Joseph H. Neale - US grants

The long term objective of the research program is the understanding of nervous system function as influenced by benzodiazepines and opiates. The immediate aims of the benzodiazepine research are: 1) production of monoclonal antibody to benzodiazepine/GABA/chloride ion channel complex proteins which have been purified using a ligand affinity method. 2) application of these antibodies to purify the individual components of this receptor complex. 3) complete purification of the receptor proteins using HPLC. 4) reconstitution of the receptor complex proteins into nerve cells. 5) use of antisera prepared against the receptor complex to study the neurophysiology and metabolism of this structure in neurons. The significance of this work is derived from the role of this receptor/ion channel as a prominent and widely distributed inhibitory conductance mechanism in the central nervous system. Further, it is the site of action of clinically important drugs, such as the benzodiazepines (i.e., Valium) and barbiturates. With respect to the study of opiate neurobiology, this research will focus upon the potential interaction between opioid peptides and the (putative) acidergic neurotransmitters, glutamate and aspartate, in spinal cord and sensory neurons. This will be achieved with the application of antisera prepared against the amino acids and the enzymes, glutaminase and aspartate aminotransferase, which may serve as markers of acidergic neuronal function. The synthesis, cellular release and immunohistochemical distribution of glycylglutamine, a dipeptide derived from beta-endorphin, will also be studied.

The long term objective of the research program is the understanding of nervous system function as influenced by benzodiazepines and opiates. The immediate aims of the benzodiazepine research are: 1) production of monoclonal antibody to benzodiazepine/GABA/chloride ion channel complex proteins which have been purified using a ligand affinity method. 2) application of these antibodies to purify the individual components of this receptor complex. 3) complete purification of the receptor proteins using HPLC. 4) reconstitution of the receptor complex proteins into nerve cells. 5) use of antisera prepared against the receptor complex to study the neurophysiology and metabolism of this structure in neurons. The significance of this work is derived from the role of this receptor/ion channel as a prominent and widely distributed inhibitory conductance mechanism in the central nervous system. Further, it is the site of action of clinically important drugs, such as the benzodiazepines (i.e., Valium) and barbiturates. With respect to the study of opiate neurobiology, this research will focus upon the potential interaction between opioid peptides and the (putative) acidergic neurotransmitters, glutamate and aspartate, in spinal cord and sensory neurons. This will be achieved with the application of antisera prepared against the amino acids and the enzymes, glutaminase and aspartate aminotransferase, which may serve as markers of acidergic neuronal function. The synthesis, cellular release and immunohistochemical distribution of glycylglutamine, a dipeptide derived from beta-endorphin, will also be studied.

One long-term goal of the PI's research program has been to understand nervous system function as influenced by GABA and by clinically significant and abused drugs. The goal of this application for a Fogarty International Research Collaboration Award is to focus on the explicit role of the GABA-A receptor complex in promoting neuronal survival and neurite branching during development. The immediate aim of this proposal is to test the hypothesis that nerve cell survival and complexity of neurite outgrowth are regulated during development by ambient GABA as well as factors which influence the activity of the GABA-A receptor, including barbiturates, benzodiazepines, and neurosteroids. This will be accomplished using primary cell cultures prepared at low nerve cell density from rat cerebellum, cortex and spinal cord. Cell survival will be assayed over 7-10 days in culture using phase contrast optics. For studies of the complexity of neurite development, nerve cells in culture will be stained with tetanus toxin fragment C and visualized using a peroxidase-diaminobenzidine development system. Cell morphometry will be determined utilizing an optical scanning system and "Image" software which will permit the fractile dimension of cells to be calculated. This work is a natural extension of: 1) the PI's research program which is focused on studies of the influence of receptor activation on the expression of GABA-A subunit message and protein during development in primary cell culture; 2) the experience of the foreign collaborator who has studied GABA-A receptor expression in situ and in vitro. While permitting the test of this important developmental hypothesis, the work will increase our understanding of the influence of selected neuroactive drugs during development and will enhance the research capabilities of the foreign coinvestigator by providing collaborative expertise as well as support for supplies and equipment.

