Urea enhances the nerve growth factor-induced neuroprotective effect on cholinergic neurons in organotypic rat brain slices.

Cholinergic neurons degenerate in Alzheimer's disease and dementia and neuroprotective substances are of high interest to counteract this cell death. The aim of the present study was to test the effect of urea and the nitric oxide synthetase inhibitor l-thiocitrulline on the survival of cholinergic neurons. Organotypic brain slices of the basal nucleus of Meynert were cultured for 2 weeks in the presence of 1-100 microM urea with or without NGF or other growth factors or with or without 1-10 microM of the NOS inhibitor L-thiocitrulline. A high number of cholinergic neurons survived in the presence of 0.1-100 ng/ml NGF. Urea or L-thiocitrulline alone did not exhibit neuroprotective activity; however, when brain slices were incubated with urea or L-thiocitrulline together with NGF there was a significant potentiating survival effect. Incubation of brain slices with NGF + urea + L-thiocitrulline did not further enhance the number of cholinergic neurons. NGF as well as urea did not stimulate expression of the enzyme choline acetyltransferase pointing to survival promoting effects. Urea did not modulate the NGF binding in PC12 cells indicating that this effect was indirect. It is concluded that urea may play a role as an indirect survival promoting molecule possibly involving the nitric oxide pathway.

Molecular interactions between neurotrophin receptors.

Neurotrophins signal via a dual-receptor system comprising receptor tyrosine kinases, the Trks, and a tumor necrosis factor (TNF) receptor like molecule, p75. Interest in these receptors was spurred on by the finding that they are employed by their neurotrophin ligands to activate opposing cellular mechanisms. Signalling via Trk receptors promotes the survival of embryonic neurons, whereas activation of p75 can trigger apoptosis. However, this antagonistic view is an oversimplification. It is more accurate to refer to this system as a signalling network in which ligands, receptors and their intracellular target proteins are linked by balanced biochemical interactions. This article reviews recent advances in our understanding of these molecular mechanisms which critically determine many cell-type-specific responses to neurotrophins. Emphasis is given to the formation of receptor complexes, the generation of receptor diversity by alternative splicing and the influence exerted by the local membrane environment on neurotrophin signalling.

Neurotrophin-3 promotes the cholinergic differentiation of sympathetic neurons.

Neurotrophins influence the epigenetic shaping of the vertebrate nervous system by regulating neuronal numbers during development and synaptic plasticity. Here we attempt to determine whether these growth factors can also regulate neurotransmitter plasticity. As a model system we used the selection between noradrenergic and cholinergic neurotransmission by paravertebral sympathetic neurons. Developing sympathetic neurons express the neurotrophin receptors TrkA and TrkC, two highly related receptor tyrosine kinases. Whereas the TrkA ligand nerve growth factor (NGF) has long been known to regulate both the survival and the expression of noradrenergic traits in sympathetic neurons, the role of TrkC and of its ligand neurotrophin-3 (NT3) has remained unclear. We found that TrkC expression in the avian sympathetic chain overlaps substantially with that of choline acetyltransferase. In sympathetic chain explants, transcripts of the cholinergic marker genes choline acetyltransferase and vasoactive intestinal polypeptide were strongly enriched in the presence of NT3 compared with NGF, whereas the noradrenergic markers tyrosine hydroxylase and norepinephrine transporter were reduced. The transcription factor chicken achaete scute homolog 1 was coexpressed with cholinergic markers. The effects of NT3 are reversed and antagonized by NGF. They are independent of neuronal survival and developmentally regulated. These results suggest a role for NT3 as a differentiation factor for cholinergic neurons and establish a link between neurotrophins and neurotransmitter plasticity.

Conserved synteny between the chicken Z sex chromosome and human chromosome 9 includes the male regulatory gene DMRT1: a comparative (re)view on avian sex determination.

Sex-determination mechanisms in birds and mammals evolved independently for more than 300 million years. Unlike mammals, sex determination in birds operates through a ZZ/ZW sex chromosome system, in which the female is the heterogametic sex. However, the molecular mechanism remains to be elucidated. Comparative gene mapping revealed that several genes on human chromosome 9 (HSA 9) have homologs on the chicken Z chromosome (GGA Z), indicating the common ancestry of large parts of GGA Z and HSA 9. Based on chromosome homology maps, we isolated a Z-linked chicken ortholog of DMRT1, which has been implicated in XY sex reversal in humans. Its location on the avian Z and within the sex-reversal region on HSA 9p suggests that DMRT1 represents an ancestral dosage-sensitive gene for vertebrate sex-determination. Z dosage may be crucial for male sexual differentiation/determination in birds.

The zinc finger protein NRIF interacts with the neurotrophin receptor p75(NTR) and participates in programmed cell death.

