Randy D. Blakely - US grants

The rapid reuptake of neurotransmitters across presynaptic neural and glial membranes constitutes the principle mechanism for the termination of chemical signals at CNS and -sympathetic synapses. Specific, Na+-driven, high-affinity transporters have been identified for L-glutamate, GABA, glycine, norepinephrine, dopamine, serotonin, adenosine, and the acetylcholine metabolite choline, among others. Defects in carrier structure or regulation are likely to exert profound effects on nervous system development and synaptic function and have been implicated in depression, hypertension, and drug addiction. The ability of transmitter-like neurotoxins, such as MPP+ and 6-hydroxydopamine, to be concentrated within nerve endings by these transporters suggests an involvement in neuropathology. Although the ability of antidepressants and cocaine to block monoamine reuptake focused attention on neurotransmitter transport over two decades ago, the majority of these proteins have yet to be purified and their genes have remained uncharacterized. Through the application of novel molecular biologic strategies, cDNA cloning of the human L-norepinephrine transporter (NET) has been achieved. Ibis breakthrough heralds new opportunities for the understanding of molecular principles involved in monoamine transporter assembly, function, and regulation, of critical importance if we are to understand the functional consequences of alterations or mutations occurring in brain disease. To achieve these goals, the present proposal first seeks to express and characterize cloned NET in transfected mammalian cells to determine whether a single mRNA encodes all the readily observable activities present in the native transporter. Secondly, mutated NET will be to produced and these constructs expressed in mammalian cells to elucidate the organization of functional domains and critical residues. These studies will be greatly advanced by the procurement of transporter and domain-specific antibodies, requiring the overexpression of NET in bacterial and eukaryotic cells and immunization with purified protein or synthetic peptides. These antibodies will permit the confirmation of gross structural integrity following transporter mutations, permit immunoprecipitation of labeled transporters for regulatory studies, and allow for tests of structural models built on primary sequence data. Finally, the present proposal evaluates the presence of transporter diversity, both within the NET family and among other monoamine carriers, utilizing sequence and expression-based cloning strategies, complemented by a determination of cellular organization by situ-hybridization. Insights gleaned should illuminate conserved features of related neurotransmitter transporters and provide a basic understanding of the consequences of altered transporter structure and function in disease.

The magnitude and duration of chemical neurotransmission is determined by reciprocal presynaptic activities of neurotransmitter release and reuptake. At sympathetic synapses of the vertebrate autonomic system, the catecholamine neurotransmitters norepinephrine (NE) and epinephrine (Epi) are rapidly cleared by transporter proteins thought to be enriched in presynaptic terminals and varicosities. The efficient recovery of NE and Epi permits repetitive sympathetic signaling without synaptic receptor desensitization, spatially limits the response to neurotransmitter, and helps maintain presynaptic catecholamine stores necessary for sustained signaling. Alterations in NE transport, as occurs with pharmacologic blockade by cocaine or tricyclic antidepressants, leads to a an augmentation of synaptic responses, a spillover of NE out of the synapse, the triggering of extrasynaptic receptors, and subsequent receptor desensitization. Whereas the kinetic behavior and drug sensitivities of catecholamine transporters are well described, molecular details of transporter structure, localization, and regulation have been unavaIlable. Recently, we have cloned the first cDNAs encoding cocaine and antidepressant-sensitive NE transporters (NETs) from the human medulloblastoma SK-N-SH, characterized the functional properties of expressed carriers in transfected cells, and developed NET specific antibodies for biochemical and immunocytochemical studies. We now propose to 1) identify and dissect homologous NETs and Epi transporters (ETs) expressed in the vertebrate heart, 2) elucidate molecular mechanisms for acute NET regulation using radiotracer flux and ligand binding, single cell electrophysiological techniques, and biochemical analysis of modified NET protein and 3) determine where and how transporters are spatially localized to presynaptic terminals using autoradiography and immunocytochemistry. To accomplish these objectives, we will perform experiments on individual gene products in reconstituted systems as well as primary tissues expressing NETs. Data from these studies should substantially augment our understanding of presynaptic control mechanisms involved in sympathetic neurotransmission.

This renewal application requests support for the Cellular and Molecular Neuroscience Training Program at Vanderbilt University. The program was established in 1991 to provide graduate and postgraduate training for individuals pursuing a research career in neuroscience. For the past decade, neuroscience has been a focus of faculty recruitment in the School of Medicine, creating a nucleus of faculty interested in interdisciplinary neuroscience training. These faculty joined together to develop the current training program that focuses on cellular and molecular neuroscience to emphasize the strengths of the faculty and the opportunities for linkage between molecular and clinical neuroscience. The initial application was funded in 1992. Since then, ten additional faculty have been recruited, a first-year interdisciplinary graduate program providing comprehensive training in biochemistry, cell biology, genetics, and molecular biology has been implemented, and a new Center for Molecular Neuroscience has been established. These initiatives as well as new graduate courses, seminar series, and several cross-departmentally-sponsored activities have created a rich environment for interdisciplinary training in neuroscience. To take advantage of the increased number of neuroscience faculty and improved training environment, this renewal requests increased funding from three to six predoctoral slots and from one to four postdoctorals. The program is designed to provide graduate students with an integrated training that consists of a strong foundation in the fundamentals of biochemistry, molecular biology, cell biology, and genetics - building on this base with advanced courses in membrane excitability, synaptic transmission, and nervous system development - combined with rigorous in-depth training in research. The principal focus of postdoctoral training is research. Although both predoctoral and postdoctoral training emphasize a research career, several training experiences have been designed to develop teaching and leadership skills. Each of the training faculty has a peer-reviewed, contemporary research laboratory offering training opportunities in chemical and electrical signaling in the nervous system, neurogenetics, neuronal development, neurotoxicity, regulation of gene expression, and biological basis of neuropsychiatric diseases.

