Habibeh Khoshbouei, PhD, PharmD - US grants

DESCRIPTION (provided by applicant): Methamphetamine (METH) is one of the most addictive and neurotoxic drugs in existence whose societal impact is on the rise. The molecular mechanisms underlying the effects of METH on the dopamine transporter (DAT), the major molecular target of several psychoactive drugs, are poorly understood. Importantly, due to structural similarities between METH and amphetamine (AMPH), METH regulation of DAT is generally inferred from studies characterizing AMPH. Therefore, the biophysical properties and underlying molecular mechanisms of METH-exposed DAT are virtually unknown. METH primarily exerts its addictive properties by producing large elevations in extracellular striatal dopamine (DA). The DAT is a neurotransmitter transporter that regulates the magnitude and duration of synaptic signaling by clearing released DA from the synapse. However, DAT also mediates DA release via reverse transport (efflux) and can operate in a channel mode, which dramatically increases DA flux. METH mediates DA efflux via DAT, and revealing the mechanisms for this efflux is critical in understanding METH addiction and neurotoxicity. The proposed studies will test the hypotheses that METH regulates extracellular DA by: stabilizing DAT channel mode activity to increase DA efflux, decreasing DA uptake, and/or modifying DAT cell surface distribution in a voltage- and phosphorylation-dependent manner, and that these coordinated events account for the highly addictive nature and neurotoxicity of METH when compared with structural congeners, like AMPH. We will test these hypotheses, all of which are supported by promising preliminary data, with the following specific aims: 1) Determine the biophysical and molecular mechanisms underlying METH-induced DA efflux relative to AMPH, 2) Test the hypothesis that METH targets a phosphorylated state of DAT to regulate DA efflux, substrate uptake, and DAT surface distribution, 3) Compare METH-induced with AMPH-induced current-to-substrate ratios 4) Measure METH-provoked DAT surface mobility as a function of DAT N-terminal phosphorylation. We will achieve these aims in midbrain dopaminergic neurons and DAT expressing oocytes using whole-cell, cell-attached, and cell-detached patch clamp with simultaneous amperometry to measure DA efflux; and use the fluorescent substrate ASP+ to monitor DAT-dependent uptake. We anticipate that our findings will identify mechanisms for novel therapeutic strategies that may prevent or reverse METH toxicity/addiction, as well as suggest unique targets for other neurological diseases whose etiology includes dysfunction of the dopaminergic system. PUBLIC HEALTH RELEVANCE: Chronic use of methamphetamine can lead to addiction, severe neurological and psychiatric impairment, as well as pronounced neurodegeneration. Due to structural similarities regulation of dopamine transporter (DAT) is generally inferred from studies characterizing other amphetamines. Our proposed studies 1) will clarify the molecular mechanisms of methamphetamine-induced dopamine efflux via DAT, and 2) differentiate methamphetamine from its well-characterized, less neurotoxic congener, amphetamine that are valuable for identification of therapeutic strategies for the treatment of methamphetamine addiction and other neurodegenerative diseases.

DESCRIPTION (provided by applicant): Methamphetamine abuse is a major public health problem with no effective treatment strategies or FDA-approved pharmacotherapies. Methamphetamine addiction is thought to be a chronic disease rooted in numerous neurobiological adaptations induced by this psychostimulant. Arguably, the greatest promise for the treatment of methamphetamine abuse lies in determining the underlying molecular mechanisms of these neuronal adaptations to generate the opportunity of developing targeted therapies. The long-term goal of this work is to identify these underlying molecular mechanisms. The dopamine transporter (DAT) is the primary target for psychostimulants such as methamphetamine and cocaine. By inhibiting dopamine transporter uptake, these drugs are able to increase extracellular dopamine concentration, which modulates dopamine-associated behaviors. However, what is not often recognized is that methamphetamine is a substrate for the dopamine transporter and a high affinity ligand at the sigma-1 receptor (sigma1R), an intracellular chaperon protein. Our preliminary data suggest that: 1) the sigma1R is expressed in the soma and dendrites of midbrain dopaminergic neurons; 2) the sigma1R is co-localized and interacts with the DAT; 3) the administration of either a sigma1R agonist or methamphetamine increases the co-localization and interaction of DAT/sigma1R; and 4) co-administration of a sigma1R agonist influences methamphetamine-regulation of dopamine uptake, dopamine efflux, and the excitability of dopaminergic neurons. In this application we will test the overarching hypothesis that methamphetamine regulation of dopamine transporter function relies in part on methamphetamine-induced increases in sigma1R/DAT interactions and their functional consequences. We will focus on determining the mechanism of DAT/sigma1R interaction (how Aim1), the cellular localization of DAT/sigma1R interaction (where, Aim 2), and the functional consequence of this interaction (what, Aim 3). If, as we postulate, the interaction with the sigma1R represents a main intracellular mechanism for mediating methamphetamine-regulation of DAT, then we will have identified a possible target for treating methamphetamine addiction and other psychiatric diseases involving dysregulation of dopamine neurotransmission.

