Recombinant Human Brain-derived Neurotrophic Factor Research Articles

A Role for Retinal Brain-Derived Neurotrophic Factor in Ocular Dominance Plasticity

Visual deprivation is a classical tool to study the plasticity of visual cortical connections. After eyelid closure in young animals (monocular deprivation, MD), visual cortical neurons become dominated by the open eye, a phenomenon known as ocular dominance (OD) plasticity . It is commonly held that the molecular mediators of OD plasticity are cortically derived and that the retina is immune to the effects of MD . Recently, it has been reported that visual deprivation induces neurochemical, structural, and functional changes in the retina , but whether these retinal changes contribute to the effects of MD in the cortex is unknown. Here, we provide evidence that brain-derived neurotrophic factor (BDNF) produced in the retina influences OD plasticity. We found a reduction of BDNF expression in the deprived retina of young rats. We compensated this BDNF imbalance between the two eyes by either injecting exogenous BDNF in the deprived eye or reducing endogenous BDNF expression in the nondeprived eye. Both treatments were effective in counteracting the OD shift induced by MD. Retinal BDNF could also influence OD distribution in normal animals. These results show for the first time that OD plasticity is modulated by BDNF produced in the retina.

Endogenous BDNF is required for long-term memory formation in the rat parietal cortex

Information storage in the brain is a temporally graded process involving different memory phases as well as different structures in the mammalian brain. Cortical plasticity seems to be essential to store stable long-term memories, although little information is available at the moment regarding molecular and cellular events supporting memory consolidation in the neocortex. Brain-derived neurotrophic factor (BDNF) modulates both short-term synaptic function and activity-dependent synaptic plasticity in hippocampal and cortical neurons. We have recently demonstrated that endogenous BDNF in the hippocampus is involved in memory formation. Here we examined the role of BDNF in the parietal cortex (PCx) in short-term (STM) and long-term memory (LTM) formation of a one-trial fear-motivated learning task in rats. Bilateral infusions of function-blocking anti-BDNF antibody into the PCx impaired both STM and LTM retention scores and decreased the phosphorylation state of cAMP response element-binding protein (CREB). In contrast, intracortical administration of recombinant human BDNF facilitated LTM and increased CREB activation. Moreover, inhibitory avoidance training is associated with a rapid and transient increase in phospho-CREB/total CREB ratio in the PCx. Thus, our results indicate that endogenous BDNF is required for both STM and LTM formation of inhibitory avoidance learning, possibly involving CREB activation-dependent mechanisms. The present data support the idea that early sensory areas constitute important components of the networks subserving memory formation and that information processing in neocortex plays an important role in memory formation.

The Presence of the EGF Family and their Effects on Cultured Rat Conjunctival Goblet Cell Proliferation

Existing evidence suggests that brain-derived neurotrophic factor (BDNF) promotes survival and proliferation of endothelial cells, stimulates mobilization of hematopoietic progenitors, and induces angiogenesis in ischemic tissues. However, the mechanisms underlying vascular protective function of BDNF are poorly understood. We hypothesized that BDNF increases antioxidant capacity of circulating angiogenic cells. Human mononuclear cells were isolated from peripheral blood of 30 healthy male volunteers (48 ± 2 years old), and cultured in endothelial growth medium-2 for 4–5 days. The attached cells (so called early endothelial progenitor cells [early EPCs], or circulating angiogenic cells) expressed BDNF receptors, tropomyosin-related kinase B and p75 neurotrophin receptor. Treatment of early EPCs with recombinant human BDNF for 24 h significantly increased manganese superoxide dismutase (MnSOD) expression, but had no effect on expression of other antioxidant enzymes including copper zinc SOD (CuZnSOD), catalase, and glutathione peroxidase-1. BDNF stimulated phosphorylation of IκB kinase (IKK)α/β and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK); however it did not activate p38, Erk, or AKT. Treatment with nuclear factor κB inhibitor, PDTC, or JNK inhibitor, SP600125, attenuated BDNF-augmented MnSOD protein expression. BDNF treatment inhibited apoptosis induced by a superoxide anion generator LY83583, and serum starvation-induced cell detachment. These findings suggest that BDNF protects EPCs by increasing expression of MnSOD thereby enhancing their antioxidant capacity.

