Regulation of GTP Cyclohydrolase I Gene Expression and Tetrahydrobiopterin Content in Cultured Sympathetic Neurons by Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor

Abstract: Cultures of neonatal rat superior cervical ganglia (SCG) were used to test the hypothesis that the cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) control GTP cyclohydrolase I (GTPCH) gene expression and 5,6,7,8-tetrahydrobiopterin (BH4) content as traits of the noradrenergic phenotype. Treatment for 7 days with 1 ng/ml of LIF was found to produce the characteristic switch in the SCG neurotransmitter phenotype reported by others, as evidenced by a 60% decline in tyrosine hydroxylase (TH) activity and a 75% increase in choline acetyltransferase activity. This LIF treatment paradigm decreased BH4 levels in a concentration-dependent manner, with a maximal decline of 60% observed at 1 ng/ml. Analysis of the time course of this response indicated that LIF decreased BH4 levels by 60% following 3–7 days of treatment. Treatment of cultures with CNTF (2 ng/ml) resulted in a decline in BH4 levels that was of equal magnitude and followed the same time course as that produced by LIF. The LIF-dependent decline in BH4 levels resulted from a reduction in GTPCH enzyme activity, which decreased by 75% following 7 days of treatment. Nuclease protection assays of RNA extracted from cells treated for 7 days with 2 ng/ml of LIF or CNTF detected a 78–96% reduction in GTPCH mRNA content relative to β-actin mRNA content. Concomitant decreases in TH and GTPCH gene expression in response to LIF or CNTF demonstrate a coordinated regulation of gene expression for this BH4-dependent enzyme and the rate-limiting enzyme in the synthesis of its essential cofactor, BH4. Moreover, these results indicate that GTPCH gene expression in SCG neurons should be regarded as a trait of the noradrenergic phenotype.     

Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor Increase Activated Ras in a Neuroblastoma Cell Line and in Sympathetic Neuron Cultures

Abstract: The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have been implicated in determination of neuronal phenotype as well as promotion of neuronal survival. However, the intracellular mechanisms by which their signals are transduced remain poorly understood. We have previously studied the regulation of vasoactive intestinal polypeptide gene expression by LIF and CNTF in the NBFL neuroblastoma cell line. Because these cytokines induce tyrosine phosphorylation that may lead to Ras activation, we explored a possible role for Ras in LIF- and CNTF-induced signal transduction. In NBFL cells LIF increases activated Ras in a rapid, transient, and concentration-dependent manner. CNTF and a related cytokine, oncostatin M, produce similar increases. CNTF and LIF also increase activated Ras in neuron-enriched dissociated cultures of sympathetic ganglia. Moreover, these cytokines rapidly and transiently induce specific tyrosine-phosphorylated proteins, p165 and p195. The protein kinase inhibitors K252a and staurosporine block LIF-induced increases in tyrosine phosphorylation, activated Ras, and vasoactive intestinal polypeptide mRNA in NBFL cells. These data support a possible role for Ras in the cell differentiation effects of LIF and CNTF.     

Binding Interactions of Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor with the Different Subunits of Their High Affinity Receptors

Abstract: Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) share common components in their multimeric receptors. Both cytokine receptors contain gp130/interleukin-6-receptor transducer as well as gp190/low-affinity LIF receptor. For CNTF, addition of a third subunit, or α subunit, defines the high-affinity CNTF receptor. In the present study, we analyzed the binding interactions of LIF and CNTF in human cell lines and showed a mutual displacement for LIF and CNTF toward the trimeric high-affinity CNTF receptor. Similar results were obtained in the JEG cell line, which only expressed the gp130/gp190 high-affinity LIF receptor, by adding a soluble form of the αCNTF receptor to the system to reconstitute the high-affinity-type CNTF receptor. The different receptor subunits were then expressed separately in transfected cells and their binding capacities analyzed. The results showed that the heterocomplex CNTF/αCNTF receptor bound to gp130 with an affinity of 3–5 × 10⁻¹⁰ M , whereas LIF interacted mainly with gp190. In summary, the observed competition between LIF and CNTF does not result from the binding to a common site or receptor subunit, but rather to the interaction of the three receptor components to create a conformational site common to both LIF and CNTF.     

