Cone photoreceptors respond to their own glutamate release in the tiger salamander.

Pulse-like currents resembling miniature postsynaptic currents were recorded in patch-clamped isolated cones from the tiger salamander retina. The events were absent in isolated cones without synaptic terminals. The frequency of events was increased by either raising the osmotic pressure or depolarizing the cell. It was decreased by the application of either glutamate or the glutamate-transport blockers dihydrokainate and D,L-threo-3-hydroxyaspartate. The events required external Na+ for which Li+ could not substitute. The reversal potential of these currents followed the equilibrium potential for Cl- when internal Cl- concentration was changed. Thus, these miniature currents appear to represent the presynaptic activation of the glutamate receptor with glutamate transporter-like pharmacology, caused by the photoreceptor's own vesicular glutamate release. Using a noninvasive method to preserve the intracellular Cl- concentration, we showed that glutamate elicits an outward current in isolated cones. Fluorescence of the membrane-permeable form of fura-2 was used to monitor Ca2+ entry at the cone terminal as a measure of membrane depolarization. The increase in intracellular Ca2+ concentration, elicited by puff application of 30 mM KCl, was completely suppressed in the presence of 100 microM glutamate. Puff application of glutamate alone had no measurable depolarizing effect. These results suggest that the equilibrium potential for Cl-, ECl, was more negative than the activation range for Ca2+ channels and that glutamate elicited an outward current, hyperpolarizing the cones.

Amyloid precursor protein processing is stimulated by metabotropic glutamate receptors.

Stimulation of muscarinic m1 or m3 receptors can, by generating diacylglycerol and activating protein kinase C, accelerate the breakdown of the amyloid precursor protein (APP) to form soluble, nonamyloidogenic derivatives (APPs), as previously shown. This relationship has been demonstrated in human glioma and neuroblastoma cells, as well as in transfected human embryonic kidney 293 cells and PC-12 cells. We now provide evidence that stimulation of metabotropic glutamate receptors (mGluRs), which also are coupled to phosphatidylinositol 4,5-bisphosphate hydrolysis, similarly accelerates processing of APP into nonamyloidogenic APPs. This process is demonstrated both in hippocampal neurons derived from fetal rats and in human embryonic kidney 293 cells transfected with cDNA expression constructs encoding the mGluR 1 alpha subtype. In hippocampal neurons, both an mGluR antagonist, L-(+)-2-amino-3-phosphonopropionic acid, and an inhibitor of protein kinase C, GF 109203X, blocked the APPs release evoked by glutamate receptor stimulation. Ionotropic glutamate agonists, N-methyl-D-aspartate or S(-)-5-fluorowillardiine, failed to affect APPs release. These data show that selective mGluR agonists that initiate signal-transduction events can regulate APP processing in bona fide primary neurons and transfected cells. As glutamatergic neurons in the cortex and hippocampus are damaged in Alzheimer disease, amyloid production in these regions may be enhanced by deficits in glutamatergic neurotransmission.

Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive channels.

Hippocampal neurons maintained in primary culture recycle synaptic vesicles and express functional glutamate receptors since early stages of neuronal development. By analyzing glutamate-induced cytosolic calcium changes to sense presynaptically released neurotransmitter, we demonstrate that the ability of neurons to release glutamate in the extracellular space is temporally coincident with the property of synaptic vesicles to undergo exocytotic-endocytotic recycling. Neuronal differentiation and maturation of synaptic contacts coincide with a change in the subtype of calcium channels primarily involved in controlling neurosecretion. Whereas omega-agatoxin IVA-sensitive channels play a role in controlling neurotransmitter secretion at all stages of neuronal differentiation, omega-conotoxin GVIA-sensitive channels are primarily involved in mediating glutamate release at early developmental stages only.

Structural conservation of ion conduction pathways in K channels and glutamate receptors.

