Brain-derived Neurotrophic Factor Binding Research Articles

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.

Interactions between Brain-derived Neurotrophic Factor and the TRKB Receptor

The extracellular domain of the human neurotrophin TRKB receptor expressed in Chinese hamster ovary cells is a highly glycosylated protein, possessing binding ability for brain-derived neurotrophic factor (BDNF). Two distinct ligand binding domains of TRKB were isolated from proteolytic digests of the receptor by affinity separation on immobilized BDNF. One of these domains consists of amino acid residues 103-181 and contains both the third leucine-rich motif and the second cysteine cluster domain. The second domain is close to the second immunoglobulin-like domain (amino acid residues 342-394). Each of these two domains can bind BDNF independently. Disulfide linkages present in the first domain are necessary for BDNF binding, probably because of preservation of the native conformation. To study the second domain in greater detail, a truncated form of TRKB containing the second immunoglobulin-like domain (residues 248-398) was expressed in Escherichia coli. This domain was cross-linked to BDNF through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling reaction. Several synthetic peptides corresponding to amino acid residues 343-379 were able to bind immobilized BDNF. Amino acid substitution and cross-linking analysis indicated that amino acids Phe347, Asp354, and Tyr361 are intimately involved in BDNF binding. These results, obtained from a variety of experimental techniques, highlight the importance of two distinct regions of the extracellular domain of the TRKB receptor in binding BDNF.

Sequential cis/trans autophosphorylation in TrkB tyrosine kinase.

TrkB, a member of the tyrosine kinase family of growth factor receptors, is activated by binding of brain-derived neurotrophic factor or neurotrophin 4/5. The intracellular kinase domain of TrkB (ICD-TrkB) was prepared by an insect cell expression system and characterized to identify the mechanism of autophosphorylation. The time course of autophosphorylation, which shows a biphasic progression with a slow nonlinear phase followed by a fast linear phase, indicates the existence of autophosphorylation-induced activation in ICD-TrkB. This is also supported by the finding that phosphorylated ICD-TrkB shows significantly higher activity than control naive ICD-TrkB. Interestingly, the autophosphorylation rate in the linear phase clearly depends on the ICD-TrkB concentration, whereas the rate of initial autophosphorylation is independent of the concentration of ICD-TrkB in the reaction mixture. This observation suggests a two-step autophosphorylation, first an intramolecular activating step and then an intermolecular step. This mechanism is confirmed by the result that only the later phase of autophos-phorylation is inhibited by addition of glycerol which interferes with intermolecular interactions. Therefore, we propose the mechanism of ICD-TrkB autophosphorylation as a sequential cis/trans phosphorylation.

Differential distribution of exogenous BDNF, NGF, and NT‐3 in the brain corresponds to the relative abundance and distribution of high‐affinity and low‐affinity neurotrophin receptors

To evaluate effective means for delivering exogenous neurotrophins to neuron populations in the brain, we compared the distribution and transport of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) following intracerebral delivery. Rats received an injection of radioiodinated or unlabeled neurotrophin into the lateral ventricle and were killed humanely after 1.5-24 hours. Other rats received continuous infusion of unlabeled neurotrophin into the lateral ventricle, the striatum, or the hippocampus for 3-14 days. The neurotrophins were detected by autoradiography or immunohistochemistry. There were striking differences between BDNF, NGF, and NT-3 in their penetration through brain tissue. These differences occurred regardless of the site or method of delivery, but were most pronounced following a bolus intracerebroventricular (ICV) injection. After ICV injection, NGF was widely distributed in tissues around the ventricles and at the surface of the brain, whereas the penetration of BDNF into brain tissue was distinctly less than that of NGF, and the penetration of NT-3 was intermediate. An ICV injection of NGF produced prominent but transient labeling of cells that contain the low-affinity NGF receptor, whereas an injection of BDNF prominently labeled the ventricular ependyma. During ICV infusion (12 micrograms/day), the distribution of BDNF was no greater than that observed after a 12-micrograms bolus injection. A sixfold increase in the amount of BDNF infused (72 micrograms/day) produced a more widespread distribution in the brain and doubled the depth of penetration into periventricular tissues near the cannula. Corresponding to their differences in penetration, NGF was retrogradely transported by basal forebrain cholinergic neurons after ICV or intrastriatal delivery, whereas NT-3 was transported by a few basal forebrain neurons after ICV delivery, and BDNF was rarely detected in neurons after ICV delivery. Delivery of BDNF directly to the striatum or the hippocampus labeled numerous neurons in nuclei afferent to these structures. In situ hybridization studies confirmed that the high-affinity BDNF receptor (TrkB) was much more widely expressed in neurons than was the high-affinity NGF receptor (TrkA). Moreover, mRNA for truncated forms of TrkB was expressed at high levels in the ependyma, the choroid epithelium, and the gray matter. It is likely that binding of BDNF to TrkB, which appears to be more abundant and ubiquitous than TrkA, restricts the diffusion of BDNF relative to that of NGF.

