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Objective: Research has implicated dysfunction of glutamatergic neurotransmission in the pathophysiology of schizophrenia. This review evaluates evidence from preclinical and clinical studies that brain glutamatergic neurotransmission is altered in schizophrenia, may affect symptom expression, and is modulated by antipsychotic drugs., Method: A comprehensive review of scientific articles published over the last decade that address the role of glutamate in the pathophysiology of schizophrenia was carried out., Results: Glutamatergic neurons are the major excitatory pathways linking the cortex, limbic system, and thalamus, regions that have been implicated in schizophrenia. Postmortem studies have revealed alterations in pre- and postsynaptic markers for glutamatergic neurons in several brain regions in schizophrenia. The N-methyl-D-aspartic acid (NMDA) subtype of glutamate receptor may be particularly important as blockade of this receptor by the dissociative anesthetics reproduces in normal subjects the symptomatic manifestations of schizophrenia, including negative symptoms and cognitive impairments, and increases dopamine release in the mesolimbic system. Agents that indirectly enhance NMDA receptor function via the glycine modulatory site reduce negative symptoms and variably improve cognitive functioning in schizophrenic subjects receiving typical antipsychotics., Conclusions: Dysfunction of glutamatergic neurotransmission may play an important role in the pathophysiology of schizophrenia, especially of the negative symptoms and cognitive impairments associated with the disorder, and is a promising target for drug development.

This study reports that insulin-like growth factor I (IGF-I) prevents cerebellar granule cells from developing sensitivity to kainate neurotoxicity. Sensitivity to kainate neurotoxicity normally develops 5-6 days after switching cultures to a serum-free medium containing 25 mM K(+). Addition of either IGF-I or insulin to the serum-free medium at the time of the switch prevented the development of sensitivity to kainate, whereas brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and nerve growth factor did not. The dose-response curves indicated IGF-I was more potent than insulin, favoring the assignment of the former as the physiological protective agent. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (10-100 nM) and LY 294002 (0.3-1 microM) abolished the protection afforded by IGF-I. The p70 S6 kinase (p70(S6k)) inhibitor rapamycin (5-50 nM:) also abolished the protection afforded by IGF-I. The activities of both enzymes decreased in cultures switched to serum-free medium but increased when IGF-I was included; wortmannin (100 nM) lowered the activity of PI 3-K from 2 to 5 days after medium switch, whereas rapamycin (50 nM) prevented the increase observed for p70(S6k) activity over the same interval. The mitogen-activated protein kinase kinase inhibitor U 0126 and the mitogen-activated protein kinase inhibitor SB 203580 did not abolish IGF-I protection. Kainate neurotoxicity was not prevented by Joro spider toxin; therefore, the development of kainate neurotoxicity could not be explained by the formation of calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. These results indicate that IGF-I functions through a signal transduction pathway involving PI 3-K and p70(S6k) to prevent the development of sensitivity to kainate neurotoxicity in cerebellar granule cells.

The present study deals with the functional interaction of antipsychotic drugs and NMDA receptors. We show that both the conventional antipsychotic drug haloperidol and the atypical antipsychotic drug clozapine mediate gene expression via intracellular regulation of NMDA receptors, albeit to different extents. Data obtained in primary striatal culture demonstrate that the intraneuronal signal transduction pathway activated by haloperidol, the cAMP pathway, leads to phosphorylation of the NR1 subtype of the NMDA receptor at (897)Ser. Haloperidol treatment is likewise shown to increase (897)Ser-NR1 phosphorylation in rats in vivo. Mutation of (896)Ser and (897)Ser to alanine, which prevents phosphorylation at both sites, inhibits cAMP-mediated gene expression. We conclude that antipsychotic drugs have the ability to modulate NMDA receptor function by an intraneuronal signal transduction mechanism. This facilitation of NMDA activity is necessary for antipsychotic drug-mediated gene expression and may contribute to the therapeutic benefits as well as side effects of antipsychotic drug treatment.

Receptor supersensitivity is an important concept for understanding neurotransmitter and receptor dynamics. Traditionally, detection of receptor supersensitivity has been performed using autoradiography or positron emission tomography (PET). We show that use of magnetic resonance imaging (MRI) not only enables one to detect dopaminergic supersensitivity, but that the hemodynamic time course reflective of this fact is different in different brain regions. In rats unilaterally lesioned with intranigral 6-hydroxydopamine, apomorphine injections lead to a large increase in hemodynamic response (cerebral blood volume, CBV) in the striato-thalamo-cortico circuit on the lesioned side but had little effect on the intact side. Amphetamine injections lead to increases in hemodynamic responses on the intact side and little on the lesioned side in the same animals. The time course for the increase in CBV after either amphetamine or apomorphine administration was longer in striatum and thalamus than in frontal cortex. (11)C-PET studies of ligands which bind to the dopamine transporter (2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane 1, 5-naphthalnendisulfonate, WIN 35, 428 or CFT) and D2 receptors (raclopride) confirm that there is a loss of presynaptic dopamine terminals as well as upregulation of D2 receptors in striatum in these same animals. Pharmacologic MRI should become a sensitive tool to measure functional supersensitivity in humans, providing a complementary picture to that generated using PET studies of direct receptor binding., (Copyright 2000 Wiley-Liss, Inc.)

