Annette E. Fleckenstein - US grants

DESCRIPTION: (Applicant's Abstract) The incidence of methamphetamine (METH) abuse has risen sharply over the past few years: hence, its neurochemical effects on central monoaminergic neurons warrant investigation. High-dose METH administration to rodents and primates results in short- and long-term deficits in monoaminergic systems. This laboratory reported recently the novel finding that METH administration causes a rapid and reversible decrease in dopamine transporter (DAT) function which is distinct from degeneration of dopamine terminals. Because DAT are essential for clearing dopamine from the extraneuronal environment, this rapid and reversible change in DAT may be a regulatory phenomenon of importance. Hence, the hypothesis will be tested that METH acutely and selectively alters the nature and function of DAT in a reversible manner by achieving the following Specific Aims: A. Assess the selectivity of the METH effect on DAT by: 1) confirming that this phenomenon is not due to dopamine neuronal degeneration; 2) comparing METH actions on DAT with effects on vesicular monoamine and glutamate transporters; and 3) assessing brain regional and species differences in the METH effect on DAT. B. Elucidate the nature of the reversible effect of METH on DAT by investigating whether: 1) METH treatment alters DAT protein structure; 2) dopamine D2 receptors modulate DAT function; 3) de novo protein synthesis is necessary for DAT recovery; and 4) antioxidants restore DAT activity after METH administration. C. Demonstrate functional consequences of METH-affected DAT by assessing the effect of METH-impaired DAT on dopamine reuptake in vivo, and subsequent uptake of METH in vitro. D. Evaluate whether the METH effect on DAT is selective to METH by assessing the response of DAT to: 1) agents that block dopamine reuptake (cocaine, nomifensine), or increase dopamine release (i.e., amphetamine, MDMA, MDE and fenfluramine); and 2) an agent which increases dopamine synthesis (L-dopa). Results of these studies will elucidate the importance of the rapid and reversible METH-induced decrease DAT activity. An understanding of this phenomenon has important implications regarding the mechanism of action of METH and other psychostimulants, as well as for the physiological regulation of dopaminergic systems. Such understanding could help develop better strategies for treating drug abuse and dopamine-related neurological and psychiatric disorders.

Since 1991, there has been a disturbing increase in the abuse of the potent stimulant, methamphetamine (METH) in both adolescents and young adults. We and others have investigated the short- and long-term effects of METH on dopamine (DA) and serotonergic (5HT) systems of young adult rats (typically 60-90 postnatal day; PND). Because of reports that younger animals (20-40 PND), corresponding to the human adolescent developmental stage, are less likely to experience METH-induced long-term changes in DA systems, little METH-related research has been conducted on this population. In preliminary studies, we confirmed that multiple high doses of METH cause deficits of 50-70 percent in striatal DA parameters after 7 d in 90 PND, but not 40 PND, male rats. In contrast, METH-induced short-term (1 h after treatment) responses by DA systems were very similar in both the 40 and 90 PND groups. Surprisingly, we did not observe an age-dependent difference in either the short- or long-term responses by the 5HT systems to METH treatment. Because of these age-dependent differential responses, it is important to study and compare the effects of METH on both young (corresponding to human adolescence) and more mature (corresponding to human adults) animals. In order to elucidate the age-dependent effects of METH abuse, this proposal will test the hypothesis that there are age-related differential long- but not short-term responses by DA systems to METH; however, the developmental pattern of response to METH is different for the 5HT system. This hypothesis will be tested by achieving the following Specific Aims: A. Determine the pattern of the age-dependent differential response by DA systems to METH, and compare these age-related effects to those observed in 5HT systems. B. Determine the mechanism responsible for the age-dependent DA differential responses to METH. C. Determine the consequences of the age-dependent differential response to METH. The studies associated with this specific aim are based on the interesting observation that exposure of adolescent rats (i.e., 40 PND), which as noted above, are refractory to the long-term DA deficits caused by METH, makes these animals also refractory when later challenged as adults (90 PND) with high doses of METH. The mechanism and selectivity of this phenomenon will be studied.

