Structural determinants of Trk receptor specificities using BNDF-based neurotrophin chimeras

Neurotrophins play very important roles in the development and maintenance of the vertebrate nervous system. In mammals, there are four members of the family: NGF, BDNF, NT-3, and NT-4/5. Members of the neurotrophin family activate different receptors that belong to a class of receptor tyrosine kinases known as “Trks.” For example, NGF is the specific ligand of TrkA, while BDNF activates TrkB. To elucidate which regions of the two neurotrophins determine the receptor specificities, chimeric neurotrophins were constructed using BDNF as the backbone, with various regions being substituted by the corresponding regions of NGF. The activity of the chimeras on the Trk receptors was assayed in transfected fibroblasts ectopically expressing the Trk receptors. Our findings revealed that, although BDNF is absolutely conserved in mammals, substitution of several small variable regions from NGF into the BDNF backbone did not lead to significant loss in TrkB activity or gain in TrkA activity. Moreover, important determinants of TrkB activation might be located in the carboxy-terminal half of BDNF. On the other hand, critical elements for TrkA activation might be located within the amino-terminal half of the mature NGF molecule. © 1996 Wiley-Liss, Inc.     

BDNF and NT-4 differentially modulate neurite outgrowth in developing retinal ganglion cells.

We show here that neurite outgrowth of ganglion cells (RGCs) was selectively enhanced following treatment with BDNF or NT-4 in short-term cultures of dissociated cells derived from the neuroretina of postnatal rats. NT-4 was more effective than BDNF. The effect of NT-3 was variable, whereas NGF and CNTF had no effects upon neurite elongation. The neuritogenic responses of RGCs to both BDNF and NT-4 were prevented by competition with soluble TrkB receptor, and abolished by K252a, a selective inhibitor of the tyrosine kinase activity of Trks. These results indicate that the differentiating effects of BDNF and NT-4 are mediated by TrkB receptors, naturally expressed by RGCs. Developing RGCs treated with these TrkB ligands displayed distinct, albeit partially overlapping, patterns of neurite morphology. BDNF supported predominantly polarized outgrowth, whereas NT-4 induced the appearance of intensely branched symmetrical arbors. The lack of RGCs showing combined morphologies (e.g., highly arborized unipolar cells) suggests distinct mechanisms underlying either elongation or branching, and implicates distinct responses of RGC subsets. We conclude that neurite growth in vitro is extensively promoted by neurotrophins in developing RGCs. Moreover, highly homologous neurotrophins such as BDNF and NT-4, presumably activating via TrkB receptors, selectively control the differentiation of distinct ganglion cell neuritic morphologies.     

The Neurotrophins BDNF, NT-3 and NT-4/5, But Not NGF, Up-regulate the Cholinergic Phenotype of Developing Motor Neurons

Although developing motor neurons express low-affinity nerve growth factor (NGF) receptors, there is no known biological effect of NGF on developing or adult motor neurons. In this study, we found that, unlike NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) stimulated cholinergic phenotype by increasing choline acetyltransferase (CAT) activity in cultures enriched with embryonic rat motor neurons. Ciliary neurotrophic factor (CNTF) also stimulated CAT activity. The effects of BDNF and NT-4/5 on CAT activity appeared to be synergistic with that of CNTF. Cotreatment with BDNF and NT-3 resulted in an additive effect, suggesting that signal transduction was mediated through different high-affinity receptors tyrosine kinases B and C (Trk B and Trk C). However, cotreatment with BDNF and NT-4/5 did not result in an increase in CAT activity greater than that of either BDNF or NT-4/5 alone, suggesting that their effects were mediated via the same receptor Trk B. Supporting our findings that spinal cholinergic neurons are responsive to trophic actions of members of the neurotrophin family, motor neuron-enriched cultures were found to express mRNA for Trk B and Trk C, which have been identified as high-affinity receptors for BDNF and NT-4/5, and NT-3, respectively.     

Neurotrophin-induced trk receptor phosphorylation and cholinergic neuron response in primary cultures of embryonic rat brain neurons.

