Ampakine CX546 increases proliferation and neuronal differentiation in subventricular zone stem/progenitor cell cultures

Ampakines are chemical compounds known to modulate the properties of ionotropic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)-subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis-enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single-cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau-positive axons co-labelled for the phosphorylated form of SAPK/JNK (P-JNK), and dendritogenesis (MAP2-positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell-based therapies.     

Dnmt3a regulates both proliferation and differentiation of mouse neural stem cells

DNA methylation is known to regulate cell differentiation and neuronal function in vivo. Here we examined whether deficiency of a de novo DNA methyltransferase, Dnmt3a, affects in vitro differentiation of mouse embryonic stem cells (mESCs) to neuronal and glial cell lineages. Early‐passage neural stem cells (NSCs) derived from Dnmt3a‐deficient ESCs exhibited a moderate phenotype in precocious glial differentiation compared with wild‐type counterparts. However, successive passaging to passage 6 (P6), when wild‐type NSCs become gliogenic, revealed a robust phenotype of precocious astrocyte and oligodendrocyte differentiation in Dnmt3a^?/? NSCs, consistent with our previous findings in the more severely hypomethylated Dnmt1^?/? NSCs. Mass spectrometric analysis revealed that total levels of methylcytosine in Dnmt3a^?/? NSCs at P6 were globally hypomethylated. Moreover, the Dnmt3a^?/? NSC proliferation rate was significantly increased compared with control from P6 onward. Thus, our work revealed a novel role for Dnmt3a in regulating both the timing of neural cell differentiation and the cell proliferation in the paradigm of mESC‐derived‐NSCs. © 2012 Wiley Periodicals, Inc.     

HIV-1-infected and/or immune-activated macrophage-secreted TNF-alpha affects human fetal cortical neural progenitor cell proliferation and differentiation

Neurogenesis, tied to the proliferation, migration and differentiation of neural progenitor cells (NPC) is affected during neurodegenerative diseases, but how neurogenesis is affected during HIV-1 associated dementia (HAD) has not been fully addressed. Here we test the hypothesis that HIV-1-infected and/or immune-activated brain macrophages affect NPC proliferation and differentiation through the regulation of cytokines. We showed that human monocyte-derived macrophages (MDM) conditioned medium (MCM) induces a dose dependent increase in NPC proliferation. Conditioned media from lipopolysaccharide (LPS)-activated MDM (LPS-MCM) or HIV-infected MCM (HIV-MCM) induced a profound increase in NPC proliferation. HIV-infected and LPS-activated MCM (HIV+LPS-MCM) induced the most robust increase in NPC proliferation. Moreover, LPS-MCM and HIV+LPS-MCM decreased beta-III-tubulin and increased GFAP expression, demonstrating an induction of gliogenesis and inhibition of neurogenesis. The increase of NPC proliferation and gliogenesis correlated with increases in production of TNF-alpha by infected/activated MDM. Although both IL-1beta and TNF-alpha induced NPC proliferation and gliogenesis, these effects were only partially abrogated by soluble TNF-alpha receptors R1 and R2 (TNF-R1R2), but not by the IL-1 receptor antagonist (IL-1ra). This indicated that the HIV-1-infected/LPS-activated MCM-mediated effects were, in part, through TNF-alpha. These observations were confirmed in severe combined immunodeficient (SCID) mice with HIV-1 encephalitis (HIVE). In these HIVE mice, NPC injected with HIV-infected MDM showed more astrocyte differentiation and less neuronal differentiation compared to NPC injection alone. These observations demonstrated that HIV-1-infected and immune-activated MDM could affect neurogenesis through induction of NPC proliferation, inhibition of neurogenesis, and activation of gliogenesis.     

