Synthesis and regulation of apolipoprotein E during the differentiation of human neuronal precursor NT2/D1 cells into postmitotic neurons

Recently, we showed expression of apolipoprotein E (apoE) in human neuronal-type cells such as neuroblastoma SK N SH-SY 5Y cells. In this model, a negative effect of neuronal differentiation on apoE synthesis was suspected. To check this hypothesis, we studied the regulation of apoE in human postmitotic neurons. The presence of apoE was investigated in undifferentiated human teratocarcinoma NT2/D1 (NT2) cells and during their differentiation into postmitotic hNT neurons induced by retinoic acid (RA) treatment. Before differentiation, apoE protein and mRNA were detected in NT2 cells by Western blotting and RT-PCR experiments. Immunofluorescence study showed that apoE was present in all cells. For longer times of RA treatment (3 weeks), the apoE labeling became heterogeneous: only some cells were immunopositive and among them were some differentiating cells in which apoE was located in both cellular body and neuritic process. Interestingly, terminally differentiated hNT cells no longer expressed apoE. These results demonstrate that neuronal precursor and differentiating cells were able to synthesize apoE while the fully neuronal differentiation exerted a negative effect on apoE neuronal expression. Our results are compatible with a weak expression of apoE in neurons of adult brains.     

Neuronal survival and resistance to HIV-1 Tat toxicity in the primary culture of rat fetal neurons

In this study we report that primary cultures of rat fetal neurons contain subpopulations of cells that may be sensitive or resistant to HIV-1 Tat neurotoxicity. We demonstrate that rapid binding/uptake of Tat 1–86 for 2 h was sufficient to trigger caspase activation and neurodegeneration in rat fetal midbrain cell cultures. The uptake of Tat was followed by an increase in MCP1 (CCL2) immunoreactivity. Approximately 70% of neurons were able to survive transient or continuous (7 days) Tat exposure. The surviving neurons did not contain bound/internalized Tat, but were able to interact with Tat after medium replacement. These neurons were resistant to Tat toxicity. In neurons that resisted the toxic effects of continuous and repeated Tat treatment, levels of NR2A subunit of the NMDA receptor complex were significantly lower than in controls. We suggest that the subunit composition of NMDAR complexes may be important for the sensitivity of neurons to Tat toxicity.     

Differentiation of the RN33B cell line into forebrain projection neurons after transplantation into the neonatal rat brain

The rat neural cell line RN33B has a remarkable ability to undergo region-specific neuronal differentiation after transplantation into the CNS. To further study its neurogenic properties in vivo, we used a recombinant lentiviral vector to genetically label the cells with the Green Fluorescent Protein (GFP) gene before implantation into the striatum/cortex, hippocampus, or mesencephalon of newborn rats. Three weeks after implantation, about 1-2% of the GFP-expressing cells had developed morphologies typical of neurons, astrocytes, or oligodendrocytes, the rest remained as either immature or undifferentiated nestin-positive cells. At 15-17 weeks postgrafting, the immature cells had disappeared in most graft recipients and only cells with neuronal or glial morphologies remained in similar numbers as at 3 weeks. The GFP distributed throughout the expressing cells, revealing fine morphological details, including dendrites with spines and extensive axonal projections. In all forebrain regions, the grafted cells differentiated into neurons with morphologies characteristic for each site, including large numbers of pyramidal-like cells in the cortex and the hippocampus, giving rise to dense projections to normal cortical target regions and to the contralateral hippocampus, respectively. In lower numbers, it was also possible to identify GFP-positive granulelike cells in the hippocampus, as well as densely spiny neurons in the striatum. In the mesencephalon by contrast, cells with astrocytic features predominated. The ability of the grafted RN33B cells to undergo region-specific differentiation into highly specialized types of forebrain projection neurons and establish connections with appropriate targets suggests that cues present in the microenvironment of the neonatal rat brain can effectively guide the development of immature progenitors, also in the absence of ongoing neurogenesis.     

