Biomedical subjects

Y A Barde

Publications and source records attributed to Y A Barde.

At least 55 records · Page 3Linked to original sources

PubMed1994

Neurotrophin receptors.

The neurotrophins are members of a family of four related proteins that allow the survival and differentiation of specific sub-sets of embryonic vertebrate neurons. On neurons, two types of neurotrophin receptors can be distinguished on the basis of their dissociation constants: low affinity receptors (Kd 10(-9) M) and high affinity receptors (Kd 10(-11) M). Several genes coding for neurotrophin receptors have been cloned and the expression in fibroblasts of the recombinant membrane proteins allows comparisons to be made between the binding properties of the neurotrophins on such cell lines and neurons. As a result, it appears that much of the low affinity binding sites detected on neurons for all neurotrophins can be attributed to a single molecular entity, the low affinity neurotrophin (or NGF) receptor. This receptor binds all known neurotrophins with similar affinity but different binding kinetics. Its role in neurotrophic signal transduction remains to be established. In addition to this receptor, three members of the trk-subfamily of tyrosine kinase receptors have recently been identified as receptors for the neurotrophins. These receptors (whose intrinsic tyrosine kinase activity can be stimulated by the various neurotrophins) bind the neurotrophins with higher affinity and higher ligand specificity when compared with the low affinity receptor. However, the observation has been made that some of the recombinant trk-receptors on cell lines bind more than one neurotrophin (though typically with lower affinity than their own ligands).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Sensory ganglia require neurotrophin-3 early in development.

The role played by neurotrophin-3 during the development of quail sensory ganglia was investigated using a monoclonal antibody that specifically blocks the biological activity of this neurotrophin. Neutralisation of neurotrophin-3 was initiated during completion of gangliogenesis. Neuronal cell counts indicate that about 30% of the neurons normally present in either the placode-derived ganglion nodosum or in a leg-innervating, neural crest-derived dorsal root ganglion are eliminated by the antibody treatment. In both ganglia, this reduction is seen early in development, and the results obtained with the ganglion nodosum indicate that neurotrophin-3 plays an essential role already during gangliogenesis. Neuronal numbers are also compared with those obtained after treatment with a monoclonal antibody to nerve growth factor, used either alone or in combination with the neurotrophin-3 antibody.

Animals↗

PubMed1994

Expression of the BDNF gene in the developing visual system of the chick.

Using a sensitive and quantitative method, the mRNA levels of brain-derived neurotrophic factor (BDNF) were determined during the development of the chick visual system. Low copy numbers were detected, and BDNF was found to be expressed in the optic tectum already 2 days before the arrival of the first retinal ganglion cell axons, suggesting an early role of BDNF in tectal development. After the beginning of tectal innervation, BDNF mRNA levels markedly increased, and optic stalk transection at day 4 (which prevents subsequent tectal innervation) was found to reduce the contralateral tectal levels of BDNF mRNA. Comparable reductions were obtained after injection of tetrodotoxin into one eye, indicating that, already during the earliest stages of target encounter in the CNS, the degree of BDNF gene expression is influenced by activity-dependent mechanisms. BDNF mRNA was also detected in the retina itself and at levels comparable to those found in the tectum. Together with previous findings indicating that BDNF prevents the death of cultured chick retinal ganglion cells, these results support the idea that the tightly controlled expression of the BDNF gene might be important in the co-ordinated development of the visual system.

Animals↗

PubMed1994

Neurotrophins: a family of proteins supporting the survival of neurons.

