Marc Caron - US grants

This application presents a comprehensive research program aimed at characterizing a hormone/neurotransmitter receptor, its mechanism of action and its physiological regulation at the molecular level. The model system chosen for study is the anterior pituitary dopamine receptor. This receptor mediates the physiological effects of dopamine to inhibit prolactin secretion. There are three specific aims. 1) To characterize the biochemical mechanisms by which the binding of dopamine to its specific anterior pituitary receptor is transduced into the physiological response inhibition of prolactin secretion; 2) To solubilize, purify and characterize the dopamine receptors as well as identify the binding subunit of the receptor by affinity and photoaffinity labelling; 3) To determine the molecular mechanisms of endocrine regulation of the dopaminergic receptors and hence of dopaminergic responsiveness. Drawing on the previous experience with purification and photo-affinity labelling of the beta-adrenergic receptors, new specific procedures and probes will be developed for purification of the receptor protein itself in order to pave the way for the eventual complete biochemical and physicochemical characterization of the dopaminee receptor. These results and procedures should be widely applicable to other receptor/neurotransmitter systems such as those in the central nervous system. Moreover, the results should shed further light on our understanding of the endocrinological regulation of receptor expression in target tissues in particular with regard to the regulation of prolactin secretion. Such information will facilitate the development of new and more efficacious forms of treatment for endocrinological and autonomic disorders, particularly those which may involve altered prolactin secretion or dopaminergic function. This project constitutes an integral part of the overall long term objective of this laboratory to characterize at the molecular level the various catecholamine receptors. The results of these and other ongoing studies should therefore provide a comprehensive and fundamental understanding of how catecholamine responsiveness is mediated and modulated at the receptor level in both physiological and pathophysiological circumstances.

This grant is requested to support a basic research program aimed at elucidating at the molecular level the nature, mode of functioning and mechanisms of regulation of the specific receptors for dopamine. Physiologically, dopamine exerts effects such as regulation of hormone synthesis and release (prolaction, alpha-melanocyte stimulating hormone and parathyroid hormone) in the periphery and control of behavioral and motor functions in the central nervous system (CNS). These effects are mediated by two distinct receptors (D1 and D2) which are coupled to stimulation (D1) and inhibition (D2) of adenylate cyclase as well as inhibition (D2) of the phosphatidylinositol/calcium signal transfer system. To accomplish the four specific goals of this proposal, studies will be conducted using three main approaches 1) to obtain detaled molecular information about the nature of D1 and D2-receptors we will purify and characterize these proteins from bovine anterior pituitary and corpus straiatum, by newly developed methods of affinity chromatography and affinity and photoaffinity labeling. 2) to understand the way in which agonist occupancy of receptors is translated into the generation of an intracellular signal and the relationship of this signal to the physiological response we will identify the components involved in these systems and perform reconstitution studies with purified components. The D2-receptor represent a unique model to study the relationship between signal transduction systems since this receptor can presumably couple to two signal transfer systems within the same cell. 3) In order to elucidate the biochemical mechanisms by which the function of these receptors might be modulated in vivo, we will examine the receptor-effector coupling in whole cells and reconsitituted systems after treatment of animals or cells with dopamine agonists, neuroleptics and steroids, agents known to modulate dopaminergic responsiveness in target tissues. These studies should provide the complete elucidation of the biochemical nature of these receptors and the mechanisms of signal transduction as well as the mechanisms by which responsiveness of target tissues is controlled. Modulation of dopaminergic responsiveness appears to be an important factor in the control of pituitary function (hyperprolactinemia/amenorrhea) as well as the normal functioning of CNS behaviour and motor functions (schizophrenia, Parkinsons disease and tardive dyskinesia). This project constitutes an integral part of the overall long term objective of this laboratory to characterize at the molecular level the various receptors for catecholamines.

