Characterization of neurotrophin receptors by affinity crosslinking

Neurotrophic factors regulate the developmental survival and differentiation of specific neuronal populations. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are members of the nerve growth factor (NGF) protein family, also known as the neurotrophins. Insights into the different roles of neurotrophins can be gained by studying the expression of their functional receptors. Here we report the development of procedures for their radiolabeling and efficient crosslinking to specific cell-surface receptors. BDNF and NT-3 receptors in cell lines and tissue preparations expressing receptors for the 2 neurotrophins have been identified using this affinity crosslinking procedure. Like NGF, BDNF and NT-3 crosslinking to the low affinity NGF receptor (p75^NGFR) on PC12 cells. BDNF and NT-3 also crosslinked to cells expressing p145^trkB protein, producing an approximately 160 kD neurotrophin-receptor complex. Crosslinking of the 2 neurotrophins in vivo to specific trk family members in many areas of the central nervous system also produced a 160 kD receptor complex. However, in all brain regions a complex of approx. 100 kD could also be identified, all or most of which represents crosslinking to a truncated from of trkB. The broad distribution of BDNF and NT-3 receptors throughout the CNS suggests that neurotrophins may have yet unrecognized functions on specific neuronal populations. BDNF and NT-3 receptors were also found in brain areas in which the neurotrophins themselves are also synthesized, suggesting that beyond long-range trophic effects, these proteins may also act as autocrine or short-range paracrine regulators. © 1993 Wiley-Liss, Inc.     

Neurotrophin gene expression by cell lines derived from human gliomas

The expression of neurotrophin (NGF, BDNF, and NT-3) mRNAs in 24 cell lines derived from human malignant gliomas was studied by Northern analysis. Widespread expression of neurotrophin genes was found with BDNF being the most abundantly expressed. Nearly all cell lines expressed BDNF, and about two-thirds of the cell lines expressed NGF and NT-3. Half of the cell lines analyzed expressed all three neurotrophins. Secretion of NGF into the medium of several cell lines could be detected by ELISA and a PC12 neurite outgrowth assay. Immuno- and bioactive NGF was isolated from conditioned medium of one cell line. No evidence of expression of the neurotrophin receptors trk and trkB by Northern analysis was found. Receptor crosslinking with radiolabeled cognate ligands failed to detect functional receptors in all but one cell line. In this cell line a receptor complex for BDNF was found that corresponded to truncated trkB receptors that lack the signal transducing tyrosine kinase domain. Neurotrophins did not stimulate mitosis of the glioma cultures. The findings suggest that production of neurotrophins by glioma cells is a general phenomenon, although neurotrophins made by gliomas lacking their receptors may not play an autocrine but rather a paracrine role. © 1993 Wiley-Liss, Inc.     

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.     

Developmental expression of neurotrophins and their receptors in postnatal rat ventral midbrain

Neurotrophins are a group of structurally related polypeptides that support the survival, differentiation, and maintenance of neuronal populations that express the appropriate high-affinity neurotrophin receptors. Two members of the neurotrophin family, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), have been shown to increase the survival of dopaminergic neurons from the ventral midbrain in vitro. Evidence suggests that ventral midbrain neurons might be able to derive support from these trophic factors in vivo through paracrine or autocrine interactions. Both BDNF and NT-3 mRNAs and their receptor mRNAs, trkB and trkC mRNAs, respectively, have been localized to the ventral mesencephalon. However, the relative expression levels of the neurotrophins and their receptor mRNAs throughout ontogeny and in adulthood have not been elucidated. In the present study, the postnatal developmental expression of BDNF, NT-3, trkB, and trkC mRNAs was analyzed via in situ hybridization to gain insight into the possible role of these factors in vivo. We found that there was a developmental decline in the expression of BDNF and NT-3 mRNAs in the ventral mesencephalon. In contrast, no alterations in the expression of midbrain trkB or trkC mRNAs could be discerned. The present results suggest a role for BDNF and NT-3 in the earlier postnatal developmental events of responsive populations. The continued, albeit lower, expression of the neurotrophins in the ventral mesencephalon in adulthood also suggests a role for these factors in mature neuronal systems.     

