Brain-derived neurotrophic factor, acting at the spinal cord level, participates in bladder hyperactivity and referred pain during chronic bladder inflammation.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin (NT) known to participate in chronic somatic pain. A recent study has indicated that BDNF may participate in chronic cystitis at the peripheral level. However, the principal site of action for this NT is the central nervous system, most notably the spinal cord. The effects of centrally-acting BDNF on bladder function in normal animals and its central role during chronic cystitis are presently unknown. The present study was undertaken to clarify this issue. For that purpose, control non-inflamed animals were intrathecally injected with BDNF, after which bladder function was evaluated. This treatment caused short-lasting bladder hyperactivity; whereas chronic intrathecal administration of BDNF did not elicit this effect. Cutaneous sensitivity was assessed by mechanical allodynia as an internal control of BDNF action. To ascertain the role of BDNF in bladder inflammation, animals with cyclophosphamide-induced cystitis received intrathecal injections of either a general Trk receptor antagonist or a BDNF scavenger. Blockade of Trk receptors or BDNF sequestration notably improved bladder function. In addition, these treatments also reduced referred pain, typically observed in rats with chronic cystitis. Reduction of referred pain was accompanied by a decrease in the spinal levels of extracellular signal-regulated kinase (ERK) phosphorylation, a marker of increased sensory barrage in the lumbosacral spinal cord, and spinal BDNF expression. Results obtained here indicate that BDNF, acting at the spinal cord level, contributes to bladder hyperactivity and referred pain, important hallmarks of chronic cystitis. In addition, these data also support the development of BDNF modulators as putative therapeutic options for the treatment of chronic bladder inflammation.

Sequestration of brain derived nerve factor by intravenous delivery of TrkB-Ig2 reduces bladder overactivity and noxious input in animals with chronic cystitis.

Brain derived nerve factor (BDNF) is a trophic factor belonging to the neurotrophin family. It is upregulated in various inflammatory conditions, where it may contribute to altered pain states. In cystitis, little is known about the relevance of BDNF in bladder-generated noxious input and bladder overactivity, a matter we investigated in the present study. Female rats were intraperitoneally (i.p.) injected with cyclophosphamide (CYP; 200 mg/kg). They received saline or TrkB-Ig(2) via intravenously (i.v.) or intravesical administration. Three days after CYP-injection, animals were anaesthetized and cystometries performed. All animals were perfusion-fixed and the spinal cord segments L6 collected, post-fixed and processed for c-Fos and phosphoERK immunoreactivity. BDNF expression in the bladder, as well as bladder histology, was also assessed. Intravesical TrkB-Ig(2) did not change bladder reflex activity of CYP-injected rats. In CYP-animals treated with i.v. TrkB-Ig(2) a decrease in the frequency of bladder reflex contractions, in comparison with saline-treated animals, was observed. In spinal sections from the latter group of animals, the number of phosphoERK and c-Fos immunoreactive neurons was lower than in sections from saline-treated CYP-animals. BDNF immunoreactivity was higher during cystitis but was not changed by TrkB-Ig(2) i.v. treatment. Evaluation of the bladder histology showed similar inflammatory signs in the bladders of inflamed animals, irrespective of the treatment. Data show that i.v. but not intravesical administration of TrkB-Ig(2) reduced bladder hyperactivity in animals with cystitis to levels comparable to those observed in unirritated rats. Since i.v. TrkB-Ig(2) also reduced spinal extracellular signal-regulated kinase (ERK) activation, it is possible that BDNF contribution to inflammation-induced bladder hyperactivity is via spinal activation of the ERK pathway. Finally, the reduction in c-Fos expression indicates that TrkB-Ig(2) also reduced bladder-generated noxious input. Our results show that sequestration of BDNF may be considered a new therapeutic strategy to treat chronic cystitis.

Characterisation of ultraviolet-B-induced inflammation as a model of hyperalgesia in the rat.

