Howard Gu - US grants

DESCRIPTION: (Applicant's Abstract) Biogenic amine transporters are the main targets of cocaine, amphetamines, and antidepressants. My broad, long-term objectives are to understand the functions and mechanisms of these transporters, specifically their roles in the addiction to drugs of abuse and the actions of therapeutic drugs. A Mentored Research Scientist Development Award will allow me to learn new techniques necessary to study the in vivo functions of the biogenic amine transporters and to gain further experience before embarking on a fully independent research program. Biogenic amine transporters have been studied extensively in tissue preparations and cultured cells, and indirectly in whole animals using compounds that block these transporters. The only direct assessment of the roles of the transporters is the recent knockout of dopamine transporter in transgenic mice. Despite the significant new information learned from this study, extensive adaptation changes have set limitations to the study. Therefore, we propose to engineer an inducible antisense suppression system to study the biogenic amine transporters. In this approach, an antisense RNA would be expressed with an improved tetracycline operon based inducible system to suppress a specific transporter in a controllable and reversible manner. We will evaluate the effectiveness of the approach in cultured cells first, and next in transgenic mice. Then we will use this approach to investigate the physiologic and behavioral changes of the animals. We hope to provide new information about the functions of the transporters in whole animals and their roles in the addiction to drugs of abuse and the therapeutic actions of antidepressants. The knowledge gained from these studies may eventually lead to better approach to treat drug addictions, depression and other related diseases.

DESCRIPTION (provided by applicant): Currently, there are no pharmacological treatments for cocaine addiction partly because the mechanism of cocaine addiction is not clear. Cocaine has 3 high affinity targets in the brain, dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). Cocaine binds and blocks the uptake functions of these transporters, resulting in prolonged and elevated transmitter levels in the synapses, which are believed to underlie the stimulating and rewarding effects of cocaine. Surprisingly, knockout mice with DAT, NET, or SERT individually disrupted still exhibit cocaine reward, suggesting that no single transporter is explicitly required for cocaine reward and drugs preventing cocaine inhibition of a single transporter may not be effective. However, compensatory changes in the knockout mice may have altered the reward pathway. To avoid the compensatory changes, we have generated a knock-in mouse line carrying a DAT mutant that retains the uptake function but is insensitive to cocaine inhibition (DAT-KI mice). In these mice, normal doses of cocaine no longer block DAT, stimulate locomotion, or produce reward. Our results indicate that DAT blockade is required for cocaine reward in mice with a functional DAT. Most significantly, it suggests drugs that antagonize cocaine inhibition of DAT should be effective in blocking cocaine reward. DAT-KI mice provide a unique novel tool to investigate the mechanism of the complex cocaine effects. We propose to continue our current studies. We will examine cocaine responses by DAT-KI mice in several other behavioral tests to dissect out the contribution of DAT in the complex effects of cocaine. In addition, we will use AAV vector to re-introduce wild type DAT back into selected brain regions of DAT-KI mice to restore cocaine-induced DA elevation in those selected regions only and study what cocaine responses are restored. This unique approach allows us to correlate cocaine actions in specific brain regions to specific cocaine effects. Our preliminary data show that we are now able to inject AAV in confined brain regions and groups of mice with varying AAV injections restored cocaine responses in either conditioned place preference test only, locomotor stimulation only, both tests, or none of the tests, demonstrating the feasibility of our approach. The success of the proposed project will significantly enhance our understanding on how cocaine produces its complex effects, which are crucial for our efforts in the development of effective treatment for cocaine addiction.

DESCRIPTION (provided by applicant): Cocaine abuse and addiction continue to be a serious problem in the world. Currently, there is no effective pharmacological treatment for this mental disorder. Our long term goal is to understand the mechanism of cocaine addiction in order to develop effective treatments for this disorder. Cocaine has three known high affinity targets, the dopamine (DA) transporter (DAT), the serotonin (5HT) transporter (SERT), and the norepinephrine (NE) transporter (NET). When cocaine enters the brain it blocks all three transporters and thus increases extracellular DA, 5HT, and NE in various brain regions, resulting in complex cocaine effects. One way to study the role of each transporter is to generate knockout mice with individual transporters deleted. The potential problem with this approach is that adaptive changes to compensate for the complete removal of the critical transporter may alter the reward pathway significantly. We are employing an alternative approach. We are generating knock-in mouse lines carrying a transporter mutant that is insensitive to cocaine inhibition while maintaining functional monoamine uptake. We have generated a knock-in mouse line carrying a cocaine-insensitive DAT mutant that retains substantial uptake activity. In these mice, cocaine no longer produces reward, suggesting that DAT blockade is necessary for cocaine reward. However, DAT mutant mice do not provide information about the roles of NET and SERT in cocaine effects. The specific aims of the proposed study is (1) to construct mutants of SERT and NET that are functional but insensitive to cocaine inhibition;(2) to generate 2 mouse lines with their SERT or NET replaced by the cocaine-insensitive transporter mutants;and (3) to analyze the cocaine responses by the knock-in mice. This study will tell us whether NET or SERT blockade is also necessary for cocaine reward, or it enhances or dampens cocaine reward. Precise knowledge on how DA, 5HT, and NE systems modulate cocaine reward and aversion may suggest potential drug targets to modulate the intensity and dynamics of cocaine reward and aversion. Compounds that dampen the cocaine-induced euphoria and prolong cocaine dysphoria may discourage cocaine seeking and abuse. These compounds would be excellent drug candidates for the development of pharmacological treatment for cocaine addiction.