Brain‐derived neurotrophic factor stimulates proliferation and differentiation of neural stem cells,possibly by triggering the Wnt/β‐catenin signaling pathway

Brain‐derived neurotrophic factor (BDNF) has critical functions in promoting survival, expansion, and differentiation of neural stem cells (NSCs), but its downstream regulation mechanism is still not fully understood. The role of BDNF in proliferation and differentiation of NSCs through Wnt/β‐catenin signaling was studied via cell culture of cortical NSCs, Western blotting, immunocytochemistry, and TOPgal (Wnt reporter) analysis in mice. First, BDNF stimulated NSC proliferation dose dependently in cultured neurospheres that exhibited BrdU incorporation and neuronal and glial differentiation abilities. Second, BDNF effectively enhanced cell commitment to neuronal and oligodendrocytic fates, as indicated by increased differentiation marker Tuj‐1 (neuronal marker), CNPase (oligodendrocyte marker), and neuronal process extension. Third, BDNF upregulated expression of Wnt/β‐catenin signaling (Wnt1 and free β‐catenin) molecules. Moreover, these promoting effects were significantly inhibited by application of IWR1, a Wnt signaling‐specific blocker in culture. The TOPgal mouse experiment further confirmed BDNF‐triggered Wnt signaling activation by β‐gal labeling. Finally, an MEK inhibition experiment showed a mediating role of the microtubule‐associated protein kinase pathway in BDNF‐triggered Wnt/β‐catenin signaling cascades. This study overall has revealed that BDNF might contribute to proliferation and neuronal and oligodendrocytic differentiation of NSCs in vitro, most possibly by triggering the Wnt/β‐catenin signaling pathway. Nevertheless, determining the exact cross‐talk points at which BDNF might stimulate Wnt/β‐catenin signaling pathway in NSC activity requires further investigation. © 2012 Wiley Periodicals, Inc.     

Tumor suppressor gene BRCA-1 is expressed by embryonic and adult neural stem cells and involved in cell proliferation

BRCA-1 is a tumor suppressor gene that plays a role in DNA repair and cellular growth control. Here we show that BRCA-1 mRNA is expressed by embryonic rat brain and is localized to the neuroepithelium containing neuronal precursor cells. The expression of BRCA-1 decreases during rat brain development, but BRCA-1 is expressed postnatally by proliferating neuronal precursor cells in the developing cerebellum. Neural stem cells (NSC) prepared from embryonic rat brain and cultured in the presence of epidermal growth factor were positive for BRCA-1. Induction of NSC differentiation resulted in down-regulation of BRCA-1 expression as shown by RNA and protein analyses. In addition to embryonic cells, BRCA-1 is also present in NSC prepared from adult rat brain. In adult rats, BRCA1 was expressed by cells in the walls of brain ventricles and in choroid plexus. The results show that BRCA-1 is present in embryonic and adult rat NSC and that the expression is linked to NSC proliferation.     

Soluble mediators from human neural stem cells play a critical role in suppression of T‐cell activation and proliferation

Human neural stem cells (hNSCs) can control inflammation in the central nervous system, although the underlying mechanisms are not understood fully. We investigated the immunomodulatory effect of hNSCs on human T cells and the underlying mechanisms. Culture supernatant from an immortalized hNSC cell line, HB1.F3, which has a therapeutic effect on acute stroke and intracerebral hemorrhage, suppressed the proliferation of allogeneically or mitogenically stimulated human peripheral T cells, including the CD3⁺CD103⁺ subpopulation. CFSE labeling and flow cytometry showed that the suppression of proliferation was caused by cell cycle arrest and induction of apoptosis. The lack of significant change in caspase‐8 levels and the significant reduction in Bcl‐2 expression in the affected T cells suggest that the intrinsic pathway plays a major role in soluble‐factor‐mediated T‐cell apoptosis. The addition of culture supernatant from hNSCs to activated T cells reduced the expression of the activation markers CD69 and CD25 at 24 hr after activation, but at 48 hr only CD69 was down‐regulated. A cytometry bead assay showed that the secretion of interleukin (IL)‐2 decreased significantly, whereas that of IL‐4, IL‐10, tumor necrosis factor‐α, and interferon‐γ increased. These results show that hNSCs can negatively affect human peripheral T cells by suppressing their activation and proliferation through soluble mediators, suggesting that hNSCs have a bystander immunomodulatory effect on T cells. © 2009 Wiley‐Liss, Inc.     

