Introduction.The amino acid glutamate is present in high concentrations in the mammalian brain, and it acts as the major excitatory neurotransmitter in the CNS. Through its actions on both ionotropic and metabotropic receptors, glutamate plays an important role in a variety of physiological functions including learning, memory, and developmental plasticity. Excessive activation of glutamate receptors or disturbances in the cellular mechanisms that protect against the adverse consequences of physiological glutamate receptor activation have been implicated in the pathogenesis of a host of neurological disorders. Although several drugs designed to attenuate the pathological consequences of excessive glutamate activation have been shown to reduce injury in experimental models of cerebral ischemia, so far none of these compounds has proven to be effective in the clinical treatment of stroke. 1 N-Acetyl-L-aspartyl-L-glutamate (NAAG) is a peptide neurotransmitter that is widely distributed in the mammalian nervous system. 2 NAAG is both an agonist at metabotropic glutamate receptors (mGluR3) 3 and a mixed agonist/antagonist at the N-methyl-D-aspartate (NMDA) receptor. 4 NAAG is hydrolyzed by the neuropeptidase glutamate carboxypeptidase II (GCPII; also known as N-acetylated R-linked acidic dipeptidase, NAALADase, or NAAG peptidase) to liberate N-acetylaspartate and glutamate both in vitro and in vivo. 5 The role of this metalloprotease GCPII is thus thought to be twofold: (1) to terminate the neurotransmitter activity of NAAG and (2) to liberate glutamate which is then able to act at the various glutamate receptor subtypes. Alterations in the levels of GCPII and NAAG have been observed in disorders that are linked to abnormalities in glutamatergic neurotransmission. 6 As a consequence of these findings, it has been hypothesized that the inhibition of GCPII might provide an effective strategy for achieving neuroprotection in cases of cerebral ischemia by increasing the levels of

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face of glia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/-mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII null mutant were V max ¼ 45 and 3 pmol/mg/min and K m ¼ 2650 nM and 2494 nM, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4¢-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) 2 inhibited the residual activity. The IC 50 value for 2-PMPA was about 1 nM for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned rat and human GCPII, and 88 nM for the activity in brain membranes of the null mutants. Keywords: N-acetylaspartylglutamate (NAAG), N-acetylaspartylglutamate-peptidase (NAAG-peptidase), glutamate carboxypeptidase II metabotropic glutamate receptor. 3

The peptide neurotransmitter N-acetylaspartylglutamate is inactivated by extracellular peptidase activity following synaptic release. It is speculated that the enzyme, glutamate carboxypeptidase II (GCPII, EC 3.14.17.21), participates in this inactivation. However, CGCPII knockout mice appear normal in standard neurological tests. We report here the cloning and characterization of a mouse enzyme (tentatively identified as glutamate carboxypeptidase III or GCPIII) that is homologous to an enzyme identified in a human lung carcinoma. The mouse peptidase was cloned from two non-overlapping EST clones and mouse brain cDNA using PCR. The sequence (GenBank, AY243507) is 85% identical to the human carcinoma enzyme and 70% homologous to mouse GCPII. GCPIII sequence analysis suggests that it too is a zinc metallopeptidase. Northern blots revealed message in mouse ovary, testes and lung, but not brain. Mouse cortical and cerebellar neurons in culture expressed GCPIII message in contrast to the glial specific expression of GCPII. Message levels of GCPIII were similar in brains obtained from wild-type mice and mice that are null mutants for GCPII. Chinese hamster ovary (CHO) cells transfected with rat GCPII or mouse GCPIII expressed membrane bound peptidase activity with similar V max and K m values (1.4 lM and 54 pmol/min/mg; 3.5 lM and 71 pmol/min/mg, respectively). Both enzymes are activated by a similar profile of metal ions and their activities are blocked by EDTA. GCPIII message was detected in brain and spinal cord by RT-PCR with highest levels in the cerebellum and hippocampus. These data are consistent with the hypothesis that nervous system cells express at least two differentially distributed homologous enzymes with similar pharmacological properties and affinity for NAAG.

Docosahexaenoic acid (DHA) is critical for maintaining normal brain structure and function, and is considered neuroprotective. Its brain concentration depends on dietary DHA content and hepatic conversion from its dietary derived n-3 precursor, α-linolenic acid (α-LNA). We have developed an in vivo method in rats using quantitative autoradiography and intravenously injected radiolabeled DHA to image net incorporation into the brain of unesterified plasma DHA, and showed with this method that the incorporation rate of DHA equals the rate of brain metabolic DHA consumption. The method has been extended for use in humans with positron emission tomography (PET). Furthermore, imaging in unanesthetized rats using DHA incorporation as a biomarker in response to acute N-methyl-D-aspartate administration confirms that regional DHA signaling is independent of extracellular calcium, and likely mediated by a calcium-independent phospholipase A2 (iPLA2). Studies in mice in which iPLA2-VIA (β) was knocked out confirmed that this enzyme is critical for baseline and muscarinic cholinergic signaling involving DHA. Thus, quantitative imaging of DHA incorporation from plasma into brain can be used as an in vivo biomarker of brain DHA metabolism and neurotransmission.

