The biological effects of endogenous nerve growth factor on adult sensory neurons revealed by a trkA-IgG fusion molecule.

Evidence suggests that nerve growth factor (NGF) may function as a mediator of some persistent pain states. We have used a synthetic protein, trkA-IgG, to sequester endogenous NGF and block the survival effects of NGF on cultured sensory neurons. We show that administration of trkA-IgG produces a sustained thermal and chemical hypoalgesia and leads to a downregulation of the sensory neuropeptide CGRP (calcitonin gene-related peptide) in treated sensory neurons. Acute administration of the molecule blocks the hyperalgesia that develops with carrageenan-induced inflammation. These data suggest that peripherally produced NGF normally acts to maintain the sensitivity of nociceptive sensory neurons and that, in some inflammatory states, an upregulation of NGF is responsible for alterations in pain-related behaviour. Antagonists of NGF may therefore be of clinical use in treating some chronic pain states.

Studies on the regulation of beta-nerve growth factor gene expression in the rat iris: the level of mRNA-encoding nerve growth factor is increased in irises placed in explant cultures in vitro, but not in irises deprived of sensory or sympathetic innervation in vivo.

Beta-nerve growth factor (NGF) is a protein necessary for the survival and maintenance of sympathetic and sensory neurons that appears to be produced by the target tissues of these neurons in vivo. Both denervation and the culture of explants of one model target, the rat iris, leads to an increase in the NGF content, suggesting that innervating neurons may regulate a step in synthesis or turnover of NGF. To determine whether there is a change in synthesis controlled at the mRNA level, the rat iris has been assayed for its content of NGF mRNA after surgical and chemical denervation and after explant into culture. Using a sensitive blot hybridization assay, a large, rapid increase in the content of NGF mRNA was observed upon explant of the rat iris. The increase was readily detectable within 1 h, reached a maximum increase of 10- to 20-fold by 6 to 12 h, and was still evident after 3 d in culture. The distribution of NGF mRNA in different areas of the iris does not change during this time. This rapid increase in NGF mRNA is also seen in the fully innervated iris in vivo after trauma to the anterior chamber. In contrast, denervation to varying degrees in situ had no effect on NGF mRNA levels. Neither removal of sympathetic innervation by surgical or chemical methods nor combined surgical removal of sympathetic and sensory innervation detectably altered NGF mRNA content. Thus, denervation of the rat iris in situ does not cause the observed accumulation of NGF by increasing the level of NGF mRNA, and the increase in NGF content must be due to other factors.

Blockade of endogenous ligands of trkB inhibits formation of ocular dominance columns.

We have examined the hypothesis that the segregation of LGN axon terminals into ocular dominance (OD) patches in layer 4 of the visual cortex requires neurotrophins, acting as signals to modulate the pattern of synaptic connectivity. Neurotrophin receptor antagonists, composed of the extracellular domain of each member of the trk family of neurotrophin receptors fused to a human Fc domain, were infused directly into visual cortex during the peak phase of OD column formation. Infusion of trkB-IgG, which binds BDNF and NT-4/5, inhibited the formation of OD patches within layer 4, while trkA-IgG and trkC-IgG, which preferentially bind NGF and NT-3, respectively, had no effect. The autoradiographic labeling of LGN terminals in cortical layer 4 was reduced by trkB-IgG, in contrast with the increased labeling observed following NT-4/5 infusion. These data suggest that an endogenous ligand of trkB, normally present in limiting amounts within visual cortex, is necessary for the selective growth and remodeling of LGN axons into eye-specific patches.

Molecular cloning and expression of a novel adhesion molecule, SC1.

