Robert Freedman - US grants

The effects of phencyclidine (PCP) on catecholaminergic neuronal pathways in the mammalian central nervous system function will be studied using extracellular electrophysiological recordings from single neurons and using in vivo electrochemistry to measure catecholamine transmitter release in vivo. We will apply drugs locally in situ and to isolated neuronal circuits transplanted in oculo by microiontrophoresis and by pressure ejection form multibarrel micropipettes. Drugs will also be applied by superfusion and by eyedrops to the intraocular brain grafts, and i.p. to the whole animal. We have characterized catecholomimetic PCP effects in the cerebellar cortex, hippocampus and prefrontal cortex electrophysiologically, and we have studied PCP effects on dopamine release in the caudate nucleus. We will electrophysiologically characterize PCP interactions with norepinephrine synapses in the prefrontal cortex and with dopamine in the accumbens. We will also investigate PCP interactions with kappa and sigma agonists, we will study changes in PCP effects with chronic exposure, and we will investigate if PCP interactions with dopamine synapses are altered in animals that self-administer PCP. Finally we will determine if the PCP effects observed in rat models are similarly produced in human brain xenographs in oculo in rats. A greater understanding of how ethanol alters CNS function should furnish new insights into the problems of PCP abuse and could lead to pharmacological approaches to eliminate the unwanted effects of this psychotomimetic drug without changing its beneficial qualities.

We have previously characterized a deficit in sensory gating in schizophrenic patients. The P50 auditory evoked potential wave is decremented in response to the second of closely paired stimuli in most normals, but not in most schizophrenics. This failure in auditory sensory gating is a familial trait that also is found in about half the first degree relatives of schizophrenics. Pyramidal neurons of the CA3 layer of rat hippocampus have similar gated responses that may serve as an animal model for neurobiological studies of this phenomenon. The objectives of the present proposal are: (1) to determine why some family members with deficits in sensory gating are clinically ill, while most are not; (2) to characterize the pharmacology of deficits in sensory gating in schizophrenics and their relatives; and (3) to investigate further the cellular neurobiology of sensory gating in animal models. For objective (1), we will continue a multiparameter comparison of schizophrenics and their siblings. We will use magnetic resonance imaging to further test our initial finding that the left anterior hippocampus is smaller in schizophrenics, compared to their siblings with sensory gating deficits. For objective (2), we will pursue a preliminary finding of the role of nicotinic cholinergic neurotransmission in the sensory gating deficit, by assessing the ability of cholinergic agents to reverse sensory gating deficits in schizophrenics and their siblings. For objective (3), we will use a newly developed strategy for multiple simultaneous single neuron recordings from neural networks and other neurobiological techniques in rats to describe the neuronal pathways responsible for sensory gating. We will continue our investigation of sensory gating in the pontine reticular formation, which may influence sensory gating in the hippocampus, via the medial septal nucleus. The three objectives are designed to increase basic knowledge of the neuronal basis of sensory gating (aim 3), to assess which neuronal mechanisms may be deficient in schizophrenics and their relatives (aim 2), and to determine under what conditions individuals with a deficit in sensory gating become ill with schizophrenia (aim 1).

biomedical equipment purchase; microscopy;

This Center application is being submitted in response to the National Institution of Health's Program Announcement "Centers for the Neuroscience of Mental Disorders (CNMD)." The overall aim of the Center is a co-ordinated investigation of the neurobiology of molecular biology of inherited risk factors in schizophrenia. Although there is extensive epidemiological evidence that schizophrenia has a heritable component, the illness itself does not have a Mendelian segregation and precisely what biological dysfunction is inherited is unknown. Because of these uncertainties, no simple neurobiological or molecular biological approach is likely to readily define the heritable elements in either genetic or neuronal terms. Investigation is further complicated by the lack of an animal model and by the general prohibition against invasive experiments in human brain. Therefore, this Center is designed to pursue a series of collaborative investigations in which various approaches converge to attempt to identify the neuronal defect and its genetic basis. The Center employs neurobiological techniques in humans and human tissue--patients and their families, in oculo transplants, and most mortem tissue--to characterize the nature of the inherited neuronal dysfunctions, models their neuronal mechanisms in animals, and then uses several different molecular biological strategies to determine whether genes coding for or otherwise linked to these functions are abnormal in schizophrenics. Thus, as neurobiological hypotheses are formulated, they are confirmed or refuted by genetic analysis. The process is interactive, so that information from genetic investigations is also used to refine the identification of the phenotype, which in turn supports further genetic analysis.

