Marc G. Caron, Ph.D. Dept. Cell Biology Duke University Medical Center Phenotype-Driven Approaches...
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Transcript of Marc G. Caron, Ph.D. Dept. Cell Biology Duke University Medical Center Phenotype-Driven Approaches...
Marc G. Caron, Ph.D. Dept. Cell Biology Duke University Medical Center
Phenotype-Driven Approaches to Understanding the Neuronal Plasticity
Associated with Drugs of Abuse
Addictive process is amenable to experimental genetic approaches
• Process can be modeled in animals because the causative agent is known and several behavioral paradigms have been established.
• These behavioral paradigms have been adapted to mice, which have the principal advantage of allowing precise genetic manipulation.
Drug abuse/Addiction
• Addiction can be considered as a loss of control over the drug-taking or compulsive drug-seeking behavior, despite adverse physiological and devastation social consequences.
(a failure in the neurobiology of decision making)
Behavioral Manifestations of the Addiction Process
• Behavioral Sensitization: Repeated exposure to a drug leads to a progressive enhancement of the response (i.e. cocaine sensitization).
• Drug Tolerance: Increasing doses of a drug become necessary to elicit an equivalent physiological response (i.e. morphine tolerance).
• Drug Dependence: An adapted state of cells, circuits or organ systems unmasked by abrupt cessation of drug exposure (i.e. opiate withdrawal).
• Drug Craving: Increased drug seeking behavior following abstinence usually occasioned by drug related cues.
• Compulsive Drug Taking: Uncontrolled drug self-administration despite noxious behavioral consequences.
• Drug Relapse: After the extinguishing of uncontrolled drug taking, reacquisition of the behavior following a conditioned cue
Dopamine System in Addiction
cocaine
amphetamine
opiates nicotine
heroin
sex
foodethanol
•All drugs of abuse affect the mesolimbic DA system leading to irreversible alterations in physiology/chemistry in the reward circuits
Approaches to Understanding the Molecular Basis of Drug Abuse
Reverse Genetics 1) Manipulation of Candidate Genes
G protein-coupled receptor kinases (GRK 2-6)Arrestins (arrestin 1 and arrestin 2)
2) ENU Mutagenesis Screens in the Mouse
Dominant Modifiers of the DAT-KOSensitized Background
1) Genome-wide Expression Profiling
Mice Genetically Sensitized to Cocaine
WT, WT+cocaine, DAT-KO,NET-KO,& VMAT2-HT
3) Phenotype-driven Neuroscience Screens
Northwestern Univ. Takahashi et. al.
Forward Genetics
Approaches to Understanding the Molecular Basis of Drug Abuse
Reverse Genetics 1) Manipulation of Candidate Genes
G protein-coupled receptor kinases (GRK 2-6)Arrestins (arrestin 1 and arrestin 2)
2) ENU Mutagenesis Screens in the Mouse
Dominant Modifiers of the DAT-KOSensitized Background
1) Genome-wide Expression Profiling
Mice Genetically Sensitized to Cocaine
WT, WT+cocaine, DAT-KO,NET-KO,& VMAT2-HT
3) Phenotype-driven Neuroscience Screens
Recessive Mutation screen Northwestern Univ. Takahashi et. al.
Forward Genetics
Psychostimulants such as cocaine and amphetamine produce their behavioral effects by raising the levels of extracellular dopamine.
Increased behavioral responses to chronic intermittent exposure to drugs.
Believed to model in animals the initial stageof the drug abuse process……in humans.
Involves long term signaling plasticity resulting insupersensitivity of dopamine receptors.
