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Biomedical subjects

H Thoenen

Publications and source records attributed to H Thoenen.

At least 109 records · Page 6Linked to original sources
PubMed1988

Nerve growth factor: cellular localization and regulation of synthesis.

1. The role of nerve growth factor (NGF) as a retrograde messenger between peripheral target tissues and innervating sympathetic and neural crest-derived sensory neurons is supported by the observations that (a) the interruption of retrograde axonal transport has the same effects as the neutralization of endogenous NGF by anti-NGF antibodies and (b) the close correlation between the density of innervation by fibers of NGF-responsive neurons and the levels of NGF and mRNANGF in their target organs. 2. In situ hybridization experiments have demonstrated that a great variety of cells in the projection field or NGF-responsive neurons is synthesizing NGF, among them epithelial cells, smooth muscle cells, fibroblasts, and Schwann cells. 3. The temporal correlation between the growth of trigeminal sensory fibers into the whisker pad of the mouse and the commencement of NGF synthesis initially suggested a causal relationship between these two events. However, in chick embryos rendered aneural by prior removal of the neural tube or the neural crest, it was shown that the onset of NGF synthesis in the periphery is independent of neurons, and is controlled by an endogenous "clock" whose regulatory mechanism remains to be established. 4. A comparison between NGF synthesis in the nonneuronal cells of the newborn rat sciatic nerve and that in the adult sciatic nerve after lesion provided evidence for the important regulatory role played by a secretory product of activated macrophages. The identity of this product is currently under investigation.

Animals↗
PubMed1988

Developmental changes of nerve growth factor levels in sympathetic ganglia and their target organs.

The predominant source of nerve growth factor (NGF) used by mature sympathetic neurons originates in their target organs (Heumann, R., Korsching, S., Scott, J., and Thoenen, H. (1984), EMBO J. 3, 3183-3189; Korsching, S., and Thoenen, H. (1985), J. Neurosci. 5, 1058-1061). We have determined the NGF content of two sympathetically innervated mouse organs, submandibular gland and heart ventricle, and of sympathetic ganglia from mouse and rat between embryonic Day 12 (E12) and adulthood. NGF levels were measured by a two-site enzyme immunassay (Korsching, S., and Thoenen, H. (1983), Proc. Natl. Acad. Sci. USA 80, 3513-3516). In heart ventricle and submandibular gland, NGF first became detectable around the time of initial innervation by sympathetic neurons (E12 and E13, respectively) and increased respectively 14- and 7-fold in the following 2 days, to reach adult levels already at E14 for heart ventricle (1.4 +/- 0.2 ng NGF/g wet wt). NGF in the superior cervical ganglion (SCG) was first detected at the same time as in its target organ, the submandibular gland. NGF content in the SCG then increased 6-fold during the next 2 days and continued to increase until the end of the third postnatal week, when adult levels were reached. Although the levels of NGF in the adult mouse submandibular gland are sexually dimorphic and six orders of magnitude higher than those in other sympathetic target organs, no sex difference in the NGF content was found in either developing submandibular gland or SCG until the end of the third postnatal week. Moreover, the steep NGF increase observed in the male submandibular gland after postnatal Day 18 (250-fold within the following 3 days and up to the 55,000-fold in the next 7 days) was not reflected in a corresponding increase in the NGF content of the male SCG. These data indicate that, in accordance with earlier findings (see Levi-Montalcini, R., and Angeletti, P. U. (1968), Physiol. Rev. 48, 534-569), SCG neurons do not have access to the large amounts of NGF synthesized during and after adolescence in the mouse submandibular gland. Our results support the concept that initial fiber outgrowth of sympathetic neurons is neither dependent on NGF nor mediated by it. The time course of NGF levels in the SCG is consistent with the concept that sympathetic neurons are provided with NGF by means of retrograde axonal transport from the innervated organs already early in development.

Aging↗
PubMed1988

The synthesis of nerve growth factor (NGF) in developing skin is independent of innervation.