The neurotransmitter disturbances which occur during alcoholism and chronic alcohol use are not well understood. Although various neuroadaptive mechanisms have been inferred from model experiments in animals, the relevance of these mechanisms to human alcoholism is not well-established. One method to test hypotheses of neurotransmitter disturbance is to assess the expression of genes involved in neurotransmission in post mortem specimens of specific regions of the human brain by in situ hybridization of mRNA. This technique has the advantage of providing very specific information about the expression of specific genes which animal and clinical research have targeted as affected in alcoholism. However, the technique also requires that the mRNA in the post mortem specimens not be degraded by non-specific factors, including the debilitation produced by alcohol abuse itself. The first aim of this study is to obtain post mortem brain tissue from 40 alcoholics and 40 normals and to analyze the mRNA content and integrity. The data will be analyzed to determine if non-specific factors, related to alcohol use or to the events surrounding death and autopsy, influence mRNA content and integrity. The second aim is to examine the expression of genes related to GABA-A receptors, including the gamma-2-L component, which conveys sensitivity to alcohol. The third aim is to examine the expression of beta adrenergic receptors, which have also been linked to the response to alcohol. This component will thus support investigations in humans of neurobiological and molecular biological mechanisms that are also the focus of research in animal models in other Center projects.

Project 1 is responsible for clinical studies of physiological phenotypes related to schizophrenia. The aim of Centers for Neuroscience of Mental Disorders is to bring basic science expertise to bear upon chronic mental disorder. However, these disorders are complex, multi-faceted syndromes that are not immediately amenable to basic science investigation. This project is responsible for identifying discrete deficits in schizophrenics that may more directly represent neuronal dysfunction, so that other projects can use these deficits for genetic and molecular studies, for study in isolated tissue, and for modeling in animals. Project 1 is also responsible for testing in human subjects any hypotheses reached from basic science research. Two physiological measures will be used for most studies: deficits in smooth pursuit eye movements and diminished inhibitory gating of the P50 auditory evoked potential response. Three experiments will be performed: (1) linkage studies using these measures in collaboration with Project 2, along with clinical diagnoses, to determine if one or more of these phenotypes identifies genes relevant to the transmission of schizophrenia; (2) studies of the effects of nicotine on the measures, to determine if nicotine normalizes the defects, as predicted by Project 3's animal model; and (3) investigations to define factors, such as increased catecholamine metabolism, that cause variability in these measures, to improve their usefulness for clinical research. In addition, Project 1 collects post mortem and aborted tissue specimens for Projects 6 and 7.

DESCRIPTION: (Adapted from investigator's abstract) Susceptibility to schizophrenia appears to be transmitted in part through a complex genetic mechanism involving interaction of multiple genes, each of relatively small effect. Detection of such loci through genetic linkage studies is likely to require a very large sample of multiply affected pedigrees. In response to RFA MH-99-005, nine investigators propose a Collaborative Study of Mental Disorders to collect in three years a sample of 517 affected sibling pairs (ASPs) with DSM-IV schizophrenia, to complete a genome scan of these pedigrees for multipoint ASP analysis to detect susceptibility loci, and to share biological materials, genotypes and blinded clinical data with the scientific community through an NIMH-sponsored mechanism. Each site will recruit families in a large geographic area, using an opportunistic ascertainment strategy and efficient assessment procedures to maximize the number of ASPs collected. Subjects suspected of having schizophrenia will be assessed with the Diagnostic Interview for Genetic Studies supplemented by information from the Family interview for Genetic studies and medical records. Diagnoses will be made by consensus best-estimate procedures. Interviewer training and quality assurance monitoring of protocol adherence will be provided for all sites. Blood specimens will be obtained from all individuals with psychotic disorders plus their parents and (when both parents are not available) up to two additional siblings to provide genetic phase information. Permanent cell lines will be created and DNA extracted at the NIMH-sponsored Center for Genetic Studies. All clinical data will be merged regularly into a central study database, and blinded data transmitted to the Center for Genetic Studies. At the end of the four-year project period, biological materials and blinded pedigree and clinical data will be made available to scientific community for genetic studies of schizophrenia and related disorders. In year 4, a genome scan will be undertaken at CIDR (if approved) or at the University of Chicago. Affected subjects and relatives needed for phase information will be genotyped using the latest screening map, currently the Weber Version 9 Linkage Mapping Set, containing 387 microsatellite markers at approximately 10 cM spacing. The primary statistical approach will be multipoint analysis of allele sharing in affected individuals, with DSM-IV schizophrenia defined as affected. Secondary analyses will also be carried out. Power analyses suggest that this study would have excellent power to detect loci associated with lambda sibs of 1.4, and moderate power for a value of 1.3, i.e., loci with relatively small etiologic effects.

The objective of this contract is to provide the National Institute of Drug Abuse (NIDA) Intramural Research Program (IRP) with human post-mortem brain tissues, patient histories, and data on nicotinic receptor polymorphisms that will further our attempts to understand the genetic predispositions and neuroadaptations associated with addiction.

