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Neurotransmitter Transporters and Flies: Tools to Study Behavior and Disease

Neurotransmitter Transporters and Flies: Tools to Study Behavior and Disease. David Krantz MD, PhD Department of Psychiatry & Biobehavrioal Sciences David Geffen School of Medicine at UCLA Gonda (Goldschmied) Center for Neuroscience and Genetics Research Dkrantz@ucla.edu. Overview.

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Neurotransmitter Transporters and Flies: Tools to Study Behavior and Disease

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  1. Neurotransmitter Transporters and Flies:Tools to Study Behavior and Disease David Krantz MD, PhD Department of Psychiatry & Biobehavrioal Sciences David Geffen School of Medicine at UCLA Gonda (Goldschmied) Center for Neuroscience and Genetics Research Dkrantz@ucla.edu

  2. Overview • What are neurotransmitter transporters? • How do changes in their function affect the nervous system? • Why use flies to study them?

  3. Neurotransmitters are chemicals that allow cells to communicate Cell 1 S y n a p s e Neurotransmitter Cell 2 Receptors

  4. Neurotransmitter types • Monoamines • Dopamine, Serotonin, Norepinephrine, Epineprine (Adrenalin) • Actetylcholine • GABA • Glutamate (Peptides, Gases- not today)

  5. Plasma membrane and vesicular transporters

  6. Why do neurotransmitters need transporters? • Oil and water don’t mix • Transmitters like to be in water • Cell and organelle boundaries are oily • Transporters bridge the water pools

  7. Plasma Membrane Neurotransmitter Transporters -Monoamine transporters are part of one gene family -GABA and glutamate transporters are in other families Dopamine DAT Noradrenaline NET Serotonin SERT Bröer S Br J Pharmacol. 2012 167, 256.

  8. Mammalian Vesicular Transporters VMAT2 in all aminergic brain cells

  9. Focus on Monoamine Neurotransmitters Noradrenalin Attention Blood pressure Histamine Gastric acid release Immune response Dopamine Reward Serotonin Appetite,Mood Gastrointestinal motility

  10. Monoamine Transporters the Major Drug Targets for Stimulants and Antidepressants Vesicular monoamine Transporter Dopamine transporter Serotonin “ “ Norepi. “ “

  11. Blockade of Plasma Membrane Amine Transporter Increases Extracellular Neurotransmitter Cocaine Ritalin Prozac Wellbutrin Zoloft Dopamine transporter Serotonin “ “ Norepi. “ “

  12. Amphetamines Reverses the Flow(Mechanism is complex) Dopamine transporter Serotonin “ “ Norepi. “ “ Amphetamines Methamphetamines Adderall- ADHD

  13. Vesicular Transporters are Drug Targets Vesicular monoamine Transporter (VMAT2)

  14. Block VMAT with reserpine: Decreases monoamine storage and thus release-Antihypertensive effects-Depression Reserpine Vesicular monoamine Transporter (VMAT)

  15. What would happen if VMAT worked better? Or there was more of it?Could that change behavior? Act as a stimulant? Antidepressant?

  16. More VMAT in vitro: Increases monoamine release • Force cells to make more VMAT • Record amine release • More amines comes out of each vesicle • (Vesicles get a little bigger) “Normal cells” Extra VMAT Sulzer lab Pothos et al, J. Neurosci. 2000

  17. What about more VMAT in vivo? • -No in vivo mammalian models • -Make fly model • -Why use flies?

  18. Why use flies? • -Cheap! (good for teaching!) • -”Conservation” of genes • e.g. VMAT, DAT • -Cool genetic tools • e.g. to make more VMAT • -Short life span • Genetic experiments in a month!

  19. How can we tell if there are more extracellular monoamines in flies? -Look at their known functions Dopamine Grooming Locomotion Octopamine Egg laying Locomotion Serotonin Aggression

  20. How can we tell if there are more extracellular monoamines in flies? -Look at their known functions Dopamine Grooming Locomotion Octopamine Egg laying Locomotion Serotonin Aggression Stimulants as positive control

  21. DVMAT overexpression in vivomimics the effects of stimulants Locomotion Grooming Chang et al 2006

  22. A New Way to Increase Extracellular Amines Wellbutrin Prozac Ritalin Adderall

  23. Could we find a drug the would make VMAT work better?Or just increase exocytosis of monoamines?Could this be used to treat ADHD? depression? Parkinson’s disease?

  24. Antidepressants and stimulantsSome mechanisms not exploited No Current Drugs: Activate VMAT Increase exocytotic release Receptor VMAT Aminergic neuron Current Drugs: Block reuptake. degradation Amine agonist Amphetamines cause efflux, not exocytotic release

  25. To find drugs that might make VMAT work better…First make it work worse! Use dVMAT mutant “Sensitized genetic background” detects drug effects better than wild type

  26. Primary ScreenTest drugs in dVMAT mutant Drug: ..1042 1 3 4 5 6 2 Mix into food, Allow larvae to feed, record movement 2 3 1 4 5 6

  27. Example of Primary Screen Data -Select drugs that cause 3-4 SDs above the mean -40 hits out of 1042 drugs -3 time points, 2 concentrations, 7 undergrads, 5 months Lawal et al, 2012

  28. Dacarbazine was one of the “hits” in the screen • Chemotherapeutic agent • Alkylating agent • Induces emesis via serotonin release • Supports fly data • Toxicity could be a problem • Limits use

  29. Parse toxic vs. active bitswith derivatives Dacarbazine AICA Methdiazonium DNA alkylation AICA: Amino-4-imidazole-carboxamide Removes the toxic bit

  30. AICA also stimulates larval locomotion Dacarbazine AICA

  31. How does Dacarbazine/AICAIncrease Locomotion? • Increase Storage? • Increase Release? • Other mechanisms? • Collaborate with Nigel Maidment’s lab

  32. Potential Clinical Relevance AICA derivatives for ADHD? Depression? Parkinson’s disease?

  33. What are neurotransmitter transporters? • Why use flies to study them? • How do changes in their function affect the nervous system?

  34. Questions?

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