The enzyme glutamate carboxypeptidase II (GCP II) has been cloned from rat brain and human prostate. This enzyme, which catabolizes the neuropeptide N-acetylaspartylglutamate, has also been known as N-acetylated alpha-linked acidic dipeptidase (NAALADase), and is identical to the prostate-specific membrane antigen and to the jejunal folylpoly-gamma-glutamate carboxypeptidase. The goals of the present study were to elucidate the cell specificity and regional pattern of GCP II expression in the rat nervous system by using Northern blots and enzymatic assays of brain and subfractionated primary neuronal and glial cultures together with in situ hybridization histochemistry (ISHH) in sections of adult rat tissue. GCP II activity was assayed in astrocyte cultures (4.4 pmol/mg protein per minute), neuronal-glial cocultures (2.5 pmol/mg protein per minute) and neuron-enriched cultures (0.38 pmol/mg protein per minute), with the activity in each preparation correlating to its astrocytic content (r = 0.99). No activity was detected in cultured oligodendrocytes or microglia. Northern blots probed with a GCP II cDNA detected mRNAs exclusively in activity-positive cell preparations. ISHH results show that GCP II is expressed by virtually all astrocytes, by Bergmann glial cells in cerebellum, by Müller cells in retina and by the satellite cells in dorsal root ganglia. Astrocytes in select groups of nuclei (e.g., habenula, supraoptic nucleus, pontine nucleus) contained pronounced levels of GCP II message. The data of the present study suggest that GCP II is expressed in the adult rat nervous system exclusively in astrocytic glial cells., (Copyright 1999 Wiley-Liss, Inc.)

Objective: D-Serine is a full agonist at the glycine site on the N-methyl-D-aspartate (NMDA) receptor. Previous administration of D-serine to schizophrenic patients taking nonclozapine antipsychotics improved positive, negative, and cognitive symptoms, whereas the partial agonist D-cycloserine improved negative symptoms of patients taking conventional antipsychotics but worsened symptoms in clozapine-treated patients. To study the difference between full and partial agonists at the NMDA receptor glycine site, the clinical effects of adding D-serine to clozapine were assessed., Method: In a 6-week double-blind trial, 20 schizophrenic patients received placebo or D-serine (30 mg/kg per day) in addition to clozapine. Clinical efficacy, side effects, and serum levels of D-serine were determined every other week., Results: The patients exhibited no improvement with D-serine, nor did their symptoms worsen, as previously reported with D-cycloserine., Conclusions: The results suggest either that clozapine may have an agonistic effect on the NMDA system or that clozapine-treated patients do not respond to D-serine.

This study reports on the regulation of kainate neurotoxicity in cerebellar granule cells by calcium entry through voltage-gated calcium channels and by calcium release from internal cellular stores. Kainate neurotoxicity was prevented by the AMPA selective antagonist LY 303070 (10 microM). Kainate neurotoxicity was potentiated by cadmium, a general voltage-gated calcium channel blocker, and the L-type voltage-gated calcium channel blocker nifedipine. The antagonists of intracellular Ca2+ ([Ca2+]i) release, thapsigargin and ryanodine, were also able to potentiate kainate neurotoxicity. Kainate treatment elevated [Ca2+]i concentration with a rapid initial increase that peaked at 1543 nM and then declined to plateau at approximately 400 nM. Nifedipine lowered the peak response to 764 nM and the plateau response to approximately 90 nM. Thapsigargin also lowered the kainate-induced increase in [Ca2+]i (640 nM peak, 125 nM plateau). The ryanodine receptor agonist caffeine eliminated the kainate-induced increase in [Ca2+]i, and reduced kainate neurotoxicity. Kainate neurotoxicity potentiated by nifedipine was not prevented by RNA or protein synthesis inhibitors, nor by the caspase inhibitors YVAD-CHO and DEVD-CHO. Neither DNA laddering nor the number of apoptotic nuclei were increased following treatment with kainate and nifedipine. Increased nuclear staining with the membrane impermeable dye propidium iodide was observed immediately following kainate treatment, indicating a loss of plasma membrane integrity. Thus, kainate neurotoxicity is prevented by calcium entry through L-type calcium channels., (Copyright 1999 Elsevier Science B.V.)