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Behavioral Sensitization
The enduring neuronal plasticity that underlies the drugs of abuse phenotype might resemble processes of learning and memory
VMAT +/- MiceDecreased DA, NE, 5HTMimics reserpine
DAT-KO MiceIncreased [DA]ex
Mimics psychostimulants
NET-KO MiceIncreased [NE]ex
Mimicsantidepressants
All 3 KO mice show enhanced DA-mediated behaviors
Striatum/NAc
Berke and Hyman 2000
3x5 WT
3x5 WT+C
3x5 DAT-/-
3x5 NET-/-
3x5 VMAT+/- mRNA isolationIVT
cRNA LabelingHybridization to Affymetrix Mu74
Wash and stainScan
Data analysis
Strategy for Isolation of Tissue mRNA andMicroarray Analysis
PFC
CPU
NAc
Hip
Amg
VTASNr
Genes commonly affected between genetically and pharmacologically sensitized mice
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Genes Commonly Affected in the Striatum of DAT-/-, NET-/-,VMAT2+/- and Cocaine-Treated Mice Identified by Microarrays
Genes Commonly Affected in the Striatum of DAT-/-, NET-/-,VMAT2+/- and Cocaine-Treated Mice Identified by Microarrays
PSD-95 is one of the commonly affected gene
• Abundant scaffolding protein at PSD of excitatory synapses• Three PDZ domains for clustering PDZ domain containing proteins• SH3 domain• GK domain: Guanylate kinase-homology domain• Interacts with more than 50 proteins including ion channels, receptors, kinases, etc forming an intricate signaling complex
• LTP and learning (Migaud et al., Nature, 1998)
• Synaptic maturation (El-Husseini et al., Science 1999)
• Synaptic strength (El-Husseini et al., Cell, 2002)
• Chronic pain (Carry et al., Current Biology, 2003)
Conclusions
• PSD-95 appears to be a common target of hyperdopaminergic responsiveness• Changes in PSD-95 correlate with the development and persistence of psychostimulant sensitization
• Enhanced NAc LTP may be a cellular correlate of psychostimulant sensitization
• Functional inactivation of PSD-95(KO mice) leads to enhanced NAc LTP and responses to psychostimulants
• Phenotype-driven approach yielded 6 commonly identified genes
• Identification of previously recognized genes validates approach
• PSD-95 provides a molecular and cellular link shared between psychostimulant-related plasticity and learning
Roles of PSD-95 in Synaptic Transmission and Plasticity
• Serves as scaffold for NMDA receptors. Sheng & Kim, Science 298,776 (2002)
• Serves as indirect docking sites for synaptic AMPA receptors Schnell et al., PNAS, 2002
• Controls bidirectional synaptic plasticity: facilitate LTD and inhibits LTP Migaud et al., Nature, 1998 (Hippocampus) Stein et al., J. Neurosci. 2003 (Hippocampus) Beique and Andrade, J. Neurophys. 2003 (Cortex) Basal ganglia? Yao et al., Neuron 2004
Cocaine Usurps the Cortico-Accumbal Glutamate System by Altering the Bi-directional Synaptic
Plasticity• Cocaine depresses cortical glutamate afferent (White el al., J.
Pharmacol. Exp. Ther. 1995)• Cocaine reduces AMPA currents (Thomas et al., Nature Neurosci.
2001)• Cocaine reduces LTD (Thomas et al., Nature Neurosci. 2001)• Cocaine enhances LTP (This study)
• Reduction of PSD-95 may provide a consistent explanation for these phenomena
GluDA
StriatalMSN
SNr/VTA Cortex
GABA
Open Questions Relating to PSD-95
• How does PSD-95 mediates the interplay between the converging DA and Glu systems in the striatum?DA release from dopaminergic terminals?
DA receptor signaling?
Enhanced modulation of glutamate transmission by DA?
• Mechanisms leading to PSD-95 reduction(transcriptional and posttranslational)
• Role of PSD-95 in reward and habit learningCPP, self administration and specificity for other drugs.
• How does PSD-95 regulate LTP and LTD in the basal ganglia?
