plasticity through its downstream signals including phosphoinositide 3-kinase (PI3K)/Akt, ERK and phospholipase Cγ pathways (Kuczewski, Porcher and Gaiarsa, 2010,Numakawa, Adachi, Richards et al., 2013). Glial cell line-derived neurotrophic factor (GDNF) promotes survival of Dopamine and other neurons, and are proposed to protect neurons in PD (Hong, Mukhida and Mendez, 2008), regulates neuronal survival through activation of cellular signaling, mRNA translation and new protein

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synthesis. GDNF also protects neurons by suppressing death receptor–caspase pathway, translocation of apoptogenic Bax, and by upregulating of anti-apoptotic Bcl-2 and Bcl-XL

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(Maruyama and Naoi, 2013).

Studies have found that neurotrophic factors, especially BDNF and GDNF, significantly increased in the cultured NSCs treated with melatonin. It suggests that melatonin could

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promote the viability and facilitate neuronal differentiation of NSCs through increasing the

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level of BDNF and GDNF (Kong et al., 2008,Niles et al., 2004,Sharma et al., 2008).

3.4 Histone acetylation

There is evidence that gene alteration by melatonin is associated with the effects on NSCs (Kim and Rosenfeld, 2010,Sarlak, Jenwitheesuk, Chetsawang et al., 2013). The epigenetic

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mechanisms that modulate DNA without altering genomic sequences involve nucleosomes, which consist of DNA and an octamer of histones. The control of transcription is crucial in cell differentiation and development of the nervous system. In the last decade, histone

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modifications have been shown to control many aspects of transcription including higher

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order chromatin structure and gene expression (Lilja, Heldring and Hermanson, 2013). Histones can be post-translationally modified in many ways, for example by acetylation, methylation, phosphorylation, ubiquitination, and sumoylation (Kouzarides, 2007).

Histone acetylation is found to associate with neuronal differentiation (Hsieh, Nakashima, Kuwabara et al., 2004,Marin-Husstege, Muggironi, Liu et al., 2002,Stockhausen, Sjolund, Manetopoulos et al., 2005). Histone hyperacetylation in progenitor cells has been shown to

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promote neuronal differentiation, while blocking the development of a glial phenotype (Balasubramaniyan, Boddeke, Bakels et al., 2006,Hsieh et al., 2004). Sharma R et al. found

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that melatonin induced the mRNA expression of HDAC3, HDAC5, and HDAC7. It is likely that enhanced HDAC mRNA levels represent a compensatory feedback mechanism following melatonin-induced histone hyperacetylation (Sharma et al., 2008). Melatonin induced

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significant increases in histone H3 and H4 acetylation in the hippocampus and striatum but not in the midbrain and cerebellum were detected by Niles LP, et al. (Niles et al., 2013),

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suggesting that hippocampus and striatum may be the targets for the epigenetic effects of melatonin, and it also shows the regional differences of melatonin signaling.

In addition to acetylation, the methylation of histone is also closely related to NSCs development. The methylation of histones especially at residues H3K4, H3K9 and H3K27 is

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emerging as some of the key epigenetic marks that control transcription in stem cells and progenitors (Lilja et al., 2013). Nevertheless, the crosstalk between melatonin and histone methylation has not been reported yet, thus the modification of histone and the enzymes

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regulating these modifications can be a focus of attention when dissecting the epigenetic

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mechanisms of melatonin influencing NSCs state and fate.

3.5 bHLH transcription factors

In neurodevelopment, determination of neuron and glia cell fates from NSCs during fetal

and adult brain development depends on some bHLH factors (Kageyama, Ohtsuka, Hatakeyama et al., 2005,Ross, Greenberg and Stiles, 2003,Zhou and Anderson, 2002). There are two types of bHLH genes, the repressor type and the activator type. The repressor-type

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bHLH genes include Hes genes, homologs of Drosophila hairy and Enhancer of split [E(spl)], while the activator-type bHLH genes include Mash1, Math and Neurogenin (Ngn), homologs

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of Drosophila proneural genes achaete-scute complex and atonal (Kageyama et al., 2005). The activator-type bHLH genes Mash1, Math and Ngn are expressed by differentiating neurons. These bHLH factors form a heterodimer with a ubiquitously expressed bHLH factor,

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E47, and activate gene expression by binding to the E box (CANNTG), they not only induce the neuronal-specific gene expression but also inhibit the glia-specific gene expression. While

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the Hes genes can represses activator-type bHLH genes expression by directly binding to the promoter E47 and antagonizes the activity (Kageyama et al., 2005).

