Telomerase and neuronal marker status of differentiated NT2 and SK-N-SH human neuronal cells and primary human neurons

Upon treatment with retinoic acid, NTera-2 (NT2) human teratocarcinoma and SK-N-SH neuroblastoma cells can be induced to terminally differentiate into postmitotic neuronal cells. The neuronal cell yield obtained from the NT-2 cells is partially dependent on the time of differentiation (24-55 days). SK-N-SH cells differentiate into a mixed population of neuronal and epithelium-like cells. Here we report modified protocols that increase the number of differentiated NT-2 and SK-N-SH cells and that establish an enriched neuronal SK-N-SH-derived cell population essentially devoid of nonneuronal cells. Differentiated cells express the cytoskeleton-associated protein tau and other typical neuronal markers, such as Map2, Ngn1, NeuroD, Mash1, and GluR which are also expressed in primary human fetal neurons. Telomerase activity is down-regulated in differentiated cells, which is consistent with the telomerase status of primary fetal human neurons. Thus, differentiated NT2 and SK-N-SH cells may represent an excellent source for studies investigating the role of telomerase or other survival-promoting activities in protecting human neuronal cells from cell death-mediating stresses associated with neurodegenerative diseases.     

Increased PKA and PKC activities accompany neuronal differentiation of NT2/D1 cells

After retinoic acid treatment, a large percentage of cells of the human embryonal carcinoma cell line NT2/D1 differentiate into neuronal cells. We demonstrate here that the differentiated cells, but not the undifferentiated cells, contain high levels of neurofilament mRNA. We have also measured mRNA, protein, and activity levels of two kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), in order to explore the role of protein kinases in the establishment of the differentiated state. RNA levels for the catalytic (C alpha and C beta) subunits of PKA increased after differentiation. Total PKA activity levels increased 7-fold in the differentiated cells. Parallel with this, a rise in the level of catalytic subunit protein occurred. A 12-fold induction of Type 2 (beta) PKC mRNA levels was observed after neuronal differentiation. Increases in PKC activity and in Type 2 (beta) and Type 3 (alpha) PKC protein levels also accompanied differentiation. These changes in PKA- and PKC-specific RNA levels and enzyme activity may be necessary for production and maintenance of the differentiated state in these cells.     

Enzymatic activities during differentiation of the human neuroblastoma cells, LA-N-1 and LA-N-2

The presence of 10(-5) M retinoic acid (RA) in the culture medium of LA-N-1, a catecholaminergic cell line, and LA-N-2, a cholinergic cell line, enhanced their morphological differentiation. Tyrosine hydroxylase (TH) activity of the LA-N-1 cells was increased in the RA-treated cells compared with control cultures at day 4 and remained elevated. Choline acetyltransferase (ChAT) activity in the LA-N-2 cells gradually increased until 8 days in vitro (DIV) both in the untreated control and the RA treated cultures. This activity in control and treated cells decreased gradually to a constant level of activity. The ChAT activity at 8 DIV of RA-treated LA-N-2 cells was increased 2.1-fold (P less than 0.001) as compared to the control cultures. This increase in ChAT activity was accompanied by a 73% decrease of acetylcholinesterase (AChE) activity in LA-N-2 cells by 8 DIV. AChE activity of LA-N-1 cells was unchanged during the time course of the experiment. Phospholipase-A2 (PL-A2) activity in RA-treated LA-N-2 cells was increased at day 4 as compared with the control cultures. There were no differences observed in phospholipase-D (PL-D), choline kinase and GPC-phosphodiesterases activities in RA-treated and -untreated LA-N-1 and LA-N-2 cells.     