DESCRIPTION (provided by applicant): Serotonin (5-HT) transporters (SERTs) are critical determinants of synaptic 5HT availability in the CMS and periphery and major targets for antidepressants and psychostimulants including cocaine and MDMA. Endogenous mechanisms that modulate SERT activity are emerging, but as yet remain ill defined. Past studies have identified SERTs as phosphoproteins, regulated acutely (minutes) by multiple signaling pathways that influence transporter trafficking and intrinsic activity. In the current proposal, we continue our investigations of SERT regulation, bridging heterologous model systems with studies using in vitro and in vivo studies of native preparations. In Specific Aim I we evaluate the role of protein kinase G (PKG)-linked signaling pathways in modulation of SERT trafficking and SERT protein/protein interactions as downstream consequences of GPCR activation in vitro and in vivo. Preliminary studies here reveal PKG-dependent changes in SERT surface density, 5-HT uptake, SERT currents and 5-HT clearance in vivo and support a direct role for SERT phosphorylation in GPCR/PKG linked modulation. In Specific Aim II, we examine the role of p38 mitogen associated protein kinase (MAPK) in modulating SERT intrinsic activities. Preliminary studies here provide evidence for catalytic modulation of SERT targeting surface transporters, dependent on PP2A function for p38 MAPK regulation, and the ability of p38 MAPK to directly phosphorylate SERT. In specific aim III, we investigate the degree to which basal and PKC/PKG/p38 MAPK-mediated alterations in SERT activity are evident in human SERT coding polymorphisms. Preliminary findings on this Aim reveal the existence of multiple SERT alleles that display altered kinase-mediated regulation, alleles that may be enriched in populations at risk for 5-HT linked disorders and which offer novel opportunities for insights into molecular mechanisms of SERT regulation/therapeutics.

Presynaptic dopamine (DA) transporters (DATs) constitute the primary mechanism for inactivation of DA in the brain. DAT proteins are high- affinity targets for important addictive and therapeutic drugs including cocaine, amphetamines and methylphenidate (Ritalin/TM). Little is known regarding how DAT proteins form a selective permeation pathway for DA and how different antagonist impact activity. Recent studies reveal DATs to be acutely regulated by coordinated mechanisms involving kinase activation, transporter phosphorylation and altered membrane trafficking/stabilization, though as yet genes responsible for this regulation remain to be identified. In rodents and man, DATs also constitute the portal through which exogenous neurotoxins (e.g. 6- OHDA, methamphetamine and MPP+) enter these neurons and affect lesions reminiscent of the selective pathology of Parkinson's disease. Controversy exits as to whether these toxic insults trigger cell death in vivo via necrotic or apoptotic pathways. We have cloned the product of the C. elegans gene T23G5.5 and demonstrated in its function as a DA transporter (CeDAT), opening the door to genetic strategies for the definition of critical DAT residues, the identification of CeDAT regulators and an understanding of molecular contributors to dopaminergic neuron sensitivity to environmental toxins. In our proposal, we seek to 1) validate the molecular and cellular specificity of CeDAT- targeted drugs and toxins, evaluate the properties of 6-OHDA-induced DA neuron degeneration in the work and determine whether CeDAT is both necessary and sufficient for toxin sensitivity, 2) to establish the cellular specificity, developmental expression and subcellular localization of CeDAT protein CeDAT mutants and CeDAT regulators using novel gain-of-function screens involving suppression of sensitivity to 6-OHDA. Together, these efforts provide important opportunities to identify and characterize regulators of transporter expression, localization and function and may provide clues to molecular determinants of dopamine- dependent psychiatric and neurodegenerative syndromes.

DESCRIPTION (provided by applicant): Support is requested for the Gordon Research Conference on Catecholamines to be held July 8-13, 2001 at Proctor Academy at Andover, NH. The Gordon Conferences on Catecholamines are held every two years and are attended by approximately 120 investigators. Catecholamine research impacts both autonomic, cardiovascular and CNS function. Disorders of catecholamine biosynthesis, secretion and signaling have been implicated in a wide spectrum of autonomic, endocrine, cognitive, mood, addictive and neurodegenerative disorders. The Catecholamine Gordon Conference has a long and successful tradition of introducing current research themes in catecholamine biology and disease to both seasoned and young investigators. Novel genetic and transgenic models relevant to aminergic development, signaling and disease will be featured. The program of this conference is intentionally broad to bring current genetic, molecular, physiologic and behavioral analyses of catecholamine biology and dysfunction to a common venue and to promote cross-fertilization of fields. The program includes formal presentations from senior leaders in the field as well as more junior investigators, poster sessions and technique-oriented workshops. The program focuses on recent, cutting-edge research, and includes ample time for both formal and informal discussions in a relaxed atmosphere. Emphasis is placed on extensive interactions between junior and senior investigators. NIH funding is requested to permit pre-and postdoctoral fellows to attend the meeting in order to foster these exchanges, and provide junior investigators with a unique and stimulating scientific experience. In addition, NIH funding will help to support participants, particularly women and underrepresented minorities, who could not otherwise attend but will enrich the mix of expertise among the participants.

DESCRIPTION: (provided by the applicant) Presynaptic dopamine (DA) transporters (DATs) constitute the primary mechanism for inactivation of DA in the brain. DAT proteins are high-affinity targets for important addictive and therapeutic drugs including cocaine, amphetamines and methylphenidate (Ritalin). DATs are subject to significant regulatory modulation but molecular mechanisms supporting DAT regulation are unknown. Recent studies indicate that DATs are acutely regulated by coordinated mechanisms involving kinase activation, transporter phosphorylation and altered membrane trafficking/stabilization, though as yet genes responsible for this regulation remain to be identified. We have established a model system permitting DAT evaluation in a native neuronal context, exploiting the powerful transgenic and genomic tools afforded in Caenorhabditis elegans. In this system, we have the unique ability to selectivelv introduce or inactivate genes in living DA neurons, allowing us to test specific hypotheses regarding DAT structure/function, drug modulation and drug and kinase triggered DAT regulation. Moreover, a novel cell culture approach has been established that permits a detailed electrophysiologic and optical analysis of DAT function and regulation in identified DA neurons and that can be scaled to support the identification of novel DAT regulatory genes. In our CEBRA proposal, we seek to 1) analyze the functional and regulatory pathways supporting C. elegans DAT (CeDAT) expression in cultured DA neurons and 2) to develop a system where a combination of transgenic and proteomic approaches can be applied to permit the evaluation of hypothesized and novel DAT regulators.

