N-Acetyl-Aspartyl-Glutamate in Brain Health and Disease.

N-acetyl-aspartyl-glutamate (NAAG) is the most abundant dipeptide in the brain, where it acts as a neuromodulator of glutamatergic synapses by activating presynaptic metabotropic glutamate receptor 3 (mGluR3). Recent data suggest that NAAG is selectively localized to postsynaptic dendrites in glutamatergic synapses and that it works as a retrograde neurotransmitter. NAAG is released in response to glutamate and provides the postsynaptic neuron with a feedback mechanisms to inhibit excessive glutamate signaling. A key regulator of synaptically available NAAG is rapid degradation by the extracellular enzyme glutamate carboxypeptidase II (GCPII). Increasing endogenous NAAG-for instance by inhibiting GCPII-is a promising treatment option for many brain disorders where glutamatergic excitotoxicity plays a role. The main effect of NAAG occurs through increased mGluR3 activation and thereby reduced glutamate release. In the present review, we summarize the transmitter role of NAAG and discuss the involvement of NAAG in normal brain physiology. We further present the suggested roles of NAAG in various neurological and psychiatric diseases and discuss the therapeutic potential of strategies aiming to enhance NAAG levels.

Phe-Gly motifs drive fibrillization of TDP-43's prion-like domain condensates.

Transactive response DNA-binding Protein of 43 kDa (TDP-43) assembles various aggregate forms, including biomolecular condensates or functional and pathological amyloids, with roles in disparate scenarios (e.g., muscle regeneration versus neurodegeneration). The link between condensates and fibrils remains unclear, just as the factors controlling conformational transitions within these aggregate species: Salt- or RNA-induced droplets may evolve into fibrils or remain in the droplet form, suggesting distinct end point species of different aggregation pathways. Using microscopy and NMR methods, we unexpectedly observed in vitro droplet formation in the absence of salts or RNAs and provided visual evidence for fibrillization at the droplet surface/solvent interface but not the droplet interior. Our NMR analyses unambiguously uncovered a distinct amyloid conformation in which Phe-Gly motifs are key elements of the reconstituted fibril form, suggesting a pivotal role for these residues in creating the fibril core. This contrasts the minor participation of Phe-Gly motifs in initiation of the droplet form. Our results point to an intrinsic (i.e., non-induced) aggregation pathway that may exist over a broad range of conditions and illustrate structural features that distinguishes between aggregate forms.

Growth promotion in Arabidopsis thaliana by bacterial cyclodipeptides involves the TOR/S6K pathway activation.

Cyclodipeptides (CDPs) are the smallest peptidic molecules that can be produced by diverse organisms such as bacteria, fungi, and animals. They have multiple biological effects. In this paper, we examined the CDPs produced by the bacteria Pseudomonas aeruginosa PAO1, which are known as opportunistic pathogens of humans and plants on TARGET OF RAPAMYCIN (TOR) signaling pathways, and regulation of root system architecture. This bacterium produces the bioactive CDPs: cyclo(L-Pro-L-Leu), cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Pro-L-Val). In a previous report, these molecules were found to modulate basic cellular programs not only via auxin mechanisms but also by promoting the phosphorylation of the S6 ribosomal protein kinase (S6K), a downstream substrate of the TOR kinase. In the present work, we found that the inoculation of Arabidopsis plants with P. aeruginosa PAO1, the non-pathogenic P. aeruginosa ΔlasI/Δrhll strain (JM2), or by direct exposure of plants to CDPs influenced growth and promoted root branching depending upon the treatment imposed, while genetic evidence using Arabidopsis lines with enhanced or decreased TOR levels indicated a critical role of this pathway in the bacterial phytostimulation.

Analysis of a bac operon-silenced strain suggests pleiotropic effects of bacilysin in Bacillus subtilis.

