Uwe Rudolph - US grants

DESCRIPTION (provided by applicant): This is an application for funding of a study examining the role of a2-containing GABAA receptors animal models of depression. It has been shown previously that mice which display reduced GABAergic inhibition (32 mice) display a phenotype reminiscent of depression. It is not known which GABAA receptor subtypes mediate antidepressant-like actions, while we have recently obtained preliminary data suggesting that 13-containing GABAA receptors mediate prodepressant-like actions. Whereas dopaminergic neurons in the ventral tegemental area express primarily a3-containing GABAA receptors, the GABAergic neurons in the nucleus accumbens primarily express a2-containing GABAA receptors. Using mice lacking the a2 subunit, we want to test the hypothesis that a2-containing GABAA receptors mediate an antidepressant-like action in the despair-based forced swim test, the conflict-based novelty-suppressed feeding test and the reward-based intracranial self-stimulation paradigm. The identification of the GABAA receptor subtype responsible for antidepressant-like effects would represent an important advance for understanding mood regulation and would provide a new avenue for the development of novel antidepressant agents. Depression and related mood disorders are among the greatest public health problems;severe forms of depression affect 2-5% of the U.S. population, and up to 20% of the population suffer from milder forms of the illness. Symptoms of depression include a reduced ability to experience reward (anhedonia), dysphoria, anxiety and despair, and a substantial number of patients do not respond satisfactorily to current treatment options. Identification of the a2-containing GABAA receptor as having antidepressant and in particular reward-enhancing functions would identify this receptor subtype as a target for the development of antidepressants with a novel mechanism of action.

DESCRIPTION (provided by applicant): Human genetic studies have linked genes encoding GABAA receptor ? subunits to mental illnesses like major depression, bipolar disorder, schizophrenia, alcohol dependence, and illicit drug dependence. However, the functions of the products of these genes with respect to depressive-like behavior and abuse potential are unknown. In this application, we propose to study the role of different GABAA receptor subtypes defined by the presence of the ?1, ?2 or ?3 subunits in reward-related behavior using the intracranial self-stimulation paradigm. Diazepam (ValiumR), a non-subtype-selective benzodiazepine decreases the reward threshold, however, the GABAA receptor subtype mediating this action is unknown. The effects of zolpidem (AmbienR), a partially ?1-selective hypnotic agent that - like diazepam - is also self-administered on reward have not been examined. We hypothesize that ?1- containing GABAA receptors on GABAergic interneurons in the ventral tegmental area and ?2-containing GABAA receptors on GABAergic neurons in the nucleus accumbens mediate a decrease of the reward threshold ("antidepressant-like" action), while ?3-containing GABAA receptors in dopaminergic neurons of the ventral tegmental area mediate an increase in the reward threshold ("pro-depressant-like action"). Using knock-in mice carrying point mutations in the ?1, ?2, or ?3 subunit rendering the respective GABAA receptors insensitive to modulation by diazepam and zolpidem, we will dissect the contribution of individual receptor subtypes to the reward-modulating action of these agents. The identification of the GABAA receptor subtypes regulating reward-related behaviors is of relevance for the development of novel antidepressant drugs and also for the prediction of the dependence and abuse liability of novel subtype-selective compounds, e.g. for the treatment of anxiety disorders, insomnia or chronic pain, based on specific efficacy at defined GABAA receptor subtypes. PUBLIC HEALTH RELEVANCE: The proposed project will investigate the roles of three GABAA receptor subtypes which are expressed in the mesolimbic dopamine system in reward-related behavior with the hypothesis that some GABAA receptor subtypes will be reward-reducing while others will be reward-enhancing. Specific agonists at reward- enhancing GABAA receptor subtypes might be suitable for the treatment of depression, whereas specific agonists at reward-reducing GABAA receptor subtypes might be suitable for the treatment of manic episodes. Moreover, as GABAA receptor subtype-selective agents are currently being developed for the treatment of anxiety, insomnia, and chronic pain, knowledge on the reward-modulating functions of individual GABAA receptor subtypes will be important for the design of compounds with a low abuse liability.