The arrival of sensory fibers in developing mouse skin has been demonstrated to coincide precisely with the initiation of nerve growth factor (NGF) synthesis in the skin (Davies et al., 1987). This temporal correlation suggested that the arrival of sensory fibers might initiate NGF synthesis in their target tissues. Here we have eliminated the sensory and motor neurons projecting to the chick leg by the removal of the neural primordia in 3-day-old embryos. The levels of mRNA NGF of intact and denervated leg skin were identical, indicating that the developmental regulation of NGF synthesis in the skin of chick embryos is independent of its innervation.

Animals↗
PubMed1988

Regulation of tyrosine hydroxylase mRNA levels in rat pheochromocytoma PC12 cells by cell-cell contact.

It has previously been shown that cell density increases the specific enzyme activity of tyrosine hydroxylase (TH) in cultures of PC12 cells. The difference in TH activity was shown to be due to cell-cell contact rather than to diffusible factors released by the PC12 cells (C. Lucas, D. Edgar, and H. Thoenen (1979) Exp. Cell Res. 121, 79-86). We have extended these studies and demonstrated that TH is regulated at the transcriptional level. Cells were harvested from confluent high-density cultures and replated at either low (2 x 10(4) cells/cm2) or high density (5 x 10(5) cells/cm2). At low density, the mRNATH and TH enzyme activity decreased to low levels within several hours. The decrease in TH activity resulting from the loss of cell-cell contact appears to be an active process, as it occurs much more rapidly than would be expected from the turnover rate of the protein in cells cultured continuously at high density. In contrast to low-density cultures the mRNATH and TH activity levels in replated high-density cultures decreased only slightly and then increased four fold at the mRNATH and five fold at the TH activity levels as compared to low-density cultures 2 days after replating. The increase in mRNATH preceded the increase in TH activity by 1 day. Since alpha-amanitin inhibited the increase in TH levels, we conclude that cell-cell contact regulates TH in PC12 cells at the transcriptional level.

Adrenal Medulla↗
PubMed1988

Structural requirements for the stimulation of neurite outgrowth by two variants of laminin and their inhibition by antibodies.

Laminin derived from the Engelbreth-Holm-Swarm (EHS) tumor and a lamininlike molecule synthesized by RN22 Schwannoma cells both stimulate rapid neurite outgrowth, consistent with a common neurite-promoting site. However, antilaminin antisera can only inhibit the activity of the EHS laminin. The blocking antibodies in such sera are directed against the terminal heparin-binding domain of the laminin long arm (Edgar, D., R. Timpl, and H. Thoenen. 1984. EMBO [Eur. Mol. Biol. Organ.] J. 3: 1463-1468). These epitopes are demonstrated by immunoblotting to be part of the A chain and to be absent in RN22 laminin, showing (through metabolic labeling) that the cells synthesized little if any 440-kD A chain. This indicates that the antibody inhibition was probably due to steric hindrance, a common neurite-promoting site, apparently not being antigenic in native molecules. Antibodies raised against a 25-kD proteolytic fragment derived from the long arm of laminin were then used as probes to identify other potential neurite-promoting structures. Although these antibodies do not cross-react with native laminin, they recognized the B chains of denatured EHS and RN22 molecules on immunoblots. The antibodies also bound to the large proteolytic fragment, derived from the long arm of laminin that contains the neurite-promoting site, thus inhibiting its activity. Taken together, these results point to the localization of normally nonantigenic, defined, B chain sequences within or close to the neurite-promoting site of laminin.

Animals↗
PubMed1988

Predicted amino acid sequence of bovine tyrosine hydroxylase and its similarity to tyrosine hydroxylases from other species.