Recombinant human neurotrophic factors accelerate colonic transit and relieve constipation in humans

The aim of this study was to assess the effects of recombinant human brain-derived neurotrophic factor (r-metHuBDNF) and recombinant human neurotrophic factor 3 (r-metHuNT-3) on gastrointestinal motor functions in healthy people and in patients with constipation. Gastrointestinal and colonic transit was measured by scintigraphy before and after 2 weeks of treatment. Daily diaries documented symptoms over 6 weeks before, during, and after treatment. In a randomized study of healthy subjects, 40 received 100 microg/kg r-metHuBDNF or placebo subcutaneously (SC) daily. In a separate study, 8 healthy subjects and 8 patients with constipation received 300 microg/kg r-metHuNT-3 SC thrice weekly. r-met-HuBDNF accelerated overall and proximal colonic emptying (P<0.05) in health. r-metHuNT-3 accelerated overall colonic transit in health and constipation (all P<0.05) and gastric and small bowel transit (both P<0.05) in health. r-metHuBDNF tended to increase stool frequency compared with placebo in health (P = 0.09). r-metHuNT-3 increased stool frequency (P = 0.05) and facilitated passage of stool (P < 0.01) in constipated patients. The effects on stool frequency started within 3 days of the beginning of neurotrophin administrations and lasted up to 5 days after treatment ended. r-metHu neurotrophic factors were well tolerated, although half of the participants in the 2 studies developed injection site reactions or paresthesiae. Exogenous neurotrophic factors stimulate human gut motility in health and constipation.

Characterization of Recombinant Human Brain-Derived Neurotrophic Factor Variants

The purpose of this work was the chemical characterization of variants of the recombinant human brain derived neurotrophic factor (rHu-BDNF), expressed in Escherichia coli. This paper also addresses the question of the in vitro activity of these variants. Chemical characterization of the variants employed peptide mapping using Glu-C protease and cyanogen bromide digestion on reduced and alkylated variants followed by the analysis of the digested peptides using mass spectrometry and Edman sequencing. The BDNF variants in this work have been designated by the order of their elution as observed from the high temperature RPLC assay. It was determined that Peaks 1 and 2, which eluted just before the predominant BDNF peak, had methionine sulfoxide instead of methionine at positions 31 and 61, respectively. Peak 4, which is chromatographically a single peak, contained three variants. Two of these variants had norleucine instead of methionine, at positions 61 and 92, respectively, while the third had methionine sulfoxide instead of methionine at position 92. Peak 5 had norleucine at position 31 instead of methionine. All of these variants showed in vitro biological activity consistent with the BDNF standard, suggesting the preservation of the trkB receptor-ligand binding domain of the variants.

Reduced expression of brain-derived neurotrophic factor protein in Parkinson's disease substantia nigra.

Several in vitro and in vivo studies have shown that brain-derived neurotrophic factor (BDNF) promotes survival of damaged mesencephalic dopaminergic neurons. Using a specific antibody directed against human recombinant BDNF, we studied the expression of the protein at the cellular level in the post-mortem mesencephalon of control subjects and patients with Parkinson's disease (PD). In control subjects, BDNF was expressed in all mesencephalic regions containing dopaminergic neurons, and in the substantia nigra pars compacta (SNpc) 65% of the melanized neurons expressed BDNF. In the PD SNpc, the total number of pigmented neurons containing BDNF was reduced to 9.6% of the corresponding control value. In contrast, the number of pigmented neurons non-immunoreactive for BDNF was reduced to 23.9% of the corresponding control value. This result appears to indicate that SNpc melanized neurons not expressing BDNF have a 2.5-fold greater probability of surviving than BDNF-positive melanized neurons. Furthermore, we found that in parkinsonian mesencephalon almost all dopaminergic neurons containing Lewy bodies were immunoreactive for BDNF. These findings demonstrate a reduced expression of BDNF in PD and suggest that BDNF protein expression does not protect melanized SNpc neurons from the degenerative process in this disease.

A common mechanism for recombinant human NGF, BDNF, NT-3, and murine NGF slow unfolding.

The recombinant human nerve growth factor (hNGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT4/5), and murine NGF (mNGF) dimers all undergo rapid unfolding and dissociation to monomer in GdnHCl. Fluorescence spectroscopy, reversed-phase high-performance liquid chromatography, and size-exclusion chromatography were used to show that this monomer M1 converts slowly to a more fully unfolded monomer, M2, by a first order process with half-lives of 22, 2.5, 1.6, and 0.73 h for hNGF, mNGF, NT-3, and BDNF, respectively, at 25 degrees C. Linear Arrhenius plots for the conversion of M1 to M2 yielded activation energies of 27, 22, 24, and 24 kcal/mol for hNGF, mNGF, NT-3, and BDNF, respectively. The refolding of these neurotrophins from 5 M GdnHCl was also first order with NT-3 the slowest to refold and BDNF the fastest. Threading of the N-terminus out through the cystine-knot loop present in each of these proteins is proposed as the slow step in unfolding. The number of amino acids in the cystine-knot loop (14 for hNGF, mNGF, NT-3, and BDNF; 21 for NT4/5), and the number and position of the proline residues in this loop (2 for hNGF; 1 for mNGF, NT-3, BDNF, and NT4/5) correlate with the relative rates of unfolding. The smaller the loop and the greater the number of prolines, the more hindered and slower the unfolding.

