PYY3-36 inhibits the action potential firing activity of POMC neurons of arcuate nucleus through postsynaptic Y2 receptors

Intracerebroventricular administration of gut peptide PYY3-36 stimulates food intake. In contrast, peripheral administration inhibits food intake, suggesting that the peptide has the opposite effect by virtue of accessing a unique subset of brain sites. A previous study suggested that peripheral PYY3-36 activates anorexigenic POMC neurons in the arcuate nucleus, and this was proposed to be the mechanism underlying the peptide's anorexigenic activity. Here, we demonstrate in an electrophysiological slice preparation that, in contrast to the original model, PYY3-36 potently and reversibly inhibits POMC neurons via postsynaptic Y2 receptors. These data show a complex role for Y2 receptors in regulation of the NPY/POMC circuitry, as they are present as inhibitory receptors on both the orexigenic NPY neurons as well as the anorexigenic POMC neurons. Secondly, these data argue against a direct role of POMC neurons in mediating the anorexigenic response to administration of peripheral PYY3-36.     

Brain-derived neurotrophic factor stimulates bone/cementum-related protein gene expression in cementoblasts

Brain-derived neurotrophic factor (BDNF), recognized as essential in the developing nervous system, is involved in differentiation and proliferation in non-neuronal cells, such as endothelial cells, osteoblasts, and periodontal ligament cells. We have focused on the application of BDNF to the regeneration of periodontal tissue and indicated that BDNF promotes the regeneration of experimentally created periodontal defects. Cementoblasts form cementum, mineralized tissue, which is key to establishing a functional periodontium. The application of BDNF to the regeneration of periodontal tissue requires elucidation of the mechanism by which BDNF regulates the functions of cementoblasts. In this study, we examined how BDNF regulates the mRNA expression of bone/cementum-related proteins (alkaline phosphatase (ALP), osteopontin (OPN), and bone morphogenetic protein-2 (BMP-2)) in cultures of immortalized human cementoblast-like (HCEM) cells. BDNF elevated the mRNA levels of ALP, OPN, and BMP-2 in HCEM cells. Small interfering RNA (siRNA) for TRKB, a high affinity receptor of BDNF, siRNA for ELK-1, which is a downstream target of ERK1/2, and PD98059, an ERK inhibitor, obviated the increase in the mRNA levels. BDNF increased the levels of phosphorylated ERK1/2 and Elk-1, and the blocking of BDNF signaling by treatment with siRNA for TRKB and PD98059 suppressed the phosphorylation of ERK1/2 and Elk-1. Furthermore, BDNF increased the levels of phosphorylated c-Raf, which activates the ERK signaling pathway. These findings provide the first evidence that the TrkB-c-Raf-ERK1/2-Elk-1 signaling pathway is required for the BDNF-induced mRNA expression of ALP, OPN, and BMP-2 in HCEM cells.     

Cloning and expression of human ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a survival factor for avian ciliary ganglion neurons and a variety of other neuronal cell types in vitro. We report here the cloning of the entire genomic sequence encoding human CNTF and its primary structure. Biologically active CNTF has been expressed in Chinese hamster ovary cells from a human genomic DNA clone. Human CNTF has no significant sequence similarity to any previously reported protein, although approximately 84% similarity exists compared with rat and rabbit CNTF. The lack of both an N-terminal signal sequence and consensus sequences for glycosylation or hydrophobic regions, and the fact that active CNTF is expressed but not released into the culture medium of transfected cells, argue in favour of human CNTF as a cytosolic protein. These data provide a basis for understanding the role of CNTF in nervous system physiology and pathology.     

Overexpression of ciliary neurotrophic factor in vivo rescues chick ciliary ganglion neurons from cell death

Ciliary ganglion (CG) neurons undergo target-dependent cell death during embryonic development. Although ciliary neurotrophic factor (CNTF) was identified in vitro by its ability to support the survival of chick CG neurons, its function as a target-derived neurotrophic factor has been questioned by those working on mammalian-derived forms of CNTF. We have purified and cloned a chicken CNTF [chCNTF; formerly growth-promoting activity (GPA)] that is expressed in CG targets during the period of cell death and is secreted by cells transfected with chCNTF. In the present study we used a retroviral vector, RCASBP(A), to overexpress chCNTF in CG target tissues. Elevation of chCNTF biological activity three- to fourfold in the embryonic eye rescued an average of 31% of the neurons that would have normally died in vivo. In some individuals, nearly all of the neurons were rescued. ChCNTF had no effect on the number of neurons observed prior to cell death, nor were there any deleterious effects of either viral infection or overexpression of CNTF. These results show that chCNTF is able to function in vivo as a trophic factor for CG neurons, and suggest that limited availability of trophic support is one of the factors regulating CG neuron survival during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 283–293, 1998     

