Human Ciliary Neurotrophic Factor Research Articles

Gene therapy for amyotrophic lateral sclerosis (ALS) using a polymer encapsulated xenogenic cell line engineered to secrete hCNTF.

The gene therapy approach presented in this protocol employs a polymer encapsulated, xenogenic, transfected cell line to release human ciliary neurotrophic factor (hCNTF) for the treatment of Amyotrophic Lateral Sclerosis (ALS). A tethered device, containing around 10(6) genetically modified cells surrounded by a semipermeable membrane, is implanted intrathecally; it provides for slow continuous release of hCNTF at a rate of 0.25 to 1.0 micrograms/24 hours. The semipermeable membrane prevents immunologic rejection of the cells and interposes a physical, virally impermeable barrier between cells and host. Moreover, the device and the cells it contains may be retrieved in the event of side effects. A vector containing the human CNTF gene was transfected into a line of baby hamster kidney cells (BHK) with calcium phosphate using a dihydrofolate reductase-based selection vector with a SV40 promoter and contains a HSV-tk killer gene. hCNTF is a potent neurotrophic factor which may have utility for the treatment of ALS. Systemic delivery of hCNTF in humans has been frustrated by peripheral side effects, the molecule's short half life, and its inability to cross the blood-brain barrier. The gene therapy approach described in this protocol is expected to mitigate such difficulties by local intrathecal delivery of a known quantity of continuously-synthesized hCNTF from a retrievable implant.

A double‐blind placebo‐controlled clinical trial of subcutaneous recombinant human ciliary neurotrophic factor (rHCNTF) in amyotrophic lateral sclerosis

Ciliary neurotrophic factor (CNTF) is a neuroactive cytokine found in Schwann cells, which appears to be released in response to nerve injury. The ALS CNTF Treatment Study (ACTS) clinical trial was a phase II-III randomized, placebo-controlled, double-blind study designed to evaluate the safety, tolerability, and efficacy of subcutaneous administration of recombinantly produced human CNTF (rHCNTF) in slowing disease progression in 730 patients with amyotrophic lateral sclerosis (ALS). Patients were randomized to receive 30 micrograms/kg or 15 micrograms/kg rHCNTF or placebo subcutaneously three times a week for 9 months. The primary endpoint of the study, the slope of decline of isometric muscle strength in treated versus placebo patients, showed no statistically significant difference between rHCNTF and placebo-treated patients, and was complicated by an initial statistically significant decrease in strength early in rHCNTF-treated patients. Mortality was similar in all three study arms. There were no statistically significant treatment effects among the secondary measures. Side effects of rHCNTF included anorexia, weight loss, and cough and were sufficient to limit dosing in many patients.

The Amyotrophic Lateral Sclerosis Functional Rating Scale

<h3>Objectives:</h3> To test the utility of a new, easy to administer instrument for assessing activities of daily living in patients with amyotrophic lateral sclerosis (ALS), to validate its accuracy, and to assess its ability to record disease progression in patients with ALS against other functional scales, quantitative isometric muscle testing, and global assessment scales. <h3>Design:</h3> Serial assessments of patients who presented to four ALS treatment centers in two multicenter studies. <h3>Patients:</h3> Study I (cross-sectional) evaluated 75 consecutive patients who presented to four ALS treatment centers during a 2-month period. Study 2 (longitudinal) evaluated the progression of 53 patients who were enrolled in a multicenter, phase I-II clinical trial of recombinant human ciliary neurotrophic factor for treatment of ALS. <h3>Outcome Measures:</h3> The ALS Functional Rating Scale (ALSFRS) was compared with quantitative myometry and with other measures of daily function in patients with ALS both cross-sectionally and longitudinally. <h3>Results:</h3> The first study of 75 patients evaluated the internal consistency, the test-retest reliability, and the construct validity of the ALSFRS. Internal consistency and test-retest reliability were high. Patient self-rating of upper- and lower-extremity—dependent tasks were highly correlated with measures of upper- and lower-extremity strength, respectively. Thus, the ALSFRS has good construct validity. In the second study, ALSFRS scores declined in tandem with deterioration in motor and pulmonary function, indicating its sensitivity to change. <h3>Conclusions:</h3> The ALSFRS is a useful instrument for evaluation of functional status and functional change in patients with ALS. Its results are in close agreement with objective measures of muscle strength and pulmonary function. The ALSFRS may be used as a screening measure for entry into clinical trials, as a surrogate measure of function in situations in which muscle strength cannot be measured directly, or as an adjunct to myometry.

