Nonneuronal cells mediate neurotrophic action of vasoactive intestinal peptide

The developmental regulation of neuronal survival by vasoactive intestinal peptide (VIP) was investigated in dissociated spinal cord-dorsal root ganglion (SC-DRG) cultures. Previous studies demonstrated that VIP increased neuronal survival in SC-DRG cultures when synaptic transmission was blocked with tetrodotoxin (TTX). This effect was further investigated to determine if VIP acted directly on neurons or via nonneuronal cells. For these studies, SC-DRG cells were cultured under conditions designed to provide preparations enriched for a particular cell type: astrocyte-enriched background cell (BG) cultures, meningeal fibroblast cultures, standard mixed neuron-nonneuron (STD) cultures, and neuron-enriched (N) cultures. Addition of 0.1 nM VIP to TTX-treated STD cultures for 5 d prevented the TTX-mediated death and the death that occurred naturally during development in culture, whereas the same treatment on N cultures did not prevent neuronal cell death. Conditioned medium from VIP-stimulated BG cultures prevented neuronal cell death when added to the medium (10% of total volume) of N cultures treated with TTX. The same amount of conditioned medium from BG cultures that were not treated with VIP had no protective action on N cultures. Conditioned medium from N or meningeal fibroblast cultures, either with or without VIP treatment, did not prevent TTX-mediated cell death in N test cultures. These data indicate that VIP increases the availability of neurotrophic survival-promoting substances derived from nonneuronal cultures, the most likely source being astroglial cells. This study suggests that VIP has a role in mediating a neuron-glia-neuron interaction that influences the trophic regulation of neuronal survival.     

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.     

Effects of vasoactive intestinal peptide genotype on circadian gene expression in the suprachiasmatic nucleus and peripheral organs

The neuropeptide vasoactive intestinal polypeptide (VIP) has emerged as a key candidate molecule mediating the synchronization of rhythms in clock gene expression within the suprachiasmatic nucleus (SCN). In addition, neurons expressing VIP are anatomically well positioned to mediate communication between the SCN and peripheral oscillators. In this study, we examined the temporal expression profile of 3 key circadian genes: Per1, Per2 , and Bmal1 in the SCN, the adrenal glands and the liver of mice deficient for the Vip gene (VIP KO), and their wild-type counterparts. We performed these measurements in mice held in a light/dark cycle as well as in constant darkness and found that rhythms in gene expression were greatly attenuated in the VIP-deficient SCN. In the periphery, the impact of the loss of VIP varied with the tissue and gene measured. In the adrenals, rhythms in Per1 were lost in VIP-deficient mice, while in the liver, the most dramatic impact was on the phase of the diurnal expression rhythms. Finally, we examined the effects of the loss of VIP on ex vivo explants of the same central and peripheral oscillators using the PER2::LUC reporter system. The VIP-deficient mice exhibited low amplitude rhythms in the SCN as well as altered phase relationships between the SCN and the peripheral oscillators. Together, these data suggest that VIP is critical for robust rhythms in clock gene expression in the SCN and some peripheral organs and that the absence of this peptide alters both the amplitude of circadian rhythms as well as the phase relationships between the rhythms in the SCN and periphery.     

Insulin-like growth factor type-1 receptor transactivation in vasoactive peptide and oxidant-induced signaling pathways in vascular smooth muscle cells

Transactivation of epidermal growth factor receptor (EGFR) is a well-documented mechanism by which vasoactive peptides and H2O2 elicit their cellular responses. However, a role for the insulin-like growth factor type-1 receptor (IGF-1R) transactivation in mediating the effects of angiotensin II (Ang II) and H2O2 in vascular smooth muscle cells from different artery types have also been recently recognized. By using a series of pharmacological inhibitors of various growth factor receptor tyrosine kinases and a direct analysis of the phosphorylation status of the beta-subunit of IGF-1R, a requirement of this growth factor receptor in Ang II and H2O2 response has been demonstrated. This review discusses some of the studies that highlight the importance of IGF-1R transactivation in mediating Ang II- and H2O2-induced mitogen-activated protein kinase and protein kinase B signaling pathways.     

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.     

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.     

Peptide T does not affect induction of pineal N-acetyltransferase by vasoactive intestinal peptide

It has been suggested that the HIV virus binds to VIP recognition sites which can be blocked by the octapeptide, peptide T. Stimulation of VIP receptors on pinealocytes activates adenylate cyclase and increases the activity of the enzyme serotonin N-acetyltransferase (NAT). We examined whether peptide T or D-Ala peptide T amide affected this induction. We found no evidence for peptide T interference with NAT induction and conclude that if peptide T inhibits attachment of HIV virus to VIP receptors, it does so at regions other than that occupied by VIP in stimulating adenylate cyclase and NAT.     