DESCRIPTION (provided by applicant): The antidepressant-sensitive norepinephrine (NE) transporters (NETs) constitute the major mode of synaptic inactivation of NE. Recent clinical genetic studies by our groups identified a coding mutation, A457P, in one NET allele of a proband with Orthostatic Intolerance (OI) presenting with reduced NE clearance, increased spillover and reduced intraneuronal NE metabolism. The A457P mutation was found to track with measures of postural tachycardia in the proband?s family and to correlate with altered synaptic NE metabolism. In Specific Aim 1, we propose to ascertain the functional impact of the A457P and other identified NET coding mutations in terms of transport and efflux, transporter trafficking and surface expression using heterologous expression systems. Evidence will be sought to support a dominant-negative interaction between mutant and wildtype subunits and whether homomultimeric complexes support NET function. In Specific Aim 2, we propose to extend our genetic evaluation of NET deficiency to evaluate additional subjects with OI and cardiomyopathy (CM) using high-throughput gene scanning techniques. These studies will focus on the NET coding exons and splice junctions and also include a recently identified intronic region that plays a critical role in NET gene expression. Methods will be implemented to allow for an evaluation of altered NET protein in biopsies tissue. Finally, attention and mood are dependent on proper noradrenergic signaling in the CNS and symptoms are present in our A457P probands indicating attention deficit, anxiety and hyperarousal. Thus, we propose in Specific Aim 3 to examine NET alleles with primary diagnoses of attention-deficit hyperactivity disorder (ADHD), attentional deficit (ADD) subtype and Major Depression, melancholic subtype, which is characterized by hyperarousal and anxiety. We will select subjects for analysis in both cases on the basis of comorbid tachycardia. Together these studies offer an opportunity for a better understanding of the molecular and behavioral manifestations of genetic NET variation.

DESCRIPTION (provided by applicant): Neuroscience, the study of brain development, function, plasticity and disease, offers perhaps the most complex challenge in all of biology, relating the structure and output of genomes to the neural processes that ultimately register as thought, memory, emotion, and mood. The complexity of the biological mechanisms involved is mirrored by the interdisciplinary training required of neurobiologists, where years of postgraduate research and education are now needed to efficiently and competitively embrace the opportunities of brain science in a genomic age. Brain diseases, many with complex genetic underpinnings, constitute a great burden on individuals, families, care-givers and society, dictating novel training mechanisms to increase the sophistication of both basic and clinical neuroscientists. Multiple genomes have been sequenced, including model systems of organisms where simpler nervous systems and powerful genetics offer an accelerated pace of discovery. New mouse and human genomic information is available that can lead both to important new disease models, to diagnostic insights and to targeted therapies of brain disorders. At the same time, "topdown" approaches in neurobiology have gained momentum, allowing a greater sophistication in behavioral phenotyping and direct visualization, via PET, SPECT and fMRI, of functional specializations in the living brain. The next generation of neuroscientists will need to be able to tap genomic sciences but at the same time recognize and investigate more complex neurobiologic processes that ultimately support brain disorders. In this context, we propose the Training Program in Neurogenomics. Our program organizes the postdoctoral training efforts of 35 senior and 16 junior faculty in basic and clinical departments at Vanderbilt and Meharry Medical College through the Vanderbilt Center for Molecular Neuroscience. Our program provides for formal research training and didactic instruction, as well as seminars and technical workshops at the interface of neuroscience and genomic research.

Acetylcholine (ACh) is a major neurotransmitter in the central and peripheral nervous system. The rate-limiting step in ACh synthesis is believed to be the presynaptic acquisition of the precursor choline, achieved by the high- affinity, Na+-dependent, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT). Recent breakthroughs in the molecular elucidation of invertebrate and mammalian CHT genes, the development of CHT-specific antibodies, and the creation of tractable in vitro and transgenic model systems have established new opportunities to define neuronal CHT subcellular distribution, mechanisms of activity- and receptor-dependent CHT regulation, and the functional consequences of genetic manipulation of CHT. Recently, we have established that CHT is )redominantly vesicular in localization, both in vivo as well as in in vitro model systems. CHT appears to reside )n a subpopulation of cholinergic synaptic vesicles that express the vesicular ACh transporter (VAChT) and which store ACh. Preliminary studies document both a change in the localization of CHT in synaptic membranes in response to depolarization in wildtype mice and a posttranslational mechanism to achieve normal levels of choline transport and HC-3 binding despite a 50% reduction in CHT protein in CHT +/- mice. In this new application, we apply biochemical, imaging and functional methodologies using in vitro and in vivo model systems to investigate the nature of the vesicular pool harboring CHT and clarify the physical requirements for vesicular targeting and synaptic CHT trafficking. Secondly, we explore plasma membrane shuttling as a major route for activity, cell signaling and behaviorally induced changes in choline uptake and implement a yeast 2-hybrid screen for novel CHT interactors. Finally, we analyze the consequences of full and partial genetic CHT ablation in the mouse for cholinergic biochemistry, pharmacology, physiology and behavior. These studies will elucidate novel aspects of CHT regulation, clarify how CHT supports cholinergic synaptic/behavioral plasticity and provide new CHT links to brain disease.