NGF, BDNF, NT-3, and NT-4/5 are all members of a structurally related family of molecules that function to prevent the death of embryonic neurons during development. The presence of these molecules in the targets of innervating neurons is likely to explain at least in part why many neurons depend on their target tissues for survival. A small family of related membrane proteins with a ligand-activable tyrosine kinase and expressed in the nervous system represents a significant part of the structural basis explaining how neurons discriminate between the neurotrophins and transduce the consequence of neurotrophin binding. Thus, much structural information has been obtained that contributes to better understand some important aspects of vertebrate neurogenesis, particularly those related to selective cell survival in a very diverse cellular system like the nervous system. Future studies will have to explain how the role of these molecules has to be understood in the context of the characteristic features of the nervous system, in particular neurotransmission and electrical activity. Finally, while the role of neurotrophins has been discussed here in the context of the developing nervous system, it will be important to understand what functions these molecules might play in the central nervous system. For example, neurotrophins might function as long term mediators of changes in cellular shapes under the influence of electrical activity, as well as in pathological situations when axonal elongation is needed to restore connections, or to maintain the well-being of neurons that are eliminated during the course of neurodegenerative diseases.

Animals↗

PubMed1993

Involvement of ras p21 in neurotrophin-induced response of sensory, but not sympathetic neurons.

Little is known about the signal transduction mechanisms involved in the response to neurotrophins and other neurotrophic factors in neurons, beyond the activation of the tyrosine kinase activity of the neurotrophin receptors belonging to the trk family. We have previously shown that the introduction of the oncogene product ras p21 into the cytoplasm of chick embryonic neurons can reproduce the survival and neurite-outgrowth promoting effects of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), and of ciliary neurotrophic factor (CNTF). To assess the potential signal-transducing role of endogenous ras p21, we introduced function-blocking anti-ras antibodies or their Fab fragments into cultured chick embryonic neurons. The BDNF-induced neurite outgrowth in E12 nodose ganglion neurons was reduced to below control levels, and the NGF-induced survival of E9 dorsal root ganglion (DRG) neurons was inhibited in a specific and dose-dependent fashion. Both effects could be reversed by saturating the epitope-binding sites with biologically inactive ras p21 before microinjection. Surprisingly, ras p21 did not promote the survival of NGF-dependent E12 chick sympathetic neurons, and the NGF-induced survival in these cells was not inhibited by the Fab-fragments. The survival effect of CNTF on ras-responsive ciliary neurons could not be blocked by anti-ras Fab fragments. These results indicate an involvement of ras p21 in the signal transduction of neurotrophic factors in sensory, but not sympathetic or ciliary neurons, pointing to the existence of different signaling pathways not only in CNTF-responsive, but also in neurotrophin-responsive neuronal populations.

Animals↗

PubMed1993

Expression and binding characteristics of the BDNF receptor chick trkB.

Previous studies using transfected cells have indicated that the mammalian receptor tyrosine kinase trkB binds the neurotrophins brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4. However, most studies demonstrating that these neurotrophins prevent the death of embryonic neurons and have specific neuronal receptors have been performed with chick neurons. In order to explore the possibility that trkB is the molecular entity representing the high-affinity receptor for brain-derived neurotrophic factor on embryonic chick neurons, we cloned and expressed a chick trkB cDNA. In situ hybridisation results indicate that the distribution of trkB mRNA in the peripheral nervous system of the developing chick embryo correlates well with the structures known to respond to brain-derived neurotrophic factor. Binding studies performed with a cell line stably transfected with the ctrkB cDNA indicate a dissociation constant for brain-derived neurotrophic factor of 9.9 x 10(-10) M, which is distinctly higher than that found on primary chick sensory neurons (1.5 x 10(-11) M). When binding of brain-derived neurotrophic factor was determined in the presence of other neurotrophins, neurotrophin-3 was found efficiently to prevent the binding of brain-derived neurotrophic factor to both the ctrkB cell line and embryonic sensory neurons. In vitro, neurotrophin-3 at high concentrations completely blocked the survival normally seen with brain-derived neurotrophic factor. Thus, unlike previous cases of receptor occupancy by heterologous neurotrophins (which resulted in agonistic effects), the interaction between the brain-derived neurotrophic factor receptor and neurotrophin-3 on sensory neurons is antagonistic.

Amino Acid Sequence↗

PubMed1993

Specific high-affinity receptors for neurotrophin-3 on sympathetic neurons.