In humans, the acute inflammatory reaction caused by ultraviolet (UV) radiation is well studied and the sensory changes that are found have been used as a model of cutaneous hyperalgesia. Similar paradigms are now emerging as rodent models of inflammatory pain. Using a narrowband UVB source, we irradiated the plantar surface of rat hind paws. This produced the classical feature of inflammation, erythema, and a significant dose-dependent reduction in both thermal and mechanical paw withdrawal thresholds. These sensory changes peaked 48h after irradiation. At this time there is a graded facilitation of noxious heat evoked (but not basal) c-fos-like immunoreactivity in the L4/5 segments of the spinal cord. We also studied the effects of established analgesic compounds on the UVB-induced hyperalgesia. Systemic as well as topical application of ibuprofen significantly reduced both thermal and mechanical hyperalgesia. Systemic morphine produced a dose-dependent and naloxone sensitive reversal of sensory changes. Similarly, the peripherally restricted opioid loperamide also had a dose-dependent anti-hyperalgesic effect, again reversed by naloxone methiodide. Sequestration of NGF, starting at the time of UVB irradiation, significantly reduced sensory changes. We conclude that UVB inflammation produces a dose-dependent hyperalgesic state sensitive to established analgesics. This suggests that UVB inflammation in the rat may represent a useful translational tool in the study of pain and the testing of analgesic agents.

NGF receptor TrkAd5: therapeutic agent and drug design target.

Biochemical studies have shown that domain 5 of the TrkA (tropomyosin receptor kinase A) receptor is involved in the binding of NGF (nerve growth factor). Crystallographic studies have confirmed this, demonstrating that one homodimer of NGF binds to two TrkAd5 molecules. TrkAd5 has been made recombinantly in Escherichia coli, purified and shown to bind NGF with picomolar affinity. We have used the co-ordinates of the crystal structure of the NGF-TrkAd5 complex to screen approximately two million compounds in silico for the identification of small molecule agonists/antagonists. Selected hits were shown to be active in an in vitro ligand-binding assay; structure-activity relationships are now being investigated. In addition, TrkAd5 has been shown to be efficacious in preclinical models of inflammatory pain and asthma by the sequestration of excess levels of endogenous NGF, and therefore represents a novel therapeutic agent.

Neurotrophins and neurodegeneration.

There is growing evidence that reduced neurotrophic support is a significant factor in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). In this review we discuss the structure and functions of neurotrophins such as nerve growth factor, and the role of these proteins and their tyrosine kinase (Trk) receptors in the aetiology and therapy of such diseases. Neurotrophins regulate development and the maintenance of the vertebrate nervous system. In the mature nervous system they affect neuronal survival and also influence synaptic function and plasticity. The neurotrophins are able to bind to two different receptors: all bind to a common receptor p75NTR, and each also binds to one of a family of Trk receptors. By dimerization of the Trk receptors, and subsequent transphosphorylation of the intracellular kinase domain, signalling pathways are activated. We discuss here the structure and function of the neurotrophins and how they have been, or may be, used therapeutically in AD, PD, Huntington's diseases, ALS and peripheral neuropathy. Neurotrophins are central to many aspects of nervous system function. However they have not truly fulfilled their therapeutic potential in clinical trials because of the difficulties of protein delivery and pharmacokinetics in the nervous system. With the recent elucidation of the structure of the neurotrophins bound to their receptors it will now be possible, using a combination of in silico technology and novel screening techniques, to develop small molecule mimetics with much improved pharmacotherapeutic profiles.

Specificity in Trk receptor:neurotrophin interactions: the crystal structure of TrkB-d5 in complex with neurotrophin-4/5.

BACKGROUND: The binding of neurotrophin ligands to their respective Trk cellular receptors initiates intracellular signals essential for the growth and survival of neurons. The site of neurotrophin binding has been located to the fifth extracellular domain of the Trk receptor, with this region regulating both the affinity and specificity of Trk receptor:neurotrophin interaction. Neurotrophin function has been implicated in a number of neurological disorders, including Alzheimer's disease and Parkinson's disease. RESULTS: We have determined the 2.7 A crystal structure of neurotrophin-4/5 bound to the neurotrophin binding domain of its high-affinity receptor TrkB (TrkB-d5). As previously seen in the interaction of nerve growth factor with TrkA, neurotrophin-4/5 forms a crosslink between two spatially distant receptor molecules. The contacts formed in the TrkB-d5:neurotrophin-4/5 complex can be divided into a conserved area similar to a region observed in the TrkA-d5:NGF complex and a second site-unique in each ligand-receptor pair-formed primarily by the ordering of the neurotrophin N terminus. CONCLUSIONS: Together, the structures of the TrkB-d5:NT-4/5 and TrkA-d5:NGF complexes confirm a consistent pattern of recognition in Trk receptor:neurotrophin complex formation. In both cases, the N terminus of the neurotrophin becomes ordered only on complex formation. This ordering appears to be directed largely by the receptor surface, with the resulting complementary surfaces providing the main determinant of receptor specificity. These features provide an explanation both for the limited crossreactivity observed between the range of neurotrophins and Trk receptors and for the high-affinity binding associated with respective ligand-receptor pairs.