Presynaptic 5HT transporters (SERT) control the availability of 5HT following release and recycle 5HT for reuse in subsequent release events, thereby contributing to presynaptic 5HT homeostasis. SERTs are targets for the most commonly prescribed antidepressant medications and genetic variation in SERT has been linked to autism, anxiety, major depressive disorder (MOD) and antidepressant response. Mirroring the network of postsynaptic genes that depend on efficient 5HT clearance to dictate 5HT response, a presynaptic network of interacting proteins and cell signaling pathways dictates appropriate SERT surface abundance and catalytic activity. The Blakely laboratory has been a leader in the field of SERT molecular biology and regulation for over a decade originating with the first identification of SERT genes in mouse and man. In Project 3: Signaling Networks Supporting Serotonin Transport, Blakely's team proposes three Aims to more fully elucidate the identity and regulation of the SERT regulatory proteome in vivo, providing new targets and models to enrich our understanding of how 5HT signaling is established and modulated. In Specific Aim I, Blakely will use both candidate and proteomic approaches to illuminate the SERT regulatory network established in platelets, a rich source for SERT in the periphery, followed by biochemical and anatomical validation of co-expression in neurons, and assessment of the stability of the network to activation of SERT regulatory kinases and phosphatases. In Specific Aim II, Blakely proposes the creation and evaluation of transgenic mouse models that limit PKG mediated regulation of SERT, either through constitutive and raph-specific loss of PKG1 or elimination of a key PKG phosphorylation site in SERT. Secondly, Blakely evaluates the activity of 5HT and antidepressants on SERT trafficking and protein associations via studies of mice harboring the lle172Met allele which reduces SSRI and cocaine recognition at SERT without loss of 5HT uptake function. The ultimate goal is to link changes observed in SERT activity in these models to the stability and organization of the SERT proteome identified in Aim I. In Specific Aim I BLJIlakely's team will examine the broader physiological impact of engineered mutations, collaborating with onte Investigators to explore their impact on 5HT homeostasis and SERT activity in vivo, the abundance If'recessing (editing) and signaling of 5HT receptors, and the physiological and behavioral effects, monitored through dialysis and chronoamperometry studies as well as established behavioral techniques. Together, these efforts will more broadly elucidate how SERT regulation and the SERT regulatory proteome establishes proper 5HT clearance capacity and how genetic variation can influence SERT regulation in vivo.

high performance liquid chromatography; laboratory mouse

Description (Provided by Applicant): This proposal seeks the creation of an NIMH Silvio O. Conte Center for Neuroscience Research at Vanderbilt University to investigate "Genes Controlling Assembly and Function of Serotonin Systems". Our central hypothesis is that functional and genetic variation in a tightly controlled network of proteins establishes modulatory capacity of the neuretransmitter serotonin (5HT) during early brain development. These variations have consequences that reverberate throughout life, impacting serotonin neuronal activity and serotonin signaling and ultimately constraining behavioral flexibility that is needed to meet the challenges of daily life. To test this hypothesis and illuminate this network, we have assembled an interdisciplinary team of established researchers at Vanderbilt University (Levitt, Blakely, Sanders-Bush, Emeson, McMahon) and Case Western Reserve (Deneris). Project 1 (Deneris) identifies embryonic and postnatal 5HT neuron transcriptomes and elucidates the serotonergic-specific function of the glucocorticoid receptor in 5HT neuron development, function and contributions to behavior. Project 2 (Levitt) explores the network of signaling pathways supporting the modulatory role of 5HT in the assembly of thalamocortical and raphe circuits. Project 3 (Blakely) investigates the protein network regulating the antidepressant-sensitive serotonin transporter (SERT) and the functional impact of altered PKG/p38 MARK signaling linked to the SERT proteome in novel mouse models. Project 4 (Sanders-Bush) elucidates the genetic underpinnings of 5HT production and signaling as well as 5HT-linked behaviors in the mouse through the analysis of quantitative traits in recombinant-inbred mouse strains. Project 5 (Emeson) utilizes novel transgenic mouse models to explore the biochemical, functional and behavioral capacities linked to editing of 5HT2C receptor RNA. Project 6 (McMahon) examines the molecular network and physiological responses specified by clock genes in both raphe neurons and raphe targets in the hypothalamus linked to the modulation of behavioral rhythms. Our program is supported by Administrative, Bioanalytical, Biobehavioral, and Bioinformatics/Biostatistics Cores as well as a significant investment of the Vanderbilt School of Medicine including staff, facilities and a pilot grant program linked to the goals of the project. Through this program, we seek to illuminate the network of genes and proteins organizing the capacity for 5HT signaling in the mouse and link this information to orthologous gene networks in humans for eventual use in translational programs that target 5HT-linked disorders by the broader neuroscience community. Using web-based tools, scientific symposia, pilot grant programs and community forums, the Vanderbilt Conte Center for Neuroscience Research will seek to disseminate its findings to the general public and excite young minds to sustain exploration in this important area of basic neurobiology underlying mental illness including anxiety, depression, obsessive-compulsive disorder, and autism as well as common and future pharmacotherapies.

3-(2-Aminoethyl)-1H-indol-5-ol; 5-HT; 5-Hydroxytryptamine; 5HT; Administrator; Apparatus and Instruments; Appointment; Area; Award; Basic Research; Basic Science; Biology; Brain; Contract Services; Data; Doctor of Philosophy; Education and Outreach; Educational workshop; Encephalon; Encephalons; Enteramine; Equipment; Equipment and Supplies; Financial Management; Funding; Funding Mechanisms; Generations; Genes; Genetic; Grant; Hippophaine; Human Resources; Institutes; Instruction and Outreach; Investigators; Letters; Link; Manpower; Medical; Mental Health; Mental Hygiene; Molecular; NIMH; National Institute of Mental Health; National Institute of Mental Health (U.S.); Nervous System, Brain; Neurosciences; Neurosciences Research; Occupational activity of managing finances; On-Line Systems; Online Systems; Pennsylvania; Ph.D.; PhD; Pharmacology; Pilot Projects; Position; Positioning Attribute; Programs (PT); Programs [Publication Type]; Progress Reports; Psychological Health; Publishing; Reports, Progress; Research; Research Personnel; Research Resources; Researchers; Resource Sharing; Resources; Scientist; Series; Serotonin; South Carolina; System; System, LOINC Axis 4; Tennessee; Time; Training and Outreach; Travel; Tutoring and Outreach; United States National Institute of Mental Health; Universities; Visit; Work; Workshop; conference; experience; member; online computer; outreach; personnel; pilot study; programs; supported housing; supportive housing; symposium; web based