When used at concentrations allowing interactions only with its high-affinity receptors, neurotrophin-3 (NT-3) promotes the survival of sensory neurons isolated from embryonic day 8 (E8) chicks, but not the survival of E11 sympathetic neurons. These sympathetic neurons (which can be rescued by the addition of NGF) display high-affinity receptors for NT-3 (Kd of 1.6 x 10(-11) M) that cannot be distinguished from the high-affinity NT-3 receptors on sensory neurons using equilibrium binding or kinetic criteria. This represents the first example of embryonic neurons that cannot be rescued by the in vitro addition of a neurotrophin in spite of the presence of corresponding neurotrophin high-affinity receptors. At elevated concentrations, beyond the saturation of its high-affinity receptors, NT-3 supports the survival of some E11 sympathetic neurons, an effect that might be mediated by the high-affinity NGF receptor. Using E7 sympathetic neurons, about 40% of the cells initially plated can be rescued in vitro by the addition of low concentrations of NT-3 (but not of NGF) and produce profuse neurites. This indicates that NT-3 may play a role in the early development of some sympathetic neurons.

Animals↗

PubMed1992

Production and characterization of recombinant mouse neurotrophin-3.

Neurotrophin-3 (NT-3) is a neurotrophic-factor that has recently been cloned on the basis of its structural similarity to other members of the nerve growth factor (NGF) gene family. In order to produce this protein, a recombinant vaccinia virus containing the coding region for mouse NT-3 was constructed. Conditioned medium harvested from cells infected with the recombinant vaccinia virus contained biologically active NT-3 that was purified by chromatography on controlled-pore glass and reversed-phase and gel-filtration high-pressure liquid chromatography. Approximately 200 micrograms purified NT-3 was obtained from a single cell-factory flask (1.6 1 conditioned medium). N-terminal protein sequencing showed that the mature factor was processed from the precursor at the expected site and its amino acid composition agreed with that predicted from the DNA sequence. The biological activity of the recombinant protein was tested on dissociated neurons prepared from chick embryos. Using spinal sensory neurons, the concentration of purified recombinant NT-3 allowing half-maximal survival was determined to be 25 pg/ml.

Amino Acids↗

PubMed1992

Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor.

Neurotrophin-3 (NT-3) has low-affinity (Kd = 8 x 10(-10) M), as well as high-affinity receptors (Kd = 1.8 x 10(-11) M) on embryonic chick sensory neurons, the latter in surprisingly high numbers. Like the structurally related proteins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), NT-3 also binds to the low-affinity NGF receptor, a molecule that we suggest to designate low-affinity neurotrophin receptor (LANR). NT-3 dissociates from the LANR much more rapidly than BDNF, and more slowly than NGF. The binding of labelled NT-3 to the LANR can be reduced by half using a concentration of BDNF corresponding to the Kd of BDNF to the LANR. In contrast, the binding of NT-3 to its high-affinity neuronal receptors can only be prevented by BDNF or NGF when used at concentrations several thousand-fold higher than those corresponding to their Kd to their high-affinity neuronal receptors. Thus, specific high-affinity NT-3 receptors exist on sensory neurons that can readily discriminate between three structurally related ligands. These findings, including the remarkable property of the LANR to bind three related ligands with similar affinity, but different rate constants, are discussed.

Animals↗

PubMed1991

Neurotrophins: structural relatedness and receptor interactions.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are structurally related proteins that allow the survival of specific populations of embryonic vertebrate neurons. The primary structure of these neurotrophins, deduced from their nucleotide sequences, indicates that all three are synthesized in the form of precursor proteins presumably allowing for appropriate folding, including the formation of disulphide bridges, cleavage and secretion. While no information is yet available on the 3-dimensional structures of the neurotrophins, results from binding studies using the three neurotrophins as ligands indicate that their receptors do recognize similarities, as well as differences, between them. High-affinity receptors, that presumably mediate the biological response, as well as low-affinity receptors are present on neurons responsive to the neurotrophins. Whereas a large excess of heterologous ligand is needed to reduce binding of a particular neurotrophin to its high-affinity receptor, the same concentration of homologous or heterologous ligand similarly reduce the binding of any of the three neurotrophins to the low-affinity receptor. For all three, the low-affinity receptor appears to be the already characterized NGF low-affinity receptor that seems to be an integral part of the high-affinity receptor complexes. These results suggest that the regulation of neuronal survival by target cells can, in part, be explained by the release from these cells of limiting quantities of the structurally related neurotrophins, each being recognized by a specific high-affinity receptor complex located on the nerve terminals of the responsive neurons.

