Kent E. Vrana - US grants

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. This pivotal enzyme is under strict regulatory control and responds to a variety of cellular stimuli by increasing its activity. In the short-term, TH is phosphorylated in vivo by at least three separate protein kinase systems (cAMP-dependent protein kinase, Ca++/phospholipid- dependent protein kinase and CA++/calmodulin-dependent protein kinase). TH can be phosphorylated in vitro by two additional kinases (cGMP-dependent protein kinase and TH-associated protein kinase from pheochromocytoma cells). Although the individual contributions of the separate kinases are unclear, the overall consequence of phosphorylation is an increase in enzyme activity. Five serine residues in rat pheochromocytoma TH have been identified as sites of phosphorylation. We propose to utilize a full-length cDNA clone (isolated in this laboratory) to test the functional significance of the phosphorylation of these serine residues. This cDNA clone has been expressed in bacteria as two different forms of beta-galactosidase fusion proteins. Both of these fusion enzymes exhibit high levels of activity. Attempts will be made to express TH as a native enzyme within bacteria. These recombinant enzymes will be purified and characterized as to their physical state, enzyme kinetics parameters and phosphorylation by protein kinases. Site specific mutagenesis will be employed to convert serine residues to a number of different amino acids. The altered enzymes will then be expressed and tested from their ability to be phosphorylated and activated by protein kinases. The role of each serine in TH activity and its post- translational regulation will be established. Deletion mutants will be constructed from each end of the TH cDNA. The prevailing hypothesis that TH is composed of an amino terminal regulatory domain and carboxyl terminal catalytic domain will be tested. This will also delineate the boundaries of the catalytic core of the enzyme. Both deletion and site specific point mutants will be constructed to test the hypothesis that leucine zippers are involved in the assemble of TH monomers into its native homotetramer form. We have identified two strong candidates for leucine zippers (with a third weaker candidate) in the carboxyl terminus of TH. These will be examined by interrupting the leucine repeat which is characteristic of the leucine zipper, expressing the enzyme and determining if it associates into multimers.

These studies propose to investigate the long-term consequences of cocaine and opiate administration on molecular indices of dopaminergic function. A central problem in substance abuse research is identifying how chronic drug administration changes the brain so as to account for the long-term problems of physical dependence, psychological addiction, and tolerance. One hypothesis is that the drugs create an epigenetic imprint. That is, the chronic administration of drug alters the pattern of gene expression- the levels of RNA and protein- such that they change the state of the brain. The experiments proposed in this portion of the center application are designed to address this issue in a multi-disciplinary way by focusing on a recognized neuroanatomical substrate of substance abuse- the mesolimbic dopamine pathway. Utilizing recombinant DNA research tools developed during the last funding cycle, in combination with the facilities and expertise of both primate and rodent investigators within the Center, the following specific aims will be undertaken. In SA #1, we will test the hypothesis that chronic cocaine regulates dopaminergic gene expression in the non-human primate. Pursuing previous studies from this laboratory and others within the Center, SA #2 will examine the long-term epigenetic consequences of administration of tropane analogs with markedly different pharmacokinetics and transporter selectivity than cocaine. Further experiments are planned to identify molecular correlates for physiological and neurochemical disparities observed based on the context of drug administration. Accordingly, SA #3 will establish the relationships between epigenetic imprinting by response-dependent (self- administration) versus response-independent cocaine administration in the rodent. Finally, SA #4 will examine the hypothesis that opiate administration can alter molecular indices f dopaminergic function. These studies will continue the progress we have made in understanding how long- term drug administration affects neuronal set-points and how this might account for long-term drug abuse liabilities.

