Biomedical subjects

T Ebendal

Publications and source records attributed to T Ebendal.

At least 55 records · Page 3Linked to original sources

PubMed1996

Design, synthesis, tandem mass spectrometric sequencing and biological activity of NGF mimetics.

Nine low molecular weight nerve growth factor (NGF)-like peptides have been designed to mimic the putative receptor-binding epitope of NGF defined by two beta-hairpin loops. Eight different spacers were used as variable links between the beta-loop amino acid residues, which from mutagenesis experiments were found to play an important role in the biological activity of NGF. These spacers were amino acids, natural or non-natural, differing in length (5-13 A) and polarity. The peptides were synthesized via the Fmoc solid-phase peptide synthesis and purified by reversed-phase HPLC. Their primary sequences were analyzed by a combination of automated Edman degradation and mass spectrometry. The peptides were tested using two different biological assays, the fibre outgrowth from chick embryonic sympathetic ganglia and the PC12 cell differentiation assay. Weak antagonistic effects could be observed for some peptides.

Adrenal Medulla↗

PubMed1995

Expression of activin receptors type I and II only partially overlaps in the nervous system.

In situ hybridization was used to localize mRNA encoding two cooperative serine/threonine kinase receptors. Activin receptor IIA mRNA in the adult rat brain is highly expressed in the dentate gyrus, in the pyramidal neurones of CA3 and CA1, in the entorhinal cortex, in the cortical amygdaloid nucleus and in the amygdalohippocampal area. In the E16 rat embryo, labelling was found in the dorsal root ganglion neurones and in the spinal cord. Activin type I receptor (ALK-2/R-1) labelling was also localized to the hippocampal formation but with an even distribution over the granular cells of the dentate gyrus, the CA3 and CA1 pyramidal neurones, while no labelling was found in the entorhinal or amygdaloid areas. In the E16 rat no neuronal labelling was found. The incomplete overlap of these two expression patterns suggests that these receptors may locally have other partners for forming signalling receptor complexes.

Activin Receptors↗

PubMed1995

Neurotrophin receptor expression during development of the chick spinal sensory ganglion.

The expression of mRNA for the trk neurotrophin receptors was studied in developing chicken dorsal root ganglion neurones using in situ hybridization histochemistry. trkC mRNA is expressed first, followed by trkB mRNA and finally trkA mRNA. The expression of each receptor begins very early during neurogenesis, and although initially quite widespread throughout the ganglion, the proportion of neurones expressing each receptor reduces as development proceeds. Expression patterns for each receptor become specifically restricted within the ganglion during this time, but during subsequent development the neurones migrate to their final site within the ganglion. From an assessment of the ganglion's ability to respond to neurotrophins in vitro, and from the results of earlier studies, both on the expression of receptors and gene targeting experiments, it is apparent that DRG neurones are dependent on these factors at very early stages of development and not, as previously thought, only after target innervation.

Animals↗

PubMed1995

Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.

The recently cloned, distant member of the transforming growth factor beta (TGF-beta) family, glial cell line-derived neurotrophic factor (GDNF), has potent trophic actions on fetal mesencephalic dopamine neurons. GDNF also has protective and restorative activity on adult mesencephalic dopaminergic neurons and potently protects motoneurons from axotomy-induced cell death. However, evidence for a role for endogenous GDNF as a target-derived trophic factor in adult midbrain dopaminergic circuits requires documentation of specific transport from the sites of synthesis in the target areas to the nerve cell bodies themselves. Here, we demonstrate that GDNF is retrogradely transported by mesencephalic dopamine neurons of the nigrostriatal pathway. The pattern of retrograde transport following intrastriatal injections indicates that there may be subpopulations of neurons that are GDNF responsive. Retrograde axonal transport of biologically active 125I-labeled GDNF was inhibited by an excess of unlabeled GDNF but not by an excess of cytochrome c. Specificity was further documented by demonstrating that another TGF-beta family member, TGF-beta 1, did not appear to affect retrograde transport. Retrograde transport was also demonstrated by immunohistochemistry by using intrastriatal injections of unlabeled GDNF. GDNF immunoreactivity was found specifically in dopamine nerve cell bodies of the substantia nigra pars compacta distributed in granules in the soma and proximal dendrites. Our data implicate a specific receptor-mediated uptake mechanism operating in the adult. Taken together, the present findings suggest that GDNF acts endogenously as a target-derived physiological survival/maintenance factor for dopaminergic neurons.

Animals↗

PubMed1995

Monitoring release of neurotrophic activity in the brains of awake rats.