SC1, an integral membrane glycoprotein of 100 kd, is uniquely and transiently expressed on spinal cord motoneurons early in development and appears in peripheral neurons and several other tissues during development. SC1 has been purified by immunoaffinity techniques, and SC1 cDNA clones have been obtained by screening an E4 chick embryo phage expression library with a rabbit polyclonal antibody produced against purified SC1. The deduced protein sequence of 588 amino acids consists of a signal peptide, five immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail. The sequence is most similar to MUC18, reported as a tumor progression marker in human melanoma. Transfection of SC1 cDNA into mammalian cells leads to cell surface expression of SC1 antigen and a subsequent increase in cell-cell adhesion. SC1 molecules bind to each other via a homophilic adhesion mechanism, independently of calcium or magnesium ions. SC1 may have a role in lateral motor column formation or neurite growth or fasciculation.

The glial cell line-derived neurotrophic factor family receptor components are differentially regulated within sensory neurons after nerve injury.

Glial cell line-derived neurotrophic factor (GDNF) has potent trophic effects on adult sensory neurons after nerve injury and is one of a family of proteins that includes neurturin, persephin, and artemin. Sensitivity to these factors is conferred by a receptor complex consisting of a ligand binding domain (GFRalpha1-GFRalpha4) and a signal transducing domain RET. We have investigated the normal expression of GDNF family receptor components within sensory neurons and the response to nerve injury. In normal rats, RET and GFRalpha1 were expressed in a subpopulation of both small- and large-diameter afferents projecting through the sciatic nerve [60 and 40% of FluoroGold (FG)-labeled cells, respectively]. GFRalpha2 and GFRalpha3 were both expressed principally within small-diameter DRG cells (30 and 40% of FG-labeled cells, respectively). Two weeks after sciatic axotomy, the expression of GFRalpha2 was markedly reduced (to 12% of sciatic afferents). In contrast, the proportion of sciatic afferents that expressed GFRalpha1 increased (to 66% of sciatic afferents) so that virtually all large-diameter afferents expressed this receptor component, and the expression of GFRalpha3 also increased (to 66% of sciatic afferents) so that almost all of the small-diameter afferents expressed this receptor component after axotomy. There was little change in RET expression. The changes in the proportions of DRG cells expressing different receptor components were mirrored by alterations in the total RNA levels within the DRG. The changes in GFRalpha1 and GFRalpha2 expression after axotomy could be largely reversed by treatment with GDNF.

A paracrine effect for neuron-derived BDNF in development of dorsal root ganglia: stimulation of Schwann cell myelin protein expression by glial cells.

Addition of neurons to cultures of non-neuronal cells derived from quail embryonic dorsal root ganglia causes a 2.5-fold increase in the proportion of cells that express the glial marker Schwann cell myelin protein (SMP) when compared to cultures devoid of neurons. This effect is mediated by BDNF because incubation with a trkB immunoadhesin that sequesters BDNF, but not with trkA or trkC immunoadhesins, abolishes this stimulation. This neuronal activity can be mimicked by treatment with soluble BDNF that stimulates specifically the conversion of SMP-negative glial cells into cells that express this phenotype. That BDNF is the endogenous neuron-derived factor affecting glial development is further supported by the observation that BDNF is extensively expressed in developing sensory neurons of the avian ganglia both in vivo and in vitro, but not by the satellite cells. These results show for the first time a paracrine role for neuronal BDNF on differentiation of peripheral glial cells. This effect of BDNF is likely to be mediated by the p75 neurotrophin receptor because: (1) p75 immunoreactive protein is expressed by a subset of satellite cells; (2) neutralization of p75 abolishes the BDNF-induced stimulation; (3) a treatment of non-neuronal cell cultures with equimolar concentrations of either soluble NGF or NT-3 also affects the proportion of cells that become SMP-positive. Whereas NGF stimulates the acquisition of this glial antigen to a similar extent as BDNF, NT-3 inhibits its expression, suggesting that distinct neurotrophins signal differentially through p75. These findings also suggest that the definitive phenotype of peripheral glia is determined by a balance between positive and inhibitory signals arising in adjacent neurons.

Development of high affinity choline uptake and associated acetylcholine synthesis in the rat fascia dentata.

