Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy

The period of natural cell death in the development of rodent motor neurons is followed by a period of sensitivity to axonal injury. In the rat this early postnatal period of vulnerability coincides with that of very low ciliary neurotrophic factor (CNTF) levels in the sciatic nerve before CNTF increases to the high, adult levels. The developmental time course of CNTF expression, its regional tissue distribution and its cytosolic localization (as suggested by its primary structure) favour a role for CNTF as a lesion factor rather than a target-derived neurotrophic molecule like nerve growth factor. Nevertheless CNTF exhibits neurotrophic activity in vitro on different populations of embryonic neurons. To determine whether the vulnerability of motor neurons to axotomy in the early postnatal phase is due to insufficient availability of CNTF, we transected the axons of newborn rat motor neurons and demonstrated that local application of CNTF prevents the degeneration of the corresponding cell bodies.     

Structure, expression and function of a schwannoma-derived growth factor

During the development of the nervous system, cells require growth factors that regulate their division and survival. To identify new growth factors, serum-free growth-conditioned media from many clonal cell lines were screened for the presence of mitogens for central nervous system glial cells. A cell line secreting a potent glial mitogen was established from a tumour (or 'schwannoma') derived from the sheath of the sciatic nerve. The cells of the tumour, named JS1 cells, were adapted to clonal culture and identified as Schwann cells. Schwann cells secrete an autocrine mitogen and human schwannoma extracts have mitogenic activity on glial cells. Until now, neither mitogen has been purified. Here we report the purification and characterization of a mitogenic molecule, designated schwannoma-derived growth factor (SDGF), from the growth-conditioned medium of the JS1 Schwann cell line. SDGF belongs to the epidermal growth factor family, and is an autocrine growth factor as well as a mitogen for astrocytes, Schwann cells and fibroblasts.     

Molecular cloning and expression of brain-derived neurotrophic factor

During the development of the vertebrate nervous system, many neurons depend for survival on interactions with their target cells. Specific proteins are thought to be released by the target cells and to play an essential role in these interactions. So far, only one such protein, nerve growth factor, has been fully characterized. This has been possible because of the extraordinarily (and unexplained) large quantities of this protein in some adult tissues that are of no relevance to the developing nervous system. Whereas the dependency of many neurons on their target cells for normal development, and the restricted neuronal specificity of nerve growth factor have long suggested the existence of other such proteins, their low abundance has rendered their characterization difficult. Here we report the full primary structure of brain-derived neurotrophic factor. This very rare protein is known to promote the survival of neuronal populations that are all located either in the central nervous system or directly connected with it. The messenger RNA for brain-derived neurotrophic factor was found predominantly in the central nervous system, and the sequence of the protein indicates that it is structurally related to nerve growth factor. These results establish that these two neurotrophic factors are related both functionally and structurally.     

Molecular cloning, expression and regional distribution of rat ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) was originally characterized as a survival factor for chick ciliary neurons in vitro. More recently, it was shown to promote the survival of a variety of other neuronal cell types and to affect the differentiation of E7 chick sympathetic neurons by inhibiting their proliferation and by inducing the expression of vasoactive intestinal peptide immunoreactivity (VIP-IR). In cultures of dissociated sympathetic neurons from newborn rats, CNTF induces cholinergic differentiation as shown by increased levels of choline acetyltransferase (ChAT). This increase is paralleled by a reduction of tyrosine hydroxylase (TH) activity. Moreover, CNTF promotes the differentiation of bipotential 02A progenitor cells to type-2-astrocytes in vitro. To help establish which, if any, of these functions CNTF exerts in vivo, it is necessary to determine its primary structure, cellular expression, developmental regulation and localization. The complementary DNA-deduced amino-acid sequence and subsequent expression of cDNA clones covering the entire coding region in HeLa-cells indicate that CNTF is a cytosolic protein. This, together with its regional distribution and its developmental expression, show that CNTF is not a target-derived neurotrophic factor. CNTF thus seems to exhibit neurotrophic and differentiation properties only after becoming available either by cellular lesion or by an unknown release mechanism.     

重组人睫状神经营养因子突变体的制备及PEG修饰

通过大肠杆菌重组表达人睫状神经营养因子突变体(CNTFm),并进行PEG修饰,旨在降低免疫原性.该突变体将天然CNTF的C端15个氨基酸删除,大肠杆菌表达的CNTFm以包含体形式存在,经复性、纯化获得纯度达到95%的目的蛋白.体内生物学活性测定结果显示,给药10 d,小鼠最大体重减少率达31%,产生的最高中和抗体滴度达到1:6 400; 经PEG修饰,CNTF突变体的生物学活性降低了34%,但最高中和抗体滴度降低到1:800.该PEG修饰后的CNTFm制备工艺有望为CNTF的临床应用开辟道路.     

