BMP enhances transcriptional responses to NGF during PC12 cell differentiation.

Bone morphogenetic proteins (BMPs) enhance neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. To investigate the mechanism of this potentiating effect, real-time PCR was used to analyze the expression of 45 selected genes. A robust increase in expression of 10 immediate early genes including Egr1-4, Hes1, Junb, Jun and Fos was observed already after 1 h treatment with NGF alone. NGF plus BMP4 further increased these transcripts at 1 h and activated 18 additional genes. BMP4 alone induced Smad6, Mtap1b and Hes1. Egr3 was the gene most strongly upregulated by NGF and BMP4. However, luciferase assays showed that the cloned Egr3 proximal promoter was not involved in the BMP4 potentiation. Blocking Egr3 and Junb function by dominant-negative constructs reduced neurite outgrowth under stimulating conditions, proving that activation of members of both the Egr and Jun families is necessary for maximal PC12 cell response to NGF and BMP4.

Bone morphogenetic protein signalling in NGF-stimulated PC12 cells.

Bone morphogenetic proteins (BMPs) are shown to potentiate NGF-induced neuronal differentiation in PC12 phaeochromocytoma cells grown on collagen under low-serum conditions. Whereas, cell bodies remained rounded in control medium or with only BMPs present, addition of BMP4 or BMP6 robustly increased the neuritogenic effect of NGF within 2 days. NGF-increased phosphorylation of p44(Erk1) and p42(Erk2) between 2 and 24h was unaffected by addition of BMP6. PC12 cells transfected with the SBE(4x)-luc reporter showed that BMP4 significantly increased receptor-activated Smad activity. Expression of constitutively active BMP receptor ALK2 activating Smad1 and Smad5 resulted in a strong increase in the SBE(4x)-luc reporter response. Adding the inhibitory Smad7 drastically reduced this signal. In contrast to wild-type (wt) Smad5, a Smad5 variant lacking five Erk phosphorylation sites in the linker region (designated Smad5/5SA) showed a strong background transcriptional activity. A fusion construct (Gal4-Smad5/5SA) was also highly transcriptionally active. Addition of the MEK inhibitor U0126 to PC12 cells expressing Gal4-Smad5/wt did not increase background transcriptional activity. However, upon activation by constitutively active ALK2 both Gal4-Smad5/wt and Gal4-Smad5/5SA strongly stimulated transcription. The data show that serine residues of the linker region of Smad5 reduce spontaneous transcriptional activity and that NGF-activated Erk does not antagonise BMP signalling at this site. Hence, NGF and BMP signals are likely to interact further downstream at the transcriptional level in neuronal differentiation of the PC12 cells.

Potentiating interactions between morphogenetic protein and neurotrophic factors in developing neurons.

mRNA for bone morphogenetic protein receptor type II (BMPR-II) was mapped to different neurons in peripheral ganglia and spinal cord of the chicken embryo. The expression of this serine/threonine kinase receptor partially overlaps with that of tyrosine kinase receptors Trk and Ret. Biological activities of osteogenic protein-1 (OP-1), a documented ligand for BMPR-II, were tested in explanted embryonic chicken ganglia and dissociated ganglionic neurons. OP-1 had only a limited stimulatory effect on neuronal survival. However, OP-1 combined with either neurotrophin-3 (NT-3, a relative of nerve growth factor) or glial cell line-derived neurotrophic factor (GDNF) potentiated neuronal survival three- to fourfold. We also show that OP-1 strongly potentiates nerve fiber outgrowth from ganglia stimulated with NT-3 or GDNF. Signaling by BMPR-II in neurons may potentiate the tyrosine kinase pathway activated by NT-3 and GDNF. The data suggest that morphogenetic proteins may modulate neurotrophic activities during neuronal development and plasticity.

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.

Molecular cloning of the chicken trkA and its expression in early peripheral ganglia.

The neurotrophin tyrosine kinase receptors trkA, trkB, and trkC have been isolated and sequenced from several mammalian species. Their cognate ligands nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and neurotrophin-3 (NT-3) act as survival and trophic factors for neurons in the peripheral nervous system (PNS). In this study we have focused on the isolation and expression of the chicken trkA homologue. In addition to a near full-length cDNA sequence described, including an extracellular six amino-acid motif earlier found in neuronal TrkA in human and rat, a novel insert of 150 base pairs (bp) between subdomains IX and X in the otherwise well-conserved intracellular kinase domain is reported. Phylogenetic analysis showed the relationship between chicken trkA and the mammalian trkA receptors. Comparisons of the extracellular domains showed some amino-acid motifs of putative NGF binding function to be well conserved in chicken TrkA. The early expression of trkA mRNA, including the alternatively spliced insert form, was localized by in situ hybridization. As early as embryonal day 3 (E3), trkA mRNA is expressed in the condensing dorsal root ganglia, and at E4 distinct trkA mRNA expression appears in the primary sympathetic chain ganglia. Finally, using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach, we found that among several tested growth factors only fibroblast growth factor-2 (FGF-2) upregulated trkA mRNA expression in E9 sympathetic ganglion explants. This upregulation of trkA was corroborated by subsequent NGF-stimulated fiber outgrowth.

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.

Monitoring release of neurotrophic activity in the brains of awake rats.

Intracerebral microdialysis of awake rats was used to monitor the possible release of neurotrophic factors from brain cells in response to injury and excitation. Perfusates were tested with ganglia bioassays and enzyme immunoassay. Trophic activity was released after implantation of the microdialysis probe into the hippocampus but not into the striatum, as assessed by increased nerve fiber outgrowth from Remak's ganglion. Kainic acid treatment significantly increased the release of trophic activity from hippocampal sites. These findings suggest that the brain responds to mechanical injury as well as to certain excitatory stimuli by regional extracellular release of neurotrophic activity that is not identical to the actions of known neurotrophic factors.

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.