NGF induction of NGF receptor gene expression and cholinergic neuronal hypertrophy within the basal forebrain of the adult rat

Chronic infusion of nerve growth factor (NGF) into the forebrain of the adult rat produced increases in NGF receptor (NGF-R) mRNA hybridization, NGF-R immunoreactivity, choline acetyltransferase (ChAT) mRNA hybridization, and neuronal hypertrophy, when compared with vehicle infusion or noninfused rat brain. In situ hybridization showed NGF induction of NGF-R gene expression, documented by increases in the number of NGF-R mRNA-positive cells within the medial septum, diagonal band, and nucleus basalis magnocellularis. NGF also produced hypertrophy of ChAT mRNA-positive neurons. These results suggest that NGF produces cholinergic neuronal hypertrophy through induction of NGF-R gene expression within the basal forebrain.     

大鼠基底前脑NGF-R及CHAT免疫反应阳性神经元的分布:免疫双标法

本文用免疫组化双标法观察了神经生长因子受体(NGF-R)及胆碱乙酰转移酶(ChAT)免疫反应阳性神经元在成鼠基底前脑内的分布,结果发现嗅结节、隔内侧核、斜角带核、腹侧苍白球及基底大细胞核均有NGF-R及ChAT免疫反应阳性神经元.免疫组化双标染色发现,大部分免疫反应阳性神经元的NGF-R与ChAT共存,部分神经元呈单纯NGF-R或ChAT阳性,但这种NGF-R和ChAT的共存情况在不同区域不完全相同.在隔内侧核和斜角带核,大多数的NGF-R阳性神经元和ChAT阳性神经元共存,但在腹侧仓白球和基底大细胞核,两者共存的神经元较前两区为少.此外ChAT阳性神经元在尾壳核中分布较均匀,而NGF-R阳性神经元较少见.研究结果表明,大多数胆碱能神经元有NGF-R,提示NGF对胆碱能神经元的保护和激活作用,部分可能是通过直接与NGF受体的结合而发生作用.     

Role of nerve growth factor in plasticity of forebrain cholinergic neurons

Neuronal plastic rearrangements during the development and functioning of neurons are largely regulated by trophic factors, including nerve growth factor (NGF). NGF is also involved in the pathogenesis of Alzheimer’s disease. In the brain, NGF is produced in structures innervated by basal forebrain cholinergic neurons and retrogradely transported along the axons to the bodies of cholinergic neurons. NGF is essential for normal development and functioning of the basal forebrain; it affects formation of the dendritic tree and modulates the activities of choline acetyltransferase and acetylcholinesterase in basal forebrain neurons. The trophic effect of NGF is mediated through its interactions with TrkA and p75 receptors. Experimental and clinical studies have shown that brain levels of NGF are altered in various pathologies. However, the therapeutic use of NGF is limited by its poor ability to penetrate the blood–brain barrier, adverse side effects that are due to the pleiotropic action of this factor, and the possibility of immune response to NGF. For this reason, the development of gene therapy methods for treating NGF deficit-associated pathologies is of particular interest. Another approach is creation of low molecular weight NGF mimetics that would interact with the corresponding receptors and display high biological activity but be free of the unfavorable effects of NGF.     

Overexpression of tumour necrosis factor alpha in the brain of transgenic mice differentially alters nerve growth factor levels and choline acetyltransferase activity

Tumour necrosis factor alpha (TNF-alpha) is a pleiotrophic cytokine synthesized primarily by macrophages and monocytes, which exerts a variety of biological activities during inflammatory responses, immune reactions, and wound healing. Within the central nervous system (CNS), the basal levels of TNF-alpha are almost undetectable, but increase after neurological insults. Using transgenic mice expressing high levels of TNF-alpha in the CNS, we investigated the effect of this cytokine on the levels of brain nerve growth factor (NGF), a neurotrophin playing a crucial role in the development, maintenance and regeneration of basal forebrain cholinergic neurons. The immunoenzymatic assay and in situ hybridization revealed that the constitutive expression of NGF decreased in the hippocampus, increased in the hypothalamus, while remained unchanged in the cortex. Moreover, septal cholinergic neurons which receive trophic support from NGF produced in the hippocampus display loss of choline acetyltransferase immunoreactivity, suggesting that the reduced availability of NGF may influence negatively the synthesis of brain cholinergic neurons. These observations indicate that the basal level of brain NGF can be influenced negatively or positively by local expression of TNF-alpha and that this cytokine, through dose-dependent regulation of NGF synthesis and release, may be involved in neurodegenerative events associated with aging.     

Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.

