Nerve Growth Factor mRNA Levels Research Articles

Regional and Temporal Pattern of Expression of Nerve Growth Factor and Basic Fibroblast Growth Factor mRNA in Rat Brain Following Electroconvulsive Shock

We have previously reported that focally evoked limbic motor seizures rapidly increase levels of mRNA encoding nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) in specific limbic system areas of the adult rat brain. The present studies examined the effect of both minimal and maximal electroconvulsive shock, applied via corneal electrodes, on NGF and bFGF mRNA levels in several limbic (entorhinal cortex, hippocampus, olfactory bulb) and extralimbic (striatum and cerebellum) brain regions. By 5 h following limbic motor seizures induced by low-intensity (minimal) electroshock (LES) (0.2 s, 50-70 mA; three times over a 1-h period), bFGF mRNA was significantly increased in entorhinal cortex and hippocampus, but not in the other regions examined. In contrast, tonic extensor seizures evoked by maximal electroshock (MES) (0.2 s, 150 mA; three times over a 1-h period) were associated with a significant increase in bFGF mRNA in all limbic and extralimbic regions examined. In the same animals, increases in NGF mRNA were limited to entorhinal cortex and hippocampus. Adrenal steroids were not required for the seizure-induced increase in NGF or bFGF mRNAs, based on the finding that adrenalectomized rats exhibited electroshock-induced increases in both NGF and bFGF mRNAs equivalent to the increase observed in sham-operated rats. It is suggested that the increase in mRNA levels for the neurotrophic factors occurs selectively in those regions which are especially activated by the specific seizure model, and represents an adaptive response to repeated noninjurious neuronal stimulation.

Positive Feedback between Acetylcholine and the Neurotrophins Nerve Growth Factor and Brain‐derived Neurotrophic Factor in the Rat Hippocampus

In the rat hippocampus, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are synthesized by neurons in an activity-dependent manner. Glutamate receptor activation increases whereas GABAergic stimulation decreases NGF and BDNF mRNA levels. Here we demonstrate that NGF and BDNF mRNA and NGF protein are up-regulated in the rat hippocampus by the activation of muscarinic receptors. Conversely, NGF and BDNF enhance the release of acetylcholine (ACh) from rat hippocampal synaptosomes containing the nerve endings of the septal cholinergic neurons. NGF also rapidly increases the high-affinity choline transport into synaptosomes. The reciprocal regulation of ACh, NGF and BDNF in the hippocampus suggests a novel molecular framework by which the neurotrophins might influence synaptic plasticity.

Effect of yohimbine on nerve growth factor mRNA and protein levels in rat hippocampus

Activation of noradrenergic receptors has been shown to increase expression of nerve growth factor (NGF) gene in brain cells in vitro. The present studies were undertaken to determine if this stimulation was effective in vivo as well. Rats were administered the norepinephrine-releasing drug. yohimbine (YOH), and had their hippocampi assayed for NGF mRNA and protein at various times after the injection. It was found that yohimbine caused a 3-fold increase of NGF mRNA levels at 24 h. Protein levels, however, were unaltered at this time. Thus norepinephrine release in vivo appears to be sufficient for increasing mRNA level but not for translation to protein.

Seizure-Induced Increases in NGF mRNA Exhibit Different Time Courses across Forebrain Regions and Are Biphasic in Hippocampus