DESCRIPTION (Applicant's Abstract): Cocaine and crack addiction remains a significant medical and social problem. While the dopamine transporter is thought to be the primary site of action for the acute reinforcing effects of cocaine, the post-synaptic consequences of cocaine use and the neurobiological mechanisms that subserve the addictive process remain to be confirmed. The identification of the genetic components underlying cocaine addiction is a critical step in identifying potential targets for therapeutic intervention. The central hypothesis of this application is that chronic drug abuse produces a metastable epigenetic imprint that may contribute to clinical issues such as tolerance, physical dependence, and withdrawal. This application proposes to use multiplex DNA hybridization arrays to examine the interface of functional genomics and behavior. - In Specific Aim # 1, DNA hybridization arrays will be used to profile the CNS epigenetic imprint induced by cocaine self-administration in rats. This will be accomplished using commercially available arrays as well as custom arrays, imprinted at this institution and designed to test specific hypotheses regarding cocaine abuse. These will then be followed by studies in Specific Aim #2 that examine a new binge abstinence model of cocaine administration. This model recapitulates several key features of human cocaine abuse (specifically, the progressive loss of behavioral control). Finally, studies at the end of the funding period (Specific Aim #3) will establish the time course for gene expression changes following cessation of cocaine self-administration. These last experiments will provide very important insights into the stable changes in gene expression that survive long-term cessation of the drug, while simultaneously identifying new genes whose expression is altered during the withdrawal period. In addition to the specific proposed experiments, efforts will be coordinated with Emory University, during the funding period, to establish a central repository for array data for general access by the NIDA research community (www.arraydata.org) as well as a tissue/RNA bank. The results of the studies described within the present application should provide a wealth of information concerning functional genomic contributions to the cocaine addiction process.

DESCRIPTION (provided by applicant): Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. During the previous funding cycle, work generated an innovative new structural model for TPH, provided insights into how the hydroxylases assemble into tetramers, identified a new phosphorylation site on TPH, and provided important information in the relative stabilities of TH and TPH. Experiments planned for the next funding period will address the need for more detailed information with a particular focus on four key areas. (1) Using the new structural model of TPH, along with available crystal coordinates for TH, sequence elements will be mapped within the active sites of the enzymes. These experiments will identify amino acid residues that influence and direct differential substrate utilization. (2) Mutagenesis will be conducted to refine the determinants of tetramer formation and the role of macromolecular assembly in regulating enzyme activity. Notably, while monomers have been demonstrated to retain activity, it remains to be determined how tetramer formation might influence the post-translational regulation of enzyme activity. (3) The hypothesis will be tested that differences in N-terminal regulatory domain sequences are responsible for enzyme stability. (4) The novel hypothesis will be explored that phosphorylation-regulation of TPH involves issues of selective stability as well as targeted interactions with the chaperone protein, 14-3-3. The proposed experiments extend the previous successes in the characterization of TH and TPH structure-function analysis. The studies will develop several novel ideas concerning the regulation of these enzymes and increase the base of knowledge from which we can interpret new information that will emanate from the human genome initiative in terms of naturally occurring polymorphisms in these important enzymes.

The rapid development of pre-clinical studies on alcohol abuse and alcoholism across many diverse disciplines presents an ever increasing training problem. Because of this multi-disciplinary data base, knowledge from molecular biology, biochemistry, neuroscience and behavior are required for appropriate synthesis and future study design. The proposed training program provides both the pre- and postdoctoral students with exposure to all facets of basic science alcohol research by a group of well-funded, committed alcohol researchers. The training faculty consists of 11 full-time members of either the Department of Biochemistry or the Department of Physiology and Pharmacology at Wake Forest University School of Medicine. Most of these members are also faculty of the interdisciplinary Neuroscience Program. The graduate trainees will have a choice between these three graduate programs. Trainee recruitment will be extensive and include an active attempt to involve minority students in the program. Given the faculty, resources and the multi-disciplinary nature of the training opportunity, be believe that each trainee will be able to continue a career in alcohol specific research following their tenure in the program. It is the primary goal of the program to assure that each trainee becomes able to understand the complex nature of alcohol's actions, with an appreciation for analysis from the molecular to the behavioral level.