Objective: Glutamatergic neurotransmission is important for memory and cognition and is severely affected in Alzheimer's disease. D-Cycloserine exhibits partial agonist activity at the glycine site of N-methyl-D-aspartate subtype glutamate receptor, facilitating activation of the receptor and improving cognition and memory., Method: Seventeen patients with Alzheimer's disease received a three-phase, double-blind, placebo-controlled trial of 50 mg and 100 mg/day of D-cycloserine., Results: D-Cycloserine was associated with significant improvement in scores on the cognitive subscale of the Alzheimer's Disease Assessment Scale (improvement of 3.0 points) when given at a dose of 100 mg/day., Conclusions: D-Cycloserine has cognitive benefits for patients with Alzheimer's disease.

Glutamate carboxypeptidase II (GCP II) catalyzes the extracellular hydrolysis of the neuromodulator N-acetyl-aspartylglutamate to N-acetyl-aspartate and glutamate. GCP II also hydrolyzes gamma-glutamyl bonds in folylpolyglutamate. The predicted amino acid sequence of GCP II displays similarities to aminopeptidases from Streptomyces griseus and Vibrio proteolyticus, whose crystal structures have been determined. These aminopeptidases are cocatalytic zinc metallopeptidases belonging to the peptidase family M28. Specific zinc and substrate ligands have been proposed in GCP II based on the amino acid sequence alignment to these M28 family members. In the present study, site-directed mutagenesis has been used to test the assignment of these putative ligands in human GCP II. Substitutions to the five putative zinc ligands resulted in severely reduced enzyme activity, although mutant protein was expressed as demonstrated by immunoblot analysis. In addition, substitutions of amino acids near the putative zinc ligands have identified other specific residues important for enzyme structure and/or function. Substitutions to putative substrate ligands were less perturbing, and increases in Km were observed for substitutions that introduced a large charge perturbation (e.g., Lys to Glu). The results from substitutions at the proposed zinc and substrate ligands are consistent with the assignment of these residues and suggest that GCP II has a three-dimensional structure similar to other members of the peptidase family M28.

Background: In a preliminary dose-finding study, D-cycloserine, a partial agonist at the glycine modulatory site of the glutamatergic N-methyl-D-aspartate (NMDA) receptor, improved negative symptoms and cognitive function when added to conventional neuroleptics at a dose of 50 mg/d., Methods: Forty-seven patients with schizophrenia meeting criteria for deficit syndrome were randomized to D-cycloserine, 50 mg/d (n=23) or placebo (n=24) added to their conventional neuroleptic for an 8-week, double-blind trial. Clinical assessments were performed at baseline and at weeks 1, 2, 4, 6, and 8. Serum concentrations of D-cycloserine, relevant amino acids, and homovanillic acid were assayed at baseline and at weeks 4 and 8. A cognitive battery was performed at baseline and at week 8., Results: Thirty-nine patients completed the 8-week trial. Seven dropouts occurred in the D-cycloserine group and 1 in the placebo group. The mean reduction in negative symptoms with D-cycloserine (23%) was significantly greater than with placebo (7%) as calculated by slopes representing Scale for the Assessment of Negative Symptoms (SANS) total scores. Improvement of negative symptoms was predicted by low neuroleptic dose and low baseline SANS total score. No differences were found in performance on any cognitive test between groups or in changes in any other clinical measure. Clinical response did not correlate significantly with serum amino acid concentrations at baseline or with concentrations of D-cycloserine at weeks 4 and 8., Conclusion: These results support the hypothesis that agents acting at the glycine modulatory site of the NMDA receptor improve primary negative symptoms.

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-D-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch-clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 microM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.

Background: Hypofunction of N-methyl-D-aspartate (NMDA) subtype glutamate receptor has been implicated in the pathophysiology of schizophrenia. D-serine is a full agonist of the glycine site of NMDA receptor, an endogenous cotransmitter enriched in corticolimbic regions and distributed in parallel with NMDA receptor. Supplementation of D-serine may improve the symptoms of schizophrenia., Methods: Thirty-one Taiwanese schizophrenic patients enrolled in a 6-week double-blind, placebo-controlled trial of D-serine (30 mg/kg/day), which was added to their stable antipsychotic regimens. Of these, 28 completed the trial. Measures of clinical efficacy, side effects, and serum levels of amino acids and D-serine were determined every other week. Wisconsin Card Sorting Test (WCST) was performed at the beginning and end of the trial., Results: Patients who received D-serine treatment revealed significant improvements in their positive, negative, and cognitive symptoms as well as some performance in WCST. D-serine levels at week 4 and 6 significantly predicted the improvements. D-serine was well tolerated and no significant side effects were noted., Conclusions: The significant improvement with the D-serine further supports the hypothesis of NMDA receptor hypofunction in schizophrenia. Given the effects of D-serine on positive symptoms, a trial of D-serine alone in schizophrenia should be considered.