In the present study, in situ hybridization and S1 nuclease protection analyses were used to evaluate the temporal and spatial parameters of changes in nerve growth factor (NGF) mRNA expression in rat forebrain following hilus lesion-induced recurrent limbic seizures. Seizure-induced increases in NGF mRNA levels were widespread with differences in the temporal parameters of change between brain areas. There were two distinct increases in NGF cRNA hybridization in dentate gyrus stratum granulosum. Hybridization was increased several-fold by 6 h after a seizure-producing hilus lesion (HL), declined to below control values by 12 h post-HL, and then increased again by 24 h post-HL, or 12 h after the termination of seizures. This biphasic increase was corroborated by S1 nuclease protection analysis. In entorhinal cortex, cingulate cortex and neocortex NGF cRNA hybridization was markedly increased first in layers H/III by 6-12 h post-HL and progressed to layers V/VI by 24 h post-HL. Striking increases in NGF mRNA were detected in the majority of amygdaloid nuclei beginning with the cortical nuclei by 12 h postlesion and extending into the more deeply placed nuclei by 24 h postlesion. Labeling was increased in sparsely distributed neurons in the caudate putamen, ventral pallidum, and tenia tecta at 24 h post-HL. In all areas, hybridization declined to control values by 48-96 h post-HL. NGF expression was not changed in some forebrain regions which normally contain NGF mRNA including the diagonal bands of Broca and select thalamic nuclei. These data demonstrate that seizures stimulate NGF expression in many different types of neurons. Moreover, regional differences in the time courses of induction suggest that distinct regulatory mechanisms subserve activity-dependent changes in NGF mRNA expression in different neuronal populations.

Nerve growth factor mRNA levels are normal in Alzheimer's disease : N. Jetté, M.S. Cole, and M. Fahnestock. Department of Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada, L8N 3Z5

Nerve growth factor mRNA levels are normal in Alzheimer's disease : N. Jetté, M.S. Cole, and M. Fahnestock. Department of Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada, L8N 3Z5

Cholinergic regulation of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) but not neurotrophin-3 (NT-3) mRNA levels in the developing rat hippocampus

In previous experiments it has been demonstrated that the synthesis of BDNF (brain-derived neurotrophic factor) and NGF in neurons of the hippocampus is regulated by neuronal activity. The glutamate system is predominantly responsible for upregulation and the GABAergic system for downregulation both in vitro and in vivo (Zafra et al., 1990, 1991). The aim of the present study is to examine the extent to which the cholinergic system is also involved in the regulation of NGF and BDNF mRNA and whether the regulatory contribution of the cholinergic system changes during development. Partial transection of the fimbria fornix bundle in the second postnatal week resulted in a reduction of BDNF and NGF mRNA levels in the hippocampus, suggesting that septal cholinergic input is involved in the regulation of hippocampal BDNF and NGF mRNA levels. Because the fimbria fornix bundle also contains fibers other than cholinergic ones, we further evaluated the importance of the cholinergic influence by injecting pilocarpine, a muscarinic agonist. Pilocarpine markedly increased hippocampal BDNF and NGF mRNA levels in both early postnatal and adult rats. In situ hybridization experiments demonstrated that pilocarpine led to an increase in BDNF expression in the CA1-CA4 regions of the hippocampus and in the dentate gyrus. However, pilocarpine increased NGF mRNA only in those neurons of the dentate gyrus and CA1-CA4 regions that also expressed NGF mRNA in the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor and p75NGFR factor receptor mRNA change in rodent CNS following stress activation of the hypothalamo‐pituitary‐adrenocortical axis

The synthesis of nerve growth factor (NGF) by the hippocampus raises the possibility that NGF may play a role in the regulation of the hypothalamic-pituitary-adrenal axis (HPAA). Subchronic cold stress has been shown to activate the HPAA in a mild noninvasive manner, to stimulate serum glucocorticoid levels, and to perturb NGF binding in hippocampus and basal forebrain. One or repeated episodes of cold stress increased NGF mRNA levels in the hippocampus and p75NGFR mRNA levels in the basal forebrain. These changes were not due to elevated serum glucocorticoid levels since treatment with exogenous corticosterone had no effect on NGF and p75NGFR mRNA levels. Adrenalectomy did not prevent the stress induced increases in NGF and p75NGFR mRNA.

Alkylcatechols regulate NGF gene expression in astroglial cells via both protein kinase C- and cAMP-independent mechanisms.

