Haley E Melikian, Ph.D. - US grants

DESCRIPTION: (provided by applicant) Monoamine reuptake is a definitive step in the termination of neural transmission and is mediated by a family of Na+-/C1-dependent plasma membrane transporters. Monoamine transporters are the molecular targets for the psychostimulants cocaine, amphetamines, and MDMA ("ecstasy"), which bind to and inhibit transporters, thereby increasing extracellular monoamine concentrations and enhancing post-synaptic responses. As the major psychostimulant targets, transporter presentation at the cell surface is paramount to psychostimulant binding and action. Mounting evidence from a number of investigators demonstrates that activation of cellular signaling pathways dynamically modulates monoamine transporter cell surface presentation. Moreover, data from our laboratory demonstrates that transporters dynamically traffic under steady state conditions. Transporter regulation and trafficking correlate with changes in transporter phosphorylation states. However, the relationship between transporter trafficking and phosphorylation is not well defined. The major goals of this project are to understand the role of phosphorylation in transporter function and trafficking. We will pursue this area of investigation by testing the following hypotheses: (1) transporters are phosphorylated and dephosphorylated as part of their constitutive trafficking process, and (2) protein kinase C (PKC) activation blocks basal transporter dephosphorylation, thereby perturbing endocytic transporter trafficking. The proposed studies will focus on a representative monoamine transporter, the dopamine transporter (DAT). We will test our hypotheses by directly analyzing basal and regulated transporter phosphorylation and trafficking in cell lines. Intrinsic domains mediating DAT phosphorylation will be identified using molecular truncation and mutagenesis approaches. It is expected that these approaches will provide a clearer picture of how transporter trafficking and phosphorylation are coupled. Such results are expected to have a significant impact on future therapeutic strategies aimed at monoamine-related psychostimulant drug abuse. moreover, the outcomes will enhance our understanding of the factors contributing to monoamine availability and signaling in the brain.

DESCRIPTION (provided by applicant): Presynaptic reuptake is the primary means to limit extracellular monoamine levels and terminate synaptic signaling. Biogenic amine transporters in SLC6 carrier gene family facilitate reuptake and are the major targets for addictive and therapeutic psychostimulants, as well as for antidepressants. These drugs potently inhibit monoamine reuptake, and thereby increase extracellular monoamine concentrations, enhance neuronal signaling and significantly modulate monoamine-related behaviors. Thus, transporter activity and availability are critical determinants of normal biogenic amine neurotransmission and psychoactive drug efficacy. A wealth of data supports that biogenic amine transporter surface presentation is not static. Rather, transporters are subject to robust constitutive endocytic trafficking. The dopamine transporter (DAT) is acutely modulated by protein kinase C (PKC) activation and exposure to psychostimulants cocaine and amphetamine (AMPH), which modulate DAT internalization and recycling rates, ultimately decreasing DAT surface availability. The major goal of these studies is to elucidate the mechanisms that control both basal and regulated biogenic amine trafficking. In this renewal application we build on our previous strengths in cellular and molecular approaches and our previous findings that Rin GTPase binds to DAT and is required for PKC-mediated DAT internalization. Specifically, we aim to (1) Elucidate the molecular determinants governing Rin-dependent DAT endocytosis and determine whether Rin GTPase is required for AMPH-mediated DAT trafficking in neuronal cell lines, (2) Determine whether PKC- and AMPH-mediated DAT trafficking are region-dependent and whether Rin GTPase is required for PKC- and AMPH-mediated DAT trafficking in situ, and (3) test whether Rin-dependent DAT trafficking is required for psychostimulant reward. These hypotheses are based on strong preliminary data that demonstrate specific Rin interactions with DAT, but not other SLC6 transporters, and a potential role for calmodulin in Rin downstream signaling. We will use chimeric proteins to define the DAT domains that confer specificity of the DAT/Rin interaction. We will use biochemical and pharmacological approaches in combination with GTPase mutants and shRNA-mediated Rin knockdown to determine the signaling pathways downstream of Rin that are necessary and sufficient for regulated DAT internalization in neuronal cell lines and mouse striatum. We will further test whether PKC- and AMPH-mediated DAT trafficking occur in a region-specific manner in the striatum, and use in vivo Rin knockdown to test the role Rin in regulated DAT endocytosis in situ. Finally, we will use in vivo shRNA approaches to knockdown Rin GTPase in mouse VTA to test whether DAT trafficking is required for psychostimulant reward. The results obtained from these endeavors will provide a clearer understanding of the mechanisms controlling transporter surface expression and the role of DAT trafficking in rewarding behaviors. We anticipate that our findings will greatly impact future strategies aimed at treating affective disorders and drug addiction. Moreover, the results will undoubtedly enhance our understanding of the molecular factors influencing monoamine availability in the brain.