This grant is requested to support a basic research program aimed at elucidating at the molecular level the nature, mode of functioning and regulation of the specific receptors for dopamine. Physiologically, dopamine exerts effects such as regulation of hormone synthesis and secretion in the periphery and control of cognitive, affective and neuroendocrine functions in the central nervous system (CNS). For a long time, these effects of dopamine were thought to be mediated by only two distinct G protein-coupled receptor subtypes Dl and D2, coupled respectively to stimulation and inhibition of adenylyl cyclase. Recent developments from several experimental approaches but in particular molecular biology now suggest that the family of dopamine receptors will be much larger than previously anticipated. The cDNAs and genes of three distinct dopamine receptor subtypes have already been isolated. In order to characterized these systems, the work proposed in this application (six specific aims) can be divided into two main lines of investigations. First, we want to determine using the molecular biology approach the extent of this receptor family and determine the physiological significance of the apparent heterogeneity of receptors for dopamine. Second, we want to examine the biochemical mechanisms of action, regulation and interaction of the various dopaminergic receptor systems as they may underlie pathophysiological situations. In order to characterize these mechanisms at the molecular level we propose to develop procedures for the over-expression and purification of every member of this receptor. family using the baculovirus-infected insect cell system. Elucidation of the basic molecular mechanism of signal transduction and regulation of the different subtypes of dopamine receptors may help in understanding the etiology of certain CNS disorders such as schizophrenia, Parkinson's disease or peripheral disturbances such as hyperprolactinemia. This project constitutes an integral part of the overall long term objective of this laboratory to characterized at the molecular level the Various receptors for catecholamines.

Drug addiction places tremendous social, medical and economic burdens on society. Recent developments in this area have contributed to the notion that drug abuse is a problem with definable biological underpinnings. Despite this progress, the molecular organizing principles responsible for susceptibility to the initiation and maintenance of drug taking behavior have remained elusive. Modalities for therapeutic management of the condition are also lacking. In this application, we propose to use novel and complementary approaches to identify molecular factors that underlie drug seeking behaviors. We will take advantage of several recently established lines of mice in which different genes involved in monoaminergic neurotransmission have been inactivated. The different lines all show a sensitization to the locomotor enhancing effects of psychostimulants that is similar to that seen after chronic drug treatment. We will use a genome- wide mutational approach (ENU mutagenesis) to screen for second site mutations that modify the phenotype of existing knockout mutations. This approach has been pioneered in other genetic systems where it has proven to be a uniquely powerful tool. We will also characterize mutant mice using neurochemical and biochemical approaches and High Density Oligonucleotide Array technology to characterize gene expression profiles in the central nervous system. This should allow identification of genes involved in stable alterations of the CNS associated with chronic drug exposure.

Abstract Not Provided.

DESCRIPTION (provided by applicant): The G protein-coupled receptors (PCRs) comprise a broad family of receptors that activate a large number of effectors in response to a variety of signals including amines, photons, lipids, peptides and proteases. Signaling through CPCRs requires the coordinated balance between proceses that govern receptor activation, desensitization and resensitization. Desensitization of GPCRs involves receptor phosphorylation by specific G protein-coupled receptor kinases (GRKs) and interaction with arrestin proteins. GRKs and arrestins play an important role not only to desensitize second messenger signaling but also contribute to the endocytosis of GPCRs and their ability to recycle back to the plama membrane and resensitize. In addition, the complex of receptor/arrestin engages a variety of signaling pathways including those associated with Src family kinases and components of the MAP kinase cascades. The overall objective of the proposed research is to define the basic molecular and cellular mechanisms that contribute to these processes with the goal of better understanding physiological and pathological conditions. The dopamine and adrenergic receptors will be used as prototypic GPCRs for many of the proposed studies. Aim 1: We will attempt to identify the molecular determinants by which the specificity of action of GRKs and arrestins is established. In addition, we will examine how constitutive phosphorylation and interaction of GPCRs with arrestin can underlie the loss-of-function phenotype of certain mutant GPCRs both in cellular and in vivo systems. Aim 2: The molecular basis of the ability of arrestins to act as endocytic switch and control the resensitization process of GPCRs will be examined. Aim 3: How the pardigm of GRKs and arrestins might apply to non-conventional GPCRS such as frizzled and smoothened will also be examined both in cellular and in vivo model systems. Aim 4: These experiments will explore the hypothesis that the behavioral and biochemical synergisms that exist between dopamine D 1 and D2 receptors results from the oligomerization of these receptors. Results from these studies should broaden our understanding of the role of GPCRs and their regulation in normal physiology and disease states and provide insight into potential new therapeutic approaches for conditions such as heart failure, drug abuse, and psychiatric disorders.

