The structure-function relationship of the nerve growth factor molecule and the regulation of its synthesis.
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Animals↗
Biomedical subjects
H Thoenen
Publications and source records attributed to H Thoenen.
Differential regulation of mRNA encoding nerve growth factor and its receptor in rat sciatic nerve during development, degeneration, and regeneration: role of macrophages.
In newborn rats the levels of nerve growth factor (NGF) mRNA (mRNANGF) and NGF receptor mRNA (mRNA(rec)) in the sciatic nerve were 10 and 120 times higher, respectively, than in adult animals. mRNA(rec) levels decreased steadily from birth, approaching adult levels by the third postnatal week, whereas mRNANGF levels decreased only after the first postnatal week, although also reaching adult levels by the third week. Transection of the adult sciatic nerve resulted in a marked biphasic increase in mRNANGF with time. On the proximal side of the cut, this increase was confined to the area immediately adjacent to the cut; peripherally, a similar biphasic increase was present in all segments. mRNA(rec) levels were also markedly elevated distal to the transection site, in agreement with previous results obtained by immunological methods [Taniuchi, M., Clark, H. B. & Johnson, E. M., Jr. (1986) Proc. Natl. Acad. Sci. USA 83, 4094-4098]. Following a crush lesion (allowing regeneration), the mRNA(rec) levels were rapidly down-regulated as the regenerating nerve fibers passed through the distal segments. Down-regulation of mRNANGF also occurred during regeneration but was slower and not as extensive as that of mRNA(rec) over the time period studied. Changes in mRNANGF and mRNA(rec) occurring in vivo after transection were compared with those observed in pieces of sciatic nerve kept in culture. No difference was found for mRNA(rec). Only the initial rapid increase in mRNANGF occurred in culture, but the in vivo situation could be mimicked by the addition of activated macrophages. This reflects the situation in vivo where, after nerve lesion, macrophages infiltrate the area of the Wallerian degeneration. These results suggest that mRNANGF synthesis in sciatic non-neuronal cells is regulated by macrophages, whereas mRNA(rec) synthesis is determined by axonal contact.
Animals↗
Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection.
The intact sciatic nerve contains levels of nerve growth factor (NGF) that are comparable to those of densely innervated peripheral target tissues of NGF-responsive (sympathetic and sensory) neurons. There, the high NGF levels are reflected by correspondingly high mRNANGF levels. In the intact sciatic nerve, mRNANGF levels were very low, thus indicating that the contribution of locally synthesized NGF by nonneuronal cells is small. However, after transection an increase of up to 15-fold in mRNANGF was measured in 4-mm segments collected both proximally and distally to the transection site. Distally to the transection site, augmented mRNANGF levels occurred in all three 4-mm segments from 6 h to 2 wk after transection, the longest time period investigated. The augmented local NGF synthesis after transection was accompanied by a reexpression of NGF receptors by Schwann cells (NGF receptors normally disappear shortly after birth). Proximal to the transection site, the augmented NGF synthesis was restricted to the very end of the nerve stump that acts as a "substitute target organ" for the regenerating NGF-responsive nerve fibers. While the mRNANGF levels in the nerve stump correspond to those of a densely innervated peripheral organ, the volume is too small to fully replace the lacking supply from the periphery. This is reflected by the fact that in the more proximal part of the transected sciatic nerve, where mRNANGF remained unchanged, the NGF levels reached only 40% of control values. In situ hybridization experiments demonstrated that after transection all nonneuronal cells express mRNANGF and not only those ensheathing the nerve fibers of NGF-responsive neurons.
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Synthesis of the trypsin fragment 10-25/75-88 of mouse nerve growth factor. II. The unsymmetrical double chain cystine peptide.
The synthesis of the unsymmetrical double chain cystine peptide corresponding to sequence 10-25/75-88 of mouse nerve growth factor is described. The synthetic product was inactive in all bioassays examined; consequently, this portion of the NGF molecule did not represent or contain a lower molecular weight form of the neurotrophic factor.
Amino Acid Sequence↗
Both short- and long-term effects of nerve growth factor on tyrosine hydroxylase in calf adrenal chromaffin cells are blocked by S-adenosylhomocysteine hydrolase inhibitors.