Association between a null mutation in the human ciliary neurotrophic factor (CNTF) gene and increased of psychiatric diseases?

We report a possible association between a null mutation in the human ciliary neurotrophic factor (CNTF) gene and psychiatric diseases. Prior findings that the mutant allele frequency is not significantly elevated in patients suffering from neurological diseases are confirmed. The frequency of the mutant allele was higher among psychiatric patients (0.192, n=297) than among healthy controls and neurological patients (0.142, n=267). This difference (one-tailed 2 × 2 chi-square test, P<0.05) might be evidence that disturbances in the neurotrophic factor system could play a crucial role in the etiopathogenesis of psychiatric disorders, mainly psycoses.

Ciliary neurotrophic factor may act in target musculature to regulate developmental synapse elimination.

During development of mammalian skeletal muscles, synaptic contacts from different motoneurons are made on to individual muscle fibers but then are progressively lost until the adult pattern of single innervation is established. Although this process of synapse elimination occurs throughout the developing nervous system, little is known about the underlying mechanisms that drive this process. Competition for target-derived trophic substances has been proposed as one mechanism whereby synapses are selectively maintained or eliminated. To directly test whether exogenous trophic substance could alter neuromuscular synapse elimination, levator ani (LA) muscles of male rats were treated during the period of synapse elimination with either human recombinant ciliary neurotrophic factor (CNTF-a putative motoneuronal survival factor), or vehicle. LA muscles were stained with tetranitroblue tetrazolium at the end of treatment and the number of axonal inputs per muscle fiber was quantified. Effects of CNTF on other parameters such as body weight, axonal sprouting, muscle fiber and motoneuronal growth were also assessed. CNTF-treated muscles contained 3 times more multiple innervation than did vehicle-treated muscles, suggesting that CNTF can regulate synapse elimination in the LA. Moreover, CNTF delivered near the LA was more potent in blocking synapse elimination than the same dose of CNTF delivered at a site distant from the LA, suggesting that the target muscle is an important site, either for direct CNTF action on synapse elimination, and/or for directed transport of CNTF to motoneuronal cell bodies.

Human CNTF and related cytokines: effects on DRG neurone survival

Ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), interleukin-6 (IL-6), and interleukin-11 (IL-11) are structurally and functionally related cytokines. We compared their survival-promoting activities on embryonic chick and newborn rat dorsal root ganglion (DRG) neurones. Human CNTF showed the well known trophic effect on both chick and rat DRG neurones. Human and murine LIF and, at unphysiologically high doses, human OSM were trophic for rat neurones, but failed to promote chick DRG cell survival. Human IL-11, murine IL-6 and human IL-6 did not improve chick or rat DRG neurone survival; soluble human IL-6 receptor α did not increase sensitivity to human IL-6. Thus, human CNTF as well as murine and human LIF had special neurotrophic properties compared with other related cytokines.

Human CNTF and related cytokines

Ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), interleukin-6 (IL-6), and interleukin-11 (IL-11) are structurally and functionally related cytokines. We compared their survival-promoting activities on embryonic chick and newborn rat dorsal root ganglion (DRG) neurones. Human CNTF showed the well known trophic effect on both chick and rat DRG neurones. Human and murine LIF and, at unphysiologically high doses, human OSM were trophic for rat neurones, but failed to promote chick DRG cell survival. Human IL-11, murine IL-6 and human IL-6 did not improve chick or rat DRG neurone survival; soluble human IL-6 receptor alpha did not increase sensitivity to human IL-6. Thus, human CNTF as well as murine and human LIF had special neurotrophic properties compared with other related cytokines.