DESCRIPTION (provided by applicant): Cocaine abuse remains a significant societal problem. A central theme of this research program, and the main hypothesis of this renewal application, is that chronic cocaine abuse produces significant changes in CNS gene expression that contribute to clinical issues such as tolerance, sensitization, physical dependence, craving, and withdrawal. Preliminary findings have added to the growing database on cocaine-responsive gene expression in the central nervous system. Experiments are proposed, within the present application, that extend these findings to identify those genes whose expression is altered following an enforced abstinence from cocaine self-administration. To accomplish this, a new behavioral paradigm has been developed in which a binge pattern of cocaine self-administration in rats, followed by a period of abstinence, produces a behavioral sensitization that displays some of the hallmarks of the addictive process in humans. This powerful behavioral model will be examined to identify those genes exhibiting persistent changes in gene expression following cessation of drug administration. The first series of experiments will examine the expression of known cocaine-responsive genes to see if they correlate with the behavioral sensitization. The studies will focus on the nucleus accumbens (core and shell), medial prefrontal cortex, hippocampus, and amygdala as neuroanatomical substrates of the behavioral perturbation. In addition, subsequent studies will concentrate on establishing the temporal course of gene expression to (a) establish the limits of the alterations (how long will they last?), and (b) determine the time at which the changes first become manifest (before or during the abstinence period?). The second series of studies will use new Affymetrix GeneChip technologies (14,280 genes at a time) to identify families of genes that may be coordinately regulated and to identify novel targets of cocaine's effects. The proposed experiments will continue to contribute to our understanding of the role of genomics in establishing and maintaining cocaine addiction.

DESCRIPTION (provided by applicant): Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin. As such, it has been implicated in a variety of mental health disorders. One of the goals of this long-standing R01 has been to better understand this pivotal enzyme. However, in the midst of the preceding cycle, other investigators (and current collaborators) made the seminal discovery of a new variant of this enzyme (encoded by a separate gene) that is responsible for central nervous system serotonin synthesis - TPH2. Given that the work in this field had to this point focused on the peripheral enzyme (now termed TPH1), and given the importance of CNS serotonin to health and disease, we propose shifting the emphasis of this upcoming renewal period to better understanding this novel and pivotal enzyme. Virtually nothing is known about the structure, function, and enzymology of hTPH2. Partnering with the discoverers of this new enzyme, we have established important new characterization studies on the human TPH2 (hTPH2). We are strongly positioned to pursue three specific aims in the present proposal. Specific Aim 1 will characterize the functional consequences of naturally-occurring polymorphisms in the hTPH2 gene. In the few short years since discovery of the TPH2 gene, seven different coding region polymorphisms have been described. Little is known concerning the consequences of these amino acid substitutions and this aim will address this gap in our knowledge. Aim 2 will use data we have obtained from tyrosine hydroxylase and hTPH1 to map the active site of hTPH2. These biochemical studies will provide important functional insights into this novel and important enzyme. Specific Aim 3 will explore the regulation of hTPH2 by its N-terminal regulatory domain. These experiments will contribute to our knowledge of the dynamic regulation of serotonin biosynthesis in health and disease. The novel hTPH2 gene was first described three years ago. Its discovery resolves several important discprepancies in the field of serotonin biosynthese. However, we must establish a deeper understanding of this pivotal enzyme to better appreciate its role in health and disease and to provide a basis for potential development of novel pharmacotherapeutics.