Neuromedin U suppresses prolactin secretion via dopamine neurons of the arcuate nucleus

Neuromedin U (NMU) has a precursor that contains one additional peptide consisting of 33 or 36 amino acid residues. Recently, we identified this second peptide from rat brain and designated it neuromedin U precursor-related peptide (NURP), showing it to stimulate prolactin release from the pituitary when injected via the intracerebroventricular (icv) route. Here, we examined whether NMU, like NURP, also stimulates prolactin release. Unlike NURP, icv injection of NMU significantly decreased the secretion of prolactin from the pituitary. This suppression of prolactin release by NMU was observed in hyper-prolactin states such as lactation, stress, pseudopregnancy, domperidone (dopamine antagonist) administration, and icv injection of NURP. Immunohistochemical analysis revealed that icv injection of NMU induced cFos expression in dopaminergic neurons of the arcuate nucleus, but not the substantia nigra. Mice with double knockout of NMU and neuromedin S (NMS), the latter also binding to NMU receptors, showed a significant increase of the plasma prolactin level after domperidone treatment relative to wild-type mice. These results suggest that NMU and NURP may play important reciprocal roles in physiological prolactin secretion.     

Characterization of two putative cytokine receptors, gp130 and ciliary neurotrophic factor receptor, from terrestrial salamanders

Cytokines of the gp130 family are fundamental regulators of immune responses and signal through multimeric receptors to initiate intracellular second-messenger cascades. Here, we provide the first characterization of two full-length gp130 cytokine receptors from the cDNA of the red-legged salamander (Plethodon shermani). The first, gp130 (2745 bp), is a common signaling receptor for several multi-functional cytokines in vertebrates. We also isolated the full-length (1104 bp) sequence of the ciliary neurotrophic factor receptor (CNTFR), which forms a heteromeric signaling complex with gp130. The open reading frames of both receptors were predicted to contain many of the conserved features found in mammalian gp130s, such as cytokine binding homology regions and residues known to form disulfide bonds. Finally, we used RT-PCR to show that gp130 and CNTFR were expressed in most P. shermani tissues, including brain, intestine and muscle. The expression profiles, along with the structural predictions, show that gp130, CNTFR, and their cytokine ligands are parts of the immune system of P. shermani and other caudate amphibians.     

睫状神经营养因子基因多态性在中国汉族女性人群中的分布

目的:研究 CNTF基因多态性在我国健康汉族人群体中的分布情况.方法:采用自行建立的口腔上皮细胞法提取核DNA,并对CNTF基因多态性进行分析;卡方检验群体代表性.结果:所选群体等位基因、基因型频率符合Hardy-Weinberg平衡.结论:CNTF基因多态性分布在一定程度上体现出人种差异.     

Effects of ciliary neurotrophic factor (CNTF) and depolarization on neuropeptide expression in cultured sympathetic neurons.

We examined the effects of ciliary neurotrophic factor (CNTF) and depolarization, two environmental signals that influence noradrenergic and cholinergic function, on neuropeptide expression by cultured sympathetic neurons. Sciatic nerve extract, a rich source of CNTF, increased levels of vasoactive intestinal peptide (VIP), substance P, and somatostatin severalfold while significantly reducing levels of neuropeptide Y (NPY). No change was observed in the levels of leu-enkephalin (L-Enk). These effects were abolished by immunoprecipitation of CNTF-like molecules from the extract with an antiserum raised against recombinant CNTF, and recombinant CNTF caused changes in neuropeptide levels similar to those of sciatic nerve extract. Alterations in neuropeptide levels by CNTF were dose-dependent, with maximal induction at concentrations of 5-25 ng/ml. Peptide levels were altered after only 3 days of CNTF exposure and continued to change for 14 days. Depolarization of sympathetic neuron cultures with elevated potassium elicited a different spectrum of effects; it increased VIP and NPY content but did not alter substance P, somatostatin, or L-Enk. Depolarization is known to block cholinergic induction in response to heart cell conditioned medium and we found that it blocked the induction of choline acetyltransferase (ChAT) and peptides by recombinant cholinergic differentiation factor/leukemia inhibitory factor (CDF/LIF). In contrast, it did not antagonize the effects of CNTF on either ChAT activity or neuropeptide expression. Thus, while CNTF has effects on neurotransmitter properties similar to those previously reported for CDF/LIF, the actions of these two factors are differentially modulated by depolarization, suggesting that the mechanisms of cholinergic and neuropeptide induction for the two factors differ. In addition, in contrast to CDF/LIF, CNTF did not alter levels of ChAT, VIP, substance P, or somatostatin in cultured dorsal root ganglion neurons. These observations indicate that CNTF and depolarization affect the expression of neuropeptides by sympathetic neurons and provide evidence for an overlapping yet distinct spectrum of actions of the two neuronal differentiation factors, CNTF and CDF/LIF.     