Bacilysin, as the simplest peptide antibiotic made up of only L-alanine and L-anticapsin, is produced and excreted by Bacillus subtilis under the control of quorum sensing. We analyzed bacilysin-nonproducing strain OGU1 which was obtained by bacA-targeted pMutin T3 insertion into the parental strain genome resulting in a genomic organization (bacA'::lacZ::erm::bacABCDEF) to form an IPTG-inducible bac operon. Although IPTG induction provided 3- to 5-fold increment in the transcription of bac operon genes, no bacilysin activity was detectable in bioassays and inability of the OGU1 to form bacilysin was confirmed by UPLC-mass spectrometry analysis. Phenotypic analyses revealed the deficiencies in OGU1 with respect to colony pigmentation, spore coat proteins, spore resistance and germination, which could be rescued by external addition of bacilysin concentrate into its cultures. 2DE MALDI-TOF/MS and nanoLC-MS/MS were used as complementary approaches to compare cytosolic proteomes of OGU1. 2-DE identified 159 differentially expressed proteins corresponding to 121 distinct ORFs. In nanoLC-MS/MS, 76 proteins were differentially expressed in OGU1. Quantitative transcript analyses of selected genes validated the proteomic findings. Overall, the results pointed to the impact of bacilysin on expression of certain proteins of sporulation and morphogenesis; the members of mother cell compartment-specific σE and σK regulons in particular, quorum sensing and two component-global regulatory systems, peptide transport, stress response as well as CodY- and ScoC-regulated proteins.

Potential anti-arthritic and anti-inflammatory effects of TNF-α processing inhibitor-1 (TAPI-1): A new approach to the treatment of S. aureus arthritis.

Treatment of septic arthritis has become more challenging due to the rise of multidrug resistant strains of Staphylococcus aureus (S. aureus) in recent years. Failure of antibiotic therapies has compelled to initiate the search for new alternatives. This study aimed to unveil the potential anti-arthritic effects of TAPI-1 (TNF-α processing inhibitor-1), an inhibitor that inhibits TACE (TNF-α converting enzyme) mediated release of soluble TNF-α and its receptors along with attenuation of other inflammatory and joint destructive factors responsible for the progression of arthritis. Male Swiss albino mice were inoculated with live S. aureus (5 × 106 cells/mouse) for the development of septic arthritis. TAPI-1 was administered intraperitoneally (10 mg/kg body weight) post S. aureus infection at regular intervals. Throughout the experiment, the severity of arthritis was obtained to be significantly low after TAPI-1 administration. Arthritis index and histopathology confirmed effectiveness of TAPI-1 in mitigating inflammation induced paw swelling and less bone-cartilage destruction in the arthritic knee joints. Lower levels of soluble tumor necrosis factor alpha (sTNF-α) and soluble tumor necrosis factor alpha receptor-1 (sTNFR-1) were detected in the TAPI-1 treated group suggesting TAPI-1 mediated blocking of TACE with subsequent inhibition of TNF-α signalling. Treatment with TAPI-1 lowered the levels of reactive species; matrix metalloproteinase-2 (MMP-2), receptor activator of nuclear factor kappa-B ligand (RANKL) and osteopontin (OPN) denoting less matrix degradation and less osteoclastic bone resorption. Together, this experimental work authenticates TAPI-1 as an alternative therapeutic intervention for the treatment of S. aureus arthritis.

pH effects on plant calcium fluxes: lessons from acidification-mediated calcium elevation induced by the γ-glutamyl-leucine dipeptide identified from Phytophthora infestans.

Cytosolic Ca2+ ([Ca2+]cyt) elevation is an early signaling response upon exposure to pathogen-derived molecules (so-called microbe-associated molecular patterns, MAMPs) and has been successfully used as a quantitative read-out in genetic screens to identify MAMP receptors or their associated components. Here, we isolated and identified by mass spectrometry the dipeptide γ-Glu-Leu as a component of a Phytophthora infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with synthetic γ-Glu-Leu revealed stimulatory effects on defense signaling, including a weak enhancement of the expression of some MAMP-inducible genes or affecting the refractory period to a second MAMP elicitation. However, γ-Glu-Leu is not a classical MAMP since pH adjustment abolished these activities and importantly, the observed effects of γ-Glu-Leu could be recapitulated by mimicking extracellular acidification. Thus, although γ-Glu-Leu can act as a direct agonist of calcium sensing receptors in animal systems, the Ca2+-mobilizing activity in plants reported here is due to acidification. Low pH also shapes the Ca2+ signature of well-studied MAMPs (e.g. flg22) or excitatory amino acids such as glutamate. Overall, this work serves as a cautionary reminder that in defense signaling studies where Ca2+ flux measurements are concerned, it is important to monitor and consider the effects of pH.