Background: Phencyclidine and ketamine induce a syndrome closely resembling schizophrenia due to their blockade of N-methyl-D-aspartate (NMDA) receptor. These findings suggested that some aspects of schizophrenia are associated with decreased NMDA--glutamatergic function. We hypothesized that structural and symptomatic deficits in schizophrenia are related to glutamatergic neurotransmission., Methods: We studied the relationships among cerebrospinal fluid (CSF) glutamatergic markers, clinical presentation of schizophrenia, and CT parameters of brain structure in drug-free schizophrenics., Results: We found no significant differences between patients with schizophrenia and controls in CSF glutamatergic markers. When patients with schizophrenia were considered as a group, significant negative correlations between glutamatergic markers and brain structural measures as well as clinical measures were observed. Cluster analysis reveals a group of lower indices of glutamatergic neurotransmission, and more prominent thought disorder as well as ventricular enlargement, and a group with increased glutamate level., Conclusions: The findings support the hypothesis that altered glutamatergic neurotransmission plays a role in the brain structure and the clinical symptoms of schizophrenia.

Objective: Tardive dyskinesia is a movement disorder affecting 20%-40% of patients treated chronically with neuroleptic drugs. The dopamine supersensitivity hypothesis cannot account for the time course of tardive dyskinesia or for the persistence of tardive dyskinesia and the associated structural changes after neuroleptics are discontinued. The authors hypothesized that neuroleptics enhance striatal glutamatergic neurotransmission by blocking presynaptic dopamine receptors, which causes neuronal damage as a consequence of oxidative stress., Method: CSF was obtained from 20 patients with schizophrenia, 11 of whom had tardive dyskinesia. Markers for oxidative stress, including superoxide dismutase, lipid hydroperoxide, and protein carbonyl groups, and markers for excitatory neurotransmission, including N-acetylaspartate, N-acetylaspartylglutamate, aspartate, and glutamate, were measured in the CSF specimens. Patients were also rated for tardive dyskinesia symptoms with the Abnormal Involuntary Movement Scale., Results: Tardive dyskinesia patients had significantly higher concentrations of N-acetylaspartate, N-acetylaspartylglutamate, and aspartate in their CSF than patients without tardive dyskinesia when age and neuroleptic dose were controlled for. The significance of the higher levels of protein-oxidized products associated with tardive dyskinesia did not pass Bonferroni correction, however. Tardive dyskinesia symptoms correlated positively with markers of excitatory neurotransmission and protein carbonyl group and negatively with CSF superoxide dismutase activity., Conclusions: These findings suggest that there are elevated levels of oxidative stress and glutamatergic neurotransmission in tardive dyskinesia, both of which may be relevant to the pathophysiology of tardive dyskinesia.

Jejunal folylpoly-gamma-glutamate carboxypeptidase hydrolyzes dietary folates prior to their intestinal absorption. The complete folylpoly-gamma-glutamate carboxypeptidase cDNA was isolated from a pig jejunal cDNA library using an amplified homologous probe incorporating primer sequences from prostate-specific membrane antigen, a protein capable of folate hydrolysis. The cDNA encodes a 751-amino acid polypeptide homologous to prostate-specific membrane antigen and rat brain N-acetylated alpha-linked acidic dipeptidase. PC3 transfectant membranes exhibited activities of folylpoly-gamma-carboxypeptidase and N-acetylated alpha-linked acidic dipeptidase, while immunoblots using monoclonal antibody to native folylpoly-gamma-glutamate carboxypeptidase identified a glycoprotein at 120 kDa and a polypeptide at 84 kDa. The kinetics of native folylpoly-gamma-carboxypeptidase were expressed in membranes of PC3 cells transfected with either pig folylpoly-gamma-carboxypeptidase or human prostate-specific membrane antigen. Folylpoly-gamma-carboxypeptidase transcripts were identified at 2.8 kilobase pairs in human and pig jejunum, human and rat brain, and human prostate cancer LNCaP cells. Thus, pig folylpoly-gamma-carboxypeptidase, rat N-acetylated alpha-linked acidic dipeptidase, and human prostate-specific membrane antigen appear to represent varied expressions of the same gene in different species and tissues. The discovery of the jejunal folylpoly-gamma-carboxypeptidase gene provides a framework for future studies on relationships among these proteins and on the molecular regulation of intestinal folate absorption.

