Combination of Melatonin and Small Molecules Improved Reprogramming Neural Cell Fates via Autophagy Activation.

Reprogramming cell fates towards mature cell types are a promising cell supply for treating degenerative diseases. Recently, transcription factors and some small molecules have turned into impressive modulating elements for reprogramming cell fates. Melatonin, a pineal hormone, has neuroprotective functions including neural stem cell (NSC) proliferative and differentiative modulation in both embryonic and adult brain. We developed a protocol that could be implemented in the direct reprogramming of human skin fibroblast towards neural cells by using histone deacetylase (HDAC) inhibitor, glycogen synthase kinase-3 (GSK3) inhibitor (CHIR99021), c-Jun N-terminal kinase (JNK) inhibitor, rho-associated protein kinase inhibitor (Y-27632), cAMP activator, and melatonin treatment. We found that melatonin enhanced neural-transcription factor genes expressions, including brain-specific homeobox/POU domain protein 2 (BRN2), Achaete-Scute Family BHLH transcription Factor 1 (ASCL1), and Myelin Transcription Factor 1 Like (MYT1L). Melatonin also increased the expression of different neural-specific proteins such as doublecortin (DCX), Sex determining region Y-box 2 (Sox2), and neuronal nuclei (NeuN) compared with other five small molecules (valproic acid (VPA), CHIR99021, Forskolin, 1,9 pyrazoloanthrone (SP600125), and Y-27632) combination in the presence and absence of melatonin. A noticeable upregulation of autophagy proteins (microtubule-associated protein 1A/1B-light chain 3 (LC3) and Beclin-1) were seen in the melatonin treatment during the induction period while these were reverted in the presence of L-leucine, an autophagy inhibitor. In addition, the expression of NeuN was also significantly reduced by L-leucine. Collectively, our findings revealed an activation of autophagy during neural induction; melatonin enhanced reprogramming efficiency for neuron induction through the modulation of autophagy activation.

Roles of autophagy in controlling stem cell identity: a perspective of self-renewal and differentiation.

Autophagy is crucial for the removal of dysfunctional organelles and protein aggregates and for maintaining stem cell homeostasis, which includes self-renewal, cell differentiation and somatic reprogramming. Loss of self-renewal capacity and pluripotency is a major obstacle to stem cell-based therapies. It has been reported that autophagy regulates stem cells under biological stimuli, starvation, hypoxia, generation of reactive oxygen species (ROS) and cellular senescence. On the one hand, autophagy is shown to play roles in self-renewal by co-function with the ubiquitin-proteasome system (UPS) to promote pluripotency-associated proteins (NANOG, OCT4 and SOX2) in human embryonic stem cells (hESCs). On the other hand, autophagy activity acts as cell reprogramming processes that play an important role for clearance fate determination and upregulates neural and cardiac differentiation. Deregulation of autophagy triggers protein disorders such as neurodegenerative cardiac/muscle diseases and cancer. Therefore, understanding of the roles of the autophagy in stem cell renewal and differentiation may benefit therapeutic development for a range of human diseases.

Melatonin attenuates methamphetamine-induced inhibition of proliferation of adult rat hippocampal progenitor cells in vitro.

Methamphetamine (METH) is an extremely addictive stimulatory drug. A recent study suggested that METH may cause an impairment in the proliferation of hippocampal neural progenitor cells, but the underlying mechanism of this effect remains unknown. Blood and cerebrospinal levels of melatonin derive primarily from the pineal gland, and that performs many biological functions. Our previous study demonstrated that melatonin promotes the proliferation of progenitor cells originating from the hippocampus. In this study, hippocampal progenitor cells from adult Wistar rats were used to determine the effects of METH on cell proliferation and the mechanisms underlying these effects. We investigated the effects of melatonin on the METH-induced alteration in cell proliferation. The results demonstrated that 500 µm METH induced a decrease (63.0%) in neurosphere cell proliferation and altered the expression of neuronal phenotype markers in the neurosphere cell population. Moreover, METH induced an increase in the protein expression of the tumor suppressor p53 (124.4%) and the cell cycle inhibitor p21(CIP) (1) (p21) (128.1%), resulting in the accumulation of p21 in the nucleus. We also found that METH altered the expression of the N-methyl-d-aspartate (NMDA) receptor subunits NR2A (79.6%) and NR2B (126.7%) and Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) (74.0%). In addition, pretreatment with 1 µm melatonin attenuated the effects induced by METH treatment. According to these results, we concluded that METH induces a reduction in cell proliferation by upregulating the cell cycle regulators p53/p21 and promoting the accumulation of p21 in the nucleus and that melatonin ameliorates these negative effects of METH.

Autophagy Promoted Neural Differentiation of Human Placenta-derived Mesenchymal Stem Cells.

