Transcriptional and Epigenetic Mechanisms of Addiction Eric J. Nestler Mount Sinai School of Medicine New York, NY
Dr. Ray Fuller There is every reason to be optimistic that in the future we will find even better ways of modifying the function of specific parts of the brain which will be useful in treating a variety of psychiatric and neurological diseases.
Drugs of Abuse Act at the Synapse Drugs mimic neurotransmitters by activating receptors: Morphine Nicotine Marijuana Drugs block the dopamine pump: Cocaine Amphetamine Drugs activate or inhibit channels: Alcohol PCP, ketamine
Drugs Addiction: Drug-Induced Neural Plasticity Mediated Via Altered Gene Expression Receptors Transporters Second messengers & protein phosphorylation Regulation of many cellular processes Drugs Channels Transcription factors Stable adaptations in neural function Target genes
Chromatin Studies Offer Major Advances Help identify drugregulated genes. First ever look at transcriptional mechanisms in vivo. Unique mechanisms of long-lasting adaptations.
Regulation of Gene Expression is Reflected at the Chromatin Level Active (open) Inactive (condensed) Basal transcription complex HDAC Repressors HMT HAT SWI-SNF DNMT Histone N-termini Transcription factors Nucleosome
Induction Induction Distinct Temporal Properties of Drug-Induced Transcription Factors in NAc c-fos FosB Other Fos family proteins CREB Mediated by CREB phosphorylation and ATF induction Stable FosB isoforms 2 6 12 2 6 12 Time (hr) Time (hr) Accumulating, persisting FosB Rapid normalization 2 4 6 8 2 4 6 8 Time (days) Time (days)
Unique Induction of FosB in the NAc by Chronic Drug Administration High levels of FosB are induced uniquely by chronic drug exposure, creating a molecular switch. 52-58 kd (c-fos) 46-50 kd (FosB) 40 kd (?Fra1, Fra2) 35-37 kd (modified FosB) 33 kd (unmodified FosB)
Behavioral Plasticity Mediated by Drug-Induced Transcription Factors Transcription factors mediate distinct aspects of the drug addiction phenotype. FosB mediates drug sensitization: Increases sensitivity to drug and natural rewards. Mediates a positive emotional and motivational state. Drives drug craving and relapse (positive reinforcement). CREB mediates drug tolerance and dependence: Reduces sensitivity to drug and natural rewards. Mediates a negative emotional state during drug withdrawal. Drives drug craving and relapse (negative reinforcement). Bill Carlezon, David Self
Drug Regulation of Gene Expression: DNA Microarrays or RNA-seq Identify mrna s and non-coding RNA s (e.g., mirna s) regulated in NAc by chronic drug exposure. Effect of chronic cocaine mrna
Drug Regulation of Chromatin: ChIP-chip Arrays or ChIP-seq Overlay chromatin modifications on regulated mrna s to improve accuracy of detection and reveal underlying mechanisms. Acetylated H3 or H4 Activational H3 methylation (K4) Repressive H3 methylation (K9, K27) DNA methylation mrna
Role of Transcription Factors in Drug Regulation of Gene Expression Overlay transcription factor binding to further reveal underlying mechanisms. Theoretical results: Acetylated H3 or H4 Activational H3 methylation (K4) Repressive H3 methylation (K9, K27) DNA methylation CREB FosB Other mrna
Histone Modifications Induced in the NAc by Chronic Cocaine Largely non-overlapping mechanisms of histone modifications associated with chronic cocaine regulation of gene expression ach3 ach4 me2k9h3 ach3/h4 783 221 471 178 31 1444 ach3 ach4 me2k9h3 ach3/h4 80 3 120 889 9 194