DESCRIPTION (provided by applicant): Acetylcholine secreting neurons in the brain are thought to control several types of distinct behaviors. Central cholinergic dysfunction is one of the hallmarks of certain brain diseases such as Alzheimer's disease, in which cholinergic hypofunction has been suggested to affect attention and memory. The physiological roles of acetylcholine in learning, memory and attention are still poorly understood, likely because there is no model system where cholinergic hypofunction can be selectively achieved in brain regions. Our long term goal is to understand the actions of acetylcholine in brain function in mammals. We would like to understand how acetylcholine secretion controls neurochemical systems in discrete brain regions and how this affects behavior. To achieve this goal, a collection of mouse lines with deficits of acetylcholine release will be generated using gene targeting techniques and the Cre/loxP system. These mouse lines will be characterized biochemically for the consequences of lack of acetylcholine secretion, particularly in the cortex, hippocampus and striatum. Several biochemical markers and the release of acetylcholine will be assessed. Suitable mouse lines that shown brain region deficits of acetylcholine release with preserved peripheral function will be used to investigate possible roles of acetylcholine in brain function, by using distinct behavioral paradigms. This approach will allow understanding the actions of acetylcholine in memory and attention and may reveal novel physiological functions for this neurotransmitter. We envision that these mice will help to answer long standing questions on the relationship between cholinergic dysfunction and Alzheimer's disease. The research proposed will be done primarily in Brazil, at the Federal University of Minas Gerais in collaboration with Dr. Marco A.M. Prado, Associate Professor of Pharmacology in the Institute of Biological Sciences, as an extension of the grant "Genetic Analysis of Dopaminergic Reward Mechanisms" #RO1-DA131511-01.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Dopamine is a neurotransmitter that has been implicated in the pathogenesis of several neuropsychiatric disorders, such as Parkinson's disease, schizophrenia, drug and alcohol abuse, Huntington's disease and attention-deficit hyperactivity disorder (ADHD). Dopamine transporter is a membrane-bound protein that reuptakes the released dopamine from the extracellular space back into presynaptic nerve terminals, and is the main regulator extracellular dopamine levels. Mice lacking the dopamine transporter have persistently elevated extracellular dopamine concentrations in the striatum, the main target region of dopaminergic innervation in the brain. In addition to displaying behavioral symptoms reminiscent of chronic psychostimulant abuse and schizophrenia, these mice sporadically develop progressive dyskinetic motor symptoms associated with striatal neurodegeneration resembling Huntingon's disease. The aim of the proposed studies is to investigate in detail the consequences of sustained hyperdopaminergia on striatal neuronal anatomy. This will be accomplished by acquiring and comparing histological data using very high resolution magnetic resonance imaging (MRI) (at 9.4 T), conventional immunohistochemistry, and ultrastructural imaging modalities including electron microscopy. The results will help to understand the effects of chronically high striatal dopamine levels, and may lead to better understanding of neurobiology of schizophrenia, drug addiction and Huntington's disease.

The various actions of dopamine (DA) on target neurons are mediated via prototypical 7-transmembrane G protein-coupled receptors (GPCR) that couple to various effectors through G protein-dependent mechanisms. However, it is now widely appreciated that GPCRs can also signal through the ability of the adaptor protein ?arrestin to scaffold signaling complexes that are distinct from canonical G protein signaling. These dual signaling modes may enable what is commonly referred to as functionally selective or biased signaling. We have shown before that the dopamine D2 receptor (D2R), which is the main target of clinically effective antipsychotics, mediates some of its physiological effects through engagement of a ?arrestin2/Akt/PP2A/GSK3? signaling complex. During the initial portion of this R37 award, we have used genetic, biochemical, and pharmacological approaches to provide concrete evidence that D2R/?arrestin2 signaling is important in behavioral responses following activation of the DA system. Interestingly, we discovered using neuronally selective deletions of ?arrestin2 in mice that an antipsychotic-like D2R/?arrestin2 biased tool compound UNC9994A behaved as an antagonist in the striatum but an agonist in the cortex. These results correlate with both higher levels of GPCR kinase and ?arrestin2 in cortex versus striatum and the ability of UNC9994A to reverse deficits in a mouse model of cognitive/sociability functions. These results suggest that ?arrestin2/D2R signaling may be an unappreciated means to control cognitive and social domains of behavior in vivo. The goals of our R37 continuation application are to use the genetic and biochemical tools we have developed, like G protein or ?arrestin2 preferring mutant D2Rs, to identify cell type specific molecular and biochemical mechanisms involved in the control of these behavioral domains. Our Specific Aims are: 1) determine the impact of D2R biased signaling on cognitive domains in mice reconstituted with biased D2R mutants; 2) identify the molecular and neuronal mechanisms underpinning the cognitive and social effects and 3) assess how D2R biased signaling in the cortex controls neuronal electrophysiology and affects downstream brain circuits to control cognitive and sociability functions.

