Faculty Bibliography

Quantitative in situ hybridization techniques were used to examine the effects of lesions which sever hippocampal cholinergic and cortical afferents on p75NGFR mRNA-expressing cells located in the medial septum (MS) and the vertical (VDB) and horizontal (HDB) limbs of the diagonal band of Broca. Animals received either bilateral transection of the fimbria/fornix, unilateral transection of the angular bundle, or sham surgery. Four days later, animals were sacrificed and sections through the MS, VDB and HDB were processed for detection of the p75NGFR mRNA using in situ hybridization techniques previously described (Mol. Brain Res., 6 (1989) 275-287). Transection of the fimbria/fornix and angular bundle differentially affected p75NGFR-expressing cells in the MS, VDB and HDB within 4 days after injury, in ways which were consistent and correlate with subsequent effects on cell survival, synaptic reorganization and growth. In particular, in the MS and VDB, transection of the fimbria/fornix resulted in a significant decrease in the size of p75NGFR-expressing cells (reductions of 25.9% and 15.1% respectively) which was accompanied by a significant reduction (37.9% and 12.7% fewer grains/cell) in relative levels of p75NGFR mRNA. In contrast, in the HDB, transection of the fimbria/fornix had no significant effect on the average size of p75NGFR-expressing cells; however, a significant increase (49%) in the mean relative level of p75NGFR mRNA was observed which may, in turn, reflect a large increase (as much as 2-3 fold) in the levels of p75NGFR mRNA expressed by a subpopulation of hippocampally projecting cholinergic neurons located in the HDB. Finally, transection of the angular bundle resulted in small, but significant increases (9.4% and 10.9%) in relative levels of p75NGFR mRNA in the MS and VDB, as well as an increase (19.6%) in the number of p75NGFR mRNA-expressing cells in the HDB, on the injured side. No increases in p75NGFR expression in the MS, VDB or HDB contralateral to the lesion were observed; however, a decrease in the size (6.9%) and message content (19.4%) of p75NGFR-expressing cells was detected in the MS contralateral to the lesion. Most importantly, all of these effects are consistent with the subsequent effects of these lesions on the survival of basal forebrain cholinergic cells, and the reorganization and growth of cholinergic afferents to the hippocampal formation.(ABSTRACT TRUNCATED AT 400 WORDS)

The trk tyrosine kinase proto-oncogene product gp140prototrk binds nerve growth factor (NGF) and is rapidly and selectively activated by this neurotrophic factor. To determine whether gp140prototrk is involved in transducing a functional NGF signal, PC12 cell mutants (PC12nnr) deficient in high affinity NGF binding and unresponsive to NGF were used. Northern analysis revealed that these mutant cells have greatly reduced levels of trk expression. PC12nnr cultures were transiently transfected with expression vectors encoding the full-length rat trk cDNA and assessed for responsiveness to NGF. Expression of exogenous trk rescued the capacity for NGF-promoted neurite outgrowth, cellular hypertrophy, and serum-free survival by these cells. These results indicate that gp140prototrk is necessary for functional NGF signal transduction

Protein tyrosine phosphorylation is a potential mechanism for initial signaling in PC12 cells during differentiation in response to nerve growth factor (NGF). NGF-induced tyrosine phosphorylation has been found to be initiated by the trk protooncogene, which participates in the formation of high-affinity NGF binding sites. In contrast to transfection of wild-type low-affinity p75 NGF receptors, transfection of p75NGFR with mutations in the cytoplasmic domain resulted in an inability of NGF to elicit tyrosine phosphorylation of intracellular substrates, indicating that p75NGFR is involved in initiating phosphorylation events by NGF. Even though the p75NGFR receptor does not possess any inherent tyrosine kinase activity, these experiments demonstrate that the p75NGFR has a potential role in NGF-induced tyrosine phosphorylation

Nerve growth factor (NGF) binds to a low affinity cell surface receptor (p75NGFR) which contains four extracellular repeats, rich in cysteine residues and negatively charged. We have made mutations in the receptor cDNA by inserting linkers in specific domains of the receptor. Nearly all the mutations caused a change in the predicted charge, and resulted in either an insertion or deletion in the primary sequence. Stably transfected fibroblasts were assayed for NGF binding by affinity cross-linking with 125I-NGF. Appropriate expression of the mutated receptors was monitored by rosetting with monoclonal antibodies and by metabolic labeling followed by immunoprecipitation. Although the mutant receptors were recognized by monoclonal antibodies, insertions and deletions in the third and fourth cysteine-rich regions of the receptor had a detrimental effect upon NGF binding. Insertions made outside the cysteine-rich region or in the cytoplasmic domain did not inhibit the ability of 125I-NGF to bind to the receptor, as assessed by affinity cross-linking. A chimeric human-rat NGF receptor transfected into fibroblasts indicates that NGF binding and monoclonal antibody recognition sites are separated but contained within the four cysteine repeats

To determine the domains of the low-affinity nerve growth factor (NGF) receptor required for appropriate signal transduction, a series of hybrid receptors were constructed that consisted of the extracellular ligand-binding domain of the human epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and cytoplasmic domains of the human low-affinity NGF receptor (NGFR). Transfection of these chimeric receptors into rat pheochromocytoma PC12 cells resulted in appropriate cell surface expression. Biological activity mediated by the EGF-NGF chimeric receptor was assayed by the induction of neurite outgrowth in response to EGF in stably transfected cells. Furthermore, the chimeric receptor mediated nuclear signaling, as evidenced by the specific induction of transin messenger RNA, an NGF-responsive gene. Neurite outgrowth was not observed with chimeric receptors that contained the transmembrane domain from the EGFR, suggesting that the membrane-spanning region and cytoplasmic domain of the low-affinity NGFR are necessary for signal transduction

The trk proto-oncogene encodes a 140-kilodalton, membrane-spanning protein tyrosine kinase (p140prototrk) that is expressed only in neural tissues. Nerve growth factor (NGF) stimulates phosphorylation of p140prototrk in neural cell lines and in embryonic dorsal root ganglia. Affinity cross-linking and equilibrium binding experiments with 125I-labeled NGF indicate that p140prototrk binds NGF specifically in cultured cells with a dissociation constant of 10(-9) molar. The identification of p140prototrk as an NGF receptor indicates that this protein participates in the primary signal transduction mechanism of NGF

Nerve growth factor (NGF) interacts with two different low-affinity receptors that can be distinguished by affinity crosslinking. Reconstitution experiments by membrane fusion and transient transfection into heterologous cells indicate that high-affinity NGF binding requires coexpression and binding to both the low-affinity NGF receptor and the tyrosine kinase trk gene product. These studies reveal a new growth factor receptor-mediated mechanism of cellular differentiation involving trk and the low-affinity NGF receptor

In order to study regulation of the nerve growth factor (NGF) receptor during embryogenesis in chick brain, we have used affinity crosslinking of tissues with 125I-NGF. NGF interacts with high- and low-affinity receptors; high-affinity receptors are required for the majority of NGF's actions. Most measurements of receptor levels do not distinguish between high- and low-affinity forms of the receptor. We have used the lipophilic crosslinking agent HSAB to identify the high-affinity, functional receptor during development of the chicken central nervous system. A peak of expression during Embryonic Days 5-10 was detected in all regions of the chicken central nervous system, but, shortly after birth, only the cerebellar region displays significant levels of NGF receptor protein. The time course of expression confirms the dramatic regulation of the NGF receptor gene during defined embryonic periods. The detection of high-affinity NGF receptors in brain and neural retina provides strong evidence that NGF is involved in essential ontogenetic events in the development of the chicken central nervous system