Expression of the trk family of neurotrophin receptors in developing and adult dorsal root ganglion neurons

Expression of trk receptors is a major determinant of neurotrophin responsiveness of sensory neurons. Although it has been apparent for some time that subpopulations of dorsal root and trigeminal ganglion neurons respond in vitro to each of the members of the neurotrophin family, the extent to which functionally distinct subclasses of sensory neurons are dependent on the actions of different neurotrophins for their development and function remains an active area of investigation. One step towards elucidating the role of various neurotrophins in development and function of sensory neurons has been to examine the distribution of trk receptors on sensory neurons. These studies have clearly revealed that members of the trk family are differentially expressed in functionally distinct populations of both developing and mature sensory neurons and, further, have provided evidence consistent with a shift in neurotrophin responsiveness during the development of sensory neurons.     

High affinity nerve growth factor binding displays a faster rate of association than p140trk binding. Implications for multi-subunit polypeptide receptors

Nerve growth factor (NGF) binds to two cell surface receptors, p140trk and p75NGFR, which are both expressed in responsive sensory, sympathetic, and basal forebrain cholinergic neurons. While p140trk belongs to the family of receptor tyrosine kinases, p75NGFR is a member of the TNF/Fas/CD40/CD30 family of receptors. Current views of neurotrophin receptor function have tended to interpret p140trk as the high affinity NGF-binding site. To assess if the binding of NGF to p140trk was distinguishable from binding to high affinity sites on neuronal cells, PC12 cell sublines were generated which expressed p140trk alone, or coexpressed both p140trk and p75NGFR. Kinetic analysis of 125I-NGF binding indicates that it has an unusually slow rate of association with p140trk (k + 1 = 8 x 10(5) M-1 s-1). When both p140trk and p75NGFR receptors are coexpressed, the rate of association of NGF is increased 25-fold to produce a higher affinity binding site. An increase in the rate of internalization was also observed. Since high affinity binding and internalization are believed to be prerequisite for the biological activities of NGF, these results suggest that the biological effects by NGF are derived from a novel kinetic binding site that requires the expression of both receptors. The implications of these results with respect to multisubunit polypeptide receptors are discussed.     

c-fos gene expression in postnatal rat retinas with light/dark cycle

We examined the diurnal variation of c-fos gene expression during a 12:12 light/dark cycle in developing rat retinas by in situ hybridization histochemistry. c-fos Gene was not expressed before postnatal day 10 (P10) but was expressed on P15 in the outer nuclear layer throughout the dark period and in the inner nuclear layer and the ganglion cell layer during the light period. These results demonstrated that the earliest c-fos gene expression occurred between P11 and P15. The good correlation between the expression of c-fos gene and the genes coding for proteins involved in phototransduction, in terms of their diurnal variation and in their development, suggested that c-fos gene may play a role in the regulation of these genes in retinal cells during the light/dark cycle.     

Neurotrophin receptor expression is induced in a subpopulation of trigeminal neurons that label by retrograde transport of NGF or fluoro-gold following tooth injury

Tissue responses to injury are regulated by neurotrophins and neurotrophin receptor levels and can involve both retrograde and paracrine/autocrine trophic signaling. To determine how neurotrophins may contribute to the injury response, the timing and the extent of the up-regulation of neurotrophins and their receptors was examined in a model system which is particularly well suited for the analysis of trophic signaling pathways in response to injury. Injury to the occlusal surfaces of rat molar cusps induces a localized increase in nerve growth factor (NGF) expression in the dental pulp within 4-6 h. Radiolabeled NGF was transported in a receptor-mediated fashion from the teeth to a subset of neurons in the trigeminal ganglion within 15 h, indicating that these neurons possess NGF receptors (trk A and/or p75NTR). To test for NGF responses in the tooth sensory afferent neurons, levels of expression of neurotrophins and their receptors were examined by in situ hybridization in the trigeminal ganglion at 0, 4, 12, 20, 28 and 52 h post-injury. Within the maxillary division of the trigeminal ganglion, trk A expression was elevated at 4 h post-injury, with a maximum increase (2-fold) after 52 h. p75NTR was increased by 28 h post-injury and was increased 1.35-fold by 52 h. BDNF mRNA was increased 12 h after injury (1.8-fold), and 2.5-3-fold at 52 h post-injury. The trk B expression was increased only late after injury (28 and 52 h). To determine the receptor/neurotrophin phenotype of trigeminal neurons with projections to the molar teeth, these neurons were double-labeled with the retrograde tracer fluoro-gold and probes for either BDNF or trk B. The results show that tooth-innervating trigeminal neurons express BDNF, but not trk B. The timing of mRNA expression after injury and the phenotype of identified trigeminal neurons suggests a complex signaling cascade in which NGF at the injury site regulates NGF receptor expression at the levels of the cell body as well as increases in BDNF expression. Upregulated BDNF may act in a paracrine fashion on neighboring trigeminal cells expressing trk B. This signaling cascade may be a common feature of the response to mild peripheral inflammatory injuries within nociceptive pathways.     