The improved treatment of cognitive disorders in schizophrenia has become a priority for the National Institute of Mental Health, because of the evidence that cognitive dysfunction is the chief cause of chronic disability from the illness. Currently available antipsychotic drugs do not effectively address this dysfunction. One promising lead comes from studies of nicotinic acetylcholine receptors. For the alpha7-receptor there is genetic and neurobiological evidence of involvement in schizophrenia. Nicotine has some neurocognitive effects, but they are not clinically significant and there is considerable health risk associated with nicotine in any form. alpha7-agonists have the promise of safer, more targeted effects and may not have nicotine's considerable tachyphylaxis. In the previous funding period, we completed a Phase 1 study of 3-2,4 dimethoxybenzylidene anabaseine (DMXB-A), a partial agonist at alpha7-nicotinic receptors, in schizophrenia. A positive neurocognitive effect was found. The proposed continuation of this project will test the drug's effectiveness using the newly available MATRICS Battery, developed by NIMH to assess neurocognitive effects of new drugs potentially useful in schizophrenia. We will also determine the time course of drug effect, with the intent of developing administration paradigms that prolong drug action throughout the day. We will also determine if the drug interacts with nicotine for schizophrenics who smoke. In a final experiment, we will determine if clinically useful of DMXB-A are sustained during periods of administration of one moYrth or longer. Persons with schizophrenia have difficulty with many aspects of their thinking, including their attention and their ability to learn. The drugs that they currently take are not entirely helpful, and many of them turn to smoking. We are studying a new, nicotine-like drug, that does not have the harmful effects of tobacco, but may help persons with schizophrenia to think better.

DESCRIPTION(Adapted from applicant's abstract) Acetylcholine is considered to be an important neurotransmitter in the mammalian brain, and behavioral phenomena such as attention, learning and memory, nociception, and nicotine addiction are all thought to involve cholinergic systems. Furthermore, cholinergic deficits have been implicated in disorders such as Alzheimer's disease and schizophrenia. Despite the fact that a great deal is known about cholinergic systems in general, very little is known about synaptic transmission in the brain mediated via nicotinic receptors. Hippocampal interneurons receive a fast, excitatory input via nicotinic synapses that are sensitive to the snake toxin, alpha-bungarotoxin, suggesting that the postsynaptic receptors at these synapses contain the nicotinic alpha-7 receptor subunit. The proposed experiments will investigate fundamental issues relating to neurotransmission at these nicotinic synapses. The first objective of these experiments will be to determine the source of the presynaptic cholinergic fibers that innervate hippocampal interneurons. Although most evidence suggests that the cholinergic synapses on CA1 interneurons arise from septal cholinergic neurons, there is evidence that some or all of these synapses could be made by putative intrinsic hippocampal cholinergic neurons. A second objective of this application will be to characterize the pharmacological and physiological properties of nicotinic synapses onto CA1 neurons. Very little is known about how these nicotinic synapses respond to repetitive activation, how rapidly they desensitize, whether they are affected by presynaptic modulators, and whether there are more persistent effects on interneurons that are mediated via CA2+ influx through these Ca2+-permeable ion channels. The final objective of these experiments will be to characterize the projections of interneurons that can be activated by alpha-7 nicotinic receptors. We will characterize the projections of interneurons that can be activated by alpha-7 nicotinic receptors both electrophysiologically, by recording responses from their target neurons (pyramidal neurons, and other interneurons), and morphologically, by labeling interneurons that receive nicotinic input with biocytin, and reconstructing their axonal projections. By answering these fundamental questions, it will be possible to integrate what we know about these nicotinic synapses into the much larger body of knowledge concerning the role played by interneurons in regulating hippocampal activity, and the way in which cholinergic activity controls the function of this key population of hippocampal neurons.

This project will replicate and extend the relationship of genetic polymorphisms in CHRNA 7 to the phenotype of inhibitory dysfunction as measured by inhibition of the P50 response to repeated auditory stimuli in schizophrenic and control subjects. Alpha7-specific agonists will be tested in schizophrenics to determine if they normalize P50 response and to what extent they also affect the neurocognitive features and clinical symptoms of schizophrenia. We have received FDA approval for the administration of 3-2,4 dimethoxybenzylidene anabaseine to humans and in an initial experiment demonstrated that the drug improves deficient P50 inhibition. The alpha7 nicotinic receptor not only has a role in neurotransmission, which we demonstrate by P50 inhibition, but it also has a major developmental role that we have characterized by observing differences in the regional distribution of hippocampal interneurons in animal models with acra7 variants. Because correction of neurotransmission deficits related to alpha7 receptors does not address this developmental role, we will also employ mouse animal models to determine if alpha7 agonist treatment in the perinatal period affects the development of interneurons. To extend the hippocampal pathophysiology we have postulated to include the deficits in learning and memory found in schizophrenia, we now show deficits in learning in mice with the acra7 (murine CHRNA 7) null mutation, as well as evidence in humans that P50 abnormalities correlate with decreased declarative memory. We present new data that perinatal choline supplementation to mice with acra7 polymorphisms results in offspring with normal inhibition of the hippocampal response to repeated sounds. In conjunction with Project by Restrepo, we will measure olfactory function in schizophrenics. For Project by Ross, we will record P50 inhibition as a basic measure of inhibitory function to be related to their measurements in children and adults.

