Regulation of cocaine- and amphetamine-regulated transcript expression by cyclic AMP response element binding protein Open Access
Rogge, George (2010)
Published
Abstract
Regulation of cocaine- and amphetamine-regulated
transcript
expression by cyclic AMP response element binding
protein
By
George A. Rogge
Drug addiction is a chronic brain disorder and recovering
addicts are subject to
drug relapse even after years of drug abstinence. A major focus of
drug abuse
research is to elucidate the molecular mechanisms of addiction
responsible for long-term
behavioral abnormalities that cause drug craving and repeated
relapse. One goal of that
basic research is to identify pharmacotherapy targets to develop
medications. Chronic
cocaine intake causes long-term genetic alterations in the brain
reward pathway that
contribute to addictive behaviors by up-regulating CREB
transcription factor activity in
the nucleus accumbens (NAc). Increases in CREB activity in the NAc
were correlated
with decreases in the reinforcing effects of cocaine during
conditioned place-preference
and drug self administration assays. The CART gene promoter was
found to contain a
cAMP response element (CRE) proposed to bind CREB, and CART mRNA
was
originally identified as a transcript in the rat NAc up-regulated
after cocaine
administration. Intra-NAc injection of CART peptides blunted the
rewarding effects of
cocaine in self administration assays. The overall hypothesis of
this dissertation is that
CART is a CREB-regulated gene. The data presented in this
dissertation demonstrated
by chromatin immunoprecipitation that CREB and P-CREB in cultured
cells were
capable of binding to a region of the CART promoter containing the
CRE site in the
nuclei of living cells. Furthermore, electrophoretic mobility shift
and antibody super shift
assays revealed that CREB and P-CREB from the rat NAc were able to
bind to a short
oligonucleotide identical in sequence to the CART promoter CRE
site. In the rat NAc,
over expression of CREB by virally mediated gene transfer increased
CART mRNA and
peptide levels. In sum, the body of data from this dissertation
strongly suggested that
CREB and P-CREB regulated CART mRNA and peptide expression in the
rat NAc by
acting directly at the CRE site in the CART proximal promoter. A
mechanism by which
CREB blunted the rewarding effects of cocaine may have been, in
part, by increased
expression of CART peptides in the NAc.
Table of Contents
TABLE OF CONTENTS
Page
Chapter 1: Overview of Cocaine- and Amphetamine-Regulated
Transcript
(CART): gene structure and regulation; peptide processing; putative G-
protein coupled receptor signaling and physiological effects on drug reward
and reinforcement. 1
1.1 Introduction 2
1.2 CART gene conservation and transcriptional regulation 3
1.3 CART RNA processing 8
1.4 CART peptides as neurotransmitters 12
1.5 CART receptors 13
1.6 The CART system and addiction 18
1.6.1 Psychostimulants activated CART peptide-containing neurons in the
nucleus accumbens 18
1.6.2 The CART system and mesolimbic dopamine 21
1.7 Goals of this dissertation 26
Chapter 2: CREB and P-CREB in pituitary-derived GH3 cells bound to the
CART promoter region containing a consensus CRE cis-regulatory element
and CREB and P-CREB from the rat pituitary bound to the same CRE site
in vitro . 27
2.1 Introduction 28
2.1.1 The role of CREB in drug addiction and pharmacodynamic tolerance 28
2.1.2 CREB transcription factors regulated cocaine reward 33
2.1.3 CART gene transcriptional regulation by CREB 37
2.1.4 Goals of this chapter 39
2.2 Methods 40
2.2.1 Primer design and quantitative, real-time polymerase chain reaction
(PCR) 40
2.2.2 Tissue culture and drug treatments 44
2.2.3 Nuclear protein extraction 45
2.2.4 Chromatin Immunoprecipitation (ChIP) assays 45
2.2.5 Animals and drug administration 47
