Q/R site editing in kainate receptor GluR5 and GluR6 pre-mRNAs requires distant intronic sequences.

RNA editing by adenosine deamination in brain-expressed pre-mRNAs for glutamate receptor (GluR) subunits alters gene-specified codons for functionally critical positions, such as the channel's Q/R site. We show by transcript analysis of minigenes transiently expressed in PC-12 cells that, in contrast to GluR-B pre-mRNA, where the two editing sites (Q/R and R/G) require base pairing with nearby intronic editing site complementary sequences (ECSs), editing in GluR5 and GluR6 pre-mRNAs recruits an ECS located as far as 1900 nucleotides distal to the Q/R site. The exon-intron duplex structure of the GluR5 and GluR6 pre-mRNAs appears to be a substrate of double-stranded RNA-specific adenosine deaminase. This enzyme when coexpressed in HEK 293 cells preferentially targets the adenosine of the Q/R site and of an unpaired position in the ECS which is highly edited in brain.

A set of genes expressed in response to light in the adult cerebral cortex and regulated during development.

Activity-dependent plasticity is thought to underlie both formation of appropriate synaptic connections during development and reorganization of adult cortical topography. We have recently cloned many candidate plasticity-related genes (CPGs) induced by glutamate-receptor activation in the hippocampus. Screening the CPG pool for genes that may contribute to neocortical plasticity resulted in the identification of six genes that are induced in adult visual cortical areas in response to light. These genes are also naturally induced during postnatal cortical development. CPG induction by visual stimulation occurs primarily in neurons located in cortical layers II-III and VI and persists for at least 48 hr. Four of the visually responsive CPGs (cpg2, cpg15, cpg22, cpg29) are previously unreported genes, one of which (cpg2) predicts a "mini-dystrophin-like" structural protein. These results lend molecular genetic support to physiological and anatomical studies showing activity-dependent structural reorganization in adult cortex. In addition, these results provide candidate genes the function of which may underlie mechanisms of adult cortical reorganization.

Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase.

This report demonstrates that the investigational prostatic carcinoma marker known as the prostate-specific membrane antigen (PSM) possesses hydrolytic activity with the substrate and pharmacologic properties of the N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is a membrane hydrolase that has been characterized in the mammalian nervous system on the basis of its catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) to yield glutamate and N-acetylaspartate and that has been hypothesized to influence glutamatergic signaling processes. The immunoscreening of a rat brain cDNA expression library with anti-NAALADase antisera identified a 1428-base partial cDNA that shares 86% sequence identity with 1428 bases of the human PSM cDNA [Israeli, R. S., Powell, C. T., Fair, W. R. & Heston, W.D.W. (1993) Cancer Res. 53, 227-230]. A cDNA containing the entire PSM open reading frame was subsequently isolated by reverse transcription-PCR from the PSM-positive prostate carcinoma cell line LNCaP. Transient transfection of this cDNA into two NAALADase-negative cell lines conferred NAAG-hydrolyzing activity that was inhibited by the NAALADase inhibitors quisqualic acid and beta-NAAG. Thus we demonstrate a PSM-encoded function and identify a NAALADase-encoding cDNA. Northern analyses identify at least six transcripts that are variably expressed in NAALADase-positive but not in NAALADase-negative rat tissues and human cell lines; therefore, PSM and/or related molecular species appear to account for NAAG hydrolysis in the nervous system. These results also raise questions about the role of PSM in both normal and pathologic prostate epithelial-cell function.

Palmitoylation of the GluR6 kainate receptor.

The G-protein-coupled metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirus-infected insect cells or in human embryonic kidney cells and were metabolically labeled with [3H]palmitic acid. The metabotropic mGluR1 alpha receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with [3H]palmitate. The [3H]palmitate labeling of GluR6 was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation at a cysteine residue via a thioester bond. Site-directed mutagenesis was used to demonstrate that palmitoylation of GluR6 occurs at two cysteine residues, C827 and C840, located in the carboxyl-terminal domain of the molecule. A comparison of the electrophysiological properties of the wild-type and unpalmitoylated mutant receptor (C827A, C840A) showed that the kainate-gated currents produced by the unpalmitoylated mutant receptor were indistinguishable from those of the wild-type GluR6. The unpalmitoylated mutant was a better substrate for protein kinase C than the wild-type GluR6 receptor. These data indicate that palmitoylation may not modulate kainate channel function directly but instead affect function indirectly by regulating the phosphorylation state of the receptor.

Plant histochemistry by correlation peak imaging.

Using a new NMR correlation-peak imaging technique, we were able to investigate noninvasively the spatial distribution of carbohydrates and amino acids in the hypocotyl of castor bean seedlings. In addition to the expected high sucrose concentration in the phloem area of the vascular bundles, we could also observe high levels of sucrose in the cortex parenchyma, but low levels in the pith parenchyma. In contrast, the glucose concentration was found to be lower in the cortex parenchyma than in the pith parenchyma. Glutamine and/or glutamate was detected in the cortex parenchyma and in the vascular bundles. Lysine and arginine were mainly visible in the vascular bundles, whereas valine was observed in the cortex parenchyma, but not in the vascular bundles. Although the physiological significance of these metabolite distribution patterns is not known, they demonstrate the potential of spectroscopic NMR imaging to study noninvasively the physiology and spatial metabolic heterogeneity of living plants.

