Biomedical subjects
Publications and source records attributed to A M Davies.
In the fifty years since its discovery, a substantial body of work has established that nerve growth factor plays a key role in promoting the survival of neurons during development; the recent demonstration that it can also promote cell death therefore comes as a surprise.
4-Hydroxytamoxifen is a major metabolite of the antiestrogenic drug tamoxifen used in the treatment of women with breast cancer. 4-Hydroxytamoxifen is broken down by a horseradish peroxidase/H2O2 system very much more rapidly than tamoxifen and causes much greater DNA damage determined by 32P-postlabelling. EPR spin trapping of 4-hydroxytamoxifen reaction products in the presence of the free radical trap 5,5-dimethyl-1-pyrroline N-oxide, together with glutathione as a hydrogen donor, resulted in the generation of a species with the characteristics of the glutathione thiyl radical (aN approximately 15.3 G, aH approximately 16.2 G). Support for the creation of thiyl radicals comes from the close to stoichiometric time dependent formation of glutathione disulfide concomitant with the loss of glutathione. Similar results were obtained using 4-hydroxytoremifene but no radical formation or glutathione loss could be detected using 3-hydroxytamoxifen (droloxifene). On-line LC-ESI MS analysis of the incubation products from 4-hydroxytamoxifen has identified three products with a protonated molecular mass of 773, consistent with the formation of dimers of 4-hydroxytamoxifen. The role that radical mechanisms have in the carcinogenic effects of tamoxifen in the endometrium or other target organs of women taking this drug remains to be established.
In vitro and in vivo studies suggest that certain populations of neurons switch their survival requirements from one neurotrophin to another during an early stage in their development. Although there is good evidence for neurotrophin switching in sensory neurons, the evidence for switching in sympathetic neurons has become more controversial, as has the identity of the factors that regulate their responsiveness to particular neurotrophins.
Mouse trigeminal neurons survive independently of neurotrophins when their axons are growing to their targets, and are then transiently supported by BDNF before becoming NGF dependent. During the stage of neurotrophin independence, transcripts encoding the BDNF receptor, TrkB, were expressed at very low levels. During the stage of BDNF dependence, high levels of a transcript encoding a receptor with the catalytic tyrosine kinase domain were expressed. Although the levels of this transcript fell as the neurons lost responsiveness to BDNF, there were concomitant increases in the expression of transcripts encoding TrkB variants lacking the kinase domain. Analysis of RNA from purified neurons showed that all of these transcripts were present in neurons. BDNF and NGF up-regulated the expression of these transcripts early in development but had little effect later on. To test whether truncated TrkB modulates BDNF signalling via catalytic TrkB, we injected TrkB expression plasmids into NGF-dependent sympathetic neurons. Whereas expression of catalytic TrkB alone conferred a BDNF survival response, co-expression of non-catalytic TrkB substantially reduced this response. Our results suggest that BDNF responsiveness in sensory neurons during development is modulated by the relative levels of catalytic and non-catalytic TrkB.
Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for central and peripheral neurons, and is essential for the development of kidneys and the enteric nervous system. Despite the potential clinical and physiological importance of GDNF, its mechanism of action is unknown. Here we show that physiological responses to GDNF require the presence of a novel glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) that is expressed on GDNF-responsive cells and binds GDNF with a high affinity. We further demonstrate that GDNF promotes the formation of a physical complex between GDNFR-alpha and the orphan tyrosin kinase receptor Ret, thereby inducing its tyrosine phosphorylation. These findings support the hypothesis that GDNF uses a multi-subunit receptor system in which GDNFR-alpha and Ret function as the ligand-binding and signalling components, respectively.
