Daniel T. Monaghan - US grants

The N-methyl-D-aspartate (NMDA) class of excitatory amino acid receptors have rapidly become an important, major area of research in neuroscience. NMDA receptors not only mediate and modulate neurotransmission at a vast number of CNS synapses, but also play a pivotal role in linking neuronal activity to synaptic plasticity during development and learning. NMDA receptors have also been shown to be a critical factor in a variety of pathological processes and have been suggested to be causal in Huntington's and Alzheimer's diseases, schizophrenia, autism, cerebral palsy, and epilepsy development. It is now becoming increasingly apparent that there are multiple populations of NMDA receptors. At physiological, toxicological, and biochemical levels of analysis, there is evidence that there are at least two distinct populations of NMDA receptors that differ in their anatomy, pharmacology, molecular composition, and development. In addition, radioligand binding studies indicate that there are at least two anatomically and pharmacologically-distinct binding site populations of NMDA recognition sites. Thus, NMDA receptors appear to be homologous to the other, genetically-related (Myers et al., 1989) ion-channel receptors (nicotinic, GABA-A, and glycine) in having multiple, genetically-related forms (isoforms or isoreceptors) which display differing distributions in the brain, differing developmental patterns of expression, and variations in agonist and antagonist sensitivities. NMDA receptor subtypes may have significant clinical implications, because the receptor form with the greater agonist sensitivity would be expected to be primarily responsible for the cell death resulting from modest elevations of extracellular glutamate following ischemia and hypoglycemia. The focus of this proposal is to identify the distinguishing properties of NMDA receptor subtypes and to determine how the differing measures of heterogeneity are inter-related. Quantitative autoradiography will be used to evaluate how the anatomically-distinct NMDA binding site populations differ in their physio-chemical ligand binding properties, their pharmacological properties, and their anatomical and ontological patterns of expression. These data are necessary for evaluating the correspondence between heterogeneity observed in physiological/toxicological studies and in radioligand binding studies. Anatomical and pharmacological properties of NMDA receptor proteins labelled by the photoaffinity ligand azido[3H]- MK801 will permit correlating NMDA receptor heterogeneity to that seen at the molecular level. Together these studies should provide a unifying classification and description of NMDA receptor subtypes. The identification of distinct receptor subpopulations is necessary not only for the understanding of NMDA receptor action but is also of fundamental relevance to the general area of NMDA-receptor mediated seizure activity and neurotoxicity. Only with the resolution of subtypes and their distinguishing properties, it is possible to determine their relative contributions to various aspects of normal and abnormal brain function and to develop subtype-specific antagonists that maximize protection while not interfering with normal functions.

DESCRIPTION (from applicant's abstract): There is now a significant body of evidence that growth factors and cytokines are involved in neuropathological and psychiatric disorders; precisely how these effects are mediated is unknown. Insulin and other growth factors are well known to modulate metabolism and differentiation/proliferation, respectively. However, on differentiated neurons, the major role of insulin and cytokines may be, to modulate neurotransmission. A necessary prerequisite for understanding growth factor/cytokine actions is to identify the molecular mechanisms by which these factors exert their biological action on neurons. The objective of this proposal is to determine the signal transduction pathway and, molecular mechanisms by which a growth factor (insulin) regulates synaptic transmission (potentiation of NMDA receptor activity)This model system was chosen because it has significant neuropathological and psychiatric implications. NMDA receptors are thought to play a key role various diseases (e.g. AIDS dementia, epilepsy, stroke, schizophrenia, ALS, and others), thus factors that regulate them may contribute to neurological and psychiatric disorders, and thus account for the observations that insulin improves symptoms in both Alzheimer's disease and schizophrenia. Our specific experimental goals are: 1) to use pharmacological agents and electrophysiological techniques to identify the receptor type and signal transduction pathways that mediate insulin potentiation of NMDA receptor responses in rat hippocampal slices and in Xenoptis oocytes expressing recombinant NMDA receptors; 2) to use mutated recombinant insulin receptors and insulin receptor substrates (IRS-1) to identify which signaling components of the insulin receptor system initiatee modulation of NMDA receptor responses; 3) to identify the insulin-induced phosphorylation (or dephosphorylation) sites on rat hippocampal NMDA receptors and then use site-directed mutagenesis to determine the role of these phosphorylation sites on NMDA receptor function in Xenopus oocytes. Together these studies win identify the specific cellular signaling pathways and molecular mechanisms by which insulin modulates NMDA receptor activity. This knowledge would serve as a basis for determining insulin's role in several aspects of brain function and neurological disorders. These studies would also generate predictions about which other growth factor / cytokine systems mediate similar effects and would generate markers that could test the role of this signaling system in neurological and psychiatric disorders.

