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-c-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. Keywords: ciliary neurotrophic factor, cytokine, gpl30, reactive oxygen species, signal transduction, tyrosine kinase. Signaling through the ciliary neurotrophic factor (CNTF) receptor has been implicated in the development, survival and maintenance of a broad range of neurons and glia in the PNS and CNS, as well as cardiac and skeletal muscle. CNTF receptors are essential for embryonic development and the receptor knockout phenotype in mice is embryonic lethal (DeChiara et al. 1995). Defects in CNTF expression result in an impaired neural injury response in mice (Masu et al. 1993;Yao et al. 1999;Linker et al. 2002) and decreased neuron numbers in embryonic chick (Ernsberger et al. 1989;Finn et al. 1998). In addition, a variety of neuronal, glial and muscle cell types show responses to CNTF in vivo or in vitro affecting neuronal phenotype, neurotransmitter receptor levels, neurotransmitter release, metabolism and survival (Adler et al.

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Atria isolated from 4-day chick embryos were much less responsive to the negative chronotropic effect of muscarinic agonists than were atria from 5- or 8-day embryos, even though the density of muscarinic acetylcholine receptors (mAChR) was similar at all these ages. The mAChR in hearts from 4-day embryos were also significantly less susceptible to regulation of receptor number by in vivo agonist treatment and required a 2-5-fold greater dose of the muscarinic agonist carbachol to achieve a decrease in receptor number equivalent to that observed in 5- or 8-day embryonic hearts. When 4-day atrial membranes were assayed in physiological buffers, agonist binding to the mAChR was not regulated by GTP unless a sulfhydryl reducing agent was present. Receptors from 5- and 8-day embryos did not require addition of a sulfhydryl reducing agent in order to see guanine nucleotide effects on agonist binding. Even in the presence of a sulfhydryl reducing agent, carbachol binding to the mAChR in 4-day membranes was much less sensitive to guanyl-5'-yl imidodiphosphate (GppNHp) than binding to mAChR in 5- or 8-day membranes. In addition, forskolin-activated adenylate cyclase activity was much less sensitive to inhibition by GppNHp in membranes from 4-day atria than from 5- and 8-day atria. The GTP-binding component (NI) which couples the mAChR to inhibition of adenylate cyclase activity was examined by covalent modification with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

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Chick ciliary ganglion neurons have a cholinergic membrane component that binds alpha-bungarotoxin with high affinity but has no known function. The component is different from the nicotinic ACh receptor on the neurons that mediates cholinergic transmission through the ganglion. Ciliary neuronotrophic factor (CNTF) has been shown to enhance the survival of ciliary ganglion neurons in cell culture and has been postulated to act as a target-derived trophic factor for the neurons in vivo. We show here that a factor indistinguishable from CNTF specifically down-regulates alpha-bungarotoxin binding sites on the neurons while increasing cell growth and the number of ACh receptors on the cells. Similar effects, though reduced in magnitude, are seen with chick sympathetic neurons. CNTF has no effect on the number of ACh receptors found on chick myotubes in culture. The down-regulation of alpha-bungarotoxin binding sites on neurons caused by CNTF occurs with a half-time of about 19 hr and is largely reversed within a 4 d period following CNTF removal. It is distinct from the down-regulation caused by cholinergic agonists. Nerve growth factor and fibroblast growth factor have no apparent effect on the number of alpha-bungarotoxin binding sites on the neurons, though fibroblast growth factor does stimulate neuronal growth. The results indicate that the effects of CNTF on the alpha-bungarotoxin binding component are both novel for a growth factor and specific, and they suggest a relationship between the component and the regulation of growth by the target tissue.