Neuregulin 1 growth factors regulate proliferation but not apoptosis of a CNS neuronal progenitor cell line

Growth factor-dependent proliferation of neuronal progenitors is an essential stage in CNS development. Although several of these growth factors have been identified, high levels of neuregulin 1 (NRG1) mRNA and protein expression in the CNS during the time of neuronal progenitor expansion suggest NRG1 growth factors may also play a key role in their proliferation. No previous studies have examined the expression of multiple NRG1 isoforms and receptors in these progenitors and their role in proliferation or apoptosis. Using a rat CNS clonal cell line with neuronal progenitor properties, we show for the first time these cells coexpress multiple NRG1 isoforms (NRGbeta1, NRGbeta3, CRD-NRGbeta, and SMDF, but not GGF2 or any alpha isoforms) and all three cognate receptors (erbB2-4). We also show for the first time the presence of mRNA for all four variants of the erbB4 receptor in a single CNS cell type. Neutralizing antibody treatments suggest NRG1 isoforms and receptors are involved in proliferation but not apoptosis of these cells. This model system should be useful in future studies of the ligand specificity and function(s) of the erbB4 receptor variants.     

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。     

Electrophysiological characterization of neural stem/progenitor cells during in vitro differentiation: study with an immortalized neuroectodermal cell line

Despite the accumulating data on the molecular and cell biological characteristics of neural stem/progenitor cells, their electrophysiological properties are not well understood. In the present work, changes in the membrane properties and current profiles were investigated in the course of in vitro-induced neuron formation in NE-4C cells. Induction by retinoic acid resulted in neuronal differentiation of about 50% of cells. Voltage-dependent Na+ currents appeared early in neuronal commitment, often preceding any morphological changes. A-type K+ currents were detected only at the stage of network formation by neuronal processes. Flat, epithelial- like, nestin-expressing progenitors persisted beside differentiated neurons and astrocytes. Stem/progenitor cells were gap junction coupled and displayed large, symmetrical, voltage-independent currents. By the blocking of gap junction communication, voltage-independent conductance was significantly reduced, and delayed-rectifying K+ currents became detectable. Our data indicate that voltage-independent symmetrical currents and gap junction coupling are characteristic physiological features of neural stem and progenitor cells regardless of the developmental state of their cellular environment.     

Glial cell line derived neurotrophic factor promotes the recovery of dopamine neurons damaged by 6-hydroxydopamine in vitro

Glial cell line derived neurotrophic factor (GDNF) has been shown to be a potent neurotrophic factor for dopamine neurons in culture and to prevent the loss of substantia nigra dopamine neurons following in vivo lesions with 6-hydroxydopamine (6-OHDA). In this study we used mesencephalic cultures containing both neurons and glia to examine whether GDNF protects dopamine neurons from 6-OHDA toxicity in vitro. Our data show that GDNF does not prevent the loss of dopamine neurons caused by treatment with 6-OHDA in vitro. However, continuous exposure to GDNF increases the high affinity dopamine uptake in cultures treated with 6-OHDA, suggesting that it enhances the growth of damaged dopamine neurons. We also show that in vitro treatment with 6-OHDA causes widespread cell death in mesencephalic cultures, which is not restricted to dopamine neurons. The lack of selectivity of 6-OHDA toxicity when applied in vitro may explain the inability of GDNF to prevent the loss of dopamine neurons in mesencephalic cultures. The stimulation of the growth of 6-OHDA damaged dopamine neurons by GDNF, observed in our study, suggests that it may prove beneficial in the treatment of injured dopamine neurons.     

Role of the gonads in sex differentiation of growth hormone-releasing hormone and somatostatin neurons in the mouse hypothalamus during postnatal development

We clarify the mechanism of sexual dimorphism of growth hormone releasing hormone (GHRH) neurons in the arcuate nucleus (ARC) and somatostatin (SS) neurons in periventricular nucleus (PeN), by studying the role of the gonads during the neonatal period and after puberty using immunohistochemical and morphometric methods. As in our previous works the numbers of ARC GHRH-ir and PeN SS-ir neurons were significantly greater in adult normal male (NM) mice than in adult normal female (NF) mice. Adult female mice that were ovariectomized neonatally (NOF) increased the expression of GHRH-ir neurons to the male pattern, but adult female mice ovariectomized after puberty (APO) did not change. Adult male mice castrated neonatally and after puberty (NCM and APC, respectively) were not significantly different from NM mice. However, NCT male mice, which were castrated neonatally and transplanted with ovary just before puberty, showed a significantly reduced number of GHRH-ir neurons compared with NCM mice, but no significant difference from NM and NF mice. On the other hand, the PeN SS-ir neuron expression in NCM mice and APC mice showed a significant reduction compared with NM mice, but no significant difference from NF mice. The number of PeN SS-ir neurons in NOF increased to match that of NM mice. Our results suggest that the presence of the ovary during postnatal life inhibits the development of ARC GHRH-ir neurons. The presence of the testis during postnatal life may stimulate the development of PeN SS-ir neurons, while the presence of the ovary during neonatal period may inhibit the development of PeN SS-ir neurons; the presence of ovary after puberty does not inhibit.     