The previously obtained cDNAs coding for bovine tyrosine hydroxylase (TH) mRNA (mRNATH) were further analyzed, and the entire nucleotide sequence was determined. The mRNATH consists of 1,706 nucleotides with an open reading frame for 491 amino acids, which corresponds to a calculated molecular weight of 55,011. The predicted amino acid sequence of bovine TH is compared with that of rat TH and shows a similarity of 66% in the amino terminal (amino acids 1-157) and 91% in the carboxy terminal (amino acids 158-491) region of the TH protein molecule. The carboxy terminal region has been shown to make up the catalytic site of TH and, therefore, is conserved to a greater extent in different species than the amino terminal region, which has been shown to be mainly responsible for the regulation of the catalytic activity of TH. Three of the four serine residues (Ser 8, 19, and 40) that have been shown to be substrates for various protein kinases in rat TH are also present in bovine TH and are located near the amino terminal end of the molecule. The amino acids from position 60 to position 66 of rat TH are not present in bovine TH, resulting in the absence of a predicted hydrophobic region as compared with rat TH. This difference could result in an altered degree of regulation by posttranslational phosphorylation and also association to cell organelle membranes of bovine TH as compared with rat TH.

Amino Acid Sequence↗
PubMed1988

Antibodies against mouse nerve growth factor interfere in vivo with the development of avian sensory and sympathetic neurones.

The monoclonal antibody 27/21 directed against mouse nerve growth factor (NGF) interferes in vivo with the survival of sensory dorsal root ganglion (DRG) neurones during the development of the quail embryo: the number of DRG neurones at embryonic day 11 (E11) was reduced by about 30% in embryos treated with the antibody between E3 and E11. Neurone numbers in the nodose ganglion were not affected. The effect of NGF antibodies on sympathetic neurones was assessed by determining the levels of the adrenergic marker enzyme tyrosine hydroxylase. Both total tyrosine hydroxylase activity and protein levels in sympathetic chains were reduced by about 30% in embryos treated with 27/21 antibody but not in embryos treated with a control antibody. The 27/21 antibody cross-reacts with chick NGF-like activity as shown in vitro by the ability of the antibody to partially block the survival activity of chick-embryo-fibroblast-conditioned medium for E9 chick DRG neurones.

Animals↗
PubMed1988

Is activation of the Na+K+ pump necessary for NGF-mediated neuronal survival?

The ability of nerve growth factor to cause rapid activation of the Na+K+ pump of its responsive cells was examined by measuring the uptake of 86Rb+. A significant increase in 86Rb+ uptake in E8 chick dorsal root ganglion sensory neurons after NGF treatment was seen only if the cells had been damaged during the preparation procedure. Such damaged cells could not survive in culture in the presence of NGF, and undamaged cells that did survive in response to NGF exhibited no increased 86Rb+ uptake rate. Furthermore, cultured calf adrenal medullary cells did not show an increase in 86Rb+ uptake after treatment with NGF, although these cells respond to NGF with an increased synthesis of catecholaminergic enzymes. These results are incompatible with the hypothesis that the mechanism of action of NGF that promotes neuronal survival and enzyme induction results from an initial stimulation of the Na+K+ pump.

Animals↗
PubMed1987

Influence of cell-cell contact on levels of tyrosine hydroxylase in cultured bovine adrenal chromaffin cells.

We have studied the regulation of tyrosine hydroxylase by cell-cell contact in primary cultures of bovine adrenal chromaffin cells. Preparation of dissociated chromaffin cells from bovine adrenal medullae or the harvesting of cultured cells resulted in a rapid decrease of the specific mRNA(TH) (defined as the amount of mRNA(TH) (where TH represents tyrosine hydroxylase) per microgram of total RNA). The decrease in mRNA(TH) levels appears to be stimulated by the loss of cell contact, as it occurs much more rapidly than would be expected from the turnover rate of mRNA(TH) in cultured cells. Similarly an enhanced rate of tyrosine hydroxylase enzyme degradation was observed in chromaffin cells when brought into low contact cultures. In contrast to the decrease in mRNA(TH) levels, however, the decrease in tyrosine hydroxylase enzyme levels was not so rapid and could be prevented in high contact cultures. The mRNA(TH) increased 4-fold in high contact cultures relative to dissociated cells within 1 day after plating. Similarly the rate of synthesis of tyrosine hydroxylase enzyme molecules was maximal after 1 day, although the increase in the absolute amount of tyrosine hydroxylase occurred only slowly. The increase in specific mRNA(TH) by cell contact was inhibited by alpha-amanitin, indicating that cell contact evokes an increase in tyrosine hydroxylase levels by increasing the transcription of mRNA(TH), in addition to inhibiting the degradation of tyrosine hydroxylase molecules.