The Administrative Core is responsible for the governance of the Center and for support of its fiscal conduct. It was also have specific responsibility for the collection of postmortem brain tissue. This activity supports research in Dr. Leonard's project and Dr. Restrepo's project, but it depends upon Dr. Freedman's efforts to secure continuing co-operation from autopsy services and from county coroners, Dr. Leonard's efforts to maintain and monitor the quality of brain tissue preservation, and Dr. Cathy Adams's (Project 0004) neuroanatomical expertise. Because of this extensive co-operation between projects and the role of Dr. Freedman, the Center director, it was considered proper for the Administrative Core. The Core also supports the educational and outreach efforts of the Center. A special fiscal issue is the administrative arrangements between the two campuses of the University of Colorado involved. Much of the material in this description necessarily duplicates material in the operational plan for the Center.

neurotransmitter agonist; human therapy evaluation; schizophrenia; nicotinic receptors; mental disorder chemotherapy; neurogenetics; clinical trials; brain disorders; neuropsychology; neurophysiology; patient oriented research; human subject; clinical research;

DESCRIPTION (provided by applicant): The etiological complexity of schizophrenia has prompted its parsing into endophenotypes, to facilitate genetic and biochemical study. The Center investigators found that the alpha7 nicotinic receptor subunit gene (CHRNA 7) is genetically linked to both a deficit in inhibitory gating of auditory response and to schizophrenia. Polymorphisms in the core CHRNA 7 promoter that decrease the gene's expression are associated with both schizophrenia and with diminished auditory gating. In the proposed Center, we will continue to identify CHRNA7 polymorphisms (Project 1). Second, we will determine if the polymorphisms reduce receptor expression and related cellular functions such as calcium flux in human olfactory neurons (Projects 1 and 2). Third, we will determine the effects of these polymorphisms on brain functions, including P50 sensory gating. (Project 0003). We will also consider effects of the polymorphisms on development of inhibitory neuronal circuitry in humans and animal models from the perinatal period through adulthood (Projects 4 and 5). Fourth, we will determine to what extent agonists directed against the alpha7 receptor reverse this pathophysiology and the symptoms of schizophrenia (Project 5). We provide new data to show: (1) diminished binding of transcription factors to the CHRNA7 promoter in schizophrenia, (2) decrease by nicotine of the hippocampal hemodynamic hyperactivity that we previously reported in fMRI studies of schizophrenics, and (3) deficits in learning in mice with the acra7 (murine CHRNA 7) null mutation, as well as evidence in humans that P50 abnormalities correlate with decreased declarative memory. We present provocative new data that perinatal choline supplementation to mice with acra7 polymorphisms results in offspring with normal inhibition of the hippocampal response to repeated sounds.

[unreadable] DESCRIPTION (provided by applicant): The improved treatment of cognitive disorders in schizophrenia has become a priority for the National Institute of Mental Health, because of the evidence that cognitive dysfunction is the chief cause of chronic disability from the illness. Currently available antipsychotic drugs do not effectively address this dysfunction. One promising lead comes from studies of nicotinic acetylcholine receptors. For the alpha7-receptor there is genetic and neurobiological evidence of involvement in schizophrenia. Nicotine has some neurocognitive effects, but they are not clinically significant and there is considerable health risk associated with nicotine in any form. alpha7-agonists have the promise of safer, more targeted effects and may not have nicotine's considerable tachyphylaxis. In the previous funding period, we completed a Phase 1 study of 3-2,4 dimethoxybenzylidene anabaseine (DMXB-A), a partial agonist at alpha7-nicotinic receptors, in schizophrenia. A positive neurocognitive effect was found. The proposed continuation of this project will test the drug's effectiveness using the newly available MATRICS Battery, developed by NIMH to assess neurocognitive effects of new drugs potentially useful in schizophrenia. We will also determine the time course of drug effect, with the intent of developing administration paradigms that prolong drug action throughout the day. We will also determine if the drug interacts with nicotine for schizophrenics who smoke. In a final experiment, we will determine if clinically useful of DMXB-A are sustained during periods of administration of one moYrth or longer. Persons with schizophrenia have difficulty with many aspects of their thinking, including their attention and their ability to learn. The drugs that they currently take are not entirely helpful, and many of them turn to smoking. We are studying a new, nicotine-like drug, that does not have the harmful effects of tobacco, but may help persons with schizophrenia to think better. [unreadable] [unreadable] [unreadable]