DESCRIPTION (provided by applicant): Methylphenidate (MPD) is a plasmalemmal dopamine (DA) transporter (DAT) inhibitor used to treat attention deficit hyperactivity disorder. In 2002, our laboratory reported a previously unidentified effect of this stimulant. Specifically, MPD administration in vivo rapidly and reversibly increases DA uptake via the vesicular monoamine transporter-2 (VMAT-2), a protein critical for vesicular DA sequestration, as assessed ex vivo in a non-synaptosomal membrane-associated (referred to herein as cytoplasmic) subcellular fraction prepared from treated rats. The impact of MPD on VMAT-2 is not restricted to vesicular trafficking. Preliminary data presented herein are the first to demonstrate that: 1) VMAT-2 co-fractionating with synaptosomal membranes after osmotic lysis (referred to throughout the proposal as VMAT-2M) display novel DA transport kinetics;and 2) MPD kinetically upregulates transport via the VMAT-2M, thus permitting increased vesicular DA accumulation. MPD enhances K+stimulated DA release as well. To better understand not only the functional role of VMAT-2M, but also the therapeutic and abuse properties of MPD, this proposal will test the hypothesis that MPD uniquely affects monoaminergic vesicular function and distribution by a mechanism that includes novel shifts in VMAT-2M-mediated DA transport kinetics. This will be accomplished by: 1) elucidating the unique features of the MPD-induced alterations in transport via the VMAT-2M;2) investigating mechanisms that regulate transport via the VMAT-2M per se, and after MPD treatment;and 3) demonstrating functional importance of MPD-induced alterations in DA transport via the VMAT-2M. Completion of the studies described in this proposal will accomplish two goals. First, the regulation and function of the VMAT-2M will be elucidated. This is important, given the large sequestration capacity and the unique kinetic profile of the VMAT-2M-associated vesicles and the role of these vesicular systems in regulating overall dopaminergic influences. Second, the impact of mechanistically dissimilar psychostimulants, particularly MPD, on the VMAT-2M will be explored. Taken together, these data will provide insight into mechanisms underlying the pharmacological, physiological and pathophysiological regulation of vesicular DA uptake and sequestration, and will thus advance understanding of a wide array of processes including those underlying addiction, and those contributing to the development of neurodegenerative diseases affecting dopaminergic systems. PUBLIC HEALTH RELEVANCE Methylphenidate (MPD) is a plasmalemmal dopamine (DA) transporter (DAT) inhibitor used to treat attention deficit hyperactivity disorder. Our laboratory reported previously that MPD also affects the function of the vesicular monoamine transporter-2 (VMAT-2). The goal of this project is to elucidate this effect, and thus advance understanding of a wide array of processes including those underlying addiction, and those contributing to the development of degenerative diseases affecting dopaminergic systems.

DESCRIPTION (provided by applicant): This is a Career Development Award proposal for Dr. Annette Fleckenstein. She is an Associate Professor of Pharmacology and Toxicology at the University of Utah. She has been active in drug abuse research for 10 years and has made important contributions to elucidating the effects of psychostimulants on dopamine (DA) transporter (DAT) and vesicular monoamine transporter-2 (VMAT-2) regulation and function, especially as related to the neurotoxic effects of the amphetamines. The applicant currently devotes 50% of her time to research while 50% of her effort is occupied by teaching, department, and college activities. This Career Development Award will allow the applicant to increase her research effort to 75%; substantially increasing her research flexibility and allow her to fully take advantage of her scientific skills as she becomes established as a leader in her academic field. During the time of support by this award, she will direct research which tests the following principal hypotheses: 1) differences in the effect of METH and cocaine on VMAT-2 and DAT contribute to their unique persistent DA consequences; and 2) methylphenidate affects vesicular DA transport and the intraneuronal localization of VMAT-2 in a manner distinct from amphetamine. The increase in research time resulting from a Career Development Award will not only allow the applicant to expand upon these studies, but also develop expertise in technologies new to her laboratory, including liquid chromatography-mass spectrometry to explore the hypothesis that METH-induces formation of differentially modified DAT oligomers. The increase in research time will also allow the applicant greater opportunity to participated in interdisciplinary efforts such as those involving the Utah Addiction Center (a translational center).

12-20 years old; 3,4-Dihydroxyphenethylamine; 4-(2-Aminoethyl)-1,2-benzenediol; Adolescence; Adolescent; Adolescent Development; Adolescent Youth; After Care; After-Treatment; Aftercare; Animals; Benzeneethanamine, N,alpha-dimethyl-, (S)-; Common Rat Strains; Crystal Meth; DA Neuron; DAT; DAT dopamine transporter; Data; Degenerative Disorder; Deoxyephedrine; Desoxyephedrine; Development; Disruption; Dopamine; Dopamine neuron; Dose; Event; Fever; Figs; Figs - dietary; Glia; Glial Cells; Hour; Human; Human, General; Hydroxytyramine; Hyperthermia; Idiopathic Parkinson Disease; Injection of therapeutic agent; Injections; Kolliker's reticulum; Lewy Body Parkinson Disease; Mammals, Rats; Man (Taxonomy); Man, Modern; Methamphetamine; Methylamphetamine; Modeling; N-Methylamphetamine; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neuroglia; Neuroglial Cells; Neurons; Non-neuronal cell; Paralysis Agitans; Parkinson; Parkinson Disease; Parkinson's; Parkinson's disease; Parkinsons disease; Primary Parkinsonism; Principal Investigator; Process; Programs (PT); Programs [Publication Type]; Protocols, Treatment; Public Health; Pyrexia; RGM; Rat; Rattus; Regimen; Resistance; Role; Staging; System; System, LOINC Axis 4; Testing; Toxic effect; Toxicities; Treatment Protocols; Treatment Regimen; Treatment Schedule; VMAT2 Protein; adolescence (12-20); adult youth; clinical hyperthermia; day; degenerative condition; degenerative disease; dopamine transporter; dopamine transporter proteins; dopaminergic neuron; febrile; febris; hyperthermia treatment; insight; juvenile; juvenile human; nerve cement; neuron toxicity; neuronal; neuronal toxicity; neurotoxic; neurotoxicity; postnatal; prevent; preventing; programs; public health medicine (field); resistant; social role; teenage; vesicular monoamine transporter 2; young adult

