Structural characterization and expression of a brain specific gene encoding chick kainate binding protein.

The gene encoding chick kainate-binding protein (c-KBP), a member of the non-NMDA ionotropic glutamate receptor family has been isolated and characterized. The c-KBP gene is at least 13 kilobases long and contains 11 exons interrupted by 10 introns. Primer extension and RNase protection studies identified a major transcription initiation site located 117 bases upstream from the initiation methionine codon ATG. Consensus TATA and CCAAT sequences were detected in the putative promoter region. The structure of the c-KBP gene is strikingly different from that of other members of neurotransmitter-gated ion-channels (cloned at present) although the topology of c-KBP consists of four membrane-spanning domains, a structural characteristic of ionotropic receptor subunits. The c-KBP gene was found to be expressed at high levels in chick cerebellar Bergmann glia and at extremely low levels in the forebrain. The limited expression of the c-KBP gene raises important questions concerning the mechanisms governing the regulation of c-KBP gene transcription.

Monoclonal antibodies to the turtle cortex reveal neuronal subsets, antigenic cross-reactivity with the mammalian neocortex, and forebrain structures sharing a pallial derivation.

The dorsal cortex of the pond turtle (Pseudemys scripta) is a relatively simple structure consisting of two principal classes of neurons that occupy three distinct layers. Morphological, pharmacological, and physiological data suggest many similarities to the mammalian neocortex, rendering it an interesting preparation for comparative studies. We prepared monoclonal antibodies to the turtle dorsal cortex by immunizing mice with cortical tissue from adult turtles. Twelve antibodies were generated that recognize specific components of the turtle cortex. Among these, eight antibodies label only neurons and four label only ependymal glial cells. Differences in tissue staining pattern and immunoglobulin class suggest a heterogeneity of antigenic specificity among the antibodies. The staining patterns of three of our antibodies are described. TC3, like all other neuron-marking antibodies generated, labels a subset of both pyramidal and stellate cell types. It also cross-reacts with a subset of mammalian cortical neurons and labels them with a pattern similar to that observed in the turtle cortex. TC5 stains ependymal cells and their glial processes in the turtle cortex, and cross-reacts with fibrous astrocytelike processes in mammalian neocortical white matter. TC9 appears to recognize antigens of neurons sharing a pallial derivation in turtle.

Properties of kainate receptor/channels on cultured Bergmann glia.

Following the localization, at the electron microscope level, of the immunoreactivity towards a putative kainate receptor on Bergmann glial cells in the chick cerebellar cortex, cultures of Bergmann glia were used to establish the presence of functional kainate receptor/channels and study their properties. Bergmann glia were identified by their fusiform morphology and their ability to bind an anti-kainate binding protein monoclonal antibody, a kainate receptor high affinity ligand--kainyl-bovine serum albumin--and a glial marker--anti-vimentin monoclonal antibody. Membranes prepared from the culture cells displayed, using 25 nM [3H]kainate, the binding of 4.1 pmol of [3H]kainate/mg protein and showed the presence in Western blots of the two polypeptides of 49 and 93 kDa attributed to the kainate binding protein. Kainate, at concentrations above 0.1 mM, was found to increase the influx into cultured Bergmann glia of 22Na+, 86Rb+, 45Ca2+ and 36Cl- ions. The traffic of 22Na+, induced by kainate and glutamate, observed only in the presence of 1 mM ouabain, was blocked by kainate receptor antagonists and by 0.01 mM quisqualate. Analysis of the kinetics of incorporation of 22Na+ and 45Ca2+ ions showed an initial accumulation of 22Na+ and 45Ca2+ ions followed by their total dissipation. The results indicate that the kainate-induced influx of Na+ ions through the kainate receptor/channel causes the reverse transport of Na+ ions, by activation of the Na+/Ca2+ and Na+/H+ exchangers which remove intracellular Na+ ions. Pre-exposure of the cells to 0.5 mM dibutyryl cAMP was found to greatly enhance the kainate-induced 22Na+ ion influx. We propose that the Bergmann glia kainate receptors modulate the efficacy of the glutamatergic synapses between the parallel fibers and Purkinje cell spines and form part of a glial machinery responsible for plastic changes in synaptic transmission.

Subcellular localization of a putative kainate receptor in Bergmann glial cells using a monoclonal antibody in the chick and fish cerebellar cortex.

