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Marc G. Caron, Ph.D. Dept. Cell Biology

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

  2. Drug abuse/Addiction Addictive process is amenable to experimental genetic approaches • 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) • 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.

  3. 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

  4. cocaine opiates nicotine heroin amphetamine ethanol food sex Dopamine System in Addiction • All drugs of abuse affect the mesolimbic DA system leading to irreversible alterations in physiology/chemistry in the reward circuits

  5. Approachesto 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) Forward Genetics 1) Genome-wide Expression Profiling Mice Genetically Sensitized to Cocaine WT, WT+cocaine, DAT-KO, NET-KO,& VMAT2-HT 2)ENU Mutagenesis Screens in the Mouse Dominant Modifiers of the DAT-KO Sensitized Background 3) Phenotype-driven Neuroscience Screens Northwestern Univ. Takahashi et. al.

  6. Approachesto 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) Forward Genetics 1) Genome-wide Expression Profiling Mice Genetically Sensitized to Cocaine WT, WT+cocaine, DAT-KO, NET-KO,& VMAT2-HT 2)ENU Mutagenesis Screens in the Mouse Dominant Modifiers of the DAT-KO Sensitized Background 3) Phenotype-driven Neuroscience Screens Recessive Mutation screen Northwestern Univ. Takahashi et. al.

  7. Activity Monitor Activity Monitor COCAINE INJ 1 2 3 4 5 6 7 days Distance Traveled (cm/15 min) 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 stage of the drug abuse process……in humans. Involves long term signaling plasticity resulting in supersensitivity of dopamine receptors. The enduring neuronal plasticity that underlies the drugs of abuse phenotype might resemble processes of learning and memory

  8. VMAT +/- Mice Decreased DA, NE, 5HT Mimics reserpine DAT-KO Mice Increased [DA]ex Mimics psychostimulants NET-KO Mice Increased [NE]ex Mimics antidepressants All 3 KO mice show enhanced DA-mediated behaviors

  9. Phenotype of pharmacogically and genetically “sensitized” mice DAT KO NET KO VMAT2 HT

  10. 3x5 WT 3x5 WT+C 3x5 DAT-/- 3x5 NET-/- 3x5 VMAT+/- PFC Hip mRNA isolation IVT cRNA Labeling Hybridization to Affymetrix Mu74 Wash and stain Scan Data analysis CPU VTA NAc SNr Berke and Hyman 2000 Amg Strategy for Isolation of Tissue mRNA and Microarray Analysis Striatum/NAc

  11. Genes commonly affected between genetically and pharmacologically sensitized mice

  12. Genes Commonly Affected in the Striatum of DAT-/-, NET-/-, VMAT2+/- and Cocaine-Treated Mice Identified by Microarrays

  13. Genes Commonly Affected in the Striatum of DAT-/-, NET-/-, VMAT2+/- and Cocaine-Treated Mice Identified by Microarrays

  14. 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)

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

  16. PSD-95 protein expression is decreased in the striatum

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

  18. Pre- and Post-synaptic markers Selectivity of changes in the expression of PSD-95

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

  20. Cellular Phenotype: Enhanced LTP in Nucleus Accumbens

  21. Nature 396 433-439, 1998 PSD-95 KO mice are impaired in learning Hippocampus

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

  23. PSD-95 KO mice are more responsive to psychostimulants

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

  25. Conclusions • Phenotype-driven approach yielded 6 commonly identified genes • Identification of previously recognized genes validates approach • 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 • PSD-95 provides a molecular and cellular link shared between • psychostimulant-related plasticity and learning

  26. 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

  27. Glu DA Striatal MSN 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 SNr/VTA Cortex GABA

  28. 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 learning CPP, self administration and specificity for other drugs. • How does PSD-95 regulate LTP and LTD in the basal ganglia?

  29. Approachesto 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) Forward Genetics 1) Genome-wide Expression Profiling Mice Genetically Sensitized to Cocaine WT, WT+cocaine, DAT-KO, NET-KO,& VMAT2-HT 2)ENU Mutagenesis Screens in the Mouse Dominant Modifiers of the DAT-KO Sensitized Background 3) Phenotype-driven Neuroscience Screens Recessive Mutation screen Northwestern Univ. Takahashi et. al.

  30. Phenotypic Screen: Locomotor Activity in Novel Environment 1. Use a two standard deviation cutoff for identification of outliers Dat +/- Outliers ≥ 6500 cm/ 2h Dat -/- Outliers ≥ 32000 cm or ≤ 6500 cm 2. Retest outliers Dat +/- Outliers > 4500 cm/ 2h Dat -/- Outlier > 32000cm or < 6500 cm

  31. A Genetically Sensitized Background For Dopamine Transmission DAT KO mice cannot actively clear synaptic dopamine. Extracellular dopamine levels are 5 times higher than wildtype. DAT HET mice have intermediate neurochemical phenotype (2 times more extracellular DA). Numerous neurochemical and behavioral consequences to persistent high levels of DA including hyperactivity in novel environment. Chronic hyperdopaminergia provides a sensitized background to uncover second site modifiers.

  32. G2 Dominant Modifier Screen X ENU G0 C57 Dat +/- C57Bl/6J G1 Dat +/-, +/M G2 Dat +/-, +/+ Dat +/-, +/M Dat -/-, +/M Dat +/-, +/+ Dat +/-, +/M Dat -/-, +/+ Dat +/+, +/M Dat +/+, +/+

  33. G1 screens X ENU G0 C57 Dat +/- C57 Dat +/- G1 Dat +/-, +/M Dat +/-, +/M Dat +/-, +/M Dat +/-, +/M Dat -/-, +/M Dat -/-, +/M 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)

  34. Distance Traveled in cm/2 hr Enhancer Phenotype Maintained on B6D2 F1 Background

  35. Dopamine-dependent behaviors 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) • 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

  36. 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)

  37. 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)

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

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

  40. PI3 kinase Phosphatases Calcium b-Catenin Inhibitor-2 Transcription Factors Receptors Potential Signaling pathways mediating the actions of dopamine

  41. Conclusions 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 under acute and chronic hyperdopaminergic conditions. Akt/GSK-3 pathway may provide new targets for understanding aberrant DA-associated behaviors. Implications 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

  42. Collaborators HHMI-DUMC Wei-Dong Yao Amy Mohn Martin Beaulieu Raul Gainetdinov Tatyana Sotnikova Michel Cyr Gonzalo Torres Aki Laakso Bruno Giros Mohamed Jaber Sara Jones Fei Xu Yan-Min Wang UNIVERSITY OF EDINBURGH/ SANGER CENTER, CAMBRIDGE Seth Grant Margaret McLean Arbuckle DUMC/ Microarray Facility Holly Dressman DUMC/Transgenic Facility Cheryl Bock Ontario Cancer Institute (Univ.Toronto) (GSK-3+/- mice) Lisa Kockeritz James R. Woodgett Support: NIDA, Zaffaroni Foundation

  43. Cellular Mechanism: Enhanced LTP in Nucleus Accumbens

  44. WT DAT-/- VMAT2-/+ NET-/- Cocaine Saline

  45. Brain Reward Pathways and Drugs of abuse Cocaine/ Amphetamine DAT/DAR Opiates muR PCP NMDAR Nicotine nAchRs Cannabinoids CB1&CB2R Ethanol GABAA/NMDARs Sex/Food

  46. PSD-95 is one of the commonly affected gene PSD-95 Sheng & Kim, Science 298,776 (2002)

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