N-Acetylated alpha-linked acidic dipeptidase (NAALADase) is a neuropeptidase that may modulate glutamatergic neurotransmission. Independent of its characterization in the nervous system, one form of NAALADase was shown to be expressed at high levels in human prostatic adenocarcinomas, and it was designated the prostate-specific membrane antigen (PSMA). The NAALADase/PSMA gene is known to produce multiple mRNA splice forms, and based on previous immunohistochemical evidence, it had been assumed that the human brain and prostate expressed different isoforms of the enzyme. Because PSMA is being actively pursued as a target for autoimmune and cytotoxic targeting strategies to treat prostate cancer, the rigorous comparison of the two forms of the enzyme remained an important but untested question. To assess similarities and/or differences between human brain NAALADase and PSMA, we compared the two molecules using criteria of activity, immunoreactivity and sequences of the corresponding mRNAs. NAALADase from human cerebellar isolates displayed a kinetic profile and pharmacological sensitivities similar to PSMA. Also, Northern hybridization to PSMA cDNA detected indistinguishable sets of 2.8-, 4.0- and 6.0-kb RNA species in human brain and the LNCaP prostatic tumor cell line. In addition, the monoclonal antibody 7E11-C5 directed against the prostatic form of the enzyme immunoprecipitated 82% of human cerebellar NAALADase activity. Moreover, reverse transcription-polymerase chain reaction cloning of cerebellar cDNAs indicated that the human brain and prostate express a common mRNA splice form. Therefore, we conclude that the form of NAALADase also known as PSMA is expressed in brain and comprises a significant fraction of brain NAALADase activity.

The cytoplasmic copper-zinc superoxide dismutase (Cu, Zn SOD; SOD-1) is an abundant and well-conserved intracellular antioxidant enzyme which has been implicated in a number of oxidative stress mediated phenomena, especially Down Syndrome, in which SOD-1 activity is increased due to triplication of chromosome 21 containing the gene and, in hereditary amyotrophic lateral sclerosis, in which the gene is mutated. Overexpression of SOD-1 could theoretically, therefore, lead to increased vulnerability to oxidative stress in two distinct manners: increasing steady-state hydrogen peroxide levels or increasing toxic side reactions. We used two mouse neuronal culture systems--one in which the murine chromosome containing SOD-1 is triplicated and one in which human SOD-1 is a transgene--to test the effect of overexpression of this enzyme on antioxidant status in general and specifically on glutamate mediated oxidative stress. We found that SOD-1 overexpression increases antioxidant status at the same time it decreases vulnerability to glutamate.

Glutamate carboxypeptidase II may modulate excitatory neurotransmission through the catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) and possibly other endogenous peptide substrates. To investigate the molecular properties of cloned human GCP II (hGCP II), we analyzed the NAAG-hydrolytic activity conveyed by transfection of a full-length hGCP II cDNA into PC3 cells, which do not express GCP II endogenously. Membrane fractions from these cells demonstrated activity with an apparent Km of 73 nM and Vmax of 35 pmol/(mg protein*min). Activity was inhibited by EDTA and stimulated by the addition of CoCl2. Addition of GCP II inhibitors beta-NAAG, quisqualic acid and 2-(phosphonomethyl)pentanedioic acid (PMPA) inhibited hydrolysis of 2.5 nM NAAG with IC50s of 201 nM, 155 nM and 98 pM, respectively. In competition experiments designed to infer aspects of hGCP II substrate selectivity, NAAG was the most potent alpha peptide tested, with an IC50 of 26 nM. Folate derivatives and some other gamma-glutamyl peptides showed comparable affinity to that of NAAG, also displaying IC50s in the low nM range. Taken together with previous evidence demonstrating their presence in GCP II-expressing tissues, these data suggest that both NAAG and folates are good candidate substrates for GCP II in vivo., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Recent scientific advances in understanding the function and plasticity of the central nervous system have opened fundamental insights into the pathophysiology of various mental disorders. This chapter reviews the basic physiology of the central nervous system, and further focuses on the applications of the new knowledge to clinical psychiatry.

Objective: Neurophysiological and pathological effects of ethanol may be mediated, to an important extent, via the glutamatergic system. Animal studies indicate the acute effects of ethanol disrupt glutamatergic neurotransmission by inhibiting the response of the N-methyl-D-aspartate (NMDA) receptor. Persistent attenuation of glutamatergic neurotransmission by chronic ethanol exposure results in the compensatory up-regulation of NMDA receptors. Whether glutamatergic neurotransmission and oxidative stress are enhanced during ethanol withdrawal in humans is unknown., Method: CSF was obtained from 18 matched comparison subjects and from 18 patients with alcohol dependence 1 week and 1 month after cessation of ethanol ingestion. CSF samples were analyzed for excitatory neurotransmitters, gamma-aminobutyric acid (GABA), and markers for oxidative stress., Results: The alcohol-dependent patients' CSF levels of aspartate, glycine, and N-acetylaspartylglutamate were all higher than those of the comparison subjects, and their concentration of GABA was lower. In addition, there were significant correlations between excitatory neurotransmitters and oxidative stress markers, which suggest that the two mechanisms may play an interactive role in neurotoxicity mediated by ethanol withdrawal., Conclusions: The data suggest that augmentation of excitatory neurotransmission may lead to enhanced oxidative stress, which, in concert with reduced inhibitory neurotransmission, may contribute to the symptoms of ethanol withdrawal and associated neurotoxicity in humans. Whether these abnormalities represent a trait- or state-dependent marker of ethanol dependence remains to be resolved.