The ontogenic development of hemicholinium-sensitive, high affinity choline uptake and the synthesis of acetylcholine from exogenous choline have been studied in particulate preparations of the rat fascia dentata. Between 6 days of age and adulthood the rate of high affinity choline uptake increases 3-fold, when expressed with respect to protein, and 125-fold, when expressed independently of protein. This process develops most rapidly during the period around 16-17 days of age, similar to the ontogenesis of choline acetyltransferase activity. This observation supports the idea that cholinergic septohippocampal boutons develop mainly at this time. Unlike choline acetyltransferase activity, the velocity of high affinity choline uptake increases to as much as 161% of the adult value at about 30 days of age. It is suggested that at 25-31 days of age a relatively high endogenous septohippocampal firing rate increases the rate of choline uptake. At 6 days of age we detected no synthesis of acetylcholine from the accumulated choline. Uptake-synthesis coupling develops mainly between 6 and 13 days of age, earlier than any other presynaptic cholinergic property. Acetylcholine synthesis from exogenous choline develops in paralled with high affinity choline uptake, but developmental increases in uptake velocity result in comparable increases in synthesis rate only after a delay of several days. Some limiting factor other than choline acetyltransferase activity appears to link the accumulation of exogenous choline to acetylcholine synthesis during development.

Lesion-induced plasticity of high affinity choline uptake in the developing rat fascia dentata.

After removal of the perforant path input to the rat fascia dentata at the age of 11 days, cholinergic septohippocampal fibers invade the denervated area. We have examined the effect of this lesion on hemicholinium-sensitive, high affinity choline uptake and its coupling to acetylcholine synthesis, specific properties of the septohippocampal input. Removal of the ipsilateral perforant path fibers increased the velocity of high affinity choline uptake by dentate particulate preparations, usually within 1 day. Studies conducted 5--104 days after operation showed a consistent 50--65% elevation in the molecular (denervated) layer. In contrast, the choline uptake rate in the granular layer eventually decreased slightly. Calculation of choline uptake rates independently of protein (per whole region) revealed that fasciae dentatae from operated and control sides accumulated choline at approximately equal rates, but on the operated side a greater percentage was transported by structures from the molecular layer and a lesser percentage by those from the granular layer. The rate of acetylcholine synthesis from exogenous choline increased to the same extent as high affinity choline uptake from 3 days after operation onwards. The changes in high affinity choline uptake and acetylcholine synthesis coincided spatially and temporally with the reactive growth of septohippocampal fibers. Our results support the view that a perforant path lesion during development permanently alters the distribution of functional septohippocampal boutons in the fascia dentata. Acetylcholine synthesis is regulated to the same extent by high affinity choline uptake in the anomalous boutons as in normally located boutons.

A novel group of nerve growth factor receptor-immunoreactive neurons in the ventral horn of the lumbar spinal cord.

During development and following axonal injury in adults, neurons in the anterior horn of the spinal cord express nerve growth factor receptor (NGF-R) messenger ribonucleic acid (mRNA) and protein. To examine whether unlesioned anterior horn neurons show signs of responsiveness to NGF in adult animals, spinal cords from control rats and monkeys, as well as animals that had received NGF intraventricularly, were processed for NGF-R immunocytochemistry using monoclonal and polyclonal antibodies against NGF-R. In all animals, neurons located in central/ventral sectors of lamina IX in lumbar segments of the spinal cord expressed NGF-R-like immunoreactivity; this population of nerve cells appeared to increase in size after treatment with NGF. Our findings suggest that, in adults, a subset of spinal motor neurons may respond to NGF.

Treatment of radiation-induced tissue injury by hyperbaric oxygen.

