Secretion of brain-derived neurotrophic factor from PC12 cells in response to oxidative stress requires autocrine dopamine signaling

Expression of brain-derived neurotrophic factor (BDNF) is sensitive to changes in oxygen availability, suggesting that BDNF may be involved in adaptive responses to oxidative stress. However, it is unknown whether or not oxidative stress actually increases availability of BDNF by stimulating BDNF secretion. To approach this issue we examined BDNF release from PC12 cells, a well-established model of neurosecretion, in response to hypoxic stimuli. BDNF secretion from neuronally differentiated PC12 cells was strongly stimulated by exposure to intermittent hypoxia (IH). This response was inhibited by N-acetyl-l-cysteine, a potent scavenger of reactive oxygen species (ROS) and mimicked by exogenous ROS. IH-induced BDNF release requires activation of tetrodotoxin sensitive Na+ channels and Ca2+ influx through N- and L-type channels, as well as mobilization of internal Ca2+ stores. These results demonstrate that oxidative stress can stimulate BDNF release and that underlying mechanisms are similar to those previously described for activity-dependent BDNF secretion from neurons. Surprisingly, we also found that IH-induced secretion of BDNF was blocked by dopamine D2 receptor antagonists or by inhibition of dopamine synthesis with alpha-methyl-p-tyrosine. These data indicate that oxidative stress can stimulate BDNF release through an autocrine or paracrine loop that requires dopamine receptor activation.     

Expression of CNTF/LIF‐receptor components and activation of STAT3 signaling in axotomized facial motoneurons: Evidence for a sequential postlesional function of the cytokines

Several lines of evidence suggest that ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are important for the survival and regeneration of axotomized motoneurons. To investigate the role of CNTF/LIF signaling in regenerative responses of motoneurons, we studied the expression of the three receptor components, CNTF receptor α (CNTFRα), LIF receptor β (LIFRβ), and gp130, and the activation of the STAT3 signal transduction pathway in the rat facial nucleus following peripheral nerve transection. As shown by in situ hybridization and immunoblotting, axotomy resulted in a rapid down‐regulation of CNTFRα mRNA expression within 24 h and a concomitant massive up‐regulation of LIFRβ mRNA and protein in the lesioned motoneurons. The altered mRNA levels were maintained for 3 weeks but had returned back to control levels by 6 weeks postlesion after successful regeneration. In contrast, mRNA levels remained in the lesioned state during the 6‐week period studied, when regeneration was prevented by nerve resection. Significant lesion‐induced changes in gp130 mRNA levels were not detectable. Rapid (within 24 h) and sustained (for at least 5 days) activation of STAT3 in axotomized facial motoneurons was revealed by demonstrating the phosphorylation and nuclear translocation of the protein using immunocytochemistry and immunoblotting. In agreement with previous studies showing a complementary regulation of CNTF and LIF in the lesioned facial nerve, our observations on the postlesional regulation of CNTF/LIF receptor components in the facial nucleus indicate a direct and sequential action of the two neurotrophic proteins on axotomized facial motoneurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 559–571, 1999     

Activity-dependent neurotrophic factor peptide (ADNF9) protects neurons against oxidative stress-induced death

Activity-dependent neurotrophic factor (ADNF) and a 14-amino acid fragment of this peptide (sequence VLGGGSALLRSIPA) protect neurons from death associated with an array of toxic conditions, including amyloid beta-peptide, N-methyl-D-aspartate, tetrodotoxin, and the neurotoxic HIV envelope coat protein gp120. We report that an even smaller, nine-amino acid fragment (ADNF9) with the sequence SALLRSIPA potently protects cultured embryonic day 18 rat hippocampal neurons from oxidative injury and neuronal apoptosis induced by FeSO4 and trophic factor withdrawal. Among the characteristics of this protection are maintenance of mitochondrial function and a reduction in accumulation of intracellular reactive oxygen species.     

Cadmium blocks receptor-mediated Jak/STAT signaling in neurons by oxidative stress

Cadmium is an environmental contaminant producing numerous pathological effects including neurological disorders. The mechanisms through which cadmium produces neurotoxicities are not completely known. We found that divalent cadmium (CdCl2) inhibited ciliary neurotrophic factor (CNTF)-mediated Jak1 and Jak2 tyrosine kinase signaling in human BE(2)-C neuroblastoma cells. CdCl2 concentrations as low as 0.1 microM and for times as brief as 2 h significantly reduced CNTF-induced tyrosine phosphorylation of both STAT1 and STAT3, the principle substrates of Jak kinases in neurons. The phosphorylation of STAT1 by interferon-gamma was also inhibited by CdCl2. However, activation of the fibroblast growth factor receptor tyrosine kinase was not inhibited by CdCl2. Jak/STAT signaling was inhibited by CdCl2 selectively in cultures of chick retina neurons and neuroblastoma cells, whereas signaling in the nonneuronal cells HepG2 and chick skeletal myotubes was not affected. Results using dichlorofluorescein indicated CdCl2 increased cellular oxidative stress, and all of these effects of CdCl2 were protected against by pretreatment with antioxidants. Neuronal inhibition of Jak kinase by CdCl2-induced oxidative stress is a new mechanism of cadmium action which may directly produce neurotoxic symptoms as well as implicate cadmium and related metals as environmental factors in the etiology of neurodegenerative diseases.     