Approaches to Understanding the Molecular Basis of Drug Abuse
Reverse Genetics 1) Manipulation of Candidate Genes
G protein-coupled receptor kinases (GRK 2-6)Arrestins (arrestin 1 and arrestin 2)
2) ENU Mutagenesis Screens in the Mouse
Dominant Modifiers of the DAT-KOSensitized Background
1) Genome-wide Expression Profiling
Mice Genetically Sensitized to Cocaine
WT, WT+cocaine, DAT-KO,NET-KO,& VMAT2-HT
3) Phenotype-driven Neuroscience Screens
Recessive Mutation screen Northwestern Univ. Takahashi et. al.
Forward Genetics
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1. Use a two standard deviation cutoff for identification of outliersDat +/- Outliers ≥ 6500 cm/ 2hDat -/- Outliers ≥ 32000 cm or ≤ 6500 cm
2. Retest outliersDat +/- Outliers > 4500 cm/ 2hDat -/- Outlier > 32000cm or < 6500 cm
Phenotypic Screen: Locomotor Activity in Novel Environment
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D3 and Nr1 mutations are dominant modifiersof the DAT knockout phenotype
A Genetically Sensitized Background For Dopamine Transmission
DAT KO mice cannot actively clear synapticdopamine. Extracellular dopamine levelsare 5 times higher than wildtype. DAT HETmice have intermediate neurochemicalphenotype (2 times more extracellular DA).
Numerous neurochemical and behavioralconsequences to persistent high levels ofDA including hyperactivity in novelenvironment.
Chronic hyperdopaminergia provides asensitized background to uncover secondsite modifiers.
G2 Dominant Modifier Screen
C57 Dat +/- C57Bl/6J
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392 Dat +/- screened, 11 outliers with enhancer phenotype in testcross (0.5X coverage)135 Dat -/- screened, 4 enhancer, 5 suppressor phenotypes in testcross (0.2X coverage)
G1 screens
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Enhancer Phenotype Maintained on B6D2 F1 Background
Enhancer phenotypemaintained on B6D2 F1
background
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Identification of the Akt/GSK-3 Cascade as a Signaling Pathway Mediating the Behavioral Actions of Dopamine
• Actions of dopamine are mediated via 5 distinct GPCRs (D1-like D1, D5 and D2-like D2, D3, D4)
Dopamine-dependent behaviors
• Classically most biochemical and behavioral actions of dopamine have been attributed to modulation of the cAMP signal transduction pathway
Adapted from Greengard et al., 2001
Lithium ions can antagonize the behavioral actions of dopamine agonists in vivo
Li antagonizes the effect of psychostimulants: (Cox C et al., 1971 Nature 232:336-8; many others reviewed in Einat et al., 2000 in Contemporary issues in modeling psychopathology).
Li antagonizes the effect of apomorphine: locomotor behavior and sniffing
behavior: (Fazli-Tabaei et al., 2002 Pharmacol Toxicol 1998 91; 135-139, Dehpour
et al., 1998 Pharmacol Toxicol 1998 82: 147-52; Dehpour et al., 1995 Gen
Pharmacol 26:1015-20).
Li antagonizes the effect of brain injection of dopamine agonists : (Barnes et
al.,1986, Psychopharmacology 89:311-6).
Lithium does not directly inhibit DA receptors, nor does it affect the cAMP signaling pathway.
Lithium interferes with cellular signaling: -Inositol depletion hypothesis: Lithium inhibits inositol monophosphatase
(Berridge and Irvine 1989 Nature 341: 197-205)
-Glycogen Synthase Kinase 3 (GSK-3) Hypothesis: Lithium is a direct inhibitor of GSK-3 (Klein and Melton 1996, PNAS 93: 8455-59)
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•In the DAT-KO Lithium inhibits the hyperactivity phenotype associated with high extracellular [DA]
•Lithium enhances the phosphorylation of both Akt and GSK-3 & in DAT-KO
•No effects on DARPP-32 phosphorylation and cAMP signaling pathway
•In DAT-KO mice both Akt(thr308) and GSK-3 & are dephosphorylated
•In vitro GSK-3 assay reveals increased activity of enzyme in DAT-KO
•D2 but not D1 class DA receptors mediate changes in Akt and GSK-3
Beaulieu et al, PNAS 101, 5109 (2004)
-CateninInhibitor-2TranscriptionFactorsReceptors
PI3 kinasePhosphatases Calcium
Potential Signaling pathways mediating the actions of dopamine
Actions of dopamine can be mediated through both cAMP-dependent and independent mechanisms.