DESCRIPTION: (Applicant's Abstract) The long term goal of this research program continues to be the understanding of mechanisms by which GABA-A receptor expression is regulated and the contribution of individual subunit proteins to receptor function. This includes analysis of the role of the receptor subunits in mediating the physiologic effects of benzodiazepines and barbiturates. Gamma-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the central nervous system. A substantial portion of the inhibitory actions of GABA are achieved via the GABA-A receptors. The GABA-A receptors appear to be constituted of 5 subunit proteins that are the products of transcription, alternative splicing and translation of at least 16 different genes. Each specific combination of these subunit proteins results in a receptor complex with subtly different characteristics in terms of the chloride conductance which GABA elicits and the modulatory influences of benzodiazepines, barbiturates, and steroids on these receptors. During years 17 through 20 of this research program, we propose to focus on four major hypotheses related to the regulation of GABA-A receptor structure and function, as well as its role in development. First, we will test the hypothesis that GABA-A subunit expression is regulated by metabotropic acidic amino acid/peptide receptors. Based on data obtained during the previous support interval, we propose that activation of metabotropic receptors by NAAG and other agonists influences alpha6 GABA-A receptor subunit expression in cerebellar granule cells. Subunit mRNA and protein expression will be examined in granule cell cultures using PCR and immunoblot methods. We will continue to develop subunit specific (alpha6, gamma2, beta2) antibodies which will be used to confirm subunit protein expression. Second, we will test the hypothesis that phosphorylation of the GABA-A receptors is influenced by the balance of excitatory and inhibitory receptor activation. We will use cerebellar granule cells in culture to define the extent of subunit phosphorylation under conditions of activation and blockade of GABA-A and 'glutamate' receptors. Third, we will test the hypothesis that individual neurons with similar structure and position in the CNS express different subsets of GABA-A receptor subunit mRNA and that these differences result in distinguishable electrophysiological responses to GABA, benzodiazepines and barbiturates. We have succeeded in identifying the mRNA for two different GABA-A subunit proteins in single cells in which the GABA-A receptor responses have been characterized under patch clamp conditions. We propose to extend this approach to the preparation of mRNA libraries from individual, electrophysiologically characterized cells. This technically demanding approach will permit us to test in primary cell culture and in brain slices, a series of hypotheses, derived from transfection studies, on the function of GABA-A subunit proteins in situ. Additionally, this work may confirm and perhaps extend hypotheses on preferential subunit association in GABA-A receptors that have been derived from in situ hybridization and transfection studies. Fourth, we will test the hypothesis that GABA-A receptors contribute to the establishing patterns of synaptic connectivity in the developing central nervous system. Rapidly, evolving transgenic methods will be applied to produce null mutations of the alpha6 and gamma2 subunit genes and conditional deletions of the alpha1 and beta2 subunit expression via the controlled expressions of ribozymes targeted for subunit transcripts.

DESCRIPTION (provided by applicant): The long-term goal of this laboratory continues to be the characterization of N-acetylaspartylglutamate (NAAG) as a major peptide co-neurotransmitter and group II metabotropic glutamate receptor agonist. NAAG peptidase activities regulate peptide levels following synaptic release. Inhibition of NAAG peptidase activities has therapeutic efficacy in animal models of inflammatory pain, allodynia, stroke, diabetic neuropathy, ALS, and schizophrenia. The immediate aims are to contrast the cellular expression of two NAAG peptidases that we have cloned, GCPII and III, and to analyze the consequences of inhibiting their activities. Among the hypotheses to be tested are: 1) GCPII and GCPIII function in distinct cell types and locations to regulate NAAG levels following synaptic release; 2) GCPII knock out mice are dependent upon GCPIII activity to retain normal neurological function; 3) increasing extracellular NAAG levels via peptidase inhibition in the brain and/or at the spinal-sensory level influences inflammatory and neuropathic pain perception via group II mGluRs; 4) NAAG acting via group II mGluRs decreases Substance P and CGRP release and EPSCs at sensory spinal synapses; 5) NAAG peptidase inhibition acts centrally to suppress pain-induced glial activation and peripherally to reduce PGE2-induced hyperalgesia and potentiation of capsaicin responses; 6) mGluRS receptors are negatively coupled to cGMP as well as cAMP; 7) activation of glial group II mGluRs by inhibition of NAAG peptidase increases expression of glutamate transporters, GLT-1 and GLAST. The effect of inhibition of GCPIII on total NAAG peptidase activity will be tested in vivo and in vitro using GCPII knock out mice. The influence of peptidase inhibition on inflammatory and neuropathic pain perception will be determined in the rat model and in wild type, heterozygous and GCPII knock out mice. The influence of elevated NAAG levels in vivo, obtained by inhibition of NAAG peptidase activity, will be tested in rat brain and spinal cord and in transgenic mice expressing GLT-1-GFP and GLAST-GFP fusion proteins.

This award provides funds to aid in purchase of an automated DNA sequencer and related equipment and software needed for sample preparation and data analysis. Use of the instrumentation in research and training efforts will be shared by a large group of faculty, students and postdocs. Research areas to be addressed include regulation of surface proteins in various protozoa, gene-flow in tropical trees, yeast gene regulation and variability in flower color. Many research questions in biology now require use knowledge of genotypes as a basic unit of observation. These questions span diverse fields and include hypotheses related to protein structure and function, gene regulation, genome organization, relationships within and among species, spatial and temporal organization of genetic polymorphism and the exchange of gametes among populations or individuals. Automated approaches to DNA sequencing and fragment genotyping are faster and more accurate than the manual methods they replace, increasing the quantity and quality of data. In addition to use in research, the faculty plan to employ the instrument in hands-on laboratory exercises, now technically beyond their laboratory capabilities, for both undergraduate and graduate courses.