BACKGROUND/AIM: Human placenta-derived mesenchymal stem cells (hPMSCs) are multipotent and possess neurogenicity. Numerous studies have shown that Notch inhibition and DNA demethylation promote neural differentiation. Here, we investigated the modulation of autophagy during neural differentiation of hPMSCs, induced by DAPT and 5-Azacytidine. MATERIALS AND METHODS: hPMSCs were treated with DAPT to induce neural differentiation, and the autophagy regulating molecules were used to assess the impact of autophagy on neural differentiation. RESULTS: The hPMSCs presented with typical mesenchymal stem cell phenotypes, in which the majority of cells expressed CD73, CD90 and CD105. hPMSCs were multipotent, capable of differentiating into mesodermal cells. After treatment with DAPT, hPMSCs upregulated the expression of neuronal genes including SOX2, Nestin, and ßIII-tubulin, and the autophagy genes LC3I/II and Beclin. These genes were further increased when 5-Azacytidine was co-supplemented in the culture medium. The inhibition of autophagy by chloroquine impeded the neural differentiation of hPMSCs, marked by the downregulation of ßIII-tubulin, while the activation of autophagy by valproic acid (VPA) instigated the emergence of ßIII-tubulin-positive cells. CONCLUSION: During the differentiation process, autophagy was modulated, implying that autophagy could play a significant role during the differentiation of these cells. The blockage and stimulation of autophagy could either hinder or induce the formation of neural-like cells, respectively. Therefore, the refinement of autophagic activity at an appropriate level might improve the efficiency of stem cell differentiation.

Melatonin increases proliferation of cultured neural stem cells obtained from adult mouse subventricular zone.

Melatonin, a circadian rhythm-promoting molecule secreted mainly by the pineal gland, has a variety of biological functions and neuroprotective effects including control of sleep-wake cycle, seasonal reproduction, and body temperature as well as preventing neuronal cell death induced by neurotoxic substances. Melatonin also modulates neural stem cell (NSC) function including proliferation and differentiation in embryonic brain tissue. However, the involvement of melatonin in adult neurogenesis is still not clear. Here, we report that precursor cells from adult mouse subventricular zone (SVZ) of the lateral ventricle, the main neurogenic area of the adult brain, express melatonin receptors. In addition, precursor cells derived from this area treated with melatonin exhibited increased proliferative activity. However, when cells were treated with luzindole, a competitive inhibitor of melatonin receptors, or pertussis toxin, an uncoupler of Gi from adenylate cyclase, melatonin-induced proliferation was reduced. Under these conditions, melatonin induced the differentiation of precursor cells to neuronal cells without an upregulation of the number of glia cells. Because stem cell replacement is thought to play an important therapeutic role in neurodegenerative diseases, melatonin might be beneficial for stimulating endogenous neural stem cells.

Beta-amyloid(1-42) induces neuronal death through the p75 neurotrophin receptor.

Alzheimer's disease is characterized by the accumulation of neurotoxic amyloidogenic peptide Abeta, degeneration of the cholinergic innervation to the hippocampus (the septohippocampal pathway), and progressive impairment of cognitive function, particularly memory. Abeta is a ligand for the p75 neurotrophin receptor (p75(NTR)), which is best known for mediating neuronal death and has been consistently linked to the pathology of Alzheimer's disease. Here we examined whether p75(NTR) is required for Abeta-mediated effects. Treatment of wild-type but not p75(NTR)-deficient embryonic mouse hippocampal neurons with human Abeta(1-42) peptide induced significant cell death. Furthermore, injection of Abeta(1-42) into the hippocampus of adult mice resulted in significant degeneration of wild-type but not p75(NTR)-deficient cholinergic basal forebrain neurons, indicating that the latter are resistant to Abeta-induced toxicity. We also found that neuronal death correlated with Abeta(1-42) peptide-stimulated accumulation of the death-inducing p75(NTR) C-terminal fragment generated by extracellular metalloprotease cleavage of full-length p75(NTR). Although neuronal death was prevented in the presence of the metalloprotease inhibitor TAPI-2 (tumor necrosis factor-alpha protease inhibitor-2), Abeta(1-42)-induced accumulation of the C-terminal fragment resulted from inhibition of gamma-secretase activity. These results provide a novel mechanism to explain the early and characteristic loss of cholinergic neurons in the septohippocampal pathway that occurs in Alzheimer's disease.

Small molecules re-establish neural cell fate of human fibroblasts via autophagy activation.