Time course and nerve growth factor dependence of inflammation-induced alterations in electrophysiological membrane properties in nociceptive primary afferent neurons.

Novel findings of changes in nociceptive dorsal root ganglion (DRG) neurons during hindlimb inflammation induced by complete Freund's adjuvant (CFA) injections in the hindpaw and hindleg are reported. These include increased maximum fiber following frequency in nociceptive C- and Adelta-fiber units by 2.7 and 3 times, respectively, and increased incidence of ongoing (spontaneous) activity by 3.3 times (to 54%) and 2.4 times (to 27%), respectively. These changes and the CFA-induced changes in somatic action potential (AP) configuration in nociceptive neurons (Djouhri and Lawson, 1999) were incomplete 24 hr after CFA. The nerve growth factor (NGF) dependence of the inflammation-induced changes was examined by injecting a synthetic NGF sequestering protein [tyrosine receptor kinase A Ig2 (trkA Ig2)] with CFA and subsequently into the CFA injection sites. NGF sequestration prevented some CFA-induced changes in nociceptive neurons including: the increased fiber following frequency (C and Adelta), the increased proportions of units with ongoing activity (C and Adelta), the decreased AP duration (C and Adelta), but not the decreased afterhyperpolarization (AHP) durations (C, Adelta, and Aalpha/beta) (Djouhri and Lawson, 1999). AP variables of nociceptive units with spontaneous activity were examined. The time course of electrophysiological changes in nociceptive units is consistent with processes involving altered protein expression and/or retrograde transport of factors. These results (1) implicate NGF in regulating inflammation-induced decreases in AP duration and in increases in firing rate and spontaneous activity but not in decreases in AHP duration and (2) suggest clinical advantages of reducing NGF in some inflammatory pain states.

Identification and structure of the nerve growth factor binding site on TrkA.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer's disease. NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains. We have expressed the second Ig-like domain as a recombinant protein in E. coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor. This domain (TrkAIg(2)) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation. We also present the three-dimensional structure of the TrkAIg(2) domain in a new crystal form, refined to 2.0 A resolution.

Expression of recombinant extracellular domain of the type II transforming growth factor-beta receptor: utilization in a modified enzyme-linked immunoabsorbent assay to screen TGF-beta agonists and antagonists.

TGF-beta is a ubiquitous protein that exhibits a broad spectrum of biological activity. The prokaryotic expression and purification of the extracellular domain of the type II TGF-beta receptor (T beta R-II-ED), without the need for fusion protein cleavage and refolding, is described. The recombinant T beta R-II-ED fusion protein bound commercially available TGF-beta 1 and displayed an affinity of 11.1 nM. In a modified ELISA, receptor binding to TGF-beta1 was inhibited by TGF-beta 3. The technique lends itself to high-throughput screening of combinatorial libraries for the identification of TGF-beta agonists and antagonists and this, in turn, may have important therapeutic implications.

Recombinant human nerve growth factor for clinical trials: protein expression, purification, stability and characterisation of binding to infusion pumps.

Nerve growth factor (NGF) has been suggested to be of therapeutic benefit to patients with Alzheimer's disease. One of the early changes in this disease is a loss of cholinergic function within the brain, and NGF is able to rescue cholinergic neurons both in vitro and in vivo. We describe the production of recombinant human beta-NGF (rhNGF), using baculovirus infection of insect cells; its purification, formulation and subsequent stability for use in clinical trials. Tests were also carried out to monitor release of protein from infusion pumps and catheters for intracerebroventricular administration (icv). Initial problems with non-specific binding were overcome using a blocking formula.