In the developing peripheral nervous system many neurons die shortly after their axons reach their target fields. This loss is thought to match the number of neurons to the size and requirements of their target fields because altering target field size before innervation affects the number of neurons that survive. The neurotrophic hypothesis provides an explanation for how target fields influence the size of the neuronal populations that innervate them. This hypothesis arose from work on nerve growth factor (NGF), the founder member of the neurotrophin family of secreted proteins. Its principal tenet is that the survival of developing neurons depends on the supply of a neurotrophic factor that is synthesized in limiting amounts in their target fields. The neurotrophic hypothesis has, however, been broadened by the demonstration that multiple neurotrophic factors regulate the survival of certain populations of neurons. For example, some neurons depend on several different neurotrophic factors which may act concurrently or sequentially during target field innervation. In addition, there are aspects of neurotrophin action that do not conform with the classic neurotrophic hypothesis. For example, the dependence of some populations of sensory neurons on particular neurotrophins before significant neuronal death takes place raises the possibility that the supply of these neurotrophins is not limiting for survival at this stage of development. There is also evidence that at stages before and after sensory neurons depend on target-derived neurotrophins for survival, neurotrophins act on at least some sensory neurons by an autocrine route. Yet despite the growing wealth of information on the multiple roles and modes of action of neurotrophic factors, the neurotrophic hypothesis has remained the best explanation for how neuronal target fields in the developing peripheral nervous system regulate their innervation density.
The expression of BDNF mRNA by a proportion of embryonic dorsal root ganglion neurons has led to the proposal that BDNF acts by an autocrine loop on these neurons. To clarify the role of BDNF expression in developing sensory neurons, we measured the level of BDNF mRNA in purified populations of cranial sensory neurons that depend on either NGF or BDNF for survival. When neuronal death is taking place, the highest levels of BDNF mRNA were detected in NGF-dependent cutaneous sensory neurons. BDNF mRNA was expressed at lower levels in BDNF-dependent cutaneous sensory neurons and was undetectable in BDNF-dependent proprioceptive neurons. In coculture, NGF-dependent neurons promoted the survival of BDNF-dependent neurons by the production and release of BDNF. Depolarizing levels of KCl increased the expression of BDNF mRNA in cultured sensory neurons and this effect was partially inhibited by calcium channel antagonists. Our results suggest that during the phase of naturally occurring neuronal death, BDNF acts by a paracrine mechanism in sensory neurons and that BDNF expression is regulated by neural activity.
Using a subtractive hybridization technique directed to cloning transcripts with compartmentalized distributions within cerebral cortex neurons, we have isolated rat s-rex mRNAs that are analogues of the human neuroendocrine-specific NSP gene transcripts. Differential splicing produces two main s-rex mRNA that have different regional distributions in the developing and mature rat nervous system. In certain populations of adult brain neurons, most of s-rexs, mRNA and a substantial amount of s-rexb mRNA are localized to the axonal pole of the cell body. The localization of S-Rex/NSP proteins in these neurons suggests that s-rex mRNA compartmentalization targets the encoded proteins to specific regions of the neuron.
Neurotrophic factors are proteins that promote the survival and growth of neurons in the vertebrate nervous system. Although it is well known that many neurons obtain these factors from the regions to which their axons project, studies of the sites of neurotrophic factor synthesis have raised the possibility that at least some neurons may obtain these factors from other sources. Alternative sources of neurotrophic factors include cells along a neuron's axon shaft and cells or other axons terminals within the vicinity of a neuron's cell body and dendritic arbour. In addition, recent experimental studies have shown that at certain stages of development neurotrophic factor autocrine loops operate in some neurons. The evidence for and the potential physiological significance of these different modes of action of neurotrophic factors will be discussed.
We report a retrospective study of MR imaging of 16 patients with histologically proven chondroblastoma, 12 at primary presentation and four clinically suspected recurrences. In all the primary cases MR imaging showed a lobulated low signal intensity (SI) rim. Low SI foci within the tumour were present in 11 out of the 12 cases and corresponded to calcification seen on radiographs or CT. Bone marrow oedema was also present in 11 out of 12 cases and an adjacent joint effusion in eight out of 12. The STIR sequence was of particular value, giving optimal delineation of marrow and soft tissue oedema. Of the suspected recurrences one showed peritumoral oedema and was subsequently proven histologically. Three showed no peritumoral oedema and subsequent histology was negative in these cases. The presence or absence of oedema may be a useful indicator of tumour activity although further study is required.