Animals↗

PubMed1991

BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra.

Brain-derived neurotrophic factor (BDNF), present in minute amounts in the adult central nervous system, is a member of the nerve growth factor (NGF) family, which includes neurotrophin-3 (NT-3). NGF, BDNF and NT-3 all support survival of subpopulations of neural crest-derived sensory neurons; most sympathetic neurons are responsive to NGF, but not to BDNF; NT-3 and BDNF, but not NGF, promote survival of sensory neurons of the nodose ganglion. BDNF, but not NGF, supports the survival of cultured retinal ganglion cells but both NGF and BDNF promote the survival of septal cholinergic neurons in vitro. However, knowledge of their precise physiological role in development and maintenance of the nervous system neurons is still limited. The BDNF gene is expressed in many regions of the adult CNS, including the striatum. A protein partially purified from bovine striatum, a target of nigral dopaminergic neurons, with characteristics apparently similar to those of BDNF, can enhance the survival of dopaminergic neurons in mesencephalic cultures. BDNF seems to be a trophic factor for mesencephalic dopaminergic neurons, increasing their survival, including that of neuronal cells which degenerate in Parkinson's disease. Here we report the effects of BDNF on the survival of dopaminergic neurons of the developing substantia nigra.

Animals↗

PubMed1990

Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family.

The survival and functional maintenance of vertebrate neurons critically depends on the availability of specific neurotrophic factors. So far, only two such factors, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have been characterized and shown to have the typical features of secretory proteins. This characterization has been possible because of the extraordinarily large quantities of NGF in some adult tissues, and the virtually unlimited availability of brain tissue from which BDNF was isolated. Both NGF and BDNF promote the survival of distinct neuronal populations in vivo and are related in their primary structure, suggesting that they are members of a gene family. Although there is little doubt about the existence of other such proteins, their low abundance has rendered their identification and characterization difficult. Taking advantage of sequence identities between NGF and BDNF, we have now identified a third member of this family, which we name neurotrophin-3. Both the tissue distribution of the messenger RNA and the neuronal specificity of this secretory protein differ from those of NGF and BDNF. Alignment of the sequences of the three proteins reveals a remarkable number of amino acid identities, including all cysteine residues. This alignment also delineates four variable domains, each of 7-11 amino acids, indicating structural elements presumably involved in the neuronal specificity of these proteins.

Amino Acid Sequence↗

PubMed1990

Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain.

Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.

Amino Acid Sequence↗

PubMed1990

Binding of brain-derived neurotrophic factor to the nerve growth factor receptor.

The neurotrophic proteins BDNF and NGF are related in their primary structures, and both have high- and low-affinity receptors on their responsive neurons. In this study, we investigate the extent to which these receptors can discriminate between BDNF and NGF. We found that a 1000-fold excess of the heterologous ligand is needed to reduce binding to the high-affinity receptor by 50%, but that the same concentrations of BDNF and NGF similarly reduce the binding of either ligand to the low-affinity receptor. Results obtained with cells transfected with the low-affinity NGF receptor gene indicate that these cells bind BDNF, in addition to NGF, whereas cells before transfection do not. These data indicate that the low-affinity NGF receptor is also a low-affinity BDNF receptor and that whatever is conferring high-affinity binding and biological response also considerably reinforces the ability of the low-affinity receptor to discriminate between NGF and BDNF.

Animals↗

PubMed1990

Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture.

