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.     

Central nervous system and peripheral nerve growth factor provide trophic support critical to mature sensory neuronal survival

Primary sensory neurones in cranial and dorsal root ganglia (DRG) of adult animals are generally thought to be maintained through connections with their peripheral (but not central) targets by trophic factor(s) other than nerve growth factor (NGF). Damage to the peripheral process of sensory neurones results in a dramatic response or even death of the neurones, whereas axotomy (cutting) of the central process does not initiate profound reaction in these neurones. The development and maintenance of neurones are highly dependent on a supply of trophic agents produced by targets and retrogradely transported via the peripheral process to the cell body. NGF deprivation in fetal rodents produced either by exogenously administered antibodies or by those of maternal origin, results in death of DRG and of some cranial sensory neurones. However, as chronic NGF deprivation in neonatal or adult rodents produces little or no cell death, it has been assumed that some other trophic factor(s) derived from the peripheral target sustains sensory neurones in postnatal life. By inducing NGF deprivation by autoimmunizing guinea pigs with mouse NGF and/or by cutting the central root (process) of a DRG, we demonstrate here that under certain conditions DRG neurones require NGF and centrally derived trophic support. Our results indicate that sensory neurones are maintained by the trophic support provided by both peripheral and central targets. This support is mediated by NGF and other as yet unidentified trophic factors. The relative importance of the two target fields and NGF compared with other trophic factors changes during development.     

Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons

Nerve growth factor (NGF) is a trophic molecule essential for the survival of sympathetic and sensory neurons during ontogeny. The extent to which NGF is involved in the maintenance or regulation of the differentiated phenotypes of mature peripheral neurons is much less clear, however. Biochemical analysis of the actions of NGF upon peripheral neurons has been hampered by the lack of a preparation of neuronal cells that are responsive to NGF but do not require it for survival. We report here that in adult dorsal root ganglion neurons, which can be isolated, enriched and maintained in culture in the absence of neuronal growth factors, the expression of mRNAs encoding the precursors of two neuropeptides, substance P and calcitonin gene-related peptide is regulated by NGF. Our results provide the first direct evidence of a continuous dynamic role for NGF in regulation of peptide neurotransmitter/neuromodulator levels in mature sensory neurons.     

对大鼠PC12细胞用于定量测定神经生长因子活性两种方案的评价

建株的大鼠肾上腺嗜铬细胞瘤ＰＣ１２细胞，在神经生长因子ＮＧＦ的作用下，会长出神经突起。这一现象被用于神经生长因子的活性测定。但就定量测定而言、目前已 方法，方案，包括判定阳性细胞的标准，均有一定差异，而我国也尚未建立起对这一重要的 活性的标准测定法。     

Short-latency drinking and increased Na appetite after intracerebral microinjections of NGF in rats

NERVE GROWTH FACTOR (NGF) is a polypeptide trophic factor for peripheral sympathetic and sensory neurones(1,2). Apparent NGF(3-5) and NGF receptors(6,7) have also been identified in the brain, and intracerebral administration of NGF in the adult rat produces marked biochemical(8) and morphologica(9,10) changes in brain tissue. These findings, taken together with the observations that central injections of NGF facilitate behavioural recovery from brain damage(11,12), indicate that this polypeptide may have an important role in brain function. It has been observed that rats given intraventricular injections of up to 2.3 microg NGF drink copiously (M.E.L. and G. Guroff, unpublished observations). Perkins et al.(13) reported that diencephalic application of crystalline NGF (1-15 microg) resulted in an intense polydipsia. The present report confirms the observations of M.E.L. and Guroff, and extends the findings of Perkins et al.(13) by using solutions of NGF instead of crystals. It also describes for the first time a second phenomenon produced by intracranial administration of NGF, namely an intense appetite for aversive concentrations of sodium solutions.     