Tyrosine phosphorylation of trk type neurotrophin receptors in primary cultures of embryonic rat brain cells was studied by immunoprecipitation and immunoblotting. In cultures containing basal forebrain cholinergic neurons, but not in cultures of cerebral cortex, nerve growth factor (NGF) treatment for 4 min induced tyrosine phosphorylation of trk family proteins. Stimulation with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), resulted in a very robust phosphorylation signal in basal forebrain and cortical cultures, suggesting actions of these neurotrophins not only on cholinergic cells but probably on most embryonic brain neurons. Trk tyrosine phosphorylation was completely abolished by 5 microM K-252b. Inhibition was rapid, being evident by 30 s following addition of the drug. Corresponding stimulatory and inhibitory effects were seen for phospholipase-C gamma 1 (PLC gamma 1) and extracellular signal-regulated kinase 1 (Erk1), two enzymes involved in second messenger mechanisms. Our findings indicate involvement of trk receptor activation in the NGF response of basal forebrain cholinergic cells and provide evidence for widespread presence of BDNF and NT-3 responsive neurons in the embryonic brain.     

Neurotrophins regulate proliferation and survival of two microglial cell lines in vitro

Microglia are thought to play a key role in the development and regeneration of the central nervous system although the mechanisms regulating their presence and activity are not fully understood. Substantial evidence suggests that members of the neurotrophin family such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 and -4 (NT-3/4) have a dramatic effect on both neurons and perineuronal cells. This study employed two murine microglial lines, BV-2 and N9, to examine the action of these neurotrophins on the mitotic activity and survival of microglia in vitro. Neurotrophins were incorporated into the media at the time of plating and cell number and levels of mitochondrial dehydrogenase activity (MTT) were determined at various time points in vitro. NGF increased cell number and MTT levels of both cell lines in a dose-dependent manner. BV-2 was more sensitive to NGF than N9. Similar responses were elicited by BDNF, although the sensitivity of each cell line was different than that found for NGF. NT-3 and NT-4 had no effect on cell proliferation. However, NT-4 had an effect on the survival of BV-2 and N9 cells. The response of these cells to neurotrophins was blocked by K252a, a tyrosine kinase inhibitor, suggesting that actions of neurotrophins were mediated by high-affinity tyrosine kinase receptors (Trk). Immunolocalization studies revealed positive Trk (pan) reactivity in the above cell lines and in primary microglia, but an absence of the low-affinity p75 neurotrophin receptor. Western blot analysis supported the above observations. These studies suggest that in addition to their neurotrophic actions, NGF and BDNF may also regulate microglial dynamics, thereby influencing the surrounding milieu during neuronal regeneration.     

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. © 1996 Wiley-Liss, Inc.     

BDN F Down-regulates Neurotrophin Responsiveness, TrkB Protein and TrkB mRNA Levels in Cultured Rat Hippocampal Neurons

Regulation of Trk receptors by their ligands, the neurotrophins, was investigated in dissociated cultures of embryonic day 18 rat hippocampal neurons. Cultures were exposed to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or NT-4/5 for 24 h upon plating followed by factor washout. As determined by immunohistochemical staining and phosphotyrosine blotting, the functional responses to acute stimulation with BDNF, NT-3 and NT-4/5, including c-Fos induction and phosphorylation of Trk and extracellular signal-regulated kinase (ERK) proteins, were significantly decreased after 6 days in culture by prior exposure to BDNF. As determined by Western and Northern blot analysis respectively, there was a parallel down-regulation of TrkB protein as well as of trkB and trkC mRNA levels in BDNF-pretreated cultures. Exposure to NT-3 or NT-4/5 at the same concentrations as BDNF did not down-regulate any of the measured cellular responses or TrkB protein and/or trkB and trkC mRNA levels. Regulation of hippocampal neuronal TrkB protein does not appear to be just a developmental phenomenon, as infusion of BDNF into the hippocampus of adult rats for 6 days produced an 80% decrease in levels of full-length TrkB protein. We thus show that exposure of hippocampal neurons to BDNF, both in culture and in the adult brain, results in down-regulation of TrkB. At least in vitro , this leads to long-term functional desensitization to BDNF. NT-3 and NT-4/5. as well as down-regulation of trkB and trkC mRNA.     