DESCRIPTION (provided by applicant): This is an application for funding of a study examining the neurobiology of fear and anxiety with a special emphasis on the role of synaptic ?2-containing GABAA receptors and extrasynaptic ?5- containing GABAA receptors. The ?2-containing GABAA receptors, which have been shown to mediate the anxiolytic-like action of diazepam in ethological tests of anxiety will be deleted from hippocampal principal neurons in the CA1, CA3, and DG subregions, respectively, and from the basolateral amygdala using cre- loxP-mediated recombination to elucidate subregion-specific functions and neuronal circuits (e.g. hippocampal trisynaptic and monosynaptic pathways) involved in modulation of anxiety. While the function of neurons in defined hippocampal subregions in learning and memory tasks has been extensively studied, the functions of these neurons in the regulation of emotions is still unknown. Preliminary results indicate a role for extrasynaptic ?5-containing GABAA receptors in extinction of conditioned fear, and based on this observation we propose experiments directed towards the development of a novel therapeutic strategy to promote extinction, which is a major goal in the treatment of posttraumatic stress disorder. Anxiety disorders are the most prevalent type of psychiatric disorders in the community, affecting approximately 10%-15% of the population. This proposal will investigate the functional role of GABAA receptor subtypes in defined neuronal circuits in the hippocampus and the amygdala for the modulation of anxiety-related behaviors. It is expected that the proposed research will identify the ?2- and the ?5- containing GABAA receptors as separate molecular targets for the treatment of distinct anxiety disorders, e.g. generalized anxiety disorder and posttraumatic stress disorder.

DESCRIPTION (provided by applicant): Human genetic studies have linked polymorphisms in the gene encoding the GABAA receptor ?2 subunit, GABRA2, to substance abuse, but neuronal mechanism and brain regions involved are still unknown. Recent experimental evidence suggests that a global deletion of ?2-containing GABAA receptors abolishes cocaine-induced behavioral sensitization;however, it was also reported independently that during cocaine sensitization the ?2 subunit is downregulated in the nucleus accumbens. We propose to use a model system utilizing our recently developed Gabra2 floxed mouse line in which recombinant adeno- associated virus expressing cre recombinase causes a knockdown of ?2-containing GABAA receptors in the nucleus accumbens in order to evaluate whether these receptors are required for cocaine sensitization and cocaine-induced conditioned place preference. PUBLIC HEALTH RELEVANCE: genetic studies have revealed that polymorphisms in the GABRA2 gene encoding the GABAA receptor ?2 subunit are associated with substance abuse. The proposed project will investigate whether ?2-containing GABAA receptors in the nucleus accumbens are required for the the expression of addiction- related behaviors. If it is determined that ?2-containing GABAA receptors promote addiction-related behaviors, ?2-selective antagonists might be useful in the treatment of drug addiction.

DESCRIPTION (provided by applicant): This is an application for funding of a study on the role of extrasynaptic alpha5-containing GABA-A receptors in the CA1 and CA3 subfields and in dentate gyrus and inhibitory hippocampal networks for the amnestic action of the general anesthetic etomidate, using a combination of genetic, behavioral and electrophysiological methods. In his previous position at the University of Zurich, by the generation and analysis of mice carrying a point mutation in the alpha5 subunit of the GABA-A receptor, which leads to a partial knockout of this subunit, the applicant's group demonstrated that GABA-A receptors containing the alpha5 subunit modulate defined memory functions. Using novel animal models that have recently been developed in the applicant's laboratory, we propose to characterize the physiological and pharmacological functions of the alpha5 subunit in CA1 and CA3 pyramidal cells and dentate gyrus granule cells of the hippocampus, respectively, using a combination of genetic, electrophysiological, pharmacological, and behavioral techniques, with the major focus on the mechanism of the amnestic actions of general anesthetics. PUBLIC HEALTH RELEVANCE: Intraoperative awareness during general anesthesia has been suggested to occur with an incidence of 0.13% translating to approximately 26,000 cases per year in the United States. The proposal is directed at elucidating the mechanism of action of general anesthetics on learning and memory and the hippocampal inhibitory networks mediating this effect and is expected to lead to novel therapeutic strategies for the development of amnestic drugs to be used in general anesthesia, for the treatment of posttraumatic stress disorder, and potentially also for the development of memory-enhancing drugs e.g. for the treatment of Alzheimer's disease.