DESCRIPTION(Adapted from applicant's abstract): NMDA receptors are involved in several critical functions of the CNS such as cellular mechanisms of learning, pain perception, motor patterns, experience-dependent synapse formation, and others. These receptors are also involved in epilepsy, narcotic adaptation, and neuronal cell death following ischemia, head and spinal cord injury, and HIV infection. Presently the roles that the various NMDA receptor subtypes play in these diverse actions are unknown. We propose to develop subtype-selective antagonists to facilitate the study of NMDA receptor subtypes in normal and abnormal CNS function. We propose to develop a new category of NMDA receptor antagonist- "cleft-binding" antagonists. Our previous antagonist development studies, as well as our molecular modeling studies, have lead us to the conclusion that antagonists of greater subtype-selectivity will require large side groups that can interact with the unique amino acid residues that lay outside the primary antagonist binding site. Such antagonists have side-groups that project further out into the cleft formed between the two major lobes (S1 and S2) that comprise the glutamate binding domain of the NMDA receptor. Thus, we are targeting antagonists that are capable of interacting with the more variable regions of the receptor. The objectives of this project are to: 1) Synthesize and evaluate novel NMDA receptor antagonists that define the structural requirements for binding within the cleft domain of the different NR2 subunits. 2) Test our recently developed molecular models of NR2B and NR2C glutamate binding sites by making point mutations and one chimera that are predicted to have specific alterations in antagonist selectivity. 3) The results from novel antagonists (Aim 1) and point mutations (Aim 2) will be used to test, and if necessary, refine our molecular models of NR2B and NR2C glutamate binding sites. We will then construct NR2A and NR2D molecular models. We will then use the refined models, as well as the structure-activity information, to generate new subtype-selective cleft-binding antagonists. This process would include using automated, computer-assisted routines, as well as, visually-aided design. Given the high homology in secondary, but not primary, structure for the various glutamate receptors, we feel this unexplored approach to antagonist design has significant implications for developing subtype selective antagonists within each of the glutamate receptor families.

Neural crest cell formation and migration is necessary for the normal development of cardiac septation. Neural crest ablation causes subsequent cardiac septation defects. Since homocysteine causes both neural crest- related midline closure defects and the neural crest-related cardiac defects, homocysteine appears to be preventing the normal migration and differentiation of neural crest cells. This hypothesis is consistent with preliminary data from in vitro experimental thus, the central hypothesis for this project is that homocysteine causes NGA conotruncal defects by altering the migration and/or differentiation of cardiac cells of neural crest origin. Based upon the following observations, we further hypothesize that the actions of homocysteine are mediated by an N- methyl-D-aspartate (NMDA) receptor. 1) NMDA receptor antagonists cause the same set of neural crest-related defects as homocysteine, 2) homocysteine is an NMDA receptor antagonist, 3) NMDA receptors play a key role in the Ca++-dependent migration of other cells during development, 4) neural crest cells display Ca++-dependent migration, and 5) neural crest cells express NMDA receptors. The following experiments are designed to test these hypotheses and to describe the key components of this system. 1. We will determine if homocysteine exposure alterations the formation, emigration, and/or migration of neural crest cells in stage 9-12 avian embryos. 2. Are homocysteine's actions on NC cells mimicked, or blocked, by NMDA receptor agents and by NMDA receptor subunit knockout? 3. We will identify the subtype of NMDA receptor that is expressed in NC cells and determine its ability to directly interact with homocysteine and to cause the developmental defects.

[unreadable] DESCRIPTION (provided by applicant): NMDA receptors are critically involved in a variety of CMS functions such as learning, pain amplification, motor pattern generation, and experience-dependent synapse formation and elimination. These receptors are also involved in various neuropathological conditions such as epilepsy, opiate addiction, and neuronal cell death following head/spinal cord injury, sroke, AIDS infection, and possibly the pathophysiology in schizophrenia, Alzheimer's, Parkinson's, and Huntingon's. However, in the absence of selective pharmacological tools, relatively little is known about the role of different NMDA receptor subtypes in these critical cellular processes and disease states. While most of the functional and pharmacological heterogeneity is due to the four genetically-distinct NR2 NMDA receptor subunits, highly-selective antagonists are only available for the NR2B subuit. Over the past few years we have generated a novel series of compounds that are the only NR2C, and NR2D agents known and have already revealed differential roles for NMDA receptor subtypes in synaptic physiology and plasticity. However, it has proven difficult to develop highly-selective agents because the central glutamate binding site is highly conserved for each of the NR2 subunits (as well as for the various AMPA and kainate receptor glutamate binding sites). We have now identified the subunit-specific structural features of the NR2 glutamate binding sites and we propose a two-step approach to developing NR2D and NR2C subtype-selective antagonists. 1) Use a combination of molecular modeling and site-directed mutagenesis of recombinant receptors tested in Xenopus oocytes to define the precise placement of a select group of NMDA antagonists that are large enough to reach the subunit-specific amino acid residues at the edge of the glutamate-binding pocket. 2) Using this structural information, design and test antagonists that are predicted to interact with amino acid residues that are specific to NR2D and other NR2 subunits. Using this approach, we have already designed several antagonists that are predicted by molecular modeling techniques to be highly-selective for NR2D subunit-containing NMDA receptors. [unreadable] [unreadable] [unreadable]