DESCRIPTION (provided by applicant): An important goal of this Center application to the National Institute on Drug Abuse (NIDA) is to foster the development of new approaches for drug addiction treatment and prevention. We propose to facilitate this strategy by providing an enabling resource for NIDA investigators that could accelerate the progress of their addiction research, including but not limited to the discovery of novel small molecule compounds. The primary area of focus of our program is the gene families represented by orphan and identified Seven Transmembrane G protein-coupled receptors (GPCRs). With NIDA support over the past three years we have established a cDNA collection containing the open reading frames for almost all human addiction associated GPCRs, and more importantly an expanding repository of off-the shelf cell-based assays for over half of the GPCR targets of interest to NIDA funded scientists, with a goal of quickly progressing to the remaining cell assays. Our efforts, formalized as the Duke University Assay Center (DUAC), have produced joint collaborations with NIH/NIDA chemists and biologists at multiple other institutions, including five joint ongoing projects that include the Molecular Libraries Probe Production Centers Network (MLPCN). To continue our program at the current level of effort we are seeking funding as a NIDA P30 Center of Excellence for four years. As a Center of Excellence the DUAC would continue to be at the forefront of drug addiction research as a consequence of enabling technology and synergizing collaborations with other NIDA scientists. The primary scope of our work would include the identification and in cellulo and in vivo characterization of novel tool compounds. Our specific alms entail: 1)Development and maintenance of receptor cDNA and cell assay libraries, particularly containing fluorescent beta-arrestins and NIDA GPCR targets, for immediately access by NIDA investigators. 2) Screening in a timely manner (days to weeks turnaround) of receptor targets at DUAC against limited libraries (1-5,000 compounds) provided by us or the collaborating scientists. And 3) Establish projects aimed towards the MLPCN for discovery of novel tool compounds This collaborative strategy should expedite the identification tool compounds to characterize the biology of addiction and provide an educational resource for collaborating scientists in drug discovery technology.

Depression is a psychiatric disorder where disturbance of mood is a prominent feature. Although the[unreadable] etiology of depression is unknown, alterations in serotonergic and noradrenergic function are[unreadable] implicated in the condition. The present Conte Center Grant proposes to examine depression from[unreadable] perspectives of functional imaging, morphometrics, and pharmacological interventions in humans, as[unreadable] well as though mouse genetic, molecular, biochemical, behavioral, and electrophysiological models[unreadable] of depression. The overall objective of the present proposal is to show how the norepinephrine (NE)[unreadable] and serotonin (5-HT) systems are interdependent in the development and amelioration of symptoms[unreadable] of depression, and to reveal novel mechanisms that contribute to the disorder. There are three major[unreadable] Aims. AIM 1: The roles in depression of the NE transporter, 5-HT transporter, and vesicular[unreadable] monoamine transporter 2 will be investigated. Effects of antidepressants on monoamine and[unreadable] metabolite levels in various brain regions will be studied. Molecular fingerprinting of signal[unreadable] transduction pathways will be used to analyze effects of genotype and antidepressant treatment. AIM[unreadable] 2: The role in depression of glycogen synthase kinase-3beta (GSK3b) will be analyzed. Mice will be[unreadable] developed that have GSK3b selectively deleted in the CNS. Animals will be evaluated according to[unreadable] behavioral, neurotransmitter, and signal transduction responses to antidepressants. AIM 3: Mice will[unreadable] be made that bear the same polymorphisms in tryptophan hydroxylase 2 (Tph2) found in human[unreadable] depressed patients. Mutants will be examined according to behavioral, neurotransmitter, and signal[unreadable] transduction responses to antidepressants and electroconvulsive therapy.