? DESCRIPTION (provided by applicant): A group of NIH funded scientists at the University of Florida, Gainesville campus, are requesting funds to purchase a Nikon A1R two-photon microscope system capable of imaging dynamic processes in thick samples. There currently is no two-photon instrument at any of the shared core facilities on the University of Florida Gainesville campus. This application describes the immediate needs of our NIH funded research faculty for an instrument capable of both two-photon imaging, providing the Z-depth resolution needed to capture protein movement in living tissue, as well as high-speed resonance scanning with an integrated perfect focus system to capture fast cellular events. The existing confocal and wide-field microscopes at the UF imaging core facility are maximally used, or are outdated and cannot be upgraded to accomplish the imaging needs of the major and minor users of this application. As with the current instruments, the proposed two- photon system will be housed and managed in the shared Cell and Tissue Analysis Core Facility (CTAC). All major and minor users will have access to the instrument and training will be provided through the established CTAC infrastructure. The usage charges and institutional funding have supported the service contract, supplies, and the CTAC personnel; therefore, we foresee no difficulty in this arrangement continuing for the Nikon A1R two-photon system. In addition, the CTAC will train the users of the two-photon instrument, as required by their research projects. The shared CTAC facility has an extensive track record of education, training and productivity with over 190 lab groups at the University of Florida. During the past seven years, the imaging core has introduced shared access to confocal microscopy, live cell imaging, and whole animal imaging. The UF research community now extensively uses these advanced microscopy techniques. For imaging of thick intact tissue, two-photon excitation is far superior to other approaches and permits high-resolution imaging at a level 5 to 10 fold deeper than with confocal microscopy. The proposed two-photon instrument will be the only generally available two-photon imaging system at the University of Florida. The availability of this technology is crucial for the mission of the University of Florida with the overall goal of finding treatments fo human disease and to train the next generation of outstanding investigators in biomedical sciences.

The communication between neurons and microglia is bidirectional. Microglia modulate neurotransmission, facilitate synapse formation and dissolution, and provide neuronal protection, but cellular/molecular mechanisms are incompletely understood. Much of the premise for interactions between dopamine neurons and microglia is supported by presence of dopamine receptors on microglial cells allowing them to respond to neuronal signals. The idea is that dopamine receptor stimulation on microglial cells alters microglial function, which in turn could then (reciprocally) affect dopamine neurotransmission. Here we will use patch clamp electrophysiology, two-photon imaging, biochemical and histological approaches to determine whether and how depleting microglia affects dopamine neurotransmission and whether HIV-1 Tat, a protein produced in microglial cells following HIV-1 infection, disrupts dopamine neurotransmission by altering microglial/DA neurons interactions (Aim 1). We will examine how microglial activity is affected by dopaminergic signaling in the presence or absence of HIV- 1 Tat, and conversely how microglial products may modulate dopamine neurotransmission (Aim 2). Finally, since there is a high comorbidity between HIV-1 infection and drug abuse, and since both methamphetamine and HIV-1 Tat alter dopamine neurotransmission and affect the immune system, in Aim 3 we will determine how the combined exposure to HIV-1 Tat and methamphetamine influences these processes. The proposed work will address two significant knowledge gaps: 1) reveal the cellular/molecular mechanisms underlying bidirectional communication between dopamine neurons and microglia, and 2) determine how HIV-1 Tat modulation of this bidirectional communication reduces dopamine neurotransmission.

The central goal of this study is to investigate the functional and physical interplay between the dopamine (DA) D2 autoreceptor (D2R) and the dopamine transporter (DAT) and the role of this interplay in the regulation of DA neurotransmission. DA neurons are functionally heterogeneous with spatially separated somatodendritic and axonal projections initiating in two neighboring brain structures; ventral tegmental area (VTA) and substantia nigra (SN). Aberrations in DA neurotransmission are implicated in neuropsychiatric disorders; including schizophrenia, attention deficit/hyperactivity disorder (ADHD), drug addiction, and Parkinson's disease. Critical mechanisms in the regulation of DA availability at the synapse include the activation of D2 autoreceptors and DA uptake via DAT. Traditionally, these two mechanisms ? D2R and DAT- have been studied individually; however, exciting new evidence from in vitro experiments suggests that D2 autoreceptor and DAT interact physically and possibly functionally. Published data and our own preliminary findings support the hypothesis that D2 autoreceptor and DAT exist as a macromolecular complex and that D2 autoreceptor activation regulates DAT activity and trafficking in DA neurons through a GIRK-mediated mechanism. To address this hypothesis we will use a multidisciplinary approach combining molecular, biochemical, electrophysiological, and optic approaches in cultured DA neurons and brain slices containing somatodendritic regions of VTA, SNc and their projection areas (dorsal striatum and nucleus accumbens) where both DAT and D2R are co- expressed. In aim 1, we will use genetic, electrophysiological and optic tools to examine functionally whether D2R activation increases DAT activity and trafficking through a mechanism involving GIRK-mediated hyperpolarizarion of the cell membrane. In aim 2, we will use molecular, biochemical, and optic tools to examine the contribution of the D2R-DAT physical interaction to the D2R-mediated regulation of DAT activity and trafficking. We will compare and contrast findings obtained from SN and VTA, projections areas in the dorsal striatum vs nucleus accumbens, as well as samples from male and female animals. Given the role of D2R and DAT as therapeutic targets for DA-related conditions, the successful completion of this work will reveal the physiological significance of the interplay between presynaptic D2 receptor and DAT. The result of this work will have wide-ranging significance, as it will reveal a unique mechanism for the FDA approved D2R agonists' and DAT antagonists' regulation of dopamine neurotransmission.