Search

The guidance cue receptor DCC controls mesocortical dopamine development in adolescence. Repeated exposure to an amphetamine regimen of 4 mg/kg during early adolescence induces, in male mice, downregulation of DCC expression in dopamine neurons by recruiting the Dcc microRNA repressor, microRNA-218 (miR-218). This adolescent amphetamine regimen also disrupts mesocortical dopamine connectivity and behavioral control in adulthood. Whether low doses of amphetamine in adolescence induce similar molecular and developmental effects needs to be established. Here, we quantified plasma amphetamine concentrations in early adolescent mice following a 4 or 0.5 mg/kg dose and found peak levels corresponding to those seen in humans following recreational and therapeutic settings, respectively. In contrast to the high doses, the low amphetamine regimen does not alter Dcc mRNA or miR-218 expression; instead, it upregulates DCC protein levels. Furthermore, high, but not low, drug doses downregulate the expression of the DCC receptor ligand, Netrin-1, in the nucleus accumbens and prefrontal cortex. Exposure to the low-dose regimen did not alter the expanse of mesocortical dopamine axons or their number/density of presynaptic sites in adulthood. Strikingly, adolescent exposure to the low-dose drug regimen does not impair behavioral inhibition in adulthood; instead, it induces an overall increase in performance in a go/no-go task. These results show that developmental consequences of exposure to therapeutic- versus abused-like doses of amphetamine in adolescence have dissimilar molecular signatures and opposite behavioral effects. These findings have important clinical relevance since amphetamines are widely used for therapeutic purposes in youth.

Disrupted-in-schizophrenia 1 (DISC1) is a genetic risk factor that has been implicated in major mental disorders. DISC1 binds to and stabilizes serine racemase to regulate production of D-serine by astrocytes, contributing to glutamate (GLU) neurotransmission. However, the possible involvement of astrocytic DISC1 in synthesis, metabolism, reuptake, or secretion of GLU remains unexplored. Therefore, we studied the effects of dominant-negative mutant DISC1 on various aspects of GLU metabolism by using primary astrocyte cultures and hippocampal tissue from transgenic mice with astrocyte-restricted expression of mutant DISC1. Although mutant DISC1 had no significant effects on astrocyte proliferation, GLU reuptake, glutaminase, or glutamate carboxypeptidase II activity, expression of mutant DISC1 was associated with increased levels of alanine-serine-cysteine transporter 2, vesicular glutamate transporters 1 and 3 in primary astrocytes and in the hippocampus, and elevated expression of the NR1 subunit and diminished expression of the NR2A subunit of N-methyl-D-aspartate (NMDA) receptors in the hippocampus, at postnatal day 21. Our findings indicate that decreased D-serine production by astrocytic mutant DISC1 might lead to compensatory changes in levels of the amino acid transporters and NMDA receptors in the context of tripartite synapse.

Antibiotic resistance is a worldwide public health threat that needs to be addressed by improved antibiotic stewardship and continuing development of new chemical entities to treat resistant bacterial infections. Compounds that work alongside known antibiotics as combination therapies offer an efficient and sustainable approach to counteract antibiotic resistance in bacteria. Guided by property-based analysis, a series of aza-tricyclic indolines (ATIs) were synthesized to optimize their physiochemical properties as novel combination therapies with β-lactams to treat methicillin-resistant S. aureus (MRSA) infections. A novel and highly efficient gold-catalyzed tandem cyclization was developed to facilitate the synthesis of these ATIs. One guanidine-containing ATI was discovered to possess both improved anti-MRSA activity and lower mammalian toxicity both in vitro and in vivo. In addition, it also showed significantly enhanced aqueous solubility and metabolic stability. These results indicated that the ATIs are a novel class of anti-MRSA agents suitable for further evaluations as adjuvant therapy in animal model studies.