Interactions of neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), and the NT-3.BDNF heterodimer with the extracellular domains of the TrkB and TrkC receptors.

Interactions of three neurotrophin dimers, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and a NT-3.BDNF heterodimer with extracellular, soluble TrkB and TrkC receptors were studied using native gels, light scattering, and sedimentation equilibrium. These three neurotrophins showed binding of two TrkB receptors per neurotrophin dimer, with a tendency to dissociate into one TrkB per dimer for NT-3 and the heterodimer, as determined by native gels, light scattering, and sedimentation equilibrium. For TrkC, native gels suggested binding of NT-3, heterodimer, and BDNF but not of nerve growth factor. Sedimentation equilibrium revealed that all three neurotrophin molecules bind to TrkC at two receptors per dimer but that BDNF binds much more weakly and that the heterodimer has an intermediate binding strength. Light scattering/size exclusion chromatography showed complexes with two TrkC receptors per NT-3 dimer and one TrkC per heterodimer but did not detect binding of BDNF to TrkC. This latter result is not inconsistent with the sedimentation data, because the weak binding of BDNF to TrkC may be easily dissociated by nonspecific interactions of BDNF with the size exclusion column. The relative binding constants for these neurotrophins and the soluble receptor extracellular domains, as determined by sedimentation equilibrium, are correlated with their biological activity. However, the magnitude of these binding constants is insufficient by approximately 3 orders of magnitude to promote receptor dimerization at physiologically active concentrations.

Neurotrophin binding to human alpha 2-macroglobulin under apparent equilibrium conditions.

alpha 2-Macroglobulin (alpha 2M) is a broad-spectrum proteinase inhibitor and a carrier of certain growth factors. The purpose of this investigation was to characterize the interaction of alpha 2M with nerve growth factor-beta (NGF-beta), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and ciliary neurotrophic factor (CNTF) under apparent equilibrium conditions. Binding in solution was assessed using the cross-linking agent bis(sulfosuccinimidyl) suberate (BS3). Noncovalent binding of NGF-beta, NT-3, NT-4, and BDNF to native alpha 2M and alpha 2M-methylamine (a conformationally modified form of alpha 2M that is recognized by the alpha 2M receptor) reached apparent equilibrium in less than 20 min at 37 degrees C. Apparent KD values for the binding of NT-4, NGF-beta, NT-3, and BDNF to alpha 2M-methylamine were 61, 110, 120, and 150 nM, respectively. Native alpha 2M bound all four neurotrophins with decreased affinity. Unlabeled NGF-beta competed with the radioiodinated neurotrophins for binding to immobilized alpha 2M-methylamine. The K1 for unlabeled NGF-beta was 120 nM, in good agreement with the apparent KD determined by the BS3 method. The number of NGF-beta binding sites per immobilized alpha 2M-methylamine was 1.0. CNTF bound minimally, if at all, to native alpha 2M and alpha 2M-methylamine as determined using a number of techniques. The extent of binding was insufficient for the determination of an affinity constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Transport of human recombinant brain-derived neurotrophic factor (BDNF) through the rat blood-brain barrier in vivo using vector-mediated peptide drug delivery.

