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.

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.

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.

Astroglial response in the excitotoxically lesioned neostriatum and its projection areas in the rat.

The anatomical distribution of the astrocytic glial reactions, following ibotenic acid-induced neuronal degeneration of the neostriatum in the rat, has been studied immunohistochemically using an antibody directed against the astrocytic marker, glial fibrillary acidic protein. The acute astroglial response to the excitotoxic lesion, determined 7 days post lesion, was compared with a sham-operated group and a chronic group that had received the excitotoxic lesion 6 months prior to histological evaluation. Total doses of 16-20 micrograms ibotenic acid injected unilaterally into the head of the neostriatum caused not only a marked neuronal cell loss but was also accompanied by a large increase in the number and size (about 5 times) of glial fibrillary acidic protein-stained astrocytes throughout the neostriatum by 7 days after lesion. Reactive astrocytes were also observed in the major neostriatal projection areas, the globus pallidus and the substantia nigra pars reticulata, at 7 days post lesion, although no neuronal cell loss could be detected in these regions using regular Cresyl Violet staining. Previous studies of lesions identical to the ones used here have shown that globus pallidus and substantia nigra are deafferented as a result of the neostriatal neuronal degeneration. The reactive astrocytes in the striatal projection areas had a 3-5 times larger size than control astrocytes from the same anatomical region. In animals that received a larger dose of ibotenic acid into the neostriatum (25 micrograms), neuronal cell loss was also observed in the neocortex and reactive glial fibrillary acidic-stained astrocytes were found in the entire neocortex of the injected hemisphere. In the chronic group, 6 months after the excitotoxic lesion, the astroglial response was clearly diminished or absent in the major neostriatal projection areas, but was still present within the lesioned neostriatum. The results suggest that focal neuronal destruction can result in widespread astrocytic glial reactions which follow the anatomical connectivity of the lesioned area. This may have implications for the understanding of the multifocal distribution of glial reactions seen in patients with striatal degeneration as a result of Huntington's disease.

Neural grafting in a rat model of Huntington's disease: striosomal-like organization of striatal grafts as revealed by acetylcholinesterase histochemistry, immunocytochemistry and receptor autoradiography.

Grafts of fetal striatum were implanted in the form of a cell suspension into the brains of rats with prior ibotenic acid lesions of the caudate-putamen. The grafts were placed in three different sites: the lesioned caudate-putamen, or the denervated (but otherwise undamaged) globus pallidus and substantia nigra. After 3-6 months survival the grafts were investigated by means of immunohistochemistry and receptor autoradiography in combination with routine histology and acetylcholinesterase histochemistry. The grafts placed within the lesioned caudate-putamen were at least 10-fold larger larger than those placed in the substantia nigra region, with the grafts placed in the globus pallidus being of intermediate size. In all locations the acetylcholinesterase staining had an uneven, patchy distribution, which was most pronounced in the grafts located within the caudate-putamen. These patches did not bear any obvious relationship to variations in density of the neuronal perikarya within the grafted tissue. Many of the neuropeptide-immunoreactive neuron types present in the normal striatum, such as those containing substance P, [Met]enkephalin, somatostatin, cholecystokinin and neuropeptide Y were also detected in the grafted striatum along with acetylcholinesterase-positive staining. Acetylcholinesterase-positive, [Met]enkephalin-positive, substance P-positive and tyrosine hydroxylase-positive markers all showed uneven, patchy distributions in the grafts. This was also the case for the distribution of dopamine D2 and opiate receptors (as revealed by [3H]spiroperidol and [3H]diprenorphine autoradiography, respectively), whereas muscarinic receptor binding was even throughout the grafts. As is the case in the so-called striosomal patches (neurochemically defined compartments) in the immature intact striatum during the early postnatal period, patches of high acetylcholinesterase staining in the grafts showed partial correspondence with patches of high [Met]enkephalin fibre staining, and dopamine receptor density, and (although to a lesser degree) also with patches of high opiate receptor density and high substance P-immunoreactivity. This correspondence of patches also occurred between tyrosine hydroxylase fibre staining and acetylcholinesterase staining as revealed by grafts placed into the substantia nigra. These results suggest that the fetal striatal cell suspension grafts will give rise to a fairly normal range of striatal neuron and receptor types and that they develop at least some of the striosomal features characteristic for the normal striatum.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Increased central 5-hydroxytryptamine receptor mechanisms in rats after chronic neuroleptic treatment.