Adrenal Medulla↗
PubMed1987

Neurotrophic factors and neuronal death.

The well-documented physiological role of nerve growth factor (NGF) in peripheral sympathetic and neural-crest-derived sensory neurons in vivo has its exact counterpart in vitro. This provided the conceptual basis for developing in vitro analytical procedures for the purification of new neurotrophic molecules. The experimental approaches used are discussed in the context of the purification of new neurotrophic factors, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF). The importance of the modulatory role played by extracellular matrix molecules, in particular laminin, on both NGF-mediated and BDNF-mediated survival effects is also delineated. BDNF is a very basic (pI approximately 10) molecule of about 12 kDa, having physico-chemical characteristics close to those of the monomer of NGF. However, the spectrum of its biological actions is distinctly different from that of NGF. In particular, BDNF supports the survival of retinal ganglion cells and placode-derived peripheral sensory neurons which are not supported by NGF. The trophic supply of primary sensory neurons projecting to both the central nervous system and the periphery is discussed. It is hypothesized that sensory neurons receive limited quantities of neurotrophic molecules from both peripheral and central axons, a mechanism ensuring the survival of neurons adequately connected with both peripheral and central targets.

Animals↗
PubMed1987

In vivo effect of brain-derived neurotrophic factor on the survival of developing dorsal root ganglion cells.

Implantation of silastic membranes between neural tube and somites at somitic levels 20-24 in 30-somite-stage chick embryos results in separation of early migrated neural crest cells of the dorsal root ganglion (DRG) anlage from the neural tube and their death within a few hours [Kalcheim and Le Douarin, (1986) Dev. Biol., 116, 451-460]. The in vivo effects of brain-derived neutrotrophic factor (BNDF) on survival of HNK-1 immunoreactive DRG cells separated from the tube were examined by implantation of laminin-treated silastic membranes (controls) or BDNF/laminin-treated membranes. In the presence of BDNF/laminin-treated membranes, 20/25 grafted embryos fixed 10 h after implantation, contained many rescued cells on the operated side. In contrast, only a few rescued cells on the operated side. In contrast, only a few rescued cells were observed in sections on the operated in 2/11 embryos implanted with laminin-treated silastic membranes, and no rescued cells at all could be detected in embryos implanted with NGF/laminin-treated (seven embryos) or untreated silastic membranes (12 embryos). The data presented support the hypothesis that early survival and differentiation of neural crest-derived sensory cells depend on central nervous system-derived factor(s). Moreover, this is the first evidence for the in vivo activity of BDNF on survival of developing DRG cells.

Animals↗
PubMed1987

Cellular localization of nerve growth factor synthesis by in situ hybridization.

A very sensitive and specific method for in situ hybridization has been developed. This method detects low copy numbers of mRNA(NGF) transcripts in both tissue sections and cultured cells using 35S-labelled cRNA and oligonucleotide probes. In order to reduce the high nonspecific background occurring with 35S-labelled probes, prehybridization in the presence of non-labelled thio alpha UTP at pH 5.5 proved to be essential, together with a series of additional changes in the standard procedures for in situ hybridization. With this improved method it was possible to demonstrate that in tissues densely innervated by sensory (whisker pad) or both sympathetic and sensory (iris) fibers, NGF is synthesized not only by Schwann cells ensheathing these fibers, but also--and even to a much larger extent--by the target cells of the sensory and sympathetic neurons, i.e. epithelial cells, smooth muscle cells and fibroblasts. Moreover, in the sciatic nerve of newborn rats (where the mRNA(NGF) levels are 15 X higher than in adults) it was demonstrated that all Schwann cells have the capacity to express mRNA(NGF), not just those ensheathing the axons of NGF-responsive neurons.

Animals↗
PubMed1987

Developmental changes of nerve growth factor and its mRNA in the rat hippocampus: comparison with choline acetyltransferase.