Transplantation of galectin‐1‐expressing human neural stem cells into the injured spinal cord of adult common marmosets

Delayed transplantation of neural stem/progenitor cells (NS/PCs) into the injured spinal cord can promote functional recovery in adult rats and monkeys. To enhance the functional recovery after NS/PC transplantation, we focused on galectin‐1, a carbohydrate‐binding protein with pleiotropic roles in cell growth, differentiation, apoptosis, and neurite outgrowth. Here, to determine the combined therapeutic effect of NS/PC transplantation and galectin‐1 on spinal cord injury (SCI), human NS/PCs were transfected by lentivirus with galectin‐1 and green fluorescent protein (GFP), (Gal‐NS/PCs) or GFP alone (GFP‐NS/PCs), expanded in vitro, and then transplanted into the spinal cord of adult common marmosets, 9 days after contusive cervical SCI. The animals' motor function was evaluated by their spontaneous motor activity, bar grip power, and performance on a treadmill test. Histological analyses revealed that the grafted human NS/PCs survived and differentiated into neurons, astrocytes, and oligodendrocytes. There were significant differences in the myelinated area, corticospinal fibers, and serotonergic fibers among the Gal‐NS/PC, GFP‐NS/PC, vehicle‐control, and sham‐operated groups. The Gal‐NS/PC‐grafted animals showed a better performance on all the behavioral tests compared with the other groups. These findings suggest that Gal‐NS/PCs have better therapeutic potential than NS/PCs for SCI in nonhuman primates and that human Gal‐NS/PC transplantation might be a feasible treatment for human SCI. © 2010 Wiley‐Liss, Inc.     

Activation of adenosine A1 receptor-induced neural stem cell proliferation via MEK/ERK and Akt signaling pathways

Adenosine, a modulator of neuronal function in the mammalian central nervous system, exerts a neuroprotective effect via the adenosine A(1) receptor; however, its effect on neural stem cells (NSCs) remains unclear. Because adenosine is released in response to pathological conditions and NSCs play a key role in neuroregeneration, we tested the hypothesis that adenosine is capable of stimulating NSC proliferation. We demonstrated that NSCs dominantly express adenosine A(1) and A(2B) receptors. Adenosine and the adenosine A(1) receptor agonist cyclopentyladenosine (CPA) increased proliferation of NSCs, and this CPA-induced cell proliferation was attenuated by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPA). CPA also induced phosphorylation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase/ERK kinase (MEK), and Akt, and their phosphorylation was inhibited by DPCPA. In addition, CPA-induced cell proliferation was inhibited by MEK and Akt inhibitors. These results suggest that activation of adenosine A(1) receptor-stimulated proliferation of NSCs occurs via MEK/ERK and Akt signaling pathways.     

Dopamine D3 receptor activation promotes neural stem/progenitor cell proliferation through AKT and ERK1/2 pathways and expands type‐B and ‐C cells in adult subventricular zone

The neurotransmitter dopamine acts on the subventricular zone (SVZ) to regulate both prenatal and postnatal neurogenesis, in particular through D₃ receptor (D₃R) subtype. In this study, we explored the cellular mechanism(s) underlying D₃R‐mediated cell proliferation and tested if systemic delivery of a D₃R agonist would induce SVZ multipotent neural stem/precursor cell (NSC/NPC) proliferation in vivo. We found that treatment with the D₃R agonist, 7‐OH‐DPAT, enhances cell proliferation in a dose‐dependent manner in cultured SVZ neurospheres from wild‐type, but not D₃R knock‐out mice. Furthermore, D₃R activation also stimulates S‐phase and enhances mRNA and protein levels of cyclin D1 in wild‐type neurospheres, a process which requires cellular Akt and ERK1/2 signaling. Moreover, chronic treatment with low dose 7‐OH‐DAPT in vivo increases BrdU⁺ cell numbers in the adult SVZ, but this effect was not seen in D₃R KO mice. Additionally, we probed the cell type specificity of D₃R agonist‐mediated cell proliferation. We found that in adult SVZ, GFAP⁺ astrocytes, type‐B GFAP⁺/nestin⁺ and type‐C EGF receptor (EGFR⁺)/nestin⁺ cells express D₃R mRNA, but type‐A Doublecortin (Dcx)⁺ neuroblasts do not. Using flow cytometry and immunofluorescence, we demonstrated that D₃R activation increases GFAP⁺ type‐B and EGFR⁺ type‐C cell numbers, and the newly divided Dcx⁺ type‐A cells. However, BrdU⁺/Dcx⁺ cell numbers were decreased in D₃R KO mice compared to wildtype, suggesting that D₃R maintains constitutive NSC/NPCs population in the adult SVZ. Overall, we demonstrate that D₃R activation induces NSC/NPC proliferation through Akt and ERK1/2 signaling and increases the numbers of type‐B and ‐C NSC/NPCs in the adult SVZ. © 2013 Wiley Periodicals, Inc.     