NO及相关因子对体外培养神经细胞损伤作用的研究

在体外培养的大鼠脑神经细胞上研究了ＮＯ及相关因子对神经细胞的损伤作用。研究表明：（１）ＮＯ的体外神经损伤作用与脂质过氧化作用关系密切；（２）ＮＯ的体外神经损伤作用是由ＮＯ本身的毒性造成的，而与其代谢产物亚硝酸根（ＮＯ２－）和硝酸根（ＮＯ３－）无关；（３）环境酸化可明显促进ＮＯ的体外神经损伤作用。这些结果将十分有助于加深对ＮＯ神经损伤作用的认识     

Hepatocyte growth factor stimulates cell motility in cultures of the striatal progenitor cells ST14A

Hepatocyte growth factor/scatter factor (HGF/SF) is a growth factor with pleiotropic effects on different cell types. It acts as a mitogen and motility factor for many epithelial cells. HGF/SF and its receptor Met are present in the developing and adult mammalian brain and control neuritogenesis of sympathetic and sensory neurons. We report that the striatal progenitor ST14A cells express the Met receptor, which is activated after binding with HGF/SF. The interaction between Met and HGF/SF triggers a signaling cascade that leads to increased levels of c-Jun, c-Fos, and Egr-1 proteins, in agreement with data reported on the signaling events evoked by HGF in other cellular types. We also studied the effects of the exposure of ST14A cells to HGF/SF. By time-lapse photography, we observed that a 24-hr treatment with 50 ng/ml HGF/SF induced modification in cell morphology, with a decrease in cell-cell interactions and increase of cell motility. In contrast, no effect on cell proliferation was observed. To investigate which intracellular pathway is primarily involved we used PD98059 and LY294002, two specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAP-kinase/ERK-kinase) and phosphoinositide 3-OH kinase (PI3-K), respectively. Cell motility in HGF/SF treated cultures was inhibited by LY294002 but not by PD98059, suggesting that PI3-K plays a key role in mediating the HGF/SF-induced dissociation of ST14A cells. Previous evidence of HGF stimulation of motility in nervous system has been obtained on postmitotic neurons, which have already acquired their specificity. Data reported here of a motogenic response of ST14A cell line, which displays properties of neuronal progenitors, seem of interest because they suggest that HGF could play a role in very early steps of neurogenesis.     

Postnatal age governs the extent of differentiation of hippocampal CA1 and CA3 subfield neural stem/progenitor cells into neurons and oligodendrocytes

While neural stem/progenitor cells (NSCs) in the dentate gyrus of the hippocampus have been extensively characterized, the behavior of NSCs in the CA1 and CA3 subfields of the hippocampus is mostly unclear. Therefore, we compared the in vitro behavior of NSCs expanded from the micro-dissected CA1 and CA3 subfields of postnatal day (PND) 4 and 12 Fischer 344 rats. A small fraction (∼1%) of dissociated cells from CA1 and CA3 subfields of both PND 4 and 12 hippocampi formed neurospheres in the presence of EGF and FGF-2. A vast majority of neurosphere cells expressed NSC markers such as nestin, Sox-2 and Musashi-1. Differentiation assays revealed the ability of these NSCs to give rise to neurons, astrocytes, and oligodendrocytes. Interestingly, the overall neuronal differentiation of NSCs from both subfields decreased with age (23–28% at PND4 to 5–10% at PND12) but the extent of oligodendrocyte differentiation from NSCs increased with age (24–32% at PND 4 to 45–55% at PND 12). Differentiation of NSCs into astrocytes was however unchanged (40–48%). Furthermore, NSCs from both subfields gave rise to GABA-ergic neurons including subclasses expressing markers such as calbindin, calretinin, neuropeptide Y and parvalbumin. However, the fraction of neurons that expressed GABA decreased between PND4 (59–67%) and PND 12 (25–38%). Additional analyses revealed the presence of proliferating NSC-like cells (i.e. cells expressing Ki-67 and Sox-2) in different strata of hippocampal CA1 and CA3 subfields of both PND4 and PND 12 animals. Thus, multipotent NSCs persist in both CA1 and CA3 subfields of the hippocampus in the postnatal period. Such NSCs also retain their ability to give rise to both GABA-ergic and non-GABA-ergic neurons. However, their overall neurogenic potential declines considerably in the early postnatal period.     