Previous experiments have demonstrated that in the septo-hippocampal system choline acetyltransferase (ChAT) is induced by nerve growth factor (NGF) (Gnahn et al. (1983) Dev. Brain Res. 9, 45-52) and that hippocampal NGF and mRNANGF levels are correlated with the density of cholinergic innervation (Korsching et al. (1985) EMBO J. 4, 1389-1393). In the present investigation we have compared the developmental changes of ChAT, NGF, and mRNANGF levels in this system. During the postnatal development of the hippocampus the time courses of NGF and ChAT were well correlated including the most rapid increase between P12 and P14. This increase in hippocampal NGF was preceded by a corresponding increase in mRNANGF. The developmental changes in hippocampal NGF levels were also closely reflected by corresponding changes in the septum. This, together with previous observations (Korsching et al., 1985) that the adult septum, in spite of relatively high NGF levels, does not contain measurable quantities of mRNANGF, suggests that the NGF levels in the septum are determined by the quantity of NGF transported retrogradely from the field of innervation rather than by local synthesis. During the prenatal period hippocampal NGF levels were relatively high, whereas the mRNANGF was below the level of detection. Since the ingrowth of septal fibers, and with that also the removal of NGF by retrograde transport, begins around birth, the relatively high prenatal NGF levels probably result from an accumulation produced by a small copy number of mRNANGF prior to the removal of NGF by retrograde axonal transport. It is concluded that the correlation of the developmental changes in NGF and mRNANGF with the ChAT activity in the hippocampus further supports the concept of a physiological role of NGF in the central nervous system.

Animals↗
PubMed1987

Distinct neurotrophic factors from skeletal muscle and the central nervous system interact synergistically to support the survival of cultured embryonic spinal motor neurons.

Motor neurons isolated from 6-day-old embryonic chick spinal cords require muscle extract for survival in culture; however, it was found, that some motor neurons, identified by retrograde labeling with rhodamine, will survive in mixed spinal cell cultures in the absence of the extract. The motor neuron survival-promoting activity produced by spinal cells is soluble and differs from the factor present in muscle extract, the two activities acting in a synergistic manner: the spinal cell activity potentiated that of muscle to decrease its ED50 by an order of magnitude, the motor neuronal survival (30%) seen in the presence of both factors being more than the sum of their individual activities. This synergism was shown to be restricted to the action of the spinal cell factor on motor neurons, no effect of the factor being noted with sympathetic neurons. As a series of defined growth and survival factors present in the central nervous system (nerve growth factor, brain-derived neurotrophic factor, acidic and basic fibroblast growth factors) had no effect on motor neuron survival, we conclude that the molecule responsible for the motor neuron survival-promoting activity of the spinal cells is a previously undefined factor.

Animals↗
PubMed1987

High K+-mediated survival of spinal sensory neurons depends on developmental stage.

Elevated concentrations of K+ (35 mM) have previously been shown to support the survival of most embryonic chick sympathetic neurons in vitro (Wakade et al., Exp cell res 144 (1983) 377, [23]) and to be interchangeable with nerve growth factor (NGF) as a survival-promoting agent for these cells (Wakade & Thoenen, Neurosci lett 45 (1984) 71 [21]). In the present study, we show that dorsal root ganglion (DRG) neurons from embryonic day 6 do not survive in the presence of high K+, although both NGF and brain-derived neurotrophic factor (BDNF) each support the survival of more than 50% of the cells at this developmental stage. At E6, high K+ appears to have a cytotoxic effect on BDNF-dependent neurons, and there is also considerable inhibition of neurite outgrowth. At a later developmental stage (E12), high K+ supports the survival of about 40% of DRG cells. This subpopulation of neurons is distinct from that supported by NGF (as evidenced by the additivity of these two agents), but partially overlaps with that supported by BDNF (i.e., the two agents are less than additive). At E12, only approx. 20% of the cells can be supported by either NGF or BDNF, with the rest depending exclusively on one or the other of these factors. This is in contrast to the situation at E6, where there is considerable overlap between NGF- and BDNF-dependent populations.

Animals↗