The levels of N-acetylaspartylglutamate (NAAG) were determined by HPLC in untreated or lipopolysaccharide (LPS) activated pure astrocyte, oligodendrocyte, and microglial cultures derived from developing rat brain. Oligodendrocyte cultures expressed 1.52+/-0.12 nmol/microg protein of NAAG, whereas astrocyte cultures (0.04+/-0.08 nmol/microg protein) and untreated microglial cultures (0.05+/-0.09 nmol/microg protein) contained only trace amounts of the dipeptide. After stimulation of microglial cultures for 24 h with LPS, NAAG levels increased significantly to 0.37+/-0.12 SD nmol/microg protein. NAAG levels in astrocyte and oligodendrocyte cultures remained unchanged after LPS treatment. The findings indicate that NAAG is localized to specific glial cell types. Further our results suggest that NAAG biosynthesis is induced in microglia, activated by specific stimuli., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Considerable evidence suggests that reactive oxygen species mediate the neurotoxic effects of ionotropic glutamate receptor activation. Accordingly, we have examined neuronal degeneration resulting from intrastriatal injection of quinolinic acid, an NMDA receptor agonist, and kainic acid in gene targeted and transgenic mice that under- or over-express copper, zinc superoxide dismutase (Cu,Zn-SOD; SOD-1). Elevated SOD-1 activity significantly protects against quinolinic acid and kainic acid neurotoxicity in the mouse striatum whereas reduced activity appears to potentiate neurotoxicity. Thus a 'gene-dose' effect of SOD-1 has been demonstrated with regard to excitotoxic mechanisms.

Somatostatin expression in trisomy 16 mouse neuronal cultures has been studied to investigate the effects of the presence of an extra copy of the pre-pro-somatostatin (ppSS) gene on mouse chromosome 16. The immunoreactivity for somatostatin (SS) was considered in mixed cultures of neurons and glia cells and in neuron-enriched cultures as well as that for neuropeptide Y, glutamic acid decarboxylase, and gamma-enolase immunoreactivity the genes of which are not present on mouse chromosome 16. ppSS and pre-pro-neuropeptide Y (ppNPY) mRNA expression was evaluated and SS immunoreactivity in neurons analyzed by a morphometrical study. The extra copy of the ppSS gene resulted in a significantly increased level of the transcript in trisomic cultures, whereas the expression of the other neuropeptides did not differ. The absence of glial cells in these cultures reduced the number of SS-positive neurons making their number comparable in the trisomic and control cultures. Thus, in spite of higher expression of the ppSS mRNA in trisomic cultures, the determination of this peptidergic phenotype was influenced by the presence of neuroglial cells.

N-acetylated alpha-linked acidic dipeptidase (NAALADase) hydrolyzes acidic peptides, such as the abundant neuropeptide N-acetyl-alpha-L-aspartyl-L-glutamate (NAAG), thereby generating glutamate. Previous cDNA cloning efforts have identified a candidate rat brain NAALADase partial cDNA, and Northern analyses have identified a family of related RNA species that are found only in brain and other NAALADase-expressing cells. In this report, we describe the cloning of a set of rat brain cDNAs that describe a full-length NAALADase mRNA. Transient transfection of a full-length cDNA into the PC3 cell line confers NAAG-hydrolyzing activity that is sensitive to the NAALADase inhibitors quisqualic acid and 2-(phosphonomethyl)glutaric acid. Northern hybridization detects the expression of three similar brain RNAs approximately 3,900, 3,000, and 2,800 nucleotides in length. In situ hybridization histochemistry shows that NAALADase-related mRNAs have an uneven regional distribution in rat brain and are expressed predominantly by astrocytes as demonstrated by their colocalization with the astrocyte-specific marker glial fibrillary acidic protein.

Recent evidence suggests that ethanol abuse produces its diverse effects on the brain to a substantial degree by disrupting the function of the major excitatory neurotransmitter, glutamate. Ethanol, at concentrations associated with behavioral effects in humans, inhibits the N-methyl-D-aspartate (NMDA) receptor, which mediates the post-synaptic excitatory effects of glutamate. Tolerance to ethanol results in up-regulation of the NMDA receptor so that abrupt withdrawal produces a hyperexcitable state that leads to seizures, delerium tremens, and excitotoxic neuronal death. Ethanol's inhibition of the NMDA receptor in the fetal brain likely contributes to the CNS manifestations of fetal alcohol syndrome. Therapeutic strategies aimed at correcting glutamatergic dysregulation in alcoholism need to be explored.