睫状神经营养因子对大鼠去神经骨骼肌的营养作用

目的：了解睫状神经营养因子（CNTF）对去神经引起的肌肉萎缩的治疗作用。方法：离断SD大鼠一侧坐骨神经,连续给予CNTF20d,观察肌肉湿重、蛋白含量、肌纤维横截面积、收缩性能和残肢程度。结果：①给予0.2mg/kg的CNTF,可使损务侧肌纤维横截面积增加35％,肌肉湿重增加38％,胫前肌总蛋白含量增加24％,腓长肌强直收缩强度提高40％,显著改善肢残程度;②0.2mg/kg的CNTF作用明显强于0.05mg/kg的CNTF;③此目鱼肌（慢肌）比伸趾长肌（快肌）对CNTF更敏感。结论：CNTF能显著改善成年大鼠坐骨神经离断后骨骼肌的萎缩和功能丧失,该效应的强弱与用药剂量和肌肉类型有关。     

Preparation and biological properties of native and recombinant ciliary neurotrophic factor

CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE). In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons. The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed. With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves. After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons. In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons. © 1992 John Wiley & Sons, Inc.     

Short- and long-term effects of ciliary neurotrophic factor on androgen-sensitive motoneurons in the lumbar spinal cord

Motoneuron death in the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN) of the lumbar spinal cord is androgen regulated. As a result, many more SNB and DLN motoneurons die in perinatal female rats than in males, whereas treatment of newborn females with androgen results in a permanent sparing of the motoneurons and their target muscles. We previously observed that a neurotrophic molecule, ciliary neurotrophic factor (CNTF), also arrests the death of SNB motoneurons and their target musculature, at least in the short term. The present study compares the short- and long-term consequences of perinatal CNTF treatment on motoneuron number in the SNB, the DLN, and the retrodorsolateral nucleus (RDLN), a motor pool in the lower lumbar cord that does not exhibit hormone-regulated cell death. Female pups were treated with CNTF or vehicle alone from embryonic day 22 through postnatal day 6 (P6). Motoneuron number in each nucleus was then determined immediately after treatment on P7, or 10 weeks later (P77). CNTF treatment significantly elevated motoneuron number in the SNB and DLN on P7; the volume of SNB target muscles on P7 was also greater in the CNTF-treated group. These effects were transient, however, as motoneuron number and ratings of muscle size were not different in CNTF- and vehicle-treated females on P77. Perinatal CNTF treatment did not alter cell number in the RDLN at either age. The finding that effects of CNTF on SNB and DLN motoneuron number are short lived contrasts with the permanent effects of early androgen treatment, and has implications for molecular models of the actions of androgen and neurotrophic factors on the developing spinal cord. © 1996 John Wiley & Sons, Inc.     

睫状神经营养因子对应激引起大鼠海马神经元损伤的保护作用及其机制的研究

本实验用Ｎｉｓｓｌ染色法、Ｂｉｅｌｓｃｈｏｗｓｋｙ－Ｇｒｏｓ－Ｌａｗｒｅｎｔｊｅｗ染色法、常规透射电镜、行为活动测定、双侧海马微量给药、海马神经元原代培养、活细胞连续照相、全细胞膜片钳记录、细胞内游离Ｃａ＾２＋浓度测定及Ｐ５３蛋白免疫组化测定等方法,观察了睫状神经营养因子（ＣＮＴＦ）对应激引起动物行为变化和海马神经元形态学变化的影响,探讨了ＣＮＴＦ的部分作用机制。结果表明,急性应激不引起大鼠海马神     

All‐trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells

Retinopathy of prematurity, a leading cause of visual impairment in low birth‐weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all‐trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose‐ and time‐dependent manner, and PKA signaling pathway is necessary for ATRA‐induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen‐induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.     

Inhibition of NADH/NADPH oxidase affects signal transduction by growth factor receptors in normal fibroblasts.