Ethanol abuse and alcoholism remain very serious societal problems. A significant problem is the inability to diagnose alcohol abuse either in the general population or within selected groups of individuals such as adolescents and the recovering alcoholic. Accordingly, this proposal seeks to develop diagnostic biomarker signatures of acute and chronic alcohol consumption for diagnosing high-risk drinking, detecting relapse to drinking, disclosing recent drinking and in high risk situations such as pregnancy. To this end, studies are proposed to examine serum proteins and protein patterns for potential signatures in a powerful non-human primate model that is not encumbered by problems of comorbid drug use, inadequate diet and unreliable assessments of drinking history. In these NIAAA-funded, ongoing, within-subject studies, monkeys have been induced to voluntarily drink large amounts of alcohol. In the course of the studies (encompassing over 100 individual animals covering years of behavior and observation), serum samples have routinely been collected and archived. Experiments are proposed to screen these samples for potential biomarkers that can then be taken forward into the human population. Serum samples from a long-standing nonhuman primate self-administration study will be used as a training set for biomarker identification using high throughput proteomics. Samples will be processedto deplete the most abundant, obscuring proteins and then subjected to 2-DIGE (2-D Fluorescence Difference In-Gel Electrophoresis) for quantitative fluorescence identification of altered serum protein expression followed by MALDI-ToF/ToF identification of protein species. Statistical validation will be conducted, in a blinded fashion, using a test set of samples from an independent colony of self-administering monkeys, which will also contain data on adolescent vulnerability. The key criteria of any putative biomarkers will be sensitivity (percentage of positive scores among drinkers) and specificity (percentage of false positives in a non-drinking population). In addition, these studies will provide initial indices of positive and negative predictive values for biomarker signatures. A clinical test for ethanol abuse and alcoholism would have many potential uses. To discover protein biomarkers of ethanol abuse and alcoholism, serum from a controlled non-human primate population self- administering ethanol will be examined by quantitative proteomic methods.

DESCRIPTION (provided by applicant): Alcohol abuse and alcoholism remain significant societal problems. Unfortunately, treatment for and monitoring of harmful alcohol consumption is hampered by the lack of a highly accurate diagnostic test of alcohol drinking behavior. Our long-term goal for this research project is to develop biomarkers of alcohol abuse for use in at- risk populations. In particular, we have identified highly sensitive and specific plasma protein biomarkers using a well-controlled nonhuman primate model of alcohol abuse. We propose, in this application to translate these findings from the nonhuman primate into potential diagnostic tools for monitoring alcohol abuse in humans. This will be undertaken in collaboration with three clinical sites around the world. Specifically, we will obtain de- identified clinical samples from he Coatesville Veterans Affairs Medical Center, the Yale University School of Medicine, and the Institute for Health and Welfare of Finland (Helsinki, Finland). Studies associated with this research program will be divided into two specific aims. Specific aim 1 will validate a novel plasma biomarker panel in recovering alcoholics. In this aim, our clinical collaborators (from Yale and the Coatesville VAMC) will provide anonymous, within-subject longitudinal samples from subjects undergoing withdrawal and early abstinence (21 to 28 days) from abusive drinking as well as from the general population. These well-annotated samples will be used to refine the list of 27 biomarkers to identify the set that is most diagnostic of the human drinking phenotype and withdrawal/ abstinence. These experiments will employ a multiplex quantitative solution-phase immunoassay designed for human analytes (Myriad RBM DiscoveryMAP v1.0). As with all biomarker discovery projects, our primary emphasis will be on identifying a plasma protein analyte panel with high sensitivity and specificity. Specific aim 2 will then refine the plasma biomarker panel to permit diagnosis of hazardous drinking in a cross-sectional population from multiple sites. Within- subject assessment of drinking behavior (Aim #1) is much easier because the test does not compare differing baseline physiologies. Requirements for general population screening are much more stringent and will be assessed in samples from three independent sites (Yale University, Coatesville PA Veterans Administration Medical Center and the Finland National Institute for Health and Welfare). These sites provide a rich number of samples and drinking behaviors from which to draw experimental cohorts. In summary, we believe this work is innovative in that it capitalizes on new concepts in biomarker development through the identification of novel targets. The specific aims will build on preceding successes in the nonhuman primate model by characterization of human samples. All of the proposed studies will involve correlation with normal clinical and psychosocial assessments of alcohol consumption (AUDIT self-reports; percentCDT, AST/ALT, GGT, etc.). In addition, because we are not using unitary biomarker measures, we will apply sophisticated biostatistical approaches (random forest, support vector machine, principle component analysis) to the data analysis.