Correlation between receptor-interacting protein 140 expression and directed differentiation of human embr yonic stem cells into neural stem cells

Overexpression of receptor-interacting protein 140 (RIP140) promotes neuronal differentiation of N2a cells via extracellular regulated kinase 1/2 (ERK1/2) signaling. However, involvement of RIP140 in human neural differentiation remains unclear. We found both RIP140 and ERK1/2 expression increased during neural differentiation of H1 human embryonic stem cells. Moreover, RIP140 negatively correlat-ed with stem cell markers Oct4 and Sox2 during early stages of neural differentiation, and positively correlated with the neural stem cell marker Nestin during later stages. hTus, ERK1/2 signaling may provide the molecular mechanism by which RIP140 takes part in neural differentiation to eventually affect the number of neurons produced.     

Primary cilia mediate sonic hedgehog signaling to regulate neuronal‐like differentiation of bone mesenchymal stem cells for resveratrol induction in vitro

Mesenchymal stem cells (MSCs) can differentiate into neuronal‐like cell types under specific conditions. The classical antioxidant inducers such as β‐mercaptoethanol (BME), butylated hydroxyanisol (BHA), and dimethylsulfoxide (DMSO) are limited in clinical because of toxicity. Resveratrol, a safer, natural antioxidant, can stimulate osteoblastic differentiation of MSCs. However, its effect of inducing MSCs to differentiate into neuronal‐like cells is less well studied, and its differentiated mechanisms are not well understood. Sonic hedgehog (Shh) signaling, mediated by the primary cilia, is crucial for embryonic development and tissue differentiation, but relatively little is known about the role of Shh signaling and primary cilia in neuronal‐like differentiation of MSCs. Here we show that primary cilia, harboring patched 1 (Ptc1), are present in growth‐arrested MSCs and that smoothened (Smo) and Gli1 are present in cytoplasm of MSCs, which are important components of the Shh signaling pathway. After resveratrol induction, MSCs acquire neuronal‐like cell morphologies and phenotypes, Smo translocates to the primary cilia, Gli1 enters the nucleus, and expressions of Smo and Gli1 proteins increase, which can be inhibited by cyclopamine, a Smo antagonist. Meanwhile, Smo agonist (SAG) attains similar effects compared with the resveratrol group. These data indicate that resveratrol can induce MSCs to differentiate into neuronal‐like cells and activate Shh signaling pathway in the primary cilia. Moreover, the primary cilia and Shh signaling are essential for resveratrol inducing neuronal‐like differentiation of MSCs. Our finding is important for understanding the neuronal‐like differentiation mechanism of MSCs for resveratrol and promoting its clinical therapeutic utility. © 2014 Wiley Periodicals, Inc.     

Erythropoietin promotes the differentiation of fetal neural stem cells into glial cells via the erythropoietin receptor‐β common receptor/Syne‐1/H3K9me3 pathway