Reactive oxygen species have been implicated as possible second messengers in mitogenic signal transduction. We demonstrate that in normal fibroblasts the treatment with the two inhibitors of phagocytic NADH/NADPH oxidase prevents tyrosine phosphorylation of platelet-derived growth factor receptor upon the exposure of serum-deprived cells to growth factors. Furthermore, the inhibition of NADH/NADPH oxidase abolishes ERKs activation and p21(waf1) accumulation that occurs when cells are exposed to growth factors. Finally, NADH/NADPH inhibitors prevent the p66(Shc) Ser-phosphorylation induced by serum and by phorbol 12-myristate-13-acetate, which suggests that the direct target(s) of reactive oxygen species is(are) located upstream from the machinery connecting growth factor receptors to Ras.     

睫状神经营养因子对NO引起海马神经元毒性反应的影响

本研究采用原代培养大鼠海马神经元,观察睫状神经营养因子（ｃｉｌｉａｒｙ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ,ＣＮＴＦ）对ＮＯ引起细胞毒性反应的影响。ＮＯ供体硝普钠与Ｓ－亚硝基－乙酰青霉胺,ＮＯＳ底物Ｌ－Ａｒｇ及钙载体ｉｏｎｏｍｙｃｉｎ,均可引起海马神经元存活率下降,ＬＤＨ漏出增加;提前２４ｈ给予不同浓度ＣＮＴＦ,均能提高神经元的存活率,减少ＬＤＨ漏出,其作用呈剂量依赖性。     

The basal level of intracellular calcium gates the activation of phosphoinositide 3-kinase-Akt signaling by brain-derived neurotrophic factor in cortical neurons

Brain-derived neurotrophic factor (BDNF) mediates survival and neuroplasticity through the activation of phosphoinositide 3-kinase-Akt pathway. Although previous studies suggested the roles of mitogen-activated protein kinase, phospholipase C-gamma-mediated intracellular calcium ([Ca2+]i) increase, and extracellular calcium influx in regulating Akt activation, the cellular mechanisms are largely unknown. We demonstrated that sub-nanomolar BDNF significantly induced Akt activation in developing cortical neurons. The TrkB-dependent Akt phosphorylation at S473 and T308 required only phosphoinositide 3-kinase, but not phospholipase C and mitogen-activated protein kinase activity. Blocking NMDA receptors, L-type voltage-gated calcium channels, and chelating extracellular calcium by EGTA failed to block BDNF-induced Akt phosphorylation. In contrast, chelating [Ca2+]i by 1,2-bis(o-aminophenoxy)ethane-N,N,N ',N '-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) abolished Akt phosphorylation. Interestingly, sub-nanomolar BDNF did not stimulate [Ca2+]i increase under our culture conditions. Together with that NMDA- and membrane depolarization-induced [Ca2+]i increase did not activate Akt, we conclude that the basal level of [Ca2+]i gates BDNF function. Furthermore, inhibiting calmodulin by W13 suppressed Akt phosphorylation. On the other hand, inhibition of protein phosphatase 1 by okadaic acid and tautomycin rescued Akt phosphorylation in BAPTA-AM and W13-treated neurons. We further demonstrated that the phosphorylation of phosphoinositide-dependent kinase-1 did not correlate with Akt phosphorylation at T308. Our results suggested novel roles of basal [Ca2+]i, rather than activity-induced calcium elevation, in BDNF-Akt signaling.     

Induction of an interferon-gamma Stat3 response in nerve cells by pre-treatment with gp130 cytokines

Many cytokines mediate their effects through Jak/STAT signaling pathways providing many opportunities for cross-talk between different cytokines. We examined the interaction between two cytokine families, gp130-related cytokines and interferon-gamma (IFN-gamma), which are coexpressed in the nervous system during acute trauma and pathological conditions. Typical nerve cells show an IFN-gamma response that is restricted to activating STAT1, with minor activation of STAT3. IFN-gamma elicited a pronounced STAT3 response in cells pre-treated for 5-7 h with ciliary neurotrophic factor (CNTF), leukemia inhibitory factor or interleukin-6. CNTF or interleukin-6 induced an IFN-gamma STAT3 response in a variety of cells including SH-SY5Y human neuroblastoma, HMN-1 murine motor neuron hybrid cells, rat sympathetic neurons and human hepatoma HepG2 cells. The enhancement was measured as an increase in tyrosine phosphorylated STAT3, in STAT3-DNA binding and in STAT-luciferase reporter gene activity. The enhanced STAT3 response was not due to an increase in overall STAT3 levels but was dependent upon ongoing protein synthesis. The induction by CNTF was inhibited by the protein kinase C inhibitor, BIM, and the MAPK-kinase inhibitor, U0126. Further, H-35 hepatoma cells expressing gp130 receptor chimeras lacking either the SHP-2 docking site or the Box 3 STAT binding sites failed to enhance the IFN-gamma STAT3 response. These results provide evidence for an interaction between gp130 and IFN-gamma cytokines that can significantly alter the final cellular response to IFN-gamma.