[unreadable] DESCRIPTION (provided by applicant): Site-directed and random mutagenesis is an indispensable tool in studying the structures and functions of proteins including those that are critical in drug addiction. However, the in vitro mutagenesis procedures are very labor intensive and of low throughput. Somatic hypermutation (SHM), one of the main mechanisms that generate the great diversity of antibodies in B lymphocytes, can be used to randomly mutate exogenous Genes Of Interest (GOI) that encode non-antibody proteins. Since mutations occur independently in each cell, millions of random mutants are easily generated and can be screened quickly when a proper selection method is set up. The SHM rate is much higher (up to 106 fold) at certain loci (such as loci for immunoglobulin V regions) than the rest of the cell genome. However, there is no convenient way to insert the GOI at loci of high SHM. Targeted homologous recombination can not be performed because the sequences at these loci are heterogeneous and different from cell to cell. Therefore, we propose to generate novel B-lymphoma cell lines that allow easy insertion of a GOI to loci of high SHM. Millions of mutants can be generated and expressed in the cell lines. Certain properties of the GOI mutants can be screened and selected for further studies. The mutagenesis procedure can be repeated multiple times to evolve the GOI to desired properties. We will generate the cell lines by integrating a DNA construct into the cell genome randomly that contains an insertion guide marker and a mutation reporter gene. We will perform a prescreening and two rounds of screening to isolate cells with the reporter gene mutated in a single cell division. Such cells should have the reporter gene integrated at loci of very high SHM rates. The insertion guide marker will allow easy and specific insertion of a GOI at the high SHM loci. In addition, the GOI will be controlled by an inducible promoter and thus SHM can be turned up and down because SHM rates are proportional to the levels of gene expression. In future studies, we plan to use this system to screen for mutants of the monoamine transporters that are insensitive to addictive drugs such as cocaine, amphetamine, methamphetamine, MDMA (ecstacy) or therapeutic drugs, such as Ritalin and Prozac. The cell lines can be used to study many other proteins on their structures, functions, drug binding sites, regulations, etc. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cocaine is a powerful psycho stimulant and one of the most addictive drugs of abuse. Despite decades of efforts, no effective pharmacological treatments have been developed for cocaine addiction or acute cocaine toxicity. There are two main approaches in developing the anti-cocaine treatments. 1) pharmacodynamic interventions: searching for compounds that inhibit cocaine binding to its targets, which have not resulted in effective drugs yet;2) pharmacokinetic interventions: strategies to limit or reduce the amount of cocaine available to act on its target proteins, such as the development of cocaine binding antibodies and improved cocaine degrading enzymes. The antibodies could be overwhelmed by large doses of cocaine while each enzyme protein can degrade many cocaine molecules. Significant progress has been made in improving the relative slow endogenous human enzymes by specific and random mutagenesis and by computation guided mutagenesis. In addition, a very active bacterial esterase has been cloned that hydrolyzes cocaine faster than any other known enzymes. However, this enzyme has no homology to the human enzymes and introducing this bacterial protein itself in the human body may cause severe immune responses. Therefore, we propose a strategy that takes advantage of the evolution power of nature. We propose to clone the cocaine degrading enzymes from Eloria Noyesi, the main insect pest of coca plants from which cocaine is extracted. These insects ingest large quantity of cocaine in its diet. The blood cocaine level in the feeding larvae is quite low and most of the ingested cocaine is degraded, suggesting the presence of super fast cocaine-degrading enzymes that have evolved to deal with the high cocaine diet. The successful cloning and additional studies of the cocaine degrading enzymes from Eloria should reveal the structural insight and catalytic mechanism of super efficient cocaine hydrolysis, which may provide novel ideas on how to modify and improve the human enzymes. Such ideas, combined with studies from other labs, may lead to further progress in engineering an ideal enzyme. Any significant improvement in the efficiency of the cocaine-degrading enzyme will make a big impact on the efficacy of an enzyme-based treatment of cocaine abuse. PUBLIC HEALTH RELEVANCE: We propose to clone the super efficient cocaine-degrading enzymes from Eloria Noyesi, the main insect pest of coca plant E. coca from which cocaine is extracted. The successful cloning and further studies of these enzymes may reveal the mechanistic insight of super efficient cocaine hydrolysis and thus provide novel ideals to further improve the human enzymes, which may eventually lead to effective and specific treatments for cocaine addiction and toxicity.