Location of the active site of allosteric chorismate mutase from Saccharomyces cerevisiae, and comments on the catalytic and regulatory mechanisms.

The active site of the allosteric chorismate mutase (chorismate pyruvatemutase, EC 5.4.99.5) from yeast Saccharomyces cerevisiae (YCM) was located by comparison with the mutase domain (ECM) of chorismate mutase/prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] (the P protein) from Escherichia coli. Active site domains of these two enzymes show very similar four-helix bundles, each of 94 residues which superimpose with a rms deviation of 1.06 A. Of the seven active site residues, four are conserved: the two arginines, which bind to the inhibitor's two carboxylates; the lysine, which binds to the ether oxygen; and the glutamate, which binds to the inhibitor's hydroxyl group in ECM and presumably in YCM. The other three residues in YCM (ECM) are Thr-242 (Ser-84), Asn-194 (Asp-48), and Glu-246 (Gln-88). This Glu-246, modeled close to the ether oxygen of chorismate in YCM, may function as a polarizing or ionizable group, which provides another facet to the catalytic mechanism.

An alternative pathway for signal flow from rod photoreceptors to ganglion cells in mammalian retina.

Rod signals in the mammalian retina are thought to reach ganglion cells over the circuit rod-->rod depolarizing bipolar cell-->AII amacrine cell-->cone bipolar cells-->ganglion cells. A possible alternative pathway involves gap junctions linking the rods and cones, the circuit being rod-->cone-->cone bipolar cells-->ganglion cells. It is not clear whether this second pathway indeed relays rod signals to ganglion cells. We studied signal flow in the isolated rabbit retina with a multielectrode array, which allows the activity of many identified ganglion cells to be observed simultaneously while the preparation is stimulated with light and/or exposed to drugs. When transmission between rods and rod depolarizing bipolar cells was blocked by the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), rod input to all On-center and briskly responding Off-center ganglion cells was dramatically reduced as expected. Off responses persisted, however, in Off-center sluggish and On-Off direction-selective ganglion cells. Presumably these responses were generated by the alternative pathway involving rod-cone junctions. This APB-resistant pathway may carry the major rod input to Off-center sluggish and On-Off direction-selective ganglion cells.

Long-term synaptic transformation of hippocampal CA1 gamma-aminobutyric acid synapses and the effect of anandamide.

Evidence is presented for a distinctive type of hippocampal synaptic modification [previously described for a molluscan gamma-aminobutyric acid (GABA) synapse after paired pre- and postsynaptic excitation]: transformation of GABA-mediated synaptic inhibition into synaptic excitation. This transformation persists with no further paired stimulation for 60 min or longer and is termed long-term transformation. Long-term transformation is shown to contribute to pairing-induced long-term potentiation but not to long-term potentiation induced by presynaptic stimulation alone. Further support for such mechanistic divergence is provided by pharmacologic effects on long-term transformation as well as these two forms of long-term potentiation by Cl- channel blockers, glutamate and GABA antagonists, as well as the endogenous cannabinoid ligand anandamide.

Role of the GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated genes.

We have isolated the NIL1 gene, whose product is an activator of the transcription of nitrogen-regulated genes, by virtue of the homology of its zinc-finger domain to that of the previously identified activator, the product of GLN3. Disruption of the chromosomal NIL1 gene enabled us to compare the effects of Gln3p and of Nil1p on the expression of the nitrogen-regulated genes GLN1, GDH2, and GAP1, coding respectively for glutamine synthetase, NAD-linked glutamate dehydrogenase, and general amino acid permease. Our results show that the nature of GATAAG sequence that serve as the upstream activation sequence elements for these genes determines their abilities to respond to Gln3p and Nil1p. The results further indicate that Gln3p is inactivated by an increase in the intracellular concentration of glutamine and that Nil1p is inactivated by an increase in intracellular glutamate.

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.

Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the glutamate receptor GluR2 subunit are Ca(2+)-permeable and exhibit inwardly rectifying current responses to kainate and AMPA. A proportion of cultured rat hippocampal neurons show similar Ca(2+)-permeable inwardly rectifying AMPA receptor currents. Inward rectification in these neurons was lost with intracellular dialysis and was not present in excised outside-out patches but was maintained in perforated-patch whole-cell recordings, suggesting that a diffusible cytoplasmic factor may be responsible for rectification. Inclusion of the naturally occurring polyamines spermine and spermidine in the recording pipette prevented loss of rectification in both whole-cell and excised-patch recordings; Mg2+ and putrescine were without effect. Inward rectification of Ca(2+)-permeable AMPA receptors may reflect voltage-dependent channel block by intracellular polyamines.

Tumor necrosis factors alpha and beta protect neurons against amyloid beta-peptide toxicity: evidence for involvement of a kappa B-binding factor and attenuation of peroxide and Ca2+ accumulation.

