p75 reduces TrkB tyrosine autophosphorylation in response to brain-derived neurotrophic factor and neurotrophin 4/5

Neurotrophins mediate their signals through two different receptors: the family of receptor tyrosine kinases, Trks, and the low affinity pan-neurotrophin receptor p75. Trk receptors show more restricted ligand specificity, whereas all neurotrophins are able to bind to p75. One important function of p75 is the enhancement of nerve growth factor signaling via TrkA by increasing TrkA tyrosine autophosphorylation. Here, we have examined the importance of p75 on TrkB- and TrkC-mediated neurotrophin signaling in an MG87 fibroblast cell line stably transfected with either p75 and TrkB or p75 and TrkC, as well as in PC12 cells stably transfected with TrkB. In contrast to TrkA signaling, p75 had a negative effect on TrkB tyrosine autophosphorylation in response to its cognate neurotrophins, brain-derived neurotrophic factor and neurotrophin 4/5. On the other hand, p75 had no effect on TrkB or TrkC activation in neurotrophin 3 treatment. p75 did not effect extracellular signal-regulated kinase 2 tyrosine phosphorylation in response to brain-derived neurotrophic factor, neurotrophin 3, or neurotrophin 4/5. These results suggest that the observed reduction in TrkB tyrosine autophosphorylation caused by p75 does not influence Ras/mitogen-activated protein kinase signaling pathway in neurotrophin treatments.     

CNS neurotrophins are biologically active and expressed by multiple cell types

Neutrotrophins are increasingly appreciated as potential modulators of neuronal function in the adult central nervous system (CNS). To describe the neurotrophin environment within the adult CNS, mRNA and protein expression patterns of neurotrophins-3 and –4 and of brain-derived neurotrophin were investigated in adult rat spinal cord and brain. Co-localization studies with CNS cell type-specific markers demonstrates that multiple cell types, including both neurons and glia, express these neurotrophins in the normal adult CNS. Although widely implicated in important CNS functions such as synaptic plasticity, biological activity of endogenous CNS neurotrophins has not been directly demonstrated. With a sensitive neurite outgrowth bioassay we demonstrate that CNS neurotrophins elicit neurite outgrowth and are biologically active. Moreover, antibody-blocking studies suggest that these three neurotrophins may comprise the bulk of adult CNS neurotrophic activity.     

Neurotrophins mediate human embryonic stem cell survival

Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family, which mediate antiapoptotic signals. We show that three TRK ligands, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins, TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation.     

Defining Responsiveness of Avian Cochlear Neurons to Brain-Derived Neurotrophic Factor and Nerve Growth Factor by HSV-1-Mediated Gene Transfer

Abstract: The importance of individual members of the neurotrophin gene family for avian inner ear development is not clearly defined. Here we address the role of two neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), for innervation of the chicken cochlea. We have used defective herpes simplex virus type 1 (HSV-1) vectors, or amplicons, to express these neurotrophins in dissociated cultures of cochlear neurons. HSV-1-mediated expression of BDNF promotes neuronal survival similar to the maximal level seen by exogenously added BDNF and exceeds its potency to produce neurite outgrowth. In contrast, cochlear neurons transduced with an amplicon producing bioactive NGF show no response. These results confirm BDNF as an important mediator of neurotrophin signaling inside avian cochlear neurons. However, these neurons can be rendered NGF-responsive by transducing them with the high-affinity receptor for NGF, TrkA. This study underlines the usefulness of amplicons to study and modify neurotrophin signaling inside neurons.     

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low-affinity neurotrophin receptor.

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.     

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. Received: 27 January 1997 / Accepted: 20 March 1997     

The structures of the neurotrophin 4 homodimer and the brain-derived neurotrophic factor/neurotrophin 4 heterodimer reveal a common Trk-binding site

The neurotrophins are growth factors that are involved in the development and survival of neurons. Neurotrophin release by a target tissue results in neuron growth along the neurotrophin concentration gradient, culminating in the eventual innervation of the target tissue. These activities are mediated through trk cell surface receptors. We have determined the structures of the heterodimer formed between brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT4), as well as the structure of homodimer of NT4. We also present the structure of the Neurotrophin 3 homodimer, which is refined to higher resolution than previously published. These structures provide the first views of the architecture of the NT4 protomer. Comparison of the surface of a model of the BDNF homodimer with the structures of the neurotrophin homodimers reveals common features that may be important in the binding between the neurotrophins and their receptors. In particular, there exists an analogous region on the surface of each neurotrophin that is likely to be involved in trk receptor binding. Variations in sequence on the periphery of this common region serve to confer trk receptor specificity.     