NRIF (neurotrophin receptor interacting factor) is a ubiquitously expressed zinc finger protein of the Krüppel family which interacts with the neurotrophin receptor p75(NTR). The interaction was first detected in yeast and then biochemically confirmed using recombinant GST-NRIF fusions and p75(NTR) expressed by eukaryotic cells. Transgenic mice carrying a deletion in the exon encoding the p75(NTR)-binding domain of NRIF display a phenotype which is strongly dependent upon genetic background. While at the F(2 )generation there is only limited (20%) embryonic lethality, in a congenic BL6 strain nrif(-/-) mice cannot survive beyond E12, but are viable and healthy to adulthood in the Sv129 background. The involvement of NRIF in p75(NTR)/NGF-mediated developmental cell death was examined in the mouse embryonic neural retina. Disruption of the nrif gene leads to a reduction in cell death which is quantitatively indistinguishable from that observed in p75(NTR)(-/-) and ngf(-/-) mice. These results indicate that NRIF is an intracellular p75(NTR)-binding protein transducing cell death signals during development.

The neurotrophin receptor p75 binds neurotrophin-3 on sympathetic neurons with high affinity and specificity.

High-affinity neurotrophin-3 (NT3) receptors have been identified on nerve growth factor (NGF)-dependent sympathetic neurons, but their occupancy by NT3 does not lead to neuronal survival. The molecular nature of these NT3 binding sites was investigated in this study. With freshly dissociated embryonic day 11 (E11) chick sympathetic neurons, cross-linking experiments revealed that the main receptor responsible for high-affinity specific binding was the neurotrophin receptor p75 (p75(NTR)), with only a small fraction corresponding to trkC. When E11 sympathetic neurons were cultured in the presence of NGF, trkC transcripts became undetectable, but high-affinity specific NT3 binding persisted. Cross-linking and antibody inhibition experiments indicated that p75(NTR) was the only detectable NT3 receptor protein. These characteristics were not observed when p75(NTR) was expressed in transformed cells. We conclude that p75(NTR) can exist in neurons in a confirmation conferring hitherto unrecognized properties to this receptor.

Signalling through the neurotrophin receptor p75NTR.

Activation specific tyrosine kinase receptors by neurotrophins accounts for the longest known biological actions of the neurotrophins, in particular the promotion of neuronal survival. However, recent studies have revealed that nerve growth factor, the neurotrophin regarded as best understood, also activates a signalling pathway by binding to the neurotrophin receptor p75(NTR). This receptor belongs to the tumor necrosis factor receptor family and lacks intrinsic catalytic activity. The p75(NTR) receptor binds all neurotrophins with nanomolar affinity; however, nerve growth factor seems to be uniquely able to activate it, causing the death of trkA-negative neurons during normal development. Thus, nerve growth factor prevents programmed cell death through its receptor TrkA, but promotes it by signalling through p75(NTR).

A splice variant of the neurotrophin receptor trkB with increased specificity for brain-derived neurotrophic factor.

The trkB gene codes for a receptor tyrosine kinase, which is essential for the development of the peripheral nervous system. This receptor can be activated by three different neurotrophins: BDNF, NT-4/5 and NT-3. The extracellular domain of trkB was found to be encoded in 10 exons corresponding to receptor subdomains previously identified on the basis of protein sequence comparisons. Exon 9 was skipped in a novel tyrosine kinase transcript of the trkB gene, designated ctrkB-Short (ctrkB-S). While the previously described trkB receptor ctrkB-Long (ctrkB-L) and trkB-S receptors were activated similarly by BDNF, trkB-S interacted poorly with NT-4/5 and NT-3 as measured by ligand binding, ligand-induced autophosphorylation and ligand-dependent activation of p21ras. Efficient activation of ctrkB-S by NT-3 was restored by a single amino acid replacement in NT-3 (D15A). Both trkB-L and trkB-S transcripts were detected in embryonic neurons.

Roles of neurotrophin-3 during early development of the peripheral nervous system.

The neurotrophins are structurally related proteins regulating cell numbers in the developing vertebrate nervous system. They are necessary survival factors preventing the death of specific neuronal populations. Previous experiments have indicated that the administration of nerve growth factor or of brain-derived neurotrophic factor during the formation of sensory ganglia and of target innervation increases the number of neurons by preventing normally occurring neuronal death. These results support the view that during development, neuronal numbers are adjusted to the size of the target tissue by the release of limiting amounts of neurotrophins. However, increasing the levels of neurotrophin-3 during the formation of sensory ganglia results in a marked decrease in neuronal numbers, possibly as a consequence of premature cessation of sensory neuroblast proliferation. In sympathetic ganglia, the application of neurotrophin-3 during the formation of the sympathetic chain causes cell numbers to increase, a result also observed following the application of nerve growth factor. It thus appears that neurotrophin-3 and nerve growth factor can regulate cell numbers well before the period of target-derived control, and that neurotrophin-3 affects neuronal numbers in sensory and sympathetic ganglia in opposite ways.

