Scott Mackler, M.D., Ph.D. - US grants

The primary objective of the research to be supported by this ADAMHA Scientist Development Award is the characterization of changes in gene expression in the nucleus accumbens of the adult rat brain induced by chronic cocaine and opiate administration. Addiction resulting from self-administration of several classes of drugs requires intact neuronal connections within the nucleus accumbens. Identification of messenger (m) RNA molecules regulated by both cocaine and morphine will be helpful in isolating the proteins which underlay the biological basis of the self-administration of addictive drugs. Specifically, this project will consist of three sections: i) the identification of cocaine-and morphine-regulated mRNAs from tissue sections containing the nucleus accumbens that encode proteins involved in signal transduction; ii) the isolation and characterization of novel mRNA from the nucleus accumbens regulated by cocaine or morphine administration; and iii) the description of the neurotransmitter receptor mRNA composition of individual dopaminergic neurons studied in live slice preparations of the nucleus accumbens. Techniques will be applied which enrich the mRNAs of interest via both in situ transcription in fixed tissue sections and patch clamp studies of live single neurons. Identification and characterization of known and novel drug-regulated mRNAs will lead to future studies investigating the functional role of the proteins encoded by these mRNAs in cocaine and morphine self- administration. The treatment of drug addiction is limited by an incomplete understanding of the molecular mechanisms underlying repeated drug use. Identification of mRNAs regulated by both cocaine and morphine in the rat will highlight factors common to many forms of drug addiction.

DESCRIPTION (Adapted From The Applicant's Abstract): This application proposes to investigate the function and role(s) of NAC-1. NAC-1 is a member of the POZ family of proteins and is present in the mammalian central nervous system (CNS). NAC-1 mRNA levels are increased in the nucleus accumbens of the rat CNS three weeks after chronic cocaine self administration. Interruption of NAC-1 expression via microinjection of antisense oligonucleotides into the rat nucleus accumbens augmented the locomotor responses to acute and chronic treatment, supporting the hypothesis that NAC-1 regulates the function of neurons in the nucleus accumbens that contribute to the behavioral sensitization and cocaine self-administration. The proposed molecular and behavioral studies are designed to understand how alterations in NAC-1 expression affect cocaine associated behaviors. The Specific Aims will: Aim 1: study NAC-1 binding to specific DNA sequences and other proteins and examine the effects of cocaine treatment on these interactions in the CNS; Aim 2: demonstrate the intracellular location (in vitro studies) and regional CNS distribution (in vivo studies) of NAC-1 protein before and after cocaine treatment; Aim 3: study the effects of diminished NAC-1 levels in the nucleus accumbens on the behavioral effects of acute cocaine and cocaine self- administration in the rat. Motor behavior or the rate of reinstatement of self administration will be measured after antisense oligonucleotide injection; Aim 4: determine the behavioral effects of acute cocaine after over- expression of NAC-1 protein in the nucleus accumbens via injection of a recombinant adeno virus vector that contains the NAC-1 cDNA. Repeated cocaine use leads to persistent molecular and behavioral changes. NAC-1 is one mRNA that demonstrates a long-term adaptation to cocaine use. Results from studies of NAC-1 function in the rat may be helpful in future studies of human subjects who abuse cocaine.