It has been previously demonstrated that, in mouse astroglial cells and fibroblast cells in culture, alkylcatechols cause a rapid increase in the nerve growth factor (NGF) mRNA level followed by a marked increase in the amount of NGF released into the medium. To understand the mechanism of this alkylcatechol effect on NGF gene expression in astroglial cells, we examined the effects of protein kinases that influence intracellular signal transduction and of their inhibitors. The reagents to increase the intracellular content of cyclic AMP (cAMP) such as dibutyryl cyclic AMP (Bt2cAMP), forskolin, or cholera toxin did not mimic alkylcatechol induction of NGF gene expression. Phorbol ester, a direct activator of protein kinase C (PKC), caused an increase in the NGF synthesis/secretion. The stimulatory effect of homocatechol (4-methylcatechol) on NGF synthesis was not completely inhibited by staurosporine, an inhibitor of PKC. The treatment of cells with homocatechol resulted in the translocation of PKC from cytosol to membrane-associated fractions, although the levels of the subcellular location of PKC were not correlated with the level of the induction of NGF gene expression. The concomitant administration of homocatechol (10(-4) M) and PMA (10(-8) - 10(-6) M) evoked a drastic and prolonged increase in the NGF mRNA level, and also markedly increased the amounts of NGF secreted by the cells (approximately 150-fold). This synergism was inhibited in part by staurosporine, but the level of increase in NGF mRNA and in NGF protein was rather greater than that of activation of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurotrophin production in the brain

The neurotrophin family includes four structurally related proteins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). All four neurotrophins are synthesized in the brain although at different levels and with different regional distributions. The highest levels of NGF mRNA correlate with innervation of cholinergic neurons in the brain. NT-3 mRNA shows the most restricted distribution with higher levels in the hippocampus and cerebellum compared to other regions. In contrast, BDNF and NT-4 mRNAs are widely distributed throughout the brain. The level of NT-4 mRNA in the brain is, however, significantly lower than the levels of mRNAs for the other three neurotrophins. All four neurotrophins are maximally expressed at different times of brain development with marked transient peak levels in certain brain regions. NGF and BDNF mRNAs increase rapidly but transiently in specific brain regions following seizures or after ischemic and hypoglycemic brain insults. Combined with knowledge regarding the distribution of neurotrophin receptors in the brain this information will facilitate studies on the in vivo role of the neurotrophins in the brand and open up possibilities to test protective effects of neurotrophins in neurons following brain insults.

Expression of the gene for nerve growth factor (NGF) in the monkey central nervous system

The expression of the gene for nerve growth factor (NGF) was examined in the central nervous system of adult and fetal monkeys. In adults, the highest level of NGF mRNA was found in the hippocampus and relatively high levels were observed in the cerebral cortices and thalamus. NGF mRNA was also detected in the cerebellum and the caudate nucleus. In the spinal cord, there was no evidence of the mRNA. The levels of NGF mRNA were closely correlated with those of NGF. At embryonic day 140 (E140), levels of NGF mRNA in the visual cortex and cerebellum were three times higher than those at the adult stage. Our previous study on the ontogeny of NGF (Hayashi, M. et al., Neuroscience, 36 (1990) 683–689) showed that the level of NGF in the visual cortex at E140 is the same as that at adult stage. Thus, at the fetal stage, NGF may be actively transported from the cerebral cortex to other regions of the brain, such as the basal forebrain area. By contrast, the levels of NGF and NGF mRNA in the cerebellum were almost the same at the adult and fetal stages, suggesting that NGF, which is synthesized in the cerebellum, may be taken up locally by cerebellar cells.