The sensory neurons of the vestibular and nodose ganglia of the chicken embryo have nearby and distant targets, respectively. In vitro studies have shown that these neurons survive independently of neurotrophins when their axons are growing to their targets and become dependent on brain-derived neurotrophic factor (BDNF) for survival when their axons reach the vicinity of their targets. Although the timing of BDNF dependence is principally controlled by an intrinsic timing mechanism in the neurons, the onset of dependence can be accelerated by BDNF exposure toward the end of the phase of neurotrophin independence. We have used quantitative reverse transcription/polymerase chain reaction to study the expression of transcripts coding for BDNF and the catalytic isoform of its receptor tyrosine kinase, TrkB, in these neurons and their targets at different stages of development. We show that the peripheral and central target tissues of these neurons express BDNF mRNA prior to the arrival of sensory axons. Vestibular neurons express trkB mRNA before nodose neurons, which accords with the earlier response of vestibular neurons to BDNF. In culture, early nodose neurons start expressing trkB mRNA after 36 h incubation, which is 36 h before these neurons become dependent on BDNF for survival. Although BDNF does not affect the timing and level of trkB mRNA expression during the first 48 h in vitro, it increases the level of trkB mRNA after this time. The timing of BDNF-induced elevation of trkB mRNA correlates with the period during which BDNF exposure accelerates the onset of BDNF dependence in nodose neurons. These results suggest that the timing of BDNF dependence in developing sensory neurons is due in part to expression of catalytic TrkB and demonstrate that a BDNF autocrine loop is not required for the survival of sensory neurons during the earliest stages of their development.
Insufficiency fractures of the pelvis are being increasingly recognized as a major cause of low back pain in elderly women with osteoporosis. Fractures in the sacrum are difficult to diagnose, as plain radiographic findings are either unhelpful or misleading. Bone scintigraphy is very sensitive for the detection of fractures in the sacrum, with demonstration of the H-shaped (or butterfly) sacral pattern or the combination of concomitant sacral and parasymphyseal uptake being considered as characteristic of insufficiency fractures. Computed tomography (CT) is helpful for confirming the presence of fractures in cases with atypical scintigraphic patterns, particularly in those with a known primary malignant neoplasm. CT is especially useful in the further evaluation of parasymphyseal and pubic rami lesions. The majority of patients respond well to periods of enforced bed rest and administration of analgesics. Recognition of the spectrum of imaging findings for this entity should lead to its correct identification and the institution of appropriate treatment.
The sensory neurons of the embryonic mouse trigeminal ganglion are supported in culture by different neurotrophins at successive stages of development. Initially the neurons survive in response to BDNF and NT3 and later switch to becoming NGF-dependent (Buchman, V. I. and Davies, A. M. (1993), Development 118, 989-1001). To determine if this in vitro switch in neurotrophin responsiveness is physiologically relevant, we studied the timing of neuronal death in the trigeminal ganglia of embryos that are homozygous for null mutations in the trkA, trkB and trkC genes, which encode receptor tyrosine kinases for NGF, BDNF and NT3, respectively. In wild-type embryos, the number of pyknotic nuclei increased from E11 to peak between E13 and E14, and decreased gradually at later ages, becoming negligible by birth. Neuronal death in the trigeminal ganglia of trkA-/- embryos also peaked between E13 and E14, but was almost threefold greater than in wild-type embryos at this stage. Whereas there was no significant difference between the number of pyknotic nuclei in trkA-/- and wild-type embryos at E11 and E12, there was a substantial increase in the number of pyknotic nuclei in the trigeminal ganglia of trkB-/- at these earlier stages. Counts of the total number of neurons in E13 trigeminal ganglia revealed a marked decrease in trkB-/- but not trkA-/- or trkC-/- embryos. Consistent with the later onset of excessive neuronal death in trkA-/- embryos, there was a marked decrease in the neuronal complement of the trigeminal ganglia of trkA-/- embryos at E15. These results demonstrate that TrkB signalling is required for the in vivo survival of many trigeminal neurons during the early stages of target field innervation before they become NGF-dependent.