Physiological identification of GABA as the inhibitory transmitter for mammalian cortical neurons in cell culture

(1) Rat cortical neurons grown in dissociated cell culture exhibit IPSPs which appear to be generated by an increase in membrane conductance to chloride.

(2) The neurons are all sensitive to GABA in micromolar concentrations and GABA mimics the inhibitory transmitter.

(3) The neurons are much less sensitive to glycine and insensitive to taurine.

(4) Bicuculline and strychnine both block essentially all IPSPs and at the same concentrations block GABA effects.

(5) It is concluded that GABA is the main, or only, inhibitory transmitter utilized by the cortical neurons in vitro. The relevance of this conclusion to in situ transmitter identification is discussed.

Interleukin-2 inhibition of oligodendrocyte progenitor cell proliferation depends on expression of the TAC receptor

Interleukin-2 (IL-2) has been shown to inhibit oligodendrocyte progenitor cell proliferation. Within the immune system, IL-2 biological action is dependent strictly on the expression of the IL-2 receptor. The antibody TAC, which specifically binds the lymphocyte IL-2 receptor, has been shown to also bind oligodendrocyte progenitor cells cultured in a serumless, chemically defined medium. The expression of the TAC antigen was found necessary for IL-2 inhibition of oligodendrocyte progenitor cell proliferation. After IL-2 induced down-regulation of the TAC antigen, the progenitor cell was unresponsive to IL-2, even 72 hr after IL-2 withdrawal. During this unresponsive period, the oligodendrocyte progenitor cell was immunocytochemically negative for the TAC antigen. Thus, in contrast to IL-2 receptors on T-cells, IL-2 does not up-regulate its receptor on oligodendrocyte progenitor cells. However, upon interleukin 1 (IL-1) addition both IL-2 responsiveness and TAC immunocytochemical staining reappeared. These data suggest that IL-2 inhibition of progenitor cell proliferation depends on the expression of the TAC antigen, which can be regulated by IL-1.     

Endogenous adenosine protects CA1 neurons from kainic acid-induced neuronal cell loss in the rat hippocampus

CA3 pyramidal neurons in the rat hippocampus show selective vulnerability to the intracerebroventricular injection of kainic acid (KA). However, the mechanism of this selective neuronal vulnerability remains unclear. In this study, we examined the contribution of endogenous adenosine, a potent inhibitory neuromodulator, to the differences in the neuronal vulnerability of the hippocampus, using microtubule-associated protein (MAP)-2, phosphorylated c-Jun, and major histocompatibility complex (MHC) class II immunoreactivities as markers for neuronal cell loss, neuronal apoptosis and glial activation, respectively. Pretreatment with 8-cyclopenthyltheophylline (CPT), an A1 adenosine receptor antagonist, significantly exacerbated KA-induced neuronal cell loss in both the CA1 and CA3. Although c-Jun phosphorylation, a critical step in neuronal apoptosis, was not detected in the vehicle-injected rat hippocampus, c-Jun phosphorylation was induced in the CA3 by the injection of KA alone. Pretreatment with CPT induced c-Jun phosphorylation in both the CA1 and CA3. MHC class II antigen was also detected in the regions of c-Jun phosphorylation. Coadministration of N6-cyclopenthyladenosine (CHA), an A1 adenosine receptor agonist, attenuated the neuronal cell loss in the CA1 and CA3 with or without pretreatment with CPT. These results strongly suggest that endogenous adenosine has neuroprotective effects against excitotoxin-induced neurodegeneration in the CA1 through its A1 receptors.     

