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.     

NGF in CNS: Experimental data and clinical implications

The presence of β-nerve growth factor (NGF) and its cell surface receptor (NGF-R) in the brain has been well established by a variety of experimental techniques in recent years. In particular, the molecular cloning of NGF and NGF-R as well as the development of sensitive two-site ELISA techniques for determining the levels of NGF and antibodies to NGF-R suitable for immunohistochemistry have led to rapid accumulation of data in this field from many laboratories. A main finding is the function of NGF in the cholinergic neurons of the basal forebrain, expressing NGF receptors and responding to the factor by increased activity of choline acetyltransferase, and the production of NGF in cortical areas and hippocampus comprising terminal areas for the cholinergic projections from the basal forebrain. In addition, findings suggest that additional neurons in the brain and spinal cord may utilize NGF, notably during development and possibly also after lesion of the adult CNS. Moreover, observations indicate that endogenous levels of NGF are lowered in the aged rat brain concomitant with losses of NGF-dependent neurons in the basal forebrain. The involvement of NGF in human neurodegenerative diseases is not established but the application of NGF to degenerating cholinergic neurons in Alzheimer patients may prove useful. A promising approach to achieve this goal is the production of biologically active, recombinant NGF.     

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.     

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.     

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.     

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.     

BDNF mRNA expression is increased in adult rat forebrain after limbic seizures: temporal patterns of induction distinct from NGF

We have localized brain-derived neurotrophic factor (BDNF) mRNA in rat brain and examined its regulation by seizure activity. In situ hybridization of BDNF 35S-cRNA most prominently labeled neurons in hippocampal stratum pyramidale and stratum granulosum, superficial olfactory cortex, pyramidal cell layers of neocortex, amygdala, claustrum, endopiriform nucleus, anterior olfactory nucleus, and ventromedial hypothalamus. Hybridization to BDNF mRNA was markedly increased in all of these regions after lesion-induced recurrent limbic seizures and within dentate gyrus granule cells following one electrically stimulated epileptiform afterdischarge. In contrast to seizure-elicited changes in nerve growth factor (NGF) mRNA expression, increases in BDNF mRNA occur in a greater number of different neuronal populations and develop several hours more rapidly in extrahippocampal loci. These results indicate that regulation by physiological activity may be an intrinsic property of this class of neurotrophic factor but that, in the recurrent seizure paradigm, different mechanisms mediate increased expression of mRNAs for BDNF and NGF outside hippocampus.     

Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motoneurons

In situ hybridization histochemistry and RNA blot analysis were used to study expression of nerve growth factor receptor (NGF-R) mRNA in rat spinal cord motoneurons. The results show that NGF-R mRNA is expressed at high levels in rat spinal cord motoneurons at the time of naturally occurring cell death. This expression is sustained, but reduced, during synapse formation and is subsequently greatly reduced in the adult spinal cord. A unilateral crush lesion of the sciatic nerve resulted in an 8-fold increase in NGF-R mRNA in adult rat spinal cord motoneurons 3 days after lesion, compared with the nonlesioned side. NGF-R mRNA induction was even more pronounced 7 and 14 days after lesion, reaching levels 12 times higher than those on the nonlesioned side. However, 6 weeks after lesion, when the motor function of the leg was largely restored, NGF-R expression had decreased to levels similar to those on the contralateral side. We therefore suggest that NGF-R mediates a trophic or axonal guidance function for developing and regenerating spinal cord motoneurons.     

Inhibitors of nitric oxide synthase attenuate nerve growth factor-mediated increases in choline acetyltransferase expression in PC12 cells

NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. To investigate the role of NO in NGF-activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor L-NAME (N (omega)-nitro-L-arginine methylester) or the inducible NOS selective inhibitor MIU (s-methylisothiourea), and the effect on NGF-stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady-state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO production. MIU and, to a lesser extent, L-NAME blocked neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. Both L-NAME and MIU attenuated NGF-mediated increases in choline transferase (ChAT)-specific activity and prevented the increase in expression of ChAT mRNA normally produced by NGF treatment of PC12 cells. The present study indicates that NO may be involved in the modulation of signal transduction pathways by which NGF leads to increased ChAT gene expression in PC12 cells.     

Non-radioactive detection of nerve growth factor receptor (NGFR) mRNA in rat brain using in situ hybridization histochemistry.

Radioactively labeled RNA probes in conjunction with in situ hybridization histochemistry have become a useful method for studying gene expression in the central nervous system. We used digoxigenin-labeled uridine triphosphate to synthesize cRNA probes for localization of nerve growth factor receptor (NGFR) mRNA in the rat basal forebrain. Detection of cells containing digoxigenin-labeled NGFR mRNA was accomplished using a digoxigenin antibody conjugated with alkaline phosphatase. NGFR mRNA-positive cells were distributed in three major cell groups in the basal forebrain: the medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca, and nucleus basalis. This technique provides a rapid and sensitive method for high-resolution detection of mRNA species in the central nervous system, as well as the potential for co-localization of two different mRNA species within individual cells.     