Galanin regulates the postnatal survival of a subset of basal forebrain cholinergic neurons.

The neuropeptide galanin colocalizes with choline acetyltransferase, the synthetic enzyme for acetylcholine, in a subset of cholinergic neurons in the basal forebrain of rodents. Chronic intracerebroventricular infusion of nerve growth factor induces a 3- to 4-fold increase in galanin gene expression in these neurons. Here we report the loss of a third of cholinergic neurons in the medial septum and vertical limb diagonal band of the basal forebrain of adult mice carrying a targeted loss-of-function mutation in the galanin gene. These deficits are associated with a 2-fold increase in the number of apoptotic cells in the forebrain at postnatal day seven. This loss is associated with marked age-dependent deficits in stimulated acetylcholine release, performance in the Morris water maze, and induction of long-term potentiation in the CA1 region of the hippocampus. These data provide unexpected evidence that galanin plays a trophic role to regulate the development and function of a subset of septohippocampal cholinergic neurons.

Identification of syntaxin 1A as a novel binding protein for presenilin-1.

Mutations in the presenilin 1 gene have been shown to result in Alzheimer's disease. Presenilin 1 is a multi-transmembrane protein with a large hydrophilic loop near the C-terminus. This region is required for known functions of presenilin 1. We have constrained this loop within the active site of the bacterial protein, thioredoxin, to mimic its native conformational state. This hybrid protein was used as bait in a yeast two hybrid screen in an attempt to identify presenilin binding proteins. By this method syntaxin 1A, a synaptic plasma membrane protein, was identified as a novel binding protein for presenilin 1. In vitro experiments confirm the two-hybrid results suggesting that PS1 binds syntaxin under physiological conditions.

Profound and selective loss of catalytic TrkB immunoreactivity in Alzheimer's disease.

Brain-derived neurotrophic factor (BDNF) is known to have trophic effects on various neurons, throughout the brain and spinal cord, via its high-affinity tyrosine kinase receptor TrkB. It has been reported that the mRNA for this neurotrophin is reduced in Alzheimer's disease (AD) brain. We have examined, by Western blotting, the catalytic (p145) and noncatalytic or truncated (p95) forms of TrkB and find that, in both the temporal and frontal cortex, there is a selective loss of immunoreactive-positive staining for the catalytic or kinase form compared with the truncated form. This may have important consequences for the neurotrophic support of vulnerable neurons in AD.

Cloning and characterization of the presenilin-2 gene promoter.

Mutations in the presenilin-2 (PS-2) have been shown to cause early onset Alzheimer's disease (AD) in a series of families known as the Volga Germans and in an unrelated Italian kindred. Expression of the PS-2 gene is regulated during AD, aging, development and brain injury. Although expressed primarily in neurons, enhanced levels of PS-2 have been reported in astrocytes activated by neuronal damage. Understanding the regulation of the PS-2 gene may thus provide an insight into its role in AD. We have isolated a 3635 bp DNA fragment that contains 2934 bp of DNA sequence upstream from the PS-2 gene. Primer extension analysis was used to map three major transcriptional start sites within the PS-2 gene. The promoter sequence, upstream of each transcriptional start site, does not contain TATA or CAAT boxes but does contain several GC rich sites (Sp-1 and AP-2). A reporter gene construct containing the PS-2 promoter (PS2P, -2934 to +702) transfected into M17 cells drives basal transcription to 20% of the levels of the SV-40 viral promoter. Addition of NGF to PC-12 cells was found to upregulate the PS2P promoter and an NGF-responsive element was localized by deletional analysis between -403 and +13 within the promoter. Since the PS-2 gene has multiple start sites and the upstream sequence is GC rich with no TATA box, the PS-2 promoter is consistent with the GC class of 'housekeeping' genes.

Immunoglobulin-like domains define the nerve growth factor binding site of the TrkA receptor.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system. NGF binds with high affinity to the extracellular region of the tyrosine kinase receptor TrkA. This domain comprises leucine and cysteine rich motifs, followed by two immunoglobulin like (Ig-like) domains. We describe the expression and purification of recombinant Ig-like domains. Fluorescence and circular dichroism spectroscopy show that the protein is folded into a compact globular structure and contains mainly beta-sheet secondary structure. Recombinant protein binds to NGF and can inhibit NGF bioactivity both in vitro and in vivo.