Suckling stimulates the expression of vasoactive intestinal polypeptide gene in rats.

The effect of suckling on vasoactive intestinal polypeptide (VIP) gene expression in the hypothalami was studied during the postpartum period in rats. Female rats were divided into two groups immediately after delivery. In one group, a mother was housed with 8 pups, and in the other, without any pups. The former group was named S(+) and the latter S(-). On days 0, 3, 6, 9 and 12 after delivery, the mothers were killed by decapitation. Hypothalamic VIP mRNA was measured by RNA dot hybridization. Although the VIP mRNA level showed no significant change after delivery in the S(-) group, VIP mRNA in the S(+) group on days 6, 9 and 12 increased to 1.6, 3.5 and 2.1 times higher than the level observed on day 0, respectively. These results suggest that suckling induces the synthesis of VIP after 6 postpartum days.     

Inducers of oxidative stress block ciliary neurotrophic factor activation of Jak/STAT signaling in neurons

Generation of reactive oxygen species (ROS) with the accumulation of oxidative damage has been implicated in neurodegenerative disease and in the degradation of nervous system function with age. Here we report that ROS inhibit the activity of ciliary neurotrophic factor (CNTF) in nerve cells. Treatment with hydrogen peroxide (H(2)O(2)) as a generator of ROS inhibited CNTF-mediated Jak/STAT signaling in all cultured nerve cells tested, including chick ciliary ganglion neurons, chick neural retina, HMN-1 motor neuron hybrid cells, and SH-SY5Y and BE(2)-C human neuroblastoma cells. H(2)O(2) treatment of non-neuronal cells, chick skeletal muscle and HepG2 hepatoma cells, did not inhibit Jak/STAT signaling. The H(2)O(2) block of CNTF activity was seen at concentrations as low as 0.1 mm and within 15 min, and was reversible upon removal of H(2)O(2) from the medium. Also, two other mediators of oxidative stress, nitric oxide and rotenone, inhibited CNTF signaling. Treatment of neurons with H(2)O(2) and rotenone also inhibited interferon-gamma-mediated activation of Jak/STAT1. Depleting the intracellular stores of reduced glutathione by treatment of BE(2)-C cells with nitrofurantoin inhibited CNTF activity, whereas addition of reduced glutathione protected cells from the effects of H(2)O(2). These results suggest that disruption of neurotrophic factor signaling by mediators of oxidative stress may contribute to the neuronal damage observed in neurodegenerative diseases and significantly affect the utility of CNTF-like factors as therapeutic agents in preventing nerve cell death.     

Inhibition of Ras extracellular-signal-regulated kinase (ERK) mediated signaling promotes ciliary neurotrophic factor (CNTF) expression in Schwann cells

Ciliary neurotrophic factor (CNTF) can prevent injury-induced motor neuron death. However, it is also evident that expression of CNTF in Schwann cells is suppressed during nerve regeneration. In this report, we have addressed the mechanism underlying the down-regulation of CNTF expression in injured nerves using a mouse Schwann cell line IMS32 and mouse sciatic nerve. In IMS32 cells, activation of the Ras extracellular-signal-regulated kinase (ERK) pathway by adenoviral vector-mediated expression of dominant active MEK1 did not alter a basal level of CNTF expression, whereas inhibition of the Ras-ERK pathway by using adenoviral vectors resulted in a marked increase in CNTF expression. This inverse relation between before and after axotomy was also observed in mouse sciatic nerve. In the axotomized sciatic nerve, the phosphorylated ERK was markedly increased; in contrast, the expression of CNTF was markedly decreased. These findings suggest that an inactive state of ERK is crucial for the CNTF expression in Schwann cells, and that activation of ERK following nerve injury critically influences the expression of CNTF. This might well explain why CNTF is highly expressed in quiescent Schwann cells in the peripheral nervous system, and also why CNTF is not abundant in axotomized nerves or cultured Schwann cells in which the proliferation signal is obviously active.     

Isolation and characterization of rat vasoactive intestinal peptide

We have used gel filtration, ion exchange chromatography, affinity chromatography and reversed-phase HPLC to isolate vasoactive intestinal peptide from rat intestine. Microsequence analysis of 1 nmole peptide indicated that the sequence was identical to the porcine octacosapeptide VIP. In radioimmunoassay with four antisera and in the turkey pancreas bioassay, rat VIP was equipotent with highly purified preparations of porcine, human and canine VIP. A less basic rat VIP-variant was also isolated and the N-terminal decapeptide region that was sequenced was identical with that of porcine VIP.