[unreadable] DESCRIPTION (provided by applicant): The understanding of how our genetic heritage intersects with the environment to impact brain development, function, plasticity and disease, remains arguably the most complex challenge in all of biology, demanding a comparable complexity in the advanced training of neuroscientists. It is simply not possible for basic neuroscientists to gather all of the tools necessary for a productive career through completion of even the best graduate training program. The doctoral graduate may have only minimal exposure to research projects that target mental illness mechanisms, may have never probed the challenges of modeling a brain disease in an animal model, or may be specialists at a single level of analysis, training that we predict will increasingly fail to support a successful research career. Conversely, clinical trainees regularly learn of the genetic contributions to brain function and disease but often cannot readily engage the process, denying in turn their basic-science peers the opportunity to understand the challenges and benefits inherent in disease-guided research. The Vanderbilt Postdoctoral Training Program in Neurogenomics supports the advanced training of both basic and clinical neuroscientists, providing a coordinated program of mentored research, technical forums, invited lectures and career development workshops throughout the Vanderbilt tenure of the trainee. Building on a significant commitment to neuroscience research and training infrastructure at Vanderbilt University through the Center for Molecular Neuroscience (CMN), we organized our first Neurogenomic training program in 2002 and here submit the program's first renewal application. Our past program offered a somewhat broad base of molecular neuroscience training and successfully recruited and mentored outstanding basic and clinical trainees, including significant attention to trainee diversity and professional development as well as to capture of the full breadth of our mentor base. With our renewal Application, we sustain the general structure of our initial program. However, we make a concerted effort to sharpen our program to provide research and training opportunities that best illustrate and apply the tools of gene-informed neuroscience, spanning invertebrate and vertebrate model systems as well as direct analysis of the human genome, activities that immerse trainees in leading edge neurogenomic models, approaches and technologies. Our program is directed by Randy D. Blakely, Ph.D., Director of the CMN, and an NIMH MERIT awardee, whose research capture opportunities in both nematode and transgenic mouse models to understand the regulation and genetics of biogenic amine signaling, and who has an outstanding track record in both pre- and postdoctoral training. The program is co-directed by James S. Sutcliffe, Ph.D., a talented neurogeneticist, who brings to the program expertise in the study of genes contributing risk to neurobehavioral disorders, including Angelman's syndrome and Autism. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this study is to capitalize on a recently identified dopamine (DA) transporter (DAT)-dependent phenotype in the nematode C. elegans, termed Swimming-Induced Paralysis (SWIP), to identify proteins involved in the localization and regulation of cocaine and amphetamine-sensitive DAT proteins at presynaptic nerve terminals. In vertebrates, DA modulates multiple behavioral processes involved with locomotion, cognition, and reward. Reuptake through DAT is the primary mechanism by which DA signaling is terminated and is a critical determinant of presynaptic DA homeostasis. Polymorphisms in human DAT have been associated with ADHD, schizophrenia, and drug addiction, and DAT is a major target of cocaine and amphetamine. Heterologous expression studies indicate that DATs are regulated via accessory proteins and kinase-linked signaling pathways that ultimately control transporter localization and activity. Moreover, signaling networks linked to PKC, CaMKII, PI3K, and Akt have been linked to the presynaptic actions of amphetamine and cocaine, revealing many potential new targets for addiction risk and therapeutic development. The technical challenges presented by the study of DAT regulation in the mammalian CNS have encouraged our laboratory to pursue a characterization of DAT-associated phenotypes in C. elegans, where forward genetic screens for DAT modulators are attainable. Previously, our laboratory showed that the C. elegans DAT (DAT-1) is 43% identical to mammalian DATs, is expressed exclusively in DA neurons and terminals, preferentially transports DA, and is sensitive to both cocaine and amphetamine. Under the support of a postdoctoral NRSA, I identified a phenotype, termed Swimming Induced Paralysis (SWIP) that is a reporter of functional DAT-1 expression. Thus, SWIP in dat-1 knockout or cocaine/amphetamine treated animals is reversed when DA synthesis, release, and post-synaptic signaling through the DA receptor DOP-3 are precluded. Based on these data, I launched a pilot forward genetic screen to identify DA-dependent SWIP mutants and have identified two lines bearing novel dat-1 point mutations that cause biosynthetic, trafficking and functional defects in vitro and in vivo as well as several lines where mutations appear to lie in other genes. Building on this effort, I propose the following Specific Aims: 1) To expand the screen for DA-dependent SWIP phenotypes, validating DAT deficiency via dopamine receptor deficiency complementation tests, responsivity to cocaine/amphetamine and DA transport assays on embryonic cultures and 2) To identify non-DAT, SWIP-generating genes using Illumina-based, high-throughput cDNA sequencing with transcriptome profiling, followed by a bioinformatics based elucidation of human homologs. These studies provide a powerful and unique opportunity for insight into presynaptic mechanisms controlling DAT-dependent DA signaling. PUBLIC HEALTH RELEVANCE: Alterations in dopaminergic (DA) neurotransmission are centrally involved in psychostimulant response and addiction. The presynaptic DA transporter (DAT) is the primary mode by which DA signaling is terminated and is a direct target for cocaine and amphetamine. This research engages a powerful genetic model system to elucidate genes that regulate DA signaling and DAT activity with opportunities to identify novel targets for addiction risk and/or treatment.