Brain-derived neurotrophic factor prevents the death of motoneurons in newborn rats after nerve section.

Motoneurons innervating the skeletal musculature were among the first neurons shown to require the presence of their target cells to develop appropriately. But the characterization of molecules allowing motoneuron survival has been difficult. Ciliary neurotrophic factor prevents the death of motoneurons, but its gene is not expressed during development. Although the presence of a neurotrophin receptor on developing motoneurons has suggested a role for neurotrophins, none could be shown to promote motoneuron survival in vitro. We report here that brain-derived neurotrophic factor can prevent the death of axotomized motoneurons in newborn rats, suggesting a role for this neurotrophin for motoneuron survival in vivo.     

GDNF提高端粒酶活性并且促进神经干细胞生长和分裂

对于多种神经细胞，胶质细胞源性神经营养因子（GDNF）具有维持生长、存活、促进分化和成熟的作用，初次在神经干细胞中发现了GDNF的这一营养效应，但这种作用并不对神经干细胞的分化方向产生影响，进而通过测定神经干细胞的端粒酶活性和端粒酶活性的抑制实验，表明端粒酶活性的增高与GDNF对神经干细胞生长和分裂的促进作用有关，因此完善了GDNF在神经细胞中的信号传导和功能实现途径的理解。     

Retrograde axonal transport of target tissue-derived macromolecules

Neurones depend on contact with their target tissues for survival and subsequent development. The protein, nerve growth factor (NGF), can be selectively taken up by sympathetic nerve terminals and reaches the neuronal perikaryon by a process of retrograde intra-axonal transport, suggesting that its role in vivo is to act as a target tissue-derived trophic factor. The development of the neurones of the chick ciliary ganglion requires the presence of structures derived from the optic cup. Several studies in vitro have shown that media conditioned by non-neuronal cells contain factors that result in the survival of neurones from ciliary ganglia. In particular, chick embryo iris, ciliary body and choroid contained large amounts of these factors indicating the presence of a target tissue-derived trophic factor for the cholinergic ciliary ganglion. This study demonstrates that neurones of the ciliary ganglion accumulate, by retrograde intra-axonal transport, proteins synthesized and released by optic tissues in culture.     

Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death.

Current ideas about the dependence of neurons on target-derived growth factors were formulated on the basis of experiments involving neurons with projections to the periphery. Nerve growth factor (NGF) and recently identified members of the NGF family of neuronal growth factors, known as neurotrophins, are thought to regulate survival of sympathetic and certain populations of sensory ganglion cells during development. Far less is known about factors that regulate the survival of spinal and cranial motor neurons, which also project to peripheral targets. NGF has not been shown to influence motor neuron survival, and whether the newly identified neurotrophins promote motor neuron survival is unknown. We show here that brain-derived neurotrophic factor (BDNF) is retrogradely transported by motor neurons in neonatal rats and that local application of BDNF to transected sciatic nerve prevents the massive death of motor neurons that normally follows axotomy in the neonatal period. These results show that BDNF has survival-promoting effects on motor neurons in vivo and suggest that BDNF may influence motor neuron survival during development.     

Brain-derived neurotrophic factor prevents neuronal death in vivo

Developing vertebrate neurons are thought to depend for their survival on specific neurotrophic proteins present in their target fields. The limited availability of these proteins does not allow the survival of all neurons initially innervating a target, resulting in the widely observed phenomenon of naturally occurring neuronal death. Although a variety of proteins have been reported to promote the survival of neurons in tissue culture, the demonstration that these proteins increase neuronal numbers and/or decrease neuronal death in vivo has only been possible with nerve growth factor (NGF). The generalization of the concept that neurotrophic proteins regulate neuronal survival during normal development critically depends on the demonstration that the survival of neurons in vivo can be increased by the administration of a neurotrophic protein different from NGF. We report here that this is the case with brain-derived neurotrophic factor, a protein of extremely low abundance purified from the central nervous system.     

Immunohistochemical localization of endogenous nerve growth factor

Nerve growth factor (NGF) has been proposed as a trophic molecule essential for the development of sympathetic and primary sensory neurones. In newborn mice and rats, administration of nerve growth factor results in an increase in the number of surviving neurones, whereas administration of antiserum to NGF decreases neuronal survival. Thus it has been proposed that the factor is produced and secreted by the relevant target tissues to provide trophic support for the ingrowing nerves. The site of synthesis of nerve growth factor is still unknown, and it has been emphasized that a precise physiological role for the molecule cannot be ascribed until the cell types that produce it are known. I report here the use of immunohistochemistry to localize endogenous NGF in the rat iris, a tissue in which there is sound biochemical evidence for the production of NGF activity. Surprisingly, the results reveal that NGF can be detected readily in Schwann cells, but not in smooth muscle cells of the iris when it is sympathetically denervated or cultured.     

Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF.

Nerve growth factor (NGF) is a neurotrophic factor responsible for the differentiation and survival of sympathetic and sensory neurons as well as selective populations of cholinergic neurons. NGF binds to specific cell-surface receptors but the mechanism for transduction of the neurotrophic signal is unknown. Several experiments using the NGF-responsive pheochromocytoma cell line, PC12, have implicated tyrosine phosphorylation in NGF-mediated responses, although no NGF-specific tyrosine kinases have been identified. Here we show that NGF induces tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product, a tyrosine kinase receptor whose expression is restricted in vivo to neurons of the sensory spinal and cranial ganglia of neural crest origin. Tyrosine phosphorylation of trk by NGF is rapid, specific and occurs with picomolar quantities of factor, indicating that the response is mediated by physiological amounts of NGF. Activation of the trk tyrosine kinase receptor provides a possible mechanism for signal transduction by NGF.     

New protein fold revealed by a 2.3-A resolution crystal structure of nerve growth factor

Nerve growth factor (NGF) is a member of an expanding family of neurotrophic factors (including brain-derived neurotrophic factor and the neurotrophins) that control the development and survival of certain neuronal populations both in the peripheral and in the central nervous systems. Its biological effects are mediated by a high-affinity ligand-receptor interaction and a tyrosine kinase signalling pathway. A potential use for NGF and its relatives in the treatment of neurological disorders such as Alzheimer's disease and Parkinson's disease requires an understanding of the structure-function relationships of NGF. NGF is a dimeric molecule, with 118 amino acids per protomer. We report the crystal structure of the murine NGF dimer at 2.3-A resolution, which reveals a novel protomer structure consisting of three antiparallel pairs of beta strands, together forming a flat surface. Two subunits associate through this surface, thus burying a total of 2,332 A. Four loop regions, which contain many of the variable residues observed between different NGF-related molecules, may determine the different receptor specificities. A clustering of positively charged side chains may provide a complementary interaction with the acidic low-affinity NGF receptor. The structure provides a model for rational design of analogues of NGF and its relatives and for testing the NGF-receptor recognition determinants critical for signal transduction.     

Brain-derived neurotrophic factor rescues developing avian motoneurons from cell death.

During normal vertebrate development, about half of spinal motoneurons are lost by a process of naturally occurring or programmed cell death. Additional developing motoneurons degenerate after the removal of targets or afferents. Naturally occurring motoneuron death as well as motoneuron death after loss of targets or after axotomy can be prevented by in vivo treatment with putative target (muscle) derived or other neurotrophic agents. Motoneurons can also be prevented from dying in vitro and in vivo (Y.Q.-W., R.W., D.P., J. Johnson and L. Van Eldik, unpublished data and refs 7, 13, 14) by treatment with central nervous system extracts (brain or spinal cord) and purified central nervous system and glia-derived proteins. Here we report that in vivo treatment of chick embryos with brain-derived neurotrophic factor rescues motoneurons from naturally occurring cell death. Furthermore, in vivo treatment with brain-derived neurotrophic factor (and nerve growth factor) also prevents the induced death of motoneurons that occurs following the removal of descending afferent input (deafferentation). These data indicate that members of the neurotrophin family can promote the survival of developing avian motoneurons.     

Nerve growth factor induces adrenergic neuronal differentiation in F9 teratocarcinoma cells

Teratocarcinoma cells have been used as a model to study differentiation and development in vertebrates. Treatment with retinoic acid (RA) and dibutyryl cyclic AMP can in some embryonal carcinoma (EC) cell lines lead to neural differentiation, as judged by neurofilament expression and by the induction of enzymes involved in cholinergic transmission. Short-term culture of F9 line cells with RA and dibutyryl cyclic AMP results in a biochemically demonstrable rise in acetylcholinesterase (AChE) activity. We now report that long-term culture of F9 cells with RA and dibutyryl cyclic AMP induces neurofilament expression, demonstrated by immunofluorescence with specific antibodies. Furthermore, if nerve growth factor (NGF) is also added, the developing neurone-like cells exhibit immunoreactivity to tyrosine hydroxylase, a rate-limiting enzyme of catecholamine synthesis specific for adrenergic neurones. Immunoreactivity for Leu-enkephalin-like peptides is also induced. These results suggest that F9 cells can differentiate into cells with adrenergic characteristics.     