NGF effects on developing forebrain cholinergic neurons are regionally specific

Nerve growth factor (NGF) has been shown to have an effect on neurons in the central nervous system (CNS). A number of observations suggest that NGF acts as a trophic factor for cholinergic neurons of the basal forebrain and the caudate-putamen. We sought to further characterize the CNS actions of NGF by examining its effect on choline acetyltransferase (ChAT) activity in the cell bodies and fibers of developing neurons of the septum and caudate-putamen. ChAT activity was increased after even a single NGF injection. Interestingly, the magnitude of the effect of multiple NGF injections suggested that repeated treatments may augment NGF actions on these neurons. The time-course of the response to NGF was followed after a single injection on postnatal day (PD) 2. NGF treatment produced long-lasting increases in ChAT activity in septum, hippocampus and caudate-putamen. The response in cell body regions (septum, caudate-putamen) was characterized by an initial lag period of approximately 24 hr, a rapid rise to maximum values, a plateau phase and a return to baseline. The response in hippocampus was delayed by 48 hr relative to that in septum, indicating that NGF actions on ChAT were first registered in septal cell bodies. Finally, developmental events were shown to have a regionally specific influence on the response of neurons to NGF. For though the septal response to a single NGF injection was undiminished well into the third postnatal week, little or no response was detected in caudate-putamen at that time. In highlighting the potency and regional specificity of NGF effects, these observations provide additional, support for the hypothesis that NGF is a trophic factor for CNS cholinergic neurons.Dedicated to Dr. E. M. Shooter and Dr. S. Varon as part of a special issue (Neurochemical Research, Vol. 12, No. 10, 1987).     

Induction of NGF synthesis in astrocytes by onjisaponins of Polygala tenuifolia, constituents of kampo (Japanese herbal) medicine, Ninjin-yoei-to.

The effect of a kampo medicine, Ninjin-yoei-to (NYT; Ren-shen-yang-rong-tang in Chinese) on nerve growth factor (NGF) secretion from the cultured rat astrocytes was examined in vitro. When rat embryo astrocytes were cultured in the presence of NYT for 24 h, the amount of NGF in the medium was significantly increased in a dose dependent manner. Among 14 kinds of component herbs in NYT, the roots of Polygala tenuifolia and roots of Panax ginseng extracts increased NGF levels from the astrocytes. Saponin fraction from the roots of P. tenuifolia enhanced the production of NGF, however phenolic glycoside fraction showed no effect. Onjisaponins A, B, E, F and G as major saponins of the root of P. tenuifolia strongly increased the NGF level, whereas ginsenosides Rb1 and Rg1 did not affect the NGF level. Onjisaponin F also induced ChAT mRNA level in rat basal forebrain cells. These results indicate the possibility that NYT and/or onjisaponins in P. tenuifolia may have potential therapeutic effects for the treatment of Alzheimer disease patients.     

Evaluation of the neurorestorative effects of the murine beta-nerve growth factor infusions in old rat with cognitive deficit

The nerve growth factor (NGF) is known to participate in the regulation of the expression levels and activity of the choline acetyltransferase (ChAT) in the nervous system. This enzyme is sensitive to the degenerative changes found in Alzheimer's disease (AD). We compared the effectiveness of intraparenchymal (ip) and intracerebroventricular (icv) administration of the murine beta-NGF (beta-NGFm) produced in our laboratories, through the determination of the expression levels and activity of the ChAT, and the evaluation of behavioral recovery in aged rat with cognitive deficit. Our results indicated that icv infusion of beta-NGFm stimulates the expression levels of ChAT gene in the striatum of old rats. Remarkable losses in the ChAT activity were observed in the septum and striatum of old rats. Exogenous administration of beta-NGFm produced a significant increase of ChAT activity in these brain regions differentially according to the administration pathway. The behavioral studies demonstrated that the administration pathway is an important factor in order to obtain the best results for a neurorestorative treatment.     

Does senile impairment of cholinergic system in rats concern only disturbances in cholinergic phenotype or the progressive degeneration of neuronal cell bodies?