A monoclonal antibody, IX-50, that was raised against a kainate binding protein (Mr = 49,000) from chicken cerebellum, was used in light and electron microscopic immunocytochemical studies to localize putative kainate receptors. Pre- and postembedding immunoperoxidase and immunogold methods were used in the cerebellar cortices of one to 26-day old chickens and adult rainbow trout. Immunoreactivity was detected only in association with Golgi epithelial/Bergmann glial cells. Intracellular immunoreactivity was present in the granular and agranular endoplasmic reticulum, Golgi apparatus and in lysosomes, representing the sites of synthesis, glycosylation and degradation of the protein. In the fish the granular endoplasmic reticulum was not immunoreactive. Extracellular immunoreactivity was associated with the plasma membrane. In the fish it was established that the epitope is on the outer surface of the membrane. The protein seems to be uniformly distributed along the membrane including the somata, the radial stem processes and the leafy lamellae surrounding Purkinje cell dendrites. Areas of the glial membrane in contact with other glial cells were also immunopositive. High-resolution light microscopy demonstrated all the Bergmann glial plasma membrane in the cortex, providing a "negative" image of Purkinje cell dendrites. It is apparent that Bergmann glial processes selectively outline the dendrites of the Purkinje cells by surrounding the parallel fibre terminal/Purkinje cell spine synaptic complexes. The parallel fiber terminals were highly immunoreactive for glutamate, as shown by an immunogold procedure. The association of Bergmann glial processes, carrying the Mr = 49,000 kainate binding protein, with the Purkinje cell dendrites and spine synapses could provide a basis for neuronal signalling to the Bergmann glia, possibly by glutamate.

The segregation and expression of glutamate receptor subunits in cultured hippocampal neurons.

The distribution and expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-selective glutamate receptor subunits (GluR1-4) were studied in cultured hippocampal neurons using antibodies generated against peptides corresponding to the C-termini of GluR1, GluR2/3 and GluR4, and with a set of oligonucleotide probes designed complementary to specific pan, flip and flop GluR1-4 messenger RNA sequences. GluR1-4 subunit proteins were localized in fixed hippocampal neurons (2 h to three weeks after plating) by immunocytochemistry with light and electron microscopy. At early stages in culture, moderate staining with antibodies to GluR1 and GluR2/3 and very light staining with antibody to GluR4 was observed in cell bodies and proximal portions of all neurites of some neurons. Upon establishment of identified axons and dendrites by seven days in culture, staining was intense with specific antibodies to GluR1 and GluR2/3 and light with anti-GluR4 antibody in cell bodies and dendrites. Little or no staining was observed in axons. Cells at seven days in culture exhibited a variety of morphologies. However, we could not assign a pattern of staining to a particular type. As the cultures matured over two and three weeks, staining was limited to the somatodendritic compartment. The intensity of glutamate receptor subunit staining increased and the extent of staining proceeded to the distal extreme of many dendrites. Moreover, antibodies to GluR1-4 subunits were co-localized in neurons. Immunocytochemistry on living neurons did not result in any significant labeling, suggesting that the epitope is either not expressed on the surface of the neurons, or is present, but inaccessible to the antibody. Electron microscopy demonstrated receptor localization similar to that found in brain, with staining of postsynaptic membrane and density, dendritic cytoplasm and cell body, but not within the synaptic cleft. We examined the possible role of "cellular compartmentation" in the pattern of glutamate receptor expression in hippocampal neurons. Compartmentalization studies of the subcellular distribution of messenger RNAs encoding GluR1-4 subunits was determined in mature cultures by in situ hybridization. Significant silver grain appearance was restricted to the cell body, indicating that the synthesis of glutamate receptor subunits is limited largely to the neuronal cell body. The expression of microtubule-associated protein 2 was studied in parallel. Microtubule-associated protein 2 expression appeared 6 h after plating, while glutamate receptor subunit expression was present at 2 h. This indicates that microtubule-associated protein 2 does not regulate the initial distribution of glutamate receptor subunits into neurites.(ABSTRACT TRUNCATED AT 400 WORDS)

HU-211, a non-psychotropic cannabinoid, rescues cortical neurones from excitatory amino acid toxicity in culture.