Regulation of Gene Expression in the NAc by FosB and CREB FosB accounts for >25% of all genes regulated in the NAc by chronic cocaine administration: Genes upor downregulated by cocaine 8% 26% CREB FosB
Chromatin Regulation Helps Identify Genes d or d by Cocaine and FosB ach3/4 me2k9h3 1355 FosB 198 129 769 Complementary use of ChIPchip and gene expression arrays identifies genes activated or repressed by chronic cocaine via FosB. FosB Enrichment FosB overexpression cjun overexpression
Examples of Target Genes for CREB and FosB in the NAc FosB CREB Glutamatergic, GABAergic, GluR2, Arc GluR1, NR1 & synaptic plasticity GABA A 2 Piccolo Neuronal excitability Na v, K v, Ca v Na v, K v, Ca v Structural plasticity CDK5, WASPs Actin BPs, Tropomodulin, MAP2 Neuromodulators & growth factor pathways Dynorphin Dynorphin, CRFR 1 NK1 CART, CCK, BDNF, TrkB Transcriptional regulators NF B, c-fos G9a MEF2, JmjC
Examples of Target Genes for CREB and FosB in the NAc FosB CREB Glutamatergic, GABAergic, GluR2, Arc GluR1, NR1 & synaptic plasticity GABA A 2 Piccolo Neuronal excitability Na v, K v, Ca v Na v, K v, Ca v Structural plasticity CDK5, WASPs Actin BPs, Tropomodulin, MAP2 Neuromodulators & growth factor pathways Dynorphin Dynorphin, CRFR 1 NK1 CART, CCK, BDNF, TrkB Transcriptional regulators NF B, c-fos G9a MEF2, JmjC
Binding to Cdk5 promoter (fold change over control) ChIP Reveals the In Vivo Mechanism of Cdk5 Gene Activation in the NAc Chronic cocaine induces H3 acetylation, SWI-SNF binding, and FosB binding to the Cdk5 promoter FosB alone induces marks of activation 3 2 * * Saline Cocaine * FosB off FosB on * * 1 0.5 0 ach3 ach4 Brg1 Brg2 FosB FosB ach3 Brg1 Histone acetylation SWI-SNF factors
FosB Induction of Cdk5 Expression ChIP reveals the mechanism of Cdk5 gene induction in the NAc in vivo: HDAC Cdk5 promoter (basal state) Cdk5 promoter
FosB Induction of Cdk5 Expression ChIP reveals that the Cdk5 gene is induced in the NAc via: Direct binding of FosB to the Cdk5 promoter. HDAC FosB Cdk5 promoter
FosB Induction of Cdk5 Expression ChIP reveals that the Cdk5 gene is activated in the NAc via: Direct binding of FosB to the Cdk5 promoter. Recruitment of HATs & coactivators, and exclusion of HDACs, causing increased H3 acetylation. HDAC HAT FosB SWI-SNF Cdk5 promoter (activated state)
FosB Repression of c-fos Expression ChIP reveals the mechanism of c-fos gene repression in the NAc in vivo: c-fos promoter (basal permissive state)
FosB Regulation of c-fos Expression ChIP reveals that the c-fos gene is repressed in the NAc via: Direct binding of FosB to the c-fos promoter. FosB c-fos promoter
FosB Regulation of c-fos Expression ChIP reveals that the c-fos gene is repressed in the NAc via: Direct binding of FosB to the c-fos promoter. FosB recruitment of HDAC1, causing H4 deacetylation. Increased methylation of K9H3 and induction of HMTs independent of FosB. HDAC1 HMT FosB c-fos promoter (repressed state)
Global Changes in Chromatin Modifications After Chronic Cocaine Chronic cocaine induces several global changes in chromatin modifications in the NAc that promote gene expression: Increased histone acetylation (ach3) in NAc. - Mediated by downregulation of HDAC5. Decreased repressive histone methylation (mek9h3) in NAc. - Mediated by downregulation of G9a, a mek9h3 HMT. - No persistent changes in K4 or K27 methylation. Decreased DNA methylation in NAc. - Mediated by downregulation of DNMT3a. In each case, these permissive changes promote cocaine s behavioral effects.