Neurotrophin receptor proteins immunoreactivity in human gastrointestinal endocrine cells

The distribution of neurotrophin receptors (p75, trkA-, trkB-, and trkC-receptor proteins) was studied by immunohistochemistry on sections of human gastrointestinal tract mucosa from esophagus through rectum. Moreover, chromogranin A (CgA) was studied in parallel to identify endocrine cells (EC). In all of the analyzed samples there was specific immunoreactivity (IR) for trkB-receptor protein in EC, the percentage of which varied between 26 ± 0.6% for the duodenum and 78 ± 3% for the sigmoid colon. EC displaying [trkC-receptor protein IR were also encountered, in some cases, in EC of the gastric fundus (9%), duodenum (12%), jejune (23%), and colon (12%); trkA-receptor protein IR was occasionally present labelling EC in the jejune (52%), ileum (25%), and sigmoid colon (18%); finally, p75 was in 21 % of EC exclusively in one case in the ileum. In addition to EC, IR for all assessed antigens was also present in the submucous blood vessels. Our results provide evidence for the occurrence of neurotrophin receptor proteins in nonneuronal tissues and suggest that neurotrophins, especially that binding trkB receptor proteins, can regulate a subpopulation of EC cells. However, whether EC expressing different trk receptor proteins represent neurochemical subtypes of EC, and whether the identified trk receptor proteins correspond to functional receptors, remain to be elucidated.     

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.     

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: A double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms. J. Comp. Neurol. 403:295–308, 1999. © 1999 Wiley-Liss, Inc.     

Cell death of adult pyramidal CA1 neurons after intraventricular injection of a novel peptide derived from trkA

Members of the nerve growth factor (NGF) family of neurotrophins bind to the second leucine-rich motif (LRM₂) within the extracellular domains of their respective receptors (trkA, trkB, trkC). Small LRM₂ peptides have been recently demonstrated to selectively bind the neurotrophins revealing similar complex binding characteristics as full-length receptors. We extend our recent findings, showing that the peptides (A and C) do not block nerve fiber outgrowth through high affinity trk receptors in a ganglia bioassay. Since the highest concentration of neurotrophins [NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3)] is found in the hippocampus, the peptides were injected into the 3rd ventricle of anesthetized adult rats. The (NGF binding) LRM₂-A peptide, but not the (BDNF binding) LRM₂-B or the (NT-3 binding) LRM₂-C peptides, caused severe apoptotic neurodegeneration of hippocampal pyramidal CA1 neurons as revealed by cresyl violet staining and the TUNEL reaction. The degeneration was protected by intrahippocampal injection of NGF-beta and by the non-N-methyl-D-aspartate (NMDA) antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), indicating a glutamatergic mechanism. In situ hybridization revealed that pyramidal CA1 neurons did not express trkA and p75 receptor mRNA in sham and LRM₂-A-lesioned animals. It is concluded that the LRM₂-A peptide represents a novel peptide with properties to induce apoptotic cell death of pyramidal CA1 neurons and may be useful as an experimental agent. J. Neurosci. Res. 50:402–412, 1997. © 1997 Wiley-Liss, Inc.     

The nerve growth factor family of receptors.

The neurotrophins, of which nerve growth factor (NGF) is the best known example, support the survival and differentiation of chick embryo sensory neurons at extremely low concentrations, 10(-12) M or less. These same neurons display two different classes of neurotrophin receptors with dissociation constants of 10(-11) M and 10(-9) M, respectively, implying that only low occupancy of the higher affinity receptor is required to mediate the biological actions of the neurotrophins. Two structurally unrelated receptors have now been characterized for NGF, and one of them, p75NGFR, serves as a receptor for all the known neurotrophins. This is the receptor with a dissociation constant of 10(-9) M. The second NGF receptor is a member of the trk family of tyrosine kinase receptors, p140trkA. Other members, p145trkB and p145trkC, are receptors for brain-derived neurtrophic factor (BDNF) and neurotrophin-4 (NT-4) and neurotrophin-3 (NT-3), respectively, when assayed in fibroblasts. The specificity of neurotrophin binding to these receptors appears to be much higher in neurons than in the non-neuronal cells. The receptor p140trkA has many of the properties of the higher affinity class of NGF receptors, and is able to mediate survival and differentiation of the PC12 cell line, and cell growth and transformation in fibroblast cells. On the other hand, expression of p75NGFR in several types of cells displaying p140trkA induces a component of higher affinity NGF binding not seen in its absence. Since it is unlikely that p75NGFR and p140trkA interact at the level of the receptors, the crosstalk between receptors probably occurs through their signal transduction mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of neurotrophins in skeletal muscle: Quantitative comparison and significance for motoneuron survival and maintenance of function