The Akt/GSK-3 pathway may be a mediator of dopamine actions underacute and chronic hyperdopaminergic conditions.
Akt/GSK-3 pathway may provide new targets for understanding aberrant DA-associated behaviors.
Conclusions
Akt and GSK-3 genes might be candidate genes for modulating the responsiveness to drugs of abuse
Inhibitors of the Akt/GSK-3 signaling pathway might be represent novel pharmacological targets for the treatment of drug abuse symptoms
Implications
HHMI-DUMCWei-Dong Yao Amy MohnMartin BeaulieuRaul GainetdinovTatyana Sotnikova Michel CyrGonzalo TorresAki Laakso
Bruno GirosMohamed JaberSara JonesFei XuYan-Min Wang
UNIVERSITY OF EDINBURGH/SANGER CENTER, CAMBRIDGE
Seth GrantMargaret McLean Arbuckle
Collaborators
DUMC/ Microarray FacilityHolly Dressman
DUMC/Transgenic FacilityCheryl Bock
Support: NIDA, Zaffaroni Foundation
Ontario Cancer Institute(Univ.Toronto)
(GSK-3+/- mice)Lisa Kockeritz
James R. Woodgett
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Ethanol GABAA/NMDARs
Nicotine nAchRs
Cannabinoids CB1&CB2R
PCPNMDAR
Cocaine/Amphetamine DAT/DAR
Opiates muR
Sex/Food
Brain Reward Pathways and Drugs of abuse
Physiological Functions of Dopamine•Brain Motor control
CognitionEmotion/affectReward mechanisms
•Retina Light/dark adaptation
•Pituitary Gland Hormone secretion
Dysfunction of the Dopaminergic System Associated with:
• Parkinson’s Disease• Schizophrenia and Affective Disorders• Attention Deficit Hyperactivity Disorder• Drugs of Abuse
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Psychostimulants such as cocaine and amphetamine produce their behavioral effects by raising the levels of extracellular dopamine.
Behavioral Sensitization: Increased behavioral responses to chronic intermittent exposure to drugs.Believed to model in animals the initial stageof the drug abuse process in humans.
Involves long term signaling plasticity resulting insupersensitivity of dopamine receptors.
Components of GPCR desensitization are good candidates!!
Nestler &Aghajanian, Science 278, 58-63, 1997
Possible Mechanisms of Drug-Induced Changes in GPCR Sensitivity
GRKs and Arrestins7 GRKs (2 visual)4 Arrestins (2 visual)
Locomotor Activity Sensitization
Dose Response
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Acute Effects of Cocaine in Various GRK and Arrestin KO Mice
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Morice E, Denis C, Giros B, Nosten-Bertrand M. Phenotypic expression of the targeted null-mutation in the dopamine transporter genevaries as a function of the genetic background.Eur J Neurosci. 2004 Jul;20(1):120-6.
Influence of modifier genes on the DAT KO Phenotype
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG. Tryptophan hydroxylase-2 controls brain serotonin synthesis.Science. 2004 Jul 9;305(5681):217.
Functional Polymorphism in TPH2
C57BL/6 and 129Xl/SvJ carry 1473 C allele (Pro)DBA/2 and BALB/cJ carry 1473 G allele (Arg)
Adapted from Greengard et al., 2001,and Waddington and Greengard et al 2003
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Inactivation of DARPP-32 in mice
• Inhibits the DA-dependent biochemical effects of DARPP-32 on its target proteins
• Has partial effects on many behaviors elicited by direct or indirect DA agonists
Apomorphine mg/kg
Fienberg et al, 1998 (Science)
Actions of dopamine can be mediated through both cAMP-dependent and independent mechanisms.