Coordinate Regulation of Choline Acetyltransferase, Tyrosine Hydroxylase, and Neuropeptide mRNAs by Ciliary Neurotrophic Factor and Leukemia Inhibitory Factor in Cultured Sympathetic Neurons

Abstract: The neurotransmitter phenotype switch that occurs in cultures of rat superior cervical ganglion neurons after treatment with leukemia inhibitory factor or ciliary neurotrophic factor is a useful model permitting investigation of the mechanisms of cytokine-mediated differentiation. Recently the actions of leukemia inhibitory factor and ciliary neurotrophic factor have been linked through their interactions with related receptor complexes. Here we compare the effects of these two cytokines on gene expression in sympathetic neuronal cultures and begin to investigate their mechanisms. We report that, as has been shown for leukemia inhibitory factor, ciliary neurotrophic factor regulates peptides and classical transmitters in these cultures at the mRNA level. In addition, we find that the induction of substance P mRNA by these cytokines is rapid, dependent on protein synthesis, and occurs in 40–50% of superior cervical ganglion neurons in dissociated culture.     

Differentiation Effects of Ciliary Neurotrophic Factor on Human Neuroblastoma Cells

Abstract: In the human neuroblastoma cell line LA-N-2, recombinant rat ciliary neurotrophic factor (CNTF) induced neurite growth and cholinergic differentiation that were both half-maximally saturated at <100 p M of the neurokine, but was not required for cell survival in serum-free conditions over a 13-day period. CNTF markedly stimulated choline acetyltransferase activity and acetylcholine synthesis, whereas high-affinity choline transport was only slightly enhanced and acetylcholinesterase activity was unchanged. Leukemia inhibitory factor had effects identical to CNTF on neurite growth and choline acetyltransferase activity, but interleukin 6 had no effect. Radioiodinated CNTF binding and affinity cross-linking studies were consistent with tripartite receptor activation as a mediator of the observed biological effects.     

转铁蛋白-PEG-睫状神经营养因子的制备及其生物活性评价

为了实现在体内更持久的药效作用,根据睫状神经营养因子CNTF第17位为游离半胱氨酸残基,而转铁蛋白Tf无游离半胱氨酸的特点,采用N-羟基琥珀酰亚胺-聚乙二醇5K-马来酰亚胺(NHS-PEG5k-MAL)作为偶联剂,实现了两者定点偶联,然后结合蛋白自身特性制定纯化方法,制备获得纯度高于90%的转铁蛋白-聚乙二醇5k-睫状神经营养因子(Tf-PEG5k-CNTF)耦合物。高效凝胶色谱和动态光散射分析显示耦合物的表观分子体积大于两蛋白之和。细胞试验结果显示耦合物的活性下降至原蛋白的65.8%。大鼠药代动力学试验显示Tf-PEG5k-CNTF耦合物在体内的代谢半衰期延长至8.2小时,与CNTF原蛋白相比提高了约17倍。小鼠动物试验显示在每周2次的给药频率,每次1.0 mg/kg的剂量下,Tf-PEG5k-CNTF能更为显著地影响小鼠对食物摄入量和减轻体重。因此,转铁蛋白偶联技术可用于脑部靶向蛋白药物的长效递送。     

Evidence that Brain-Derived Neurotrophic Factor Neuroprotection Is Linked to Its Ability to Reverse the NMDA-Induced Inactivation of Protein Kinase C in Cortical Neurons