DESCRIPTION (provided by applicant): The principal determinant of synaptic serotonin (5-hydroxytryptamine;5-HT) inactivation is the antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). Common polymorphisms in the SERT promoter impact SERT expression and have been associated with anxiety, obsessive- compulsive disorder and autism. SERT is also subject to powerful, posttranslational regulatory mechanisms. Recently, inflammatory cytokines, including IL12 and TNF-1, have been shown to enhance SERT activity in nerve terminal preparations, regulation that can be recapitulated in vivo by systemic cytokine elevation. These findings raise the possibility that SERT may be a convergence point for gene/environment interactions that conspire to alter 5-HT signaling and elevate disease risk. Because SERT is expressed early in brain development, of greatest interest are 5-HT linked disorders of juvenile onset. In this regard, both disrupted 5-HT signaling and disrupted immune function have been advanced as determinants of autism risk. Recently, we identified multiple, functional SERT variants at highly conserved amino acid positions in autism subjects. The most common SERT variant, Gly56Ala, leads to 1) enhanced hSERT catalytic activity, 2) elevated hSERT basal phosphorylation, and 3) a lack of responsiveness to multiple SERT posttranslational regulatory pathways including p38 MAPK pathways activated by inflammatory cytokines. Importantly, the Ala56 hSERT variant was associated with two well-described autism traits, rigid-compulsive behavior and sensory aversion. We have successfully targeted 129S6 mouse embryonic stem cells with a SERT Ala56 "knock-in" construct and obtained germ-line transmission. The SERT Ala56 mice display whole blood hyperserotonemia, hyper-responsiveness to 5HT2A/2C agonist stimulation, and evidence of alterations in social and sensory behaviors. In our current project, we propose a 2-year program to 1) determine the ability of maternal, juvenile and adult SSRIs administration to attenuate the altered behavior and drug responses of 129S6 SERTAla56 mice, 2) to place the 56Ala allele on a C57Bl/6J background using speed-congenic approaches and thereby expand the testable behavioral repertoire of the Ala56 mice, and 3) to establish the sensitivity of SERT Ala56 brain preparations on the C57BL/6J background to in vitro PKG/p38 MAPK stimulation and in vivo stimulation of the native immune system. These studies will enhance our understanding of the contributions of altered SERT activity and regulation to autism heterogeneity and provide a novel platform for the preclinical evaluation of therapeutic interventions. PUBLIC HEALTH RELEVANCE: Rare, functional gene variants that lead to hyperfunction of the serotonin transporter (SERT) have been associated with a specific subset of autism spectrum traits, rigid-compulsive behaviors and sensory aversion. We have produced a novel, transgenic mouse model expressing the most common of these SERT variants, Ala56. Based on exciting preliminary evidence that mice expressing Ala56 SERT exhibit multiple behavioral and biochemical phenotypes, we propose a two-year project to extend our studies and dissect the impact of SERT hyperfunction in vivo.

DESCRIPTION (provided by applicant): The role of pro-inflammatory cytokines in the pathogenesis of neuropsychiatric disorders has drawn a significant interest over the last decade. Cytokines are also known to modulate serotonergic activity. The 5-HT transporter (SERT), one of the major targets of antidepressants, which supports 5-HT inactivation and recycling, is tightly regulated by multiple signaling pathways including those stimulated by the proinflammatory cytokines IL-1[unreadable] and TNF-a. We have shown that IL-1[unreadable] stimulates SERT activity in a raphe cell line as well as in mouse synaptosomes ex vivo. Our preliminary data now demonstrate that peripheral injection of LPS induces an acute (1hr) increase in central 5-HT uptake in wild type mice but not in IL-1R knockouts. We hypothesizes that a presynaptic IL-1Rs communicates local and systemic inflammatory stress (and other stressors) via modulation of SERT activity. As the distribution of IL-1Rs in the CNS is not limited to the raphe neurons and their terminals, dissecting the contribution of serotonergic modulation will require restricted elimination of IL-1[unreadable] action. Thus, the objectives of the current proposal are to generate IL-1R floxed mice, further develop serotonergic neuron specific IL-1R knockout using FloxP/Cre technologies and to characterize the 5-HT homeostasis and related behaviors in these animals. My hypothesis is that deletion of IL-1 receptors in the raphe serotonergic neurons will eliminate the impact of IL-1[unreadable] / LPS on central SERT activity. The validation of this hypothesis will (1) provide critical data for an enlarged study examining cytokine-5HT interactions and (2) help to advance our understanding towards the contribution of modulated 5HT signaling networks to depression-like traits that emerge in sickness syndrome paradigms. To achieve the goal of this proposal, we plan to conduct the following studies: 1. Generate floxed IL-1R mice;2 Develop and characterize serotonergic neuron-specific IL-1R knockout mice. The essential goal of this project is to achieve germ-line transmission of a floxed allele of the IL-1R with no inherent impact on native IL-1R production in the absence of a Cre driver, and eventually develop conditional IL-1R knockouts. Together, these efforts support the long-term goal of elucidating in vivo mechanisms through which pro-inflammatory cytokines modulate brain function and behavior. PUBLIC HEALTH RELEVANCE: This project investigates the link between Immunological challenges and a key gene controlling the neurotransmitter serotonin for insights into mechanisms that may impact risk for mood disorders including depression, anxiety and autism. This project specifically investigates the immune mediator Interleukin-1 and its receptor in regulation of the brain serotonin transporter (SERT), a major target for antidepressant medications. These studies seek to develop a novel transgenic mouse model that can address the sensitivity of brain serotonin neurons and SERT to IL-1[unreadable] and establish an experimental framework to more precisely link the immune system to mood regulatory mechanisms.

