Marc G. Caron, Ph.D. Dept. Cell Biology Duke University Medical Center Phenotype-Driven Approaches to Understanding the Neuronal Plasticity Associated.

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

Phenotype of pharmacogically and genetically “sensitized” mice

NET KO VMAT2 HTDAT KO

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)

PSD-95 transcript is decreased in the basal ganglia of behaviorally sensitized mice

PSD-95 protein expression is decreased in the striatum

Decreased in PSD-95 is selective to the striatum in cocaine sensitized mice

Selectivity of changes in the expression of PSD-95

Pre- and Post-synaptic markers

Persistent PSD-95 Down-regulation in Cocaine-Sensitized Mice

Cellular Phenotype:

Enhanced LTP in Nucleus Accumbens

Nature 396 433-439, 1998

PSD-95 KO mice are impaired in learning

Hippocampus

PSD-95 KO mice display NAc LTP similar to sensitized mice

PSD-95 KO mice are more responsive to psychostimulants

PSD-95 KO mice cannot be further sensitized by chronic cocaine

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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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

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G1 screens

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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)

Inhibitors of GSK-3 reduce the hyperactivity of DAT-KO mice

GSK-3 +/- mice show a diminished response to the psychostimulant Amphetamine

-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

Cellular Mechanism:

Enhanced LTP in Nucleus Accumbens

WT DAT-/-

NET-/- VMAT2-/+

Saline Cocaine

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

PSD-95 is one of the commonly affected gene

Sheng & Kim, Science 298,776 (2002)

PSD-95

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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)

GRK6 Expression in DopaminoceptiveMedium Spiny Striatal Neurons

Locomotor Activity Sensitization

Dose Response

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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

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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

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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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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

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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(+)

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Alsterpaullone reduces hyperactivity in DAT-KO mice

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