Project 3 identified CHRNA7 as the gene that is linked to the PSO sensory gating abnormality in schizophrenia and found functional SNPs in the core promoter of the gene that are associated with this abnormality. Genetic linkage to 15q13.3, the locus of CHRNA7 has been found in multiple ethnic groups, and recent evidence suggests that rare deletions of the gene are associated with schizophrenia. The human molecular biology studies in this Project characterize mutation, function, and regulation of CHRNA7. To fulfill the need for clinically useful genomics, the human molecular biology project has undertaken extensive screening of CHRNA7 to find its pathogenic mutations. A critical finding is that the amino acid structure of the protein is generally normal in schizophrenia and thus most abnormalities involve regulation of its expression. Project 1 has used that information to design a new therapeutic. Project 3 will continue to support drug development by identifying new polymorphisms, one of which already shows preliminary evidence of a pharmacogenetic effect. Investigation of the 5' and 3' regulatory regions in Aim 1 interacts with similar efforts of Project 4 in DBA/2 mice, which also have CHRNA7 mutations. Functional mutations in human CHRNA7 \N'\\\ be introduced into transgenic mouse models in Project 5. Genotypes may eventually identify individuals who are likely to have genetically determined pathobiology involving a7nAChRs. Project 2's preventive intervention in infancy similarly requires information about CHRNA7 and other genes that convey risk for schizophrenia such as NRG1, a gene associated with risk for schizophrenia that is involved in the developmental expression of aTnAChRs. Aim 2 will determine how NRG1 and CHRNA7 polymorphisms both act to increase risk for schizophrenia. Psychiatric molecular biology in the Center includes more than genomics. Project 2 is now involved in perinatal treatment with choline as an a7nAchR agonist. In Aim 3, Project 3 will contribute its microarray technology to characterize the changes in gene expression that result in animal models from Projects 4-6 that receive this treatment. Results will be compared with our previous experience in characterizing gene expression in postmortem brain from persons who had schizophrenia. Project 3 provides basic research support to Projects 1 and 2 and interacts with basic researchers in Proiects 4, 5, and 6. It receives statistical qenetics support from Core B. RELEVANCE (See instructions): New therapeutic strategies for schizophrenia are needed to improve cognitive dysfunction and negative symptoms and to prevent the development of psychosis. The Center investigates a nicotinic acetylcholine receptor as a new therapeutic target. Investigational results are used to design a new drug treatment for schizophrenia and a preventative nutrient intervention during infant development, both of which activate this ri^r.e^riirsr

The Statistical Genetics and Treatment Analysis Core is designed to provide support in study design, data analysis, and interpretation. All Core members participate in preparation of reports, presentations, and manuscnpts. In addition. Core members will continue their efforts to develop new statistical approaches that will improve the analytic techniques available to Center investigators. Specific developments include methods to increase the power for association analysis in an area with a known linkage and methods to assign a false discovery rate to multiple gene expression measurements in a microarray experiment. Core B provides services to all five Projects. RELEVANCE (See instructions): New therapeutic strategies for schizophrenia are needed to improve cognitive dysfunction and negative symptoms and to prevent the development of psychosis. The Center investigates a nicotinic acetylcholine receptor as a new therapeutic target. Investigational results are used to design a new drug treatment for schizophrenia and a preventative nutnent intervention dunng infant development, both of which activate this rpr.pntr>r

DMXB-A is a prototypic selective alpha7 nicotinic receptor agonist that has been tested extensively both preclinically and in Phasel and 2 clinical tests in schizophrenia. This compound is a weak partial agonist with relatively short plasma half-life. It has been shown to enhance various measures of cognition including increases in attention and sensory gating. The purpose of this Core project is to synthesize DMXB-A in sufficient amounts and purity for further clinical and pre-clinical tests. The FDA has stipulated that the synthetic DMXB-A display a chemical purity equivalent to a commercial product made under Good Manufacturing Practice (GMP) standards. This will require most of the requested effort of the synthetic chemist for Year 1 of the project. A second function of this Core will be to measure plasma samples from animals and humans that have been administered DMXB-A to determine its concentration and those of its active metabolites. This will be done according to previously reported HPLC protocols that allow concurrent measurement of parent compound and its 4-OH, 2-OH, and 2,4-Dihydroxy metabolites. These metabolites are potent full agonists and may be involved in the neurobiological response to DMXB-A administration. These data will allow tests of correlation between clinical effect and plasma drug levels. The final function of the Core will be to identify and synthetically provide additional related compounds for clinical tests. Core C provides clinical research support to Project 1 and basic research support to Projects 4, 5, and 6. RELEVANCE (See instructions): New therapeutic strategies for schizophrenia are needed to improve cognitive dysfunction and negative symptoms and to prevent the development of psychosis. The Center investigates a nicotinic acetylcholine receptor as a new therapeutic target. Investigational results are used to design a new drug treatment for schizophrenia and a preventative nutrient intervention during infant development, both of which activate this r(=^r.e^rifnr

Data generated largely by investigators of this Conte Center application provide compelling evidence that the alpha 7 nicotinic acetylcholine receptor subunit is a potential target for therapeutic intervention in schizophrenia. These data include the observations that the expression of the alpha7 subunit is reduced in the hippocampus of schizophrenics (Project 1), genetic variants in CHRNA7, the gene that encodes the alpha7 subunit, are associated with schizophrenia and auditory sensory gating deficits (Project 3) and auditory gating deficits are common among schizophrenics and the selective alpha7 agonist DMXB-A improves gating deficits (Project 1). In addition, the alpha7 selective agonist choline has been shown to improve auditory gating in human infants when administered perinatally (Project 2). Strikingly similar data have been obtained in mice. For example, we have shown in mice that 1) auditory gating deficits are correlated with reduced alpha7 receptor expression, 2) genetic variability in Chrna7 is linked to reduced expression of alpha7 receptors and auditory gating deficits, 3) the alpha7 receptor selective agonist DMXB-A improves gating deficits and, 4) perinatal choline improves auditory gating in a gating deficient mouse strain. In Project 4 we will take advantage of the similarities between human and mouse with respect to alpha7 receptors and auditory gating to address fundamental biological questions regarding the specific role of alpha7 receptors and Chrna7 in normal and deficient auditory gating. The specific questions that will be addressed in Project 4 are 1) what is the molecular mechanism(s) through which genetic variability in Chrna7 leads to reduced expression of alpha7 recptors and auditory gating deficits 2) what is the neurobiological mechanism by which reduced expression of alpha7 receptors might lead to gating deficits? and 3) what is the mechanism through which perinatal choline improves auditory gating? Project 4 supports the clinical research of Projects 1 and 2. It performs molecular genetics experiments in parallel with Project 3, and it supports the phenotyping of mice in Projects 5 and 6.