DESCRIPTION (provided by applicant): Excitatory amino acid-induced toxicity has been implicated in the nerve cell loss associated with several significant clinical conditions, including stroke, mechanical trauma to the nervous system, amyotrophic lateral sclerosis, and epilepsy. Strategies aimed at reducing this excitotoxicity have focused on blocking excitatory amino acid receptors, particularly the NMDA receptor and group I metabotropic glutamate receptors (mGluRs), activation of group II and III mGluRs, and reducing synaptic glutamate. This research program focuses on the latter two strategies to design, synthesize and test new compounds that may have therapeutic potential for the treatment of excitotoxicity. Central to this approach is N-acetylaspartylyglutamate (NAAG), the most prevalent and widely distributed peptide transmitter in the mammalian central nervous system. NAAG is a selective agonist at group II metabotropic glutamate receptors. Activation of these receptors has been shown to be neuroprotective in vitro and in vivo. NAAG and related group II agonists inhibit transmitter release by a presynaptic mechanism. Equally important, NAAG is inactivated by peptidase activity in the synaptic space to produce glutamate and N-acetylaspartate The program will extend preliminary work in which NAAG analogues with a central urea group have been synthesized and tested for their ability to inhibit enzymes that hydrolyze NAAG, particularly glutamate carboxypeptidase II (GCPII). An aim is to identify novel NAAG-peptidase inhibitors that will increase the peptide concentration following synaptic release, thus increasing activation of group II receptors, while decreasing the release of glutamate from NAAG. In our current SAR work, we have identified several urea-based structures that exhibit nM potency in the inhibition of this peptidase. Based upon these lead compounds, and using rational drug design, we will prepare a library of compounds that serve to further delineate structural features relevant to enhancing inhibitory potency and bioavailability. The program aims to acquire a better understanding of the typography of the enzyme active site of GCPII, and ultimately to produce compounds for use in vivo. Homology-based modeling methods will be employed to create a 3D structure of the enzyme, and the model will be used in turn to assist in ligand design. Since this drug design program has used the peptide as lead compound, it is anticipated that some of the peptidase inhibitors that are identified also may function as group II receptor agonists, an activity that would enhance their neuroprotective potential.

[unreadable] DESCRIPTION (provided by applicant): Schizophrenia affects nearly one percent of the human population. These individuals express a range of debilitating positive, negative and cognitive symptoms. Phencyclidine (PCP), a dissociative anesthetic and drug of abuse, induces schizophrenia-like behaviors in normal humans and in animal models. PCP exacerbates these symptoms in schizophrenic individuals. Current therapies for schizophrenia also moderate PCP-induced behaviors in animals. For these reasons, PCP administration in animals is a widely accepted model in which to study novel therapeutic approaches to schizophrenia and PCP abuse. Acute and chronic administration of d-amphetamine also induces some schizophrenia-like symtoms in humans and has been used in animal models of this disorder. N-acetylaspartylglutamate (NAAG) is a peptide transmitter that selectively activates group II metabotropic glutamate receptors (mGluRs) and is inactivated by extracellular peptidases. Agonists of group II mGluRs reduce some behavioral and neurochemical consequences of PCP in animal models. The PI's group demonstrated that inhibition of enzymes that inactivate synaptically released NAAG reduces the locomotor activity and stereotypic behaviors induced by PCP and MK-801 (open channel NMDA receptor ion channel antagonists) in rats and mice. Inhibition of NAAG peptidase activity also reduces PCP-induced negative behaviors in a resident-intruder assay in mice. NAAG activation of group II mGluRs mediates the effects of peptidase inhibition in these studies. The research proposed here will test the hypothesis that NAAG peptidase inhibitors increase extracellular NAAG levels following synaptic release with a resulting reduction in PCP- and d- amphetamine-induced schizophrenia-like behaviors in rats and mice. Neurochemical and behavioral assays will address the relationship among PCP treatment, peptidase inhibition, and transmitter release in prefrontal cortex. Possible compensation to chronic peptidase inhibition will be assessed in assays of PCP-induced positive (stereotypy), negative (resident-intruder) and cognitive (delayed alternation test of working memory) behaviors. If compensation is detected, NAAG- related neurochemical markers will be examined. The hypothesis that atypical antipsychotics and NAAG peptidase inhibition affect related but not identical behavioral pathways will be tested using NAAG peptidase inhibition as adjuvant therapy with clozapine for PCP-induced behaviors. This research is aimed at the development of an entirely novel therapeutic approach to treatment of schizohrenia and PCP as a drug of abuse. PUBLIC HEALTH RELEVANCE: Current treatments for shizophrenia are not effective in all patients. Better pharmacotherapies, particularly adjuvant therapies, are needed for treatment of this debilitating disorder. Proof of the concept that inhibition of NAAG peptidase activity reduces the behavioral effects of PCP and d-amphetamine in animal models of schizophrenia represents a major step in translating basic research on NAAG and NAAG peptidase inhibitors into human therapies for schizophrenia and acute PCP intoxication. [unreadable] [unreadable] [unreadable]