Fu J et al. found that melatonin treatment induce up-regulation of Mash1, NeuroG1, and NeuroD2, which are involved in vertebrate neurogenesis, in the differentiated NSCs compared

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with untreated cells in hypoxia. But other bHLH factors such as Hes1, Hes5, and Id which can regulate the specification of gliogenesis (Jen, Manova and Benezra, 1997,Ohtsuka, Ishibashi, Gradwohl et al., 1999,Ohtsuka, Sakamoto, Guillemot et al., 2001) are not affected by

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melatonin (Fu et al., 2011). It is possible that increased neurogenesis caused by melatonin

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supplementation is the consequence of enhanced expression of these transcription factors.

3.6 Anti-apoptotic Bcl-2 family proteins

The anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein family act as key regulators in the

intrinsic or “mitochondrial” apoptosis pathway, they are classified into three different classes depending on their structural and functional properties: (a) anti-apoptotic Bcl-2 proteins including Bcl-2 itself, Bcl-XL (Bcl-extra long), Mcl-1 and Bcl-W; (b) pro-apoptotic proteins

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Bax (Bcl-2-associated × protein), Bak (Bcl-2-antagonist/killer-1) and potentially Bok (Bcl-2 related ovarian killer); and (c) BH3-only proteins including Bim (Bcl-2 interacting mediator),

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PUMA (p53 upregulated modulator of apoptosis), Bid (BH3 interacting domain death agonist), Bik (Bcl-2 interacting killer), Bad (Bcl-2 associated death promoter), Bmf (Bcl-2 modifying factor), Hrk (Hara-kiri) and Noxa (Latin for “damage”) (Anilkumar and Prehn, 2014).

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Anti-apoptotic Bcl-2 family proteins as well as Bax and Bak are often constitutively expressed in cells and activation of the mitochondrial apoptosis pathway through

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pro-apoptotic Bcl-2 proteins is able to activate different cell death pathways including apoptosis (Kilbride and Prehn, 2013). Numerous literatures show that Bcl-2 plays an important role in initiating or inhibiting apoptosis during neuronal development and injury. For example, overexpression of Bcl-2 inhibited Bax-mediated cytochrome-c release, caspase

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activation and cell death (Putcha, Deshmukh and Johnson, 1999); Bcl-2 overexpression protected hippocampal neurons against glutamate mediated excitotoxicity (Wong, Ralph, Walmsley et al., 2005). Interestingly, melatonin was also found to induced a significant

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increase in the ratio of Bcl-2/Bax and inhibition of caspase-3 activation (Fu et al., 2011) which indicate that the overexpression of the Bcl-2 family proteins may contributes to the

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protection effects of melatonin on injured NSCs.

3.7 Nrf2 signaling

The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a primary sensor and a master regulator of oxidative stress via its ability to modulate the expression of hundreds of antioxidant and detoxifying genes (Johnson, Johnson,

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Kraft et al., 2008). Nrf2 is one of the cap'n'collar (CNC) transcription factors and ubiquitously expressed in all human tissues including the brain (Moi, Chan, Asunis et al., 1994). Nrf2

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binds to the ARE, which is an enhancer element located in the 5 flanking region of many phase II detoxifying and antioxidant genes, to regulate this pathway. Under basal conditions, Nrf2 is negatively regulated in the cytoplasm by kelch-like ECH associating protein 1

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(Keap1). Keap1 prevents the nuclear translocation of Nrf2 and functions as an adaptor protein to E3 ligase to promote the degradation of Nrf2 via the ubiquitin proteasome system (UPS)

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(Gan and Johnson, 2014). Upon disruption of Keap1-Nrf2 binding, Nrf2 translocates to the nucleus and binds with small Maf proteins. The formed heterodimer binds to the ARE and coordinates the transcription of genes involved in phase II detoxification and antioxidant defense as so to maintain redox balance (Itoh, Chiba, Takahashi et al., 1997).

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A number of studies have shown the dynamic changes of Nrf2/ARE pathway in disease demonstrating the accumulation of oxidative damage and exert neuroprotection effect against oxidative stress and protect against protein oxidation and misfolding, such as Aβ and tau

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neurofilament tangles (NFTs) in AD (Gan and Johnson, 2014). Recently, some studies also

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suggest the protection effect against oxidative stress of Nrf2 pathway on NSCs/NPCs (Abdanipour, Tiraihi, Noori-Zadeh et al., 2014,Li, Johnson, Calkins et al., 2005,Ni, Liu, Ren et al., 2014). Kärkkäinen V., et al. found that Nrf2 regulates neurogenesis and protects NPCs against Aβ toxicity. Nrf2 prevents the ischemia-induced decrease in newborn neurons in the SVZ of the DG; lentivirus-mediated overexpression of Nrf2 gene or by treatment with pyrrolidine dithiocarbamate (PDTC), an Nrf2 activating compound, increased the growth of NPC neurospheres as well as the neuronal differentiation of NPCs. Aβ-induced toxicity and

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reduction in neurosphere proliferation were prevented by Nrf2 overexpression, conversely, Nrf2 deficiency enhanced the Aβ-induced reduction of neuronal differentiation (Karkkainen,

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Pomeshchik, Savchenko et al., 2014).