Effect of radial gradient of temperature on the performance of large-diameter high-performance liquid chromatography columns. I. Analytical conditions

1. The X organ-sinus gland system is a conglomerate of 150-200 neurosecretory cells in the eyestalk of crustaceans. It is the source of a host of peptide neurohormones which partake in the control of a wide range of physiological functions. Distinct families of X organ peptides have been chemically characterized: (a) two chromatophorotropic hormones of small sizes, one of 8 residues and the other of 15-20 residues; and (b) three metabotropic hormones of high molecular weight (70-80 residues), related to the control of blood sugar levels, molting, and gonad activity. Some of these hormones have been identified only in crustaceans; others are common to various arthropod groups. A number of peptides orginally described in other zoological groups are also present in the X organ-sinus gland system; such is the case for members of the FMRF-amide family, enkephalins, and other peptides. 2. Cells specifically containing each hormone have been located in the X organ and some information is available on the cellular and molecular substrate of the biosynthesis, transport, storage, and release of various hormones. The electrical activity of X organ neurons has been recorded at the cell soma, arborizations, axons, and neurosecretory terminals. Conspicuous regional differences have been defined for the various patterns of activity, as well as the distribution of their underlying ion currents. 3. The release of hormones and the electrical activity of X organ neurons are regulated by environmental and endogenous influences, such as light and darkness, stress, and circadian rhythms. These influences appear to be mediated by a host of neurotransmitters/modulators, most noticeably, gamma-aminobutyric acid, 5-hydroxytryptamine and other amines, and enkephalins. Each of these mediators acts upon a definite ionic substrate(s) and exerts specific regulatory effects on X organ cell activity. A given neuron may be under the control of more than one neurotransmitter, and a transmitter may mediate different and even opposite influences on different neurons.     

An epithelial serine protease activates the amiloride-sensitive sodium channel

Sodium balance, and ultimately blood pressure and extracellular fluid volume, is maintained by precise regulation of the activity of the epithelial sodium channel (ENaC). In a Xenopus kidney epithelial cell line (A6), exposure of the apical membrane to the protease inhibitor aprotinin reduces transepithelial sodium transport. Sodium-channel activity can be restored by subsequent exposure to the nonspecific protease trypsin. Using A6 cells and a functional complementation assay to detect increases in ENaC activity, we have cloned a 329-residue protein belonging to the serine protease family. We show that coexpression of this protein with ENaC in Xenopus oocytes increases the activity of the sodium channel by two- to threefold. This channel-activating protease (CAP1) is expressed in kidney, gut, lung, skin and ovary. Sequence analysis predicts that CAP1 is a secreted and/or glycosylphosphatidylinositol-anchored protein: ENaC activity would thus be regulated by the activity of a protease expressed at the surface of the same cell. This previously undiscovered mechanism for autocrine regulation may apply to other ion channels, in particular to members of the ENaC family that are present in neurons and epithelial cells.     

Dror, a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family of Trk-related receptor tyrosine kinases

We have identified a Drosophila gene, Dror, which encodes a putative receptor tyrosine kinase (RTK) and maps to cytological location 31B/C on the second chromosome. In embryos, this gene is expressed specifically in the developing nervous system. The Dror protein appears to be a homolog of two human RTKs, Ror1 and Ror2. Dror and Ror1 proteins share 36% amino acid identity in their extracellular domains and 61% identity in their catalytic tyrosine kinase (TK) domains. Ror1 and Ror2 were originally identified on the basis of the similarity of their TK domains to the TK domains of members of the Trk family of neurotrophin receptors. The Dror protein shows even greater similarity to the Trk proteins within this region than do the human Ror proteins. In light of its similarity to trk and its neural-specific expression pattern, we suggest that Dror may encode a neurotrophic receptor that functions during early stages of neural development in Drosophila.     