Nerve growth factor is a mitogen for cultured chromaffin cells

Nerve growth factor (NGF) is essential for the survival and differentiation of a number of neural crest derivatives, including sympathetic and sensory neurones. While early studies suggested that NGF might also have a mitogenic effect on these neurones, subsequent work has favoured the interpretation that NGF promotes cell survival or differentiation rather than proliferation. We have addressed the issue of a mitogenic effect of NGF using adrenal chromaffin cells, which are endocrine cells derived from the neural crest, and are closely related to sympathetic neurones. Adrenal chromaffin cells respond to NGF in vitro by expressing neuronal traits. We now report that NGF elicits a mitotic response in cultured chromaffin cells from young rats, and that this response is blocked by an antiserum to 2.5S NGF. The chromaffin cells that divided in response to NGF can subsequently become neuronal in the continued presence of NGF.     

Renin-like effects of NGF evaluated using renin-angiotensin antagonists

Intracranial injection of angiotensin II (AII) or activation of the cerebral isorenin-angiotensin system with intracranial renin causes an immediate thirst and a delayed sodium appetite in the rat. Nerve growth factor (NGF), a polypeptide trophic factor for peripheral sympathetic and sensory neurones, has also been reported to be a potent stimulus to thirst and sodium appetite when injected into the brain of the rat. Lewis et al. drew attention to the marked similarity between the effects of 2.5S NGF and renin on thirst and sodium appetite and suggested that the NGF responses were mediated by the cerebral isorenin-angiotensin system. We report here that NGF-induced thirst and sodium appetite, as well as increased blood pressure and increase ornithine decarboxylase activity in the brain and liver, depend on the formation of AII (see also ref. 6).     

Sarcoma viruses carrying ras oncogenes induce differentiation-associated properties in a neuronal cell line

The growth-promoting and/or differentiation-blocking activities of Kirsten (Ki-MSV) or Harvey murine sarcoma virus (Ha-MSV) on various types of cells in vitro are well documented. Here we report an unexpected effect of these viruses on a rat phaeochromocytoma cell line, PC12. PC12 cells, which multiply indefinitely in growth medium, are known to respond to nerve growth factor (NGF) by cessation of cell division and expression of several properties resembling those of differentiated sympathetic neurones. We have found that Ki- and Ha-MSV mimic some, if not all, of the activities of NGF in PC12 cells, and there is evidence that the viral oncogenes, v-Ki-ras and v-Ha-ras, are responsible for this phenomenon. This system may be of value for studying the mechanism of action of the v-ras genes as well as the regulatory mechanism of growth and differentiation in neuronal cells.     

Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21

The protein products (p21) of the ras cellular proto-oncogenes are thought to transduce membrane signals necessary for the induction of cell division. However, there is uncertainty as to the precise role of ras p21 in mediating ligand-membrane receptor signals leading to cell differentiation. Treatment of rat phaeochromocytoma cells (PC12) with nerve growth factor (NGF) results in the induction of a number of phenotypic characteristics of sympathetic neurones, including cessation of cell division and outgrowth of neuronal processes (neurites). Here we report that microinjection of antibody to ras p21 into PC12 cells inhibited neurite formation and resulted in temporary regression of partially extended neurites, an effect which was observed up to 36 h after initiation of NGF treatment. Neurite formation induced by cyclic AMP was unaffected by injection of anti-p21 antibody. These results indicate that p21 is involved in the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in hormone-mediated cellular responses distinct from cell proliferation.     

Stimulation of the pituitary-adrenocortical axis by nerve growth factor.

Nerve growth factor (NGF) is a protein essential for the development and maintenance of the peripheral sympathetic nervous system, causing responsive neurones to increase in size and to extend neurites. Biochemically, the selective induction of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase key enzymes in catecholamine biosynthesis is one of its most characteristic effects. Both the morphological and biochemical effects are modulated by glucocorticoids, suggesting a close relationship between specific effects of NGF and hormone action. NGF has been shown to induce an increase in adrenal cyclic AMP in intact but not in hypophysectomised rats, and so we have looked directly at the effect of systemic administration of NGF on the hypothalamo-pituitary-adrenal axis. We report here that NGF induced an enhanced secretion of adrenocorticotropin (ACTH) and a prolonged increase in plasma glucocorticoid concentration after intravenous (i.v.) injection. Such effects could have important implications for the biological activity of NGF.     