DESCRIPTION (provided by applicant): This is a Career Development Award proposal for Dr. Annette Fleckenstein. She is a Professor of Pharmacology and Toxicology at the University of Utah. She has been active in drug abuse research for 13 years and has made important contributions to elucidating the effects of psychostimulants on dopamine (DA) transporter (DAT) and vesicular monoamine transporter-2 (\/MAT-2) regulation and function, especially as related to the neurotoxic effects of the amphetamines. The applicant is a current K02 recipient, and this has permitted the applicant to dramatically decrease her institutional responsibilities and devote 75% of her time to research and professional development as described in her application. Renewal of this award is requested so that the applicant can continue directing research which test the hypotheses that: 1) methamphetamine (METH)-induced DAT complex formation is associated with, and may contribute to, the dopaminergic deficits caused by the stimulant; 2) methylphenidate uniquely affects monoaminergic vesicular function and distribution by a mechanism that includes novel shifts in VMAT-2M-mediated DA transport kinetics; and 3) latter-stage events related to intracellular DA management are critical for the persistent dopaminergic deficits caused by METH, and that these events are absent in both adolescent rats and those treated with METH during development. Continued support of this K02 award will not only allow the applicant to expand upon these studies, but also develop expertise in technologies new to her laboratory. In particular, funding of the K02 will facilitate the incorporation of self-administration technology into her laboratory that will permit investigation as to whether transporters are altered in that clinically relevant paradigm. Further, renewal of the K02 will allow the applicant to continue and expand interdisciplinary collaborative efforts with researchers both within and outside of the University of Utah as delineated in the proposal. PUBLIC HEALTH RELEVANCE: Psychostimulant abuse is a significant public health issue. The vesicular monoamine transporter-2 and the dopamine transporter are principal targets of these agents. Accordingly, support for this K02 award will provide support to allow the applicant to study the impact of stimulants such as methamphetamine, cocaine and methylphenidate on transporter function.

? DESCRIPTION (provided by applicant): The abuse of an increasing number of structurally diverse synthetic cathinones is a serious public health problem. The abuse of these agents is of concern for many reasons; including the fact that abuse of structurally related agents such as methamphetamine (METH) and methylenedioxymethamphetamine (MDMA) can cause persistent neurochemical and cognitive deficits. Thus, it is likely that some synthetic cathinones might likewise cause detrimental effects. Still, surprisingly little is known concerning the persistent impact of these agents on monoaminergic neuronal function. An important consideration in evaluating the impact of synthetic cathinones is that as increasing numbers of these agents are banned, illicit operations introduce chemically novel replacements to evade regulatory restrictions. Thus, as described in PAR 14-106, it is important to develop interaction profiles of common synthetic cathinones with entities such as transporters in order to classify them according to their potential abuse liability and toxicity profiles. Such templates will also b useful to anticipate the impact of these novel compounds at various developmental stages, another important focus of this PAR. As one contributor to this template, we will test the hypothesis that differential alterations in dopamine transporter (DAT) and vesicular monoamine transporter-2 (VMAT2) function predict differences in the persistent neurochemical, post-synaptic and functional consequences of synthetic cathinones and related agents; an effect impacted by the developmental stage of initial analog exposure. This will be tested by completing the following aims: 1) Evaluate the acute impact of non-contingent exposure to representatives of each of three established classes of synthetic cathinones (mephedrone, methcathinone and 3,4-methylenedioxypyrovalerone) on DAT and VMAT2 localization and function. We will conduct these studies using both adolescent and young adult rats as others and we have established that these transporters' functions can be differentially regulated as a function of age. Additionally, we will evaluate the impact of these agents on parameters demonstrated to contribute to psychostimulant-induced neurotoxicity, including reactive species formation and glutamate release. 2) Investigate the relationship between drug-induced alterations in monoamine transporter function with behavioral and neurochemical functional outcomes, including the likelihood that these agents will: a) cause persistent monoaminergic deficits; b) alter neurotensin levels, and c) be self-administrated. If self-administered, we will evaluate consequent persistent neurochemical effects as recent studies from others and we demonstrate important distinctions between the impact of contingent and non-contingent stimulant exposure. 3) Evaluate the persistent impact of co-administration of synthetic cathinones with each other, cocaine, METH and/or MDMA on the persistent neurochemical impact on dopaminergic neurons. Others and we have demonstrated that dual or sequential exposure to psychostimulants can profoundly alter their neurotoxic profiles, and thus this issue will be evaluated.