Analysis of regulatory regions of the ciliary neurotrophic factor gene in transgenic mice

In order to study the regulatory regions of the human ciliary neurotrophic factor (CNTF) gene we made constructs containing sequences upstream and downstream of CNTF coding regions and the lacZ gene and analysed their expression in transgenic mice. We show that 240 bp upstream of the translation start codon are sufficient for the transcription of the lacZ gene. A further 4 kb upstream sequence is required for the expression of the transgene in Schwann cells. These two upstream regions together with a 2 kb downstream fragment drive high level of expression of the lacZ gene in the sciatic nerve. Our results indicate that these three fragments contain regulatory regions able to mimic the CNTF expression pattern in the mouse peripheral nervous system.

The Pharmacokinetics of Subcutaneously Administered Recombinant Human Ciliary Neurotrophic Factor (rHCNTF) in Patients with Amyotrophic Lateral Sclerosis

Fifty-seven patients with amyotrophic lateral sclerosis (ALS) were randomly assigned to receive single subcutaneous doses of 0.5, 1, 3, 7, 10, or 30 micrograms/kg recombinant human ciliary neurotrophic factor (rHCNTF) or placebo. Peak plasma concentrations occurred 180 to 260 min after dosing. rHCNTF plasma concentrations then appeared to decay, with an apparent elimination half-life of 120-400 min. Eight of 12 patients who received the highest dose had measurable plasma rHCNTF levels 24 h after dosing. Peak plasma concentrations of rHCNTF in patients receiving 30 micrograms/kg rHCNTF were in the range that has been shown to support the survival of embryonic rat and human motor neurons in tissue culture. Systemic exposure to rHCNTF increased with increasing dose. The elimination of rHCNTF, over the dose range evaluated, appeared to follow first-order processes. Elevations in body temperature and evidence of activation of the acute phase response were observed.

A Phase I Study of Recombinant Human Ciliary Neurotrophic Factor (rHCNTF) in Patients with Amyotrophic Lateral Sclerosis

Fifty-seven patients with amyotrophic lateral sclerosis (ALS) were randomly assigned to receive 0.5, 1, 3, 7, 10, or 30 micrograms/kg recombinant human ciliary neurotrophic factor (rHCNTF) or placebo subcutaneously 3 times a week for 2 weeks. Dose-limiting toxicity, consisting of febrile reactions in some patients, fatigue, and nonproductive cough, was observed at a dose level of 30 micrograms/kg. Dose-related changes in parameters of the acute-phase response were noted, consistent with the relationship of CNTF and its receptor system to the cytokine interleukin-6 (IL-6) and its receptor. No adverse neurologic consequences of rHCNTF administration were observed. Antibodies to rHCNTF were observed in sera of most patients tested after 2 weeks of continuous treatment and 4 weeks' withdrawal period. rHCNTF was safe and tolerated within acceptable limits when administered to patients with ALS in this study at doses of up to 30 micrograms/kg 3 times a week for 2 weeks. Further studies to explore the efficacy of rHCNTF in the treatment of human motor neuron diseases are justified.

Preparation and a structure-function analysis of human ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a trophic protein that promotes survival and/or differentiation of a variety of neuronal cell types including sensory, sympathetic, and motor neurons. CNTF, leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and oncostatin M (OSM) share a predicted common helical framework and partially identical receptor components. In this study, we present the preparation and structure — functional analysis of recombinant human CNTF. The human CNTF gene was expressed under the control of the P L promoter in Escherichia coli, and the mutants were constructed by insertion, deletion and site-directed mutagenesis. The recombinant proteins were purified from bacteria via DEAE A-50 and Sephacryl S-200 chromatography, and their survival promoting activities were determined using cultures of embryonic chick dorsal root ganglion (DRG) neurons. Insertion at position 23 with APGL, or at position 79 with PRGA, or substitution of 162L163Q for PIDG resulted in proteins with no neurotrophic activity. However, insertion at position 186 with PRGI did not alter human CNTF activity. Deletion of the carboxy-terminal amino acid 186-200 did not reduce the biological activity, but elimination of the amino acid 162-186 abolished the activity. The mutant substituting of 17 Cys for Ser was found to display a biological activity equivalent to that of the wild type. Our data provided experimental confirmation for the structural prediction of CNTF.