The Vanderbilt Conte Center for Neuroscience Research Enduring Impact of Early-Life Serotonin Signaling involves a complementary and interconnected series of basic science projects involving internationally recognized leaders in their respective fields at Vanderbilt, Case Western Reserve University and the University of Southern California (USC). The Administrative Core provides financial oversight for the activities of all Conte Projects and Cores, including personnel management, equipment and supply purchases, generation of progress reports, and coordination with other Vanderbilt shared resources. The Core coordinates all Conte Center meetings, workshops and symposia, and manages the Vanderbilt Conte Pilot Projects program, an institutionally funded mechanism to develop young scientists through sponsored research that target Conte-allied research areas. Data and resources of the Investigators and Cores are shared via the website http://vandvconte.orq with information for scientists and the lay public on biology, genetics and pharmacology of serotonin as it relates to mental health and illness. Two new elements extend our ongoing and successful education/outreach activities. First, we sponsor a Summer Undergraduate Research Experience (SURE) that pairs students with faculty working on Conte-related projects at Vanderbilt, Case Western and USC to provide a mentored research experience. The SURE effort is greatly strengthened by Vanderbilt's well-established framework for summer undergraduate training (Vanderbilt Summer Science Academy), as well as by the excellent opportunities for recruiting students from underrepresented backgrounds at Vanderbilt, USC and CWRU. Second, we sustain and expand our public outreach program that provides educational experiences for the Nashville community, building upon ARRA-funded, adult-targeted neuroscience education exhibits at Vanderbilt's One Hundred Oaks clinical campus. Third, we support a new cadre of Elaine Sanders-Bush Fellows within Vanderbilt's highly regarded Neuroscience Training Program, launching a program tailored for graduate students interested in careers both in research and public advocacy/education. These students will also help mentor undergraduates in the SURE program, and will work as in teams with faculty and postdoctoral fellows to provide a program of core neuroscience and mental health concepts to Nashville youth. Finally, we implement a Neuroscience Cafe series whereby Vanderbilt neuroscientists, including Conte team members, provide informal discussions on brain science and health topics at local community sites.

Presynaptic, serotonin (5-hydroxytryptamine, 5-HT) transporters (SERTs) limit the signaling potential of 5-HT and recycle 5-HT for further release. SERTs are targets for antidepressants and psychostimulants. Altered SERT expression and function may contribute to multiple neuropsychiatric disorders, including anxiety, OCD, depression and suicide. The majority of studies that explore the contribution of SERT to mental illness have focused on disorders of adult onset. However, increasing evidence demonstrates that 5-HT signaling in the developing brain is critical to establish normal connectivity and behavior and. A key example, autism spectrum disorder (ASD), has been linked to abnormal 5-HT homeostasis for 50 years. The Blakely lab identified and characterized five rare, functional SERT coding variants in ASD subjects, all of which display enhanced 5-HT transport capacity. Blakely's team has developed a transgenic (knock-in) mouse expressing the most common of the ASD SERT variants, Gly56Ala. SERT Ala56 mice display hyperserotonemia and multiple behavioral phenotypes that support the SERT Ala56 model as a powerful platform to understand how compromised 5-HT signaling during development can generate lifelong behavioral deficits. Efforts to capture this opportunity are embraced by Project 3: Modeling the Serotonin Contribution to Autism Spectrum Disorders, In Specific Aim I, Blakely capitalizes on powerful RNA sequencing approaches to elucidate transcriptional networks impacted by SERT Ala56 in raphe neurons, placental tissues and B cells of the immune system. In Specific Aim II, Blakely seeks to reverse phenotypes associated with SERT Ala56 expression using 5-HT receptor agonists and other molecules suggested from gene network alterations. In Specific Aim III, Blakely's group develops novel, transgenic mouse models that provides for conditional expression of the SERT 56Ala variant, with the goal of understanding the contribution of specific sites and timing of SERT Ala56 expression to the phenotypes found in mice and humans harboring this variant.

DESCRIPTION (provided by applicant): Dopamine (DA) is a neurotransmitter used throughout phylogeny to modulate circuits controlling movement, attention, reward and cognition. Alterations in DA signaling in man have been implicated in Parkinson's Disease, Attention-Deficit Hyperactivity Disorder (ADHD), addiction and schizophrenia. DA signaling is tightly controlled by a presynaptic, DA transporter (DAT) that is a major target for addictive psychostimulants such as cocaine and amphetamine (AMPH), as well as agents used in the treatment of ADHD. Our recent identification of multiple, functional alleles of human DAT in ADHD and Bipolar disorder subjects adds translational significance to our efforts to decipher the presynaptic mechanisms that control DA release and inactivation. To date, these mechanisms have largely been studies through pharmacological and genetic manipulation of genes identified two decades or more ago. In the current application, we capitalize on a robust, forward genetic approach to identify and characterize novel presynaptic regulators of DA signaling using the powerful model system Caenorhabditis elegans. Over the past 15 years, the Blakely lab has developed skills in the C. elegans model with a focus on DAT and DA signaling, initiated by the identification of the C. elegans DAT gene (T23G5.5, dat-1). The present effort arises from our discovery of a simple DA and dat-1 dependent phenotype termed Swimming-Induced Paralysis (SWIP). Whereas wildtype animals thrash in water at ~1 Hz for up to 30 minutes, dat-1 (ok157) (DAT-deficient) animals paralyze in 3-5 minutes. This phenotype is dependent on DA synthesis, vesicular DA packaging, and DA release and is overcome by DAT activity. We request support to advance our screen to identify and characterize genes acting presynaptically to regulate DA release and reuptake, localize their mode of contribution to DA signaling, assess their impact on the actions of AMPH, and initiate an analysis of conserved vertebrate homologs. Together our efforts provide an opportunity to identify novel and conserved regulators of DA signaling that would be extremely difficult to elucidate in vertebrate models.