DESCRIPTION (provided by applicant): The relevance of copy number variations (CNVs), i.e. deletions and duplications in psychiatric disorders has recently been discovered. Large-scale genome surveys show an association between deletions on chromosomes 1q21.1 and 15q13.3 and schizophrenia with odds ratios of 7 to 18. We now propose generating two preclinical etiological models of schizophrenia using chromosome engineering to model these hemizygous deletions in murine embryonic stem cells and live animals. Mice carrying hemizygous deletions corresponding to those found in schizophrenic patients will undergo an initial behavioral characterization with an emphasis on examining sensorimotor gating and working memory deficits and are expected to be valuable tools for investigating the phenotypic potential of strong risk factors of schizophrenia and in particular the biological mechanisms underlying this increased risk. PUBLIC HEALTH RELEVANCE: Schizophrenia is a severe mental disorder affecting approximately 1% percent of the population worldwide but our knowledge about the pathogenesis of the disease is critically limited. Based on human large-scale genome-wide surveys identifying an association between specific copy number variations on chromosomes 1q21.1 and 15q13.3 and schizophrenia, we will generate two novel etiological models of schizophrenia using chromosome engineering modeling these hemizygous deletions in mice. Examination of these mice will be important for increasing our understanding of the pathogenesis and pathophysiology of the disease and for the development of novel treatment strategies.

DESCRIPTION (provided by applicant): There is converging preclinical and clinical evidence that GABAergic deficits are an important factor in major depressive disorders (MDD). However, the neuronal circuits in which these deficits exist and the individual GABAA receptor subtypes that modulate depressive-like behavior are unknown. By analyzing the response to chronic social defeat stress, a rodent model of depression with construct, face, and predictive validity, we propose to study how ?2- and ?3-containing GABAA receptors in defined neuronal circuits play a role in the behavioral transition from a normal state to a down (i.e. depressed) state and vice versa. Using a novel genetic-pharmacological approach we will further assess whether a highly ?2- specific [or ?3-specific] agonist administered during social defeat can prevent [or promote] the transition from the normal state to the down state. We will also assess whether such an agonist can acutely promote [or prevent] the transition from the depressed state back to the normal state when administered after cessation of chronic social defeat. The proposed studies are expected to demonstrate that ?2-specific agonism generates an acute antidepressant-like effect and will provide proof-of-concept for the development of a novel class of antidepressant agents.

DESCRIPTION (provided by applicant): GABAA receptors are the target of clinically used benzodiazepines, which modulate multiple GABAA receptor subtypes non-selectively. While some progress has been made in identifying differential functions of GABAA receptor subtypes based on loss of function approaches which opens up the possibility to design novel treatments for CNS diseases such as anxiety disorders and depression, until now truly subtype-specific compounds are not available, thus limiting the information that is available on the function of individual GABAA receptor subtypes. Here we propose to use a novel combined genetic and pharmacological approach to create a model system (triple point-mutated mice + the non-selective drug diazepam) in which diazepam is a true ?1-specific, ?2-specific, ?3-specific, or ?5-specific full agonist, respectively, enabling the highly selective modulation of the activity of specific GABAA receptor subtypes in order to define the physiological and pharmacological functions of these receptor subtypes, in particular in the regulation of anxiety- and depression-related behaviors. PUBLIC HEALTH RELEVANCE: GABAA receptors are the target structures of clinically used benzodiazepines; however, we have only an incomplete knowledge of the function of individual GABAA receptors subtypes, in large part due to the lack of fully subtype-specific compounds. We are proposing a novel genetic and pharmacological approach to assess the anxiety-reducing and antidepressant-like properties of specific GABAA receptor subtypes. The proposed experiments are expected to clarify the potential role of specific GABAA receptor subtypes as targets for the development of novel antidepressant drugs.