Accordingly, some literatures demonstrated that melatonin protect cells against oxidative stress, brain injury and neurotoxicity through the activation of Nrf2/ARE signaling pathway

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(Deng, Zhu, Mi et al., 2014,Ding, Wang, Xu et al., 2014,Wang, Ma, Meng et al., 2012), yet few literature indicate the involvement of Nrf2 in the mechanism of melatonin in NSCs/NPCs,

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raising the question whether the pathway play a role in the mechanism of melatonin regulating the NSCs survival and development.

3.8 Others

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Interestingly, studies showed that melatonin ameliorates dexamethasone-induced inhibitory effects on the proliferation of cultured progenitor cells obtained from adult rat hippocampus. Dexamethasone (the glucocorticoid receptor agonist) increased the glucocorticoid receptor

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protein but decreased the level of MT1 melatonin receptor, whereas melatonin increased the

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level of MT1 melatonin receptor but decreased the glucocorticoid receptor protein, suggesting the crosstalk and cross regulation between the melatonin receptor and the glucocorticoid receptor on hippocampal progenitor cell proliferation (Ekthuwapranee et al., 2014).

4. Melatonin derivative and analogues

Since melatonin plays an important role in NSC growth and differentiation, melatonin-based compounds could show a neurogenic profile of paramount importance in the

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search of new therapies for AD or other neurodegenerative diseases and neurodevelopmental diseases. However, pharmacokinetic issues such as limited oral bioavailability and short

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half-life time limit its usage. Thus, to design and synthesis of melatonin analogues, especially, the multi-targeted derivatives with improved PD/PK characters is evoking the interest of the scientific community.

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Recently, patents and patent applications from 2012 to September 2014 of which melatonin or synthetic analogues were claimed for the prevention or treatment of pathological conditions (Rivara, Pala, Bedini et al., 2015). For example, a series of novel

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had been well reviewed

hybrid molecules obtained by linking melatonin, or its oxidation products, to a tetrahydroacridine unit via a carbamate bond, which are described as potent cholinesterase enzyme inhibitors, antioxidants and able to prevent the aggregation of β-amyloid (Rivara et

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al., 2015). Here the melatonin derivatives that are supposed to play a role in stem cells were summarized below (also summarized in Table 2).

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Agomelatine (S20098), a synthetic melatonin analogue that binds to MT1 and MT2 receptors (Audinot, Mailliet, Lahaye-Brasseur et al., 2003), have been reported to increases

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progenitor cell proliferation in the DG, which requires an intact diurnal corticosterone rhythm (AlAhmed and Herbert, 2010). SSH-BM-I was synthesized from tryptamine by using a newly developed synthetic method, and it has structural similarity to melatonin. It had been reported that SSH-BM-I increases osteoblasts in scales of gold fish (Suzuki, Somei, Kitamura et al., 2008) and stimulates terminal osteoblast differentiation in a cell differentiation stage–dependent manner (Mikami, Somei and Tsuda, 2011). Data showed that SSH-BM-I

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increased the formation of mineralized nodules in mature osteoblast ROS17/2.8 cells yet suppressed bone morphogenetic protein 2 (BMP-2, which is one of the most powerful

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cytokines to promote mesenchymal progenitors differentiate into particular cell types) induced osteoblast differentiation in mesenchymal progenitor ROB-C26 cells. Several 2-vinyl-8-hydroxyquinoline derivatives were found as potential antioxidants and regulators of

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H2O2-induced oxidative stress in rat bone marrow mesenchymal stem cells, and one of these compounds, Compound 4, which has similar structure with melatonin, shows better

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proliferative activities and exhibited strong protection effects against oxidative stress in MSCs (Wang, Zeng, Li et al., 2010). Pradoldej Sompol et al. reported that N-acetylserotonin (NAS), the immediate precursor of melatonin, regulates an early event of neurogenesis by increasing NPCs proliferation in both active and sleeping phases of the mice and it significantly

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enhances NPCs proliferation in sleep-deprived mice (Sompol, Liu, Baba et al., 2011).