Our proposal, Enduring Effects of Early-Life Serotonin Signaling, explores the hypothesis that tight control of developmental determinants of serotonin (5-HT) signaling is required to achieve normal patterns of behavioral flexibility and to minimize risk for life-long neuropsychiatric disorders. To test our hypothesis, and to identify opportunities for reversal of disrupted early-life 5-HT signaling, we assemble a highly collaborative team of leading neuroscientists with experience in the development and molecular plasticity of 5-HT signaling. In Project 1, Evan Deneris tackles the support that CNS-synthesized 5-HT signaling plays in the elaboration of raphe neuron gene expression that can support stress-modulated, epigenetic programming. In Project 2, Pat Levitt builds upon his group's discovery of the placenta as a major source of forebrain 5-HT during embryonic development, Levitt's efforts assess how placental-specific disruption of 5-HT synthesis and metabolism leads to enduring effects on brain development and function and whether alterations are reversible. In Project 3, Randy Blakely elucidates the molecular and functional consequences, and potential for reversal, of an autism-associated 5-HT transporter (SERT) mutation (SERT Ala56), and how both either/or CNS and peripheral sites of expression contribute to life-long behavioral deficits, while developing novel conditional SERT mutation expression models. In Project 4, Ron Emeson brings his group's advanced understanding in 5HT2c receptor expression and signaling to bear on the timing and regional specificity of stress-dependent 5HT2c editing, elucidating their mechanisms, biochemical and behavioral consequences and possibilities for reversal. Finally, Mark Wallace leads novel education and outreach programs, extending ARRA-funded efforts to enhance community understanding of neuroscience research and mental illness and that train young scientists in the research and outreach missions of the Conte Center.

DESCRIPTION (provided by applicant): Neuroscientists and physicians continue to struggle to understand the pathophysiological mechanisms that underlie mental illness, and even more so to translate this information into the personalized therapies needed for diseases. Although research over the past five decades has uncovered genes, gene variants, molecular networks, cellular interactions and brain pathways supporting brain disorders, our translation of this information remains limited. A new generation of neuroscientists is needed that understand, and can exploit, 1) the functional impact of genetic and epigenetic variation in vivo, 2) the developmental and regulatory context within which genes produce alterations in synaptic physiology, 3) the circuit-level pathologies that lead to changes in behavior and 4) the best practices in translating research findings into novel therapies. We propose The Vanderbilt Postdoctoral Training Program in Functional Neurogenomics as a vehicle to insure a merger of disciplines, where trainees gain experience in the opportunities afforded by genetic model systems, gain expertise in the translation of human genetic findings into construct-valid animal models, learn to manipulate molecules, cells and circuits with advanced approaches in vivo, and gain experience in capturing the physiological and behavioral consequences of such manipulations. We build our program on a significant investment over the past two decades by Vanderbilt in neuroscience faculty, educational programs, technological expertise and core facilities. Our Program Director is Randy D. Blakely, Ph.D., an NIMH MERIT awardee and Director of the Vanderbilt Silvio O. Conte Center for Neuroscience Research. Dr. Blakely's research ranges from forward and reverse genetic studies in C. elegans to the modeling of mental illness-associated gene variation in transgenic mice, to the identification of functional human genetic variation underlying autism and ADHD. Two established neuroscientists with a long-standing interest in advancing the careers of junior scientists oversee the Program. The Program Co-Director is Roger D. Cone, Ph.D., Chair of the Department of Molecular Physiology & Biophysics at Vanderbilt, and an elected member of the National Academy of Sciences. Dr. Cone's research incorporates the tools of modern genetics, molecular biology, biochemistry and behavior, with a focus on CNS control of appetite and eating disorders. Similar to Dr. Blakely, the paradigms used in Dr. Cone's research ranges across powerful genetic model systems, including transgenic zebrafish and mice, to research probing for functional gene variation underlying eating disorders. Like Dr. Blakely, Dr. Cone has trained dozens of predoctoral and postdoctoral fellows, bringing significant mentorship experience to the Program.

DESCRIPTION (provided by applicant): Genetic, pharmacological and imaging studies point to altered dopamine (DA) signaling as a risk determinant of Attention-deficit Hyperactivity Disorder (ADHD) and ADHD comorbid disorders. The presynaptic DA transporter (DAT, SLC6A3) has drawn particular interest due to its interactions with the most commonly prescribed ADHD medications, methylphenidate (MPH, Ritalin) and amphetamine (AMPH) formulations (e.g. Adderall). MPH is a DAT antagonist, whereas AMPH competes with DA for transport by DAT and induces DAT-mediated, non-vesicular DA release. The molecular/cellular basis for the effectiveness of these agents in ADHD treatment, and their relationship to a DA-based etiology of ADHD is unclear, owing in large part to a lack of understanding of the etiology of the disorder. This fact has also contributed to a lack of animal models with construct validity, necessitating the use of models with trait similarities (e.g., hyperactivity) but where origins likly have little or no relationship to disease mechanisms. The genetic risk for neuropsychiatric disorders, including ADHD, is believed to arise from contributions of both common and rare genetic variation. Whereas common genetic variation is often difficult to model, owing to a lack of penetrance and conservation across species, rare variation, particularly polymorphisms that impact protein coding sequence, offers the prospect of targeting conserved elements of proteins to achieve functional changes in molecules, cells and circuits. Recently, we identified a rare, coding variant (Val559) in the SLC6A4 gene of two male siblings with ADHD, a variant also identified in subjects with bipolar disorder and autism. Our studies of DAT Val559 expressed in transfected cells and neurons revealed that although DAT protein expression and DA uptake are normal, the variant displays a striking, voltage-dependent, DAT-mediated, anomalous DA efflux (ADE) phenotype. Remarkably, AMPH suppresses the ADE of DAT Val559, whereas it induces DA efflux from WT DAT (Ala559). DAT Val559 is also phosphorylated at N-terminal sites that are normally phosphorylated only after AMPH application, and the mutation of these sites in DAT Val559 to preclude phosphorylation eliminates ADE. These studies led us to the novel hypothesis that tonic DA leak, arising from perturbations of a DAT regulatory network that normally sustains concentrative DA uptake, leads to DA-linked traits prominently featured in multiple neurobehavioral disorders. To test this hypothesis, we have generated DAT Val559 knock-in mice and initiated a multi-disciplinary program to profile their biochemical, physiologica and behavioral features. Our Preliminary Studies provide evidence of ADE in vivo that leads to anomalous actions of local and systemically administered AMPH. In the current proposal, we seek to extend these studies to establish molecular, physiological and behavioral perturbations that arise in the model, studies that can redirect the field toward a novel framework for RDoc-responsive diagnoses and treatments underlying multiple psychiatric disorders.