DESCRIPTION (provided by applicant): Benzodiazepines are among the most widely abused drugs, and like other drugs of abuse, they hijack the brain's dopaminergic reward system, in which several GABAA receptor subtypes are expressed in different neuronal cell types. The modulation of the reward system and of benzodiazepine self-administration by distinct GABAA receptor subtypes is only poorly understood. Here, we want to test the hypothesis that GABAA receptors bidirectionally modulate these behaviors. In the absence of chemical compounds which are truly specific for a GABAA receptor subtype, we propose to use a novel combined genetic and pharmacological approach to create a model system which will enable highly specific modulation of the activity of individual GABAA receptor subtypes. This will be achieved by using the non- selective benzodiazepine drugs diazepam and midazolam in triple point-mutated mice, in which these drugs are a true ?1-specific, ? 2-specific, ? 3-specific, or ? 5-specific full agonists, respectively. This system will make it possible to test whether potentiatio of a particular GABAA receptor subtype is sufficient for reward enhancement and benzodiazepine self-administration.

DESCRIPTION (provided by applicant): The genetics of developmental disorders like bipolar disorder and schizophrenia are complex, and an overlap in the genetic factors contributing to these two disorders has been found. While common variants are typically associated with small effect sizes, individually rare variants in particular copy number variants (CNVs), i.e., deletions or duplications of genomic segments, have been associated with large effect sizes, even up to an odds ratio of 63. Potentially, the relatively large effect size of CNVs could result from severa genes in the CNVs contributing to the phenotypic changes, potentially in converging pathways. We have used chromosome engineering techniques (gene targeting and cre-loxP-mediated trans-allelic recombination in vivo) to develop mice modeling a 9p24.1 duplication/triplication found in a family affected by bipolar disorder and schizoaffective disorder. As overlapping deletions have been found in other families affected by schizophrenia, we propose to study mice with 1 (deletion), 2 (wt), 3 (duplication) and 4 (triplication) copies of the 15 9p24.1 genes (collectively called 9p24.1 CNV mice). The basic hypothesis is that changes in 9p24.1 copy number, in particular increases, are sufficient to elicit phenotypic changes reminiscent of major neuropsychiatric disorders. The CNV region includes GLDC, UHRF2 and the micro RNA miR-4665. GLDC is a smoking gun. Increased expression of GLDC could result in increased degradation of glycine, a co-agonist at the NMDA receptor. NMDA receptor hypofunction has been shown to result in schizophrenia-related phenotypes in mice. We will investigate the level of glycine and D-serine, another co-agonist at the NMDA receptor at the same site, and schizophrenia-like behavior in the 9p24.1 CNV mice to test the hypothesis that duplication or triplication of GLDC may contribute to neuropsychiatric phenotypes. UHRF2, which is expressed in hippocampus and subcortical plate, encodes an ubiquitine modifier (SUMO) E3 ligase which is an important mediator of E2F1-mediated apoptosis. As increased expression of apoptotic genes has been found in bipolar disorder, an increase in UHRF2 copy number could result in increased apoptosis. miR-4665, specifically miR-4665-5p is poorly characterized so far, but has computationally predicted target genes (MECP2, ANK3, PAX5) that have been linked to neurodevelopmental disorders: MECP2 to Rett Syndrome, and ANK3 and PAX5 actually to bipolar disorder. By monitoring changes in gene expression globally, we will examine whether changes in 9p24.1 copy number will lead to changes in the expression of genes outside of this region which may be mediated by miR-4665-5p. In summary, we propose to determine copy number effects in the 9p24.1 region with specific hypotheses concerning how they may contribute to neuropsychiatric-related phenotypes. The studies form the basis for even more targeted interrogation of the role of individual genetic factors in the 9p24.1 region in the development and expression of major neuropsychiatric disorders.