Regulation of Nerve Growth Factor mRNA in the Hippocampal Formation: Effects of N-Methyl- d-aspartate Receptor Activation

In the hippocampal formation, nerve growth factor (NGF) is produced in granule cells of the dentate gyrus and a few pyramidal cells of Ammon's horn. Both neuronal populations express N-methyl- d-aspartate (NMDA) receptors and receive putative glutamatergic afferents originating in the entorhinal cortex and projecting via the perforant path. We report in this study that intrahippocampal or intraventricular injections of NMDA increase NGF mRNA levels in dentate gyrus granule cells as determined using in situ hybridization histochemistry and a solution hybridization assay. NGF mRNA induction is detected within 2 h following NMDA treatment and returns to control levels within 24 h. This NMDA effect is dose-dependent and blocked by pretreatment with 2-amino-5-phosphonopentanoic acid, a competitive NMDA antagonist. Finally, the induction of NGF mRNA is observed in the absence of detectable neurotoxicity or seizure activity. We postulate that normal physiological events associated with the activation of hippocampal NMDA receptors may regulate mRNA expression of this neurotrophic factor.

Glucocorticoid modulation of neurotrophin expression in immortalized mouse hippocampal neurons

To see whether glucocorticoid hormones can influence the regulation of neurotrophin expression in hippocampal neurons, we have used reverse-transcription followed by polymerase chain reaction to investigate changes in the mRNA levels of nerve growth factor (NGF) and neurotrophin-3 (NT-3) in immortalized hippocampal neurons after dexamethasone application. Our results show that NGF mRNA levels rise in both embryonic and postnatal neurons, but with different time courses, while NT-3 levels rise in the embryonic but not in the postnatally derived cell line. Modulation of NT expression by glucocorticoids may therefore be developmentally regulated.

Increased NGF mRNA expression in denervated rat skin

Using a coupled reverse transcription and competitive polymerase chain reaction protocol, we have measured Nerve Growth Factor (NGF) mRNA expression in adult rat skin following its denervation. By 2-4 days, levels of NGF mRNA were increased approximately 5-10 fold over levels in innervated skin, remaining increased for up to at least 2 weeks. In situ hybridization carried out on skin samples revealed that the NGF message was expressed not only in the distal nerve pathways but also in non-nerve associated cells, such as dermal fibroblasts, and in the basal epidermal layer. Denervated skin evokes a NGF-dependent collateral sprouting from neighbouring intact sensory axons. The new findings indicate that an increased NGF production is associated with the increased availability of NGF in such skin, and that cutaneous nerves have a role in regulating this NGF production.

Interleukin-4 and -5 as modulators of nerve growth factor synthesis/secretion in astrocytes.

To examine the regulation of nerve growth factor (NGF) gene expression with respect to neural trauma, we examined the effects of T cell-derived lymphokines on NGF synthesis/secretion in cultured mouse astrocytes. Interleukin (IL)-4 and IL-5 significantly increased the amount of NGF secreted by astrocytes, whereas IL-2, IL-3, and IL-6 had no significant effect. IL-4 and IL-5 produced marked increases in NGF mRNA levels in astrocytes as demonstrated by the reverse transcription-polymerase chain reaction (RT-PCR) method. The effect of IL-4 and IL-5 was greater in quiescent astrocytes than in growing cells. Neither increase in thymidine incorporation nor any morphological change was observed during the treatment with IL-4 and IL-5. The stimulatory effect of IL-4 and IL-5 on NGF synthesis was completely inhibited by the addition of anti-IL-4 monoclonal antibody and anti-IL-5 monoclonal antibody, respectively. The results indicate that IL-4 and IL-5 specifically trigger a cascade of events to regulate NGF synthesis in astrocytes, independent of cell growth.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and low-affinity nerve growth factor receptor (LNGFR) mRNA levels in cultured rat Schwann cells; differential time- and dose-dependent regulation by cAMP

Steady-state mRNA levels for NGF, BDNF, and NT-3, and their receptors, LNGFR, trk, trkB, and trkC, were examined in cultured rat Schwann cells. NGF, BDNF, and LNGFR mRNAs were expressed in control Schwann cells, but NT-3, trk, trkB and trkC mRNAs were not. As previously reported, when Schwann cells were treated with forskolin or 8-bromo-cAMP, NGF mRNA expression was enhanced, while BDNF and LNGFR mRNA levels were suppressed. The optimal concentration of forskolin for NGF mRNA enhancement and BDNF mRNA suppression was similar to that for LNGFR mRNA suppression, which coincided with the optimal concentration for Schwann cells to differentiate. However, the lag-time for forskolin-induces down-regulation of LNGFR mRNA was far greater than that for NGF mRNA increase and BDNF mRNA suppression.