A discrete domain of the human TrkB receptor defines the binding sites for BDNF and NT-4

TrkB is a member of the Trk family of tyrosine kinase receptors. In vivo, the extracellular region of TrkB is known to bind, with high affinity, the neurotrophin protein brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). We describe the expression and purification of the second Ig-like domain of human TrkB (TrkBIg(2)) and show, using surface plasmon resonance, that this domain is sufficient to bind BDNF and NT-4 with subnanomolar affinity. BDNF and NT-4 may have therapeutic implications for a variety of neurodegenerative diseases. The specificity of binding of the neurotrophins to their receptor TrkB is therefore of interest. We examine the specificity of TrkBIg(2) for all the neurotrophins, and use our molecular model of the BDNF-TrkBIg(2) complex to examine the residues involved in binding. It is hoped that the understanding of specific interactions will allow design of small molecule neurotrophin mimetics.     

Activity-Dependent Dendritic Release of BDNF and Biological Consequences

Network construction and reorganization is modulated by the level and pattern of synaptic activity generated in the nervous system. During the past decades, neurotrophins, and in particular brain-derived neurotrophic factor (BDNF), have emerged as attractive candidates for linking synaptic activity and brain plasticity. Thus, neurotrophin expression and secretion are under the control of activity-dependent mechanisms and, besides their classical role in supporting neuronal survival neurotrophins, modulate nearly all key steps of network construction from neuronal migration to experience-dependent refinement of local connections. In this paper, we provide an overview of recent findings showing that BDNF can serve as a target-derived messenger for activity-dependent synaptic plasticity and development at the single cell level.     

Novel roles for neurotrophins are suggested by BDNF and NT-3 mRNA expression in developing neurons.

The results of our in situ hybridization experiments demonstrate that sensory neurons, sympathetic neurons, and motoneurons express brain-derived neurotrophic factor and/or neurotrophin-3 mRNAs during development in mouse. In accordance with previous data, we also find neurotrophins in the targets of sensory neurons (skin) and motoneurons (muscle) and the neurotrophin receptors p75, trkA, and trkB in sensory and sympathetic ganglia. These results suggest that neurotrophins have roles other than being target-derived factors that support neuron survival during developmental cell death (neurotrophic hypothesis), but may be transported in an orthograde fashion in neurons and released from axon terminals. We discuss several novel roles for neurotrophins, including autocrine/paracrine regulation of neuron survival, regulation of Schwann cell activity, and neuron to target signaling.     

From visual experience to visual function: roles of neurotrophins.

Recently, a role for neurotrophins in regulating cortical developmental plasticity has clearly emerged. We present in this review a summary of the early data on the action of nerve growth factor (NGF) in visual cortical development and plasticity in the rat and of other neurotrophins in the visual cortex of other mammals. In addition, to clarify the differences in the results obtained with the various neurotrophins in different animal preparations, we also report new data on the action of NGF, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)3, and NT4 in the same preparation-namely, the visual cortex of the rat. We discuss old and new results in a physiological model in which different neurotrophins play different roles in regulating visual cortical development and plasticity by acting on different neural targets, such as lateral geniculate nucleus (LGN) afferents, intracortical circuitry, and subcortical afferents, and propose a tentative scheme summarizing these actions.     

Critical periods during sensory development

Recent studies have made progress in characterizing the determinants of critical periods for experience-dependent plasticity. They highlight the role of neurotrophins, NMDA receptors and GABAergic inhibition. In particular, genetic manipulation of a single molecule, brain-derived neurotrophic factor (BDNF), has been shown to alter the timing of the critical period of plasticity in mouse visual cortex, establishing a causal relation between neurotrophin action, the development of visual function, and the duration of the critical period.     

Identification of a switch in neurotrophin signaling by selective tyrosine phosphorylation

Neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and recruitment of intracellular signaling molecules. The intracellular pathways used by neurotrophins share many common protein substrates that are used by other receptor tyrosine kinases (RTK), such as Shc, Grb2, FRS2, and phospholipase C-gamma. Here we describe a novel RTK mechanism that involves a 220-kilodalton membrane tetraspanning protein, ARMS/Kidins220, which is rapidly tyrosine phosphorylated in primary neurons after neurotrophin treatment. ARMS/Kidins220 undergoes multiple tyrosine phosphorylation events and also serine phosphorylation by protein kinase D. We have identified a single tyrosine (Tyr(1096)) phosphorylation event in ARMS/Kidins220 that plays a critical role in neurotrophin signaling. A reassembled complex of ARMS/Kidins220 and CrkL, an upstream component of the C3G-Rap1-MAP kinase cascade, is SH3-dependent. However, Tyr(1096) phosphorylation enables ARMS/Kidins220 to recruit CrkL through its SH2 domain, thereby freeing the CrkL SH3 domain to engage C3G for MAP kinase activation in a neurotrophin dependent manner. Accordingly, mutation of Tyr(1096) abolished CrkL interaction and sustained MAPK kinase activity, a response that is not normally observed in other RTKs. Therefore, Trk receptor signaling involves an inducible switch mechanism through an unconventional substrate that distinguishes neurotrophin action from other growth factor receptors.     

