Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon tract formation in vivo.

The EphA4 receptor tyrosine kinase regulates the formation of the corticospinal tract (CST), a pathway controlling voluntary movements, and of the anterior commissure (AC), connecting the neocortical temporal lobes. To study EphA4 kinase signaling in these processes, we generated mice expressing mutant EphA4 receptors either lacking kinase activity or with severely downregulated kinase activity. We demonstrate that EphA4 is required for CST formation as a receptor for which it requires an active kinase domain. In contrast, the formation of the AC is rescued by kinase-dead EphA4, suggesting that in this structure EphA4 acts as a ligand for which its kinase activity is not required. Unexpectedly, the cytoplasmic sterile-alpha motif (SAM) domain is not required for EphA4 functions. Our findings establish both kinase-dependent and kinase-independent functions of EphA4 in the formation of major axon tracts.

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.

Expression of neurotrophins and trk receptors in the avian retina.

Using the RNase protection assay, we have found that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are expressed in the avian retina during development. The expression peaks around embryonic days 12-15, with decreasing levels at later stages of development. Abundant levels of NGF and BDNF but low levels of NT-3 mRNA were found in the adult retina. We also found that light/darkness regulated the levels of NGF and BDNF mRNAs but not the levels of NT-3 mRNA in the 5-day-old chicken retina. It was demonstrated that NGF and BDNF mRNA levels were up-regulated by light exposure. The cellular localization of mRNA expression for the neurotrophins and neurotrophin receptors TrkA, TrkB, and TrkC in the retina was studied using in situ hybridization. The patterns of NGF and trkA mRNA expression were very similar and were localized to the external part of the inner nuclear layer on the border with the outer plexiform layer and corresponded to the localization of horizontal cells. NT-3 labeling was also found over the external part of the inner nuclear layer, whereas trkC mRNA was found over all layers in the retina. BDNF labeling was found over all layers in the retina, whereas TrkB labeling was intense over cells in the ganglion cell layer, which is in agreement with the response of ganglion cells to BDNF stimulation. Functional neurotrophin receptors were suggested by the response of retinal explants to neurotrophin stimulation. These data indicate that the neurotrophins play local roles in the retina that involve interactions between specific neuronal populations, which were identified by the localization of the Trk receptor expression. The data also suggest that NGF and BDNF expression is regulated by normal neuron usage in the retina.

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.

The synaptic protein encoded by the gene Slc10A4 suppresses epileptiform activity and regulates sensitivity to cholinergic chemoconvulsants.

The expanding number of disease-causing dysfunctions of synaptic proteins illustrates the importance of investigating newly discovered proteins involved in neuronal transmission. The gene Slc10A4 encodes a recently described carrier protein present in pre-synaptic terminals of cholinergic and monoaminergic neurons. The biological significance of this recently described transporter protein is currently unknown. We here investigated whether absence of the Slc10a4 protein has any impact on function of the cholinergic system. We first investigated the sensitivity of Slc10a4 null mice to cholinergic stimulus in vitro. In contrast to wild type mice, gamma oscillations occurred spontaneously in hippocampal slices from Slc10a4 null mice. Furthermore, moderate treatment of Slc10a4 null slices with the cholinergic agonist carbachol induced epileptiform activity. In vivo, 3-channel EEG measurements in freely behaving mice revealed that Slc10a4 null mice had frequent epileptiform spike-activity before treatment, and developed epileptic seizures, detected by EEG and accompanied by observable behavioral components, more rapidly after injection of the cholinergic agonist pilocarpine. Similar results were obtained on non-operated mice, as evaluated by behavioral seizures and post mortem c-Fos immunohistochemistry. Importantly, Slc10a4 null mice and wild type control mice were equally sensitive to the glutamatergic chemoconvulsant kainic acid, demonstrating that absence of Slc10a4 led to a selective cholinergic hypersensitivity. In summary, we report that absence of the recently discovered synaptic vesicle protein Slc10a4 results in increased sensitivity to cholinergic stimulation.

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.

The Src homology 2 domain protein Shb transmits basic fibroblast growth factor- and nerve growth factor-dependent differentiation signals in PC12 cells.