2.2.6 Electrophoretic mobility shift assay (EMSA) and antibody
super shifts (SS) 48
2.2.7 Western blot analysis 49
2.2.8 Quantification of data and statistical analyses 50
2.3 Results 54
2.3.1 Chromatin immunoprecipitation (ChIP) assays identified appropriate chromatin
fragments from the CART, c-Fos and GAPDH genes 54
2.3.2 Verification that GH3 cells contained CREB proteins and the CART
gene promoter region containing the CRE site 55
2.3.3 Enrichment of the CART promoter CRE-containing region in GH3 cells
by Chromatin Immunoprecipitation (ChIP) with a CREB specific antibody 59
2.3.4 Western blot analyses verified that forskolin time-dependently
increased P-CREB protein levels in GH3 cells 63
2.3.5 Enrichment of the CART promoter CRE-containing region in GH3
cells by ChIP with a P-CREB-specific antibody; effects of forskolin 66
2.3.6 Enrichment of the c-Fos promoter CRE-containing region in GH3
cells by ChIP with a P-CREB-specific antibody; effects of forskolin 71
2.3.7 CREB and P-CREB from the rat pituitary gland bound the rat CART
promoter CRE cis-element in EMSA/SS assays 74
2.4 Discussion 82
Chapter 3: Regulation of CART mRNA and peptide expression by CREB
in the rat nucleus accumbens in vivo. 86
3.1 Introduction 87
3.1.1 CART gene transcriptional regulation by CREB 87
3.1.2 Psychostimulant regulation of CREB and its "transcriptome" 88
3.1.3 The CREB superfamily of transcription factors 89
3.1.4 Mechanisms of CREB-regulated transcription 91
3.1.5 Intracellular signaling cascades regulated CREB activity 92
3.1.6 The goals of this chapter 93
3.2 Methods 93
3.2.1 Cell culture and Transfections 94
3.2.2 Animals and intra-accumbal injections 94
3.2.3 Nissl staining 97
3.2.4 In situ hybridization and autoradiogram image analysis 98
3.2.5 EMSA and antibody super shift analyses 100
3.2.6 Western blot analysis 100
3.2.7 Quantification of data and statistical analyses 101
3.3 Results102
3.3.1 EMSA/SS analyses of the CART promoter CRE and NAc nuclear
proteins 102
3.3.2 Effects of CREB over expression by viral vectors on CART mRNA
and peptide levels in the rat NAc 108
3.3.3 Effects of mCREB over expression by viral vectors on CART
mRNA and peptide levels in the rat NAc 124
3.4 Discussion 131
Chapter 4: Effects of binge cocaine administration on CART gene
expression and transcription factor binding to the CART promoter CRE
cis -regulatory element. 145
4.1 Introduction146
4.2 Methods148
4.2.1 Animals and drug administration 148
4.2.2 Western blot analysis 148
4.2.3 EMSA and antibody super shift analyses 148
4.2.4 Quantification of data and statistical analyses 149
4.3. Results150
4.3.1 Effects of binge cocaine on rat NAc CREB, P-CREB and CART
peptide levels as well as TF binding to the CART promoter CRE site
in vitro. 150
4.3.2 Effects of binge cocaine on rat NAc AP1 protein binding to a
consensus AP1 cis-regulatory element 150
4.4. Discussion161
Chapter 5: EMSA and antibody super shift analyses of other putative CART
promoter cis-regulatory elements identified by database analysis in the rat and
mouse genes. 170
5.1 Introduction 171
5.2 Methods 172
5.2.1 Animals 172
5.2.2 Cell culture 172
5.2.3 EMSA/SS assays 172
5.2.4 Western blot analysis 173
5.3 Results 173
5.3.1 EMSA/SS analysis of the CART gene promoter Pit1 and Ptx1
cis-regulatory elements 173
5.3.2 EMSA/SS analysis of the mouse and rat CART promoter AP1-like
cis-regulatory elements 190
5.4 Conclusions and brief discussion 207
Chapter 6: General Discussion 210
6.1 CART gene regulation 211
6.2 Chromatin immunoprecipitation identified CREB and P-CREB interactions
with the CART promoter CRE cis-regulatory element in the gDNA of GH3 cells 212
6.3 CREB regulation of the CART gene in vivo, in the rat NAc 217
6.4 General conclusion 221
References 225
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