The Akt/GSK-3 pathway may be a mediator of dopamine actions underacute and chronic hyperdopaminergic conditions.
Akt/GSK-3 pathway may provide new targets for understanding aberrant DA-associated behaviors.
Conclusions
What is the mechanism by which D2-like receptors modulate Akt phosphorylation?
What are the substrates of GSK-3 contributing to the actions of dopamine?
What is the role of the Akt/GSK-3 pathway in manifestations of hyperdopaminergic functions…. sensorimotor gating, drug reward and neuronal cell loss…?
Are there differential behavioral effects of cAMP-dependent and independent pathways?
Questions
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wildtype n=16
dat (-/-) n=27dat (+/-) n=10
1. Use a two standard deviation cutoff for identification of outliersDat +/- Outliers ≥ 6500 cm/ 2hDat -/- Outliers ≥ 32000 cm or ≤ 6500 cm
2. Retest outliersDat +/- Outliers > 4500 cm/ 2hDat -/- Outlier > 32000cm or < 6500 cm
Phenotypic Screen: Locomotor Activity in Novel Environment
Lithium inhibits behavioral activity without affecting extracellular dopamine levels in DATKO mice
Acute dose
• No effects on DARPP-32 phosphorylation and and cAMP signaling pathway
Attractive potential mechanism
SubstratesCohen and Frame 2001 Nat Rev Mol Cell Biol 2:769-76
Li(+/-)
Li(-)
P
PI3K
P
Can we find biochemicalcorollaries in this pathwayfor the effects of Li in DAT KO mice?
Akt/GSK-3 Signaling Pathway
• Insulin signaling in glycogen synthesis
• Wnt signaling in cell fate & proliferation
• In vitro GSK-3 assay reveals increased activity of enzyme in DAT-KO
• In DAT-KO mice both Akt(thr308) and GSK-3 & are dephosphorylated
• Lithium enhances the phosphorylation of bothAkt and GSK-3 & in DAT-KO
Status of Akt/GSK3 in DAT-KO and in response to Li
DADA
THTH
DATDAT
DADA
THTH
Normal Normal NeurotransmissionNeurotransmission
Lack of Dopamine Transporter:Lack of Dopamine Transporter:the DAT-KO micethe DAT-KO mice
Amphetamine
MPT
Elimination of DA tone reverses Akt/GSK-3 state of DAT-KO
Increasing DA tone in WT mice reproduces DAT-KO Akt/GSK-3 state
• D2 but not D1 class DA receptors mediate changes in Akt and GSK-3
GSK-3 inhibitors
SB216763Glaxo Smith Kline(ATP binding)
IndirubinesQing Dai, Chinese medicine(ATP binding)
Valproic acid(mechanism?)
Paullones(ATP binding)
ThiadiazolidinonesTDZD(Substrate binding)
DA
AKT
GSK-3
MPTRaclopride
Hyperactivity?
Not responsive to:- SCH23390- 8-Br-cAMP
D2R
Chronic/DAT-KO
Li(+)
(-)
(?)
GSK-3 knockout is lethal due to a disruption of NF-k-B. (Hoeflich et al., 2000 Nature 406:86-90)
GSK-3+/- develop normally and do not display overt phenotype. (Hoeflich et al., 2000)
GSK-3 Transgenic exhibit developmental deficits and neuropathies associated with Tau hyperphosphorylation.(Spittaels et al.,2002 Neuroscience 113:797-808, Lucas et al., 2001, EMBO 20: 27-39; Brownlees J et al.,1997 Neuroreport : 3251)
Animal models of GSK-3 dysfunction