Chronic Cocaine Induces a Permissive State of Gene Regulation More genes are induced after chronic than acute cocaine: # Significantly upregulated genes 0 50 100 150 200 250 300 Acute Chronic + acute Chronic + 1-wk-wd + acute Acute Chronic + acute Chronic + 1-wk-wd + acute Acute Chronic + acute Chronic + 1-wk-wd + acute
Fold change Fold change Chronic Cocaine, via FosB, Represses mek9h3 and G9a in NAc Cocaine decreases levels of me2k9h3 and G9a expression 1 Acute Chronic me3k9h3 Control -2 * * me2k9h3 G9a Chronic cocaine These effects are mediated by FosB 1-2 * * me2k9h3 G9a FosB off FosB on me3k9h3 is a marker of heterochromatin: chronic cocaine alters the amount of heterochromatin in NAc neuronal nuclei.
Drug side - saline side (sec) AAV-Cre AAV-GFP Drug side - saline side (sec) Repression of mek9h3 Enhances Behavioral Responses to Cocaine G9a inhibition in NAc enhances cocaine s behavioral effects: floxed G9a mice 400 * GFP G9a Merge 400 * 300 300 200 200 100 me2k9h3 -tubulin GFP Cre 100 0 GFP Cre AAV vectors in floxed G9a mice me2k9h3 -tubulin Veh BIX 0 Veh BIX 01294
Drug side - saline side (sec) Fold me2k9h3 Induction of mek9h3 Suppresses Behavioral Responses to Cocaine G9a overexpression in NAc reduces cocaine s behavioral effects: 400 ac 300 400 200 NAc 100 0 * GFP G9a mutg9a HSV vectors in wildtype mice 4 3 2 1 0 * me2k9h3 -tubulin me2k9h3 -tubulin G9a - + mutg9a - +
Examples of Target Genes for CREB and FosB in the NAc FosB CREB Glutamatergic, GABAergic, GluR2, Arc GluR1, NR1 & synaptic plasticity GABA A 2 Piccolo Intrinsic excitability Na v, K v, Ca v Na v, K v, Ca v Structural plasticity CDK5, WASPs Actin BPs, Tropomodulin, MAP2 Neuromodulators & growth factor pathways Dynorphin Dynorphin, CRFR 1 NK1 CART, CCK, BDNF, TrkB Transcriptional regulators NF B, c-fos G9a MEF2, JmjC
Mining ChIP-chip and Expression Data to Understand Structural Plasticity Numerous FosB targets are implicated in dendritic growth Cocaine FosB CREB, others Actin-binding proteins Wasps, Waves NF B CDK5 Ubiquitin signaling Arc Many others MEF2 Regulation of the actin cytoskeleton and induction and stabilization of dendritic spines
# spines/10 m FosB Mediates Cocaine-Induced Structural Changes in NAc Neurons Viral expression of FosB in NAc mimics cocaineinduced increases in spine density, while JunD blocks cocaine action 30 25 20 * * * Saline Cocaine 15 10 GFP FosB 5 0 GFP FosB JunD
Mining ChIP-chip and Expression Data to Understand Structural Plasticity Numerous FosB targets are implicated in dendritic growth Cocaine FosB CREB, others Actin-binding proteins Wasps, Waves NF B CDK5 Ubiquitin signaling Arc Many others MEF2 Regulation of the actin cytoskeleton and induction and stabilization of dendritic spines
Role for FosB in Cocaine Induced Structural Changes in NAc Neurons Repeated drug exposure (e.g., via FosB & numerous target genes Normal responses to drugs Use-dependent plasticity causing sensitized responses to drugs and environmental cues
Summary and Future Directions Gene and chromatin arrays provide an unprecedented view of the transcriptional mechanisms underlying chronic drug action in the NAc. This work is defining complex biochemical pathways underlying drug action. It is crucial to carry out equivalent studies of other brain regions to define circuit level links between cells and behavior. Look at multiple adaptations in concert, not individually. Eventually use this information to develop fundamentally novel diagnostic and treatment approaches for drug addiction.
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