Remyelination of the Adult Demyelinated Mouse Brain by Grafted Oligodendrocyte Progenitors and the Effect of B-104 Cografts

The 4e transgenic mouse is characterized by overexpression of the PLP gene. Heterozygous littermates containing three PLP gene copies develop and myelinate normally. However, a progressive CNS demyelination begins at 3-4 months of age. Despite focal demyelination, these animals survive for one year with hind limb paralysis. We used this CNS demyelination model to determine if grafts of CG4 oligodendrocyte progenitors would survive and myelinate the adult CNS. Either CG4 cells, or co-grafts of CG4/B104 cells 11:1 ratio respectively) were performed. Grafted cells survived and migrated in the normal and transgenic brain. Non-treated transgenic animals revealed extensive lack of myelin. Three months post-transplant hosts with CG4 or co-transplants displayed a near normal myelin pattern. Double immunofluorescence for neurofilament and myelin basic protein revealed the presence of many naked axons in non-grafted transgenic animals. Those grafted with progenitor CG4 cells or cografts displayed a clear increase in remyelination. This data provides a new direction for the development of cell replacement therapies in demyelinating diseases.     

Interferon-β Is a Potent Promoter of Nerve Growth Factor Production by Astrocytes

Abstract: Recent clinical evidence has suggested that interferon-β is efficacious in the treatment of the demyelinating disease, multiple sclerosis. The mechanism of its efficacy remains unclear, and suggested modes of action have focused on immune modulation. Nonimmune effects of interferon-β may also contribute to its efficacy. Given that astrocytes produce a range of neurotrophic factors, we examined the possibility that interferon-β could increase the astrocytic production of nerve growth factor (NGF), which has been reported to cause oligodendrocytes to proliferate and to extend their processes; these phenotypes can impact favorably on remyelination. When the recombinant form of mouse interferon-β was added to mouse astrocyte cultures, a dose-dependent increase in NGF mRNA was obtained. The 40-fold increase in NGF mRNA elicited by 1,000 U/ml interferon-β was far more potent than that produced by other NGF-elevating agents in this study. In concordance, the protein for NGF was elevated by interferon-β. The production of NGF by interferon-β may be relevant to its clinical efficacy in multiple sclerosis. Furthermore, we suggest the potential utility of interferon-β in Alzheimer's disease.     

Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target-derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF-specific ligand-binding α-subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well-defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina-specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα-expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.     

Differential Expression of Ciliary Neurotrophic Factor Receptor in Skeletal Muscle of Chick and Rat After Nerve Injury

Abstract: The activities of ciliary neurotrophic factor (CNTF) were initially thought to be restricted to cells in the nervous system. However, the recent identification of its receptor specificity-conferring α component (CNTFRα) in skeletal muscle has provided the clue to the unexpected actions of CNTF in the periphery. In the present study, we demonstrated that the mRNA expression of CNTFRα in chick skeletal muscle was decreased by ∼10-fold after nerve transection; this finding is in sharp contrast to the dramatic up-regulation observed in denervated rat muscle. As a first step toward investigating the differential regulation of CNTFRα in chick and rat, we examined the mRNA expression of CNTFRα in different types of muscle following nerve injury in young and adult animals. Our findings demonstrated that the differential expression of CNTFRα observed in denervated skeletal muscle of the chick and rat was not dependent on age or muscle type. The temporal profile of the changes in CNTFRα expression was, however, dependent on the age of the chick as well as the types of muscle. Furthermore, the low level of CNTFRα expression observed in denervated chick muscle recovered to almost control levels in regenerating skeletal muscle. Taken together, our findings provided the first extensive analysis on the mRNA expression of CNTFRα and the α subunit of the acetylcholine receptor in various skeletal muscles of the chick following nerve injury and regeneration.     

Ciliary Neurotrophic Factor (CNTF) Genotypes and CNTF Contents in Human Sciatic Nerves as Measured by a Sensitive Enzyme-Linked Immunoassay

Abstract: To study the level of ciliary neurotrophic factor (CNTF) in human nervous tissues, we developed a sensitive enzyme-linked immunoassay using a specific antibody against human CNTF. This method allowed us to detect as little as 0.3 ng/ml of human CNTF with good linearity and accuracy. Using this method, CNTF levels were determined in human sciatic nerves obtained at autopsy from 21 amyotrophic lateral sclerosis (ALS) patients and 48 subjects who had died of other neurological diseases. CNTF genotypes were also determined. The results indicated that CNTF levels were high in the normal homozygotes and approximately halved in the heterozygote subjects. There was, however, no significant difference in CNTF levels in the sciatic nerves between ALS and other neurological disease patients, indicating that the CNTF level was mainly determined by its genotypes and that the level in the sciatic nerves was not reduced in ALS patients.     