The present study examined potential neuroprotective effects of HU-211, a synthetic non-psychotropic cannabinoid with non-competitive NMDA antagonist properties on neurones exposed to various excitotoxins in culture. HU-211 was found to protect neurones from NMDA and quisqualate-induced toxicity but not that produced by AMPA or kainate. NMDA-mediated neurotoxicity was blocked by HU-211 in a dose dependent manner with an EC50 = 3.8 +/- 0.9 microM. Radioligand binding studies have shown that HU-211 inhibits the binding of [3H]MK-801 to rat forebrain membranes (KI = 11.0 microM +/- 1.323) in a competitive manner, but was unable to displace [3H]kainate and [3H]AMPA binding. These data suggest that the neuroprotective activity of HU-211 is directly associated with the NMDA receptor channel and possibly with the quisqualate receptor of the metabotropic class. Thus, HU-211 appears to act as an NMDA open channel blocker and shows promise as a novel neuroprotectant for clinical use.

Novel m1 muscarinic agonists in treatment and delaying the progression of Alzheimer's disease: an unifying hypothesis.

M1 selective agonists from the AF series (e.g. AF102B, AF150(S)), via m1 muscarinic receptors, activate distinct signal transductions, enhance amyloid precursors proteins secretion from transfected cells and primary cell cultures, show neurotrophic effects and are beneficial in a variety of animal models for Alzheimer's disease. Such m1 agonists may be effective in the treatment and therapy of Alzheimer's disease.

A microtest plate assay of functional excitatory amino acid receptors.

A microtest plate assay of functional excitatory amino acid receptors present on cultured chick embryo retinal cells has been developed. It is based on measurements of excitatory amino acid-mediated increase in 22Na+ influx into retinal cells adhering to each of the 96 wells of microtest plates. Dose-dependent responses to L-glutamate, kainate and N-methyl-D-aspartate but not to quisqualate can be measured. These responses are selectively inhibited by antagonists of excitatory amino acids. The assay is reliable, fast to perform and parsimonious in terms of the volume and thus of amount of the drug applied. It allows a single investigator to perform, in one day, measurements of the effects of known or putative glutamatergic ligands in more than 100 different conditions.

Factor(s) from goldfish brain induce neuritic outgrowth from explanted regenerating retinas.

The extensive outgrowth from explanted goldfish retina in vitro stimulated by the axotomy of the optic nerve several days prior to the explantation, enables a more direct examination of the events which accompany optic nerve regeneration. Thus far neuritic outgrowth has been obtained in serum-containing medium. In the present report we demonstrate for the first time that factor(s) from adult goldfish brain can support neuritic outgrowth from retinal explant and replace the serum. This finding suggests that goldfish neuronotrophic factor(s) are active on retinal ganglia cells. Under the same experimental conditions nerve growth factor from submaxillary gland did not exhibit outgrowth stimulatory activity.

Kainyl-bovine serum albumin: a novel ligand of the kainate sub-type of glutamate receptor with a very high binding affinity.

Bovine serum albumin has been conjugated with kainylaminooxyacetylglycine to afford a multivalent kainylated protein called kainyl-bovine serum albumin (KA-BSA). This derivative, radiolabelled with 125I to more than 5000 Ci/mmol, was found to interact in the chick, goldfish and rat brain to specific membranous sites displaying the pharmacological properties attributed to the kainate sub-type of glutamate receptor. Measurements of the kinetics of association and dissociation of KA-BSA showed a quasi-irreversible binding with dissociation constants in the subpicomolar and nanomolar range. The chemical properties and the binding characteristics of KA-BSA suggest that it interacts mainly with kainate binding sites present in clusters in the membrane. Localization of the KA-BSA binding sites, by autoradiography in the chick cerebellum and by immunoperoxidase staining in the goldfish cerebellum, revealed an exclusive association with the molecular layer.

Neuroprotective and antioxidant activities of HU-211, a novel NMDA receptor antagonist.