Cation-exchange displacement chromatography for the purification of recombinant protein therapeutics from variants

Removal of low level impurities that are closely related to the bioproduct is a commonly encountered challenge in the purification of biopharmaceuticals. These separations are typically carried out by using shallow linear salt gradients at relatively low column loadings, significantly limiting the throughput of the purification process. In this manuscript we examine the utility of displacement chromatography for the purification of recombinant human brain-derived neurotrophic factor, rHuBDNF. The utility of displacement chromatography is compared to gradient elution for the removal of variants of the rHuBDNF. The results demonstrate that displacement chromatography is capable of achieving high yields and purity at high column loadings. Displacements developed on 20 μm and 50 μm cation-exchange resins are shown to provide 8-fold and 4.5-fold increases in production rates, respectively, when compared to an existing linear gradient elution operation. These results demonstrate the efficacy of displacement chromatography for the purification of therapeutic proteins from complex feed streams.

Intraventricular injection of NGF, but not BDNF, induces rapid motor activation that is inhibited by nicotinic receptor antagonists.

The acute and subacute effects of intracerebroventricularly (ICV) administered nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) on locomotor activity were evaluated in awake adult rats. Immediately after ICV injection through an implanted cannula, locomotor activity was measured by a computerized system using infrared photocells, which allowed us to record locomotion, motility, and rearing simultaneously. A single dose of 5 microg mouse beta-NGF produced significant increases in horizontal ambulatory components of locomotor activity (locomotion and motility), but not vertical movement (rearing) 30-45 min after ICV administration. These increases lasted for at least 3-4 h. Systemic injection of 2.0 mg/kg mecamylamine, a central nicotinic receptor antagonist, inhibited the hyperactivity induced by NGF. Systemic injection of 0.5 mg/kg scopolamine, a muscarinic receptor antagonist, did not interfere with the NGF effects. Thus, while scopolamine induced marked increases in all three measures of behavior in both NGF and cytochrome-c-treated animals, locomotion and motility remained significantly higher in the NGF group. Immunohistochemistry demonstrated that NGF diffused readily from the ventricular space into brain parenchyma on the injected side and could be visualized 1 h after ICV injection. These results suggest that ICV administration of NGF increases locomotor activity by inducing acetylcholine release, and that nicotinic receptors are involved in the hyperactivity induced by NGF. ICV administration of 5 microg recombinant human BDNF had no significant effect on locomotor activity during the 0- to 4-h period after ICV injection. However, it produced significant decreases in locomotion, motility, and rearing 24-26 h later. Hence ICV administration of BDNF has entirely different effects on animal behavior from those evoked by NGF. While NGF elicits increases in ambulatory behavior within hours, BDNF causes delayed decreases in ambulatory behavior.

Distribution of brain-derived neurotrophic factor in rats and its changes with development in the brain.

A newly established, sensitive, two-site enzyme-immunoassay system for brain-derived neurotrophic factor (BDNF) is described. Using this system, we investigated the tissue distribution of BDNF and developmental changes in tissue levels of BDNF in rats. The minimal limit of detection of the assay was 3 pg/0.2 ml of assay mixture. BDNF was successfully solubilized from tissues in the presence of guanidine hydrochloride but not in any of the other buffers examined. In the rat brain at 1 month of age, the highest level of BDNF was detected in the hippocampus (5.41 ng/g of wet weight), followed by the hypothalamus (4.23 ng/g) and the septum (1.68 ng/g). In other regions, levels of BDNF ranged between 0.9 and 1.7 ng/g. The level of BDNF in the posterior lobes of the cerebellum from rats at 30 days of age was slightly higher than that in the anterior lobes. The concentration of BDNF increased in all regions of the brain with postnatal development. In peripheral tissues, BDNF was found at very low concentrations (0.65 ng/g in the spleen, 0.21 ng/g in the thymus, and 0.06 ng/g in the liver). The subfractionation of the hippocampal homogenate indicated that approximately 50% of BDNF was contained in the crude nuclear fraction. Immunoblots of BDNF-immunoreactive proteins extracted from the hippocampus, hypothalamus, and cerebellum contained doublet bands of protein of approximately 14 kDa, a value close to the molecular mass of recombinant human BDNF. Immunocytochemical investigations showed that, in the hippocampus, BDNF was localized in the nucleus of the granule cells in the dentate gyrus and of the cells in the pyramidal cell layer. The frequency of cells that were stained in the dentate gyrus was greater than that of cells in the pyramidal cell layer.