DESCRIPTION (Adapted From The Applicant's Abstract): This application proposes to investigate the function and role(s) of NAC-1. NAC-1 is a member of the POZ family of proteins and is present in the mammalian central nervous system (CNS). NAC-1 mRNA levels are increased in the nucleus accumbens of the rat CNS three weeks after chronic cocaine self administration. Interruption of NAC-1 expression via microinjection of antisense oligonucleotides into the rat nucleus accumbens augmented the locomotor responses to acute and chronic treatment, supporting the hypothesis that NAC-1 regulates the function of neurons in the nucleus accumbens that contribute to the behavioral sensitization and cocaine self-administration. The proposed molecular and behavioral studies are designed to understand how alterations in NAC-1 expression affect cocaine associated behaviors. The Specific Aims will: Aim 1: study NAC-1 binding to specific DNA sequences and other proteins and examine the effects of cocaine treatment on these interactions in the CNS; Aim 2: demonstrate the intracellular location (in vitro studies) and regional CNS distribution (in vivo studies) of NAC-1 protein before and after cocaine treatment; Aim 3: study the effects of diminished NAC-1 levels in the nucleus accumbens on the behavioral effects of acute cocaine and cocaine self- administration in the rat. Motor behavior or the rate of reinstatement of self administration will be measured after antisense oligonucleotide injection; Aim 4: determine the behavioral effects of acute cocaine after over- expression of NAC-1 protein in the nucleus accumbens via injection of a recombinant adeno virus vector that contains the NAC-1 cDNA. Repeated cocaine use leads to persistent molecular and behavioral changes. NAC-1 is one mRNA that demonstrates a long-term adaptation to cocaine use. Results from studies of NAC-1 function in the rat may be helpful in future studies of human subjects who abuse cocaine.

DESCRIPTION (provided by applicant): Drug addiction is associated with long-term behavioral changes. Identifying molecules that contribute to these behaviors is an important goal. NAC1 is a member of the POZ/BTB family of transcription factors that demonstrated increases in gene expression weeks after cocaine self-administration in the rat. Manipulation of NAC 1 levels in the rat nucleus accumbens caused changes in cocaine-regulated behaviors, yet the endogenous function of NAC 1 remains unknown. The proposed molecular and behavioral studies are designed to examine the role of NACI in the mammaliml brain and NAC1 's impact on cocaine-induced-behaviors. The specific aims will demonstrate: Aim 1. where NACI is expresseEand how cocaine administration will affect NAC1 's distribution; Immunokigtology will be performed using a polyclonal antibody that detects which cellular nuclei contain NAC 1 and the expression pattern will be determined in those CNS regions known to be involved in cocaine-induced behaviors. Aim 2. what other proteins interact with NAC1; Both POZ-BTB and non-OZ BTB proteins will be examined for their interactions with NAC 1. NAC 1, either full-length or in truncated forms, will be used as the "bait" to screen a mouse brain library, using a yeast two-hybrid screen. The protein-protein interactions will be confirmed by several biochemical tests and each cDNA will be sequenced to determine the identity of the encoded protein. Aim 3. the effects of manipulating NAC1 gene expression? Mice will be bred which do not express NAC1. They will be examined for: their phenotype and the behavioral responses to cocaine. The characterization of NAC 1, a gene that eMaibited long-term changes in expression after cocaine use, will further the understanding of cocaine addiction. The hnman NAC 1 proteinis similar to that of rat and mouse, therefore these experiments may provide important insights into cocaine abuse in humans.

[unreadable] DESCRIPTION (provided by applicant): Addiction is associated with long-term behavioral changes produced by repeated exposure to drugs of abuse. The enduring nature of addiction implies equally enduring changes in brain biochemistry. Identifying molecules that contribute to enduring behavioral changes elicited by addictive drugs may provide novel pharmacotherapeutic targets for treating addiction. During the previous tenure of this application NAC1 was characterized as a member of the POZ/BTB family of transcription factors that demonstrates increases in gene expression in the nucleus accumbens weeks after discontinuing cocaine self-administration in rats. Using antisense oligonucleotide and viral transfection strategies, the level of NAC1 in the rat nucleus accumbens was shown to be inversely related to the expression of locomotor sensitization elicited by repeated cocaine administration. However, the physiological function of endogenous NAC1 remains unknown. This application will examine the physiological role of NAC1 in the mammalian brain and its potential pathological role in cocaine-induced behaviors. It is proposed to examine the role of NAC1 as a transcriptional regulator, and as a proteasome binding partner in protein degradation. The relevance of these functions in cocaine addiction will be examined in vivo, using AAV transfection of NAC1 cDNA or siRNA and animals models of cocaine-induced behavioral plasticity and addiction, such as locomotor sensitization and the reinstatement of drug-seeking. The overall hypothesis of this application is that the regulation of NAC1 by withdrawal from cocaine is an important compensatory physiological response consisting of changes in gene transcription and mobilization of proteasome activity. [unreadable] [unreadable]