Reduced cholinergic function in normal and Alzheimer's disease brain is associated with apolipoprotein E4 genotype.

Apolipoprotein E (ApoE) is a potent risk factor for Alzheimer's disease. Since the loss of cholinergic function in Alzheimer's disease is known to occur at an early stage in the disease we have examined this function in normal subjects with an Apoepsilon4 allele to see if the deficit occurs in the absence of Alzheimer pathology or symptoms. We report that brain tissue obtained post-mortem from normal subjects and Alzheimer patients with an Apoepsilon4 allele has a lower cholinergic activity than tissue from those subjects without this allele. This has important significance for the interpretation of the cholinergic deficits found in Alzheimer's disease.

Cloning of a non-catalytic form of human trkB and distribution of messenger RNA for trkB in human brain.

A truncated form of the human trkB gene has been cloned and sequenced. This gene is related to the trk family of tyrosine kinases, the products of which act as receptors for the neurotrophins. Of these, brain-derived neurotrophic factor and mammalian neurotrophin-4 are the known ligands for the TrkB receptor. Catalytic and non-catalytic (or truncated) forms of the trkB gene have been cloned for rat and mouse. In this study, using in situ hybridization, we describe the distribution of trkB messenger RNA in fetal and adult human brain.

Normal beta-NGF content in Alzheimer's disease cerebral cortex and hippocampus.

Nerve growth factor (beta-NGF) is known to have beneficial effects on cholinergic cell survival and to function both in vivo and in vitro. It has been speculated that this protein, or the lack of it, may be involved in the aetiology of Alzheimer's disease (AD). We describe the measurement of beta-NGF content in 4 regions of the cerebral cortex and the hippocampus in AD brain compared with brain tissue from age-matched normal subjects using a sensitive sandwich immunoassay (ELISA). There was no difference in beta-NGF content in any region examined in AD compared with normal values despite the marked loss of cortical cholinergic function.

Low affinity nerve growth factor receptor binding in normal and Alzheimer's disease basal forebrain.

The binding characteristics of radiolabelled beta-nerve growth factor ([125I]NGF) have been determined on membrane preparations of basal forebrain from Alzheimer's disease (AD) brain and age-matched normal brains. [125I]NGF binds in a specific fashion indicative of a single receptor and is not displaced with microM concentrations of cytochrome c, insulin or epidermal growth factor (EGF). The mean dissociation constant (Kd) and the mean capacity (Bmax) of the NGF receptor were not significantly different between the 5 AD and 5 normal basal forebrain samples examined. Choline acetyltransferase (ChAT) activity was significantly reduced (P less than or equal to 0.001) in AD cerebral cortical samples compared with normal tissue.

Distribution of beta-nerve growth factor receptors in the human basal forebrain.

The distribution of neurons expressing the receptor for beta-nerve growth factor has been examined immunohistochemically in serial coronal sections of basal forebrain from aged normal human subjects. Neurons expressing the receptor were observed in the nucleus of the diagonal band of Broca and in the anterior, the intermediate, and the posterior portions of the nucleus basalis of Meynert. Neurons could also be seen in the medial septal nucleus and embedded in myelinated fibre tracts such as those of the external capsule, cingulum, medullary laminae of the globus pallidus, ansa penduncularis, ansa lenticularis, and anterior commissure. In situ hybridization with a 35S cDNA probe to the human beta-nerve growth factor receptor confirms a neuronal location as the site of synthesis of beta-nerve growth factor receptors in the nucleus basalis of Meynert in a fifth brain. A high percentage of Nissl-stained hyperchromic magnocellular neurons expressed the receptor for beta-nerve growth factor, suggesting that most neurons in the human cholinergic magnocellular basal forebrain system express these receptors. Recent data suggest that beta-nerve growth factor functions as a neurotrophic factor in basal forebrain cholinergic neurons. In Alzheimer's disease there is known to be a reduction in cholinergic function and an apparent loss of neurons in the cholinergic nucleus basalis of Meynert. For this reason we have examined the distribution of receptors for beta-nerve growth factor in the normal human basal forebrain in order to form a basis for comparison to those with Alzheimer's disease.