The blood-brain barrier (BBB) transport of brain-derived neurotrophic factor (BDNF) in anesthetized rats was examined in the present studies using vector-mediated peptide drug delivery. Following tritiation, the BDNF was biotinylated via a disulfide linker and was coupled to a covalent conjugate of neutral avidin (NLA), which binds the biotinylated peptide with a high affinity, and the murine OX26 monoclonal antibody to the rat transferrin receptor. Owing to the abundance of transferrin receptors on brain capillary endothelium, the OX26 monoclonal antibody undergoes receptor-mediated transcytosis through the BBB, and the NLA-OX26 conjugate transports biotinylated peptide therapeutics through the BBB. The present studies show that while unconjugated BDNF was not transported through the BBB in vivo, the conjugation of biotinylated BDNF to the NLA-OX26 vector resulted in a marked increase in the brain delivery of BDNF, as defined by measurements of the percentage of the injected dose (ID) delivered per gram of brain. Although BDNF was not transported through the BBB in vivo, this cationic peptide was avidly bound by isolated human brain capillaries via a low-affinity, high-capacity system that was inhibited by protamine and by serum protein binding of BDNF. In conclusion, these studies show that the delivery of unconjugated BDNF to brain is nil owing to the combined effects of negligible BBB transport and rapid systemic clearance of intravenous administered BDNF. The brain delivery of BDNF may be augmented by conjugation of BDNF to BBB drug delivery vectors, such as the NLA-OX26 conjugate.

Expression and binding characteristics of the BDNF receptor chick trkB.

Previous studies using transfected cells have indicated that the mammalian receptor tyrosine kinase trkB binds the neurotrophins brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4. However, most studies demonstrating that these neurotrophins prevent the death of embryonic neurons and have specific neuronal receptors have been performed with chick neurons. In order to explore the possibility that trkB is the molecular entity representing the high-affinity receptor for brain-derived neurotrophic factor on embryonic chick neurons, we cloned and expressed a chick trkB cDNA. In situ hybridisation results indicate that the distribution of trkB mRNA in the peripheral nervous system of the developing chick embryo correlates well with the structures known to respond to brain-derived neurotrophic factor. Binding studies performed with a cell line stably transfected with the ctrkB cDNA indicate a dissociation constant for brain-derived neurotrophic factor of 9.9 x 10(-10) M, which is distinctly higher than that found on primary chick sensory neurons (1.5 x 10(-11) M). When binding of brain-derived neurotrophic factor was determined in the presence of other neurotrophins, neurotrophin-3 was found efficiently to prevent the binding of brain-derived neurotrophic factor to both the ctrkB cell line and embryonic sensory neurons. In vitro, neurotrophin-3 at high concentrations completely blocked the survival normally seen with brain-derived neurotrophic factor. Thus, unlike previous cases of receptor occupancy by heterologous neurotrophins (which resulted in agonistic effects), the interaction between the brain-derived neurotrophic factor receptor and neurotrophin-3 on sensory neurons is antagonistic.

Binding of brain-derived neurotrophic factor to the nerve growth factor receptor

The neurotrophic proteins BDNF and NGF are related in their primary structures, and both have high- and low-affinity receptors on their responsive neurons. In this study, we investigate the extent to which these receptors can discriminate between BDNF and NGF. We found that a 1000-fold excess of the heterologous ligand is needed to reduce binding to the high-affinity receptor by 50%, but that the same concentrations of BDNF and NGF similarly reduce the binding of either ligand to the low-affinity receptor. Results obtained with cells transfected with the low-affinity NGF receptor gene indicate that these cells bind BDNF, in addition to NGF, whereas cells before transfection do not. These data indicate that the low-affinity NGF receptor is also a low-affinity BDNF receptor and that whatever is conferring high-affinity binding and biological response also considerably reinforces the ability of the low-affinity receptor to discriminate between NGF and BDNF.