The effect of brain-derived neurotrophic factor on elongation of axons after transection with suture-morphometric evaluation

Brain-derived neurotrophic factor (BDNF) is considered to play an important role in survival, maintenance, development and repair of the peripheral neuron. In this study, the effect of human recombinant BDNF on sprouting and elongation of axons, the early phase of regeneration of nerve fibers, was morphometrically evaluated 7 days following the sciatic nerve transection and juxtaposition of proximal and distal stumps with suture in Sprague-Dawley rats. In the experimental group (test), 20 mg/kg of BDNF was injected subcutaneously every day for seven days in eight rats, starting 30 minutes after the transection. In the control group (control), phosphate buffered-saline alone was injected in nine rats as in the experimental group. The various morphometric parameters were evaluated in the sciatic nerve of each rat of the control and test, 3 mm distal to the site of the transection on light and electron microscopy of Epon-embedded sections. On both light and electron microscopy only a few myelinated fibers with a very thin myelin sheath were found only in one nerve in each of the control and test. However, significant numbers of unmyelinated axons were found in each nerve of the control and test. There were no statistically significant differences in the total fascicular area per nerve, the numbers of myelinated and unmyelinated axons per mm2 of fascicular area and per nerve, their maximum and median diameters and their size distribution histograms between control and test. In addition, there were no statistically significant differences in the numbers of myelin ovoids per mm2 of the fascicular area and per nerve, their maximum and median diameters and their size distribution histograms between control and test. Therefore, we concluded that there was no definite evidence that BDNF promoted the sprouting and elongation of axons, the early phase of the regeneration of nerve fibers, at least under this experimental conditions.

Retrograde transport of brain-derived neurotrophic factor (BDNF) following infusion in neo- and limbic cortex in rat: relationship to BDNF mRNA expressing neurons.

Brain-derived neurotrophic factor (BDNF) was the second member of the nerve growth factor (NGF) family to be isolated. The ability of BDNF to be retrogradely transported following intraparenchymal infusion represents a unique neurobiological tool to determine the location of putative neuron-specific BDNF-responsive neuronal systems. In the present study, we infused recombinant human (rh) BDNF into the rodent neo- and limbic cortex and used a turkey anti-BDNF antibody to determine specific populations of neurons which retrogradely transport this neurotrophin. Frontal cortex infusion retrogradely labeled neurons within the ipsilateral and contralateral frontal cortex, basal forebrain, lateral hypothalamus, centrolateral, mediodorsal, ventrolateral, ventromedial, ventral posterior, rhomboid, reuniens, and medial geniculate thalamic nuclei, and locus coeruleus. Occipital cortex infusion retrogradely labeled neurons in the frontal, temporal, occipital, and perirhinal cortices as well as the claustrum, basal forebrain, thalamus, epithalamus, hypothalamus, and raphe nuclei. Dorsal hippocampal infusion retrogradely labeled neurons within the septal diagonal band, supramammillary nucleus, and entorhinal cortex and was also transported within various hippocampal subfields. Entorhinal cortex infusion retrogradely labeled neurons within the perirhinal cortex, endopiriform nucleus, piriform cortex, dentate gyrus, presubiculum, parasubiculum, CA1-CA4 fields, amygdaloid nuclei, basal forebrain, thalamus, hypothalamus, periaqueductal gray, raphe nuclei, and locus coeruleus. Amygdala infusion labeled neurons in the endopiriform nucleus, temporal cortex, piriform cortex, paralimbic cortex, hippocampus, subiculum, entorhinal cortex, amygdala, basal forebrain, thalamus, hypothalamus, substantia nigra, pars compacta, raphe, and pontine parabrachial nuclei. In situ hybridization experiments demonstrated that virtually all areas which retrogradely transport BDNF also express its message. Neuroanatomical distributional studies of BDNF will unravel specific central nervous system neurotrophic-responsive systems.