[unreadable] DESCRIPTION (provided by applicant): Psychostimulant addiction is a widespread problem in the United States that affects millions of individuals at a substantial cost in both lost wages and medical expenses. Amphetamines and cocaine act primarily by blocking monoamine reuptake facilitated by sodium- and chloride-dependent plasma membrane transporters for serotonin, norepinephrine and dopamine. Although these psychostimulants have equimolar affinity for the monoamine transporters, the reinforcing properties of these drugs are thought to be largely mediated by their interaction with the dopamine transporter (DAT). Recent in vitro and in vivo studies demonstrate that, in addition to inhibiting DAT function, amphetamines and cocaine also modulate DAT cell surface levels via membrane trafficking. Preliminary studies from our laboratory demonstrate that amphetamine- induced loss of cell surface DAT occurs via enhancing DAT internalization rates. However, it is unknown whether psychostimulant-induced changes in DAT surface expression are integral to the addictive process. This is due to a lack of available animal models to test this hypothesis. We recently identified a ten amino acid region spanning residues 587-596 in the DAT carboxy terminus that is necessary for basal and protein kinase C-stimulated DAT endocytosis. We propose to test whether psychostimulant- mediated DAT trafficking is required for the rewarding properties of psychostimulants. To this end, we aim to generate DAT "knock in" mice expressing endocytic-defective DATs. The resulting mice will be assessed for amphetamine self-administration, conditioned place preference and sensitization, to test whether DAT trafficking plays a role in the rewarding properties of psychostimulants. Moreover these animals will be an invaluable tool for future studies examining the role of DAT trafficking in synaptic transmission and dopaminergic plasticity. Psychostimulant abuse and addiction is a growing problem in the United States, reaching near epidemic proportions over the past several years. Despite numerous investigations into the mechanisms underlying psychostimulant addiction, there are still large gaps in our knowledge. The current proposal aims to develop an animal model in which the targets of amphetamine in the brain are modified, to determine whether the actions of amphetamine on these target proteins contribute to the addictive process. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Extracellular dopamine (DA) levels in the brain directly impact a variety of physiological functions, including movement, cognition and reward. Following synaptic release, DA availability is limited by presynaptic reuptake, mediated by the plasma membrane DA transporter (DAT). DAT is a member of the SLC6 carrier gene family and is the primary target for addictive and therapeutic psychostimulants, such as amphetamine, cocaine and methylphenidate (Ritalin) as well as for antidepressants, such as bupropion (Wellbutrin). These drugs potently inhibit DA uptake, and thereby increase extracellular DA concentrations, enhance neuronal signaling and significantly modulate DA-related behaviors. Thus, DAT activity and availability critically determine normal DA neurotransmission and psychoactive drug efficacy. DAT plasma membrane presentation is not static. Rather, DAT is dynamically shuttled to and from the plasma membrane by constitutive endocytic trafficking. Protein kinase C (PKC) activation and amphetamine (AMPH) exposure modulate DAT internalization and recycling rates, ultimately decreasing DAT surface availability. Recent studies in an ADHD pedigree have identified a DAT mutant that has altered DAT trafficking kinetics, both under basal and AMPH-stimulated conditions, and altered targeting to membrane raft microdomains, thereby implicating altered DAT trafficking in human disease. However, studies investigating DAT endocytic mechanisms have yielded conflicting results and DAT cell surface behavior is not well defined. The major goal of these studies is to directly examine DAT surface dynamics by TIRF microscopy in living cells and to elucidate the endocytic mechanisms that mediate basal and regulated DAT internalization. Specifically, we aim to (1) characterize the surface