Recently, a new family of melatonin−N,N-dibenzyl (N-methyl) amine hybrids with a balanced multifunctional profile including neurogenic, antioxidant, cholinergic, and

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neuroprotective properties at low micromolar concentrations were synthesized by linking the

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melatonin framework and the protonable N,N-dibenzyl (N-methyl) amine, which is present in the well-known acetylcholinesterase (AChE) inhibitor AP2238 and could interact with the AChE catalytic active site (Piazzi, Cavalli, Belluti et al., 2007,Piazzi, Rampa, Bisi et al., 2003,Rizzo, Bartolini, Ceccarini et al., 2010). They promoted the maturation of NSCs into a neuronal phenotype and protected neural cells against mitochondrial oxidative stress. They also have antioxidant properties, and could inhibit human AChE (Lopez-Iglesias, Perez, Morales-Garcia et al., 2014). A series of melatonin analogues resulting from the hybridization

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of both pinoline and melatonin structures was synthesized and pharmacological evaluation indicated that pinoline at trace concentration and compound 2 were able to stimulate early

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neurogenesis and neuronal maturation in an in vitro model of neural stem cells isolated from the adult rat SVZ (de la Fuente Revenga, Perez, Morales-Garcia et al., 2015). In addition, several melatonin based compounds were synthesized by replacing the acetamido group with

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a series of reversed amides and azoles, and were found to promote differentiation of rat NSCs to a neuronal phenotype in vitro (de la Fuente Revenga, Fernandez-Saez, Herrera-Arozamena

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et al., 2015).

As the complexity of AD pathology, the therapeutic paradigm one-compound-one-target has failed till now. Thus, the design of molecules that are able to interact with two or more complementary targets is catching much more people’s attention, with the expectation that

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these multi-targeted ligands (MTLs) may represent important advantages in the treatment of AD and other neurodegenerative pathologies (Cavalli, Bolognesi, Minarini et al., 2008,Leon,

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Garcia and Marco-Contelles, 2013,Morphy and Rankovic, 2005).

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5. Perspective and conclusions

NSCs are self-renewing progenitors that are capable of acquiring a neuronal or glial

phenotype. As such, NSC transplantation may serve as a potential therapeutic approach for several neurodegenerative diseases. Yet engrafted stem cells in neurodegenerative diseases’ models have poor survival rates and may not acquire the desired phenotype (Arenas, 2002). Therefore, stem cell transplantation could benefit from combined treatment with compounds that improve graft integration and differentiation. Varies data in the literature have shown that

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melatonin modulates NSCs proliferation, survival and differentiation. Because melatonin can cross the blood brain barrier and is soluble in both lipid and water (Reiter, Tan, Manchester et

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al., 2007), this endogenous modulator might be beneficially used for modulating NSCs, pro-proliferation and pro-survival of endogenous NSCs as well as engrafted NSCs for CNS diseases.

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Signaling and transcriptional responses elicited by the exposure of NSCs to melatonin are now partially disclosed (see Fig. 1), but obviously further work is needed to provide a more

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precise picture of melatonin-mediated regulatory and differentiation mechanisms. For instance, the correlation between melatonin and microRNAs (miRNAs) on influencing the cell fate of NSCs, since an emerging hypothesis is that miRNAs play a central role in controlling stem cell-fate determination (Stevanato and Sinden, 2014). miRNAs are integrated

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within networks that form both positive and negative feedback loops to promote and stabilize cell fate choice at multiple levels (Shenoy and Blelloch, 2014) and channelize the cellular physiology toward neuronal differentiation or indirectly influence neurogenesis by

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regulating the proliferation and self-renewal of NSCs and are dysregulated in several

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neurodegenerative diseases (Iyengar, Choudhary, Sarangdhar et al., 2014). Meza-Sosa KF et al. found that, by regulating different target genes, microRNAs let-7, microRNA-9 and microRNA-124 have been shown to promote the differentiation of NSCs/NPCs into specific neural cell types while microRNA-25, microRNA-134 and microRNA-137 have been characterized as microRNAs that induce the proliferation of NSCs/NPCs (Meza-Sosa, Pedraza-Alva and Perez-Martinez, 2014). Interestingly, recent studies found that melatonin significantly modulates the expression of miRNAs (Fu, Zhao, Zheng et al., 2014,Lee, Kim,

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Youn et al., 2011), including let-7a and miR-124, which are involved with neurogenesis, indicating that regulating the expression of miRNAs may be involved in the mechanism of

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melatonin to modulate the development of NSCs, which is so far poorly investigated. Further studies should be carried out to uncover the roles of miRNAs in the effects of melatonin on NSCs.