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on the ciliary neurotrophic factor receptor alpha chain

Ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin are members of the four-helix bundle cytokine family. These proteins signal through a common tripartite receptor composed of leukemia inhibitory factor receptor, gp130, and ciliary neurotrophic factor receptor alpha. Binding to ciliary neurotrophic factor receptor alpha occurs through an interaction site located at the C terminus of the cytokine AB loop and alphaD helix, known as site 1. In the present study, we have generated a model of neuropoietin and identified a conserved binding site for the three cytokines interacting with ciliary neurotrophic factor receptor alpha. To identify the counterpart of this site on ciliary neurotrophic factor receptor alpha, its cytokine binding domain was modeled, and the physicochemical properties of its surface were analyzed. This analysis revealed an area displaying properties complementary to the site 1 of ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin. Based on our computational predictions, residues were selected for their potential involvement in the ciliary neurotrophic factor receptor alpha binding epitope, and site-directed mutagenesis was carried out. Biochemical, cell proliferation, and cell signaling analyses showed that Phe(172) and Glu(286) of ciliary neurotrophic factor receptor alpha are key interaction residues. Our results demonstrated that ciliary neurotrophic factor, cardiotrophin-like cytokine, and neuropoietin share a conserved binding site on ciliary neurotrophic factor receptor alpha.     

TGF-β regulates the survival of ciliary ganglionic neurons synergistically with ciliary neurotrophic factor and neurotrophins

We investigated putative roles of transforming growth factor (TGF)-β expressed in peripheral ganglia in the regulation of neuronal cell survival during the period of ontogenetic neuron death (OD). The chick ciliary ganglion (CG), where OD occurs between embryonic days (E) 6 and 10, was employed as a model system. We show that CG neurons (E8) are immunoreactive (ir) for TGF-β2 and -β3 as well as the TGF-β receptor TβR-II, but are not ir for TGF-β1. Ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF)-2, established neurotrophic molecules for CG neurons, up-regulate TGF-β3 mRNA and TGF-β biological activity in cultures of E8 CG neurons. None of the TGF-β isoforms—β1, β2, or β3—has a trophic, survival-promoting effect on cultured CG neurons. However, all isoforms enhance CG neuron survival mediated by CNTF or FGF-2, significantly and over a wide range of concentrations. In combination with the neurotrophins (NT) nerve growth factor (NGF) and NT-3, which are not neurotrophic for CG neurons, TGF-β significantly promotes CG neuron survival. However, TGF-β does not act synergistically with the neuropoietic cytokines oncostatin M, leukemia inhibiting factor, or interleukin-6. Immunoneutralization of endogenous TGF-β released from CG neurons using an antibody to TGF-β1/-β2/-β3 significantly reduces the potency of CNTF or FGF-2 to promote CG neuron survival. The blocking effect of the anti–pan-TGF-β antibody could be rescued by adding exogenous TGF-β. Together, these data suggest that para-/autocrine TGF-β signaling has an important effect on the regulation of neuron survival in a model system of peripheral neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 563–572, 1998     

Sequence and structural organization of the human gene encoding ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a potent polypeptide hormone whose actions appear to be restricted to the nervous system where it promotes survival, neurotransmitter synthesis and neurite outgrowth in certain neuronal populations. We have cloned the gene encoding human CNTF (hCNTF) and have characterized its structure and organization. The hCNTF gene appears to be a unique-copy gene with a simple genetic organization, since only a single intron interrupts the coding domain. The hCNTF gene is located on chromosome 11, as determined using human-hamster somatic cell hybrids. The CNTF protein is highly conserved in evolution. The amino acid (aa) sequences of rat and rabbit CNTF translated from cDNAs display approx. 85% homology with the deduced aa sequence encoding hCNTF.     

Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo.