The trophic effect of continuous intraventricular infusion of nerve growth factor (NGF) on morphology of the basal forebrain (BF) cholinergic neurons was tested in 4- and 28-month-old male Wistar rats. All studies were conducted using behaviorally uncharacterized animals from the same breeding colony. Immunohistochemical procedure for choline acetyltransferase (ChAT) and p75NTR receptor has been applied to identify cholinergic cells in the structures of basal forebrain (BF). Using a quantitative image analyzer, morphometric and densitometric parameters of ChAT- and p75NTR-positive cells were measured immediately after cessation of NGF infusion. In 28-month-old non-treated rats the number of intensively ChAT-positive cells in all forebrain structures was reduced by 50-70% as compared with young animals. The remaining ChAT-positive cells appeared shrunken and the neuropil staining was NTR markedly reduced. In contrast, the same neurons when stained for p75 were numerous and distinctly visible with perfect morphology. Analysis of Nissl stained sections also showed that 28-month-old rats did not display significant losses of neuronal cell bodies. NGF restored the number of intensely stained ChAT-positive cells to about 90% of that for young controls and caused a significant increase in size of those cells in 28-month-old rats as compared with the control, age-matched group. NGF did not influence the morphology of p75NTR-positive neurons, which were well labeled, irrespective of treatment and age of the rats. In 4-month-old rats, NGF infusion decreased the intensity of both ChAT and p75NTR immunostaining. These data provide some evidence for preservation of BF cholinergic neurons from atrophy during aging and indicate that senile impairment of the cholinergic system in rats concerns decrease in ChAT-protein expression rather than an acute degeneration of neuronal cell bodies. Treatment with NGF resulted in restoration of cholinergic phenotype in the BF neurons of aged rats. However, the present study also rises issue of possible detrimental effects of NGF in young normal animals.     

Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells

Recent work has shown that estrogen receptor mRNA and protein co-localize with neurotrophin receptor systems in the developing basal forebrain. In the present study we examined the potential for reciprocal regulation of estrogen and neurotrophin receptor systems by their ligands in a prototypical neurotrophin target, the PC12 cell. using in situ hybridization histochemistry, RT-PCR and a modified nuclear exchange assay, we found both estrogen receptor mRNA and estrogen binding in PC12 cells. Moreover, while estrogen binding was relatively low in naive PC12 cells, long-term exposure to NGF enhanced estrogen binding in these cells by sixfold. Furthermore, concurrent exposure to estrogen and NGF receptor mRNAs deifferentially regulated the expression of the two NGF receptor mRNAs. The expression of trkA mRNA was up-regulated, while p75^NGFR mRNA was down-regulated transiently. The present data indicate that NGF may increase neuronal sensitivity to estrogen, and that estrogen, by differentially regulating p75^NGFR and trkA mRNA, may alter the ratio fo the two NGF receptors, and, conseuqnetly, neurotrophin responsivity. In view of the widespread co-localization of estrogen and neurotrophin receptor systems in the developing CNS, the reciprocal regulation of these receptor systems by NGF and estrogen may have important implications for processes governing neural maturation and the maintenance of neural funciton. 1994 John Wiley & Sons, Inc.     

Evidence for a physiological role of nerve growth factor in the central nervous system of neonatal rats

Forebrain cholinergic neurons have been shown to respond in vivo to administration of nerve growth factor (NGF) with a prominent and selective increase of choline acetyltransferase (ChAT) activity. This has suggested that NGF can act as a trophic factor for these neurons. To test this hypothesis directly, anti-NGF antibodies (and their Fab fragments) were intracerebroventricularly injected into neonatal rats to neutralize endogenously occurring NGF. The anti-NGF antibody administration produced a decrease of ChAT activity in the hippocampus, septal area, cortex, and striatum of rat pups. This finding was substantiated by a concomitant decrease of immunopositive staining for ChAT in the septal area. These effects indicate that the occurrence of endogenous NGF in the CNS is physiologically relevant for regulating the function of forebrain cholinergic neurons.     

Differential Effects of Nerve Growth Factor on Expression of Choline Acetyltransferase and Sodium-Coupled Choline Transport in Basal Forebrain Cholinergic Neurons in Culture

Abstract: Nerve growth factor (NGF) treatment of primary cultures of embryonic day 17 rat basal forebrain differentially altered activity of choline acetyltransferase (ChAT) and high-affinity choline transport; ChAT specific activity was increased by threefold in neurons grown in the presence of NGF for between 4 and 8 days, whereas high-affinity choline transport activity was not changed relative to control. Dose-response studies revealed that enhancement of neuronal ChAT activity occurred at low concentrations of NGF with an EC₅₀ of 7 ng/ml, with no enhancement of high-affinity choline transport observed at NGF concentrations up to 100 ng/ml. In addition, synthesis of acetylcholine (ACh) and ACh content in neurons grown in the presence of NGF for up to 6 days was increased significantly compared with controls. These results suggest that regulation of ACh synthesis in primary cultures of basal forebrain neurons is not limited by provision of choline by the high-affinity choline transport system and that increased ChAT activity in the presence of NGF without a concomitant increase in high-affinity choline transport is sufficient to increase ACh synthesis. This further suggests that intracellular pools of choline, which do not normally serve as substrate for ACh synthesis, may be made available for ACh synthesis in the presence of NGF.