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observed that rats given intraventricular injections of up to 2.3 microg NGF drink copiously (M.E.L. and G. Guroff, unpublished observations).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.     

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).     

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.     

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.     

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.     

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.     

眼镜蛇毒神经生长因子对成年猫坐骨神经损伤治疗效果的实验室观察

目的 :探讨从眼镜蛇毒分离纯化出的神经生长因子(nervegrowthfactor,NGF)对成年猫坐骨经损伤后的影响。方法 :本实验制成成年猫坐骨神经损伤模型 ,损伤局部注射蛇毒NGF(2μg/kg/d) ,分别治疗10d和30d ,并与对照组(损伤坐骨神经 ,不给药物)比较。结果 :眼镜蛇毒NGF在神经损伤早期应用能减轻神经纤维发生的溃变 ,促进神经纤维再生。结论 :损伤局部长时间注射NGF会导致神经纤维增生过度 ,丧失传导功能     

Requirement for nerve growth factor in the development of myelinated nociceptors in vivo

In adult animals, sensory neurons innervating the skin are phenotypically diverse. We have now investigated whether nerve growth factor (NGF) has a physiological role in the development of this diversity. We gave antisera against NGF to rats from postnatal day 1 (PND 1) to adulthood (5 weeks). We found a virtually complete depletion of high threshold mechanoreceptors conducting in the A delta range (2-13 ms-1) in the sural nerve. This afferent type, normally present in large numbers, appeared to have been replaced by D-hair afferents, sensitive mechanoreceptors which normally are relatively rare. NGF deprivation had this effect only in early postnatal life; treatment from postnatal day 14 to adulthood had no effect. We conclude that the presence of NGF postnatally in skin is necessary for the proper phenotypic development of A delta cutaneous nociceptors.     

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.     

Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor.

Nerve growth factor (NGF) is involved in a variety of processes involving signalling, such as cell differentiation and survival, growth cessation and apoptosis of neurons. These events are mediated by NGF as a result of binding to its two cell-surface receptors, TrkA and p75. TrkA is a receptor with tyrosine kinase activity that forms a high-affinity binding site for NGF. Of the five domains comprising its extracellular portion, the immunoglobulin-like domain proximal to the membrane (TrkA-d5 domain) is necessary and sufficient for NGF binding. Here we present the crystal structure of human NGF in complex with human TrkA-d5 at 2.2 A resolution. The ligand-receptor interface consists of two patches of similar size. One patch involves the central beta-sheet that forms the core of the homodimeric NGF molecule and the loops at the carboxy-terminal pole of TrkA-d5. The second patch comprises the amino-terminal residues of NGF, which adopt a helical conformation upon complex formation, packing against the 'ABED' sheet of TrkA-d5. The structure is consistent with results from mutagenesis experiments for all neurotrophins, and indicates that the first patch may constitute a conserved binding motif for all family members, whereas the second patch is specific for the interaction between NGF and TrkA.     

Amelioration of cholinergic neuron atrophy and spatial memory impairment in aged rats by nerve growth factor

In aged rodents, impairments in learning and memory have been associated with an age-dependent decline in forebrain of cholinergic function, and recent evidence indicates that the cholinergic neurons in the nucleus basalis magnocellularis, the septal-diagonal band area and the striatum undergo age-dependent atrophy. Thus, as in Alzheimer-type dementia in man, degenerative changes in the forebrain cholinergic system may contribute to age-related cognitive impairments in rodents. The cause of these degenerative changes is not known. Recent studies have shown that the central cholinergic neurons in the septal-diagonal band area, nucleus basalis and striatum are sensitive to the neurotrophic protein nerve growth factor (NGF). In particular, intraventricular injections or infusions of NGF in young adult rats have been shown to prevent retrograde neuronal cell death and promote behavioural recovery after damage to the septo-hippocampal connections. It is so far not known, however, whether the atrophic cholinergic neurons in aged animals are responsive to NGF treatment. We report here that continuous intracerebral infusion of NGF over a period of four weeks can partly reverse the cholinergic cell body atrophy and improve retention of a spatial memory task in behaviourally impaired aged rats.     

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.     

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.     

Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition.