Brain-derived neurotrophic factor (BDNF) was found to promote the survival of E17 rat embryo septal cholinergic neurons in culture, as assessed by a histochemical stain for acetylcholinesterase (AChE). A 2.4-fold increase in neuronal survival was achieved with 10 ng/ml BDNF. After initial deprivation of growth factor for 7 days, BDNF failed to bring about this increase, strongly suggesting that BDNF promotes cell survival and not just induction of AChE. BDNF was also found to increase the levels of cholinergic enzymes; choline acetyltransferase (ChAT) and AChE activities were increased by approximately 2-fold in the presence of 50 ng/ml BDNF. BDNF produced a 3-fold increase in the number of cells bearing the NGF receptor, as detected by the monoclonal antibody IgG-192. Although NGF had no additive effect with BDNF in terms of neuronal survival, suggesting that both act on a similar neuronal population, the combination of both produced an additive response, approximately a 6-fold increase, in ChAT activity.

Acetylcholinesterase↗

PubMed1990

The nerve growth factor family.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are small, basic, secretory proteins that allow the survival of specific neuronal populations. In their biologically active form, after cleavage from their biosynthetic precursors, these three neurotrophic proteins, or neurotrophins, show about 50% amino acid identities. The genes coding for the neurotrophins are not only expressed during development, but also in the adult, in a variety of tissues including the central nervous system. In the adult brain, the hippocampal formation is the site of highest expression of the three neurotrophin genes. These genes are expressed in neurons, and the mRNA levels of two of them (NGF and BDNF) have been shown to be regulated by neurotransmitters. There are also convincing indications that the administration of NGF prevents the atrophy and death of axotomized cholinergic neurons in the adult central nervous system, and improves the performance of rats selected for their poor memory retention in simple behavioral tasks.

Amino Acid Sequence↗

PubMed1989

Molecular cloning and expression of brain-derived neurotrophic factor.

During the development of the vertebrate nervous system, many neurons depend for survival on interactions with their target cells. Specific proteins are thought to be released by the target cells and to play an essential role in these interactions. So far, only one such protein, nerve growth factor, has been fully characterized. This has been possible because of the extraordinarily (and unexplained) large quantities of this protein in some adult tissues that are of no relevance to the developing nervous system. Whereas the dependency of many neurons on their target cells for normal development, and the restricted neuronal specificity of nerve growth factor have long suggested the existence of other such proteins, their low abundance has rendered their characterization difficult. Here we report the full primary structure of brain-derived neurotrophic factor. This very rare protein is known to promote the survival of neuronal populations that are all located either in the central nervous system or directly connected with it. The messenger RNA for brain-derived neurotrophic factor was found predominantly in the central nervous system, and the sequence of the protein indicates that it is structurally related to nerve growth factor. These results establish that these two neurotrophic factors are related both functionally and structurally.

Amino Acid Sequence↗

PubMed1989

The survival of chick retinal ganglion cells in response to brain-derived neurotrophic factor depends on their embryonic age.

The survival effects of brain-derived neurotrophic factor (BDNF) on the ganglion cells of the chick retina were studied in vitro at different embryonic ages. We found these effects to be strongly age-dependent: at E5, when the first ganglion cell axons have crossed the optic chiasm, but not yet reached the tectum, ganglion cells survived on a laminin substrate irrespective of the presence or absence of BDNF. At E6, when the axons of the first-generated ganglion cells reached the rostral pole of the tectum, the ganglion cells began to show a dependency on BDNF for survival, but the majority of them were alive after 2 days in vitro in the absence of BDNF. With increasing age, the BDNF dependency for survival increased, and at E11, the majority of the ganglion cells plated were dependent on BDNF for survival. It is at this age that the maximal number of axons can be found in vivo in the optic nerve, the subsequent elimination of ganglion cells and their axons resulting in the loss of hundreds of thousands of them over the next few days. Taken together, these data indicate that retinal ganglion cells depend on BDNF for survival only when their axons have reached their target in vivo. This situation is reminiscent of that described in the peripheral nervous system for the nerve growth factor responsiveness of mouse trigeminal sensory neurons during the period of innervation of their target.

Age Factors↗