Maternal Immune Activation (MIA) results from viral infection during pregnancy and is the best characterized non-genetic risk factor for schizophrenia. We and others have developed a mouse model of MIA that produces neurobiological and behavioral deficits that resemble those of schizophrenia. The MIA model, unlike the Center's other animal modes, makes no suppositions about the role of alpha? nicotinic acetylcholine receptors. Therefore, it is an excellent model to test the effects of perinatal choline to determine if this intervention is effective in a model that does not pre-suppose diminished alpha? nicotinic receptors. MIA is often hypothesized to interact with genetic risk for schizophrenia, so that its most marked effects are in genetically vulnerable individuals. Therefore, in a second aim, we will test whether its effects are enhanced in dams and fetuses who are heterozygous for the Chrna? null mutation. We hypothesize that there may be additive effects of fetal genotype and the MIA insult. In addition, the dam's genotype may be influential in regulating MIA, because alpha? nicotinic receptors have been shown to play a role in the moderation of inflammatory responses. Project 6 thus introduces a new model to the Center, which will influence Project 2's clinical research on the possible maternal causes of sensory gating abnormalities in infants, as well as Project 1's investigation of which adult patients respond to nicotinic agonist therapies. Project 6 will receive genetic analysis support from Project 3, phenotyping support from Project 4, and will assess MIA in humanized animals of Project 5. RELEVANCE (See instructions): New therapeutic strategies for schizophrenia are needed to improve cognitive dysfunction and negative symptoms and to prevent the development of psychosis. The Center investigates a nicotinic acetylcholine receptor as a new therapeutic target. Investigational results are used to design a new drug treatment for schizophrenia and a preventative nutrient intervention during infant development, both of which activate this rpr.pntnr

Primary prevention is a goal for the investigation of any chronic illness. Project 2 focuses on translating basic neurobiological findings about the role of alpha 7 nicotinic receptors in early brain development into clinical assessments of early human developmental abnormalities that can progress to schizophrenia later in life. It is also conducting initial tests of a specific perinatal intervention to reduce this risk. We have systematically examined the development of psychophysiological and neuropsychological abnormalities associated with the genetic liability to schizophrenia in adults. These include auditory sensory gating deficits demonstrated with PSO auditory evoked potentials, abnormalities in smooth pursuit eye movements, mismatch negativity, and various measures of attention. Abnormalities in these measures appear in some children of parents with schizophrenia as soon as testing is developmentally possible. Basic science research points to the perinatal period as a critical window for the role of alpha 7 nicotinic receptors, with their maximal expression occurring then as the adult forms of GABA and glutamate synapses are expressed. We developed techniques to record PSO sensory gating within several weeks of birth and showed that this function is already normally present. In the proposed aims, we will extend our initial observation that parental history of psychosis is associated with diminished PSO auditory sensory gating. Project 3 will genotype our subjects to test whether the genetic associations of these deficits in early development are the same as the associations in adults. Similar genetic association may support the hypothesis that the deficits in children at risk for schizophrenia have the same molecular basis as deficits observed in schizophrenia itself. We will also extend a second observation that maternal nicotine use during pregnancy is associated with diminished PSO auditory sensory gating. These effects are additive with the effects of apparent genetic risk and implicate nicotinic receptors in the deficit, as is also true in adults with schizophrenia who respond to chronic nicotine exposure with loss of PSO gating. Based on Project 3's demonstration that perinatal choline, an alpha 7 nicotinic receptor agonist, improves sensory gating in the DBA/2 mouse model of genetic deficiencies in alpha 7 nicotinic receptors and sensory gating, we have obtained FDA permission to administer perinatal choline to mothers and their infants. We will continue this clinical investigation, which shows initial promise of early enhancement of sensory gating. Project 2 receives clinical research support from Project 1 and basic research support from Projects 3,4,5,6. RELEVANCE (See instructions): New therapeutic strategies for schizophrenia are needed to improve cognitive dysfunction and negative symptoms and to prevent the development of psychosis. The Center investigates a nicotinic acetylcholine receptor as a new therapeutic target. Investigational results are used to design a new drug treatment for schizophrenia and a preventative nutrient intervention during infant development, both of which activate this rprpntnr