Reduction of connexin43 expression and dye-coupling during neuronal differentiation of human NTera2/clone D1 cells

Gap junctions are plasma membrane specializations that allow direct communication among adjoining cells. We used a human pluripotential teratocarcinoma cell line, NTera-2/clone D1 (NT2/D1), as a model to study gap junctions in CNS neurons and their neuronal precursors. These cells were differentiated following retinoic acid (RA) treatment for 4 weeks and antiproliferative agents for 3 weeks, respectively, to yield post-mitotic CNS neuronal (NT2-N) cells. The cytoplasmic RNA was isolated from NT2/D1 cells both before and during RA treatment and from differentiated neurons (NT2-N cells). These RNA samples were examined using Northern blot analysis with cDNA probes specific for connexin26, −32, and −43. Connexin26 and −32 mRNAs were absent in NT2/D1 and NT2-N cells. Connexin43 mRNA was expressed at high levels in NT2/D1 cells before RA treatment, but it decreased significantly during RA induction. There was no detectable connexin43 mRNA in NT2-N cells. Western blot analysis confirmed the expression of connexin43 protein in NT2/D1 cells before and during RA treatment. The protein profile detected in Western blot analysis indicated two bands representing different phosphorylation states of connexin43. Our immunocytochemistry results did not show connexin26 and −32 immunoreactivity in NT2/D1 and NT2-N cells. However, we detected connexin43 immunoreactivity in NT2/D1 cells with a decreasing pattern upon RA induction. Both Western blotting and immunocytochemistry confirmed the absence of connexin43 protein in NT2-N cells. NT2/D1 cells passed calcein readily to an average of 18 cells, confirming the functionality of gap junctions in these cells. The extent of dye-coupling decreased about 78% when NT2/D1 cells were RA treated for 4 weeks. NT2-N differentiated neurons did not pass dye to the adjacent cells. We conclude that both connexin43 expression and dye coupling capacity decrease during neuronal differentiation of NT2/D1 cells. J. Neurosci. Res. 49:19–31, 1997. © 1997 Wiley-Liss Inc.     

The role of receptor for advanced glycation end products (RAGE) in neuronal differentiation

The receptor for advanced glycation end products (RAGE) is a multiligand receptor protein thought to play an important role in neuronal differentiation. RAGE can bind a number of ligands and activate a variety of signalling pathways that lead to diverse downstream effects. Amphoterin and S100B are endogenous ligands, the interaction of which with RAGE is known to be involved in defined physiological processes. The present study investigated the spatiotemporal pattern of the expression for RAGE and its ligands, amphoterin and S100B, during neuronal differentiation of NT2/D1 cells. In this study, all three proteins were shown to increase with progression of neuronal differentiation as determined by Western blotting, raising the possibility that both amphoterin and S100B may interact with RAGE and have important functions during the process of cell differentiation. Moreover, blocking the activation of RAGE with neutralizing antibody in the presence of retinoic acid disrupted the progression of normal neuronal differentiation. Immunocytochemistry (ICC) studies showed that amphoterin partially colocalized with RAGE within differentiating NT2 cells, whereas S100B showed a high degree of colocalization. This result suggests that S100B is more likely to be the principal ligand for RAGE during the differentiation process and that RAGE and amphoterin might have both independent and combined roles. Moreover, RAGE was expressed only in cells that were committed to a neuronal phenotype, suggesting direct involvement of RAGE in mediating cellular changes within differentiating neuronal cells. Further detailed studies are now required to characterize fully the role of RAGE during the neuronal differentiation period.     

Neuronal Differentiation of NT2/D1 Teratocarcinoma Cells Is Accompanied by a Loss of Lamin A/C Expression and an Increase in Lamin B1 Expression