Tissue injury generates endogenous factors that heighten our sense of pain by increasing the response of sensory nerve endings to noxious stimuli. Bradykinin and nerve growth factor (NGF) are two such pro-algesic agents that activate G-protein-coupled (BK2) and tyrosine kinase (TrkA) receptors, respectively, to stimulate phospholipase C (PLC) signalling pathways in primary afferent neurons. How these actions produce sensitization to physical or chemical stimuli has not been elucidated at the molecular level. Here, we show that bradykinin- or NGF-mediated potentiation of thermal sensitivity in vivo requires expression of VR1, a heat-activated ion channel on sensory neurons. Diminution of plasma membrane phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) levels through antibody sequestration or PLC-mediated hydrolysis mimics the potentiating effects of bradykinin or NGF at the cellular level. Moreover, recruitment of PLC-gamma to TrkA is essential for NGF-mediated potentiation of channel activity, and biochemical studies suggest that VR1 associates with this complex. These studies delineate a biochemical mechanism through which bradykinin and NGF produce hypersensitivity and might explain how the activation of PLC signalling systems regulates other members of the TRP channel family.     

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.     

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.     

Sortilin is essential for proNGF-induced neuronal cell death

Sortilin (approximately 95 kDa) is a member of the recently discovered family of Vps10p-domain receptors, and is expressed in a variety of tissues, notably brain, spinal cord and muscle. It acts as a receptor for neurotensin, but predominates in regions of the nervous system that neither synthesize nor respond to this neuropeptide, suggesting that sortilin has additional roles. Sortilin is expressed during embryogenesis in areas where nerve growth factor (NGF) and its precursor, proNGF, have well-characterized effects. These neurotrophins can be released by neuronal tissues, and they regulate neuronal development through cell survival and cell death signalling. NGF regulates cell survival and cell death via binding to two different receptors, TrkA and p75NTR (ref. 10). In contrast, proNGF selectively induces apoptosis through p75NTR but not TrkA. However, not all p75NTR-expressing cells respond to proNGF, suggesting that additional membrane proteins are required for the induction of cell death. Here we report that proNGF creates a signalling complex by simultaneously binding to p75NTR and sortilin. Thus sortilin acts as a co-receptor and molecular switch governing the p75NTR-mediated pro-apoptotic signal induced by proNGF.     

Nerve growth factor activity of several immunal related serine proteases

Rabbit was immuned by the previously purified protein with high nerve growth factor (NGF) bioactivity (NGF_like protease) from \%Agkistrodon halys Pallas\% and the antisera were collected. The polyclonal antibodies were tentatively purified and then used as ligands of an affinity column. The \%A.h.Pallas\% crude venom was fractionated by this affinity column and then by Mono Q on fast protein liquid chromatography (FPLC). As a result, fraction Ⅱ and fraction Ⅲ were purified respectively, whose N_terminal amino acid sequences show high homology with the serine proteases in snake venoms, as well as the previous NGF_like protease. However, they possessed different levels of NGF bioactivity. The NGF activity of the previous NGF_like protease is equivalent to that of NGF, while the activity of fraction Ⅱ seems relatively low in contrast to fraction Ⅲ which had no NGF activity.     

NGF诱导PC12细胞神经元样细胞分化

目的:建立PC12细胞的神经元样细胞分化模型,并探讨ERK蛋白在PC12细胞神经元样细胞分化中的可能机制.方法:以10、20、50 g/L的NGF(NGF溶于PBS,培养基中PBS的终浓度不超过2%)培养PC12细胞,应用倒置相差显微镜、显微镜测微尺及流式细胞仪鉴定PC12细胞的分化,以确定NGF使用剂量.运用免疫印迹检测不同浓度、不同作用时间时ERK蛋白在PC12细胞中的表达,并进行统计学分析.结果:随NGF剂量的升高,PC12细胞的体积、最长突起长度和突起数目均会增大或增多。统计学分析证明50 g/L的NFG作用48 h足以诱导PC12细胞的交感神经样改变。尽管ERK总蛋白水平在NFG作用前后无明显改变,但在NFG作用5 min后磷酸化的ERK蛋白水平即显著升高,达到峰值,并持续约1 h.50 g/L的NFG作用于PC12细胞48 h可使细胞发生明显的G1期阻滞.结论:PC12细胞可在NGF作用下出现交感神经样改变,并且细胞的分化程度依赖于NGF的使用剂量和作用时间.在NGF诱导的PC12细胞神经元样分化中ERK蛋白的磷酸化对NGF呈现剂量和时间依赖性,提示ERK蛋白在分化的早期发挥重要作用.