Pharmacological activation of group II metabotropic glutamate receptors (mGluR2/3) inhibits cocaine self-administration and reinstatement of drug-seeking behavior, suggesting a possible use of mGluR2/3 agonists in the treatment of cocaine dependence. In this study, we investigated whether elevation of the endogenous mGluR2/3 ligand N-acetyl-aspartatylglutamate (NAAG) levels by the N-acetylated-alpha-linked-acidic dipeptidase inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) attenuates cocaine self-administration and cocaine-induced reinstatement of drug seeking. N-acetylated-alpha-linked-acidic dipeptidase is a NAAG degradation enzyme that hydrolyzes NAAG to N-acetylaspartate and glutamate. Systemic administration of 2-PMPA (10-100 mg/kg, i.p.) inhibited intravenous self-administration maintained by low unit doses of cocaine and cocaine (but not sucrose)-induced reinstatement of drug-seeking behavior. Microinjections of 2-PMPA (3-5 microg/side) or NAAG (3-5 microg/side) into the nucleus accumbens (NAc), but not into the dorsal striatum, also inhibited cocaine-induced reinstatement, an effect that was blocked by intra-NAc injection of LY341495, a selective mGluR2/3 antagonist. In vivo microdialysis demonstrated that 2-PMPA (10-100 mg/kg, i.p.) produced a dose-dependent reduction in both extracellular dopamine (DA) and glutamate, an effect that was also blocked by LY341495. Finally, pre-treatment with 2-PMPA partially attenuated cocaine-enhanced extracellular NAc DA, while completely blocking cocaine-enhanced extracellular NAc glutamate in rats during reinstatement testing. Intra-NAc perfusion of LY341495 blocked 2-PMPA-induced reductions in cocaine-enhanced extracellular NAc glutamate, but not DA. These findings suggest that 2-PMPA is effective in attenuating cocaine-induced reinstatement of drug-seeking behavior, likely by attenuating cocaine-induced increases in NAc DA and glutamate via pre-synaptic mGluR2/3s.

Chronic glutamate toxicity is implicated in the pathogenesis of ALS. The neuropeptide N-acetyl-aspartyl glutamate (NAAG) appears to function both as a storage form for glutamate and as a neuromodulator at glutamatergic synapses. Catabolism of NAAG by N-acetylated-α-linked acidic dipeptidase (NAALADase; also termed glutamate carboxypeptidase II), yields N-acetyl aspartate (NAA) and glutamate. Since prior studies demonstrate an up-regulation of NAALADase in motor cortex and increased levels of NAA and glutamate in the CSF of ALS patients, we hypothesized that inhibition of NAALADase could protect against neuronal degeneration in ALS. Neuroprotective effects of two NAALADase inhibitors were assessed. 2-(Phosphonomethyl)pentanedioic acid (2-PMPA) decreased motor neuron loss and prevented loss of choline acetyltransferase (ChAT) activity in an in vitro model of ALS wherein chronic glutamate toxicity was induced by blocking glutamate transport. Gross morphology was preserved in 2-PMPA-treated cultures. In a SOD-1 transgenic mouse model of ALS, oral administration of a structurally different NAALADase inhibitor (GPI 5693) increased survival by 29 days and delayed onset of clinical symptoms by 17 days. Preliminary analysis of spinal cord pathology revealed severe neuronal depletion and astrocytosis with white matter changes in control mice. In mice treated with GPI 5693, normal neuronal populations with modest vacuolar changes were observed. These data suggest that NAALADase inhibition may provide an exciting therapeutic approach to the devastating disease, ALS.

Membrane-bound glutamate carboxypeptidase II (GCPII) is a zinc metalloenzyme that catalyzes the hydrolysis of the neurotransmitter N-acetyl-L-aspartyl-L-glutamate (NAAG) to N-acetyl-L-aspartate and L-glutamate (which is itself a neurotransmitter). Potent and selective GCPII inhibitors have been shown to decrease brain glutamate and provide neuroprotection in preclinical models of stroke, amyotrophic lateral sclerosis, and neuropathic pain. Here, we report crystal structures of the extracellular part of GCPII in complex with both potent and weak inhibitors and with glutamate, the product of the enzyme's hydrolysis reaction, at 2.0, 2.4, and 2.2 Å resolution, respectively. GCPII folds into three domains: protease-like, apical, and C-terminal. All three participate in substrate binding, with two of them directly involved in C-terminal glutamate recognition. One of the carbohydrate moieties of the enzyme is essential for homodimer formation of GCPII. The three-dimensional structures presented here reveal an induced-fit substrate-binding mode of this key enzyme and provide essential information for the design of GCPII inhibitors useful in the treatment of neuronal diseases and prostate cancer.