Dlx-2 homeobox gene controls neuronal differentiation in primary cultures of developing basal ganglia

Homeodomain-containing genes of theDlx family are expressed in the developing basal ganglia. To investigate the role ofDlx genes during development, we studied their cellular localization in primary cultures of embryonic basal telencephalon, and examined the changes in cellular phenotypes resulting from blockade ofDlx-2 expression. Cells containingDlx-1, Dlx-2, andDlx-5 mRNAs are immature cells of the neuronal lineage expressing the microtubule-associated proteins (MAPs) MAP1B and MAP2, but not glial fibrillary acidic protein (GFAP). Treatment of these cells with antisense oligonucleotides targeted toDlx-2 caused a specific decrease ofDlx-2 mRNA and protein. This decrease in theDlx-2 gene product was associated with a decrease in the expression of MAP2, a protein localized in neuronal dendrites, along with a smaller decrease in the 200-kDa neurofilament subunit (NF-H). Proteins expressed preferentially in axons were unchanged. This reduction in MAP2 expression was associated with a decrease in dendrite outgrowth and an increased level of cell proliferation. None of these changes were elicited by antisense oligonucleotides targeted toDlx-1. We suggest that theDlx-2 gene product regulates two interrelated aspects of neuronal differentiation: the exit from the mitotic cycle and the capability to grow MAP2-positive dendrites. As such, this gene product may be important for the establishment of neuronal polarity, setting the stage for afferent synaptic connectivity.     

Pathway for interferon-gamma to promote the differentiation of cholinergic neurons in rat embryonic basal forebrain/septal nuclei

BACKGROUND: The supernatant of interferon-gamma (IFNγ) co-cultured with neonatal rat cortical glia can promote the cells in embryonic basal forebrain/septal nuclei to differentiate into cholinergic neurons, but the mechanism is still unclear. OBJECTIVE: To analyze the pathways for IFNγ to promote the differentiation of primarily cultured cholinergic neurons in rat embryonic basal forebrain/septal nuclei through culture in different conditioned medium. DESIGN: A controlled experiment taking cells as the observational target. SETTINGS: Department of Biochemistry and Molecular Biology, Youjiang Medical College for Nationalities; Department of Cell Biology, Beijing University Health Science Center. MATERIALS: Sixty-four pregnant Wistar rats for 16 days (250–350 g) and 84 Wistar rats (either male or female, 5–7 g) of 0–1 day after birth were provided by the experimental animal department of Beijing University Health Science Center. Rat IFNγ were provided by Gibco Company; Glial fibrillary acidic protein by Huamei Company. METHODS: The experiments were carried out in the Department of Cell Biology, Beijing University Health Science Center and Daheng Image Company of Chinese Academy of Science from July 1995 to December 2002. ① Interventions: The nerve cells in the basal forebrain/septal nuclei of the pregnant Wistar rats for 16 days were primarily cultured, and then divided into four groups: Blank control group (not any supernatant and medium was added); Control group (added by mixed glial cell or astrocyte conditioned medium); IFNγ group (added by mixed glial cell or astrocyte conditioned medium+IFNγ). Antibody group (added by mixed glial cell or astrocyte conditioned medium+IFNγ+Ab-IFNγ). Mixed glial cell or astrocyte conditioned medium was prepared using cerebral cortex of Wistar rats of 0–1 day after birth. ② Evaluation: The immunohistochemical method was used to perform the choline acetyltransferase (ChAT) staining of cholinergic neurons. The ChAT positive cells were counted. MAIN OUTCOME MEASURES: Comparison of ChAT positive cells in rat basal forebrain and septal nuclei in different conditioned medium. RESULTS: ① ChAT positive cells in mixed glial cell conditioned medium: The ChAT positive cells in the IFNγ group and antibody group were significantly more than those in the control group (P < 0.01). ② ChAT positive cells in astrocyte conditioned medium: The ChAT positive cells in the IFNγ group were significantly more than those in the control group, but there was no significant difference between the antibody group and control group (P > 0.05). CONCLUSION: IFNγ cannot directly promote the differentiation of cholinergic neurons, but plays a role through activating glial cells (except astrocytes) to produce IFNγ like molecules.