Early BDNF, NT-3, and NT-4 signaling events.

Much more is known about nerve growth factor (NGF) signaling than that initiated by brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or NT-4. We sought to study early BDNF, NT-3, and NT-4 signaling events. Using TrkB-expressing cells, we found that BDNF and NT-4 individually induced tyrosine phosphorylation of TrkB in a dose-dependent fashion. At maximally effective concentrations, BDNF or NT-4 induced robust TrkB tyrosine phosphorylation at 5 min; this progressively declined at 15, 30, and 60 min. Using immunoprecipitation, PI3-kinase and tyrosine phosphorylated PLC-gamma1 and SHC were shown to be associated with tyrosine phosphorylated TrkB in response to both BDNF and NT-4. BDNF and NT-4 induced similar intensities of phosphorylation of TrkB and signaling intermediates at equivalent doses. NT-3 treatment of TrkC-expressing cells induced very similar patterns for induction of TrkC tyrosine phosphorylation and recruitment of signaling intermediates. BDNF, NT-3, and NT-4 caused rapid tyrosine phosphorylation of ERK and SNT. These data suggest that the earliest signaling events for BDNF, NT-3, and NT-4 are very similar to those for NGF.     

Neurotrophins and neuronal versus glial differentiation in medulloblastomas and other pediatric brain tumors

Medulloblastomas are highly malignant and poorly understood childhood neoplasms. To determine if neurotrophins might influence the phenotypic properties of medulloblastoma in a paracrine or autocrine manner, 51 pediatric brain tumors including 20 biopsy specimens of these primitive neuroectodermal tumors (PNETs) and 31 other pediatric brain tumors were studied. Immunohistochemistry was used with antibodies to nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and NT-3, their cognate high affinity receptors as well as to neuronal and glial markers. TrkA, TrkB, and TrkC were observed in 5 (25%), 8 (40%), and 17 (85%), respectively, of these medulloblastomas while NGF, BDNF, and NT-3 were observed in 6 (30%), 8 (40%), and 3 (15%), respectively, and antibodies to neurofilament (NF) and glial fibrillary acidic proteins (GFAP) stained 16 (80%) and 11 (55%), respectively. TrkA and NGF were not observed in the same biopsy samples, while TrkB and BDNF were co-distributed in 6 of the cases, all of which expressed NF proteins. TrkC and NT-3 were co-distributed in 3 of the medulloblastomas, and these areas overlapped with NF protein-positive tumor cells in all 3 cases. In contrast to medulloblastomas, TrkA and NGF co-distributed in other pediatric brain tumors, and both Trk receptors and their neurotrophins co-distributed with GFAP-positive tumor cells in 13 (42%) of the non-PNET pediatric brain tumors. The absence of medulloblastomas that contain NGF and TrkA is consistent with in vitro data demonstrating that NGF-mediated TrkA signaling induces apoptosis. Finally, this study also suggests that BDNF and NT-3 may act in a paracrine or autocrine manner through TrkB and TrkC receptors, respectively, to induce neuronal differentiation in medulloblastomas. Received: 2 October 1997 / Revised, accepted: 29 December 1997     

Gangliosides prevent excitotoxicity through activation of TrkB receptor

Gangliosides protect cerebellar granule cells from excitotoxicity; however, their mechanism of action remains to be fully characterized. GM1 ganglioside has been shown to activate Trk, the tyrosine kinase receptor implicated in the neuroprotective properties of the neurotrophins. In these studies, we used primary cultures of cerebellar granule cells to determine whether gangliosides exert neuroprotective effect via the activation of Trk receptors. We first examined the relative potency of the neurotrophins, brain derived neurotrophic factor (BDNF), neurotrophin-3 and nerve growth factor to prevent glutamate-mediated apoptosis. BDNF was the only neurotrophin that elicited a complete neuronal protection against glutamate. GM1 and its semisynthetic compound LIGA20 also prevented glutamate toxicity, however, LIGA20 was more potent than GM1. Both LIGA20 and BDNF blocked glutamate-mediated activation of caspase-3 and consequently apoptosis; however, the anticaspase-3 activity was seen only when these compounds were added to the cultures several hours before glutamate, suggesting that LIGA20 and BDNF share an identical molecular mechanism. To test this hypothesis, we compared the ability of LIGA20 and BDNF to activate TrkB. Both compounds elicited a similar time-dependent increase in Trk tyrosine phosphorylation. Moreover, the neuroprotective effect of BDNF and LIGA20 was abolished in neurons exposed to the Trk tyrosine kinase inhibitor k252a, demonstrating a relationship between neuroprotection and activation of Trk receptors. Our data suggest that by activating the Trk neurotrophin receptors, gangliosides may be used as neuroprotective agents.     