Human glutamate carboxypeptidase II (GCPII) is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. In humans, the enzyme is expressed predominantly in the nervous system and the prostate. The prostate form, termed prostate-specific membrane antigen, is overexpressed in prostate cancer and is used as a diagnostic marker of the disease. Inhibition of the form of GCPII expressed in the central nervous system has been shown to protect against ischemic injury in experimental animal models. Human GCPII consists of 750 amino acids, and six individual domains were predicted to constitute the protein structure. Here, we report the analysis of the contribution of these putative domains to the structure/function of recombinant human GCPII. We cloned 13 mutants of human GCPII that are truncated or extended at one or both the N- and C-termini of the GCPII sequence. The clones were used to generate stably transfected Drosophila Schneider's cells, and the expression and carboxypeptidase activities of the individual protein products were determined. The extreme C-terminal region of human GCPII was found to be critical for the hydrolytic activity of the enzyme. The deletion of as few as 15 amino acids from the C-terminus was shown to completely abolish the enzymatic activity of GCPII. Furthermore, the GCPII carboxypeptidase activity was abrogated upon removal of more than 60 amino acid residues from the N-terminus of the protein. Overall, these results clearly show that amino acid segments at the N- and C-termini of the ectodomain of GCPII are essential for its carboxypeptidase activity and/or proper folding.

The sodium-vitamin C co-transporter SVCT2 is primarily responsible for the accumulation of the important antioxidant ascorbate into brain cells. In vitro studies have demonstrated strong expression of this transporter in cultured astrocytes, whereas in situ hybridization analysis has so far detected SVCT2 only in neurons. In the present study, we examined the response of SVCT2 mRNA expression in the brain to focal ischemia induced for 2 h by unilateral middle cerebral artery occlusion. The mRNA expression patterns of SVCT2 and the glutamate-activated immediate early gene Arc were investigated at 2 and 22 h after ischemia. SVCT2 and Arc mRNA expression was lost in the ischemic core at both time points. In areas outside the core, Arc was strongly up-regulated, primarily at 2 h, whereas SVCT2 showed an increase at 2 and 22 h. SVCT2 expression was increased in neurons as well as in astrocytes, providing the first evidence for SVCT2 expression in astrocytes in situ. These findings underscore the importance of ascorbate as a neuroprotective agent and may have implications for therapeutic strategies. In addition, the increase of SVCT2 in astrocytes after ischemia suggests that cultured astrocytes are exposed to chronic oxidative stress.

In this study we examined the effect of 2-(phosphonomethyl)pentanedioic acid (2-PMPA) and GPI 5693, selective inhibitors of the enzyme N-Acetylated-α-Linked-Acidic Dipeptidase (NAALADase; glutamate carboxypeptidase II; EC no. 3.4.17.21), which cleaves glutamate from the dipeptide N-acetyl-aspartyl-glutamate (NAAG), on the conditioned place preference (CPP) response to cocaine in male rats. The i.p. administration of 15 mg/kg of cocaine produced a significant CPP response. The acquisition and expression of the CPP response to cocaine was blocked by the i.p. administration of 100 mg/kg of 2-PMPA and the p.o. administration of 30 mg/kg of GPI 5693. In contrast, neither 2-PMPA nor GPI 5693 produced a CPP or conditioned place aversion response when administered alone. Furthermore, neither 2-PMPA or GPI 5693 altered the expression of the CPP response to food. These results indicate that NAALADase inhibitors block the incentive motivational value of cocaine, suggesting that such agents may be of use in treating cue-induced craving in cocaine addicts. Synapse 41:22–28, 2001. © 2001 Wiley-Liss, Inc.