DESCRIPTION (provided by applicant): The brain serotonin (5HT) system plays a critical role in numerous neuronal functions and dysregulation of its homeostasis contributes to many psychiatric disorders. Numerous conditions such as major depression, obsessive compulsive disorder, anxiety and panic disorder, are effectively treated by raising the extracellular concentrations of brain 5HT using selective serotonin re-uptake inhibitors (SSRI). Tryptophan hydroxylase (TPH2) the rate-limiting enzyme for the synthesis of 5HT is expressed selectively in neurons while TPH1 is expressed peripherally. There is a high degree of sequence conservation between TPH1 and TPH2 and other mono-oxygenases for tyrosine (TH) and phenylalanine (PAH). We initially identified a single nucleotide polymorphism (SNP) in the mouse Tph2 gene that results in a ~ 50% decrease in the in vitro activity of the enzyme that correlates with a similar reduction in brain 5HT synthesis and content in inbred mice carrying the SNP. A similar mutation in the human TPH2 (R441H) was then identified in a cohort of elderly unipolar depressed patients, which decreased the activity of the enzyme by >80% and was associated with reduced responsiveness to therapeutic effects of SSRI in the patients. A mutation at the exact same codon in PAH represents the most severe and prevalent mutation in phenylketonuria where more than 300 coding mutations have been identified. Several additional SNPs in the coding regions of human TPH2 have recently been identified but not yet characterized. We have developed a knockin mouse carrying a (R439H) mutation in mTph2, equivalent to the (R441H) mutation in humans. We are also generating congenic mouse lines carrying the functional (C1473G) SNP in mTph2 in both C57BI/6J and BALB/cJ backgrounds. The overall goal of this proposal is to determine the neurochemical and behavioral consequences of this mutation and establish the biological significance of such mutations in the human TPH2 gene. There are three Specific Aims: 1) Neurochemical and behavioral characterization of Tph2(R439H) knockin and congenic mouse lines;2) Sequence anlysis of TPH2 in different cohorts of 5-HT-related neuropsychiatric conditions and 3) Characterization of novel functional human TPH2 mutants in cellular systems and genetically modified animals. Results from these experiments should provide potential genetic determinants for 5-HT-related disorders as well as useful animal models to explore the actions of and responsiveness to SSRI and test new therapeutic approaches.

The goal of the Program in Cell Regulation and Transmembrane Signaling is to foster scientific interactions between members of the Duke Comprehensive Cancer Institute (DCCI) who have interest in understanding the biology involved in regulating cell proliferation and death. The Program continues to attract individuals interested in aspects of cell signaling. Currently, the Program includes 36 members from 9 different basic and clinical departments. To foster an outstanding forum for scientific discourse and collaboration, the Program remains organized into one of four interest groups that include 1) transmembrane signaling; 2) intracellular signaling; 3) cell proliferation and death; and 4) signaling and disease. The program sponsors three distinct educational activities, the University-wide weekly seminar series The Signal Transduction Colloquium (STQ), the most attended series on campus; a graduate course in Cellular Signaling, taken by the majority of biomedical graduate students; and an Annual Offsite Retreat. The last two years' retreats each featured more than 20 platform and 100 poster presentations by members, students and fellows. Speakers for the STQ selected by a panel of Program members come primarily from outside the Duke University Medical Center to represent the broad interests of the membership (See list of invited speakers since 2004, Appendix PI). Each colloquium speaker meets with interested members of the Program and lunches with students in the Molecular Cancer Biology Graduate Program immediately after completion of the seminar. The diversity of experimental approaches used by the members of this Program represents an effective asset for promoting cross-fertilization of ideas aimed at understanding cell growth and regulation. During the last grant period, members of the Program have published 800 papers in primary peer-reviewed journals of which 732 or 90% bear directly on cancer-related problems. Finally neariy 15% of the cancerrelated publications (98) are the result of intra-programmatic or inter-programmatic collaboration among DCCI members. At the present time, there are 36 program investigators.