Single channel recordings demonstrate that ion channels switch stochastically between an open and a closed pore conformation. In search of a structural explanation for this universal open/close behavior, we have uncovered a striking degree of amino acid homology across the pore-forming regions of voltage-gated K channels and glutamate receptors. This suggested that the pores of these otherwise unrelated classes of channels could be structurally conserved. Strong experimental evidence supports a hairpin structure for the pore-forming region of K channels. Consequently, we hypothesized the existence of a similar structure for the pore of glutamate receptors. In ligand-gated channels, the pore is formed by M2, the second of four putative transmembrane segments. A hairpin structure for M2 would affect the subsequent membrane topology, inverting the proposed orientation of the next segments, M3. We have tested this idea for the NR1 subunit of the N-methyl-D-aspartate receptor. Mutations that affected the glycosylation pattern of the NR1 subunit localize both extremes of the M3-M4 linker to the extracellular space. Whole cell currents and apparent agonist affinities were not affected by these mutations. Therefore it can be assumed that they represent the native transmembrane topology. The extracellular assignment of the M3-M4 linker challenged the current topology model by inverting M3. Taken together, the amino acid homology and the new topology suggest that the pore-forming M2 segment of glutamate receptors does not transverse the membrane but, rather, forms a hairpin structure, similar to that found in K channels.

D-serine, an endogenous synaptic modulator: localization to astrocytes and glutamate-stimulated release.

Using an antibody highly specific for D-serine conjugated to glutaraldehyde, we have localized endogenous D-serine in rat brain. Highest levels of D-serine immunoreactivity occur in the gray matter of the cerebral cortex, hippocampus, anterior olfactory nucleus, olfactory tubercle, and amygdala. Localizations of D-serine immunoreactivity correlate closely with those of D-serine binding to the glycine modulatory site of the N-methyl-D-aspartate (NMDA) receptor as visualized by autoradiography and are inversely correlated to the presence of D-amino acid oxidase. D-Serine is enriched in process-bearing glial cells in neuropil with the morphology of protoplasmic astrocytes. In glial cultures of rat cerebral cortex, D-serine is enriched in type 2 astrocytes. The release of D-serine from these cultures is stimulated by agonists of non-NMDA glutamate receptors, suggesting a mechanism by which astrocyte-derived D-serine could modulate neurotransmission. D-Serine appears to be the endogenous ligand for the glycine site of NMDA receptors.

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer’s disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer’s disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer’s disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid β-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

The C terminus of β-tubulin regulates vinblastine-induced tubulin polymerization

Oligoanions such as sodium triphosphate or GTP prevent and/or reverse vinblastine-induced polymerization of tubulin. We now show that the anions of glutamate-rich extreme C termini of tubulin are similarly involved in the regulation of the vinblastine effect. Cleavage of the C termini by limited proteolysis with subtilisin enhances vinblastine-induced tubulin polymerization and abolishes the anion effect. Only the β-tubulin C terminus needs to be removed to achieve these changes and the later cleavage of the α-tubulin C terminus has little additional effect. In fact, vinblastine concentrations >20 μM block cleavage of the α-tubulin C terminus in the polymer, whereas cleavage of the β-tubulin C terminus proceeds unimpeded over the time used. The vinblastine effect on tubulin polymerization is also highly pH-dependent between pH 6.5 and 7.5; this is less marked, but not absent, after subtilisin treatment. A working model is proposed wherein an anionic domain proximal to the extreme C terminus must interact with a cationic domain to permit vinblastine to promote polymerization. Both exogenous and extreme C-terminal anions compete for the cationic domain with the proximal anionic domain to prevent vinblastine-induced polymerization. We conclude that the electrostatic regulation of tubulin polymerization induced by vinblastine resides primarily in the β-tubulin C terminus but that additional regulation proximal in the tubulin molecule also plays a role.

In vivo evidence that erythropoietin protects neurons from ischemic damage

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from N-methyl-d-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report in vivo evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca2+, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.