To assess a possible role for the Src homology 2 (SH2) domain adaptor protein Shb in PC12 cell signal transduction and differentiation, we have investigated the expression of Shb in PC12 cells and found that the differentiation factors nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), as well as the PC12 cell mitogen epidermal growth factor, increased Shb protein expression and Shb mRNA steady-state levels. Two PC12 cell clones stably overexpressing the Shb cDNA exhibited enhanced NGF- or bFGF-induced differentiation, assessed as neurite outgrowth. This effect showed no direct correlation to mitogen-activated protein kinase activation, although the mitogen-activated protein kinase/kinase inhibitor PD-98059 caused a partial inhibition of neurite outgrowth. Furthermore, it was found that the Shb-overexpressing cells extended neurites in response to epidermal growth factor. The effects of Shb overexpression on neurite outgrowth required a functional SH2 domain because PC12 cells expressing Shb with an inactivated SH2 domain did not differentiate more readily in response to NGF. Tyrosine phosphorylation of the p13 Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target protein in response to bFGF was strongly inhibited by Shb overexpression, without correlating with the corresponding rate of neurite outgrowth. NGF-induced tyrosine phosphorylation of phospholipase Cgamma, TrkA, and Shc was unaltered in the Shb-overexpressing cells, whereas that of Shb was greatly enhanced in these cells compared with control PC12-neo cells. In addition, an NGF-activated Mr 140,000 phosphotyrosine protein was found to be associated with Shb in immunoprecipitation experiments. The data in this study suggest that Shb is involved in transmission of NGF- and bFGF-dependent differentiation signals in PC12 cells.

Two restricted sites on the surface of the nerve growth factor molecule independently determine specific TrkA receptor binding and activation.

Nerve growth factor (NGF) and neurotrophin-3 (NT-3) mediate activities such as survival, differentiation, and proliferation in various subsets of neurons. In this report, we define precisely the residues in human NGF responsible for NGF biological activity and binding specificity to the neurotrophin receptor TrkA. In earlier studies we defined five amino acid residues of NGF which confer NGF-like activity to NT-3 when replacing corresponding residues in the 120-amino acid long NT-3 molecule. Using this gain-of-function strategy we report the further dissection of this functional epitope. We also define another motif separated topographically in the NGF dimer and determined to be independently responsible for NGF specificity. The first of the two motifs determined to elicit NGF specificity is defined by the residues Val-48, Pro-49, and Gln-96, which are situated in the two top beta-loops of NGF. The second motif is represented by residues Pro-5 and Phe-7 situated in the proximal part of the NH2 terminus. Both motifs contain structurally important residues revealing a novel principle, where specificity for neurotrophin ligand-receptor interactions could be determined by variable residues modifying the conformation of the neurotrophin backbone. These findings will enhance further the possibility of mimicking NGF with low molecular weight compounds.

Do adolescents' own intentions regarding healthy behaviours affect outcome? A two-year prospective study.

Adolescents' own intentions regarding health behaviours, in addition to their context, are believed to be important for the health habits they chose. This was studied prospectively over a 2-y period. A total of 552 students, 391 aged 13 y and 161 aged 15 y, reported their health and problem behaviours, socioeconomic background and intentions regarding health behaviours through questionnaires in 1991 and in 1993. Outcome dealt with three domains: health habits; acquisition of adult lifestyles; and problem behaviours. The material was analysed for correlations. Significant results were entered into multiple regression stepwise procedures. As expected, already having initiated adult lifestyles or problem behaviours were the most important factors associated with such behaviours 2 y later. Further analyses were then limited to those students who had not started such lifestyles, in order to determine what factors kept them from doing so in a 2-y span. Key predictors for healthy behaviours were adolescents' own decisions not to engage in adult lifestyles or risky behaviours, family processes consistent with support and school satisfaction. Association with peer groups where smoking and drinking were commonplace predicted less optimal behaviours. Gender or socioeconomic factors were not predictive. The results support a comprehensive approach to health promotion during adolescence.

Specificity of neurotrophin-3 determined by loss-of-function mutagenesis.

Neurotrophin-3 (NT-3) is a member of the family of neurotrophic factors, which also includes nerve growth factor (NGF) and which have specific activities on different subsets of vertebrate neurons. The aim of this study was to determine which residues in NT-3 direct its specificity to the cognate TrkC receptor. It was possible to replace 80% of the residues in NT-3 with NGF residues without loss of specific activity. Residues D72, Y85, R87, W101, S107, and A111, together with either the residues F12, V18, V20, M37, V42, F54, and K57 or the variable regions IV and V, accounted for the specificity of NT-3. It is concluded that NGF and NT-3 use overlapping as well as separated regions for determination of specificities for their cognate receptors TrkA and TrkC, respectively.

Gender differences in early adolescence in factors related to outcome of healthy behaviours two years later.

In this study, 390 students comprising 89.7% of the student population answered a questionnaire on health and health behaviours, socioeconomic status (SES), school, family and peer relationships, and intentions regarding behaviours at 13.5 and 15.5 y of age. The aim was to investigate whether there were gender differences in predictors of outcome of behaviours with impact on health at 15.5 y of age. Outcome dealt with three domains: health habits, acquisition of adult lifestyles, and problem behaviours. The material was analysed for correlations. Significant results were entered into multiple regression stepwise procedures. As expected, having already initiated adult lifestyles or exhibiting problem behaviours at 13.5 y were the most important factors associated with such behaviours 2 y later. Further analyses were then limited to those students who had not started such lifestyles to see what factors kept them from doing so in a 2-y period and what gender differences existed, if any. Results differed for the genders, with more boys being more concrete and behaviour-oriented, representing a sample of boys with late to normal puberty. Girls more often expressed intentions regarding behaviour, which predicted positive outcomes. Both genders were prudent in most areas studied: they were well adapted at school, had positive self-esteem and were not moving in circles where smoking and drinking were abundant. The conclusion is that young adolescents who do not start risky behaviours between the ages of 13 and 15 y live in a supportive context, which allows for positive interactions. The next step would be to investigate if alternative strategies may be used to implement similar options for those who lack such support in their natural environment.