Abstract : Several lines of evidence indicate that a rapid loss of neuronal protein kinase C (PKC) activity is a characteristic feature of cerebral ischemia and is a necessary step in the NMDA-induced death of cultured neurons. Exposing embryonic day 18 primary rat cortical neurons to 50 μ M NMDA or 50 μ M glutamate for 10 min caused ~80% cell death over the next 24 h, but excitotoxic death was largely averted, i.e., by 70-80%, in cells pretreated with brain-derived neurotrophic factor (BDNF). An 8-h preexposure to BDNF (50-100 ng/ml) maximally protected cortical cells from the effects of NMDA and glutamate, although the transient application of BDNF between 8 and 4 h before NMDA was equally protective. These effects of BDNF were abolished at supralethal, i.e., >100 μ M , NMDA concentrations. It is significant that BDNF pretreatment prevented the inactivation of PKC in cortical cells normally seen 30 min to 2 h following lethal NMDA or glutamate exposure. This BDNF effect did not arise from changes in NMDA channel activity because neither whole-cell NMDA current amplitudes nor increases in intracellular free Ca²⁺ concentration were altered by the 8-h BDNF pretreatment. Furthermore, BDNF offered no neuroprotection to cells treated with the PKC inhibitors staurosporine (10-20 n M ), calphostin C (1-2.5 μ M ), or GF-109203X (100 n M ) at the time of NMDA addition. These results underscore the importance of PKC inactivation in glutamate-induced neuronal death. They also suggest that BDNF neuroprotection arises, at least in part, via its ability to block the mechanism by which pathophysiological Ca²⁺ influx through the NMDA receptor causes membrane PKC inactivation.     

Construction and Characterization of Ciliary Neurotrophic Factor (CNTF) Antagonists: Microenvironmental Difference in the CNTF Receptor Between Rat and Chicken Cells for Recognizing the D1 Cap Region

Abstract: Antagonistic mutants of ciliary neurotrophic factor (CNTF) were constructed and their properties characterized. K155A and K155W mutants lost cell survival promoting activity for chicken dorsal root ganglion (DRG) neurons and inhibited the activity of the wild type. However, they retained slight agonistic activity for the survival of rat DRG neurons, indicating there is a difference between chicken and rat cells for receptor recognition around the D1 cap region including K155 residue. The chicken receptor recognizes the D1 cap region more strictly than does the rat receptor. The substitution of F152, which locates at the top of the D1 cap region, was combined with the K155A mutation. A combination of the two mutations gave an antagonistic feature to not only chicken but also rat cells. Both F152S/K155A and F152D/K155A mutants lacked cell survival promoting activity and had an antagonistic effect on rat DRG neurons. The three-dimensional structure of CNTF suggests the following. F152 and K155 bind to the receptor with hydrophobic and electrostatic interactions, respectively. F152 locates close to L156 with a van der Waals contact, and K155 contacts with Q42 through a hydrogen bond. Both interactions play indispensable roles in maintaining the structure around the D1 cap region of CNTF.     

Expression of Tyrosine Hydroxylase Gene in Cultured Hypothalamic Cells: Roles of Protein Kinase A and C

Abstract: In hypothalamic cells cultured in serum-free medium, the quantity of tyrosine hydroxylase mRNA increases after treatment with an activator of the protein kinase A pathway (8-bromoadenosine cyclic AMP, 3-isobutyl-1-methylxanthine, or forskolin) or an activator of protein kinase C (12- O -tetradecanoylphorbol 13-acetate or sn -1,2-diacylglycerol). The tyrosine hydroxylase mRNA level decreases in the cells after inhibition of protein kinase C with calphostin C or after depletion of protein kinase C by extended phorbol ester treatment. These data suggest that both protein kinase pathways regulate tyrosine hydroxylase gene expression in hypothalamic cells. As simultaneous activation of both pathways has less than an additive effect on the tyrosine hydroxylase mRNA level, they appear to be interrelated. Compared with the rapid and dramatic increase of the tyrosine hydroxylase mRNA level in pheochromocytoma cells, activation of the protein kinase A or protein kinase C pathway in the cultured hypothalamic cells induces slow changes of a small magnitude in the amount of tyrosine hydroxylase mRNA. The slow regulation of tyrosine hydroxylase gene expression in hypothalamic dopaminergic neurons corresponds to the relatively high stability of tyrosine hydroxylase mRNA (half-life = 14 ± 1 h) in these cells.     