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DESCRIPTION (provided by applicant): Alterations in serotonin (5-hydroxytryptamine, 5-HT) neurotransmission have long been theorized to play an important role in the pathogenesis of psychiatric disorders, particularly depression. This theory stems from the facts that drugs (e.g., SSRIs) increasing levels of extracellular 5-HT (5-HTExt) treat depression with moderate efficacy and that anomalies in putative biomarkers of central 5-HT function have been repeatedly reported in depression. However, brain 5-HT dysfunction has never been directly demonstrated in depression patients and whether low 5-HT can elicit or predispose to depression remains unclear. The rate-limiting step in brain 5-HT synthesis is the conversion of tryptophan to 5-hydroxytryptophan (5-HTP) by tryptophan hydroxylase 2 (Tph2). The recent identification of functional mutations in several of the genes involved in 5-HT homeostasis, including tph2, and their associations with depression or impaired therapeutic responses to SSRIs have stimulated renewed interest in the 5-HT deficiency theory of depression. We have generated a mouse line carrying a rare Tph2(R439H) mutation originally identified in a depression cohort. In these mutant mice brain 5- HT synthesis and tissue storage are decreased by 80% and 5-HTExt levels by 60-80%, while evoked 5-HTExt responses are qualitatively preserved. The mice recapitulate several anomalies in putative 5-HT biomarkers reported in severe depression and exhibit depression-, anxiety-, and aggressive-like behaviors, seemingly providing a model of the behavioral alterations associated with 5-HT deficits in humans. Thus, our mutant (henceforth 5-HThypo) mice may represent a unique naturalistic model of 5-HT deficiency and, possibly, depression. Plausibly, multiple diverse insults to 5-HT homeostasis could each result in 5-HT deficiency, thus the 5-HThypo mouse likely represents a useful model of 5-HT deficiency in general as well as a model of impaired Tph2 catalytic function. The overall goal of our continued research is to use the 5-HThypo mouse to better understand how 5-HT deficiency contributes to depression etiology and affects antidepressant treatment, including the consequences for stress susceptibility and responses to current and novel therapies. For this goal, we propose four specific aims. Aim 1 will define whether 5-HT deficiency alters susceptibility to stress, as tested in the social defeat and chronic mild stress paradigms. Aim 2 will test whether antidepressant- like responses to SSRI and ketamine are affected by 5-HT deficiency. Aim 3 will pre-clinically test a novel 5- HTP-based antidepressant augmentation concept under 5-HT-deficient and normal conditions. In Aim 4 we will use conventional and state-of-the-art approaches to identify the cellular signaling pathway changes underlying the depression-like behaviors arising consequent to 5-HT deficiency. Collectively, the proposed experiments will address long outstanding questions in depression neurobiology and test a new treatment concept.

DESCRIPTION (provided by applicant): Understanding the genetic mechanisms which underlie tumor development will provide a foundation for developing new generations of better and more effective cancer therapies. To address this fundamental issue, new technologies are needed that are superior to those currently available. Current state of the art mouse models enable testing the stochastic activation of oncogenes in a cell type specific fashion and provide a mechanism for testing targeted therapies. Despite these desirable features, their limited accessibility and feasibility precludes their implementation in most laboratories. Moreover, as the list of oncogenes continues to grow, reliable and efficient technologies are needed that can assess several oncogenes simultaneously in order to facilitate rapid analysis of tumorigenicity and their responses to therapy. This application provides a solution to all these issues by developing an innovative transgenic mouse platform (Crainbow) to rapidly, efficiently, and cost effectively create mouse models of tumorigenesis. The Crainbow technology will enable multiple user selectable oncogenes to be incorporated into a single transgenic mouse whereby each oncogene will be identifiable on the basis of a unique epitope tag and a separately expressed spectrally resolvable fluorescent protein. Using a strategy of Cre induced stochastic activation, the Crainbow technology enables the contribution of each oncogene to be assessed coincidentally in the same tissue with single cell resolution. Additionally, clonally derived tumor cell populations can be studied in any tissue and cell type by breeding to the plethora of validated cell type specific Cre mouse lines. Underlying the flexibility of this technology is a novel plasmid construct for incorporating the oncogenes of interest. The Crainbow plasmid can be easily modified for oncogene targeting using the molecular biology skills available in most all biology laboratories. Crainbow transgene mice can then be produced rapidly on site or obtained from other investigators or repositories. The utility and versatility of this Crainbow platform technology is demonstrated through the following specific aims which: (1) Establish a flexible and rapid strategy for cancer rainbow transgene design and construction, (2) Generate and validate cancer rainbow transgenic mice, & (3) Generate tissue specific tumors using cancer rainbow transgenic mice. The Cancer rainbow technology addresses in a single mouse shortcomings of current technology by providing a comprehensive method to simultaneously monitor the activity of multiple genes, and a means to also rapidly screen novel targeted cancer therapies. Consequently, the Cancer rainbow technology will provide a paradigm shift for studying tumorigenesis.