Intracerebral microdialysis of awake rats was used to monitor the possible release of neurotrophic factors from brain cells in response to injury and excitation. Perfusates were tested with ganglia bioassays and enzyme immunoassay. Trophic activity was released after implantation of the microdialysis probe into the hippocampus but not into the striatum, as assessed by increased nerve fiber outgrowth from Remak's ganglion. Kainic acid treatment significantly increased the release of trophic activity from hippocampal sites. These findings suggest that the brain responds to mechanical injury as well as to certain excitatory stimuli by regional extracellular release of neurotrophic activity that is not identical to the actions of known neurotrophic factors.

Animals↗

PubMed1995

The levels of neurotrophin-3 protein in the rat brain determined by enzyme immunoassay show a pattern distinct from nerve growth factor.

Using the monoclonal antibody 3W3 a two-site enzyme immunoassay (EIA) for neurotrophin-3 (NT-3) was established. The assay was sensitive to levels of NT-3 below 10 pg/ml and does not recognize nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF). In the adult rat brain, highest levels of NT-3 (1-1.5 ng/g tissue) were found in hippocampus and piriform cortex. Also the cerebellum contained detectable NT-3 protein. In the postnatal (P1 and P5) rat brain very high levels of NT-3 (7-9 ng/g) were found in the cingulate cortex consistent with previous observations of a local, transient NT-3 mRNA expression. NT-3 was also found prenatally in hippocampus, telencephalon and cerebellum. The EIA shows that the pattern of NT-3 protein in brain tissues is distinct from that of NGF.

Aging↗

PubMed1995

The effect of mercury vapour on cholinergic neurons in the fetal brain: studies on the expression of nerve growth factor and its low- and high-affinity receptors.

The effects of mercury vapour on the production of nerve growth factor during development have been examined. Pregnant rats were exposed to two different concentrations of mercury vapour during either embryonic days E6-E11 (early) or E13-E18 (late) in pregnancy, increasing the postnatal concentration of mercury in the brain from 1 ng/g tissue to 4 ng/g tissue (low-dose group) or 11 ng/g (high-dose group). The effect of this exposure in offspring was determined by looking at the NGF concentration at postnatal days 21 and 60 and comparing these levels to age-matched controls from sham-treated mothers. Changes in the expression of mRNA encoding NGF, the low- and high-affinity receptors for NGF (p75 and p140 trk, respectively) and choline acetyltransferase (ChAT) were also determined. When rats were exposed to high levels of mercury vapour during early embryonic development there was a significant (62%) increase in hippocampal NGF levels at P21 accompanied by a 50% decrease of NGF in the basal forebrain. The expression of NGF mRNA was found to be unaltered in the dentate gyrus. The expression of p75 mRNA was significantly decreased to 39% of control levels in the diagonal band of Broca (DB) and to approximately 50% in the medial septal nucleus (MS) whereas no alterations in the level of trk mRNA expression were detectable in the basal forebrain. ChAT mRNA was slightly decreased in the DB and MS, significantly in the striatum. These findings suggest that low levels of prenatal mercury vapour exposure can alter the levels of the NGF and its receptors, indicating neuronal damage and disturbed trophic regulations during development.

Animals↗

PubMed1995

Glial cell line-derived neurotrophic factor stimulates fiber formation and survival in cultured neurons from peripheral autonomic ganglia.

Human recombinant glial cell line-derived neurotrophic factor (GDNF) was tested for its ability to stimulate fiber formation and neuron survival in primary cultures of peripheral ganglia dissected from the chicken embryo. GDNF, first characterized by its actions on central nervous system (CNS) neurons, had a marked stimulatory effect on fiber outgrowth in sympathetic and ciliary ganglia. Weaker responses were evoked in sensory spinal and nodose ganglia and in the ganglion of Remak. In addition, survival of neurons from the sympathetic and ciliary ganglia was stimulated by GDNF at 50 ng/ml. The effects were not mimicked by the distant but related protein transforming growth factor beta 1 (TGF beta 1). The profile of neurons stimulated by GDNF is also distinct from the patterns of stimulation shown by nerve growth factor (NGF), stimulating strongly sympathetic but not ciliary ganglia, and ciliary neurotrophic factor (CNTF), stimulating mainly the ciliary ganglion. Moreover, using in situ hybridization histochemistry, GDNF was demonstrated to be present in the pineal gland in the newborn rat, a target organ for sympathetic innervation. The present results suggest that GDNF is likely to act upon receptors present in several autonomic and sensory neuronal populations. GDNF may serve to support fiber outgrowth and cell survival in peripheral ganglia, adding yet one more trophic factor to the list of specific proteins controlling development and maintenance of the peripheral nervous system.