We examined the effects of N-acetylaspartylglutamate (NAAG), an endogenous peptide thought to be involved in neurotransmission and neuromodulation, on striatal quinolinate lesions, a rodent model of Huntington's disease. We found that NAAG (500 and 1000 nmol) co-injected with quinolinic acid significantly reduced lesion volumes (by 50% and 65%, respectively). A 1000 nmol dose of the non-hydrolyzable analogue, beta-NAAG, also reduced quinolinic acid lesion volumes by 78.4%, indicating that the protection observed was not secondary to cleavage of NAAG into N-acetyl-aspartate (NAA) and glutamate. Likewise, co-injection of both NAA and glutamate (1000 nmol each) with quinolinic acid did not significantly alter the size of lesions. NAAG's protective effect may be mediated through actions on N-methyl-D-aspartate receptors or metabotropic glutamate receptors.

The dipeptide N-acetylaspartylglutamate (NAAG) may be involved in the process of glutamatergic signaling by both acting at glutamate receptors and as a glutamate protransmitter. In the present study we determined the cellular localization and distribution of NAAG-like immunoreactivity (NAAG-LI) in normal human brain and in neurodegenerative disorders to ascertain the degree of NAAG's colocalization to putative glutamatergic pathways. Immunohistochemistry with an antibody against NAAG was performed on control, Huntington's disease (HD) and Alzheimer's disease (AD) human autopsy and biopsy brain sections from the cerebral cortex, hippocampus, amygdala, neostriatum, brainstem and spinal cord. In normal human brain, NAAG-LI was widespread localized to putative glutamatergic pyramidal neurons of the cerebral cortex and hippocampus. Punctate NAAG-LI was present in areas known to receive neuronal glutamatergic input, such as layer IV of the cerebral cortex, striatal neuropil, and the outer portion of the molecular layer of the hippocampal dentate gyrus. In the two pathologic brain regions examined, the HD neostriatum and the AD temporal cortex, we observed a widespread loss of NAAG-LI neurons. In addition NAAG-LI reactive microglia surrounding plaques were seen in AD temporal cortex but not in the HD striatum. Our results suggest that NAAG is substantially localized to putative glutamatergic pathways in human brain and that NAAG-LI neurons are vulnerable to the neurodegenerative process in HD and AD.

There is mounting evidence, primarily from research in experimental animals, that the dipeptide N-acetylaspartylglutamate (NAAG) and its metabolic enzyme, N-acetylated alpha-linked acid dipeptidase (NAALADase), are involved in glutamatergic neurotransmission. Previous studies in neuropsychiatric disorders associated with the dysregulation of glutamatergic neurotransmission, such as schizophrenia, seizure disorders, and amyotrophic lateral sclerosis (ALS), have revealed region-specific alterations in the levels of NAAG and in the activity of NAALADase. To establish better the cellular localization of these and related parameters in human brain, we have examined their alterations in two well-characterized selective neurodengenerative disorders, Huntington Disease (HD) and Alzheimer Disease (AD). Brain regions from postmortem controls and HD- or AD-affected individuals were assayed to determine the activity of NAALADase as well as the levels of NAAG, N-acetylaspartate (NAA), and several amino acids. The relationships between changes in these neurochemical parameters and changes in neuronal and glial cell density were determined. The present report demonstrates that the decreases in the levels of NAAG and NAA and in the activity of NAALADase in AD and HD brain correlate primarily with neuronal loss. By inference, the results suggest that NAAG and NAA have primarily a neuronal localization in human brain and that there is a close relationship between NAAG and the dipeptidase NAALADase in populations of affected neurons.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide found in millimolar concentrations in brain that is localized to subpopulations of glutamatergic, cholinergic, GABAergic, and noradrenergic neuronal systems. NAAG is released upon depolarization by a Ca(2+)-dependent process and is an agonist at mGluR3 receptors and an antagonist at NMDA receptors. NAAG is catabolized to N-acetylaspartate and glutamate primarily by glutamate carboxypeptidase II, which is expressed on the extracellular surface of astrocytes. The levels of NAAG and the activity of carboxypeptidase II are altered in a regionally specific fashion in several neuropsychiatric disorders.

Growing evidence suggests that non-N-methyl-D-aspartate receptor activation may contribute to neuronal death in both acute and chronic neurological diseases. The intracellular processes that mediate this form of neuronal death are poorly understood. We have previously characterized a model of kainic acid neurotoxicity using cerebellar granule cell neurons in vitro and we sought to determine the mechanism of kainic acid-induced neuronal degeneration. We found DNA laddering by agarose gel electrophoresis, cellular DNA fragmentation by in situ end labeling of DNA, and chromatin condensation using a fluorescent DNA intercalating dye, in cerebellar granule cells following exposure to kainic acid (100 microM). Aurintricarboxylic acid protected cerebellar granule cells from kainic acid-induced death. While the morphological and biochemical features of neuronal death induced by kainic acid resembled low K(+)-induced apoptosis in cerebellar granule cells, the time interval from the institution of the death promoting condition to neuronal death was shorter with kainic acid and did not require new protein or RNA synthesis. These results demonstrate that kainic acid receptor activation can induce transcription-independent apoptosis in neurons. This in vitro model should be useful in identifying the intracellular pathways that link kainic acid receptor activation with apoptosis.