Nuclear lamins are prominent elements of the nuclear matrix and are expressed in cell type-specific and differentiation state-specific patterns. A few observations have indicated that nervous tissue may display unusual patterns of lamin expression, in that some neurons appear to lack A-type lamins, which are generally prominently expressed in terminally differentiated, postmitotic cells. To investigate lamin expression patterns during the differentiation of a teratocarcinoma cell line into neurons, NT2/D1 cells were induced to differentiate with retinoic acid treatment. Lamin expression and organization during differentiation in vitro were examined by quantitative immunofluorescence and immunoblotting methods. Undifferentiated NT2/D1 cells were all strongly labeled with an anti-lamin B1 antibody, but displayed marked variation in A/C lamin immunoreactivity. After differentiation, neuronal nuclear envelopes were significantly more strongly labeled by anti-lamin B1 antibody than those of undifferentiated cells, but completely lacked A/C lamin immunoreactivity. In contrast, nonneuronal cells displayed a slight reduction in B1 lamin immunoreactivity, along with a distinct increase in A/C lamin levels. The loss of lamin A/C expression in NT2/D1 neurons is contrary to the pattern normally observed in most somatic cell types during early development and indicates that the nuclear matrix of some neurons, along with certain neuroendocrine and hematopoietic cells, is uniquely specialized in this regard.     

Differentiation-dependent progesterone synthesis and metabolism in NT2-N human neurons

Human embryonic teratocarcinoma-derived Ntera2/cl.D1 (NT2) cells recapitulate many features of embryonic neuronal progenitor cells. Upon retinoic acid (RA) treatment they terminally differentiate into post-mitotic neuron-like cells (NT2-N), akin to human fetal neurons, thus representing an in vitro model of human neuron terminal differentiation. Experimental evidence also indicate NT2-N cultures as a potential source for cell transplantation therapy. The neurosteroids progesterone and its metabolite 3α-hydroxy-5α-pregnan-20-one (3α,5α-THP) promote neurogenesis and show anti-neurodegenerative properties. This study's aim was to assess the neurosteroidogenic competence of NT2 cells during RA-induced neuronal differentiation. Radioimmunoassay measurements revealed progesterone only in NT2-N cultures (4 week RA). Accordingly, progesterone synthesis from ³H-pregnenolone was absent in NT2 cells and increased during RA exposure, being highest in NT2-N. [³H]-pregnenolone metabolism, yielding [³H]-progesterone and [³H]-5α-dihydroprogesterone ([³H]-5α-DHP), was time-dependent and inhibited by trilostane, a 3β-hydroxysteroid-dehydrogenase (3β-HSD) inhibitor. Conversely, ³H-progesterone metabolism, which yielded [³H]-5α-DHP > [³H]-3β,5α-THP > [³H]-3α,5α-THP, occurred at all time points examined, though showing a nadir in cultures treated with RA for 1 and 2 weeks. The differentiation-dependent increase of progesterone accumulation matched 3β-HSD type I mRNA expression and 3β-HSD immunoreactivity, that co-localized with Map2a/b- and GAD67 in NT2-N. Hence, in vitro differentiated human neurons, while retaining progesterone metabolic activity, also become competent in progesterone synthesis. These findings suggest an autocrine/paracrine role of neuronal progesterone, either on its own or through its 5α-reduced metabolites, in fetal brain development and allow speculation that NT2-N-produced neurosteroids may contribute to the encouraging results of NT2-N transplants in animal models of neurodegenerative diseases.     

Characterization of the NT2‐derived neuronal and astrocytic cell lines as alternative in vitro models for primary human neurons and astrocytes

Primary human fetal neurons and astrocytes (HFNs and HFAs, respectively) provide relevant cell types with which to study in vitro the mechanisms involved in various human neurological diseases, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease. However, the limited availability of human fetal cells poses a significant problem for the study of these diseases when a human cell model system is required. Thus, generating a readily available alternative cell source with the essential features of human neurons and astrocytes is necessary. The human teratoma‐derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal and glial cells can be generated. Nevertheless, a direct comparison of NT2 neurons and primary HFNs in terms of their morphology physiological and chemical properties is still missing. This study directly compares NT2‐derived neurons and primary HFNs using immunocytochemistry, confocal calcium imaging, high‐performance liquid chromatography, and high‐content analysis techniques. We investigated the morphological similarities and differences, levels of relevant amino acids, and internal calcium fluctuations in response to certain neurotransmitters/stimuli. We also compared NT2‐derived astrocytes and HFAs. In most of the parameters tested, both neuronal and astrocytic cell types exhibited similarities to primary human fetal neurons and astrocytes. NT2‐derived neurons and astrocytes are reliable in vitro tools and a renewable cell source that can serve as a valid alternative to HFNs/HFAs for mechanistic studies of neurological diseases. © 2014 Wiley Periodicals, Inc.     