We have previously shown that primary cultures of calf chromaffin cells respond to nerve growth factor (NGF) treatment with a selective induction of tyrosine hydroxylase (TH), which takes 48 h to be manifested. In the present study, we report that short exposure of calf chromaffin cells to NGF (5-60 min) results in TH activation, which involves a change in the Vmax of the enzyme with no change in the number of enzyme molecules, similar to an effect that has been previously reported in PC12 cells. This activation is markedly potentiated when the chromaffin cells are plated on a laminin substrate, such that after 5 min of NGF exposure, there is an approximately fourfold increase in the TH activity. Both short-term activation and long-term TH induction brought about by NGF treatment are blocked by 5'-deoxy-5'-methylthioadenosine and other drugs that act as S-adenosylhomocysteine (SAH) hydrolase inhibitors to block methylation by end-product inhibition. These drugs did not inhibit cyclic AMP-mediated TH activation or increases in the levels of TH. However, measurements of the degree of blockade of methylation in cells treated with these drugs, taken together with conceptual information regarding the nonregulatory nature of methylation in eukaryotic cells, were not consistent with inhibition of methylation as the crucial effect of the drugs to block the effects of NGF. Nonetheless, since SAH hydrolase inhibitors selectively inhibited NGF-mediated effects, and not comparable effects triggered by other stimuli, these compounds provide useful tools in future studies of the biochemical signalling mechanism of NGF.
8-Bromo Cyclic Adenosine Monophosphate↗
N-terminal sequence of pig brain choline acetyltransferase purified by a rapid procedure.
A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.
Amino Acid Sequence↗
Relationship between differentiation and terminal mitosis: chick sensory and ciliary neurons differentiate after terminal mitosis of precursor cells, whereas sympathetic neurons continue to divide after differentiation.
A population of undifferentiated cells has been characterized during the early development of nodose and ciliary ganglia. This population is defined by the absence of surface markers specific for neurons (tetanus toxin receptor, Q211 antigen) and for glial cells (O4 antigen). These undifferentiated cell populations were isolated from the ganglia and were shown to contain neuronal precursor cells that were able to differentiate in vitro into neurons, as characterized by morphology and surface antigens. Undifferentiated cells were detected during the period of neuronal birth, indicating that dividing neuronal precursor cells do not express neuron-specific surface markers. This was directly shown by 3H-thymidine-labeling studies using nodose ganglia, ciliary ganglia, and dorsal root ganglia. In sympathetic ganglia, however, no undifferentiated neuronal precursor cells were detectable at developmental stages when sympathetic neurons are born. 3H-Thymidine injected during that stage at E7 was incorporated into cells expressing the neuronal markers tetanus toxin receptor and Q211 antigen. Quantitative fluorimetric determination of the DNA content of dissociated sympathetic ganglion cells demonstrated the presence of a population of Q211-positive sympathetic ganglion cells in the G2 phase of the cell cycle. E7 sympathetic ganglion cells expressing neuronal surface markers were also shown to be able to divide in vitro. We have concluded that the relationship between terminal mitosis and the onset of differentiation differs between ganglia of the chick peripheral nervous system: Sympathetic ganglion cells continue to divide after the acquisition of neuronal properties, whereas neuronal precursor cells from other autonomic and sensory ganglia start to differentiate after a terminal mitosis.
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Comparison between the time course of changes in nerve growth factor protein levels and those of its messenger RNA in the cultured rat iris.
In previous experiments, it has been demonstrated that, in rat irides in culture, a rapid increase in nerve growth factor (NGF) levels occurred (see Barth, E.-M., Korsching, S., and Thoenen, H. (1984) J. Cell Biol. 99, 839-843). We have now determined the levels of mRNANGF in rat irides as a function of time in culture as well. After an initial lag period of 2 h, mRNANGF levels were transiently increased, so that after 12 h, they had increased 35-fold with respect to zero time. In contrast, poly(A)+ RNA levels dropped to 55% of the zero time values within 5 h, recovered to 85% after 24 h, and remained constant until the end of the observation period. Total ribosomal RNA was found to remain constant, indicating that there was no nonspecific decline of overall metabolic function. Actinomycin D prevented the increase in mRNANGF without reducing the basic mRNANGF levels over a 5-h time period, indicating that the enhanced synthesis of NGF in the rat iris in culture is primarily mediated by an augmented production of mRNANGF. The increases of mRNANGF, cellular NGF, and NGF released into the medium were found to be strictly sequential. Monensin selectively abolished the increased production of mature NGF (see Barth et al.) but not of mRNANGF, suggesting that the processing of NGF precursor is prevented.
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Cholinergic denervation of the rat hippocampus by fimbrial transection leads to a transient accumulation of nerve growth factor (NGF) without change in mRNANGF content.
Unilateral stereotaxic fimbrial transection was used to disrupt the cholinergic pathway from the medial septum to the hippocampus in adult rats. The resulting alterations in hippocampal nerve growth factor (NGF) and mRNANGF levels were then analysed by two-site enzyme immunoassay and quantitative Northern blots, respectively. NGF levels did not change up to one week after lesion but had increased to 145% of contralateral control values by two weeks. In contrast, mRNANGF levels were not changed at this time point. Neither bilateral fimbrial transection, nor removal of the sympathetic innervation of hippocampal vasculature by excision of the superior cervical ganglia, led to more pronounced increases in NGF levels. These results are consistent with the concept that the magnocellular cholinergic neurons in the medial septum are constantly reducing hippocampal NGF levels by retrograde axonal transport.