Cultured basal forebrain cholinergic neurons from postnatal rats show both overlapping and non-overlapping responses to the neurotrophins

Basal forebrain cholinergic neurons respond in vitro and in vivo to nerve growth factor (NGF) and to brain-derived neurotrophic factor (BDNF). It is not clear to what extent the neurons that respond to these two factors, or to neurotrophin-3 or−45 (NT-3;NT-45) are identical or only partially overlapping populations. We have addressed this issue in cultures of basal forebrain neurons derived from 2-week-old postnatal rats, using choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) as cholinergic markers. Cholinergic neuron survival was enhanced in the presence of NGF, BDNF andNT-45.NT-45 was as effective as BDNF. NT-3 was without effect at this age, although in cultures derived from embryonic forebrain, cholinergic differentiation was induced by NT-3. Cotreatment with NGF and BDNF resulted in small, but consistent, increases in the number of ChAT-positive neurons, compared with either factor alone.NT-45 was also found to be additive with NGF, whereas cotreatment with BDNF andNT-45 showed no addivity. NT-3 had no additive effects with any other neurotrophin on any cholinergic parameters in postnatal cultures. Taken together, the results indicate the existence in postnatal rat brain of a large overlapping population of cholinergic neurons that are responsive to ligands for the neurotrophin receptors TrkA (NGF) and TrkB (BDNF andNT-45), but not TrkC (NT-3), and small distinct populations that show specificity for NGF or BDNF but not both. We hypothesize that cholinergic neurons projecting into different regions of the hippocampus may derive trophic support from distinct neurotrophins.     

Highly selective effects of nerve growth factor,brain-derived neurotrophic factor,and neurotrophin-3 on intact and injured basal forebrain magnocellular neurons

Cholinergic neurons of the basal nucleus complex (BNC) respond to nerve growth factor (NCF), the first member of a polypeptide gene family that also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), NGF, BDNF, and NT-3 are enriched in hippocampus. In addition, NGF and, more recently, BDNF have been shown to stimulate the cholinergic differentiation and enhance the survival of BNC cells in vitro. The present investigation was designed to test, in a comparative fashion, the in vivo effects of human recombinant NGF, BDNF, and NT-3 with confirmed activities in vitro on cholinergic and γ-aminobutyric acid (GABA)-ergic BNC neurons. The specific questions asked were whether and, to what extent, biologically active recombinant neurotrophins stimulate the transmitter phenotypes of intact cholinergic and GABAergic neurons of the BNC, and whether, and to what extent, recombinant neurotrophins protect the transmitter phenotypes of axotomized cholinergic and GABAergic neurons of the BNC following complete transections of the fimbria-fornix (measured by ChAT mRNA hybridization). Our results confirm the profound stimulatory and p^75NGFR expression in both intact and axotomized cholinergic neurons and to exert minor effects on some cholinergic markers (e.g., ChAT immunoreactivity). NT-3 had no influence on GABAergic neurons. Taken together, these results indicate that, despite their significant sequence homologies and their shared abundance in target fields of BNC neurons, NGF, BDNF, and NT-3 show striking differences in their efficacies as cholinergic trophic factors. GABAergic neurons of the BNC are resistant to neurotrophins. The result of the present investigation establish that NGF excels among neurotrophins as a trophic factor for intact and injured basal forebrain cholinergic neurons. © 1994 Wiley-Liss, Inc.     