Effects of neurotrophin-3 on the differentiation of neural stem cells into neurons and oligodendrocytes

In this study,cells from the cerebral cortex of fetal rats at pregnant 16 days were harvested and cultured with 20 μg/L neurotrophin-3.After 7 days of culture,immunocytochemical staining showed that,22.4% of cells were positive for nestin,10.5% were positive for β-III tubulin(neuronal marker),and 60.6% were positive for glial fibrillary acidic protein,but no cells were positive for O4(oligodendrocytic marker).At 14 days,there were 5.6% nestin-,9.6% β-III tubulin-,81.1% glial fibrillary acidic protein-,and 2.2% O4-positive cells.In cells not treated with neurotrophin-3,some were nestin-positive,while the majority showed positive staining for glial fibrillary acidic protein.Our experimental findings indicate that neurotrophin-3 is a crucial factor for inducing neural stem cells differentiation into neurons and oligodendrocytes.     

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。     

Different apolipoprotein E,apolipoprotein A1 and prostaglandin-H2 D-isomerase levels in cerebrospinal fluid of schizophrenia patients and healthy controls

Abstract

Objectives. To identify proteins differentially expressed in schizophrenia patients, we collected 50 µl cerebrospinal fluid from 17 first-episode schizophrenia patients and 10 healthy controls. Methods. Their proteins were separated by two-dimensional gel electrophoresis without using any depletion method and identified by mass spectrometry. Results. Approximately 550 spots were detected, six of which had significantly different intensities in schizophrenia compared to control specimens. We were able to validate in individual samples the upregulation of apolipoprotein E, apolipoprotein A1 and prostaglandin-H2 D-isomerase by Western blot analyses and detect the downregulation of transthyretin, TGF-β receptor type-1 and coiled-coil domain-containing protein 3 precursor. Conclusions. These findings may help to elucidate the disease mechanisms and confirm the hypothesis of disturbed cholesterol and phospholipid metabolism in schizophrenia, and thus reveal the final role players. Moreover, a grouped protein expression analysis of apolipoprotein E, apolipoprotein A-I, and prostaglandin-H2 D-isomerase in cerebrospinal fluid from patients might be a potential diagnostic tool for schizophrenia.     

Cyclin D1/D2/D3在大鼠脑胶质瘤组织中的表达

目的 探讨cyclin D1/D2/D3在大鼠脑胶质瘤组织中的表达.方法 将体外培养的大鼠C6胶质瘤细胞(细胞数为3×105个)借助动物立体定向仪接种于Wistar大鼠左侧尾状核区,解剖标本,行组织病理学检查及GFAP、cyclin D1/D2/D3免疫组化检测.结果 cyclinD1/D2/D3均呈阳性表达.cyclin D1/D3主要在细胞胞核表达,cyclin D2主要在胞浆表达,部分在胞核表达,不同存活期荷瘤鼠瘤组织中cyclin D1/D2/D3表达量不同.结论 cyclinD1/D2/D3过分表达参与调节大鼠脑胶质瘤增殖.荷瘤鼠生存期与cyclin D家族蛋白的表达呈负相关关系.     