Early expression of the nerve growth factor receptor ctrkA in chick sympathetic and sensory ganglia.

Avian sympathetic and sensory ganglia are useful models to study the biological effects of nerve growth factor (NGF) in vitro as well as in vivo. In order to examine the expression pattern of the NGF tyrosine kinase receptor during embryogenesis, we cloned a full-length cDNA encoding chick trkA (ctrkA). Compared with human trkA, the sequence identity is 46% in the extracellular domain and is unevenly distributed between the subdomains. Between embryonic Days 6.5 and 16 (E6.5 and E16), a single 3-kb ctrkA transcript is detected in sympathetic and sensory ganglia, and in situ hybridization experiments reveal the presence of ctrkA mRNA in both ganglia from E4.5 onward. The detection of ctrkA in the primary sympathetic chain is unexpected in view of previous experiments with cultured sympathetic neurons indicating a lack of a survival response to NGF at early developmental stages. However, it fits with the observation that, in vivo, the administration of NGF markedly affects cell numbers substantially before the period of target control of neuronal survival in the sympathetic chain.

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)

Molecular control of neuronal survival in the chick embryo.

Neurotrophins are structurally related proteins which promote the survival and differentiation of specific neuronal populations during the development of vertebrate embryos. Like many growth factors, the neurotrophins mediate their actions by binding to membrane proteins that have a ligand-activated tyrosine kinase activity. The interactions of the neurophins with their neuronal receptors have been mostly studied using chick embryonic neurons. These neurons are also extensively used to characterise biological responses to neurotrophins in physiologically relevant systems. We have recently cloned and expressed the chick homologue of trkB (ctrkB), thought to be a receptor for BDNF, and examined by in situ hybridisation the pattern of expression of the ctrkB gene during development of the chick embryo. We found that whereas the sequence of ctrkB shows a high degree of conservation with the mammalian homologues in the intracellular tyrosine kinase domain, the extracellular binding domain is less well conserved. As in mammals, ctrkB mRNAs appear to exist in differentially spliced forms that result in a full length and a truncated receptor lacking the tyrosine kinase domain. These two forms are differentially expressed in neurons and non-neuronal cells respectively. The binding characteristics of ctrkB expressed in a transfected cell line are similar, but not identical to those of the BDNF binding sites on primary chick neurons, specially with regard to the affinity of BDNF.

A crucial role for neurotrophin-3 in oligodendrocyte development.

The neurotrophins nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 promote the survival of subpopulations of vertebrate neurons in vitro, but so far only nerve growth factor has been demonstrated to be essential for normal neuronal development; no neurotrophin has previously been shown to function in normal glial cell development. We found recently that neurotrophin-3 promotes the survival of pure oligodendrocyte precursor cells in vitro, and, although by itself it induces only a small percentage of these cells to synthesize DNA, in combination with platelet-derived growth factor it induces the majority of them to do so. Neither of these factors, however, has been shown to contribute to oligodendrocyte precursor cell proliferation in vivo or to stimulate pure populations of these cells to proliferate (as opposed to synthesize DNA) in vitro. Here we show that neurotrophin-3 and platelet-derived growth factor collaborate to promote clonal expansion of oligodendrocyte precursor cells in vitro and to drive the intrinsic clock that times oligodendrocyte development. We also show that neurotrophin-3 helps stimulate the proliferation of oligodendrocyte precursor cells in vivo and is thus required for normal oligodendrocyte development.

Brain-derived neurotrophic factor is a survival factor for cultured rat cerebellar granule neurons and protects them against glutamate-induced neurotoxicity.

We have studied the effects of different neurotrophins on the survival and proliferation of rat cerebellar granule cells in culture. These neurons express trkB and trkC, the putative neuronal receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) respectively. Binding studies using iodinated BDNF and NT-3 demonstrated that both BDNF and NT-3 bind to the cerebellar granule neurons with a similar affinity of approximately 2 x 10(-9) M. The number of receptors per granule cell was surprisingly high, approximately 30 x 10(-4) and 2 x 10(5) for BDNF and NT-3, respectively. Both NT-3 and BDNF elevated c-fos mRNA in the granule neurons, but only BDNF up-regulated the mRNA encoding the low-affinity neurotrophin receptor (p75). In contrast to NT-3, BDNF acted as a survival factor for the granule neurons. BDNF also induced sprouting of the granule neurons and significantly protected them against neurotoxicity induced by high (1 mM) glutamate concentrations. Cultured granule neurons also expressed low levels of BDNF mRNA which were increased by kainic acid, a glutamate receptor agonist. Thus, BDNF, but not NT-3, is a survival factor for cultured cerebellar granule neurons and activation of glutamate receptor(s) up-regulates BDNF expression in these cells.

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.

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.

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.

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.