A role for N-acetylaspartylglutamate (NAAG) and mGluR3 in cognition.

The peptide transmitter N-acetylaspartylglutamate (NAAG) and its receptor, the type 3 metabotropic glutamate receptor (mGluR3, GRM3), are prevalent and widely distributed in the mammalian nervous system. Drugs that inhibit the inactivation of synaptically released NAAG have procognitive activity in object recognition and other behavioral models. These inhibitors also reverse cognitive deficits in animal models of clinical disorders. Antagonists of mGluR3 block these actions and mice that are null mutant for this receptor are insensitive to the actions of these procognitive drugs. A positive allosteric modulator of this receptor also has procognitive activity. While some data suggest that drugs acting on mGluR3 achieve their procognitive action by increasing arousal during acquisition training, exploration of the procognitive efficacy of NAAG is in its early stages and thus substantial opportunities exist to define the breadth and nature of this activity.

Plasticity of gastrointestinal vagal afferent satiety signals.

The vagal link between the gastrointestinal tract and the central nervous system (CNS) has numerous vital functions for maintaining homeostasis. The regulation of energy balance is one which is attracting more and more attention due to the potential for exploiting peripheral hormonal targets as treatments for conditions such as obesity. While physiologically, this system is well tuned and demonstrated to be effective in the regulation of both local function and promoting/terminating food intake the neural connection represents a susceptible pathway for disruption in various disease states. Numerous studies have revealed that obesity in particularly is associated with an array of modifications in vagal afferent function from changes in expression of signaling molecules to altered activation mechanics. In general, these changes in vagal afferent function in obesity further promote food intake instead of the more desirable reduction in food intake. It is essential to gain a comprehensive understanding of the mechanisms responsible for these detrimental effects before we can establish more effective pharmacotherapies or lifestyle strategies for the treatment of obesity and the maintenance of weight loss.

[Calpain mediated pulmonary vascular remodeling in hypoxia induced pulmonary hypertension].

OBJECTIVE: To explore the role of calpain in pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension and the underlying mechanisms.
 METHODS: Sprague-Dawley rats were randomly divided into the hypoxia group and the normoxia control group. Right ventricular systolic pressure (RVSP) and mean pulmonary artery pressure (mPAP) were monitored by a method with right external jugular vein cannula. Right ventricular hypertrophy index was presented as the ratio of right ventricular weight to left ventricular weight (left ventricle plus septum weight). Levels of calpain-1, -2 and -4 mRNA in pulmonary artery were determined by real-time PCR. Levels of calpain-1, -2 and -4 protein were determined by Western blot. Primary rat pulmonary arterial smooth muscle cells (PASMCs) were divided into 4 groups: a normoxia control group, a normoxia+MDL28170 group, a hypoxia group and a hypoxia+MDL28170 group. Cell proliferation was detected by MTS and flow cytometry. Levels of Ki-67 and proliferating cell nuclear antigen (PCNA) mRNA were determined by real-time PCR.
 RESULTS: RVSP, mPAP and right ventricular remodeling index were significantly elevated in the hypoxia group compared to those in the normoxia group. In the hypoxia group, pulmonary vascular remodeling was significantly developed, accompanied by up-regulation of calpain-1, -2 and -4. MDL28170 significantly inhibited hypoxia-induced proliferation of PASMCs concomitant with the suppression of Ki-67 and PCNA mRNA expression.
 CONCLUSION: Calpain mediates vascular remodeling via promoting proliferation of PASMCs in hypoxia-induced pulmonary hypertension.

Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells.

Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicate that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.

Cyclic dipeptides: from bugs to brain.