Tumor necrosis factor stimulates the synthesis and secretion of biologically active nerve growth factor in non-neuronal cells.

Tumor necrosis factor-alpha (TNF) markedly stimulates the synthesis and secretion of immunoreactive nerve growth factor (NGF) in quiescent mouse fibroblasts, which is a result of increase in the NGF mRNA level. NGF produced by TNF-treated fibroblasts has a molecular mass of 13 kDa on SDS-polyacrylamide gel electrophoresis, which is consistent in size with the subunit of mouse beta-NGF, and induces neurite outgrowth in paravertebral sympathetic neurons. Several peptide growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor also stimulate NGF production in the cells, but not platelet-derived growth factor. The dose responses of TNF and bFGF to stimulate NGF production in the cells are, respectively, similar to those to induce cell proliferation. However, no correlation is observed between the ability of these growth factors to stimulate NGF production and that to induce cell proliferation. Thus, the stimulation of NGF production in the cells seems to be a specific activity of TNF and some other growth factors. TNF stimulates the synthesis and secretion of NGF also in other cells such as human glioblastoma cells. These findings suggest that TNF plays a role in regulating neuronal cell function through an indirect mechanism by which it stimulates NGF production in glial cells and fibroblasts.

Expression of nerve growth factor and nerve growth factor receptor genes in human tissues and in prostatic adenocarcinoma cell lines.

Nerve growth factor (NGF) mRNAs were detected and quantified in a variety of normal and neoplastic human tissues by northern blot hybridization. Human heart contained the highest NGF mRNA levels, whereas lower but comparable levels were found in the placenta, prostate, and kidney. All tissues examined coexpressed the low-affinity NGF receptor (LNGFR), whereas none of these tissues expressed the high-affinity NGF receptor encoded by the trk protooncogene. The widespread distribution of the LNGFR suggests that it plays a role in the regulation of normal cell growth. No overexpression of NGF or LNGFR mRNA was detected in neoplastic tissues, whereas LNGFR-like immunoreactivity was localized outside of tumor cells. Transforming growth factor-alpha and protooncogene c-fos expression in these tissues did not show a systematic correlation with NGF/LNGFR expression. Furthermore, regulation of the human NGF gene was studied in DU145 cells, a prostatic adenocarcinoma cell line that synthesizes significant NGF mRNA levels. Serum induced, whereas dexamethasone inhibited, NGF mRNA synthesis in these cells. Serum induction was preceded by a rapid and transient activation of the c-fos protooncogene.

Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are molecules which regulate the development and maintenance of specific functions in different populations of peripheral and central neurons, amongst them sensory neurons of neural crest and placode origin. Under physiological conditions NGF is synthesized by peripheral target tissues, whereas BDNF synthesis is highest in the CNS. This situation changes dramatically after lesion of peripheral nerves. As previously shown, there is a marked rapid increase in NGF mRNA in the nonneuronal cells of the damaged nerve. The prolonged elevation of NGF mRNA levels is related to the immigration of activated macrophages, interleukin-1 being the most essential mediator of this effect. Here we show that transsection of the rat sciatic nerve also leads to a very marked increase in BDNF mRNA, the final levels being even ten times higher than those of NGF mRNA. However, the time-course and spatial pattern of BDNF mRNA expression are distinctly different. There is a continuous slow increase of BDNF mRNA starting after day 3 post-lesion and reaching maximal levels 3-4 wk later. These distinct differences suggest different mechanisms of regulation of NGF and BDNF synthesis in non-neuronal cells of the nerve. This was substantiated by the demonstration of differential regulation of these mRNAs in organ culture of rat sciatic nerve and Schwann cell culture. Furthermore, using bioassays and specific antibodies we showed that cultured Schwann cells are a rich source of BDNF- and ciliary neurotrophic factor (CNTF)-like neurotrophic activity in addition to NGF. Antisera raised against a BDNF-peptide demonstrated BDNF-immunoreactivity in pure cultured Schwann cells, but not in fibroblasts derived from sciatic nerve.