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.     

Functional regeneration of sensory axons into the adult spinal cord

The arrest of dorsal root axonal regeneration at the transitional zone between the peripheral and central nervous system has been repeatedly described since the early twentieth century. Here we show that, with trophic support to damaged sensory axons, this regenerative barrier is surmountable. In adult rats with injured dorsal roots, treatment with nerve growth factor (NGF), neurotrophin-3 (NT3) and glial-cell-line-derived neurotrophic factor (GDNF), but not brain-derived neurotrophic factor (BDNF), resulted in selective regrowth of damaged axons across the dorsal root entry zone and into the spinal cord. Dorsal horn neurons were found to be synaptically driven by peripheral nerve stimulation in rats treated with NGF, NT3 and GDNF, demonstrating functional reconnection. In behavioural studies, rats treated with NGF and GDNF recovered sensitivity to noxious heat and pressure. The observed effects of neurotrophic factors corresponded to their known actions on distinct subpopulations of sensory neurons. Neurotrophic factor treatment may thus serve as a viable treatment in promoting recovery from root avulsion injuries. I     

BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra

Brain-derived neurotrophic factor (BDNF), present in minute amounts in the adult central nervous system, is a member of the nerve growth factor (NGF) family, which includes neurotrophin-3 (NT-3). NGF, BDNF and NT-3 all support survival of subpopulations of neural crest-derived sensory neurons; most sympathetic neurons are responsive to NGF, but not to BDNF; NT-3 and BDNF, but not NGF, promote survival of sensory neurons of the nodose ganglion. BDNF, but not NGF, supports the survival of cultured retinal ganglion cells but both NGF and BDNF promote the survival of septal cholinergic neurons in vitro. However, knowledge of their precise physiological role in development and maintenance of the nervous system neurons is still limited. The BDNF gene is expressed in many regions of the adult CNS, including the striatum. A protein partially purified from bovine striatum, a target of nigral dopaminergic neurons, with characteristics apparently similar to those of BDNF, can enhance the survival of dopaminergic neurons in mesencephalic cultures. BDNF seems to be a trophic factor for mesencephalic dopaminergic neurons, increasing their survival, including that of neuronal cells which degenerate in Parkinson's disease. Here we report the effects of BDNF on the survival of dopaminergic neurons of the developing substantia nigra.     

Disoriented pathfinding by pioneer neurone growth cones deprived of filopodia by cytochalasin treatment

A major question in developmental neurobiology is how developing nerve cells accurately extend processes to establish connections with their target cells. This problem involves both the nature of cues for growth cone guidance and also the question of how growth cones survey their environment for cues and respond by altering their direction of migration. The filopodia which normally extend from neuronal growth cones have been shown to affect growth cone steering in vitro and it has been proposed that they function in vivo in the detection of and response to guidance cues. This hypothesis could be tested in vivo if growth cones which normally have filopodia could be induced to migrate in their absence. The pair of Ti1 neurones are the first neurones to extend axons through the limb buds of embryonic grasshoppers. We report here an examination of the migration of Ti1 pioneer growth cones deprived of filopodia by culture in agents which disrupt actin microfilaments. Under these conditions, axons continue to extend but a large percentage of growth cones are highly disoriented. Our results indicate that Ti1 filopodia are not necessary for axonal elongation in vivo but that they are important for correctly oriented growth cone steering.     

Interleukin-1 regulates synthesis of nerve growth factor in non-neuronal cells of rat sciatic nerve

The Schwann cells and fibroblast-like cells of the intact sciatic nerve of adult rats synthesize very little nerve growth factor (NGF). After lesion, however, there is a dramatic increase in the amounts of both NGF-mRNA and NGF protein synthesized by the sciatic non-neuronal cells. This local increase in NGF synthesis partially replaces the interrupted NGF supply from the periphery to the NGF-responsive sensory and sympathetic neurons, whose axons run within the sciatic nerve. Macrophages, known to invade the site of nerve lesion during wallerian degeneration, are important in the regulation of NGF synthesis. Here we demonstrate that the effect of macrophages on NGF-mRNA levels in cultured explants of sciatic nerve can be mimicked by conditioned media of activated macrophages, and that interleukin-1 is the responsible agent.     