Cyclic dipeptides (CDPs) are a group of hormone-like molecules that are evolutionarily conserved from bacteria to humans. In bacteria, CDPs are used in quorum sensing (QS) to communicate information about population size and to regulate a behavioural switch from symbiosis with their host to virulence. In mammals, CDPs have been shown to act on glial cells (macrophage-like cells) to control a conceptually homologous behavioural switch between homeostatic and inflammatory modes, with implications for the control of neurodegenerative disease. Here we argue that, because of their capacity to regulate inflammation via glial cells and induce a protective response in neuronal cells, CDPs have potential therapeutic utility in an array of inflammatory diseases.

Calpains are downstream effectors of bax-dependent excitotoxic apoptosis.

Excitotoxicity resulting from excessive Ca(2+) influx through glutamate receptors contributes to neuronal injury after stroke, trauma, and seizures. Increased cytosolic Ca(2+) levels activate a family of calcium-dependent proteases with papain-like activity, the calpains. Here we investigated the role of calpain activation during NMDA-induced excitotoxic injury in embryonic (E16-E18) murine cortical neurons that (1) underwent excitotoxic necrosis, characterized by immediate deregulation of Ca(2+) homeostasis, a persistent depolarization of mitochondrial membrane potential (Δψ(m)), and insensitivity to bax-gene deletion, (2) underwent excitotoxic apoptosis, characterized by recovery of NMDA-induced cytosolic Ca(2+) increases, sensitivity to bax gene deletion, and delayed Δψ(m) depolarization and Ca(2+) deregulation, or (3) that were tolerant to excitotoxic injury. Interestingly, treatment with the calpain inhibitor calpeptin, overexpression of the endogenous calpain inhibitor calpastatin, or gene silencing of calpain protected neurons against excitotoxic apoptosis but did not influence excitotoxic necrosis. Calpeptin failed to exert a protective effect in bax-deficient neurons but protected bid-deficient neurons similarly to wild-type cells. To identify when calpains became activated during excitotoxic apoptosis, we monitored calpain activation dynamics by time-lapse fluorescence microscopy using a calpain-sensitive Förster resonance energy transfer probe. We observed a delayed calpain activation that occurred downstream of mitochondrial engagement and directly preceded neuronal death. In contrast, we could not detect significant calpain activity during excitotoxic necrosis or in neurons that were tolerant to excitotoxic injury. Oxygen/glucose deprivation-induced injury in organotypic hippocampal slice cultures confirmed that calpains were specifically activated during bax-dependent apoptosis and in this setting function as downstream cell-death executioners.

[Characteristics of the pineal gland and thymus relationship in aging].

The review presents the interference between thymus and pineal gland during their involution. The research data of thymus peptides influence on pineal gland and pineal peptides on thymus are summarized. Analysis of these data showed that pineal peptides (Epithalamin, Epitalon) had more effective geroprotective effect on thymus involution in comparison with geroprotective effect of thymic peptides (Thymalin, Thymogen) on involution of pineal gland. The key mechanisms of pineal peptides effect on thymus dystrophy is immunoendocrine cooperation, which is realized as transcription's activation of various proteins.

Advances in understanding the peptide neurotransmitter NAAG and appearance of a new member of the NAAG neuropeptide family.

A substantial body of data was reported between 1984 and 2000 demonstrating that the neuropeptide N-acetylaspartylglutamate (NAAG) not only functions as a neurotransmitter but also is the third most prevalent transmitter in the mammalian nervous system behind glutamate and GABA. By 2005, this conclusion was validated further through a series of studies in vivo and in vitro. The primary enzyme responsible for the inactivation of NAAG following its synaptic release had been cloned, characterized and knocked out. Potent inhibitors of this enzyme were developed and their efficacy has been extensively studied in a series of animal models of clinical conditions, including stroke, peripheral neuropathy, traumatic brain injury, inflammatory and neuropathic pain, cocaine addiction, and schizophrenia. Considerable progress also has been made in defining further the mechanism of action of these peptidase inhibitors in elevating synaptic levels of NAAG with the consequent inhibition of transmitter release via the activation of pre-synaptic metabotropic glutamate receptor 3 by this peptide. Very recent discoveries include identification of two different nervous system enzymes that mediate the synthesis of NAAG from N-acetylaspartate and glutamate and the finding that one of these enzymes also mediates the synthesis of a second member of the NAAG family of neuropeptides, N-acetylaspartylglutamylglutamate.