dynamics of wildtype and trafficking mutant DATs, (2) test the hypothesis that basal, PKC- and AMP-stimulated DAT endocytosis are clathrin-independent and Cdc42-dependent, and (3) test the hypothesis that DAT internalization is a dynamin-independent process. These hypotheses stem from strong preliminary data that demonstrate 1) DAT surface localization primarily to clathrin-independent foci, 2) dynamin-independent DAT trafficking and 3) Cdc42-dependent DAT endocytosis. For live TIRF imaging studies, we capitalize on a novel chemical biology approach that directly couples fluorophore to cell surface DAT. This innovative technique will facilitate the first direct examination of DAT surface dynamics without binding to bulky antibodies or inhibitory ligands. Complementary biochemical studies will be performed both in neuronal cell lines and mouse striatal slices, utilizing small molecule clathrin, dynamin and Cdc42 inhibitors to acutely perturb membrane trafficking, as well as standard shRNA and GTPase mutant approaches. The information gleaned from these studies will provide a clearer understanding of DAT surface dynamics and the mechanisms mediating DAT endocytosis, both for wildtype and trafficking mutants. We anticipate that our findings will greatly impact future strategies aimed at treating affective disorders and drug addiction. Moreover, the results will undoubtedly enhance our understanding of the molecular factors influencing DA availability in the brain.

Dopamine (DA) is a major neurotransmitter with diverse physiological impact, and is required for movement, sleep, working memory, and reward. Following evoked release, DA extracellular half-life is determined by presynaptic reuptake, mediated by the SLC6 plasma membrane DA transporter (DAT). Addictive and therapeutic psychostimulants, such as amphetamine, cocaine and methylphenidate (Ritalin), potently inhibit DA uptake, sustain DA signaling and impact DA-dependent behaviors. DAT coding variants are implicated in a variety of neuropsychiatric disorders, and transgenic mouse studies clearly demonstrate that DAergic signaling and behaviors, as well as psychostimulant efficacy, are highly sensitive to the level of DAT expression. DAT is not static at the plasma membrane, but is subject to robust constitutive and regulated endocytic recycling. However, it remains unclear whether regulated DAT internalization impacts DAergic function and/or DA-dependent behaviors. Our central hypothesis is that regulated DAT trafficking is likely a critical and influential determinant of DA signaling and DA-dependent behaviors. To test this hypothesis directly, we aim to replace endogenous mouse DAT with trafficking dysregulated DAT mutants in adult mice. However, germline DAT perturbations have clear developmental compensatory issues, underlining the need for a system that evaluates DAT mutants in vivo, but circumvents the pitfalls of germline mutant expression. To this end, we have developed a mouse model that integrates mouse with a floxed DAT gene (DATfl/fl) with a DAergic TET-OFF mouse (Pitx3IRES2-tTA). The resulting DATfl/fl;Pitx3IRES2-tTA mouse will facilitate AAV-mediated mouse DAT excision with Cre recombinase, and transgene replacement, driven by the Tet-responsive element, for in vivo molecular replacement studies. We contend that this system offers considerable advantages over germline knock-in approaches to test mutant protein function, as it circumvents developmental compensation and additionally allows for circuit-specific replacement using AAVs. Feasibility for this approach is strongly supported by strong preliminary data. The main aims of this two-year project are: 1) to optimize conditions for in vivo DAT molecular replacement, and 2) to test whether DAT ?gain-of-function? and ?loss of function? endocytic mutants impact DAT trafficking, DAergic signaling, and DA-dependent behaviors in male and female mice. We anticipate that at the completion of these studies we will have developed a powerful mouse system to interrogate the impact of DAT mutants in vivo in a manner that, heretofore, was not feasible. Moreover, we expect that our new mouse model will have broad utility for a number of researchers aiming to evaluate mutant function in adult mouse DAergic circuits.