Bcl-2, Bcl-x and Bax are members fo a family of cytoplasmic proteins that influence cell survival. Whereas increased expression of Bcl-2 or Bcl-x promotes cell survival following withdrawal of survival factors, increased expression of Bax is thought to suppress survival. To investigate the potential roles of these proteins in regulating the survival of developing neurons, we compared the effects of overexpressing these proteins in embryonic neurons deprived of different neurotrophic factors in vitro. Surprisingly, overexpression of Bax rescued populations of sensory neurons deprived of nerve growth factor, as did overexpression of Bcl-2 and two Bcl-x variants, Bcl-XL and Bcl-Xbeta. Bax also enhanced the survival of ciliary neurons deprived of ciliary neurotrophic factor, although this effect was short-lived. Whereas Bcl-2 overexpression did not affect the survival response of neurons to neurotrophic factors, Bax overexpression partially inhibited the action of neurotrophic factors. Co-injection of Bcl-2 and Bax expression vectors promoted the survival of neurotrophic factor-deprived neurons if either was in excess, but failed to rescue neurons if they injected at a 1:1 ratio. Our findings demonstrate that Bax can promote the survival of neurotrophic factor-deprived neurons and that its effect on survival is dominant to that of neurotrophic factors. Our results also argue that the relative amounts of Bcl-2 and Bax are critical in regulating neuronal survival.
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Studies of cell lines and some cultured neurons have demonstrated potential cross-talk between neurotrophins and their receptors; high concentrations of neurotrophins can exhibit either agonist or antagonistic actions on heterologous neurotrophin receptors. We have studied neurotrophin discrimination among the sensory neurons of the embryonic chicken trigeminal system. We show that nerve growth factor (NGF) at a concentration that is six orders of magnitude greater than that required to promote the survival of NGF-dependent dorsomedial trigeminal ganglion (DMTG) neurons has no effect on the survival of brain-derived neurotrophic factor (BDNF)-dependent trigeminal mesencephalic nucleus (TMN) neurons and does not affect the dose-response relationship of these neurons to BDNF. A similar high level of neurotrophin-3 neither promotes the survival of BDNF-dependent ventrolateral trigeminal ganglion neurons nor affects the dose response of these neurons to BDNF. High levels of BDNF have a negligible effect on the survival of mid-embryonic DMTG neurons. These results show that some neurons are able to discriminate completely between neurotrophins at very high concentrations, indicating that neurotrophin responses can be far more highly specific than previously appreciated.
Neurotrophins promote neuronal survival by signalling through Trk receptor tyrosine kinases: nerve growth factor signals through TrkA, brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)4 through TrkB and NT3 through TrkC. Although studies in some, but not all, cell lines indicate that NT3 can also signal through TrkA and TrkB, it is not known if such signalling can occur in neurons. We show that NT3 can promote the in vitro survival of sensory and sympathetic neurons isolated from embryos that are homozygous for a null mutation in the trkC gene. During the mid-embryonic period, NT3 promoted the survival of as many trigeminal and nodose neurons as the preferred neurotrophins, NGF and BDNF. However, later in development, these neurons lost their ability to respond to NT3. NT3 also promoted the survival of almost all sympathetic neurons, but no decrease in effectiveness was observed during development. Trigeminal neurons from trkC-/- trkA-/- embryos did not respond to NT3 and nodose neurons from trkB-/- embryos likewise failed to respond to NT3. These results show that NT3 can signal through TrkA and TrkB in neurons at certain stages of development and may explain why the phenotype of NT3-/- mice is more severe than that of trkC-/- mice.