? DESCRIPTION (provided by applicant): A Cancer rainbow mouse for simultaneous assessment of multiple oncogenes Genetically engineered mouse models of cancer hold significant promise for studying the basic cellular and molecular mechanisms underlying tumor formation and then evaluating prospective therapies. However, several factors have limited the success of current models and development of next-generation technologies. First, the conceptualization, engineering, and generation of a new mouse strain is outside the scope of most laboratories. These projects encounter high rates of failure and continue to carry significant risk for even the most seasoned and prestigious research programs. Second, establishing roles for tumor driver genes in vivo in important cellular behaviors such as differentiation, proliferation, and migration often requires extensive compound breeding, making these experiments expensive and time consuming. Third, deep-sequencing continues to identify new tumor driver genes at a rate in which the utilization of a one driver gene per mouse paradigm is unable to keep pace. Therefore to meet these demanding needs, we have developed the Cancer rainbow (Crainbow) mouse platform to generate user-defined, reliable, and multiplexed models of tumorigenesis in a single genetically tractable system. In our R21 IMAT funded project, we have demonstrated in vivo proof-of-principle for this technology and are now beyond the initial phases of this project. With this system we are able to stochastically express multiple tumor driver genes in a diversity of tissues and cell-types and simultaneously monitor their effects at a single cell level using spectrally resolvable fluorescent protein reporters. The objective of this R33 proposal is to perform extensive and rigorous testing of our emerging technology to ensure its reliability and accessibility for the scientific community. To achieve this objective, our specific aims will 1) Optimize Crainbow Generation and Expression, 2) Validate Crainbow technology In Vivo, and 3) Validate Quantitative Multiplex Analysis of Driver Gene Activity In Vivo. We expect the Crainbow platform to transform cancer research by enabling all investigators to reliably build mouse models of cancer on-demand and with minimal inherent risk. The flexible and forward-thinking design of Crainbow will also enable robust delivery of genome-editing tools to provide multiplexing of endogenously edited driver genes with cellular and temporally precise resolution. Through multiplex driver gene analysis and enabling of a decentralized user-base, the Crainbow platform will enable synergistic modeling of a diversity of cancers and provide future models for testing personalized therapeutic intervention.

Parkinson?s disease (PD) is a neurodegenerative disorder characterized by severe motor deficits caused by the progressive loss of striatal dopamine (DA) input and it is commonly treated with the DA precursor L-DOPA or by DA D2 receptor (D2R) agonists. Although L-DOPA ameliorates the motor deficits, prolonged use leads to motor abnormalities, termed L-DOPA-induced dyskinesias. Despite these limitations, L-DOPA is the mainstay for PD treatment. Various animal models of PD suggest that dyskinesias are associated with enhanced G-protein mediated signaling at DA receptors (DARs) and this leads to changes in gene expression and uncontrolled neuronal excitability. Research over the past decade has shown that DARs can signal not only through G-proteins, but also also through ?- arrestin2 (?arr2) scaffolds containing signaling complexes that initiate intracellular signals distinct from those of G- proteins. Recently we have demonstrated in PD models that over-expression of ?arr2 in the striata of mouse, rat, or macaque reduces L-DOPA-induced dyskinesias. This novel approach facilitated locomotion and simultaneously desensitized G-protein signaling, thereby reducing the dyskinesia without potentially affecting other neurotransmitter systems. The Overall Goal of the proposed research is to provide preclinical results for taking novel D1R and D2R ?arr-biased compounds into clinical PD trials. Our Central Hypothesis is that small molecule drugs that selectively activate the ?arr, but not the G-protein, pathway at D1Rs/D2Rs will have anti-parkinsonian activities without inducing dyskinesias. We have three Specific Aims. Aim 1 will develop ?arr-biased D1R and D2R ligands from the leads/hits we recently discovered. Aim 2 will assess the in vitro profile of the newly synthesized ?arr-biased compounds. Aim 3 will determine the in vivo effects of D1R and D2R ?arr-biased compounds in animal models of PD.