1 The behavioural responses of drugs known to act through central 5-hydroxytryptamine (5-HT) mechanisms have been investigated in rats receiving a neuroleptic (trifluoperazine) in their drinking water for 4 to 6 months.2 5-Hydroxytryptophan (5-HTP) induced 5-HT-dependent behaviours including head bobbing and lateral head weaving, reciprocal forepaw treading, tremor, backward walking, body writhing and ;wet-dog' shakes. In doses of 50 to 150 mg/kg, 5-HTP induced more intense behavioural effects in neuroleptic-treated rats than in the control animals.3 Similarly the putative 5-HT agonist, quipazine (1 to 20 mg/kg) and the 5-HT releasing drug, fenfluramine (5 to 20 mg/kg), both induced significantly greater motor responses in the chronically neuroleptic-treated rats.4 A 5-HT uptake inhibitor (femoxetine, 2.5 to 10 mg/kg) had little behavioural effect in either control or trifluoperazine-treated rats.5 Total specific high-affinity binding of radiolabelled 5-HT was significantly increased in crude membrane fractions prepared from the cortex, striatum and substantia nigra of neuroleptic-treated rats compared to control animals.6 High-affinity uptake of radiolabelled 5-HT into striatal slices was similar in experimental and control animals.7 Behavioural and biochemical data would indicate that postsynaptic 5-HT mechanisms are enhanced in rats treated chronically with trifluoperazine. Chronic neuroleptic therapy may thereby induce cerebral 5-HT receptor supersensitivity in addition to the well-documented cerebral dopamine receptor supersensitivity.

A comparison of stereospecificity at central and peripheral 'muscarine-sensitive' acetylcholine receptors: observations with the enantiomeric forms of procyclidine and tricyclamol.

1 Procyclidine resembles hyoscine in enhancing the effects of amphetamine on ipsiversive turning by mice with a unilateral central dopamine lesion. 2 The stereospecific index for procyclidine is not greater than 10, in contrast to 173 for acetylcholine receptors in ileum from the same mice. 3 This suggests that although the central effects of procyclidine in this test involve acetylcholine receptors similar to those at peripheral sites, they cannot be identical with them unless there are differences at some secondary site, for example, if the weaker enantiomer were a stronger inhibitor of dopamine uptake or if there were a stereoselective uptake process for procyclidine itself.

Nerve growth factor receptor mRNA distribution in human brain: normal levels in basal forebrain in Alzheimer's disease.

Nerve growth factor (NGF) receptor mRNA was found to be widely distributed throughout the human central nervous system, with the highest levels in the basal forebrain; this suggests that NGF may function as a retrograde trophic messenger for basal forebrain magnocellular cholinergic nerve cells. The degeneration of the latter constitutes one of the main features of Alzheimer's disease and it may be responsible for some of the cognitive impairment that characterizes the disease. No evidence was obtained for an insufficient synthesis of NGF receptor mRNA in the basal forebrain in Alzheimer's disease, where NGF receptor-like immunoreactivity was confined to neuronal cell bodies. NGF could thus be therapeutically beneficial. It could be expected to induce basal forebrain cholinergic cells to hypertrophy, synthesize more choline acetyltransferase and extend neurites.

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.

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.

Lesions of the superior colliculus in the rat differentiate between nigrostriatal and mesolimbic dopamine systems.

Bilateral electrolytic lesions of the superior colliculus in rats increased spontaneous locomotor activity, enhanced amphetamine-induced hyperactivity and attenuated apomorphine-induced biting. These lesions were associated with an increased rate of turnover of dopamine in the nucleus accumbens, but not in the striatum. Similarly concentrations of the dopamine metabolites homovanillic acid and 3,4-dihydroxyphenylacetic acid were elevated in accumbens tissue but not in striatum in rats with bilateral collicular lesions. The results indicate that lesions of the superior colliculus cause differentiation between hyperactivity and stereotypy, and that this may be related to blockade of a nigrostriatal outflow, and relief of inhibition on mesolimbic systems.

Morphometric immunochemical analysis of neurons in the nucleus basalis of Meynert in Alzheimer's disease.

In Alzheimer's disease there is a reported loss of large cells in the cholinergic nucleus basalis of Meynert. It has been suggested, however, that there may be neurons in the nucleus basalis in Alzheimer's disease which are atrophied and therefore difficult to distinguish from neuroglia by size. This has important therapeutic implications and we have attempted to clarify the situation using a neuron-specific antiserum directed against neuron-specific enolase (NSE). Sections of nucleus basalis were stained using this antiserum and the neuronal cross-sectional area was measured. A profile of neuronal distribution with area was obtained, by image analysis, and compared in controls and patients with Alzheimer's disease. A significant 29% overall loss of neurons was found in Alzheimer's disease with a much greater loss (61%) of large neurons and concurrent increase (59%) in small neurons. Analysis of variance showed significant reduction in mean cross-sectional neuronal area as a consequence of this shift in frequency towards a preponderance of small cells. It is suggested that in the nucleus basalis in Alzheimer's disease, large neurons are not completely lost; many are shrunken and thus excluded from the previous studies of large cells counted in Nissl-stained material. That there is partial preservation of these neurons makes it more likely that cholinergic dysfunction, characteristic of Alzheimer's disease, will be amenable to neurotrophic influence.

Neuropeptide Y: regional distribution chromatographic characterization and immunohistochemical demonstration in post-mortem human brain.