Animals↗

PubMed1995

Developmentally regulated expression of mRNA for neurotrophin high-affinity (trk) receptors within chick trigeminal sensory neurons.

To investigate the distribution of neurons within the developing trigeminal sensory system which express mRNA for each of the three known high-affinity neurotrophin receptors (trk, trkB and trkC), we have performed in situ hybridization histochemistry on serial sections through the trigeminal ganglion and trigeminal mesencephalic nucleus at various ages of development using specific antisense oligonucleotide probes. We show that trkC mRNA is first expressed in the chicken embryo at stage 13, in presumptive neurons prior to the formation of the ganglion, that trkB mRNA labelling is initially observed within peripheral neurons slightly later, at stage 19, and that trk mRNA expression is not detectable until around embryonic day 3.5 (stage 21/22). The neurons which exhibit mRNA labelling for each of the high-affinity receptors occupy discrete regions within the ganglion, indicating that the ganglion comprises distinct neuronal subpopulations, each of which has a different capacity to respond to the different neurotrophins. Neurons which express trk mRNA are confined to the proximal region of the ganglion, whereas those which express trkB mRNA and trkC mRNA are located in two distinct regions within the distal aspect and also within the trigeminal mesencephalic nucleus. From the estimation of the number of neurons which exhibit labelling between embryonic days 9 and 18, we determined that the expression of mRNA for the high-affinity receptors changes during embryonic development of the ganglion. This is consistent with the observed differences in the response to neurotrophins in vitro.

Age Factors↗

PubMed1995

Retinoic acid-mediated increase in TrkA expression is sufficient to elicit NGF-dependent survival of sympathetic neurons.

Sympathetic neurons depend on the classical neurotrophin NGF for survival by the time they innervate their targets, but the mechanisms controlling the onset of NGF responsiveness in developing neuroblasts have not been defined. Immature chick sympathetic neurons are unresponsive to NGF, but express low mRNA levels of the high-affinity NGF receptor trkA. Treatment with retinoic acid (RA) leads to increased levels of both trkA mRNA and protein, a response mediated through retinoic acid receptor alpha (RAR alpha). Ectopic expression of trkA in these cells results in the ability to survive with NGF, suggesting that RA-induced trkA expression is sufficient to elicit NGF-dependent survival. Our data establish a mechanism controlling NGF responsiveness and implicate a function for RA at defined late stages of neuron development.

Animals↗

PubMed1995

Effects of transferrin receptor antibody-NGF conjugate on young and aged septal transplants in oculo.

The purpose of this study was to investigate the effects of nerve growth factor (NGF) conjugated to a monoclonal transferrin receptor antibody (OX-26) on septal transplants in oculo. Three different doses of OX-26-NGF conjugate (0.3, 3, and 50 micrograms/injection) were injected into the tail vein of young adult hosts 2, 4, and 6 weeks following intraocular transplantation of fetal forebrain tissue containing septal nuclei. Intravenous injections of OX-26 alone, NGF alone, and saline served as controls. An increase in intraocular tissue growth, as well as an increase in the intensity of immunoreactivity for p75 receptors and acetylcholinesterase, was observed following peripheral OX-26-NGF administration at the two highest doses tested. In addition, aged host rats with 16-month-old intraocular septal grafts were injected intravenously with OX-26 or OX-26-NGF (10 micrograms NGF/injection) every 2 weeks until the transplants were 24 months old. The intensity of choline acetyltransferase-like (ChAT) staining appeared to be greater and the cell bodies were larger with more processes in aged transplants in hosts treated with the OX-26-NGF conjugate than in aged OX-26-treated subjects. The present results suggest that peripheral OX-26-NGF can deliver biologically active NGF across the blood-brain barrier and have dose-dependent positive effects on both aged and developing cholinergic neurons in septal transplants.

Acetylcholinesterase↗

PubMed1995

Specific lesions in the extrapyramidal system of the rat brain induced by 3-nitropropionic acid (3-NPA).