Macrophages express neurotrophins and neurotrophin receptors. Regulation of nitric oxide production by NT-3

In this study we examined the expression of neurotrophins and their receptors in mouse macrophages and the effects of the neurotrophins on nitric oxide secretion. Macrophages expressed TrkB and TrkC but not BDNF, NT-3 or NT-4. LPS induced up-regulation of TrkB and TrkC and of BDNF and NT-3 expression. Treatment of macrophages with NT-3 increased the secretion of nitric oxide in LPS-treated macrophages and this increase was blocked by K252a, a Trk kinase inhibitor. In contrast, BDNF and NT-4 had no significant effects on the induction of nitric oxide. Our results suggest that NT-3 play important roles in the function of macrophages during inflammatory responses and in tissue repair.     

Neurotrophins: The therapeutic potential and concept of minipeptides

Neurotrophins belong to a family of polypeptides that exert control over many aspects of the survival, development, and functioning of structures within the central and peripheral nervous system. Neurotrophins, the nerve growth factor (NGF), the brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), as well as extracellular tyrosine kinase receptors (TrkA, TrkB, and TrkC) are specific targets for therapeutic intervention during different diseases. All these neurotrophins also bind to the p75 receptor, which has many functions depending on the type of cell where it is present. The diversity of neurotrophin effects is determined by ligand-receptor binding and the type of signaling responses that are specific for this interaction. NGF and other neurotrophins are involved in many pathological conditions. It has been shown that an insufficient level of neurotrophins in certain brain structures may be not only an initial cause of Alzheimer??s disease but also may be typical for cerebrovascular dysfunction, brain trauma, cognitive impairments, etc. The therapeutic potential of neurotrophins has been shown in many studies in the last decade but the effectiveness of neurotrophic therapy is limited by the poor diffusion of molecules across the blood-brain barrier and toxic adverse effects. The solution to this problem may be the creation of minipeptides and peptidomimetics that affect the activity of tyrosine-kinase receptors. Some of these structures are combinations of cyclic pentapeptides that facilitate interaction with Trk receptors and exert neuroprotective activity. In this review, we discuss the clinical and experimental data on the results of an alternative strategy that uses these peptidomimetics. These compounds comprise a new group of perspective agents in the therapy of neurodegenerative disorders.     

Axotomy alters neurotrophin and neurotrophin receptor mRNAs in the vagus nerve and nodose ganglion of the rat

Neurotrophins and neurotrophin receptors play an important role in survival and growth of injured peripheral nerves. To study the injury-mediated neurotrophic response in autonomic nerves, we investigated changes in mRNA expression of neurotrophins and their receptors in the transected vagus nerve and nodose ganglion. Studies using in situ hybridization histochemistry showed that axotomy of the cervical vagus nerve resulted in increased expression of mRNAs for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and for TrkA, TrkB, and TrkC receptors in non-neuronal cells at both the proximal and distal segments of the transected cervical vagus nerve. Moreover, NGF protein was increased in the distal end, and NT-3 protein was increased in both the proximal and the distal ends of the transected nerve 3 days after axotomy. No change of p75(NTR) mRNA was detected in the transected vagus nerve. The induction of each neurotrophin and Trk receptor mRNA was apparent within 1 day after the axotomy and was sustained at least 14 days. By 45 days after the axotomy, a time when axonal reconnection with target tissue is made (integrity of the nerve-target connection was confirmed by the retrograde transport of FluoroGold from the stomach to vagal cell bodies), the levels of neurotrophin and Trk mRNAs in the vagus nerve declined to pre-axotomy levels. TrkA, TrkC, and p75(NTR) mRNA-containing vagal sensory neurons in the nodose ganglion were reduced in number after cervical vagotomy. Neurotrophin-mRNA-containing neurons were not found in the nodose ganglia from either intact or vagotomized rats. The axotomy-induced up-regulation of neurotrophins and Trk receptors mainly in the non-neuronal cells at or near the site of transection suggests that neurotrophins are involved in the survival and regeneration process of the vagus nerve after injury.     