AimsTo investigate the effect of erythropoietin (EPO) on the differentiation of neural stem cells (NSCs)/neural progenitors (NPs) in the treatment of hypoxic–ischemic injury and its potential mechanisms.MethodsFetal NSCs/NPs were treated with EPO after oxygen and glucose deprivation/reoxygenation (OGD/R). Cell viability, proliferation, and differentiation of NSCs/NPs were detected by CellTiter‐Glo, Edu assay, flow cytometry, and quantitative real‐time PCR (qPCR). Immunofluorescence staining, co‐immunoprecipitation (Co‐IP), and western blotting were used to test the existence of EPO receptor/β common receptor (EPOR/βCR) heterodimer on NSCs/NPs and the possible pathway.ResultsEPO treatment at different time points increased cell viability without affecting proliferation. EPO treatment immediately after OGD/R promoted oligodendrocyte and astrocyte differentiation, while decreasing neuronal differentiation of NSCs/NPs. EPOR/βCR heterodimer existed on the cell surface of the fetal cortical NSCs/NPs, EPO treatment significantly increased the mRNA expression of βCR and elevated the correlation between EPOR and βCR levels. In addition, mass spectrometry analysis identified Syne‐1 as a downstream signaling molecule of the EPOR/βCR heterodimer. Immunofluorescence staining and western blotting indicated that the βCR/Syne‐1/H3K9me3 pathway was possibly involved in the differentiation of fetal neural stem cells into the glial cell effect of EPO.ConclusionEPO treatment immediately after OGD/R could not facilitate fetal NSCs/NPs neurogenesis but promoted the formation of the EPOR/βCR heterodimer on fetal NSCs/NPs, which mediates its function in glial differentiation.     

Function of death‐associated protein 1 in proliferation,differentiation, and apoptosis of chicken satellite cells

Introduction: Muscle growth and regeneration are processes closely associated with proliferation, differentiation, and apoptosis of muscle cells. Death‐associated protein 1 (DAP1) has been identified as a negative regulator of autophagy. Little is known about the function of DAP1 in the regulation of myogenesis and satellite cells. Methods: Chicken satellite cells were transfected with DAP1 cloned into the pCMS–enhanced green fluorescent protein vector or pcDNA3.1 vector, or a small interference RNA against the endogenous DAP1 gene. The cells were assayed for proliferation, differentiation, and apoptosis. Results: The overexpression of DAP1 increased proliferation, differentiation, and myotube diameter, but it had no effect on satellite cell apoptosis. In contrast, knockdown of DAP1 significantly decreased proliferation, differentiation, and number of nuclei per myotube, and it increased apoptosis of the cells. Conclusion: DAP1 is required for regulating myogenesis and apoptosis of satellite cells, which may affect muscle mass accretion and regeneration, and ameliorate muscle sarcopenia. Muscle Nerve 48:777–790, 2013     

Nurr1对小胶质细胞联合神经干细胞共培养促进神经干细胞向多巴胺神经元分化作用研究

目的探讨联合过表达核受体相关因子1(Nurr1)基因的小胶质细胞(MG)和神经干细胞(NSC)共培养对神经干细胞向多巴胺神经元分化的影响。方法原代培养SD大鼠神经干细胞和小胶质细胞,并过表达Nurr1基因。CCK-8法检测Nurr1过表达对神经干细胞以及小胶质细胞活率的影响。Transwell系统共培养神经干细胞和小胶质细胞,实验分为NSC组、NSC+MG组和N(NSC+MG)组。ELISA检测共培养后第3天、第6天和第9天各组脑源性神经营养因子(BDNF)、血小板源性神经营养因子(PDNF)和胶质细胞源性神经营养因子(GDNF)表达变化;RT-PCR和Western Blot检测各组第9天酪氨酸羟化酶(TH)、多巴胺转运蛋白(DAT)DAT和Nurr1的表达变化;细胞免疫荧光鉴定神经干细胞的分化,并对TH和DAT阳性细胞计数,计算各组神经干细胞向多巴胺神经元的分化效率。结果原代培养小胶质细胞以及神经干细胞并成功过表达Nurr1基因。CCK-8法检测结果表明,Nurr1过表达对神经干细胞以及小胶质细胞活率无明显影响。ELISA检测结果表明,N(NSC+MG)组在不同时间点神经营养因子(BDNF、PDNF和GDNF)表达量明显高于其他各组(P0.05)。RT-PCR和Westen Blot检测结果表明,N(NSC+MG)组TH、DAT和Nurr1的表达水平明显高于其他各组(P0.05)。细胞免疫荧光鉴定结果表明,N(NSC+MG)组TH阳性细胞率明显高于其他各组(P0.05)。结论Nurr1基因可促进神经干细胞和小胶质细胞共培养系统神经营养因子的分泌。过表达Nurr1基因的神经干细胞和小胶质细胞共培养可促进神经干细胞向多巴胺神经元的分化。