Objective: The effects of D-cycloserine added to clozapine were assessed and compared with previous results for D-cycloserine plus conventional neuroleptics., Method: Ten schizophrenic outpatients receiving clozapine entered consecutive 2-week trials of placebo and D-cycloserine at 5, 15, 50, and 250 mg/day. Clinical evaluations were videotaped and scored by a rater blind to the sequence of assessments., Results: There was a significant dose effect of D-cycloserine on scores on the Scale for the Assessment of Negative Symptoms (SANS); the 50-mg dose produced a mean 21% increase in SANS score. The patients had significantly higher baseline serum glutamate concentrations than the patients receiving typical neuroleptics in the previous trial. Baseline glutamate level and change in glycine level significantly correlated with response of negative symptoms to 50-mg D-cycloserine., Conclusions: The improvement of negative symptoms with D-cycloserine previously observed in patients receiving typical neuroleptics did not occur in patients treated with clozapine.

Growing evidence suggests that non-N-methyl-D-aspartate receptor activation may contribute to neuronal death in both acute and chronic neurological diseases. The intracellular processes that mediate this form of neuronal death are poorly understood. We have previously characterized a model of kainate neurotoxicity using cerebellar granule cell neurons in vitro and we sought to determine the mechanism of kainate-induced neurons degeneration. We found DNA, and chromatin condensation using a fluorescent DNA intercalating dye, in cerebellar granule cells following exposure to kainate (100 microM). Aurintricarboxylic acid protected cerebellar granule cells from kainate-induced death. While the morphological and biochemical features of neuronal death induced by kainate resembled low-K(+)-induced apoptosis in cerebellar granule cells; the time interval from the institution of the death-promoting condition to neuronal death was briefer with kainate and did not require new protein or RNA synthesis. These results demonstrate that kainate receptor activation can induce transcription-independent apoptosis in neurons. This in vitro model should be useful in identifying the intracellular pathways that link kainate receptor activation with apoptosis.

One hundred patients with clinically diagnosed Huntington's disease (HD) were randomized to either idebenone, an antioxidant and enhancer of oxidative metabolism, or placebo, in a 1-year, double-blind, parallel-group study aimed at slowing the rate of progression of the disease. Ninety-one patients completed the study. There were no significant differences between groups on the primary outcome measures of the Huntington's Disease Activities of Daily Living Scale (ADL-an index of functional status) and the Quantified Neurologic Examination (QNE). Sample size calculations based on progression of the ADL and QNE in this study group revealed that a larger study group is necessary to detect any differences less than an almost complete halting of the disease. This argues for multicenter efforts for future therapeutic trials in HD.

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.

Oxidative stress refers to the cytopathologic consequences of a mismatch between the production of free radicals and the ability of the cell to defend against them. Growing data from experimental models and human brain studies suggest oxidative stress may play an important role in neuronal degeneration in diseases such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Mitochondrial oxidative metabolism, nitric oxide, phospholipid metabolism, and proteolytic pathways are potential sources of intracellular free radicals. Alterations in free radical defense systems may also contribute to oxidative stress. A net increase in reactive oxygen species can produce damage to lipids, proteins, and DNA and induce necrosis or apoptosis. Elucidating the pathways important in the production of and defense from free radicals may be important in devising new pharmacologic strategies to slow or halt neuronal degeneration.

Schizophrenia is a syndrome, undoubtedly with multiple etiologies, that variably exhibits several features including positive and negative symptoms, cognitive deficits, onset in young adulthood, and deterioration from the previous level of function. This review will examine the growing evidence that dysfunction of corticolimbic glutamatergic neurotransmission may contribute to or account for the manifestations of schizophrenia. Glutamatergic neurons represent the primary excitatory afferent and efferent systems innervating the cortex, limbic regions, and striatum. The postsynaptic actions of glutamate are mediated by a family of glutamate-gated ion channels that permit the influx of sodium and calcium, thereby depolarizing (exciting) neurons. One of these receptors, the N-methyl-D-aspartate (NMDA) receptor, is the site of action of psychotomimetics such as phencyclidine and related anesthetics, which can reproduce in normal individuals most of the symptomatic features of schizophrenia. An endogenous antagonist at the NMDA receptor, N-acetyl-aspartyl glutamate, appears to have enhanced activity in the frontal cortex and hippocampal formation in persons with this disorder. Glutamatergic dysfunction may be particularly relevant to those forms of schizophrenia in which negative symptoms, cognitive deficits, and deterioration are prominent features. In support of this inference, clinical studies have shown that drugs that enhance NMDA-receptor function reduce negative symptoms and cognitive deficits in persons with chronic schizophrenia who are receiving neuroleptics. Thus, dysfunction of glutamatergic neurotransmission represents an important organizational focus for research on the complex manifestations of schizophrenia.