Interleukin 1-beta upregulates brain-derived neurotrophic factor,neurotrophin 3 and neuropilin 2 gene expression and NGF production in annulus cells

The relationship between disc cells, nerves and pain production in the intervertebral disc is poorly understood. Neurotrophins, signaling molecules involved in the survival, differentiation and migration of neurons, and neurite outgrowth, are expressed in non-neuronal tissues including the disc. We hypothesized that three-dimensional exposure of human disc cells to the proinflammatory cytokine IL-1ß in vitro would elevate neurotrophin gene expression levels and production of nerve growth factor (NGF). Cells isolated from Thompson grade III and IV discs were cultured for 14 days under control conditions or with addition of 10² pM IL-1ß; mRNA was isolated and conditioned media assayed for NGF content. IL-1ß exposure in three-dimensional culture significantly increased expression of neurotrophin 3, brain-derived neurotrophic factor, and neuropilin 2 compared to controls. IL-1ß-exposed cells showed significantly increased NGF production compared to controls. Findings support our hypothesis, expand previous data concerning expression of neurotrophins, and provide the first documented expression of neurotrophin 3 and neuropilin 2. Our results have direct translational relevance, because they address the primary clinical issue of low back pain and open the possibility of novel analgesic therapies using specific small-molecular antagonists to neurotrophins.     

An extended surface of binding to Trk tyrosine kinase receptors in NGF and BDNF allows the engineering of a multifunctional pan-neurotrophin.

Neurotrophin-mediated cell survival and differentiation of vertebrate neurons is caused by ligand-specific binding to the Trk family of tyrosine kinase receptors. However, sites in the neurotrophins responsible for the binding to Trk receptors and the mechanisms whereby this interaction results in receptor activation and biological activity are unknown. Here we show that in nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), discontinuous stretches of amino acid residues group together on one side of the neurotrophin dimer forming a continuous surface responsible for binding to and activation of TrkA and TrkB receptors. Two symmetrical surfaces are formed along the two-fold axis of the neurotrophin dimer providing a model for ligand-mediated receptor dimerization. Mutated neurotrophins inducing similar levels of receptor phosphorylation showed different biological activities, suggesting that structural differences in a ligand may result in dissimilar responses in a given tyrosine kinase receptor. Our results allowed us to combine structural elements from NGF, BDNF and neurotrophin-3 to engineer a pan-neurotrophin that efficiently activates all Trk receptors and displays multiple neurotrophic specificities.     

The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells

Previous studies have demonstrated local functions for neurotrophins in the developing and mature testis of rodents. To examine whether these signaling molecules are present and also potentially active in the human testis, we characterized immunohistochemically the expression and cellular localization of the known neurotrophins and their receptors during prenatal testicular development as well as in the adult human testis. Results obtained revealed the presence of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and 4, as well as neurotrophin receptors p75^NTR, TrkA, TrkB, and TrkC during testis morphogenesis. These proteins were also detectable in the adult human testis, and their local expression could be confirmed largely by immunoblot and RT-PCR analyses. Remarkably, the Leydig cells were found to represent the predominant neurotrophin/receptor expression sites within both fetal and adult human testes. Functional assays performed with a mouse tumor Leydig cell line revealed that NGF exposure increases cellular steroid production, indicating a role in differentiation processes. These findings support previously-recognized neuronal characteristics of Leydig cells, provide additional evidence for potential roles of neurotrophins during testis morphogenesis and in the mature testis, and demonstrate for the first time a neurotrophin-induced functional activity in Leydig cells.     

Neurotrophin dependence mediated by p75NTR: contrast between rescue by BDNF and NGF

During development, neurons pass through a critical phase in which survival is dependent on neurotrophin support. In order to dissect the potential role of p75NTR, the common neurotrophin receptor, in neurotrophin dependence, we expressed wild-type and mutant p75NTR in cells that do not express endogenous p75NTR or Trk family members (NIH3T3). Expression of wild-type p75NTR created a state of neurotrophin dependence: cells could be rescued by nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-3 (NT-3), but not by a mutant NGF that binds well to Trk A but poorly to p75NTR. Similarly, expression of p75NTR in human prostate cancer cells in culture rendered a metastatic tumor cell line (PC-3) sensitive to the availability of neurotrophins for survival. Moreover, expression of mutant p75NTR's in another neurotrophin-insensitive cell line (HEK293T) showed that a domain within the intracellular domain governs alternate responses to neurotrophins: the carboxy terminus of the intracellular domain of p75NTR including the sixth alpha helix domain is necessary for rescue by BDNF, but not NGF. These results, when considered with previous studies of the timing of p75NTR expression, support the hypothesis that p75NTR is a mediator of neurotrophin dependence during the critical phase of developmental cell death and during the progression of carcinogenesis in prostate cancer.     