The levels of nerve growth factor (NGF) and its mRNA in the rat central nervous system were determined by two-site enzyme immunoassay and quantitative Northern blots, respectively. Relatively high NGF levels (0.4-1.4 ng NGF/g wet weight) were found both in the regions innervated by the magnocellular cholinergic neurons of the basal forebrain (hippocampus, olfactory bulb, neocortex) and in the regions containing the cell bodies of these neurons (septum, nucleus of the diagonal band of Broca, nucleus basalis of Meynert). Comparatively low, but significant NGF levels (0.07-0.21 ng NGF/g wet weight) were found in various other brain regions. mRNANGF was found in the hippocampus and cortex but not in the septum. This suggests that magnocellular cholinergic neurons of the basal forebrain are supplied with NGF via retrograde axonal transport from their fields of innervation. These results, taken together with those of previous studies showing that these neurons are responsive to NGF, support the concept that NGF acts as trophic factor for magnocellular cholinergic neurons.     

Nerve growth factor increases stimulus-evoked metabolic activity in acetylcholine-depleted barrel cortex

Lesions of the basal forebrain deplete the neocortex of cholinergic fibers. Acetylcholine depletion in the somatosensory cortex of rats results in reduced stimulus-evoked activity in response to whisker stimulation. Previous studies demonstrate that embryonic basal forebrain transplants improve functional activity toward normal. It is not clear if the activity increase is due to cholinergic replacement or other factors present in the graft. In this study, we examined the possibility that nerve growth factor (NGF), a neurotrophin known as a survival factor and a specific protectant for cholinergic basal forebrain neurons, can preserve basal forebrain cells after a lesion and restore functional activity in the somatosensory cortex. We report that NGF alone is capable of restoring functional activity in the barrel cortex of animals with basal forebrain lesions, while vehicle injections of saline do not alter activity. Both high (10 mug) and low (5 mug) doses of NGF unilaterally injected into the lateral ventricle improved stimulus-evoked functional activity during bilateral whisker stimulation. The mechanism of NGF action is not clear since the restoration of functional activity in cortex was not accompanied by increased cholinergic activity as detected by acetylcholinesterase fiber staining. NGF may act directly on cortical neurons, although its site of action is not well defined.     

Neurotrophins and the primate central nervous system: A minireview

The central nervous system (CNS) of primates is more complex than the CNS of other mammals. Details of the development and aging of the primate CNS have recently been revealed by various neurobiological techniques. It has become clear that the primate CNS has unique characteristics, for example, the capacity for the overproduction and elimination of fibers and synapses. Some differences have also been found in the distribution of and changes with development in levels of various neuroactive substances. Recent discoveries of a variety of neurotrophins in the mammalian CNS have led to research on the neurobiology of these molecules in the primate CNS. The distribution of and changes with development in levels of nerve growth factor (NGF) in the primate CNS are closely correlated with the cholinergic system of the basal forebrain. The administration of NGF into the monkey brain prevents the degeneration of the cholinergic neurons of the basal forebrain after axotomy, a result that suggests that neurotrophins might be very valuable agents for the future treatment of neurological diseases, such as Alzheimer's and Parkinson's diseases. This review is dedicted to Dr. Hans Thoenen.     

Immunohistochemical localization of nerve growth factor (NGF) and NGF receptor (NGF-R) in the developing first molar tooth of the rat

Nerve growth factor (NGF) is a well established target-derived trophic factor supporting sympathetic and sensory innervation in the peripheral tissues as well as cholinergic innervation in the brain. Despite its name, NGF may have broader biological functions early in development in a wide range of non-neuronal differentiating cells. The many effects of NGF are directly dependent on initial binding of NGF to specific plasma membrane receptors on target cells. Here we use immunohistochemical methods to show that NGF and its receptor (NGF-R) are localized in a variety of embryonic epithelial and mesenchymal cells in the rat developing molar tooth. Dental cells known to play important roles in morphogenesis and inductive tissue interactions show NGF-like reactivity. Thus, labelling is seen in epithelial preameloblasts and mesenchymal odontoblasts. We also show a transient expression of NGF-R in restricted parts of the dental epithelium (inner dental epithelium) and dental mesenchyme differentiating cells (post-mitotic, polarizing odontoblasts). The expression patterns of NGF are different to those of NGF-R during embryogenesis and this is illustrated in detail in the developing tooth. The histochemical findings reported here support the notion that NGF may have multiple roles during morphogenetic and cytodifferentiation events in the tooth.     

p140trk mRNA marks NGF-responsive forebrain neurons: evidence that trk gene expression is induced by NGF.

Nerve growth factor (NGF) appears to act as a neurotrophic factor for basal forebrain and caudate-putamen cholinergic neurons. The mechanism by which NGF transduces its signal in these neurons is yet to be defined. Recent data indicate that the product of the trk gene, p140trk, is a critical component of the NGF receptor. Herein, we show that p140trk mRNA is highly restricted in its distribution in the adult rat forebrain, that it is present in cholinergic neurons, and that most if not all cholinergic neurons contain p140trk mRNA. Furthermore, induction of trk expression by NGF suggests that neurotrophin-mediated up-regulation of their receptor tyrosine kinases is an important feature of their actions and that neurotrophins may regulate the activity of responsive neurons through increasing the level of their receptors.     