聚乙二醇20k修饰与转铁蛋白偶联睫状神经营养因子的生物活性对比研究

为了延长重组睫状神经营养因子在体内的保留半衰期,基于CNTF中天然的游离半胱氨酸残基,在前期工作中采用聚乙二醇修饰和转铁蛋白偶联的两种方式对CNTF进行了改造。此后又采用常规分析手段对PEG20k-CNTF和Tf-PEG5k-CNTF进行对比表征。高效凝胶过滤和动态光散射分析结果显示两者拥有相近的表观分子体积。细胞试验结果显示两种耦合物的活性分别下降至未修饰CNTF的50.6%和65.8%。抗体CNTF抗体亲和力结果显示PEG20k修饰后亲合力下降至原蛋白的3.8%,转铁蛋白偶联后保留89.9%原蛋白亲合力。药代动力学结果显示PEG20k-CNTF和Tf-PEG5k-CNTF在SD大鼠血液中的保留半衰期分别为5.34±0.26和8.65±0.60小时,与未修饰rh CNTF相比延长了约21.4倍和34.6倍。药效学结果显示在每周两次每次1.0 mg/kg(rh CNTF等量)的给药频率和剂量下,PEG20k-CNTF比Tf-PEG5k-CNTF更显著地降低实验小鼠体重。     

转铁蛋白-PEG-睫状神经营养因子的制备及其生物活性评价

为了实现在体内更持久的药效作用,根据睫状神经营养因子CNTF第17位为游离半胱氨酸残基,而转铁蛋白Tf无游离半胱氨酸的特点,采用N-羟基琥珀酰亚胺-聚乙二醇5K-马来酰亚胺(NHS-PEG5k-MAL)作为偶联剂,实现了两者定点偶联,然后结合蛋白自身特性制定纯化方法,制备获得纯度高于90%的转铁蛋白-聚乙二醇5k-睫状神经营养因子(Tf-PEG5k-CNTF)耦合物。高效凝胶色谱和动态光散射分析显示耦合物的表观分子体积大于两蛋白之和。细胞试验结果显示耦合物的活性下降至原蛋白的65.8%。大鼠药代动力学试验显示Tf-PEG5k-CNTF耦合物在体内的代谢半衰期延长至8.2小时,与CNTF原蛋白相比提高了约17倍。小鼠动物试验显示在每周2次的给药频率,每次1.0 mg/kg的剂量下,Tf-PEG5k-CNTF能更为显著地影响小鼠对食物摄入量和减轻体重。因此,转铁蛋白偶联技术可用于脑部靶向蛋白药物的长效递送。     

Release of ciliary neurotrophic factor from cultured astrocytes and its modulation by cytokines

CNTF rescues various types of lesioned neurons in vivo, and it needs to be released from astrocytes into the extracellular space to have the effect. However, direct evidence for CNTF release has not been unequivocally demonstrated. We hypothesized that the rapid sequestration by CNTF receptor present on cultured astrocytes might be the cause of the inability to detect CNTF released into astrocyte-conditioned medium (ACM). Therefore, we measured CNTF immunoreactivity in medium conditioned by astrocytes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) which was used to prevent released CNTF from binding to the CNTF receptor, since PI-PLC cleaves glycosyl-phosphatidylinositol anchor of CNTFR, the unique component involved in CNTF binding. CNTF was not detectable in untreated ACM, but was detectable in PI-PLC-treated ACM. These results together with the evidence that PI-PLC treatment did not have a toxic effect on astrocytes prove the fact that CNTF can be released from astrocytes without cell lysis. Subsequently, the effect of cytokines such as IL-1, TNF-, and EGF on CNTF release was examined. These cytokines increased CNTF protein levels in ACMs without increasing CNTF protein levels in astrocyte-extracts, indicating that they enhanced CNTF release from astrocytes.     