ABSTRACT Most mental disorders have strong underlying genetic component, with many disease-associated variants occurring in genes involved in neuronal development and synaptic transmission. This suggests that deficits in cellular mechanisms may ultimately alter brain-circuit connectivity to yield dysfunctions in specific behavioral domains. Consistent with the RDoC framework, several independent circuit deficits likely synergize to create a distinct emergent state in large-scale networks ultimately yielding mental disorders. While multiple rodent models have begun to link circuit function with behavior, modeling these complex network-level alterations has been largely intractable with current optogenetic and chemogenetic tools that only target individual circuit elements of complex networks. This proposal presents a solution to this problem by developing and validating a new tool to simultaneously and bidirectionally control distinct brain circuits via chemogenetics. We then apply these reagents to two dopamine (DA) circuits in the brain, the nigrostriatal and mesocorticolimbic circuits. The resulting model will be characterized behaviorally and with state-of-the-art electrophysiological techniques to measure the functional consequences of concurrent bidirectional modulation of DA circuity. In Aim 1, we present the design and validation experiments necessary to generate and verify the correct function of these reagents. In Aim 2, we will use these reagents to associate DA circuit functional status with specific behavioral domain dysfunctions through the use of electrophysiological ensemble recordings during behavioral tasks. After completing these aims, we will have established proof-of-concept that a mouse model can recapitulate distinct and simultaneous modifications in two brain circuits that can be used to identify synergistic or antagonistic interactions between these brain circuits at the network level.

ABSTRACT The prefrontal cortex (PFC), part of the cortico-striato-thalamo-cortical (CSTC) circuit, is a critical regulator of cognition/executive function and social processes. Disruptions to this circuit and these behavioral domains are often associated with several neuropsychiatric disorders. Despite the debilitating nature of these impairments, the challenge to develop therapies that effectively manage this myriad of deficits remains unmet. Although current therapeutics to treat neuropsychiatric conditions primarily target dopamine receptors (DARs), specifically the D2R, they also interact with other D2 subclass of DARs (D3R, D4R). The D4R is of particular interest as it is expressed at higher levels than the D2R in key PFC neuronal populations. Additionally, some of the more clinically effective agents also exhibit high D4R affinity. However, previous attempts to develop clinically effective therapies targeting the D4R have proved unsuccessful. Intriguingly, these efforts occurred prior to recent advances in our understanding of the concepts of GPCR biased signaling i.e. signaling through G proteins or ?-arrestins, which elicit different cellular and physiological outcomes. This, coupled with the ability to engineer cell specific genetic manipulations and the capacity to interrogate brain neural network function in real time and in response to pharmacological or behavioral manipulations, all provide us with an exciting opportunity to address this important challenge. Our collaborators have recently developed a purely D4R-selective partial agonist (UCSF924), and we have discovered that it exhibits remarkable antipsychotic-like properties in pharmacological and genetic animal models. The objective of this proposal is to provide proof-of-concept that functionally-selective targeting of the D4R is necessary to mitigate both the cognitive/executive function and social impairments observed in neuropsychiatric disorders. This is accomplished in the following Specific Aims. Aim 1: Systematic determination of D4R-mediated signaling in PFC neuronal subtypes. Aim 2: Elucidating the effect of selective D4R signaling in the regulation of neuronal networks. In Aim 1, we will utilize our validated CRISPR/Cas9 intersectional system to selectively and systematically delete D4R or ?-arrestin2 in PFC pyramidal neurons or interneurons, and assess how UCSF924 modulates select behavioral domains. This will be expanded in Aim 2, where our co-investigator?s laboratory will utilize in vivo electrophysiological ensemble recordings to assess how UCSF924 engages neural networks and modulates behavioral outcomes. Addressing these aims will test our central hypothesis that selective targeting of D4R-mediated signaling in key PFC neurons has therapeutic potential. Our proposed work should provide a proof-of-concept for the development of a strategy to correct behavioral domains that are currently refractory to existing therapies.