Differential regulation by MK801 of immediate-early genes, brain-derived neurotrophic factor and trk receptor mRNA induced by a kindling after-discharge

Transient changes in immediate-early genes and neurotrophin expression produced by kindling stimulation may mediate secondary downstream events involved in kindling development. Recent experiments have demonstrated conclusively that both kindling progression and mossy fibre sprouting are significantly impaired by administration of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801. To further examine the link between kindling, changes in gene expression and the NMDA receptor, we examined the effects of MK801 on neuronal induction of immediate-early genes, brain-derived neurotrophic factor (BDNF) and trk receptor mRNA expression produced by a single electrically induced hippocampal after-discharge in rats. The after-discharge produced a rapid (after 1 h) increase in Fos, Jun-B, c-Jun, Krox-24 mRNA and protein and Krox-20 protein in dentate granule neurons and a delayed, selective expression of Fos, Jun-D and Krox-24 in hilar interneurons. MK801 pretreatment produced a very strong inhibition of Fos, Jun-D and Krox-20 increases in dentate neurons but had a much smaller effect on Jun-B and c-Jun expression. MK801 did not inhibit Krox-24 expression in granule neurons or the delayed expression of Fos, Jun-D and Krox-24 in hilar interneurons. BDNF protein and trk B and trk C mRNA expression were also strongly induced in dentate granule cells 4 h following an after-discharge. MK801 abolished the increase in BDNF protein and trk B, but not trk C mRNA in granule cells at 4 h. These results demonstrate that MK801 differentially regulates the AD-increased expression of a group of genes previously identified as being likely candidates for an involvement in kindling. Because MK801 significantly retards the development of kindling and mossy fibre sprouting, it can be argued that those genes whose induction is not significantly attenuated by MK801 are unlikely to play an important role in the MK801-sensitive component of kindling and the changes in neural connectivity (mossy fibre sprouting) associated with kindling. Conversely, the role in kindling of those genes whose expression was significantly attenuated by MK801 (Fos, Jun-D, Krox-20, trkB and BDNF) requires further examination.     

Rapid suppression of free radical formation by nerve growth factor involves the mitogen-activated protein kinase pathway

Neurotrophins such as nerve growth factor (NGF) regulate neuronal survival during development and are neuroprotective in certain models of injury to both the peripheral and the central nervous system. Although many effects of neurotrophins involve long-term changes in gene expression, several recent reports have focused on rapid effects of neurotrophins that do not involve synthesis of new gene products. Because enhanced formation of reactive oxygen species (ROS) represents one consequence of many insults that produce neuronal death, we hypothesized that neurotrophins might influence neuronal function and survival through acute alterations in the production of ROS. Using an oxidation-sensitive compound, dihydrorhodamine, we measured ROS formation in a central nervous system-derived neuronal cell line (GT1-1 trk) and in superior cervical ganglion neurons, both of which express the transmembrane NGF receptor tyrosine kinase, trkA. There was enhanced production of ROS in both cell types in the absence of NGF that was rapidly inhibited by application of NGF; complete inhibition of ROS generation in GT1-1 trk cells occurred within 10 min. NGF suppression of ROS formation was prevented by PD 098059, a specific inhibitor of MEK (mitogen/extracellular receptor kinase, which phosphorylates mitogen-activated protein kinase). The observation that NGF acutely blocks ROS formation in neurons through activation of the mitogen-activated protein kinase pathway suggests a novel mechanism for rapid neurotrophin signaling, and has implications for understanding neuroprotective and other effects of neurotrophins.     

In vitro cytotoxicity and DNA damage production in Chinese hamster ovary cells and topoisomerase II inhibition by 2-[2''-(dimethylamino)ethyl]-1, 2-dihydro-3H-dibenz[de,h]isoquinoline-1,3-diones with substitutions at the 6 and 7 positions (azonafides)

The p53 tumor suppressor gene encodes a phosphoprotein which when overexpressed can induce growth arrest at the G1 and G2/M phases of the cell cycle, promote differentiation and apoptosis. This paper demonstrates that p53 can associate with trk tyrosine kinase. Expression of a murine temperature-sensitive (ts) p53 mutant in PC12 cells overexpressing trk (a model system to analyse cellular differentiation and signal transduction induced by NGF) induces morphological changes in the absence of NGF stimulation at 32 degrees C but not at 37 degrees C. In cells differentiated by p53, trk, but not EGFr, was hyperphosphorylated on tyrosine. Furthermore trk was not phosphorylated when expressed in Saos-2 cells (human osteosarcoma cells that lack expression of both endogenous trk and p53) at either temperature. However, transfection of ts p53 into these cells induces trk phosphorylation at 32 degrees C in the absence of NGF stimulation. Association of trk and p53 can be detected in NIH3T3 and PC12 cells co-expressing trk and the ts p53 mutant, in NIH3T3 and PC12 cells transfected with trk alone, and in untransfected PC12 cells, showing that overexpressed and/or endogenous trk associates with endogenous, low levels of p53. These data suggest a novel function for p53 which involves the stimulation of signal transduction pathways (mediating morphological properties of cells), possibly through association with and hyperphosphorylation of trk.     