Hyperbaric oxygen therapy appears to be a beneficial adjunctive treatment modality in the management of radionecrosis of bone and soft tissue in the head and neck. The mechanism of such wound healing enhancement appears to be related to oxygen stimulation of fibroblastic activity and neovasculation. In this pilot study, involving 13 cases of refractory mandibular radionecrosis treated with hyperbaric oxygen, complete healing of soft tissue disease with covering of exposed bone has been noted in seven cases 11 to 27 months posttreatment; transient healing was seen in three cases; moderate to marked improvement in soft tissue disease has been noted in three cases. Pain relief was marked in six cases, moderate in four cases, and slight in one case of the 11 patients with significant pretreatment pain. Radiographic improvement was slight to moderate in ten cases. Four of the five patients with pathologic fractures developed a firm fibrous union of the mandibular segments during or shortly after treatment. Three additional cases of head and neck radionecrosis of other sites have noted significant improvement in their lesions during treatment. Three other patients with radionecrosis of the foot, hip, and vagina have also been treated with good results only in the vaginal case. None of the 19 patients treated with hyperbaric oxygen developed persistent or significant complications.

Antibody binding regions on human nerve growth factor identified by homolog- and alanine-scanning mutagenesis.

The binding specificities of a panel of mouse monoclonal antibodies (MAbs) to human nerve growth factor (hNGF) were determined by epitope mapping using chimeric and point mutants of NGF. Subsequently, the MAbs were used to probe NGF structure-function relationships. Six MAbs, which recognize distinct or partially overlapping regions of hNGF, were evaluated for their ability to block the binding of hNGF to the TrkA and p75 NGF receptors in various in vitro assays, which included blocking of TrkA autophosphorylation and blocking of NGF-dependent survival of dorsal root ganglion sensory neurons. Three MAbs (911,912,938) were potent blockers of all activities. Potent blocking of p75 binding occurs only with MAb 909, which recognizes an NGF region identified by mutagenesis as important for NGF-p75 binding. These results are consistent with recently proposed models of binding regions involved in NGF-TrkA and NGF-p75 interactions generated through mutagenic analysis and structure determination of the NGF-TrkA complex. These studies provide insight to the epitope specificities and potency of MAbs that would be useful for physiological NGF blocking studies.

Sciatic nerve regeneration is not inhibited by anti-NGF antibody treatment in the adult rat.

Elevated nerve growth factor (NGF) is believed to play a role in many types of pain. An NGF-blocking antibody (muMab 911) has been shown to reduce pain and hyperalgesia in pain models, suggesting a novel therapeutic approach for pain management. Since NGF also plays important roles in peripheral nervous system development and sensory nerve outgrowth, we asked whether anti-NGF antibodies would adversely impact peripheral nerve regeneration. Adult rats underwent a unilateral sciatic nerve crush to transect axons and were subcutaneously dosed weekly for 8weeks with muMab 911 or vehicle beginning 1day prior to injury. Plasma levels of muMab 911 were assessed from blood samples and foot print analysis was used to assess functional recovery. At 8-weeks post-nerve injury, sciatic nerves were prepared for light and electron microscopy. In a separate group, Fluro-Gold was injected subcutaneously at the ankle prior to perfusion, and counts and sizes of retrogradely labeled and unlabeled dorsal root ganglion neurons were obtained. There was no difference in the time course of gait recovery in antibody-treated and vehicle-treated animals. The number of myelinated and nonmyelinated axons was the same in the muMab 911-treated crushed nerves and intact nerves, consistent with observed complete recovery. Treatment with muMab 911 did however result in a small decrease in average cell body size on both the intact and injured sides. These results indicate that muMab 911 did not impair functional recovery or nerve regeneration after nerve injury in adult rats.

CGRP function-blocking antibodies inhibit neurogenic vasodilatation without affecting heart rate or arterial blood pressure in the rat.

