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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 [email protected] Overview • What are neurotransmitter transporters? • How do changes in their function affect the nervous system? • Why use flies to study them? Synapse Neurotransmitters are chemicals that allow cells to communicate Cell 1 Neurotransmitter Cell 2 Receptors Neurotransmitter types • Monoamines – Dopamine, Serotonin, Norepinephrine, Epineprine (Adrenalin) • Actetylcholine • GABA • Glutamate (Peptides, Gases- not today) Plasma membrane and vesicular transporters 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 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. Mammalian Vesicular Transporters VMAT2 in all aminergic brain cells Focus on Monoamine Neurotransmitters Noradrenalin Dopamine Reward Attention Blood pressure Serotonin Histamine Gastric acid release Immune response Appetite,Mood Gastrointestinal motility Monoamine Transporters the Major Drug Targets for Stimulants and Antidepressants Vesicular monoamine Transporter Dopamine transporter Serotonin “ “ Norepi. “ “ Blockade of Plasma Membrane Amine Transporter Increases Extracellular Neurotransmitter Dopamine transporter Serotonin “ “ Norepi. “ “ Cocaine Ritalin Prozac Wellbutrin Zoloft Amphetamines Reverses the Flow (Mechanism is complex) Dopamine transporter Serotonin “ “ Norepi. “ “ Amphetamines Methamphetamines Adderall- ADHD Vesicular Transporters are Drug Targets Vesicular monoamine Transporter (VMAT2) Block VMAT with reserpine: Decreases monoamine storage and thus release -Antihypertensive effects -Depression Reserpine Vesicular monoamine Transporter (VMAT) What would happen if VMAT worked better? Or there was more of it? Could that change behavior? Act as a stimulant? Antidepressant? More VMAT in vitro: Increases monoamine release “Normal cells” Extra VMAT Sulzer lab Pothos et al, J. Neurosci. 2000 • Force cells to make more VMAT • Record amine release • More amines comes out of each vesicle • (Vesicles get a little bigger) What about more VMAT in vivo? -No in vivo mammalian models -Make fly model -Why use flies? 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! How can we tell if there are more extracellular monoamines in flies? -Look at their known functions Dopamine Grooming Locomotion Serotonin Aggression Octopamine Egg laying Locomotion How can we tell if there are more extracellular monoamines in flies? -Look at their known functions Dopamine Grooming Locomotion Serotonin Aggression Stimulants as positive control Octopamine Egg laying Locomotion DVMAT overexpression in vivo mimics the effects of stimulants Locomotion Grooming Chang et al 2006 A New Way to Increase Extracellular Amines Wellbutrin Prozac Ritalin Adderall 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? Antidepressants and stimulants Some mechanisms not exploited No Current Drugs: Activate VMAT Increase exocytotic release Receptor Aminergic neuron Current Drugs: Block reuptake. degradation Amine agonist Amphetamines cause efflux, not exocytotic release 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 Primary Screen Test drugs in dVMAT mutant Drug: 1 2 3 4 5 6 ..1042 Mix into food, Allow larvae to feed, record movement 1 2 3 4 5 6 Example of Primary Screen Data Number of grids crossed by P/P; UAS-VMAT. Screen on Drugs 105-122 (2A5-2B7) 7 6 0 hr Ave 5 2 hr Ave 24 hr Ave 4 3 2 1 -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 VEH 2B7-L 2B7-H 2A7-L 2A7-H 2H6-L 2H6-H 2G6-L 2G6-H 2F6-L 2F6-H 2E6-L 2E6-H 2D6-L 2D6-H 2C6-L 2C6-H 2B6-L 2B6-H VEH 2A6-L 2A6-H 2H5-L 2H5-H 2G5-L 2G5-H 2F5-L 2F5-H 2E5-L 2E5-H 2D5-L 2D5-H 2C5-L 2C5-H 2B5-L 2B5-H 2A5-L 2A5-H 0 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 Parse toxic vs. active bits with derivatives Dacarbazine AICA Methdiazonium DNA alkylation AICA: Amino-4-imidazole-carboxamide Removes the toxic bit AICA also stimulates larval locomotion Dacarbazine AICA How does Dacarbazine/AICA Increase Locomotion? • • • • Increase Storage? Increase Release? Other mechanisms? Collaborate with Nigel Maidment’s lab Potential Clinical Relevance AICA derivatives for ADHD? Depression? Parkinson’s disease? • What are neurotransmitter transporters? • Why use flies to study them? • How do changes in their function affect the nervous system? Questions?