Dilysine retrieval signal-containing p24 proteins collaborate in inhibiting γ-cleavage of amyloid precursor protein.

γ-Secretase mediates intramembranous γ-cleavage and ε-cleavage of ß-amyloid precursor protein (APP) to liberate ß-amyloid peptide (Aß) and APP intracellular domain respectively from the membrane. Although the regulatory mechanism of γ-secretase cleavage remains unresolved, a member of the p24 cargo protein family, named p24δ(1) or TMP21, has been identified as an activity-modulating component. The p24 family proteins are divided into four subfamilies (p24α, ß, δ and γ). In contrast to p24δ(1), p24ß(1) has reportedly no effect on γ-cleavage. In this study, we determined whether p24α(2), p24γ(3) or p24γ(4) modulates APP processing. Knockdown of cellular p24α(2) induced a significant increase in Aß generation but not in APP intracellular domain production in cell-based and cell-free assays, whereas p24α(2) over-expression suppressed Aß secretion. By contrast, Aß secretion was not altered by p24γ(3) or p24γ(4) knockdown. Endogenous p24α(2) co-immunoprecipitated with core components of the γ-secretase complex, and the anti-p24α(2) immunoprecipitate exhibited γ-secretase activity. Mutational disruption of the conserved dilysine ER-retrieval motifs of p24α(2) and p24δ(1) perturbed inhibition of γ-cleavage. Simultaneous knockdown, or co-over-expression, of these proteins had no additive or synergistic effect on Aß generation. Our findings suggest that dilysine ER-retrieval signal-containing p24 proteins, p24α(2) and p24δ(1), bind with γ-secretase complexes and collaborate in attenuating γ-cleavage of APP.

[Gastrointestinal hormones in food intake control]. / Las hormonas gastrointestinales en el control de la ingesta de alimentos.

The discovery of gut hormones regulating the energy balance has aroused great interest in the scientific community. Some of these hormones modulate appetite and satiety, acting on the hypothalamus or the solitary tract nucleus in the brainstem. In general, the endocrine signals generated in the gut have direct or indirect (through the autonomous nervous system) anorexigenic effects. Only ghrelin, a gastric hormone, has been consistently associated with the initiation of food intake and is regarded as the main orexigenic signal both in animal models and humans. In this review, we provide a brief description of the major gastrointestinal hormones implicated in the regulation of food intake. Given the increased importance of food intake disturbances, especially obesity, a better understanding of the underlying mechanisms of action of the gastrointestinal hormones might contribute to the development of new molecules that could increase the therapeutic arsenal for treating obesity and its associated comorbidities.

Nonproteolytic induction of catalytic activity into the single-chain form of urokinase-type plasminogen activator by dipeptides.

Serine proteases are initially synthesized as single-chain proenzymes with activities that are many orders of magnitude lower than those of the mature enzyme. Proteolytic cleavage of an exposed loop liberates a new amino terminus that inserts into a hydrophobic pocket and forms a stabilizing salt bridge with a ubiquitously conserved aspartate residue, resulting in a conformational change organizing the mature oxyanion hole. In a decisive 1976 work, Huber and Bode [Bode, W., and Huber, R. (1976) FEBS Lett. 68, 231-236] demonstrated that peptides sequentially similar to the new amino terminus in combination with a catalytic site inhibitor could specifically induce a trypsin-like conformation in trypsinogen. We now demonstrate that an Ile-Ile or Ile-Val dipeptide can induce limited enzyme activity in the single-chain zymogen form of urokinase-type plasminogen activator (uPA) or its K158A variant, which cannot be activated proteolytically. Furthermore, the slow formation of a covalent serpin-protease complex between single-chain uPA and PAI-1 is significantly accelerated in the presence of specific dipeptide sequences. The technique of using a dipeptide mimic as a surrogate for the liberated amino terminus further provides a novel means by which to covalently label the immature active site of single-chain uPA with a fluorescent probe, permitting fluorescence approaches for direct observations of conformational changes within the protease domain during zymogen activation. These data demonstrate the structural plasticity of the protease domain, reinforce the notion of "molecular sexuality", and provide a novel way of studying conformational changes of zymogens during proteolytic activation.