Using a neuropeptide Y (NPY)-directed radioimmunoassay the post-mortem stability of NPY was assessed in both rat and human brain samples. The regional distribution of NPY-like immunoreactivity in human brain was determined. The NPY-like immunoreactivity in human brain separated on Sephadex G-50 columns and 18C reverse phase at the position expected for NPY. Immunohistochemical staining using the NPY-directed antiserum revealed a characteristic population of cortical and striatal neurons containing NPY-like immunoreactivity.

Immunohistochemical localization of beta-nerve growth factor receptors in the forebrain of the rat.

Using a monoclonal antibody (192-IgG) directed against the rat beta-nerve growth factor (beta-NGF) receptor the distribution of beta-NGF receptors in the forebrain of the rat has been determined. beta-NGF receptor-containing cells were located in presumed cholinergic cells of the medial septal nucleus, vertical and horizontal limbs of the diagonal band of Broca and the nucleus basalis of Meynert.

Survival of basal ganglia neuropeptide Y-somatostatin neurones in Huntington's disease.

The content of somatostatin-like immunoreactivity (SRIF-LI) and neuropeptide Y-like immunoreactivity (NPY-LI) has been measured in both control post-mortem human brains and in Huntington's disease brains. The content of both SRIF-LI and NPY-LI was found to be significantly increased in the basal ganglia of Huntington's disease brains compared with a control group. The nature of the SRIF-LI and NPY-LI in both control and Huntington's disease brains was investigated after separation on Sephadex G25 and G50 columns. Using a C-terminal-directed SRIF radioimmunoassay (RIA), 3 peaks of immunoreactivity were measured, whilst an N-terminal-directed SRIF RIA detected two peaks of immunoreactivity. In each case, the elution profile did not differ between control and Huntington's disease caudate nucleus. The content of immunoreactivity in each peak was found to be increased in Huntington's disease brains compared with controls. Only one peak of NPY-LI was detected in both control and Huntington's disease caudate after separation on Sephadex G25 and G50 columns. Immunohistochemical staining of the caudate and putamen of control and Huntington's disease brains revealed a population of neurones containing NPY-LI. The number of NPY-positive neurones was found to be increased in both the caudate and putamen of Huntington's disease brains compared to control caudate and putamen.

Peptides derived from prodynorphin are decreased in basal ganglia of Huntington's disease brains.

The contents of methionine-enkephalin-Arg-Gly-Leu, dynorphin A, dynorphin B and alpha-neoendorphin have been measured in both control and Huntington's disease brains obtained postmortem. All 4 peptides were significantly reduced in the caudate nucleus and putamen of Huntington's disease compared with the control group. No differences were observed in frontal cortex or hypothalamus. Immunocytochemistry showed a marked depletion of dynorphin-like immunoreactivity in Huntington's disease substantia nigra.

Behavioural and biochemical changes following chronic administration of L-dopa to rats.

Rats were given powdered diet containing L-DOPA (together with the peripheral decarboxylase inhibitor carbidopa) for a period of 6 months. The estimated daily intake was in the range 20-30 mg/kg. Initially, at 1 week and 1 month, L-DOPA-fed rats exhibited enhanced spontaneous locomotor activity, but this fell to within the control range by 3 and 6 months, although (+)-amphetamine-induced hyperactivity was greater at 6 months in L-DOPA-treated animals than in control rats. Six months after receiving L-DOPA in their diet rats showed enhanced stereotypy scores to a series of dopamine agonists administered acutely including (+)-amphetamine, nomifensine, L-DOPA, apomorphine and piribedil compared with the control animals. In another behaviour test L-DOPA administration reduced the cataleptic potency of both fluphenazine and haloperidol was increased. Biochemically 6 months treatment of rats with L-DOPA was associated with significantly increased plasma concentrations of L-DOPA, enhanced striatal levels of L-DOPA, dopamine and dopamine metabolites, enhanced specific binding (as indicated by increased Bmax values) of [3H] spiroperidol, [3H] ADTN and [3H] 5-HT to striatal membranes, and increased basal and dopamine-stimulated striatal adenylate cyclase activity. The results are discussed in the light of changes of sensitivity of cerebral dopamine receptors, an increase in receptor numbers, and the tolerance to L-DOPA which often develop in the treatment of Parkinson's disease.

Acute motor effects of N-methyl-D-aspartic acid and kainic acid applied focally to mesencephalic dopamine cell body regions in the rat.

Bilateral application of N-methyl-D-aspartate (NMDA) and kainic acid to the substantia nigra, pars compacta (SNc) and the ventral tegmental area (VTA) induced enhanced motor responses in the rat. A greater locomotor response was elicited from the VTA; sniffing was also observed following SNc injections. Both motor activities were blocked by systemic fluphenazine. Application of NMDA and kainate to the substantia nigra pars reticulata caused sedation and catalepsy. The experiments illustrate a dual motor response for excitatory amino acids when applied to either pars compacta or pars reticulata of the rat SN.