BACKGROUND Prostate-specific membrane antigen (PSMA) is a glutamate carboxypeptidase that cleaves terminal carboxy glutamates from both the neuronal dipeptide N-acetylaspartylglutamate (NAAG) and gamma-linked folate polyglutamate. The prostate enzyme has activity in both the membrane and cytosolic fractions termed PSMA and PSMA′, respectively. METHODS Using a NAAG hydrolytic radioenzymatic assay, we quantitated the enzymatic activity of PSMA and PSMA′ in normal, benign prostatic hyperplasia (BPH), and prostate cancer (PC) tissues from radical prostatectomies. PSMA enzyme activity was evaluated in each tissue type and expressed per milligram protein and epithelial cell content. RESULTS PSMA and PSMA′ enzyme activities were significantly elevated in prostate cancer when compared to normal prostate tissue and BPH. Ratios of PSMA to PSMA′ were also decreased in BPH as compared to cancerous and normal tissue. CONCLUSIONS Prostate carcinogenesis is associated with an elevation in PSMA and PSMA′ enzyme activity. In contrast, no such enhancement in PSMA activity is observed with benign neoplastic changes in BPH. Thus, the enhancement observed in prostate cancer is not simply related to a generalized prostatic hyperplasia, but is specific to its malignancy. Prostate 45:350–354, 2000. © 2000 Wiley-Liss, Inc.

Sensitization to cocaine has been attributed to alterations in excitatory amino acid and dopamine neurotransmission in the mesolimbic system. The present study sought to determine whether inhibition of NAALADase, an enzyme that cleaves glutamate from the endogenous neuropeptide, N-acetyl-aspartyl-glutamate (NAAG), attenuates sensitization to the psychomotor stimulant effects of cocaine. Rats received daily injections of cocaine (20.0 mg/kg/day; i.p.) or saline for 5 days. Fifteen minutes prior to these injections they received an i.p. injection of the NAALADase inhibitor, 2-PMPA (50.0-100 mg/kg), or vehicle. Locomotor activity and stereotypy produced by a challenge dose of cocaine (15.0 mg/kg) were assessed 3 days later. Acute cocaine administration increased locomotor activity in control animals. In animals with a prior history of cocaine administration, the behavioral response to cocaine was significantly enhanced. In animals that had received 2-PMPA in combination with cocaine, the enhancement of cocaine-induced locomotor activity was attenuated. No alteration in cocaine-evoked activity was observed in animals that had received once daily injections of 2-PMPA, alone. Acute administration of 2-PMPA also did not modify saline-induced locomotor activity or activity produced by an acute cocaine challenge. These data demonstrate that NAALADase inhibition attenuates the development of sensitization to the locomotor-activating effects of cocaine. Furthermore, this action cannot be attributed to an antagonism of the acute effects of cocaine.

BACKGROUND The polysulfonated napthlyurea suramin has shown significant antitumor activity in patients with hormone-refractory metastatic prostate cancer. The mechanism by which suramin exerts this effect is unknown. In 1993, prostate-specific membrane antigen (PSM) was identified as a prostate biomarker that is elevated in hormone-refractory and metastatic prostate cancer. PSM is a glutamate exocarboxypeptidase capable of cleaving the terminal alpha-linked glutamate from the dipeptide N-acetyl-aspartyl-glutamate (NAAG) and the gamma-linked glutamates from folate polyglutamate. METHODS Using a NAAG hydrolytic radioenzymatic assay, we tested whether suramin had any effect on the enzymatic activity of PSM. RESULTS We demonstrate that suramin potently inhibits the enzymatic activity of PSM with a K(i) = 15 nM and 68 nM for the membrane-associated and soluble forms of PSM, respectively. In addition, we show that suramin inhibition of PSM enzyme activity displays the kinetics of a classic competitive inhibitor. CONCLUSIONS This is one of the most potent activities described for suramin to date and may represent a portion of its pharmacologic and/or toxicological mechanism of action.

