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Dopamine Transporter Genetics: From Model Systems to Man

This study explores the role of dopamine transporter genetics in various model systems, including C. elegans, to better understand its function in humans and its implications for brain disorders. Key findings include the identification of critical residues in the transporter protein and the involvement of other genes in its regulation and sensitivity to neurotoxins.

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Dopamine Transporter Genetics: From Model Systems to Man

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  1. Dopamine Transporter Genetics: From Model Systems to Man Randy D. Blakely Department of Pharmacology Center for Molecular Neuroscience www.blakelylab.org

  2. P P Regulated Trafficking of Biogenic Amine Transporters S P P Syn 1A, PP2Ac dissociate P Phosphorylation R e c y c l i n g R P P Internalization

  3. Studying Transporters in C. elegans Why use a worm model? *small size (~1mm) * numerous progeny, self-fertilization * ease of laboratory cultivation * anatomically simple (1) 959 somatic cells (2) 302-cell nervous system * transparent * all synaptic connections identified * completed genome sequence * methods for generating gene KO’s are well-developed * methods for generating trangenic animals are well-developed

  4. Dopaminergic Neurotransmissionin C. elegans Anatomy Function Mechanosensation Egg-laying Locomotion Pharyngeal pumping Mating (male) DA Histofluorescence Genes CAT-1 Tyrosine Hydroxylase CAT-2 VMAT CAT-4GTP Cyclohydrolase BAS-1 AADC DAT-1 DA Transporter Courtesy, J. Duerr

  5. Assessment of Sites of C.elegans DA Transporter Gene Expression DAT-1 Promoter Green Fluorescent Protein Inject into Gonad Under DIC Optics Co-inject Dominat Marker (rol-6) Screen for GFP Expression in Rollers Integrate After Radiation-Induced DNA Strand Breaks and Host Repair

  6. Imaging DAergic Neurons in Living Animals Head Head ab ADE terminal bulb NR metacorpus NR cb CEP Tail Tail PDE vulva pb

  7. a d BY200 BY200 + DA 100 BY200 + DA+ IMP 6 80 DAT-1 ko + DA 5 [3H]DA Uptake (% Control) 60 4 [3H]DA transport (cpmx1000) 40 3 Km = 17 nM 20 2 Control IMIP DA 1 0 wt wt-Imip ko ko-Imip 0 0 10 20 30 40 50 Dopamine [nM] 10 pA 100 ms 2 µm b e c f

  8. Expression of TAP-Tagged DAT-1 for Purification of Associated Proteins

  9. Affinity Purification of DAT-1Associated Proteins

  10. Transporters Support the Accumulation of Neurotoxins

  11. 6-OHDA Treatment Results in the Loss of CeDAT::GFP Expression Nass et. al., PNAS, 2002 Control 6-OHDA 6-OHDA/Imipramine 6-OHDA/Amphetamine

  12. dat-1 KO in C. elegans PCR primers for deletion analysis N2 dat-1(0) 500 bp Region of deletion in dat-1 (0) Loss of exons 4-12 dat-1 Deletion Single Worm PCR

  13. DAT-1 KO Blocks 6-OHDA Toxicity wt/PDAT-1::GFP (6-OHDA) wt/PDAT-1::GFP dat-1/PDAT-1::GFP dat-1/PDAT-1::GFP (6-OHDA)

  14. Genetic Screen for Genes SupportingToxin-Induced DA Neuron Death Discard Treat with DA Neuron Toxin Keep!!! DAT Mutants Regulation Mutants Toxicity Mutants Mutagenize

  15. 6-OHDA Insensitive 100 6-OHDA Resistant Wildtype (BY-200) 80 60 40 20 0 200 2 6 10 11 13 16 25 29 32 39 41 44 50 51 52 60 62 Lines Harvested from EMS Screenfor 6-OHDA Resistance Normal CEPs (% worms) Line Designation

  16. 100 80 60 40 20 0 200 2 6 10 11 13 16 25 29 32 39 41 44 50 51 52 60 62 6-OHDA Screen Yields Both DAT and Non-DAT Suppressors of Toxicity DAT Suppressor Normal CEPs (% worms) 6-OHDA Resistant Wildtype (BY-200) Non-DAT Suppresor Line Designation