The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective agonist at the type 3 metabotropic glutamate receptor (mGluR3) where it acts to decrease cAMP levels. Rat cortical interneurons express both NAAG and glutamic acid decarboxylase, as well as mGluR3 mRNA. In the presence of ionotropic glutamate receptor antagonists, both NAAG and the group II metabotropic glutamate receptor agonist, DCG-IV, reduced the calcium-dependent, KCl-induced [(3)H]-GABA release from rat cortical neurons by 35%. This release process was unaffected by tetrodotoxin. The group II antagonist, ethyl glutamate, reversed the effects of DCG-IV and NAAG. The mGluR3-selective antagonist, beta-N-acetylaspartylglutamate, reversed the effect of NAAG. While pretreatment of cortical neurons with forskolin alone did not significantly affect KCl-stimulated [(3)H]-GABA-release, forskolin abolished the inhibition of release produced by NAAG. The protein kinase A inhibitor, H-89, decreased [(3)H]-GABA release while NAAG produced no additional inhibition in the presence of H-89. In contrast, the protein kinase C inhibitor, Ro 31--8220, had no effect on KCl-stimulated release, nor did it affect the inhibition of release produced by NAAG. The L-type calcium channel blocker, nifedipine, also inhibited the release of [(3)H]-GABA and coapplication with NAAG resulted in no significant additional inhibition of release. These data support the hypothesis that the inhibition of KCl-stimulated [(3)H]-GABA release by NAAG is mediated via presynaptic mGluR3 on GABAergic cortical neurons and that this effect is obtained by decreasing cAMP with a consequent decrease in protein kinase A activity and L-type calcium channel conductance.

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Human immunodeficiency virus (HIV)-associated infection involves the entry of virus-bearing monocytes into the brain, followed by microglial activation, neuroinflammation, and upregulated arachidonic acid (AA) metabolism. The HIV-1 transgenic (Tg) rat, a noninfectious HIV-1 model, shows neurologic and behavioral abnormalities after 5 months of age. We hypothesized that brain AA metabolism would be elevated in older HIV-1 Tg rats in vivo. Arachidonic acid incorporation from the plasma into the brain of unanesthetized 7-to-9-month-old rats was imaged using quantitative autoradiography, after [1-14 C]AA infusion. Brain phospholipase (PLA 2 ) activities and eicosanoid concentrations were measured, and enzymes were localized by immunostaining. AA incorporation coefficients k* and rates J in , measures of AA metabolism, were significantly higher in 69 of 81 brain regions in HIV-1 Tg than in control rats, as were activities of cytosolic (c)PLA 2 -IV, secretory (s)PLA 2 , and calcium independent (i)PLA 2 -VI, as well as prostaglandin E 2 and leukotriene B 4 concentrations. Immunostaining of somatosensory cortex showed elevated cPLA 2 -IV, sPLA 2 -IIA, and cyclooxygenase-2 in neurons. Brain AA incorporation and other markers of AA metabolism are upregulated in HIV-1 Tg rats, in which neurologic changes and neuroinflammation have been reported. Positron emission tomography with [1-11 C]AA could be used to test whether brain AA metabolism is upregulated in HIV-1-infected patients, in relation to cognitive and behavioral disturbances.

. GABA A receptor ␤3 subunit deletion decreases ␣2/3 subunits and IPSC duration. J Neurophysiol 89: 128 -134, 2003Neurophysiol 89: 128 -134, . 10.1152Neurophysiol 89: 128 -134, /jn.00700.2002. Deletion of the ␤3 subunit of the GABA A receptor produces severe behavioral deficits and epilepsy. GABA A receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in cortical neurons in cultures from ␤3 Ϫ/Ϫ mice were significantly faster than those in ␤3 ϩ/ϩ mice and were more prolonged by zolpidem. Surface staining revealed that the number of ␤2/3, ␣2, and ␣3 (but not of ␣1) subunit-expressing neurons and the intensity of subunit clusters were significantly reduced in ␤3 Ϫ/Ϫ mice. Transfection of ␤3 Ϫ/Ϫ neurons with ␤3 cDNA restored ␤2/3, ␣2, and ␣3 subunits immunostaining and slowed mIPSCs decay. We show that the deletion of the ␤3 subunit causes the loss of a subset of GABA A receptors with ␣2 and ␣3 subunits while leaving a receptor population containing predominantly ␣1 subunit with fast spontaneous IPSC decay and increased zolpidem sensitivity.