Accounting; Brain; CRISP; Cell Communication and Signaling; Cell Death; Cell Signaling; Code; Coding System; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Condition; Encephalon; Encephalons; Funding; Grant; Individual; Institution; Intracellular Communication and Signaling; Investigators; Laboratories; Long-Term Depression (Neurophysiology); Long-Term Depression (Physiology); Long-Term Potentiation; Long-Term Synaptic Depression; Mass Spectrum; Mass Spectrum Analysis; Mediating; N-Methyl-D-Aspartate Receptors; NIH; NMDA Receptor-Ionophore Complex; NMDA Receptors; National Institutes of Health; National Institutes of Health (U.S.); Nervous System, Brain; Pathway interactions; Photometry/Spectrum Analysis, Mass; Play; Process; Protein Binding Domain; Protein Binding Motif; Protein-Protein Interaction Domain; Proteins; Receptor Activation; Receptor Signaling; Receptors, N-Methylaspartate; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Signal Pathway; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Molecule; Silver Staining; Source; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Synapses; Synaptic; Synaptic plasticity; United States National Institutes of Health; biological signal transduction; gene product; long term depression; necrocytosis; pathway; silver impregnation; social role

DESCRIPTION (provided by applicant): The N-methyl-D-aspartate (NMDA) receptor family regulates various CNS functions such as synaptic plasticity; however, hypo or hyper-activation of NMDA receptors (NMDARs) is critically involved in many neurological and psychiatric conditions. To date, most therapeutic efforts have focused on inhibitors of NMDA receptor activity. However, there are other clinical indications for which an NMDA receptor potentiator is more likely to be appropriate. Presently, there are no preclinical studies of positive allosteric modulators for NMDA receptors. Recently, the joint laboratories of the two PIs discovered a family of compounds that are positive allosteric modulators (PAMs) as well as negative allosteric modulators (NAMs) of NMDARs. These agents have novel sites of action, novel mechanisms of action, and several novel patterns of activity. Their members include the first general NMDAR PAMs, and PAMs that are selective for each of the four GluN1/GluN2 subtypes. NMDAR PAMs have important therapeutic applications, such as for treating schizophrenia or enhancing cognition. Thus, these agents represent significant opportunities as neurobiological tools and are unique lead compounds for developing agents that can modulate NMDAR activity and synaptic plasticity for therapeutic benefit. Further progress in this critical field requires a muc better understanding of the mechanism of action and how that is related to pharmacological activity and receptor modulation. We propose that NMDAR PAMs act at two closely related sites to modulate receptor deactivation and desensitization. Experiments will identify these mechanisms of action of the NMDA receptor PAMs and characterize the respective pharmacophores responsible for these actions. Our goal is to resolve these relationships to provide firm ground for further drug development.

ABSTRACT Despite advances in basic and translational neuroscience research, the development of new therapeutics remains in want. The National Institutes of Health has recognized the need to translate bench research to therapies that improve human disease outcomes and initiated programs that train researchers who can effectively conceptualize neurological disease processes. One critical ?in need? area is in the discipline of neuroimmunity. This research area remains understudied despite its close linkage to the pathobiology of degenerative, infectious, developmental, and psychiatric disorders. To these ends, our training goal is to provide talented students with a fundamental understanding of peripheral and central immunology as it affects neuronal injury, differentiation, regeneration, and protection. The program is designed to provide the student with broad exposure to research methods that facilitate technical proficiency. The program ensures that the student will acquire broad knowledge in neuroimmunity. This would allow critical thinking for how inflammation affects the pathogenesis and treatment of neurological disorders. Several approaches are proposed to achieve this goal. First, is the use of our newly published textbook Neuroimmune Pharmacology (2nd Ed.) designed specifically as a coursework guide in neuroscience, immunology, and pharmacology. Second, is in developing a cross discipline mentorship training to provide the student with opportunities to intersect studies of immunity and neural function. Third, are ?unique? research experiences in systems biology, cell signaling, glial and neuronal biology, human disease models and synaptic physiology. These opportunities serve to complement research in neural genetics, development, repair, and pharmacology. Fourth, are formal student presentations to interdisciplinary basic neuroscientists and supervisory committees to acquire research feedback in design, interpretation and conceptualization of ongoing research activities. This serves to challenge existing paradigms and existing student perceptions. Fifth, are uniquely offered cross-discipline team mentoring, teaching, and clinical neurological experiences. Sixth, are cross-disciplinary internships where students will complete thesis component(s) in another laboratory using a different research approach and mentor. Seventh, are sustained community, university and logistical support. By coordinating the training efforts of divergent research groups linked by common interests in neuroimmunity trainees will develop deeper understandings of innate and adaptive immunity in relationship to neurologic disease. Such trainees will be better prepared to develop successful careers in studies of disease pathobiology and therapeutic interventions for human nervous system disorders.