Possible implication of ciliary neurotrophic factor (CNTF) and beta-synuclein in the ammonia effect on cultured rat astroglial cells: a study using DNA and protein microarrays

Astrocytes are considered the key cell in hepatic encephalopathy; although their precise role in the disease has not yet been determined, exposure to ammonia appears to have an important pathogenic effect. We exposed confluent cultures of rat astroglial cells to ammonia (5 mM NH₄Cl) for 1, 3, 5 and 7 days, and determined astroglial levels of actin, glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), GLAST glutamate transporter, 25 kDa heat-shock protein (HSP25), HSP60 and HSP70 by Western blot; the glutamine content in culture medium was measured by mass spectrometry. Significant increases were observed for GS, HSP60 and glutamine, and significant reductions for actin and GFAP.

Astrocytes exposed to ammonia for 4 days were used to analyze the effect of ammonia in protein and DNA microarrays. After protein microarray data filtration by signal intensity, x-fold change and z-score, 11 proteins were selected, among which the significant increase in β-synuclein was confirmed by Western blot. DNA microarray data filtration by intensity signal, x-fold change and p-value selected almost 600 genes. The significant increase in -synuclein mRNA was confirmed by quantitative RT-PCR, but no change was observed in -synuclein protein levels. A notable decrease in ciliary neurotrophic factor (CNTF) was demonstrated by Western blot after ammonia treatment, concurring with the reduction in CNTF mRNA observed in DNA microarrays. We discuss the possibility of a pathogenic role for CNTF and a protective role for β-synuclein in experimental hyperammonemia. This study demonstrates the use of microarrays as tools to ascertain the possible implication of previously unidentified proteins in the pathogenesis of hepatic encephalopathy.     

V170 Area Plays an Essential Role in the Biological Activity of Human Ciliary Neurotrophic Factor

Abstract: The structure-function relationships of human ciliary neurotrophic factor (CNTF) were analyzed by mutagenic means. Amino acid substitutions at helix D caused marked changes in the biological activity of CNTF, suggesting that the residues at helix D of CNTF participate in receptor recognition. In particular, both the cell survival-promoting activity and receptor binding ability of V170 mutant CNTF proteins correlated well with the hydrophobicity of amino acids at position 170. The reduction of hydrophobicity at position 170 resulted in a loss of biological activity, indicating that the hydrophobicity of V170 is essential for the receptor binding and cell survival-promoting activity. Substitutions of R171 or D175 evoked very little folding ability and negated the biological activity of CNTF. As R171 and D175 interact electrostatically with each other and with E75 and R72, respectively, these interactions would be indispensable for stabilizing the whole CNTF protein and for maintaining the structure of the receptor binding epitope.     

Inhibition of p42 and p44 Mitogen-Activated Protein Kinase Activity by PD98059 Does Not Suppress Nerve Growth Factor-Induced Survival of Sympathetic Neurones

Abstract: Nerve growth factor (NGF) induces persistent p42 and p44 mitogen-activated protein kinase (MAPK) activity in sympathetic neurones in parallel to its survival-promoting activity. To investigate whether these MAPK activities are necessary for NGF-induced survival, we have inhibited NGF-stimulated p42/p44 MAPK activity over extended periods using the compound 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059). Despite attaining up to 95% inhibition of p42/p44 MAPK activity in cultures treated with NGF and PD98059, neuronal survival is maintained undiminished, although a decrease in the density of the neuritic network is observed. Because p21Ras activity is essential for NGF-induced survival, we conclude that p21Ras-linked activities other than p42 and p44 MAPKs are responsible for mediating NGF-dependent survival of rat sympathetic neurones.     

Ciliary neurotrophic factor upregulates ubiquitin-proteasome components in a rat model of neuronal injury

Neuronal injury triggers the release of ciliary neurotrophic factor (CNTF), promoting local neuronal repair but producing systemic effects of anorexia and lean body weight loss. Due to the rapid rate of systemic protein loss stimulated by CNTF, we hypothesized involvement of the hepatic ubiquitin-proteasome proteolytic (UPP) pathway in CNTF-induced proteolysis. To assess the role of central CNTF in systemic UPP regulation, we measured hepatic UPP mRNA and proteasome activity in a rat model of neuronal injury and determined alterations induced by intracerebroventricular (ICV) administration of CNTF-neutralizing antibody or additional exogenous CNTF. We also assessed proteolytic parameters and nutritional status by measuring caloric intake, body weight, and protein levels. We produced neuronal injury by implanting a lateral ventricle cannula and giving daily ICV saline bolus injections, which increased hepatic 20S proteasome mRNA and enzymatic activity while reducing caloric intake, body weight, and protein levels compared to controls. Administration of ICV anti-CNTF antibodies (but not control antibodies) prevented these effects. Addition of exogenous CNTF augmented the weight loss along with the increases in 20S proteasome mRNA and proteolytic activity induced by neuronal injury. We conclude that CNTF decreases lean body weight through a combination of appetite inhibition and UPP pathway activation.     