Short increase of BDNF messenger RNA triggers kainic acid-induced neuronal hypertrophy in adult mice.

Neurotrophin gene expression in adult brain varies according to physiological activity and following brain injury, suggesting a role in neuronal maintenance and plasticity. However, the exact roles and mechanisms of action of neurotrophins in the adult brain are still poorly understood. We have recently demonstrated that neurons of the adult mouse dentate gyrus can develop a conspicuous morphogenetic response to intrahippocampal injection of kainic acid. This response is correlated with long-lasting overexpression of the brain-derived neurotrophic factor gene, suggesting a causal relationship between molecular and structural changes. To test this hypothesis, brain-derived neurotrophic factor messenger RNA were sequestered in vivo by administration of antisense oligodeoxynucleotides. When administered before 20 h post-kainate, antisense oligodeoxynucleotides totally prevented the kainate-induced neuronal hypertrophy, while sense or missense sequences had no effect. On the other hand, the hypertrophic response was observed when antisense administration was begun 24 h post-kainate, indicating an involvement of brain-derived neurotrophic factor messenger RNA in the initiation of structural changes, but not in their evolution. The hypertrophy was blocked by inhibition of tyrosine kinase activities by K252a, suggesting an involvement of Trk high affinity receptors. Administration of human recombinant brain-derived neurotrophic factor without previous treatment by kainate failed to induce any morphogenetic response. These results show that a short activation of the brain-derived neurotrophic factor gene can, in association with neuronal activation by kainate, trigger dramatic and long-lasting morphological changes in adult neurons. A physiological role of brain-derived neurotrophic factor in adult brain could therefore be to link, by autocrine/paracrine action, activation of glutamate receptors and neuronal morphological adaptive responses.

The effect of brain-derived neurotrophic factor on regeneration of nerve fibers after crush injury--morphometric evaluation

Brain-derived neurotrophic factor (BDNF) is produced in Schwann cells and fibroblasts in the peripheral nerve, and is considered to play an important role in survival, maintenance, development and repair of the peripheral neuron. In this study, the effect of human recombinant BDNF on the regeneration of nerve fibers following a crush injury to the sciatic nerves of Sprague-Dawley rats was evaluated. In the experimental group, 20 mg/kg of BDNF was injected subcutaneously three times a week for 4 weeks in seven rats. In the control group, only the dissolving solution used in the experimental group was injected similarly in seven rats as in the experimental group. The various morphometric parameters were evaluated in the peroneal, sural and tibial nerves and the nerve to soleus muscle distal to the crush site. There were no statistically significant differences in the change of body weight, the improvement of the width between the first and fifth toes on the side of the crush, the density of total myelinated fibers (numbers per mm2), the total number of myelinated fibers (number per nerve), maximum and median diameters of myelinated fibers, the density of unmyelinated fibers, and the electron microscopic parameters of axon and myelin sheath. On the other hand, the number of myelin lamellae in relation to the transverse axonal area was significantly greater in the experimental group than in the control group. Therefore, BDNF may promote the myelination. However, such an effect on myelination seems not to be clinically significant, because such an effect was not demonstrated in other morphometric evaluations reflecting the myelination condition. Therefore, taking all the data obtained in this study into consideration, we concluded that there was no definite evidence that BDNF promoted the regeneration of nerve fibers at least under these experimental conditions.

Brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 maintain functional tolerance to ethanol

Neurotrophins and growth factors not only affect neuronal development, but also maintain neuronal survival and influence neuronal function in the adult brain, and affect various cognitive processes related to learning and memory. Functional tolerance to ethanol represents an adaptive change in the central nervous system that has been hypothesized to have mechanisms in common with those underlying learning or memory. In the present work, the effects of neurotrophins on ethanol tolerance were compared to the effect of the neuropeptide, arginine vasopressin, which maintains (reduces the rate of dissipation of) both ethanol tolerance and memory. Functional tolerance to ethanol was induced in C57BL 6J mice by feeding them an ethanol-containing liquid diet, and the effect of neurotrophins on the rate of dissipation of tolerance to the hypnotic effect of ethanol was assessed. Human recombinant brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5, injected intracere-broventricularly once daily following ethanol withdrawal, maintained ethanol tolerance, while tolerance dissipated in ethanol-fed mice injected with vehicle (artificial cerebrospinal fluid) or with basic fibroblast growth factor. The results demonstrate that some neurotrophins can modulate neuroadaptation to ethanol, supporting the hypothesis that these factors can influence the function of postmitotic neurons in the adult brain.