D1 cap region involved in the receptor recognition and neural cell survival activity of human ciliary neurotrophic factor.

Human ciliary neurotrophic factor (hCNTF), which promotes the cell survival and differentiation of motor and other neurons, is a protein belonging structurally to the alpha-helical cytokine family. hCNTF was subjected to three-dimensional structure modeling and site-directed mutagenesis to analyze its structure-function relationship. The replacement of Lys-155 with any other amino acid residue resulted in abolishment of neural cell survival activity, and some of the Glu-153 mutant proteins had 5- to 10-fold higher biological activity. The D1 cap region (around the boundary between the CD loop and helix D) of hCNTF, including both Glu-153 and Lys-155, was shown to play a key role in the biological activity of hCNTF as one of the putative receptor-recognition sites. In this article, the D1 cap region of the 4-helix-bundle proteins is proposed to be important in receptor recognition and biological activity common to alpha-helical cytokine proteins reactive with gp130, a component protein of the receptors.

Human ciliary neurotrophic factor: a structure-function analysis.

Ciliary neurotrophic factor (CNTF) promotes survival in vitro and in vivo of several neuronal cell types including sensory and motor neurons. The primary structure of CNTF suggests it to be a cytosolic protein with strong similarity to the alpha-helical cytokine family which is characterized by a bundle of four anti-parallel helices. CNTF exerts its activity via complexation with CNTF receptor (CNTF-R). This complex consists of a CNTF-binding protein (CNTF-R) and two proteins important for signal transduction [gp130 and leukaemia inhibitory factor receptor (LIF-R)]. We have shortened the cDNA coding for CNTF at both the 5' and the 3' end and expressed the truncated proteins in bacteria. Biological activities of the protein preparations were determined by their ability to induce proliferation of BAF/3 cells that were stably transfected with CNTF-R, gp130 and LIF-R cDNAs. CNTF proteins with 14 amino acid residues removed from the N-terminus were biologically active whereas the removal of 23 amino acids resulted in an inactive protein. In addition, 18 amino acid residues could be removed from the C-terminus of the CNTF protein without apparent loss of bioactivity, but further truncation at the C-terminus yielded biologically inactive proteins. The introduction of two point mutations into the CNTF protein at a site that presumably interacts with one of the two signal-transducing proteins resulted in a CNTF mutant with no measurable bioactivity. In addition, a model of the three-dimensional structure of human CNTF was constructed using the recently established structural co-ordinates of the related cytokine, granulocyte colony-stimulating factor. CD spectra of CNTF together with our mutational analysis and our three-dimensional model fully support the view that CNTF belongs to the family of alpha-helical cytokines. It is expected that our results will facilitate the rational design of CNTF mutants with agonistic or antagonistic properties.

Crystal structure of dimeric human ciliary neurotrophic factor determined by MAD phasing.

Ciliary neurotrophic factor (CNTF) promotes the survival and differentiation of developing motor neurons and is a potential therapeutic for treating neurodegeneration and nerve injury. The crystal structure of human CNTF has been determined at 2.4 A resolution using multi-wavelength anomalous diffraction (MAD) phasing from a single Yb3+ ions. The structure reveals that CNTF is dimeric, with a novel anti-parallel arrangement of the subunits, not previously observed for other cytokines. Each subunit adopts a double crossover four-helix bundle fold, in which two helices contribute to the dimer interface, whilst two different helices show pronounced kinks. Analysis of the electrostatic surface of CNTF identified residues within these kinked helices that may contact the CNTF receptor-alpha. Solution experiments show that CNTF dimerizes at concentrations > 40 microM. Such dimers are likely to be relevant to the storage of CNTF in the peripheral nerve given the high concentrations present in this tissue. However, it is unlikely that they play a role in engaging the three distinct receptor subunits that comprise the CNTF receptor, given the low concentration of extracellular CNTF and its high potency.