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP) receptor antagonists effectively abort migraine headache and inhibit neurogenic vasodilatation in humans as well as rat models. Monoclonal antibodies typically have long half-lives, and we investigated whether or not function-blocking CGRP antibodies would inhibit neurogenic vasodilatation with a long duration of action and therefore be a possible approach to preventive therapy of migraine. During chronic treatment with anti-CGRP antibodies, we measured cardiovascular function, which might be a safety concern of CGRP inhibition. EXPERIMENTAL APPROACH: We used two rat blood flow models that measure electrically stimulated vasodilatation in the skin or in the middle meningeal artery (MMA). These vasomotor responses are largely dependent on the neurogenic release of CGRP from sensory afferents. To assess cardiovascular function during chronic systemic anti-CGRP antibody treatment, we measured heart rate and blood pressure in conscious rats. KEY RESULTS: Treatment with anti-CGRP antibodies inhibited skin vasodilatation or the increase in MMA diameter to a similar magnitude as treatment with CGRP receptor antagonists. Although CGRP antibody treatment had a slower onset of action than the CGRP receptor antagonists, the inhibition was still evident 1 week after dosing. Chronic treatment with anti-CGRP antibodies had no detectable effects on heart rate or blood pressure. CONCLUSIONS AND IMPLICATIONS: We showed for the first time that anti-CGRP antibodies exert a long lasting inhibition of neurogenic vasodilatation in two different rat models of arterial blood flow. We have provided strong preclinical evidence that anti-CGRP antibody may be a suitable drug candidate for the preventive treatment of migraine.

Are there more members of the CNTF-GPA family?

Ciliary neurotrophic factor (CNTF) and growth promoting activity (GPA) are two members of a family of structurally and functionally related cytokines. Although the primary sequences of these proteins are only distantly related, many share striking functional similarities. The question of the potential existence of more, as yet undiscovered, members of this family, especially those most related to CNTF, is discussed. There are several biological systems which exhibit unexplained CNTF-like activities. This has led to speculation that there are indeed other CNTF-like proteins to be found. Because of the poor primary sequence conservation among known members of this family, even those sharing strong functional similarities, it is unlikely that a cloning approach based on sequence homology will find these putative new members of the family. Instead, a more biological approach, based on functional similarities, is more likely to succeed.

Studies on the expression of the beta nerve growth factor (NGF) gene in the central nervous system: level and regional distribution of NGF mRNA suggest that NGF functions as a trophic factor for several distinct populations of neurons.

Beta nerve growth factor (NGF), a target-derived protein necessary for survival and development of sympathetic and sensory neurons, can also affect subpopulations of neurons in the central nervous system (CNS). Using a blot hybridization assay capable of detecting 10 fg of mRNA, we measured the levels of NGF mRNA in the major brain regions, including those innervated by NGF-responsive neurons. NGF mRNA was detected unambiguously in each major region of the CNS. The levels were comparable to those in sympathetic effector organs. Discrete areas contained very different amounts of NGF mRNA. Up to 40-fold differences were seen, a range comparable to the differences between richly and sparsely innervated sympathetic effector organs. The highest concentrations of NGF mRNA were found in the cortex and hippocampus, which are the major targets of the NGF-responsive cholinergic neurons of the basal forebrain nuclei. Significant amounts of NGF mRNA were also found in areas that contain the central processes of NGF-responsive sensory neurons, such as the pons, medulla, and spinal cord. The presence of NGF mRNA in these areas suggests that brain NGF may act as a target-derived trophic factor for both populations of neurons. NGF mRNA was also found in the striatum, suggesting that locally derived NGF may act there as a trophic factor for a recently identified population of NGF-responsive cholinergic local circuit neurons. However, high levels of NGF mRNA were also found in some regions, such as the diencephalon, that have no relation to any identified population of NGF-responsive neurons. This suggests that there may be additional populations of NGF-responsive neurons in the CNS that have not yet been discovered.

Expression of the beta-nerve growth factor gene correlates with the density of sympathetic innervation in effector organs.