Animals↗
Molecular cloning of bovine and chick nerve growth factor (NGF): delineation of conserved and unconserved domains and their relationship to the biological activity and antigenicity of NGF.
Previous experiments with purified mouse and bovine nerve growth factor (NGF) have shown that the biological activities of these two NGFs are identical, whereas the immunological cross-reactivity of antibodies produced against the two NGF molecules is very limited. This observation, together with the fact that antibodies to mouse NGF do not affect the development of sympathetic and sensory neurons in chick embryos, suggests that the domain of the NGF molecules responsible for the biological action has been highly conserved during evolution, whereas other domains determining the immunological properties were under less rigorous evolutionary constraint. The nucleotide sequences of bovine and chick NGF were determined from a cDNA clone prepared from mRNA of bovine seminal vesicles and from cloned chick genomic DNA, and the amino acid sequences deduced therefrom were compared with the available sequences of mouse and human NGF. All six cysteine residues were conserved in agreement with the previous finding that the biological activity of NGF is conformation-dependent requiring intact disulfide bridges. Amino acid changes are mainly confined to hydrophilic regions expected to be potential antigenic determinants, thus providing an explanation for the poor immunological cross-reactivities between the different NGFs. One single hydrophilic region is conserved in all NGFs and this region could be involved in the biological activity. The carboxy termini of bovine and chick NGF differ from that of mouse NGF, the changes in the amino acid sequences suggest that chick and bovine NGF are probably not processed by the gamma-subunit and that no 7S complex can be formed as in the mouse submandibular gland.
Amino Acid Sequence↗
Methyltransferase inhibitors block NGF-regulated survival and protein phosphorylation in sympathetic neurons.
Nerve growth factor (NGF) and elevated K+ concentrations (35 mM) support the survival of the same population of chick embryonic sympathetic neurons. We have used methyltransferase inhibitors, which block protein methylation in intact cells, to investigate the mechanism(s) by which NGF and high K+ exert their effects. Methyltransferase inhibitors selectively blocked NGF-but not high K+-mediated survival of neurons. The ability of neurons, plated on laminin, to respond rapidly to NGF with neurite outgrowth was used to demonstrate that the blockade of the effects of NGF by methyltransferase inhibitors was reversible. At the molecular level, we studied the rapid decrease in phosphorylation of p70, a 70-kd phosphoprotein of sympathetic neurons regulated by both NGF and high K+. Methyltransferase inhibitors blocked the decrease in p70 phosphorylation induced by NGF but not that by high K+. We conclude that the early molecular events of NGF-mediated neuronal survival differ from those of high K+-mediated neuronal survival in that they involve protein methylation, whereas at a later step, possibly at the level of protein phosphorylation, the two pathways leading to survival of sympathetic neurons converge.
Adenosine↗
Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture.
The purposes of the experiments reported is to provide an unambiguous demonstration that embryonic skeletal muscle contains factors that act directly on embryonic spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonic chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone had no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).
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Laminin increases both levels and activity of tyrosine hydroxylase in calf adrenal chromaffin cells.
We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of laminin can be blocked by antibodies directed against a fragment of the heparin-binding domain of the molecule, whereas antibodies directed against other fragments do not block the increase in tyrosine hydroxylase. Thus the laminin domain involved in enzyme regulation in chromaffin cells is apparently the same as that previously implicated in laminin's interactions with neurons to potentiate survival and stimulate neurite outgrowth (Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468). The increase in chromaffin cell tyrosine hydroxylase levels is preceded by an activation of the enzyme in which the Vmax (but not the Km) is altered. The effects of laminin appear to be developmentally regulated, since neither activation nor increased levels of tyrosine hydroxylase occur in adult adrenal chromaffin cells exposed to laminin.
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Selective induction of tyrosine hydroxylase by cell-cell contact in bovine adrenal chromaffin cells is mimicked by plasma membranes.
As a first step towards the identification and purification of the molecule(s) that are involved in cell contact-mediated tyrosine hydroxylase (TH) induction in cultures of bovine adrenal chromaffin cells, we have prepared plasma membranes (PM) from bovine adrenal medulla and tested their ability to mimick cell contact-mediated TH induction in low density chromaffin cultures. PM indeed induced TH in a manner similar to that observed in high density cultures. The maximal TH induction reached by PM corresponded to 69% of that of high density cultures, and half-maximal TH induction was obtained with 12 micrograms of PM per ml of medium. The induction of TH by PM was blocked by alpha-amanitin as observed in high density cultures. Since acetylcholinesterase was neither induced in high density nor in PM-treated low density cultures, an induction of TH as a result of a general increase in protein synthesis was excluded. The cell contact molecule(s) appear to be intrinsic membrane proteins. They were not removed by high or low salt extraction, but solubilized by 50 mM octylglucoside. They were resistant to 0.1% trypsin and heat denaturation but inactivated by 0.01% chymotrypsin. PM isolated from the adrenal cortex, kidney, and liver also induced TH in low density chromaffin cell cultures, although to a smaller extent than PM of the adrenal medulla. In contrast, muscle and erythrocyte PM were inactive. This shows that the cell contact molecule(s) are not restricted to the adrenal medulla, but are also present in some other but not all tissues.