The generation of neural cells is of great interest in medical research because of its promising in neurodegenerative diseases. Small chemical molecules have been used for inducing specific cell types across lineage boundaries. Therefore, to direct neural cell fate, small molecule is a feasible approach for generating clinically relevant cell types without genetic alterations. Human fibroblasts have been directly induced into neural cells with different combinations of small molecules; however, the mechanism underlying neural induction is still not fully understood. In this study, human fibroblasts were induced into neural cells by using only 4 small molecules in a short time period, 5 d. Small molecules used in this study included WNT activator, DNMT inhibitor, Notch inhibitor, and retinoic acid. Neural-specific genes, including NESTIN, TUJ1, and SOX2, were upregulated upon the induction for 5 d. Noteworthy, this neural induction process by small molecules coincided with the activation of autophagy. Autophagy-related genes, such as LC3, ATG12, and LAMP1, were enhanced upon neural induction, and the number of induced-neural cells decreased when autophagy was suppressed by chloroquine. The activation of autophagy was found to reduce ROS generation within the induced-neural cells, and the inhibition of autophagy by chloroquine suppressed the expression of antioxidant genes, CATALASE, SOD, and GPX. This implied that autophagy maintained the optimal level of ROS for neural induction of human fibroblasts. Altogether, this study presented the effective and convenient condition to induce neural cells from human fibroblasts and revealed the positive roles of autophagy in controlling neural cell induction.

p75 neurotrophin receptor expression defines a population of BDNF-responsive neurogenic precursor cells.

Although our understanding of adult neurogenesis has increased dramatically over the last decade, confusion still exists regarding both the identity of the stem cell responsible for neuron production and the mechanisms that regulate its activity. Here we show, using flow cytometry, that a small population of cells (0.3%) within the stem cell niche of the rat subventricular zone (SVZ) expresses the p75 neurotrophin receptor (p75(NTR)) and that these cells are responsible for neuron production in both newborn and adult animals. In the adult, the p75(NTR)-positive population contains all of the neurosphere-producing precursor cells, whereas in the newborn many of the precursor cells are p75(NTR) negative. However, at both ages, only the neurospheres derived from p75(NTR)-positive cells are neurogenic. We also show that neuron production from p75(NTR)-positive but not p75(NTR)-negative precursors is greatly enhanced after treatment with brain-derived neurotrophic factor (BDNF) or nerve growth factor. This effect appears to be mediated specifically by p75(NTR), because precursor cells from p75(NTR)-deficient mice show a 70% reduction in their neurogenic potential in vitro and fail to respond to BDNF treatment. Furthermore, adult p75(NTR)-deficient mice have significantly reduced numbers of PSA-NCAM (polysialylated neural cell adhesion molecule)-positive SVZ neuroblasts in vivo and a lower olfactory bulb weight. Thus, p75(NTR) defines a discrete population of highly proliferative SVZ precursor cells that are able to respond to neurotrophin activation by increasing neuroblast generation, making this pathway the most likely mechanism for the increased neurogenesis that accompanies raised BDNF levels in a variety of disease and behavioral situations.

Comparison of melatonin with growth factors in promoting precursor cells proliferation in adult mouse subventricular zone.

Melatonin, secreted mainly by the pineal gland, plays roles in various physiological functions including protecting cell death. We showed in previous study that the proliferation and differentiation of precursor cells from the adult mouse subventricular zone (SVZ) can be modulated by melatonin via the MT1 melatonin receptor. Since melatonin and epidermal growth factor receptor (EGFR) share some signaling pathway components, we investigated whether melatonin can promote the proliferation of precursor cells from the adult mouse SVZ via the extracellular signal-regulated protein kinase /mitogen-activated protein kinase (ERK/MAPK) pathways in comparison with epidermal growth factor (EGF). Melatonin-induced ERK/MAPK pathways compared with EGF were measured by using in vitro and vivo models. We used neurosphere proliferation assay, immunocytochemistry, and immuno-blotting to analyze significant differences between melatonin and growth factor treatment. We also used specific antagonist and inhibitors to confirm the exactly signaling pathway including luzindole and U0126. We found that significant increase in proliferation was observed when two growth factors (EGF+bFGF) and melatonin were used simultaneously compared with EGF + bFGF or compared with melatonin alone. In addition, the present result suggested the synergistic effect occurred of melatonin and growth factors on the activating the ERK/MAPK pathway. This study exhibited that melatonin could act as a trophic factor, increasing proliferation in precursor cells mediated through the melatonin receptor coupled to ERK/MAPK signaling pathways. Understanding the mechanism by which melatonin regulates precursor cells may conduct to the development of novel strategies for neurodegenerative disease therapy.

Rapamycin regulates autophagy and cell adhesion in induced pluripotent stem cells.

BACKGROUND: Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined. METHODS: Human iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with bafilomycin to block autophagy during iPSC maintenance. High concentrations of rapamycin treatment unexpectedly resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with rapamycin treatment. RESULTS: We demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5, and LC3B. Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs) with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of rapamycin in mediating iPSC detachment and differentiation. CONCLUSIONS: High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products. This research therefore highlights the potential of rapamycin in producing uniform EBs and in shortening iPSC differentiation duration.