The irreversible mitochondrial toxin 3-nitropropionic acid (3-NPA) is a specific inhibitor of succinate dehydrogenase. We performed stereotaxic unilateral injections of 3-NPA into the nigrostriatal dopaminergic pathway in rats in order to examine its specific effects on the dopamine system. The 3-NPA-treated rats displayed unidirectional apomorphineinduced rotations, suggesting that 3-NPA selectively damages dopaminergic neurons when injected into the nigrostriatal pathway. In situ hybridization 7 weeks postinjection indicated a decrease in tyrosine hydroxylase (TH) mRNA to 30% of the noninjected side in the substantia nigra pars compacta (P < 0.05) and decreased to 62% of the noninjected side in the ventral tegmental area (VTA) (nonsignificant) of 3-NPA-lesioned rats. The number of TH mRNA positive cells showed statistically significant decreases in substantia nigra and VTA (P < 0.001) within the lesioned side. In contrast, expression of mRNAs encoding choline acetyltransferase, p75 low-affinity NGF receptor, neurotrophin tyrosine kinase receptors Trk and TrkB, and brain-derived neurotrophic factor showed neuronal sparing in several other regions of the brain. The results suggest that the nigrostriatal dopaminergic system might be selectively vulnerable to 3-NPA and demonstrate that it is possible to employ 3-NPA in a model of partial lesion of the nigrostriatal dopaminergic system resembling early stages of Parkinson's disease.

Animals↗

PubMed1995

In vitro toxicity of methyl mercury: effects on nerve growth factor (NGF)-responsive neurons and on NGF synthesis in fibroblasts.

The effect of methyl mercury chloride (MeHgCl) on the chick sympathetic and sensory dorsal root ganglia was studied in a biological in vitro assay. These cultures were not affected by the addition of MeHg up to a concentration of 2 microM. However, after an addition of 4-5 microM MeHg the capability of the neurons to respond to added nerve growth factor (NGF) was completely inhibited. The effect of MeHg was also examined in a fibroblast cell line, mouse 3T3 cells. After the addition of mercury to the culture medium at concentrations as low as 0.1 microM, an elevated production of the NGF protein was observed. However, NGF mRNA measured in the individual fibroblast cells by in situ hybridization was found to be reduced to about 80% of the control in the low level at day 2 of exposure. These results suggest that the release of NGF is actively enhanced from the 3T3 cells by addition of low levels of mercury. The results thus show that MeHg at low to moderate concentrations has adverse effects on NGF responses in cultured neurons and moreover alter levels of NGF production in cells, suggestive of mechanisms for mercury toxicity in the developing nervous system.

3T3 Cells↗

PubMed1995

Noradrenergic neurons in the locus coeruleus of birds express TrkA, transport NGF, and respond to NGF.

The chicken locus coeruleus contains a population of noradrenergic neurons which express the neurotrophin receptor p75 (von Bartheld and Bothwell, 1992). To determine which neurotrophin may regulate the development of noradrenergic neurons in the chicken locus coeruleus, expression of trk receptors, retrograde transport of neurotrophins, and responses to NGF were examined. P75-expressing noradrenergic neurons were found to project to the basal forebrain. They transport radio-iodinated NGF after injections into this target. The retrograde transport of NGF is specific to the noradrenergic neuronal population as evidenced by double labeling with antibodies against dopamine-beta-hydroxylase. The same neuronal population expresses trkA receptor mRNA. The size of noradrenergic neurons in the locus coeruleus proper, but not in the nucleus subcoeruleus, is significantly increased after injections of NGF into the telencephalon, consistent with the hypothesis that target-derived NGF provides trophic support. Noradrenergic coeruleus neurons are rescued from toxic effects of 6-hydroxydopamine injected into the telencephalon when NGF is injected into the midbrain. NGF has no rescue effect when it is coinjected with 6-hydroxy-dopamine into the telencephalon. In explant or dissociated cultures, noradrenergic coeruleus neurons do not respond to elevated levels of NGF with increased neurite outgrowth. Taken together, these results suggest that NGF plays a role in the development and maintenance of noradrenergic coeruleus neurons in the chick brain. The data also support our previous conclusion that major species differences exist between birds (chicken) and mammals with regard to trophic regulation of presumptive homologous neuronal populations.

Animals↗

PubMed1995

Neurotrophins and their receptors in chicken neuronal development.

A review on current studies of chicken neurotrophins and their receptors is given. Chicken NGF, BDNF and NT-3 have been cloned and sequences have been used to synthesize oligonucleotides for specific localization of expression during development. Also, chicken TrkA, TrkB and TrkC have been cloned, sequenced and studied by in situ hybridization. Recombinant NT-3 was applied to chicken ganglia at different developmental stages to examine acquirement of responsiveness to NT-3 compared to NGF. Phylogenetic analyses of the chicken neurotrophins and Trk receptors were carried out based on parsimony. Finally, some data on apoptosis in chicken embryo sympathetic ganglia are presented.

Animals↗

PubMed1994

Neurotrophin-3 acquires NGF-like activity after exchange to five NGF amino acid residues: molecular analysis of the sites in NGF mediating the specific interaction with the NGF high affinity receptor.

