Rationale: Glutamate Carboxypeptidase II (GCPII; EC 3.4.17.21) was identified as an enzyme that catalyzes in brain the hydrolysis of N-acetyl- aspartyl-glutamate (NAAG) to acetyl-aspartate (NAA) and glutamate with 200 nM Km for NAAG. Given the role of NAAG as an endogenous antagonist at NMDA receptors and an agonist at mGluR3 receptors in brain reduced GCPII activity would be predicted to attenuate NMDA receptor. Since the dissociative anaesthetics, phenylcyclidine and ketamine, produce the symptoms of schizophrenia by antagonizing the NMDA receptor, NAAG could serve as endogenous psychotogen. Indeed, post-mortem neurochemical studies indicated significant reductions in the activity of GCPII in temporal, cortex, hippocampus and frontal cortex in schizophrenics (Tsai et al., 1995). Consistent with this post-mortem finding, several studies have independently demonstrated by MR spectroscopy reductions in the levels of NAA, the product of GCPII activity, in temporal cortex and frontal cortex in schizophrenia, the very regions shown to have reduced GCPII activity. Folate reduction and its surrogate elevated homocysteine, have been shown to be common in schizophrenia. Studies of enzymatic activity and, more recently, molecular cloning have demonstrated that GCPII accounts for folypoly- gamma-glutamate carboxy peptidase activity in the small intestine in humans, which is responsible for effective absorption of folate. While we have demonstrated that the GCPII protein expressed in human cerebellum is identical to that of jejunal peptidase as cell as the cell surface marker for metastatic prostate cerebellum is identical to that of jejunal peptidase as well as the cell surface marker for metastatic prostate cancer, Prostate Specific Membrane antigen (PSM) a complete characterization of human brain GCPII, possible allelic variants and the nature of the loss of activity in schizophrenia needs to be performed in order to understand more fully its possible role in schizophrenia and how that may related to reported impairments in folate absorption in schizophrenia.
Specific Aim 1. Identify common allelic variants of GCPII targeting both normal subjects and patients suffering from schizophrenia studied in Project VI.
Specific Aim 2. Carry out Northern blots,, in situ hybridization, Western blots and immunocytochemical studies to define the expression for the GCPII in selected regions of control and schizophrenic brains as previously described in rat brain (Berger et al., 1999).
Specific Aim 3. Measure the levels of NAA, NAAG, glutamate and aspartate in temporal cortex, hippocampus, frontal cortex and other regions in post-mortem brain studies from individuals suffering from schizophrenia and suitable controls. Through microdissection, we will distinguish between gray matter and white matter as NAA is present in axons. Levels of glycine, D-serine and folates will also be measured. Potential alterations in amino acid levels and folate levels will be correlated with the expression GCPII in these samples.
Specific Aim 4. Develop a conditional knock-out of GCPII to determine its effects on behavior and hippocampal neurophysiology in Project III.
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