In Alzheimer disease (AD) the amyloid beta-peptide (A beta) accumulates in plaques in the brain. A beta can be neurotoxic by a mechanism involving induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels ([Ca2+]i). In light of evidence for an inflammatory response in the brain in AD and reports of increased levels of tumor necrosis factor (TNF) in AD brain we tested the hypothesis that TNFs affect neuronal vulnerability to A beta. A beta-(25-35) and A beta-(1-40) induced neuronal degeneration in a concentration- and time-dependent manner. Pretreatment of cultures for 24 hr with TNF-beta or TNF-alpha resulted in significant attenuation of A beta-induced neuronal degeneration. Accumulation of peroxides induced in neurons by A beta was significantly attenuated in TNF-pretreated cultures, and TNFs protected neurons against iron toxicity, suggesting that TNFs induce antioxidant pathways. The [Ca2+]i response to glutamate (quantified by fura-2 imaging) was markedly potentiated in neurons exposed to A beta, and this action of A beta was suppressed in cultures pretreated with TNFs. Electrophoretic mobility-shift assays demonstrated an induction of a kappa beta-binding activity in hippocampal cells exposed to TNFs. Exposure of cultures to I kappa B (MAD3) antisense oligonucleotides, a manipulation designed to induce NF-kappa B, mimicked the protection by TNFs. These data suggest that TNFs protect hippocampal neurons against A beta toxicity by suppressing accumulation of ROS and Ca2+ and that kappa B-dependent transcription is sufficient to mediate these effects. A modulatory role for TNF in the neurodegenerative process in AD is proposed.

Aromatic-L-amino-acid decarboxylase, a pyridoxal phosphate-dependent enzyme, is a beta-cell autoantigen.

Different autoantigens are thought to be involved in the pathogenesis of insulin-dependent diabetes mellitus, and they may account for the variation in the clinical presentation of the disease. Sera from patients with autoimmune polyendocrine syndrome type I contain autoantibodies against the beta-cell proteins glutamate decarboxylase and an unrelated 51-kDa antigen. By screening of an expression library derived from rat insulinoma cells, we have identified the 51-kDa protein as aromatic-L-amino-acid decarboxylase (EC 4.1.1.28). In addition to the previously published full-length cDNA, forms coding for a truncated and an alternatively spliced version were identified. Aromatic L-amino acid decarboxylase catalyzes the decarboxylation of L-5-hydroxytryptophan to serotonin and that of L-3,4-dihydroxyphenylalanine to dopamine. Interestingly, pyridoxal phosphate is the cofactor of both aromatic L-amino acid decarboxylase and glutamate decarboxylase. The biological significance of the neurotransmitters produced by the two enzymes in the beta cells remains largely unknown.

7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide (IDRA 21), a congener of aniracetam, potently abates pharmacologically induced cognitive impairments in patas monkeys.

We report here on the ability of IDRA 21 and aniracetam, two negative allosteric modulators of glutamate-induced DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization, to attenuate alprazolam-induced learning deficit in patas monkeys working in a complex behavioral task. In one component of a multiple schedule (repeated acquisition or "learning"), patas monkeys acquired a different four-response chain each session by responding sequentially on three keys in the presence of four discriminative stimuli (geometric forms or numerals). In the other component (performance) the four-response chain was the same each session. The response chain in each component was maintained by food presentation under a fixed-ratio schedule. When alprazolam (0.1 or 0.32 mg/kg p.o.) was administered alone, this full allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors produced large decreases in the response rate and accuracy in the learning component of the task. IDRA 21 (3 or 5.6 mg/kg p.o.) and aniracetam (30 mg/kg p.o.) administered 60 min before alprazolam, having no effect when given alone, antagonized the large disruptive effects of alprazolam on learning. From dose-response studies, it can be estimated that IDRA 21 is approximately 10-fold more potent than aniracetam in antagonizing alprazolam-induced learning deficit. We conclude that IDRA 21, a chemically unrelated pharmacological congener of aniracetam, improves learning deficit induced in patas monkeys by the increase of GABAergic tone elicited by alprazolam. Very likely IDRA 21 exerts its behavioral effects by antagonizing AMPA receptor desensitization.

Molecular design of the N-methyl-D-aspartate receptor binding site for phencyclidine and dizolcipine.

The N-methyl-D-aspartate receptor (NMDAR), a pivotal entity for synaptic plasticity and excitotoxicity in the brain, is a target of psychotomimetic drugs such as phencyclidine (PCP) and dizolcipine (MK-801). In contrast, a related glutamate receptor, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor GluR1, is weakly sensitive to these drugs. Three point mutations on GluR1, mimicking homologous residues on the NMDAR, confer the PCP and MK-801 blockade properties that are characteristic of the NMDAR--namely, high potency, voltage dependence, and use dependence. The molecular determinants that specify the PCP block appear confined to the putative M2 transmembrane segment, whereas the sensitivity to MK-801 requires an interplay between residues from M2 and M3. Given the plausible involvement of the NMDAR in the etiology of several neurodegenerative diseases and in excitotoxic neuronal cell death, tailored glutamate receptors with specific properties may be models for designing and screening new drugs targeted to prevent glutamate-mediated neural damage.