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 gp140^trk. 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 β-loops at one end of the NGF molecule, forming a cleft that could specifically interact with high affinity to the signalling NGF receptor gp140^trk.Copyright © 1994 Wiley-Liss, Inc.     

Regionally specific effects of BDNF on oligodendrocytes

To define the effects of neurotrophins on oligodendrocytes, we monitored NGF, BDNF and NT-3 actions on basal forebrain (BF) and cortical populations. NGF, BDNF and NT-3 applied to BF oligodendrocytes elicited increases in expression of myelin basic protein (MBP) and enhanced the numbers of MBP+ cells, without affecting total cell numbers. In the cortex, however, while NGF and NT-3 influenced MBP expression, BDNF was without effect. To explore this apparent regional difference in BDNF action, we compared expression of the neurotrophin receptors trkA, trkB and trkC. While BF cells expressed all three trks, cortical cells did not express the full-length BDNF receptor, trkB. Interestingly, in no case was any receptor expressed by all oligodendrocytes, indicating that oligodendrocytes may be heterogeneous within a brain region. The data suggest that BF oligodendrocytes are influenced by BDNF to express MBP and are distinct in this ability from cortical cells.     

Enhanced survival and morphological features of basal forebrain cholinergic neurons in vitro: role of neurotrophins and other potential cortically derived cholinergic trophic factors

The present study examined survival- and growth-enhancing effects of cortical cells on basal forebrain cholinergic neurons (BFCNs) in culture and the degree to which endogenous nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) contribute to those trophic effects. When fetal (17 days of gestation) basal forebrain (BF) cells were grown for 5 days in coculture with cortical neurons, staining for acetylcholinesterase (AChE) showed a threefold increase in the number of BFCNs relative to BF cultures without cortex. Most of these labeled cells also displayed enhanced somatic, dendritic, and axonal growth. Coculturing cortical neurons with BF cells taken from postnatal animals produced similar results but with a somewhat greater degree of morphologic enhancement. Function-neutralizing antibodies to NGF, BDNF, and NT-3 were employed to determine whether they would block the trophic effects of cortical neurons on postnatal BFCNs. Although no significant changes in numbers or morphological features of AChE(+) neurons were observed with treatment with individual antibodies, cocultures treated with a combination of all three antibodies displayed fewer morphologically enhanced AChE(+) cells and more nonenhanced cells; the total number of AChE(+) neurons was not significantly changed. Treatment of pure BF cultures with exogenous NGF, BDNF, and NT-3 increased the number of AChE(+) neurons but did not reproduce the morphologic enhancement of cortical cells on BFCNs. These results suggest that neurotrophins by themselves can increase survival of postnatal BFCNs in culture and may work in concert with other unknown cortically derived factors to enhance BFCN morphologic differentiation. The unidentified cortical factors may also have strong survival-enhancing effects on BFCNs that are independent of the known neurotrophins.     

Synergistic effects of BDNF and NT-3 on postnatal spiral ganglion neurons

Neurotrophins have profound effects on the development and maintenance of neurons that compose the VIIIth cranial nerve. In the auditory division of the nerve, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been localized to the sensory epithelium, and their respective high-affinity tyrosine kinase receptors (TrkB and TrkC) are expressed within the neuronal population. By using a culture methodology that allows evaluation of single neurons, we determined that BDNF and neurotrophin-4 (NT-4), which both bind to the TrkB high-affinity receptor, greatly enhanced neuron survival above control cultures. NT-3, which acts via the TrkC high-affinity receptor, also increased survival, but to a lesser extent. By testing a variety of neurotrophin concentrations and combinations, we observed that simultaneous activation of the TrkB and TrkC receptors synergistically promoted neuron survival compared to cultures that contained either neurotrophin alone at the same total concentration. Antibody labeling showed that the high-affinity Trk receptors were localized predominantly to the neurons and not to the surrounding satellite cells; furthermore, TrkB- and TrkC-specific antibodies each labeled 100% of the cultured neurons. These results suggest that synergistic interactions between BDNF and NT-3 may be crucial for spiral ganglion neuron survival during the final stages of development. J. Comp. Neurol. 386:529–539, 1997. © 1997 Wiley-Liss, Inc.