Neurotrophins play a crucial role in the regulation of survival and maintenance of specific functions of various populations of neurons. Brain-derived neurotrophic factor (BDNF), newrotrophin-3 (NT-3) and neurotrophin 4/5 (NT-4) have been shown to support motoneuron survival during embryonic development and, after birth, to protect motoneurons from degeneration after nerve lesion. We have compared the levels of these neurotrophins in skeletal muscle by quantitative Northern blot analysis, both during embryonic development and postnatally. We localized the sites of expression of these neurotrophins by in situ hybridisation and analysed the expression of trkB in the spinal cord by in situ hybridisation and immunohistochemistry. NT-3 is most abundantly expressed both during embryonic development and in the postnatal phase, followed by NT-4. The levels of BDNF are very low, in particular after birth. After nerve lesion, NT-3 mRNA essentially remained unchanged, whereas NT-4 mRNA rapidly decreased. The slow increase in BDNF expression seems to be essentially due to the expression in Schwann cells rather than skeletal muscle, demonstrated by in situ hybridisation. Our data indicate that motoneurons can receive trophic support from several members of the neurotrophin gene family during the period of naturally occurring cell death. Postnatally, the predominant ligand acting via trkB on motoneurons is NT-4, whereas BDNF expression seems to play a role mainly after nerve lesion. © 1995 Wiley-Liss, Inc.     

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.     

Brain-derived neurotrophic factor-, neurotrophin-3-, and tyrosine kinase receptor-like immunoreactivity in lingual taste bud fields of mature hamster

The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), as well as their respective tyrosine kinase (Trk) receptors, TrkB and TrkC, influence peripheral target cell innervation, survival, and proliferation. In the mature taste system the role of neurotrophins and their receptors is not known. The mature hamster is an intriguing model because anterior lingual fungiform, unlike posterior lingual foliate and circumvallate, taste buds survive denervation. In light of this difference, we examined whether the degree of neurotrophin- or neurotrophin receptor-like immunoreactivity (IR) normally differs among lingual gemmal fields. In single- and double-labeled immunofluorescent experiments, 3,209 taste bud sections (profiles) from 13 hamsters were examined for immunopositive gemmal cells or nerve fibers using antibodies to BDNF and NT-3, their respective receptors TrkB and TrkC, and the neural marker ubiquitin c-terminal hydrolase L-1 [protein gene product (PGP) 9.5]. In each gemmal field, more than 75% of taste bud profiles showed immunopositivity to BDNF, NT-3, and TrkB. Across bud fields, BDNF-, TrkB-, and BDNF/TrkB-like IR, as well as PGP 9.5 and PGP 9.5/BDNF-like IR in centrally located, fungiform bud cells was greater (P < 0.0001 to P < 0.002) than in circumvallate or foliate buds. Within bud fields, the number of BDNF-like, labeled bud cells/bud profile was greater than that for NT-3-like IR in fungiform (P < 0.0002) and foliate (P < 0.0001) buds. TrkC was immunonegative in gemmal cells. The average density of TrkB- and TrkC-like fiber IR was more pronounced in fungiform than posterior gemmal-bearing papillae. Thus, fungiform papillae, whose taste buds are least affected by denervation, exhibit specific neurotrophin and receptor enrichment.     

A potential interaction of p75 and trkA NGF receptors revealed by affinity crosslinking and immunoprecipitation

Nerve growth factor binds independently to two transmembrane receptors, the p75 neurotrophin receptor and the p140^trk (trkA) tyrosine kinase receptor, which are both co-expressed in the majority of neuronal cells that respond to NGF. Previous findings have suggested that appropriate co-expression of the two receptors gives rise to high affinity NGF binding sites and increased neurotrophin responsiveness; however, evidence demonstrating a direct interaction between the two receptors in cell lines has been lacking. Here we have utilized affinity crosslinking agents with ¹²⁵I-NGF to detect an association of trkA and p75 receptors in embryonic spinal cord and brain tissues enriched in the two receptors. Although multimeric complexes of trkA and p75 were not detected by affinity crosslinking, immunoprecipitation of cross-linked NGF-receptor complexes with trk-specific antibodies resulted in selective immunoprecipitation of crosslinked p75. Our results indicate that the trkA and p75 receptors can potentially interact, and that such an association may be responsible for the generation of high affinity NGF binding sites. © 1995 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.     

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.