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.     

Glial cells express N-CAM/D2-CAM-like polypeptides in vitro

The joining together of neurites to form fascicles and the growth of axons along glial surfaces during early development suggest that neurone-neurone and neurone-glial adhesion interactions are of considerable importance for defining nerve tracts. In vitro studies have indicated that adhesion between neurones involves a glycoprotein that has been independently studied under the names of N-CAM (for neural cell adhesion molecule), D2-CAM and BSP-2 (refs 10, 11). As N-CAM/D2-CAM appears to be a homophilic ligand that binds to N-CAM/D2-CAM polypeptide on adjacent cells, this glycoprotein is potentially important in adhesion interactions between any two N-CAM/D2-CAM-expressing cells. While it has been suggested that neurone-glial adhesion involves molecules other than N-CAM/D2-CAM, it is known that N-CAM/D2-CAM antigenic determinants are expressed by glial cells in vivo and that injection of anti-N-CAM antibodies into the eye-cup of chick embryos disrupts normal patterns of neuritic apposition to glial endfeet in the developing optic stalk. Do the molecules expressed by glia share restricted antigenic determinants, or binding domains, with N-CAM/D2-CAM, or are N-CAM/D2-CAM polypeptides expressed by glia? Here we present immunocytochemical evidence which suggests that all classes of macroglia express N-CAM/D2-CAM antigenic determinants on their surfaces and immunochemical analyses which indicate that the molecules expressed by purified astrocytes are closely similar, or identical, to at least some forms of N-CAM/D2-CAM obtained from whole brain or purified neurones. However, our results also suggest that different N-CAM/D2-CAM polypeptides may be separately expressed by neurones and astrocytes.     

Role of ion flux in the control of c-fos expression

There has been much interest in the biochemical and biophysical processes that couple extracellular signals to alterations in gene expression. While many early events associated with the treatment of cells with growth factors have been described (for example, ion flux and protein phosphorylation), it has proved difficult to establish biochemical links to gene expression. Recently, the study of such genomic control signals has been facilitated by the demonstration that the c-fos proto-oncogene is rapidly and transiently induced by treatment of several cell types with polypeptide growth factors and other growth modulating substances. In one particular system it has been shown that nerve growth factor (NGF) causes a transient induction of c-fos in the phaeochromocytoma cell line PC12, within 15 min. Furthermore, the magnitude of this induction can be modulated with pharmacological agents such as peripheral-type benzodiazepines (BZDs). Thus, the study of c-fos expression in PC12 cells could yield valuable clues to the coupling mechanisms linking cell surface activation to genomic events. Here we demonstrate that c-fos is induced in PC12 cells either by receptor-ligand interaction or by agents or conditions that effect voltage-dependent calcium channels.     

多壁碳纳米管-神经生长因子复合物的制备及生物活性测定

为探讨采用羧基化多壁碳纳米管（MWCNTs—COOH）非共价接枝神经生长因子（NGF）制备碳纳米管神经生长因子（MWCNTsNGF）复合物,考察复合物的生物活性。采用透射电子显微镜（TEM）表征MWCNTs—NGF复合物的微观形貌,酶联免疫吸附法（ELISA法）测定MWCNTs—NGF复合物载带NGF的量,MTT法测定了MWCNTs—NGF复合物的对嗜铬细胞瘤细胞（PCI2细胞）的毒性,PCI2细胞培养法评价复合物的生物活性,TEM表征复合物与细胞的分布情况。结果：TEM图像表明NGF连接到了MWCNT上,EI．ISA法测得MWCNTsNGF复合物载带NGF的量为797．63pg／mg,MWCNTs—NGF复合物对PCI2细胞有一定的毒性,生物活性试验表明NGF浓度相同的情况下,MwCNTs—NGF复合物组PCI2细胞的分化率明显高于NGF组。TEM图像表明碳纳米管能进入细胞。结论：碳纳米管能载带NGF进入细胞,使NGF能更好的表达生物活性。