Different factors from the central nervous system and periphery regulate the survival of sensory neurones

Work on nerve growth factor has established that the survival of developing vertebrate neurones depends on the supply of a neurotrophic factor from their target field. The discovery of several new neurotrophic factors has raised the possibility that neurones which innervate multiple target fields require several different neurotrophic factors for survival. Here we show that two distinct neurotrophic factors, one in the central nervous system (CNS) and the other in skeletal muscle, promote the survival of proprioceptive neurones in culture. At saturating concentrations, either factor alone supported most neurones and there was no additional survival in the presence of both factors, but at subsaturating concentrations the combined effect was additive. The neurotrophic activity of each factor was greatest during the period of natural neuronal death. Our results demonstrate that each cultured proprioceptive neurone responds to two distinct neurotrophic factors present in its respective central and peripheral target fields, and suggest that these factors cooperate in regulating survival during development.     

Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family

The survival and functional maintenance of vertebrate neurons critically depends on the availability of specific neurotrophic factors. So far, only two such factors, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been characterized and shown to have the typical features of secretory proteins. This characterization has been possible because of the extraordinarily large quantities of NGF in some adult tissues, and the virtually unlimited availability of brain tissue from which BDNF was isolated. Both NGF and BDNF promote the survival of distinct neuronal populations in vivo and are related in their primary structure, suggesting that they are members of a gene family. Although there is little doubt about the existence of other such proteins, their low abundance has rendered their identification and characterization difficult. Taking advantage of sequence identities between NGF and BDNF, we have now identified a third member of this family, which we name neurotrophin-3. Both the tissue distribution of the messenger RNA and the neuronal specificity of this secretory protein differ from those of NGF and BDNF. Alignment of the sequences of the three proteins reveals a remarkable number of amino acid identities, including all cysteine residues. This alignment also delineates four variable domains, each of 7-11 amino acids, indicating structural elements presumably involved in the neuronal specificity of these proteins.     

Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1

B-cell stimulatory factor-1 (BSF-1), formerly designated B-cell growth factor, is a T-cell-derived factor required for entry into the S phase of the cell cycle by B cells stimulated with low concentrations of anti-IgM antibodies. BSF-1 acts directly on resting B cells to prepare them to synthesize DNA more promptly on subsequent exposure to competent stimuli and to strikingly enhance their expression of class II molecules of the major histocompatibility complex. Previous studies have shown that murine BSF-1 can be separated physically from interleukin-2 (IL-2) and that the molecule has an apparent relative molecular mass (Mr) of approximately 15,000 and pI values of 6.4-6.7 and 7.4. Here, we report the production of a monoclonal antibody to BSF-1, its use in characterizing BSF-1, and functional studies demonstrating that this molecule is distinct from IL-1, IL-2 and IL-3.     

Developmentally regulated production of platelet-derived growth factor-like molecules

Platelet-derived growth factor (PDGF) is thought to mediate the proliferation of smooth muscle cells in injured arteries, and may be involved in the pathogenesis of atherosclerosis. PDGF-like molecules from non-platelet sources may also play a role in the regulation of cell activity in other circumstances. Transformation of cells by a wide range of oncongenic agents appears to activate a cellular gene encoding a PDGF-like molecule, possibly accounting for the ability of transformed cells to grow without addition of exogenous mitogens. We show here that a molecule (PDGF-c) which can compete with 125I-PDGF for binding to PDGF receptors is secreted by cultured rat aortic smooth muscle cells (rASMC) isolated from 13 to 18-day-old rats (pups) but not from three-month-old animals (adults). Thus, production of PDGF-c appears to be developmentally regulated and may be a factor in the more rapid proliferation of rASMC and synthesis of connective tissue components which occurs during growth of the aorta in vivo.     

PF18-3, a 35-ku molecule, expresses specifically on apoptotic subset of mouse thymocytes

PF18-3 monoclonal antibody (mAb), one of the rat mAbs against mouse thymic stromal cells (MTSC), has been found to inhibit thymocyte apoptosis induced by a mouse thymic dendritic cell line, MTSC4, in previous co-culture study. The aim of this research is to investigate the character of PF18-3 mAb recognized molecule ( PF18-3 molecule) and its role in MTSC4-induced thymocyte apoptosis. The characterization of PF18-3 molecule expression has been conducted by FACS analysis. PF18-3 molecules have been found to express on MTSC4 as well as on Con A activated but not freshly isolated thymocytes. Up-regulated expression of PF18-3 molecules has been also observed on thymocytes after being co-cultured with MTSC4 for 48 h. The results from FACS analyses by PI staining for detecting apoptosis-related hypodiploid and by PF18-3 mAb staining reveal that PF18-3 molecules expresss specifically on the apoptotic subgroup of thymocytes with high hypodiploid content. The PF18-3 molecule expressed on apoptotic thymocytes with 35 ku of molecular weight, identified by immunoprecipitation and western blotting, is thus likely to be a molecule involved in thymocyte apoptosis.