Effects of brain-derived neurotrophic factor on motor dysfunction in wobbler mouse motor neuron disease.

Brain-derived neurotrophic factor (BDNF) has been shown to promote the survival of developing motor neurons in vitro and to rescue motor neurons from axotomy-induced cell death in vivo. In this study, we examined the effects of exogenous BDNF on the progression of wobbler mouse motor neuron disease (MND). After clinical diagnosis at age 3 to 4 weeks, 20 affected mice received subcutaneous injections of recombinant human BDNF (5 mg/kg, n = 10) or vehicle (n = 10), three times a week for 4 weeks. In a separate experiment done to conduct a histometric analysis of the C-5 and C-6 ventral roots and to determine the number of myelinated nerve fibers, 7 wobbler mice received identical BDNF treatment. In the 10 BDNF-treated wobbler mice, grip strength declined at a slower rate (p < 0.03) and was twice as great as that of vehicle-treated animals at the end of treatment (p < 0.01). In vivo biceps (p < 0.01) and in vitro muscle twitch tensions (p < 0.02) were also greater than those of vehicle-treated mice. The biceps muscle weight was 20% greater (p < 0.05) and the mean muscle fiber diameter was significantly larger in BDNF-treated mice (p < 0.001) because the number of small (denervated) muscle fibers was markedly reduced. The number of myelinated motor axons at the cervical ventral roots studied in the additional 7 affected mice was 25% greater with BDNF treatment (p < 0.0001). This study establishes that exogenous BDNF administration can retard motor dysfunction in a natural MND and diminish denervation muscle atrophy and motor axon loss.

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.

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.

Refolding of brain-derived neurotrophic factor from guanidine hydrochloride: Kinetic trapping in a collapsed form which is incompetent for dimerization

We have studied the pathway and kinetics of refolding of recombinant human brain-derived neurotrophic factor (BDNF), which is a very tightly-associated dimer in its native state. When BDNF unfolded in 6 M guanidine hydrochloride is diluted 20-fold into phosphate-buffered saline, a partially folded intermediate is rapidly formed (< 1 min). Circular dichroism and fluorescence spectroscopy show that this intermediate has extensive secondary structure, but no well-defined tertiary structure. Size-exclusion chromatography with light scattering detection shows that it is compact and monomeric, and therefore corresponds to what is often called a "collapsed form" or "molten globule". This collapsed form disappears with a half-time of approximately 30 min, simultaneously with the appearance of native dimers, without accumulation of monomeric species with a native tertiary structure. Remarkably, the monomer-dimer association constant of the collapsed form is approximately 10(10) weaker than the native structure, and it has a low tendency to form large aggregates. Given the very large hydrophobic surface present at the dimer interface of nerve growth factor (and presumably in BDNF), these results indicate that these hydrophobic groups are not exposed in the collapsed form, and that it is therefore quite dissimilar from the native structure. A significant conformational change in the collapsed form is necessary to re-expose these hydrophobic groups to form the dimer interface, making this the rate-limiting step in reaching the native conformation.

Acid-induced unfolding of brain-derived neurotrophic factor results in the formation of a monomeric "A state"

Recombinant human brain-derived neurotrophic factor in acid undergoes a slow loss of tertiary structure as monitored by both near-UV circular dichroism and fluorescence, and appears to retain some secondary structure, as monitored by far-UV circular dichroism and Fourier transform infrared spectroscopy. This loss of tertiary structure parallels a decrease in the weight average molecular weight, from dimer to monomer, when examined using light scattering. Increasing the temperature accelerates this slow reaction. This process may be described most simply as N2 in equilibrium with 2D where N and D are the native and denatured forms of the protein, respectively. However, the acid denaturation strongly depends on the protein concentration, with higher concentration resulting in a lower rate and extent of denaturation. This suggests that the more complicated mechanism N2 in equilibrium with 2N in equilibrium with 2D more accurately describes the denaturation, where the dissociation into a native monomer is the rate-limiting step, and the conversion of N to D occurs relatively rapidly. Size-exclusion chromatography (at neutral pH) at several points during denaturation further demonstrated that the amount of tertiary structure remaining paralleled the dimer concentration and also that the monomer form was long-lived, remaining as monomer during the course of the chromatography. Size-exclusion chromatography and sedimentation velocity determination indicated that the acid-denatured form is a compact molecule. On the basis of the above data, the acid-denatured form may be considered to be a monomeric compact intermediate A state with no tertiary structure but considerable secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)