Adult rat sciatic nerve is known to express high levels of ciliary neurotrophic factor (CNTF) mRNA and protein. Here we examine the cellular localization of CNTF protein and mRNA in peripheral nerve and the regulation of CNTF expression by peripheral axons. In intact nerve, CNTF immunoreactivity is found predominantly in the cytoplasm of myelin-related Schwann cells. After axotomy, CNTF immunoreactivity and mRNA levels fall dramatically and do not recover unless axons regenerate. This behavior is similar to the pattern of myelin gene expression in these nerves. We conclude that the expression of CNTF in Schwann cells depends on axon-Schwann cell interactions.     

Intraocular gene transfer of ciliary neurotrophic factor rescues photoreceptor degeneration in RCS rats

Ciliary neurotrophic factor (CNTF) is known as an important factor in the regulation of retinal cell growth. We used both recombinant CNTF and an adenovirus carrying the CNTF gene to regulate retinal photoreceptor expression in a retinal degenerative animal, Royal College of Surgeons (RCS) rats. Cells in the outer nuclear layer of the retinae from recombinant-CNTF-treated, adenoviral-CNTF-treated, saline-operated, and contralateral untreated preparations were examined for those exhibiting CNTF photoreceptor protective effects. Cell apoptosis in the outer nuclear layer of the retinae was also detected. It was found that CNTF had a potent effect on delaying the photoreceptor degeneration process in RCS rats. Furthermore, adenovirus CNTF gene transfer was proven to be better at rescuing photoreceptors than that when using recombinant CNTF, since adenoviral CNTF prolonged the photoreceptor protection effect. The function of the photoreceptors was also examined by taking electroretinograms of different animals. Adenoviral-CNTF-treated eyes showed better retinal function than did the contralateral control eyes. This study indicates that adenoviral CNTF effectively rescues degenerating photoreceptors in RCS rats.S.-P.H. and P.-K.L. contributed equally to this work.     

Proliferation and differentiation of embryonic chick sympathetic neurons: effects of ciliary neurotrophic factor

At early developmental stages (embryonic day 7, E7), chick paravertebral sympathetic ganglia contain a cell population that divides in culture while expressing various neuronal properties. In an attempt to identify factors that control neuronal proliferation, we found that ciliary neurotrophic factor (CNTF) specifically inhibits the proliferation of those cells expressing neuronal markers. In addition, CNTF affects the differentiation of sympathetic ganglion cells by inducing the expression of vasoactive intestinal peptide immunoreactivity (VIP-IR). After 1 day in culture, tyrosine hydroxylase immunoreactivity (TH-IR) was expressed by about 86% of the cells whereas VIP-IR was virtually absent. In the presence of CNTF, 50%-60% of the cells expressed VIP-IR after 4 days in culture; however, none of the cells expressed VIP-IR in the absence of CNTF. These results, and the demonstration of cells that express both VIP and TH-IR, indicate that VIP is induced in cells that initially express tyrosine hydroxylase. The findings suggest a potential role for CNTF as a factor affecting the proliferation and differentiation of developing sympathetic neurons.     

Neuroprotective properties of ciliary neurotrophic factor for cultured adult rat dorsal root ganglion neurons

We observed that recombinant ciliary neurotrophic factor (CNTF) enhanced survival and neurite outgrowth of cultured adult rat dorsal root ganglion (DRG) neurons. Among other neurotrophic factors (NGF and GDNF) and interleukin (IL)-6 cytokine members [IL-6, LIF, cardiotrophin-1, and oncostatin M (OSM)] at the same concentration (50 ng/ml), CNTF, as well as LIF and OSM, displayed high efficacy for the promotion of the number of viable neurons and neurite-bearing cells. CNTF enhanced the number of neurite-bearing cells in both small neurons (soma diameter <30 mum) and large neurons (soma diameter >/=30 mum), whereas NGF and GDNF promoted that in only small neurons. Western blot analysis revealed that CNTF induced phosphorylation of STAT3, Akt, and ERK1/2 in the neurons. Furthermore, the neurite outgrowth-promoting activity of CNTF was diminished by co-treatment with Janus kinase (JAK) 2 inhibitor, AG490; STAT3 inhibitor, STA-21; phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor, LY294002; and mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, in a concentration-dependent manner. Its survival-promoting activity was also affected by AG490, STA-21, and LY294002 at higher concentrations, but not by PD98059. These findings suggest the involvement of JAK2/STAT3, PI3K/Akt, and MEK/ERK signaling pathways in CNTF-induced neurite outgrowth, where the former two pathways are thought to play major roles in mediating the survival response of neurons to CNTF.