  17. S Mutant #52 (Splicing DK584R) Identification of DAT-1 Mutants Supporting 6-OHDA Resistance Mutant #25 G55E (G75E) Mutants #13, 62 G90E (G110E)

  18. Transport Analysis ofDAT-1 Mutations50nM [3H] Dopamine

  19. ImmunoblotAnalyses of HA-Tagged DAT-1 and Mutants in COS-7 Cells T=Total, S=Surface (Biotinylated) DAT-1 pcDNA3 DAT-1 ∆K584R DAT-1 DAT-1 pcDNA3 DAT-1 G55E G90E M.W. (KDa) M.W. (KDa) 108- 97-- 90- 48 -- 50.7- T S T S T S T S T S T S T S

  20. Summary C. elegans DA neurons are sensitive to acute application of 6-OHDA in a dat-1 dependent manner. Forward genetic strategies can be used to identify molecular determinants of 6-OHDA-induced neurodegeneration. The most frequently recovered suppressors are in dat-1, the C. elegans DA transporter and alter the coding sequence. These mutations reduce DA transport activity in transfected cells. Mutations recovered reveal critical contributions of conserved residues in DAT-1 TM1 and 2 and the C-terminus for transporter biosynthesis and trafficking. Preliminary studies indicate decreased localization of dat-1 mutants to nerve ring and processes. Non-dat-1 genes participate in dat-1 function, dat-1 regulation or in the sensitivity to neurotoxin.

  21. Human Diseases Supported by Catecholamine Transporter Dysfunction? Issues of Functional Compensation? Can Phenotypes of Rare Transporter Variants Illuminate Convergent Pathways Supporting Brain/Behavioral Disorders?

  22. Monoamine Transporter Gene Organization NET 16q12.2 DAT 5p15.3 SERT 17q11.1-12

  23. Orthostatic Intolerance: Symptoms • Lightheadedness, palpitations, pre-syncope, and dizziness. (80% of patients) • Syncope (56% of patients) • Fatigue (44% of patients) • Symptoms consistent with decreased cerebral perfusion.

  24. S-S A 4 5 7 P P P P P P P A Mutation in the Human Norepinephrine Transporter DNA: GCC CCC Protein: Alanine Proline

  25. Inheritance of A457P Mutation in Three Generations of a Family Male Female

  26. A457P Tracks with Elevated Heart Rate

  27. Keeping the Periphery in Mind OI heart rate DHPG:NE tyramine Anxiety Depression ADHD

  28. ADHD Pharmacology Methylphenidate: DAT Blocker Increases extracellular DA/NE by blocking catecholamine reuptake (DAT and NET) D-Amphetamine: DA/NE Releaser Increases extracellular DA by triggering transporter (DAT and NET) reversal Atomoxetine (Strattera): NET Blocker Increases extracellular DA/NE via reuptake blockade (nonstimulant)

  29. Hypothesis ADHD may represent a clinical phenotype enriched for functional DA transporter coding variants Study of individuals and families with DAT coding variants may clarify role of central DA systems in facets of ADHD

  30. Patient DNA + heteroduplexes Control DNA homoduplexes Heat/cool homoduplex G G C C + + A A to reanneal G C T A Control heteroduplexes homoduplexes T T SNP Load reannealed DNA samples into the RevealTM Mutation Discovery System CCD camera laser capillaries Automated SNP Discovery via TGCE

  31. SNP Detection via TGCE 242 C/C - Control Wild-type sequence 242 C/T - A028-ADHD Heterozygous SNP 242 T/T - A027-ADHD Homozygous SNP

  32. SNP Analysis in ADHD Subjects ADHD Subjects Recruited :Combined ADHD: 40/46 - 87% Male: 40/46 - 87% Hyperactive/Impulsive: 6/46 - 13% Female: 6/46 - 13%

  33. Nonsynonymous SNP Identified in hDAT Exon 13 A559V Future Directions Pedigree genotype/phenotype assessment Physiology and regulation vs reference seq Additional subject screening Extension of effort to bipolar disorder TMD 11 TMD 12

  34. Acknowledgements C.elegansH. Sapiens Francesca Binda Steve Couch Lucia Carvelli Maureen Hahn Tammy Jessen Denise Malone Paul McDonald Michelle Mazei Richard Nass Angela Steele Lou DeFelice, Aurelio Galli NIDA, NHLBI, NIDDK, NIMH

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