人睫状神经营养因子基因及突变体在大肠杆菌中的表达

人睫状神经营养因子（ｈｕｍａｎ ｃｉｌｉａｒｙ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ,ｈＣＮＴＦ）ｃＤＮＡ克隆入具有ＰＬ启动子的表达载体,转化大肠杆菌ＨＢ１０１,构建成表达ｈＣＮＴＦ菌株。热诱导后,ｈＣＮＴＦ的表达量达菌体总蛋白量的２５％。用离子交换层析、凝胶过滤层析从菌体裂解液中纯化了ｈＣＮＴＦ。ＳＤＳ－ＰＡＧＥｈＣＮＴＦ的分子量约为２４ｋｄ,Ｎ端氨基酸序列分析结果与依基因核苷酸推导疗列相符。     

Ciliary Neurotrophic Factor (CNTF) Genotypes and CNTF Contents in Human Sciatic Nerves as Measured by a Sensitive Enzyme-Linked Immunoassay

Abstract: To study the level of ciliary neurotrophic factor (CNTF) in human nervous tissues, we developed a sensitive enzyme-linked immunoassay using a specific antibody against human CNTF. This method allowed us to detect as little as 0.3 ng/ml of human CNTF with good linearity and accuracy. Using this method, CNTF levels were determined in human sciatic nerves obtained at autopsy from 21 amyotrophic lateral sclerosis (ALS) patients and 48 subjects who had died of other neurological diseases. CNTF genotypes were also determined. The results indicated that CNTF levels were high in the normal homozygotes and approximately halved in the heterozygote subjects. There was, however, no significant difference in CNTF levels in the sciatic nerves between ALS and other neurological disease patients, indicating that the CNTF level was mainly determined by its genotypes and that the level in the sciatic nerves was not reduced in ALS patients.     

Activation of Protein Kinase C by Purified Bovine Brain 14-3-3: Comparison with Tyrosine Hydroxylase Activation

Abstract: In the course of the purification of 14-3-3 protein (14-3-3) we found that 14-3-3 isolated from bovine forebrain activates protein kinase C (PKC), rather than the previously reported protein kinase C inhibitory activity (KCIP). We have characterized the 14-3-3 activation of PKC. The physical properties of purified PKC activator are the same as those previously reported for 14-3-3 and KCIP; i.e., (1) it is composed of subunits of molecular weight 32,000, 30,000, and 29,000; (2) it is homogeneous with respect to molecular weight, as judged by native gradient-gel electrophoresis, with a molecular weight of 53,000; and (3) it is composed of at least six isoforms when analyzed by reverse-phase HPLC. The concentration dependence of PKC activation by 14-3-3 is in the same range as that shown previously for KCIP inhibition of PKC, and as that required for 14-3-3 activation of tyrosine hydroxylase; a maximal stimulation of two- to three-fold occurs at 40–100 µg/ml. 14-3-3's activation of PKC is sensitive to α-chymotrypsin digestion but is not heat labile. Activation is specific to PKC; at least two other protein kinases, cyclic AMP- and calcium/calmodulin-dependent protein kinases, are not activated. The activation of PKC by 14-3-3 is independent of phosphatidylserine and calcium and, as such, is an alternative mechanism for the activation of PKC that obviates its translocation to membranes.     

Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.     

Recombinant Cholinergic Differentiation Factor (Leukemia Inhibitory Factor) Regulates Sympathetic Neuron Phenotype by Alterations in the Size and Amounts of Neuropeptide mRNAs

The cholinergic differentiation factor (CDF) in heart cells is identical to leukemia inhibitory factor (LIF). Recombinant CDF/LIF was shown to alter dramatically neurotransmitter production as well as the levels of several neuropeptides in cultured rat sympathetic neurons. Here it is shown that these changes are likely to be caused by alterations in the mRNA for these proteins and peptides. Growth in 1 nM recombinant CDF/LIF induces mRNA for acetyl CoA: choline-O-acetyltransferase [EC 2.3.1.6; choline acetyltransferase (ChAT)], somatostatin (SOM), substance P, and vasoactive intestinal polypeptide while lowering mRNA levels of tyrosine hydroxylase (EC 1.14.16.2) and neuropeptide Y (NPY). In addition, the sizes of the mRNAs for ChAT, SOM, and NPY are larger after recombinant CDF/LIF treatment.     