Expression of neuronal-NOS in developing basal forebrain cholinergic neurons: Regulation by NGF

Nerve growth factor (NGF) acts through the receptor tyrosine kinase trkA to serve as a trophic factor for cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band. We have previously shown that the neuronal isoform of nitric oxide synthase (NOS) is selectively expressed in a large fraction of trkA-expressing cholinergic neurons in these brain regions in the adult rat, and that NGF induces the expression of neuronal-NOS in these cells. Herein, we show that: 1) neuronal-NOS is also localized to these neurons in the developing septum; 2) the expression of neuronal-NOS is regulated in the developing medial septal nucleus and vertical limb of the diagonal band; 3) neuronal-NOS regulation parallels that for other markers of basal forebrain cholinergic neuron differentiation, such as cholineacetyltransferase; and 4) NGF infusion in the postnatal period induces robust increases in neuronal-NOS mRNA and in NOS activity in the basal forebrain. Taken together with earlier findings, our results suggest that neuronal-NOS has a role in the differentiation and mature function of septal cholinergic neurons. Through enhancing neuronal-NOS synthesis, endogenous NGF is likely to regulate NO functions in vivo. Special issue dedicated to Dr. Hans Thoenen.     

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.     

Cholinergic Control of Nerve Growth Factor in Adult Rats: Evidence from Cortical Cholinergic Deafferentation and Chronic Drug Treatment

Abstract: It is well documented that nerve growth factor (NGF) plays an important role in maintaining functions of cholinergic basal forebrain neurons. In the present study, we tested the hypothesis that cholinergic activity controls NGF levels in cholinoceptive neurons of the cerebral cortex and hippocampus. To address that question, we used both cholinergic deafferentation of cerebral cortex and hippocampus by cholinergic immunolesion with 192IgG-saporin and chronic pharmacological treatment of sham-treated and immunolesioned rats with the cholinergic agonist pilocarpine and the cholinergic antagonist scopolamine. We observed an increase in NGF protein levels in the cortex and hippocampus after cholinergic immunolesions and also after muscarinic receptor blockade by chronic intracerebroventricular scopolamine infusion in sham-treated rats after 2 weeks. There was no further increase in the accumulation of NGF after scopolamine treatment of immunolesioned rats. Chronic infusion of pilocarpine had no effect on cortical and hippocampal NGF protein levels in sham-treated rats. In rats with cholinergic immunolesions, however, pilocarpine did prevent the lesion-induced accumulation of NGF. There was no effect of cholinergic lesion and drug treatment on cortical or hippocampal NGF mRNA levels, consistent with the importance of NGF retrograde transport as opposed to its de novo synthesis. This study provides strong evidence for the hypothesis that there is cholinergic control of cortical and hippocampal NGF protein but not mRNA levels in adult rats.     

Streptozotocin-induced diabetes is associated with changes in NGF levels in pancreas and brain

Type 1 diabetes mellitus (DM), a "classical" result of a pancreatic-beta cell damage, is associated with various metabolic, neuronal, endocrine and immune alterations at cellular, tissue and organ levels. Nerve growth factor (NGF) is one of the most extensively studied neurotrophic factors, which is produced and released by numerous cells including the pancreatic beta cells. NGF plays an important role during brain development and may be able to delay or even reverse damaged forebrain cholinergic neurons that undergo degeneration in aged animals and in Alzheimer's disease (AD). Recent reports indicate that experimentally induced DM in rodents can cause brain biochemical and molecular alterations similar to those observed in sporadic AD. Given the importance of NGF in the pathophysiology of brain cholinergic neurons, we looked for NGF changes in the pancreas and brain of diabetic rats. The aim of this study was, therefore, to investigate the effect of streptozotocin-induced DM on NGF and NGF receptor expression in pancreas and brain. The results showed that DM is associated with altered NGF, NGF-receptor expression in both pancreas and brain.     

On the molecular basis linking Nerve Growth Factor (NGF) to Alzheimer's disease

1. Alzheimer's disease (AD) is pathologically defined by the deposition of amyloid peptide and neurofibrillary tangles and is characterized by a progressive loss of cognition and memory function, due to marked cortical cholinergic depletion. 2. Cholinergic cortical innervation is provided by basal forebrain cholinergic neurons. The neurotrophin Nerve Growth Factor (NGF) promotes survival and differentiation of basal forebrain cholinergic neurons. 3. This assertion has been at the basis of the hypothesis developed in the last 20 years, whereby NGF deprivation would be one of the factor involved in the etiology of sporadic forms of AD. 4. In this review, we shall summarize data that lead to the production and characterization of a mouse model for AD (AD11 anti-NGF mice), based on the expression of transgenic antibodies neutralizing NGF. The AD-like phenotype of AD11 mice will be discussed on the basis of recent studies that have posed NGF and its precursor pro-NGF back to the stage of AD-like neurodegeneration, showing the involvement of the precursor pro-NGF in one of the cascades leading to AD neurodegeneration.     

Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues

Nerve growth factor (NGF) is a polypeptide important for normal development of the nervous system and promotion of survival and differentiation of sensory and sympathetic neurons in culture. The cellular effects of NGF are mediated by a specific cell surface molecule, nerve growth factor receptor (NGF-R). In the present study we have used a monoclonal antibody against human NGF-R to examine, by the avidin-biotin-immunoperoxidase method, the receptor distribution in a wide range of normal tissues and in more than 200 malignant tumors. Our results show that (a) human NGF-R is expressed in the peripheral nervous system but not in any of the central nervous system areas tested; (b) NGF-R expression is not restricted to neural tissues but is also found in a number of normal epithelial, mesenchymal, and lymphoid tissues; (c) NGF-R expression changes during normal development; and (d) NGF-R expression in malignant tumors generally parallels its normal tissue distribution. Thus, NGF-R is detected in a proportion of neuroectoderm-derived tumors, carcinomas, and lymphomas, and also in a characteristic group of small round-cell tumors (Ewing's sarcomas and embryonal rhabdomyosarcomas). These findings suggest a normal regulatory role for NGF in both neuronal and non-neuronal cells and identify a range of human tumors in which the NGF/NGF-R system may contribute to the malignant phenotype.     

Distribution and characteristics of nerve growth factor binding on cholinergic neurons of rat and monkey forebrain

In sections of rat forebrain, perikarya labeled radioautographically with¹²⁵I-NGF resembled cholinesterase-positive neurons in their distribution within striatum and basal forebrain. Neurons with NGF receptors were also visualized in radioautographs prepared from the basal forebrain of a cerebrus monkey. Present techniques fail to detect axons projecting from basal forebrain to hippocampus or cortex which have been shown to take up NGF selectively in retrograde transport studies. In studies with membrane-enriched preparations from rat, high-affinity binding of¹²⁵I-NGF (half maximal saturation in the 15–30 pM range) was detected in basal forebrain and striatum; lower levels of high-affinity binding were seen in hippocampus and neocortex. The binding and molecular properties of these receptors are similar to those described in other NGF-responsive tissues. These observations are further evidence supporting a biological role for NGF on some forebrain cholinergic neurons in adult rat.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Nerve growth factor

Nerve growth factor (NGF) is widely recognized as a target-derived factor responsible for the survival and maintenance of the phenotype of specific subsets of peripheral neurons and basal forebrain cholinergic nuclei during development and maturation. Other NGF-responsive cells are now known to belong to the hemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. The concentration of NGF is elevated in a number of inflammatory and autoimmune states in conjunction with increased accumulation of mast cells. Mast cells and NGF appear to be involved in neuroimmune interactions and tissue inflammation. Mast cells themselves are capable of producing and responding to NGF, suggesting that alterations in mast cell behavior may trigger maladaptive neuroimmune tissue responses, including those of an autoimmune nature. Moreover, NGF exerts a modulatory role on sensory nociceptive nerve physiology in the adult, and appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. NGF can thus be viewed as a multifactorial modulator of neuroimmune-endocrine functions.     

On the Molecular Basis Linking Nerve Growth Factor (NGF) to Alzheimer’s Disease

SUMMARY 1. Alzheimer’s disease (AD) is pathologically defined by the deposition of amyloid peptide and neurofibrillary tangles and is characterized by a progressive loss of cognition and memory function, due to marked cortical cholinergic depletion.2. Cholinergic cortical innervation is provided by basal forebrain cholinergic neurons. The neurotrophin Nerve Growth Factor (NGF) promotes survival and differentiation of basal forebrain cholinergic neurons.3. This assertion has been at the basis of the hypothesis developed in the last 20 years, whereby NGF deprivation would be one of the factor involved in the etiology of sporadic forms of AD.4. In this review, we shall summarize data that lead to the production and characterization of a mouse model for AD (AD11 anti-NGF mice), based on the expression of transgenic antibodies neutralizing NGF. The AD-like phenotype of AD11 mice will be discussed on the basis of recent studies that have posed NGF and its precursor pro-NGF back to the stage of AD-like neurodegeneration, showing the involvement of the precursor pro-NGF in one of the cascades leading to AD neurodegeneration.     

K-252a Promotes Survival and Choline Acetyltransferase Activity in Striatal and Basal Forebrain Neuronal Cultures

Abstract: The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 n M K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (>60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleukin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neurodegenerative diseases.