Expression of neurotrophins and their receptors in peripheral lung cells of mice

Neurotrophins play an essential role in nerve systems. Recent reports indicated that neurotrophins [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5)] have numerous effects on non-neural cells, especially on immune cells. However, whether lung cells express neurotrophins and/or their receptors (TrkA for NGF, TrkB for BDNF and NT-4/5, and TrkC for NT-3) has never been systematically investigated. We investigated constitutive expression of neurotrophin family and their Trk receptor family in alveolar macrophages and other peripheral lung cells of mice. New findings were: (1) RT-PCR for neurotrophins and their receptors detected NT-3 and NT-4/5 in alveolar macrophages, BDNF, NT-4/5, trkA, the truncated form of trkB, and trkC in lung homogenate, but no trks in alveolar macrophages, (2) immunohistochemistry for neurotrophin receptors detected TrkA in capillary cells, the truncated form of TrkB, and TrkC in interstitial macrophages, (3) immunoelectron microscopy for TrkC revealed expression of TrkC on the surface of interstitial macrophages, and (4) in situ hybridization for neurotrophins detected BDNF in interstitial macrophages and alveolar type I cells, NT-3 in alveolar macrophages, and NT-4/5 in alveolar and interstitial macrophages. These findings indicate that a previously unknown signal trafficking occurs through neurotrophins in peripheral lung.     

Making and breaking the innervation of the ear: neurotrophic support during ear development and its clinical implications

Analyses of single and double mutants of members of the neurotrophin family and their receptors are reviewed. These data demonstrate that the two neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3), and their high-affinity receptors trkB and trkC, are the sole support for the developing afferent innervation of the ear. Neurotrophins are first expressed in the otocyst around the time afferent sensory neurons become postmitotic. They are crucial for the survival of certain topologically distinct populations of sensory neurons. BDNF supports all sensory neurons to the semicircular canals, most sensory neurons to the saccule and utricle, and many sensory neurons to the apex and middle turn of the cochlea. In contrast, NT-3 supports few sensory neurons to the utricle and saccule, all sensory neurons to the basal turn of the cochlea and most sensory neurons to the middle and apical turn. Some topologically restricted effects reflect the pattern of neurotrophin distribution as revealed by in situ hybridization (e.g., loss of all innervation to the semicircular canal sensory epithelia in BDNF or trkB mutants). However, other topologically restricted effects cannot be explained on the basis of current knowledge of neurotrophin or neurotrophin receptor distribution. Data on mutants also support the notion that BDNF may play a role in neonatal plastic reorganization of the pattern of innervation in the ear and possibly the brainstem. In contrast, data obtained thus far on the ability of neurotrophins to rescue adult sensory neuron after insults to cochlear hair cells are less compelling. The ear is a model system to test the interactions of the two neurotrophins, BDNF and NT-3, with their two high-affinity receptors, trkB and trkC.     

K-252 Compounds: Modulators of Neurotrophin Signal Transduction

K-252 compounds, which share a common polyaromatic aglycon structure, are rather general and potent inhibitors of various protein kinases, including protein kinase C and tyrosine-specific protein kinases, and possibly act by interfering at or near the ATP binding site. However, chemical modifications in their sugar moiety can result in high specificity of the inhibitory action and, furthermore, can induce other stimulatory and inhibitory effects on nerve cells. These compounds are of particular interest because, in intact cells, they inhibit the actions of NGF and other neurotrophins without diminishing comparable actions of other growth factors. This effect seems to reflect a direct inhibitory action on trk neurotrophin receptor proteins. At concentrations lower than those necessary to inhibit neurotrophin actions, K-252a and K-252b have been shown to potentiate the stimulatory effects of NT-3 on different neurons in culture and on PC12 cells. The structural requirements for this effect seem to be different from those for the inhibition of neurotrophin actions. These findings raise the possibility of development of compounds of high selectivity, able to inhibit or potentiate the transduction mechanisms of individual neurotrophins, and identify K-252a and K-252b as lead compounds for the development of such selective molecules. Specific inhibitors and stimulators of neurotrophins would be valuable tools to investigate biological functions of the neurotrophins in vitro and in vivo. Furthermore, it is possible that, in the future, highly selective drugs with agonistic or antagonistic actions on neurotrophin mechanisms could become therapeutically useful in the treatment of neurological disease and injury.