This study examines the ability of (+)-(3S,4S)-7-hydroxy-delta 6-tetrahydrocannabinol-1,1-dimethylheptyl (HU-211), a non-competitive NMDA receptor antagonist to: (1) rescue neurons in culture from injury evoked by sodium nitroprusside, hydrogen peroxide (H2O2) and oxygen glucose deprivation; and (2) scavenge reactive oxygen species in vitro. Qualitative and quantitative assessments of cell survival have indicated that: (1) Neuronal cell injury produced following deprivation of oxygen and glucose was significantly attenuated by 5 microM HU-211. (2) Glial and neuronal cell damage induced by sodium nitroprusside was markedly ameliorated by 10 microM HU-211. (3) HU-211 reduced protein oxidation initiated by gamma irradiation, and scavenged peroxyl radicals. (4) HU-211 carries an oxidation potential of 550 mV. These findings suggest that HU-211 holds a unique position among putative neuroprotectant agents in that it combines NMDA receptor antagonistic activity and free radical scavenging abilities in a single molecule.

Early regenerative responses induced by monoclonal antibodies directed against a surface glycoprotein of goldfish retinal ganglion cells.

Monoclonal antibodies directed primarily against antigenic determinants associated with the goldfish optic nerve were prepared and characterized. One selected clone 23-4-C(IgG2a), detected antigenic determinants of glycoprotein nature with an apparent mol. wt. of 140 000. Following injury the level of these molecules increased with a peak at 5-7 days after the lesion (2- to 3-fold higher than the basal level). The results strongly suggest that the increase derives, at least partially, from a real increment in the level of these molecules in the retinal ganglion cells rather than from changes in accessibility. Immunofluorescence studies indicate that the retinal ganglion cells carry the antigenicity. Intraocular injection of the monoclonal antibodies, concomitantly with crush injury, resulted in an earlier and higher regenerative response, reflected by sprouting capacity, protein synthesis and accumulation of radiolabeled material in the tectum contralateral to the side of injury. This may indicate that the antibodies directly activate retinal cells via interaction with surface molecules. Alternatively, the antibodies may be directed against surface molecules which are associated with axonal growth inhibitors, and may therefore mask these surface antigens from further interaction with their native substrate.

Isolation, immunochemical characterization and localization of the kainate sub-class of glutamate receptor from chick cerebellum.

The low-affinity kainate binding sites, present at high density in chick cerebellar membranes, were solubilized with Triton X-100 and purified 41-fold. The purified kainate binding sites, therein referred to as the kainate receptor, displayed the expected pharmacological specificity: domoate = kainate much greater than L-glutamate much greater than D-glutamate, quisqualate, N-methyl-D-aspartate. Analysed by SDS-PAGE under reducing conditions, a single polypeptide with a Mr = 49,000 was observed. Western blots of membranes prepared from different brain areas and animal species were analysed using a monoclonal antibody, named mAb IX-50, raised against the purified kainate receptor. The mAb IX-50 stained the 49,000 polypeptide in chick, goldfish and mammalian brain tissues indicating its conservation during evolution. The staining intensity correlated with the density of kainate binding sites. The mAb IX-50 stained also a 93,000 polypeptide but the latter did not copurify with the 49,000 polypeptide. The kainate binding activity was selectively immunoadsorbed on mAb IX-50 coupled to Sepharose which, upon elution, released a 49,000 polypeptide. The immunohistochemical localization of mAb IX-50 binding sites in the chick cerebellar molecular layer coincided with that of the kainate receptor. We conclude that the 49,000 polypeptide is part of the kainate receptor and carries the kainate recognition site.

Immune response potential to poly(Tyr,Glu)-poly(DLAla)--poly(Lys) of human T cells of different donors.

Human peripheral blood T cells of normal donors were activated in vitro with autologous adherent cells pulsed with poly(LTyr,LGlu)-poly(DLAla)--poly(LLys) [abbreviated (T,G)-A--L]. The "educated" T cells were tested: (i) for their ability to produce a (T,G)-A--L-specific T cell-replacing factor in the cooperation with B cells for antibody responses in vivo or in vitro and (ii) for their ability to proliferate in the presence of a second stimulus of (T,G)-A--L. Results of screening of 66 donors demonstrated that educated T cells of about 50% of the donors produced an active (T,G)-A--L-specific factor, whereas activated cells of only half of the factor producers were capable of proliferating in the presence of the antigen. Thus, as reported for all other species studied, human individuals differ in their response potential to (T,G)-A--L.

Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the m1 muscarinic receptor to beta-amyloid precursor protein secretion.

Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)-dependent and mitogen-activated protein kinase (MAPK)-dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S-trans, trans-farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12-myristate 13-acetate-induced APPs secretion, enhances both muscarinic-stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras-dependent but PKC-independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.