Long-term potentiation and dual-component quantal signaling in the dentate gyrus

Long-term potentiation (LTP) of excitatory transmission is an important candidate cellular mechanism for the storage of memories in the mammalian brain. The subcellular phenomena that underlie the persistent increase in synaptic strength, however, are incompletely understood. A potentially powerful method to detect a presynaptic increase in glutamate release is to examine the effect of LTP induction on the rate at which the use-dependent blocker MK-801 attenuates successive N-methyl-d-aspartic acid (NMDA) receptor-mediated synaptic signals. This method, however, has given apparently contradictory results when applied in hippocampal CA1. The inconsistency could be explained if NMDA receptors were opened by glutamate not only released from local presynaptic terminals, but also diffusing from synapses on neighboring cells where LTP was not induced. Here we examine the effect of pairing-induced LTP on the MK-801 blocking rate in two afferent inputs to dentate granule cells. LTP in the medial perforant path is associated with a significant increase in the MK-801 blocking rate, implying a presynaptic increase in glutamate release probability. An enhanced MK-801 blocking rate is not seen, however, in the lateral perforant path. This result still could be compatible with a presynaptic contribution to LTP in the lateral perforant path if intersynaptic cross-talk occurred. In support of this hypothesis, we show that NMDA receptors consistently sense more quanta of glutamate than do α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. In the medial perforant path, in contrast, there is no significant difference in the number of quanta mediated by the two receptors. These results support a presynaptic contribution to LTP and imply that differences in intersynaptic cross-talk can complicate the interpretation of experiments designed to detect changes in transmitter release.

R-type Ca2+ currents evoke transmitter release at a rat central synapse

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and γ-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

Structural homologies with ATP- and folate-binding enzymes in the crystal structure of folylpolyglutamate synthetase

Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-Å resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined Ω loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.

Aurintricarboxylic acid prevents GLUR2 mRNA down-regulation and delayed neurodegeneration in hippocampal CA1 neurons of gerbil after global ischemia

Aurintricarboxylic acid (ATA), an inhibitor of endonuclease activity and other protein–nucleic acid interactions, blocks apoptosis in several cell types and prevents delayed death of hippocampal pyramidal CA1 neurons induced by transient global ischemia. Global ischemia in rats and gerbils induces down-regulation of GluR2 mRNA and increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced Ca2+ influx in CA1 before neurodegeneration. This result and neuroprotection by antagonists of AMPA receptors suggests that formation of AMPA receptors lacking GluR2, and therefore Ca2+ permeable, leads to excessive Ca2+ influx in response to endogenous glutamate; the resulting delayed neuronal death in CA1 exhibits many characteristics of apoptosis. In this study, we examined the effects of ATA on expression of mRNAs encoding glutamate receptor subunits in gerbil hippocampus after global ischemia. Administration of ATA by injection into the right cerebral ventricle 1 h before (but not 6 h after) bilateral carotid occlusion prevented the ischemia-induced decrease in GluR2 mRNA expression and the delayed neurodegeneration. These findings suggest that ATA is neuroprotective in ischemia by blocking the transcriptional changes leading to down-regulation of GluR2, rather than by simply blocking endonucleases, which presumably act later after Ca2+ influx initiates apoptosis. Maintaining formation of Ca2+ impermeable, GluR2 containing AMPA receptors could prevent delayed death of CA1 neurons after transient global ischemia, and block of GluR2 down-regulation may provide a further strategy for neuroprotection.

Protein thermostability above 100°C: A key role for ionic interactions

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100°C. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100°C and 88°C, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104°C. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104°C over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.

Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity

Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1S12OG and nm23-H1P96S. Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1P96S or nm23-H1S120G, and another was phosphorylated much more slowly by nm23-H1P96S and by nm23-H1S120G than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1S120G and nm23-H1P96S were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1S120 was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1P96S was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.

Endocrine modulation of the neurotoxicity of gp120: Implications for AIDS-related dementia complex

HIV infection often involves the development of AIDS-related dementia complex, a variety of neurologic, neuropsychologic, and neuropathologic impairments. A possible contributor to AIDS-related dementia complex is the HIV envelope glycoprotein gp120, which damages neurons via a complex glutamate receptor- and calcium-dependent cascade. We demonstrate an endocrine modulation of the deleterious effects of gp120 in primary hippocampal and cortical cultures. Specifically, we observe that gp120-induced calcium mobilization and neurotoxicity are exacerbated by glucocorticoids, the adrenal steroids secreted during stress. Importantly, this deleterious synergy can occur between gp120 and synthetic glucocorticoids (such as prednisone or dexamethasone) that are used clinically in high concentrations to treat severe cases of the Pneumocystis carinii pneumonia typical of HIV infection. Conversely, we also observe that estradiol protects neurons from the deleterious actions of gp120, reducing toxicity and calcium mobilization.