Glial Cell Line-Derived Neurotrophic Growth Factor Inhibits Apoptotic Death of Postnatal Substantia Nigra Dopamine Neurons in Primary Culture

Abstract: Glial cell line-derived neurotrophic factor (GDNF) was identified on the basis of its ability to enhance the development of embryonic mesencephalic dopamine neurons. It remains unknown whether GDNF is a physiologically relevant trophic factor for these neurons. We have shown that natural cell death among dopamine neurons of the substantia nigra occurs largely postnatally. To investigate whether GDNF may have the ability to support these neurons during their period of natural cell death, we have used a postnatal primary culture model. We find that GDNF is able to support the viability of postnatal nigral dopamine neurons by inhibiting apoptotic death. This ability of GDNF shows both regional specificity for the nigra and cellular specificity for the dopamine phenotype. Among eight other neurotrophic factors previously reported to support embryonic dopamine neurons, GDNF was unique in this ability. Thus, GDNF meets this criterion for a physiologically relevant trophic factor for dopamine neurons of the substantia nigra.     

Construction and Characterization of Ciliary Neurotrophic Factor (CNTF) Antagonists: Microenvironmental Difference in the CNTF Receptor Between Rat and Chicken Cells for Recognizing the D1 Cap Region

Abstract: Antagonistic mutants of ciliary neurotrophic factor (CNTF) were constructed and their properties characterized. K155A and K155W mutants lost cell survival promoting activity for chicken dorsal root ganglion (DRG) neurons and inhibited the activity of the wild type. However, they retained slight agonistic activity for the survival of rat DRG neurons, indicating there is a difference between chicken and rat cells for receptor recognition around the D1 cap region including K155 residue. The chicken receptor recognizes the D1 cap region more strictly than does the rat receptor. The substitution of F152, which locates at the top of the D1 cap region, was combined with the K155A mutation. A combination of the two mutations gave an antagonistic feature to not only chicken but also rat cells. Both F152S/K155A and F152D/K155A mutants lacked cell survival promoting activity and had an antagonistic effect on rat DRG neurons. The three-dimensional structure of CNTF suggests the following. F152 and K155 bind to the receptor with hydrophobic and electrostatic interactions, respectively. F152 locates close to L156 with a van der Waals contact, and K155 contacts with Q42 through a hydrogen bond. Both interactions play indispensable roles in maintaining the structure around the D1 cap region of CNTF.     

The N-terminal cytokine binding domain of LIFR is required for CNTF binding and signaling

Ciliary neurotrophic factor (CNTF) forms a functional receptor complex containing the CNTF receptor, gp130, and the leukemia inhibitory factor receptor (LIFR). However, the nature and stoichiometry of the receptor-mediated interactions in this complex have not yet been fully resolved. We show here that signaling by CNTF, but not by LIF or oncostatin M (OSM), was abolished in cells overexpressing a LIFR mutant with the N-terminal cytokine binding domain deleted. Our results illustrate molecular differences between the CNTF active receptor complex and those of LIF and OSM and provide further support for the hexameric model of the CNTF receptor complex.     

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.     

具有NGF活性通过二硫键连接的 2个小分子多肽的制备及鉴定(英文)

神经生长因子 (NGF)作用广泛 ,β NGF是神经生长因子的活性部分 .由于 3对二硫键的影响 ,体外表达NGF很难形成正确折叠的肽链 .由于神经系统血脑屏障的存在 ,大分子肽链的给药途径是一个不可忽视的难题 .根据NGF的氨基酸序列及其晶体构象资料 ,选择CNBr在 9位Met处 ,胰蛋白酶在Arg或Lys处裂解 β NGF .经过SephadexG 5 0层析、DE 5 2纤维素离子交换层析和反相高压液相层析分离纯化后获得NGF活性片段 .氨基酸组成分析及氨基酸序列分析结果表明 ,此片段由 16肽GEFSVCDSVSVWVGDK与 14肽HWNSYCTTTHTFVK通过 1对二硫键连接而成 .8日龄鸡胚背根神经节生物活性分析表明 ,其最佳作用浓度为 0 0 0 1～ 0 1μg L .它的成功分离和鉴定为合成或表达高活性小分子神经营养物质奠定了关键而重要的基础 .