In vitro induction and in vivo expression of bcl-2 in the hNT neurons

Bcl-2 encodes membrane-associated proteins that suppress programmed cell death in cells of various origins. Compelling evidence suggests that bcl-2 is also involved in neuronal differentiation and axonal regeneration. The human Neuro-Teratocarcinoma (hNT) neurons constitute a terminally differentiated human neuronal cell line that is derived from the Ntera-2/clone D1 (NT2) precursors upon retinoic acid (RA) treatment. After transplantation into the central nervous system (CNS), the hNT neurons survive, engraft, maintain their neuronal identity, and extend long neurite outgrowth. We were particularly interested in the intracellular determinants that confer these post-transplant characteristics to the hNT neurons. Thus, we asked whether the hNT neurons express bcl-2 after transplantation into the rat striatum and if RA induction of the neuronal lineage is mediated by bcl-2. The grafted hNT neurons were first identified using three different antibodies that recognize human-specific epitopes, anti-hMit, anti-hNuc, and NuMA. After a 1-month post-transplant survival time, NuMA immunostaining revealed that 12% of the hNT neurons survived the transplantation. These neurons extended long neuritic processes within the striatum, as demonstrated using the human-specific antibody against the midsize neurofilament subunit HO14. Importantly, we found that 85% of the implanted hNT neurons expressed bcl-2 and that the in vitro induction of the neuronal lineage from the NT2 precursors with RA resulted in an upregulation of bcl-2 expression. Together, these data suggest that the differentiation of the hNT neurons to a neuronal lineage could be mediated at least partially by bcl-2.     

Expression of α5 integrin rescues fibronectin responsiveness in NT2N CNS neuronal cells

The extracellular matrix protein fibronectin is implicated in neuronal regeneration in the peripheral nervous system. In the central nervous system (CNS), fibronectin is up‐regulated at sites of penetrating injuries and stroke; however, CNS neurons down‐regulate the fibronectin receptor α5β1 integrin during differentiation and generally respond poorly to fibronectin. NT2N CNS neuron‐like cells (derived from NT2 precursor cells) have been used in preclinical and clinical studies for treatment of stroke and a variety of CNS injury and disease models. Here we show that, like primary CNS neurons, NT2N cells down‐regulate α5β1 integrin during differentiation and respond poorly to fibronectin. The poor neurite outgrowth by NT2N cells on fibronectin can be rescued by transducing NT2 precursors with a retroviral vector expressing α5 integrin under the control of the murine stem cell virus 5′ long terminal repeat. Sustained α5 integrin expression is compatible with the CNS‐like neuronal differentiation of NT2N cells and does not prevent robust neurite outgrowth on other integrin ligands. Thus, α5 integrin expression in CNS neuronal precursor cells may provide a strategy for enhancing the outgrowth and survival of implanted cells in cell‐replacement therapies for CNS injury and disease. © 2009 Wiley‐Liss, Inc.     

Dopamine D1/D5 receptor signaling regulates synaptic cooperation and competition in hippocampal CA1 pyramidal neurons via sustained ERK1/2 activation

Synaptic cooperation and competition are important components of synaptic plasticity that tune synapses for the formation of associative long‐term plasticity, a cellular correlate of associative long‐term memory. We have recently reported that coincidental activation of weak synapses within the vicinity of potentiated synapses will alter the cooperative state of synapses to a competitive state thus leading to the slow decay of long‐term plasticity, but the molecular mechanism underlying this is still unknown. Here, using acute hippocampal slices of rats, we have examined how increasing extracellular dopamine concentrations interact and/or affect electrically induced long‐term potentiation (LTP) in the neighboring synapses. We demonstrate that D1/D5‐receptor‐mediated potentiation at the CA1 Schaffer collateral synapses differentially regulates synaptic co‐operation and competition. Further investigating the molecular players involved, we reveal an important role for extracellular signal‐regulated kinases‐1 and 2 (ERK1/2) as signal integrators and dose‐sensors. Interestingly, a sustained activation of ERK1/2 pathway seems to be involved in the differential regulation of synaptic associativity. The concentration‐dependent effects of the modulatory transmitter, as demonstrated for dopaminergic signaling in the present study, might offer additional computational power by fine tuning synaptic associativity processes for establishing long‐term associative memory in neural networks. © 2015 The Authors Hippocampus Published by Wiley Periodicals, Inc.