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Given small molecules are more druggable, it evokes a great interest to develop potential orally administrated small molecules to modulate NSCs/NPCs in vivo (Rishton,

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2008,Swaminathan, Kumar, Halder Sinha et al., 2014). As we know, CNS diseases, including AD and PD, are multifactorial pathologies. MTLs should be more efficiency to combat these diseases. To design of MTLs-based melatonin derivatives or analogues could be a new frontier of melatonin in near future. Further investigations into the targets and functions of

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melatonin-based MTLs will be beneficial to develop new strategies for the therapy of neurodegenerative diseases.

study

was

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This

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Acknowledgments

supported

by

Guangdong

Provincial

International

Cooperation Project of Science & Technology (No. 2013B051000038) and National Science

Foundation

of

China

(No.31371070) to Pi R.,

and

Natural

Scientific

and

Technological Cooperation between the Italian Republic and the People’s Republic of China for Year 2013-2015 (No. MAE-M00705\CN13MO9) to Pi R. and Macchia M..

Conflict of interests

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None.

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Legend:

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Fig.1. Involved signaling pathways in melatonin mechanism to promote NSCs survival and modulate NSCs proliferation and differentiation. PARP, poly ADP-ribose polymerases EIK-1, ets like gene-1

c-fos, EIK-1 target gene

histone acetyl transferase

MSK, mitogen

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Nuclear factor erythroid2 related factor-2

ribosomal s6 kinase

and stress activated kinase

HAT, CREB,

Mash, Basic Helix-Loop-Helix Factors

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cAMP-response element binding protein

RSK, p90

Keap1, Kelch-like ECH-associated protein 1.

Nrf2,

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Table 1. The effects and most effective concentrations of melatonin in different neural stem cells.

Effects

C17.2 Cortical NSCs

Differentiation survival

Most effective concentration 1 nM 100 nM

NSCs obtained from pregnant rat midbrain

survival

1 nM

Proliferation

1 µM

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Differentiation

25 µM

Proliferation

1 µM

Sotthibundhu, A., et al. 2010

Survival

100 nM

Fu, J., et al. 2011

Proliferation

1 µM

Tocharus, C., et al. 2014

Survival

100 nM

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Ischemic-stroke mice

MSCs exposed to H2O2

1 µM

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Differentiation

New neurons in the hippocampus of adult mice

Kong, X., et al. 2008

Moriya, T., et al. 2007

NSCs obtained from adult mouse SVZ NSCs subjected to hypoxia Adult rat hippocampal progenitor cell

Sharma, R., et al. 2008 Song, J., et al. 2015

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NSCs derived from the mouse embryo striatum

Reference

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Cells

Ramirez-Rodriguez, G., et al. 2009

Differentiation

1 nM

Neurogenesis

10 mg/kg

Proliferation protection

10 mg/kg 100 µM

Chern, C., et al. 2012 Zhou, L., et al. 2015

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Name

Structure

Reference

Agomelatine (S20098)

Increase NPCs proliferation

AlAhmed, S., et al. 2010

SSH-BM-I

Stimulate terminal osteoblast differentiation

Mikami, Y., et al. 2011

2-vinyl-8-hydroxyquinoline derivatives (Compound 4)

Increase proliferation; Protection effects against oxidative stress in MSCs

Wang, T., et al. 2010

Increase NPCs proliferation

Sompol, P., et al. 2011

Differentiation Protection Inhibitary of AChE

Lopez-Iglesias B., et al. 2014

Stimulate early neurogenesis and neuronal maturation

de la Fuente Revenga, M., et al. 2015

Stimulate early neurogenesis and neuronal maturation

de la Fuente Revenga, M., et al. 2015

promote differentiation of rat NSCs to a neuronal phenotype

de la Fuente Revenga, M., et al. 2015

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Activity

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Table 2. The effects of Melatonin derivatives/analogues on neural stem cells.

Melatonin−N,Ndibenzyl (Nmethyl) amine hybrids

Pinoline

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Melatonin-pinoline hybrid (compound 2)

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N-acetylserotonin (NAS)

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N‑Acetyl Bioisosteres of Melatonin (compound 16)

Note: MSCs, mesenchymal stem cells; NPCs, neural progenitor cells.

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Highlights： ： Melatonin modulates cell fate of neural stem cells (NSCs).




Melatonin modulates cell fate of NSCs via multiple mechanisms.




Melatonin analogues or derivatives show good effects on NSCs.




The multi-targeted compounds show therapeutic potential in Alzheimer’s disease.

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