Acetylcholinesterase↗
Brain-derived neurotrophic factor supports the survival of cultured rat retinal ganglion cells.
Brain-derived neurotrophic factor (BDNF) is a small, basic protein purified from the mammalian brain that has been shown previously to support the survival of cultured spinal sensory neurons (Barde et al., 1982). In current studies, BDNF was tested for its ability to support the survival of cultured CNS cells isolated from the perinatal rat retina. Both immunofluorescent labeling of Thy-1 and prior retrograde labeling with HRP were used as retinal ganglion cell markers in vitro. With embryonic day (E) 17 retinas, it was found that BDNF allowed the survival of a small subpopulation of neurons (about 7% of the cells plated at this age) identified by the immunofluorescent labeling of Thy-1. No detectable effects were seen when either the total number of cells or the number of tetanus toxin-positive neurons was measured. BDNF also had an effect on cultured neurons retrogradely labeled after HRP injections in the superior colliculi of neonatal rats. The BDNF-responsive population was therefore detected only in retinal cultures with specific markers and identified as consisting of retinal ganglion cells. These cells could be enriched about 80-fold by density gradient centrifugation, and purified ganglion cell cultures were shown to be responsive to BDNF. Whereas with E17 retinas, the number of surviving Thy-1 positive neurons could be kept constant for at least 4 d, the survival of postnatal neurons was only transiently increased by BDNF. We conclude that in the retina, BDNF affects only the survival of ganglion cells in vitro by a direct action on these cells. The results are discussed in terms of target-derived neurotrophic support during development.
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The response of chick sensory neurons to brain-derived neurotrophic factor.
To determine the spectrum of activity of brain-derived neurotrophic factor (BDNF) among first-order sensory neurons, explants of the nine distinct populations of sensory neurons from embryonic chicks of 3-14 d incubation (E3-E14) were grown in collagen gels with and without BDNF in the culture medium. The explants responded to BDNF with profuse neurite outgrowth and comprised those in which neurons are derived from neural crest (the dorsomedial part of the trigeminal ganglion, rostromedial part of the trigeminal mesencephalic nucleus, jugular ganglion, and lumbosacral dorsal root ganglia) and from epibranchial placodes (the ventrolateral part of the trigeminal ganglion and the geniculate, petrosal, and nodose ganglia). This response was first clearly observed in the ventrolateral trigeminal and nodose ganglia as early as E4, but did not appear until later in other explants. The dorsomedial trigeminal, jugular, and geniculate ganglia were the latest to develop a response, which was not apparent until E8. In all explants the response was maximal between E10 and E12, and there was a decline in the magnitude of the response from E12 to E14. Although explants of the vestibular ganglion failed to respond to BDNF, the survival and growth of vestibular neurons in dissociated neuron-enriched cultures were promoted by BDNF. To investigate whether all first-order sensory neurons respond to BDNF or whether BDNF-responsive neurons comprise a distinct subset, we studied the influence of BDNF and NGF on the survival and growth of the placode-derived and the neural crest-derived neurons of the trigeminal ganglion in dissociated neuron-enriched culture.(ABSTRACT TRUNCATED AT 250 WORDS)
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Developmental potential of quail dorsal root ganglion cells analyzed in vitro and in vivo.
The cell types present in quail dorsal root ganglia during early development were identified using markers for neurons, glial cells, and fibroblasts (Rohrer et al., 1985). Using the quail-chick transplantation technique, the potential of quail dorsal root ganglion cells to differentiate to adrenergic chromaffin cells, as identified by tyrosine hydroxylase immunostaining, was analyzed. A population of undifferentiated cells, which is present in quail dorsal root ganglia at embryonic day 5, was separated from neurons and glial cells. We show that this population contains cells that differentiate to adrenergic chromaffin cells after back-transplantation into 2-d-old chick embryos. A large proportion of these undifferentiated cells also differentiates to neurons in vitro. Precursors for adrenal chromaffin cells and for neurons are present in dorsal root ganglia in significant numbers only during early development.
Adrenal Glands↗
Neurotrophic factors.
In addition to nerve growth factor (NGF), many proteins present in soluble tissue extracts and in the extracellular matrix influence the survival and development of cultured neurons. The structure, synthesis, and mechanism of action of NGF as a neurotrophic factor are considered along with the experiments on the new putative trophic molecules.
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