Molecular phylogeny and evolution of the neurotrophins from monotremes and marsupials.

We have investigated the phylogenetic relationships of monotremes and marsupials using nucleotide sequence data from the neurotrophins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The study included species representing monotremes, Australasian marsupials and placentals, as well as species representing birds, reptiles, and fish. PCR was used to amplify fragments encoding parts of the neurotrophin genes from echidna, platypus, and eight marsupials from four different orders. Phylogenetic trees were generated using parsimony analysis, and support for the different tree structures was evaluated by bootstrapping. The analysis was performed with NGF, BDNF, or NT-3 sequence data used individually as well as with the three neurotrophins in a combined matrix, thereby simultaneously considering phylogenetic information from three separate genes. The results showed that the monotreme neurotrophin sequences associate to either therian or bird neurotrophin sequences and suggests that the monotremes are not necessarily related closer to therians than to birds. Furthermore, the results confirmed the present classification of four Australasian marsupial orders based on morphological characters, and suggested a phylogenetic relationship where Dasyuromorphia is related closest to Peramelemorphia followed by Notoryctemorphia and Diprotodontia. These studies show that sequence data from neurotrophins are well suited for phylogenetic analysis of mammals and that neurotrophins can resolve basal relationships in the evolutionary tree.

Lampetra fluviatilis neurotrophin homolog, descendant of a neurotrophin ancestor, discloses the early molecular evolution of neurotrophins in the vertebrate subphylum.

We have isolated a neurotrophin from the lamprey that permitted us to perform a phylogenetic analysis of the neurotrophin gene family that dates back more than 460 million years to the early vertebrate ancestors. The results show that the neurotrophin gene family was originally formed by two subsequent duplications. The duplication that formed nerve growth factor, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 occurred after the split of lampreys but before the split of cartilaginous fish from the main vertebrate lineage. Compilation of chromosomal gene maps around the neurotrophins shows that they are located in paralogous regions, suggesting that the genes were formed at major duplication events possibly by complete genome doubling. Analysis of two isolated Trk receptor sequences shows similar results as for the lamprey neurotrophin. Multiple neurotrophin and Trk genes, including neurotrophin-6 and -7, have been found in bony fish, and we suggest that the extra genes were formed by an additional duplication in the bony fish lineage. Analysis of lamprey Trk mRNA expression in the adult brain shows that the genes are expressed in all regions analyzed so far. Together, the results suggest that the duplications of ancestral neurotrophin and Trk genes at an early vertebrate stage have permitted evolution to bring about differential neurotrophin and Trk expression, thereby allowing the formation of specific functions in selective neuronal populations.

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.

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.

Ephrin-B3 is the midline barrier that prevents corticospinal tract axons from recrossing, allowing for unilateral motor control.

Growing axons follow highly stereotypical pathways, guided by a variety of attractive and repulsive cues, before establishing specific connections with distant targets. A particularly well-known example that illustrates the complexity of axonal migration pathways involves the axonal projections of motor neurons located in the motor cortex. These projections take a complex route during which they first cross the midline, then form the corticospinal tract, and ultimately connect with motor neurons in the contralateral side of the spinal cord. These obligatory contralateral connections account for why one side of the brain controls movement on the opposing side of the body. The netrins and slits provide well-known midline signals that regulate axonal crossings at the midline. Herein we report that a member of the ephrin family, ephrin-B3, also plays a key role at the midline to regulate axonal crossing. In particular, we show that ephrin-B3 acts as the midline barrier that prevents corticospinal tract projections from recrossing when they enter the spinal gray matter. We report that in ephrin-B3(-/-) mice, corticospinal tract projections freely recross in the spinal gray matter, such that the motor cortex on one side of the brain now provides bilateral input to the spinal cord. This neuroanatomical abnormality in ephrin-B3(-/-) mice correlates with loss of unilateral motor control, yielding mice that simultaneously move their right and left limbs and thus have a peculiar hopping gait quite unlike the alternate step gait displayed by normal mice. The corticospinal and walking defects in ephrin-B3(-/-) mice resemble those recently reported for mice lacking the EphA4 receptor, which binds ephrin-B3 as well as other ephrins, suggesting that the binding of EphA4-bearing axonal processes to ephrin-B3 at the midline provides the repulsive signal that prevents corticospinal tract projections from recrossing the midline in the developing spinal cord.