Site-directed mutagenesis of human CNTF: functional analysis of recombinant variants.

Ciliary neurotrophic factor (CNTF), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M (OSM) share functional properties, a predicted common helical framework, and partially identical receptor components. CNTF is a survival promoting factor for various types of neurons in vitro and in vivo. In the present study, structural features essential for the biological function of human CNTF were investigated. Several recombinant CNTF variants were constructed by PCR and expressed in E. coli. Their survival promoting activities were determined using cultures of embryonic chick and newborn rat dorsal root ganglion cells. Deletion of 14 N-terminal and 18 C-terminal amino acids significantly increased bioactivity compared to wild-type (wt) CNTF. Further truncation of the CNTF molecule at the N- or C-terminus resulted in a significant reduction or complete loss of activity. Substitution of two amino acids (Lys154Glu and Trp157Pro) abolished the survival promoting effect. Recently described analogous substitutions in IL-6 had resulted in a partial IL-6 receptor antagonist. However, the double substitution variant had no significant inhibitory effect on wtCNTF activity in assays with both wt and mutant factor. The CNTF variants constructed had almost identical effects on both chick and rat neurons indicating a close similarity of the avian and the mammalian CNTF receptor complex. The present results also demonstrate that a core segment of the CNTF molecule is indispensable for biological function. Analogous segments important for activity have already been identified in the related molecules IL-6, LIF, and OSM. Thus, our data confirm the close structural relationship of CNTF to these "neuropoietic" cytokines. In addition, they demonstrate that site-directed mutagenesis of recombinant human CNTF can yield molecules which show increased survival promoting activity on mammalian neurons.

CDNA cloning and chromosomal localization of the human ciliary neurotrophic factor gene

Full-length cDNA for human ciliary neurotrophic factor (CNTF) was isolated from a human sciatic nerve cDNA library. Sequence analysis revealed that the longest cDNA was comprised of a 48-bp 5′-untranslated region, a 600-bp coding region and a 1207-bp 3′-untranslated region containing four ATTA pentamer motifs and a polyadenylation sequence. The transcription starting point was assigned at 81 bp upstream of the initiation methionine by 5′ RACE analysis. Using the cDNA and genomic DNA fragment including the entire intron region as mixed probes, the human CNTF gene was localized to the long arm of chromosome 11 at region q12 by fluorescence in situ hybridization.

Histometric effects of ciliary neurotrophic factor in wobbler mouse motor neuron disease

We investigated the histological effects of ciliary neurotrophic factor on degenerating motor neurons, their axons, and skeletal muscles in 68 wobbler mice with motor neuron disease. Treatment consisted of recombinant rat or human ciliary neurotrophic factor (or a vehicle solution), 1-mg/kg subcutaneous injection, three times per week for 4 weeks after the clinical diagnosis. The number of motor neurons immunoreactive for calcitonin gene-related peptide was higher in mice receiving rat ciliary neurotrophic factor (p < 0.03), although the number of choline acetyltransferase-reactive neurons was the same in both treated and untreated control groups. Treatment did not prevent vacuolar degeneration of motor neurons. In mice treated with human ciliary neurotrophic factor, the percentage of axons undergoing acute axonal degeneration (myelin ovoids) was smaller in the entire C5 ventral root (p < 0.02) and in the musculocutaneous nerve (p < 0.04), and the number of myelinated nerve fibers was 30% higher in both nerves (p < 0.01 and p < 0.04, respectively) than in controls. In ciliary neurotrophic factor-treated mice, the biceps muscle weight was 20% greater, the mean muscle fiber diameter was 30% larger, and the number of atrophied muscle fibers was 75% lower than that in the vehicle-treated wobbler mice (p < 0.001 for all three results). The number of terminal axonal branching points and the mean length of motor end-plates were also higher in the ciliary neurotrophic factor-treated mice (p < 0.001 and p < 0.02, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding characteristics of ciliary neurotrophic factor to sympathetic neurons and neuronal cell lines.