Melatonin ameliorates dexamethasone-induced inhibitory effects on the proliferation of cultured progenitor cells obtained from adult rat hippocampus.

Glucocorticoids, hormones that are released in response to stress, induce neuronal cell damage. The hippocampus is a primary target of glucocorticoids in the brain, the effects of which include the suppression of cell proliferation and diminished neurogenesis in the dentate gyrus. Our previous study found that melatonin, synthesized primarily in the pineal, pretreatment prevented the negative effects of dexamethasone, the glucocorticoid receptor agonist, on behavior and neurogenesis in rat hippocampus. In the present study, we attempted to investigate the interrelationship between melatonin and dexamethasone on the underlying mechanism of neural stem cell proliferation. Addition of dexamethasone to hippocampal progenitor cells from eight-week old rats resulted in a decrease in the number of neurospheres; pretreatment with melatonin precluded these effects. The immunocytochemical analyses indicated a reduction of Ki67 and nestin-positive cells in the dexamethasone-treated group, which was minimized by melatonin pretreatment. A reduction of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and G1-S phase cell cycle regulators cyclin E and CDK2 in dexamethasone-treated progenitor cells were prevented by pretreatment of melatonin. Moreover, luzindole, a melatonin receptor antagonist blocked the positive effect of melatonin whereas RU48, the glucocorticoid receptor antagonist blocked the negative effect of dexamethasone on the number of neurospheres. Moreover, we also found that dexamethasone increased the glucocorticoid receptor protein but decreased the level of MT1 melatonin receptor, whereas melatonin increased the level of MT1 melatonin receptor but decreased the glucocorticoid receptor protein. These suggest the crosstalk and cross regulation between the melatonin receptor and the glucocorticoid receptor on hippocampal progenitor cell proliferation.

Melatonin attenuates methamphetamine-induced inhibition of neurogenesis in the adult mouse hippocampus: An in vivo study.

Methamphetamine (METH), a highly addictive psychostimulant drug, is known to exert neurotoxic effects to the dopaminergic neural system. Long-term METH administration impairs brain functions such as cognition, learning and memory. Newly born neurons in the dentate gyrus of the hippocampus play an important role in spatial learning and memory. Previous in vitro studies have shown that METH inhibits cell proliferation and neurogenesis in the hippocampus. On the other hand, melatonin, a major indole secreted by the pineal gland, enhances neurogenesis in both the subventricular zone and dentate gyrus. In this study, adult C57BL/6 mice were used to study the beneficial effects of melatonin on METH-induced alterations in neurogenesis and post-synaptic proteins related to learning and memory functions in the hippocampus. The results showed that METH caused a decrease in neuronal phenotypes as determined by the expressions of nestin, doublecortin (DCX) and beta-III tubulin while causing an increase in glial fibrillary acidic protein (GFAP) expression. Moreover, METH inhibited mitogen-activated protein kinase (MAPK) signaling activity and altered expression of the N-methyl-d-aspartate (NMDA) receptor subunits NR2A and NR2B as well as calcium/calmodulin-dependent protein kinase II (CaMKII). These effects could be attenuated by melatonin pretreatment. In conclusion, melatonin prevented the METH-induced reduction in neurogenesis, increase in astrogliogenesis and alteration of NMDA receptor subunit expression. These findings may indicate the beneficial effects of melatonin on the impairment of learning and memory caused by METH.

Abeta(1-42) stimulates adult SVZ neurogenesis through the p75 neurotrophin receptor.

The generation of amyloid-beta peptide (Abeta) and its accumulation in amyloid plaques are generally recognized as key characteristics of Alzheimer's disease. A number of reports have indicated that Abeta can regulate the proliferation of neural precursor cells and adult neurogenesis, suggesting that this may underpin the cognitive decline and compromised olfaction also associated with the condition. Here we report that Abeta(1-42) treatment both in vitro and in vivo, as well as endogenous generation of Abeta in C100 and APP/PS1 transgenic models of Alzheimer's disease, stimulate neurogenesis of young adult subventricular zone precursors. The neurogenic effect of Abeta(1-42) was found to require expression of the p75 neurotrophin receptor (p75(NTR)) by the precursor cells, and activation of p75(NTR) by metalloprotease cleavage. However, precursors from 12-month-old APP/PS1 mice failed to respond to Abeta(1-42). Our results suggest that overstimulation of p75(NTR)-positive progenitors during early life might result in depletion of the stem cell pool and thus a more rapid decline in basal neurogenesis. This, in turn, could lead to impaired neurogenic function in later life.