The mouse models of alpha? nicotinic receptor dysfunction of Project 4 have led to significant understanding of the role of allelic variants in the gene for this receptor in development and function of the hippocampus, particulary in regard to its role in auditory sensory gating;the possible therapeutiuc effects of nicotinic agonists on sensory gating dysfunction in schizophrenia;and the possible preventive effect of perinatal intervention with choline supplementation. As the discoveries of Project 4 are translated into clinical studies by Projects 1 and 2, possible limitations reflecting the differences between human CHRNA? and mouse Chrna? must be considered. Therfore Project 5 will attempt to produce a mouse that has the human CHRNA? in place of the mouse gene, using homologous recombination in mouse embyonic stem cells. This technique will allow us to study differences between the mouse and human gene in Experiment 1, to assess the effects of variants in the gene found in schizophrenia by Project 3 in Experiment 2, and to determine the effect of choline on the development of neurons that have human versions of CHRNA? and the alpha 7 nicotinic receptor in Experiment 3 to help Project 2 design the safest and most effective human treatment. Although production of a transgenic mouse is always a high risk project, the Center's history of successful translational use of research findings in animal models to shape clinical trials now supports this enhancement of its animal model of the genetic basis of a? nicotinic receptor dysfunction. Project 5 supports clinical research in Projects 1 and 2 and receives genetic resources frim Project 3, phenotyping support from Project 4, and will interact with Project 6 to examine effects of maternal immune activation. RELEVANCE (See instructions): New therapeutic strageties for schizophrenia are needed to improve cognitive dysfunction and negative symptoms and to prevent the development of psychosis. The Center examines a dysfunctioning nicotinic acetylcholine receptor as a new therapeutic target. It investigates a new drug treatment for schizophrenia and a preventative nutrient intervention during early infant development, both of which activate this receptor.

Nicotinic agonist therapy for schizophrenia arose from the Center's discovery of the role of the alpha 7 nicotinic acetylcholine receptor and its gene CHRNA7 in the pathophysiology and genetic transmission of risk for schizophrenia. Although nicotine itself has many undesirable properties, its effects on neurocognition and sensory gating in schizophrenia prompted the search for a safer, more effective agonist. 3-2,4 dimethoxybenzylidene anabaseine (DMXB-A), first synthesized by William Kem in Core C, can be administered orally and produces less tachyphylaxis than nicotine. It also has a more favorable safety profile. Phase 1 and Phase 2 trials conducted by the Center showed promising effects on neurocognition in patients with schizophrenia. Nevertheless, the full possibilites of this target have not yet been determined. DMXB-A's relatively short half life may limit its ability to achieve maximal effects on neurocognition and clinical symptoms. Therefore, we will test a sustained release preparation to assess if more robust effects can be obtained. Imaging of the neurobiological effects using fMRI shows that DMXB-A diminishes hyperactivation of the hippocampus, a trait associated with schizophenia, and increases activity in the medial septal nucleus, the source of cholinergic innervation to the hippocampus, including the alpha 7 nicotinic receptors on inhibitiory interneurons. This imaging strategy will be used to determine if longer acting DMXB-A has increased neurobiological effects or whether its effects are limited by tachyphylaxis. Our studies have been performed in non-smokers to avoid interference from the possible desensitizing effects of nicotine from schizophrenics' heavy chronic nicotine abuse. With the new sustained release preparation, we can test whether DMXB-A will substitute for nicotine in the contextof a smoking cessation treatment program. We will use fMRI to help assess the degree to which nicotine interferes with DMXB-A's effects as persons with schizophrenia who are trying to stop smoking are treated with DMXB-A. Project 1 receives basic research support from Projects 3, 4, 5, and 6. Core C provides DMXB-A. RELEVANCE (See instructions): New therapeutic strategies for schizophrenia are needed to improve cognitive dysfunction and negative symptoms and to prevent the development of psychosis. The Center investigates a nicotinic acetylcholine receptor as a new therapeutic target. Investigational results are used to design a new drug treatment for schizophrenia and a preventative nutrient intervention during infant development, both of which activate this r(arp>ntnr

The aims of this Translational Conte Center are (1) to enhance the basic molecular and neurobiological characterization of the role of the alpha7 nicotinic acetylcholine receptor in the brain and its dysfunction in schizophrenia;(2) to use this biology to continue development of a new therapeutic strategy to improve the treatment of cognitive dysfunction and negative symptoms in persons who have schizophrenia (Project 1), and (3) to use emerging information about the developmental role of the receptor to initiate a perinatal intervention for pregnant women and their children to decrease the risk of schizophrenia (Project 2). The Center investigates the potential therapeutic effects of selective alpha 7 nicotinic agonists in animal models of neuronal dysfunction in schizophrenia and then in early human clinical trials in patients with schizophrenia. It examines CHRNA7, the gene for this receptor, to determine how its expression becomes aberrant in schizophrenia (Project 3) and in animal models where the gene is naturally or intentionally mutated (Project 4). It determines in humans and animal models how specific variants in the gene result in dysfunction in the development of inhibitory neuronal circuitry in the hippocampus and other forebrain regions. It then examines the effects of nicotinic agonists that can potentially alter these genetic effects on development. These agonists include selective alpha 7 nicotinic receptor agonists in animal models and nicotine itself in both humans and animal models because some pregnant women smoke cigarettes. Because the endogenous ligand during fetal brain development appears to be choline, the Center also examines the potential beneficial effects of choline supplementation on alpha 7 nicotinic receptor function and neuronal development in a clinical trial in pregnant women and their newborn infants and in related animal models. To support these investigations, the Center develops strategies to image inhibitory function hemodynamically and electrophysiologically, in both adults and infants, and it develops new animal models, including the transfer of human CHRNA7 into mice (Project 5) and models of developmental abnormalities induced by the immune response to infections (Project 6).