Ciliary neurotrophic factor (CNTF) is a cytokine whose actions are largely restricted to the nervous system because of the predominant neuronal distribution of its receptor, CNTFR alpha. In this study, we sought to define the binding characteristics of CNTF to cultured sympathetic neurons and cell lines of neuronal origin. We report that 125I-CNTF binds to cultured sympathetic neurons, MAH, PC12, and EW-1 cells via high and low affinity receptors that can be distinguished on the basis of their dissociation constants (KD1 approximately 10(-12) M and KD2 approximately 10(-9) M). Competition experiments showed that the IC50 for rat and human CNTF were, respectively, 65 pM and 5 nM for sympathetic neurons and 75 pM and 1.2 nM for EW-1 cells. Interestingly, leukemia inhibitory factor (LIF) did not compete for CNTF binding even at 100 nM concentration. The binding of 125I-CNTF to sympathetic neurons involved all three components of the CNTF receptor complex, namely CNTFR alpha, LIFR, and gp130, as shown by cross-linking experiments. CNTF and LIF treatments down-regulated CNTF binding to sympathetic neurons and EW-1 cells, suggesting that heterologous ligands can regulate CNTF receptor levels, which may in turn modulate the efficacy of CNTF in vitro and in vivo.

Production, characterization and cytotoxic properties of a diphtheria toxin-ciliary neurotrophic factor fusion protein.

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. This study describes the genetic construction, expression, purification and properties of a diphtheria toxin-related CNTF fusion gene in which the native receptor binding domain of diphtheria toxin was genetically replaced with a synthetic gene encoding human CNTF. The fusion protein expressed from the chimeric tox gene was designated DAB389-CNTF. This fusion toxin has a deduced molecular weight of 67 440 and is formed by the fusion of the first 389 amino acids of diphtheria toxin to amino acids 15-200 of mature human CNTF (Cys17-->Ser), using a bridge of 34 additional amino acids including six consecutive histidine residues. This latter span allows for a single-step purification of the fusion protein by Ni(2+)-immobilized metal ion affinity chromatography, and provides a degree of flexibility which facilitates polypeptide refolding. DAB389-CNTF was selectively cytotoxic for clonal cells bearing CNTF receptors and for CNTF-responsive spinal sensory ganglion neurons in primary culture. The cytotoxic action of DAB389-CNTF, like that of native diphtheria toxin, required receptor-mediated endocytosis, passage through an acidic compartment and delivery of an ADP-ribosyltransferase to the cytosol of target cells. The delivery of the catalytic domain to the target cell cytosol results in inhibition of protein synthesis and cell death. This latter point was confirmed by the observation that both CNTF and DAB389-CNTF increased c-fos mRNA expression, but only CNTF induced Fos protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional phage display of ciliary neurotrophic factor

We report the display of human ciliary neurotrophic factor (hCNTF), a survival factor for neuronal cells belonging to the α-helical cytokine superfamily, on the surface of the filamentous bacteriophage fd. The h CNTF cDNA was fused to a DNA sequence encoding the C-terminal domain of pIII, a minor coat protein exposed at one end of fd. Gene fusions were cloned into a plasmid containing the ColE1 plasmid and fd origins of replication, and were packaged into phagemid particles upon superinfection with M13KO7 helper phage. The resulting fusion phage bound specifically to anti-CNTF antibodies and to the recombinant soluble CNTF α-receptor. Moreover, phage-displayed hCNTF was found to possess biological activity at concentrations comparable to those of the soluble cytokine. These results demonstrate that CNTF can be displayed on phage in a correctly folded and functionally active form. Binding of fusion phage to immobilized CNTF α-receptor and subsequent elution at low pH resulted in affinity purification of CNTF-displaying virions. Utilization of this technology should enable the selection of high-affinity variants from libraries of CNTF mutants displayed on phage.