Production and secretion of nerve growth factor by clonal striated muscle cell line, G8-1.

Two specific methods, Northern blot analysis using a 50 nucleotides probe to the conserved region of the nerve growth factor (NGF) gene, and enzyme immunoassay using a monoclonal biotinylated rat anti-NGF IgG-avidin conjugated peroxidase system, were used to demonstrate the production and secretion of NGF by mouse striated muscle cell line G8-1. Calcium ionophore, A23187 (0.1-1 microM), forskolin (0.1-100 microM) and dibutyryl cyclic AMP (0.1-10 mM) strongly decreased the secretion of ir-NGF. The level of NGF mRNA was decreased by veratridine, A23187, forskolin and cyclic AMP but not by cyclic GMP. Consequently, we conclude that the secretion of NGF molecules paralleled the changes of NGF mRNA levels in the cells induced by all agents tested. Carmamylcholine also decreased the level of NGF mRNA. Immunoblot analysis suggested that denatured ir-NGF molecules exist in a higher molecular weight form (22 KDa) than those of mouse submaxillary gland (13 KDa). Both Ca(2+)- and cAMP mediated mechanisms contribute to the decreased production of NGF mRNA in the cells and the consequent inhibition of secretion of NGF molecules. Finally, molecular cloning of NGF of G8-1 cells was conducted and confirmed the structure of the gene that consists of 1, 3, and 4 exons deleting exon 2. Thus, G8-1 NGF is derived from transcript B.

Mechanisms of nerve growth factor mRNA regulation by interleukin-1 beta in hippocampal cultures: role of second messengers.

Cytokines such as interleukin-1, which are found in the brain after trauma, regulate expression of nerve growth factor (NGF) mRNA and protein in hippocampal cultures. We have investigated possible mechanisms by which Il-1 beta regulates NGF in hippocampal cells. The induction of NGF mRNA by Il-1 beta was blocked by a receptor antagonist indicating that this effect is receptor mediated. Il-1 beta elicited a dramatic induction of c-fos mRNA and a slight elevation of c-jun mRNA in a time dependent manner which may allow for a role in the induction of NGF mRNA expression. We examined whether specific second messenger pathways were involved in mediating the action of Il-1 beta in the hippocampus. Activation of cAMP with forskolin or treatment with 8-Br-cAMP had no effect on NGF mRNA levels. Moreover, exposure of hippocampal cultures to Il-1 beta evoked no change in cAMP levels, indicating that this second messenger system played little or no role in the regulation of NGF expression by Il-1 beta in these cells. Further, interleukin-1 elicited no change in membrane inositol phosphate turnover, nor did it affect intracellular calcium levels. Treatment of cell cultures with the phorbol ester PMA elicited an increase in NGF mRNA, suggesting that activation of protein kinase C (PKC) may mediate NGF mRNA expression. However, prolonged treatment of cultures with PMA to desensitize PKC did not eliminate the Il-1 beta induction of NGF mRNA. Il-1 beta, therefore, did not appear to activate NGF expression via cAMP, Ca2+, or a PKC isoform that is downregulated by prolonged PMA treatment. However, a phosphorylation event may be involved in the signal transduction mechanism, as treatment with okadaic acid to inhibit protein phosphatase 2a potentiated the induction of NGF mRNA by Il-1 beta. The results presented indicate that Il-1 beta acts via its receptor to induce a rise in NGF expression. Identification of the specific second messenger pathway has remained elusive; however, a phosphorylation event appears to be intermediary. Moreover, the induction of c-fos and c-jun may represent a final common path in activation of NGF gene expression by different signals such as Il-1 beta and PMA.