The Administrative and Database Core is charged with governance of the Center, education outreach to professionals and the community, and overseeing databases for the Center's investigations. The Core will arrange external advisory visits and regular governance meetings of the investigators. The first meeting will be a special meeting of the investigators and external advisors to discuss the review of the Center and to implement any directions or changes from the review. Thereafter, it will organize an annual meeting that will bring together the investigators, the advisory board, and all trainees to fulfill the Center's special education role. Other meetings at the Society for Neuroscience and in Denver will ensure that the investigators meet at quarteriy intervals, either face to face or by teleconference. Databases to support genotypic and phenotypic analyses of human subjects and mice are maintained by the Center. The Core oversees these databases and provides for transmission of genotypic and phenotypic and clinical trials information to Core B for analyses, while maininting appropriate blind conditions for investigators Core A provides administrative, educational, and database services to all Projects and Cores.

DESCRIPTION (provided by applicant): The alpha 7-nicotlnic acetylcholine receptor is an investigational target for the development of new drugs to improve brain function in schizophrenia. The target is supported by (1) electrophysiological evidence for its participation in the sensory gating disturbances that are an endophenotype found in persons with schizophrenia, (2) genetic evidence for abnormalities involving CHRNA7, the gene for the alpha7-nicotinic receptor subunit, and (3) pharmacological evidence for possible therapeutic effects of nicotine as well as more specific alpha7-nicotinic agonists. The hypothesis that emerges is that persons with schizophrenia have diminished expression of the alpha7-nicofinic receptor on inhibitory interneurons in the hippocampus and thalamus. Increased activation of these inhibitory interneurons, by enhanced pharmacological stimulation of this diminished population of alpha7-nicotinic receptors, would improve patients' neurocognitive abilities, particularly their characteristic problems in sustained attention. UCI-40083, an allosteric modulator at the alpha7- nicotinic receptor, has unique properties as a candidate therapeutic at this site. UCI-40083 has the ability to selectively increase ion currents through the a7-nicotinic receptor channel while retaining fidelity to the agonist-induced kinetics of channel opening and closing, making it a safe and potentially effective drug to increase cholinergic neurotransmission at this receptor. It has shown promising safety and efficacy in animal models. This National Cooperative Drug Discovery Development Group will take the next step to evaluate UCI-40083 as a potential therapeutic for schizophrenia by performing a first-in-humans Phase 1 pharmacokinetics and safety trial, a Phase 1b preliminary dose-finding trial in schizophrenia, and an initial Phase 2 proof-of-principle clinical trial in schizophrenia to determine effects on neurocognition, with additional assessments involving brain imaging, pharmacogenomics, psychosocial function, and positive and negative symptoms. The drug will be synthesized through the University of California Irvine, where it was discovered, and tested at the University of Colorado Denver, which has previous experience with the clinical evaluation of agonists of the alpha7-nicotinic receptor for neurocognitive dysfunction in schizophrenia.

Aim 1 is to perform a Phase 1 safety and pharmacokinetic study of UCI-40083 in healthy persons. The study will test an escalating range of single oral doses to determine if adequate plasma levels are reached and what side effects are encountered. Specific EEG and neurocognitive monitoring will be performed, because the primary target is brain receptors. The study will be performed first in non-smokers and then extended to persons who smoke to assess possible interaction with nicotine. Aim 2 is to perform a Phase lb safety, pharmacokinetic, and preliminary efficacy study of UCI-4083 in persons with schizophrenia. This single oral dose double-blind placebo-controlled, study will be modeled on the results ofthe Aim 1 study, with a dose range identified by Aim 1 as safe and adequate to achieve putatively therapeutic levels. In previous work with the a7-nicotinic receptor selective partial agonist 3-2,4 dimethxoybenzylidene anabaseine (DMXB-A), we performed a similar Phase lb single dose study. In addition to the primary outcome of improvement in neurocognition, we used the inhibition of auditory cerebral evoked response to repeated sounds, measured by the P50 wave ofthe auditory evoked potential, to assess the neurobiological response to the drug. Animal model studies with UCI-40083 have shown positive effects on this same inhibitory function (Project 3). Thus, this project is the human translation equivalent of Project's 3's animal modeling ofthe effects of UCI-40083. Aim 3 is to perform an initial Phase 2 proof-of-principle clinical study of UCI-40083 in schizophrenia. Persons with schizophrenia will receive UCI-4083 for 4 weeks in an outpatient, placebocontrolled double-blind study. The primary outcome will be performance on the NIMH MATRICS Consensus Cognitive Battery. To assess neurobiological effects, functional MRl studies during eye tracking and the default mode will be added to the P50 auditory evoked potentials recorded in Aim 2. Pharmacogenomic effects of single nucleotide polymorphisms in the CHRNA7 promoter were observed with DMXB-A and we will therefore investigate whether similar effects are seen for UCI-40083.