[Possible relation of BDNF and GDNF to neuropsychiatric disorders].

Structural abnormalities are demonstrated in various neuropsychiatric disorders, including Alzheimer's disease, Parkinson's disease, and even major depression. On the other hand, recent studies have demonstrated the structural and functional modifications in the adult brain that are associated with synaptic plasticity and neurogenesis. Accordingly, regulation of synaptic plasticity and neurogenesis may lead to the development of novel treatments for neuropsychiatric disorders. Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) have important roles not only in neuronal survival and differentiation, but also in the formation and maintenance of neural circuits and synapse plasticity. Accumulating evidence suggests that these neurotrophic factors may be applied to the treatment of neuropsychiatric disorders. In addition, compounds that increase the expression of BDNF and/or GDNF in the brain should have potential therapeutic values. We have demonstrated that systemic administration of dipeptide Leu-Ile increases BDNF and GDNF production in the brain, and has a protective role in methamphetamine and morphine dependence. In this review, we discuss the potential role of BDNF, GDNF and their inducers in the treatment for neuropsychiatric disorders.

Effects of N-acetylaspartylglutamate (NAAG) at group II mGluRs and NMDAR.

A group II metabotropic glutamate receptor (mGluR) agonist was recently reported to be clinically efficacious against symptoms of schizophrenia [Patil, S.T., Zhang, L., Martenyi, F., Lowe, S.L., Jackson, K.A., Andreev, B.V., Avedisova, A.S., Bardenstein, L.M., Gurovich, I.Y., Morozova, M.A., Mosolov, S.N., Neznanov, N.G., Reznik, A.M., Smulevich, A.B., Tochilov, V.A., Johnson, B.G., Monn, J.A., Schoepp, D.D., 2007. Activation of mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized phase 2 clinical trial. Nature Med 13, 1102-1107]. The endogenous neuropeptide N-acetylaspartylglutamate (NAAG) has been described as an agonist at mGluR2 and mGluR3 [Wroblewska, B., Wroblewski, J.T., Pshenichkin, S., Surin, A., Sullivan, S.E., Neale, J.H., 1997. N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells. J. Neurochem. 69, 174-181; Cartmell, J., Adam, G., Chaboz, S., Henningsen, R., Kemp, J.A., Klingelschmidt, A., Metzler, V., Monsma, F., Schaffhauser, H., Wichmann, J., Mutel, V., 1998. Characterization of [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes. Br. J. Pharmacol. 123, 497-504] and is degraded by the enzyme glutamate carboxypeptidase II (also known as N-acetyl-alpha-linked acidic dipeptidase or NAALADase). Hence, elevating the concentration of endogenous NAAG by inhibition of NAALADase represents a potential strategy for the treatment of schizophrenia via group II mGluR activation. We therefore investigated the activity of NAAG at both rat native and human recombinant mGluRs. We found that NAAG had no effect on synaptic transmission at the medial perforant pathway inputs to the rat dentate gyrus which is known to be sensitive to group II mGluR activation. We proceeded to examine the effects of NAAG at human recombinant mGluR2 and mGluR3 in a cellular G protein-activated K+ channel electrophysiology assay. Furthermore, due to discrepancies in the literature concerning the activity of NAAG at the N-methyl-d-aspartate receptor [NMDAR; Westbrook, G.L., Mayer, M.L., Namboodiri, M.A., Neale, J.H., 1986. High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture. J. Neurosci. 6, 3385-3392; Losi, G., Vicini, S., Neale, J., 2004. NAAG fails to antagonize synaptic and extrasynaptic NMDA receptors in cerebellar granule neurons. Neuropharmacology 46, 490-496], we also tested NAAG at NMDARs in rat hippocampal neurons in culture. We found that a purified NAAG preparation had no effect at mGluR2, mGluR3 or NMDAR. Taken together, these findings do not support a rationale for targeting NAALADase and increasing extracellular NAAG levels as a therapeutic strategy for the treatment of schizophrenia.