Despite the large sequence similarity around 55-60% among the known NGF-related neurotrophins, the members display different activities on different subset of neurons. Recent studies have shown that the various neurotrophins are ligands with high affinity to different receptors of the Trk family of tyrosine kinase receptors. We wanted to elucidate what specific parts of NGF replaced in neurotrophin-3 (NT-3) would result in NGF-like receptor binding and biological activity. By studying evolutionarily conserved amino acid sequences not shared by NT-3 and NGF and excluding parts which have been examined in earlier work with NGF and BDNF chimeras as well as taking advantage of the crystallographic data available for NGF, we decided to exchange three specific blocks of two or three amino acids in the human NT-3 backbone for the corresponding residues in NGF. The NGF residues Asn-Ile-Asn (43-45), Val-Phe (48,49) and Gln-Ala-Ala (96-98) were combined in pairs and are all shown to contribute NGF-like activity in the context of NT-3. The most efficient NGF-like transformation was obtained by the exchange of Pro-Val and Leu-Val-Gly in NT-3 to the NGF residues Val-Phe and Gln-Ala-Ala. This mutant reached 90% NGF activity, based on survival of sympathetic neurons, stimulation of fibre outgrowth from sympathetic ganglia, the ability to block high affinity NGF binding to PC12 cells and phosphorylation of gp140trk. Thus, the three mutants with paired combinations of the NGF residues as well as the NT-3 housing all three blocks of NGF residues were able to mimic NGF activity. This activity is gained, although the mutated neurotrophin proteins do not lose the original NT-3 activity as ascertained by the stimulation of neurite outgrowth from the Remak ganglion. The three mutated sites are situated in two beta-loops at one end of the NGF molecule, forming a cleft that could specifically interact with high affinity to the signalling NGF receptor gp140trk.

Amino Acid Sequence↗

PubMed1994

Unilateral stimulation or removal of rat vibrissae: analysis of nerve growth factor and tyrosine hydroxylase mRNA in the brain.

Previous work has shown that unilateral manipulation of vibrissae in the rat can lead to behavioral asymmetries and to neuronal changes in the basal ganglia: in brief, vibrissae stimulation led to increases in neostriatal dopamine release, whereas unilateral removal of vibrissae led to asymmetries in striatal afferents and to bilateral changes in mesencephalic dopamine mechanisms which were related to the occurrence of behavioral asymmetries and the later recovery therefrom. In the present study, the analysis of neuronal mechanisms possibly affected by vibrissae manipulation was extended to the nerve growth factor and the expression of tyrosine hydroxylase mRNA. Unilateral stimulation or removal of the vibrissae did not lead to significant changes in tissue levels of nerve growth factor in the neostriatum, parietal cortex (including the barrel cortex) or the hippocampus. In contrast, tyrosine hydroxylase mRNA in the substantia nigra and ventral tegmental area was affected by vibrissae removal but not by stimulation, as a bilateral increase in labeling was observed on the level of individual neurons. This effect was only observed in animals tested 4 h after vibrissae removal but not after 10 days. The results are discussed with respect to the interaction of vibrissae function with the basal ganglia, the neurotransmitter dopamine and mechanism of functional recovery.

Afferent Pathways↗

PubMed1994

Engineering cells to secrete growth factors.

The neutrotrophins stimulate survival and differentiation of a range of target neurons. A wealth of evidence suggests that central cholinergic neurons depend on nerve growth factor (NGF) for trophic support. Grafts of NGF-producing cells rescue axotomized basal forebrain cholinergic neurons and reduce cholinergic cell death in the medial septum. Skeletal muscle cells, immortalized from embryonic day 15 (E15) rat embryos for transplantation purposes, were transfected with a human NGF construct and individual clones tested for NGF production by a biological assay using embryonic sympathetic ganglia. Clone RM22 showed a consistent ability to produce human recombinant NGF in high concentration; RM22 cells were grafted to the rat brain, following fimbria-fornix lesions, in order to examine the influence of these cells on basal forebrain cholinergic neurons. The results suggest that implantation of genetically modified cells, engineered by the introduction of expression plasmids or viral constructs to produce NGF or other neurotrophins may have therapeutic applications in rescuing damaged central cholinergic neurons in senile dementia of the Alzheimer type as well as in providing trophic support for chromaffin tissue grafts in Parkinson's disease. Moreover, the use of genetically engineered cells may be used to study the effects of administering tailor-made neurotrophins with novel activity profiles.

Animals↗