Characterization of TrkB Receptor-Mediated Signaling Pathways in Rat Cerebellar Granule Neurons: Involvement of Protein Kinase C in Neuronal Survival

Abstract: TrkB belongs to the Trk family of tyrosine kinase receptors and mediates the response to brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5). Here, we report that both truncated and full-length forms of TrkB receptors are expressed in developing cerebellar granule neurons. BDNF and NT-4/5 increased the survival of cultured cerebellar granule neurons. BDNF and NT-4/5 also induced an autophosphorylation of TrkB receptors and subsequently resulted in a phosphorylation and binding of phospholipase C-γ (PLC-γ) and SH2-containing sequence to the autophosphorylated TrkB receptors. Both contain src homology 2 (SH2) regions. In keeping with a signaling function of PLC-γ, BDNF increased the phosphatidylinositol (PI) turnover and elevated intracellular calcium levels. To investigate the involvement of protein kinase C (PKC) in the survival of granular neurons, we show here activation of PKC after BDNF or TPA treatment and blocking of the observed survival-promoting effects of BDNF and TPA with calphostin C, a specific PKC inhibitor. In addition, BDNF activated c- ras in a concentration-dependent manner. These results suggest that two different pathways, the c- ras and the PLC-γ pathway, are activated by TrkB receptors in primary neurons and that PKC activation is involved in the survival promoting effect of BDNF.     

Protein Phosphorylation in Astrocytes Mediated by Protein Kinase C: Comparison with Phosphorylation by Cyclic AMP-Dependent Protein Kinase

The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), has been found recently to transform cultured astrocytes from flat, polygonal cells into stellate-shaped, process-bearing cells. Studies were conducted to determine the effect of PMA on protein phosphorylation in astrocytes and to compare this pattern of phosphorylation with that elicited by dibutyryl cyclic AMP (dbcAMP), an activator of the cyclic AMP-dependent protein kinase which also affects astrocyte morphology. Exposure to PMA increased the amount of 32P incorporation into several phosphoproteins, including two cytosolic proteins with molecular weights of 30,000 (pI 5.5 and 5.7), an acidic 80,000 molecular weight protein (pI 4.5) present in both the cytosolic and membrane fractions, and two cytoskeletal proteins with molecular weights of 60,000 (pI 5.3) and 55,000 (pI 5.6), identified as vimentin and glial fibrillary acidic protein, respectively. Effects of PMA on protein phosphorylation were not observed in cells depleted of protein kinase C. In contrast to the effect observed with PMA, treatment with dbcAMP decreased the amount of 32P incorporation into the 80,000 protein. Like PMA, treatment with dbcAMP increased the 32P incorporation into the proteins with molecular weights of 60,000, 55,000 and 30,000, although the magnitude of this effect was different. The effect of dbcAMP on protein phosphorylation was still observed in cells depleted of protein kinase C. The results suggest that PMA, via the activation of protein kinase C, can alter the phosphorylation of a number of proteins in astrocytes, and some of these same phosphoproteins are also phosphorylated by the cyclic AMP-dependent mechanisms.     

Evidence that Protein Kinase C Activities Involved in Regulating Neurite Growth Are Localized to Distal Neurites

Abstract: Previously, we observed that long-term treatment of distal nerve fibers of rat sympathetic neurons in compartmented cultures with phorbol 12-myristate 13-acetate (PMA) caused a reduction in the rate of neurite elongation by >50%. In the present report we show that protein kinase C (PKC) activity could be measured in extracts of distal neurites by an assay of the Ca²⁺-dependent phosphorylation of a PKC-specific octapeptide substrate. We found that local application of 1 µ M PMA for 24 h to distal neurites caused nearly complete down-regulation of Ca²⁺-dependent PKC activity measured in this manner. We determined that the inhibition of neurite elongation by PMA was mediated by local mechanisms in the neurites because local application of PMA to center compartments containing cell bodies and proximal neurites did not inhibit the rate of elongation of distal neurites. We then investigated the effects of the recently available PKC inhibitors, calphostin C and chelerythrine, finding that, like PMA, these inhibited the growth of distal neurites when applied locally to them, and had no effect when applied to cell bodies and proximal neurites. However, the inhibition of neurite growth by calphostin C occurred at a concentration far below its IC₅₀ value for protein kinase inhibition, and both calphostin C and chelerythrine inhibited distal neurite growth even in neurons pretreated with PMA. Thus, it appears that these agents do not all inhibit neurite growth through the same mechanisms. Although the PKC activities involved in neurite elongation in sympathetic neurons have not been precisely defined, these data presented in this study indicate that protein kinases localized to growth cones play a complex and important role in regulating axonal growth.