Although beta-nerve growth factor (NGF), a protein necessary for survival and development of sympathetic neurons, is believed to be a trophic factor that is produced by sympathetic effector organs, its synthesis by these tissues has never been conclusively demonstrated. Using an assay capable of detecting 10 fg of mRNA, we measured the level of NGF mRNA in tissues innervated by sympathetic neurons. NGF mRNA was detected unambiguously in each tissue at a level that appeared to be more than enough to account for the low levels of NGF protein previously detected. Tissues that were densely innervated had comparatively high levels of NGF mRNA, while those with sparser innervation had lower levels. There was a strong positive correlation between the NGF mRNA level and norepinephrine content, a measure of the density of sympathetic innervation. NGF gene expression in one of these tissues, the iris, was shown to be induced by denervation. NGF mRNA was also found in other areas, including elements of the adult peripheral nervous system--the sciatic nerve and the sympathetic and sensory ganglia. In the central nervous system, levels of NGF mRNA were found that are too high to be attributed entirely to the vasculature, suggesting a role for NGF in adult central nervous system function.

Expression and regulation of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in distinct avian motoneuron subsets.

We performed a detailed study of the expression of neurotrophin-3 and brain-derived neurotrophic factor transcripts in spinal motoneurons using in situ hybridization of serially sectioned chick embryos aged 3 to 8 days (E3 to E8). Neurotrophin-3 mRNA is detected in motoneuron subsets from E3.5 to E4 only in brachial segments of the neural tube and from E5 in both brachial and lumbar regions. Expression of brain-derived neurotrophic factor mRNA is first evident on E5 in a subset of brachial level motoneurons and from E6 also in motoneurons located in the rostral-most portion of the lateral motor column, as well as in the tail-innervating region of the spinal cord. Analysis along the rostrocaudal extent of the brachial lateral motor column reveals an overlap zone of expression of both neurotrophins of about two segments. In transverse sections of this region, it is observed that neurotrophin-3-positive motoneurons preferentially occupy the lateral part of the column, whereas brain-derived neurotrophic factor-producing motoneurons are localized in a more medial position. These results show that the two factors are synthesized at discrete axial levels of the spinal cord by distinct motoneuron subpopulations. Since brain-derived neurotrophic factor mRNA is expressed within the brachial but not the lumbar lateral motor column, we tested the possibility that brain-derived neurotrophic factor expression is regulated by the type of peripheral target, that is, the wing or the leg. Unilateral transplantation of a wing bud instead of a leg bud and vice versa, prior to the onset of peripheral innervation, failed to alter the original pattern of brain-derived neurotrophic factor mRNA observed in either level of the axis. Thus, the early synthesis of brain-derived neurotrophic factor by subsets of spinal motoneurons is independent of the type of peripheral target and may instead reflect intrinsic differences between motoneuron populations.

Adult mammalian sensory and motor neurons: roles of endogenous neurotrophins and rescue by exogenous neurotrophins after axotomy.

We have tested the ability of neurotrophins to reverse axotomy-induced changes in adult motor and sensory neurons, using the physiological measure of conduction velocity. Five weeks after axotomy, sensory and motor conduction velocities were greatly reduced. NT-3 at 60 microg/d, pumped directly onto the cut nerve stump, largely prevented the change in sensory fibers. Lower doses were less effective, and NT-4/5 was without effect. In contrast, both NT-3 and NT-4/5 were effective at rescuing motoneurons, with similar dose dependencies. This amelioration of physiological deficits in adult mammalian neurons suggests possible therapeutic application of neurotrophins. We have also studied the physiological effects of neurotrophin deprivation on intact peripheral neurons. After 2 weeks of sequestration of trkB ligands (BDNF and NT-4/5), motor, but not sensory, neuron conduction was significantly slowed. Sequestration of NT-3 was found to affect both motor and sensory fiber velocities but more modestly and only with higher doses of sequestering agent. These data therefore suggest that peripherally produced neurotrophins are necessary for the maintenance of normal functional properties of peripheral neurons.