Developmental regulation of apoptosis in dorsal root ganglion neurons

The survival of dorsal root ganglion (DRG) neurons, both in vivo and in vitro, is dependent on the availability of nerve growth factor (NGF) for a transient period early in development after which these neurons become independent of NGF for survival. The precise molecular mechanism by which developing DRG neurons gain independence from NGF has not been determined. We used an in vitro model of DRG neuronal development to test hypotheses that independence from NGF in mature DRG neurons could be caused by developmental regulation of either elements of the NGF withdrawal signal transduction pathway or of proteins important for activation of the apoptosis output pathway. Interruption of phosphotidylinositol-3 kinase activation, by treatment with the specific inhibitor LY294002, resulted in apoptosis in immature but not mature DRG neurons in a manner similar to that observed with NGF withdrawal. Further downstream along the signal transduction pathway, c-JUN phosphorylation occurred in both immature and mature DRG neurons after NGF withdrawal or treatment with LY294002, despite the fact that the older neurons did not undergo apoptosis. In contrast, the ratio of expression of the proapoptotic gene bax to antiapoptotic gene bcl-xL was many times higher in immature than mature neurons, both in vivo and in vitro. Taken together, these results strongly suggest that developmental regulation of NGF withdrawal-induced apoptosis in DRG occurs via control of the relative level of expression of members of the bcl-2 gene family.     

Expression of the apoptosis-effector gene, Bax, is up-regulated in vulnerable hippocampal CA1 neurons following global ischemia

The observation that delayed death of CA1 neurons after global ischemia is inhibited by protein synthesis inhibitors suggests that the delayed death of these neurons is an active process that requires new gene expression. Delayed death in CA1 has some of the characteristics of apoptotic death; however, candidate proapoptotic proteins have not been identified in the CA1 after ischemia. We studied the expression of Bax protein and mRNA, a member of the bcl-2 family that is an effector of apoptotic cell death, after global ischemia in the four-vessel global ischemia model in the rat and compared these results with the expression of the antiapoptotic gene bcl-2. Bax mRNA and protein are both expressed in CA1 before delayed death, whereas bcl-2 protein is not expressed. Bcl-2 protein expression, but not that of Bax, is increased in CA3, a region that is ischemic but less susceptible to ischemic injury. In the dentate gyrus, both Bax and bcl-2 proteins are expressed. The selective expression of Bax in Ca1 supports the hypothesis that Bax could contribute to delayed neuronal death in these vulnerable neurons by an independent mechanism or by forming heterodimers with gene family members other than bcl-2.     

PIPkins1, their substrates and their products: new functions for old enzymes

The phosphatidylinositolphosphate kinases (PIPkins) are a unique family of enzymes that catalyse the production of phosphorylated inositol lipids. Recent advances have revealed that, due to their ability to utilise a number of different lipid substrates (at least in vitro), this family is potentially able to generate several distinct, physiologically important inositol lipids. Despite their importance, however, our understanding of the regulation of the PIPkins and of their physiological role in cellular signalling and regulation is still poor. Here we describe in turn the diverse physiological functions of the known substrates and major products of the PIPkins. We then examine what is known about the members of the PIPkin family themselves, and their characteristics and regulation.     

Changes in the neurons of the lateral geniculate body under the effect of prolonged darkness (cytochemical study)

With the aid of the cytospectrophotometry method the author studied the influence of light deprivation (the exposure of animals to darkness during 8 weeks following birth) on neurons of the dorsal nucleus of the external geniculate body of rats according to the index of glutamatedehydrogenase activity. It was established that unlike the visual cortex, separate groups of neurons which react differently to light deprivation, the majority of nervous cells in the external geniculate body demonstrate similar changes: a drop in the medium activity of glutamatdehydrogenase in neurons and its neurological satellites, a shift of neuron histograms according to this index to the left. These changes are accompanied by an increase of smaller neurons as compared to normal, as well as a drop in the amount of neurons with satellites. The found differences of reactions to light deprivation between the cortical neurons and switch areas of the visual analyzer are discussed in the light of functional traits of such neurons.