BACKGROUND The prostate cancer marker prostate-specific membrane antigen (PSM) is highly homologous to the brain enzyme N-acetylated alpha-linked acidic dipeptidase (NAALADase). NAALADase is known to cleave terminal carboxy glutamates from both the neuronal peptide N-acetylaspartylglutamate (NAAG) and folate polyglutamate. In this report, we compare the NAAG hydrolyzing activity of NAALADase and the prostate enzyme PSM. METHODS Using a NAAG hydrolytic radioenzymatic assay, we compared the pharmacological and kinetic properties of the brain and prostate enzymes. RESULTS Eight normal prostate tissues from different species exhibited NAAG hydrolyzing activity. Among 14 cancer cell lines examined, activity was observed in human LNCaP, PC-82, and rat Dunning G and AT-1 cells. Brain exhibited membrane-localized activity exclusively, while the prostate enzyme had activity in both membrane and cytosolic fractions. The only observed pharmacological difference was the sensitivity to their putative substrates, folate polyglutamate and NAAG. Kinetically, the soluble form of the prostate enzyme had two catalytic sites, while the membrane-bound form exhibited single site kinetics with a lower Vmax than the brain enzyme, which may suggest a less active hydrolase in the prostate. CONCLUSIONS The brain enzyme NAALADase and the prostate enzyme PSM are remarkably similar. The importance of the differences in substrate specificities and kinetic parameters remains to be elucidated. Prostate 39:28–35, 1999. © 1999 Wiley-Liss, Inc.

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as N-acetylated-α-linked-acidic dipeptidase, NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. We report the mapping of the entire coding region of GCPII and identification of the region sufficient and necessary for the production of active recombinant protein. Extracellular portion of human glutamate carboxypeptidase II (amino acids 44–750) was expressed in Drosophila Schneider's cells and purified to homogeneity. A novel assay for hydrolytic activity of GCPII, based on fluorimetric detection of released alpha-amino groups was established, and used for enzymological characterization of GCPII. The potential of this assay for high-throughput inhibitor testing was evaluated and pH dependence for the enzymatic activity have been analysed. Using a complete set of protected dipeptides, substrate specificity of recombinant GCPII was elucidated. Ac-Glu-Met, Ac-Asp-Met and surprisingly Ac-Ala-Met were identified as novel substrates for GCPII. The glycosylation has been found indispensable for the activity of the enzyme. A series of point mutants of the enzyme has been expressed and purified and the glycosylation sites critical for the proteolytic activity have been identified.

N-acetylated alpha-linked acidic dipeptidase (NAALADase) is a membrane bound enzyme that cleaves glutamate from the endogenous neuropeptide N-acetyl-aspartyl-glutamate (NAAG). We report the immunocytochemical localization of NAALADase in rat brain and kidney by using specific anti-NAALADase antiserum. NAALADase-immunoreactivity (NAALADase-IR) was widely distributed, abundant in neuropil, absent from neuronal cytoplasm, and displayed regional heterogeneity. Staining was selectively enriched in several structures previously reported to contain NAAG-immunoreactivity (NAAG-IR) including the amygdala, caudate-putamen, central gray, dorsal raphe, globus pallidus, hippocampus, hypothalamus, locus coerulus, medial and lateral geniculate, olfactory bulb, periaqueductal gray, solitary nucleus, spinal trigeminal nucleus, substantia nigra, superior colliculus, and thalamus. Staining within these structures was enriched in neuropil; no intracellular staining was detected, even after colchicine treatment. In addition, NAALADase-IR was observed in some NAAG-containing fiber tracts including the corpus callosum, fornix, habenular commissure, solitary tract, stria medularis, and stria terminalis. The co-localization of NAALADase-IR and NAAG-IR support the hypothesis that NAALADase is responsible for the catabolism of NAAG in vivo. NAALADase-IR was also detected in brain regions that, to date, have not revealed NAAG-IR, including the bed nucleus of the stria terminalis and the median eminence. In addition, NAALADase-IR was detected in the rat kidney cortex, specifically in the brush border of the proximal convoluted tubules. The observation that NAALADase-IR was more widespread than NAAG-IR suggests that NAALADase may also be involved in the catabolism of other structurally related neural and renal peptides.

The peptide N-Acetylaspartylglutamate (NAAG) is hydrolyzed by N-Acetylated-alpha-linked-acidic dipeptidase (NAALADase, glutamate carboxypeptidase II) into N-Acetylated aspartate (NAA) and glutamate. Hydrolysis can be measured as described in this unit by employing radiolabeled NAAG (NAA-[(3)H]glu) as the substrate. The occurrence of NAALADase activity in a wide range of tissues has implications for a variety of physiological purposes. The assay described here is useful for the analysis of NAALADase activity and its inhibition in brain synaptosomal preparations, tissue homogenates and tissue culture cell pellets.

Glutamate carboxypeptidase II (GCPII, also known as N-acetylated-alpha-linked acidic dipeptidase or NAALADase) knockout (KO) mice were generated by inserting a GCPII targeting cassette containing a PGK-Neo resistance marker and stop codons in exons 1 and 2, and removal of exons 1 and 2 intron/exon boundary sequence. Embryonic stem cells were injected into C57BL6 blastocysts, and chimeric offspring born. Germline transmission was confirmed by mating the chimeras to generate heterozygous KO mice. Crossing heterozygous mice generated F2 generation mice homozygous for the null mutant, as confirmed by loss of GCPII protein. NAAG hydrolyzing activity was minimal (0.07 pmol/mg/min) in KO tissue, with normal levels (4.82 pmol/mg/min) in wild types and intermediate levels (1.73 pmol/mg/min) in heterozygotes. Preliminary neuropathy experiments showed KO mice are less affected by nerve-crush and recover faster from the damage-induced neuropathy, as indicated by EMG recording and nerve morphology. Similarly, GCPII KO mice subjected to high dose vitamin B6 displayed less severe neuropathy than wild types, as indicated by reduced sensory nerve conduction velocity and morphological deficits. Also, in a transient middle cerebral artery occlusion model, GCPII KO mice were significantly more resistant to the effects of cerebral ischemia than their wildtype littermates. Findings support GCPII involvement in stroke and in mediating chronic neuropathic conditions and suggest GCPII inhibitors may be useful in treatment of brain ischemia as well as peripheral neuropathies.

NAALADase (N-acetylated-α-linked-acidic dipeptidase; also termed glutamate carboxypeptidase II) is an enzyme that catalyzes hydrolysis of the abundant brain peptide N-acetyl-aspartyl-glutamate (NAAG) to N acetyl-aspartate (NAA) and glutamate. We have synthesized many potent and selective NAALADase inhibitors from distinct chemical classes. In rodents, selective inhibitors have been shown to increase NAAG and decrease glutamate extracellularly and provide therapeutic benefits in animal models of neurological diseases where excess glutamate neurotransmission has been implicated. Specifically, NAALADase inhibitors exhibit neuroprotection in rats following transient middle cerebral artery occlusion, attenuate neuropathic pain following chronic constrictive injury, prevent nerve conduction velocity deficits and sural nerve degeneration following chronic diabetes in STZ and BB/W rats, and delay clinical symptoms and mortality in the mouse SOD transgenic model of ALS. These biological effects are likely mediated by increases in extracellular NAAG and decreases in extracellular glutamate as a consequence of NAALADase inhibition. NAALADase inhibition may be useful in neurological disorders in which excessive excitatory amino acid transmission is pathogenic. Also, NAALADase inhibition may represent a novel glutamate regulating strategy devoid of the side-effects that have hampered development of postsynaptic glutamate receptor antagonists. A lead NAALADase inhibitor termed GPI 5693 is currently completing Phase I clinical testing.