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
Overview
• What are neurotransmitter transporters?
• How do changes in their function affect the nervous system?
• Why use flies to study them?
Cell 2
Neurotransmitters are chemicals that allow cells to communicate
Cell 1
Receptors S y
n a
p s
e
Neurotransmitter
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
Bröer S Br J Pharmacol. 2012 167, 256.
-Monoamine transporters are part of one gene family
-GABA and glutamate transporters are in other families
DopamineDAT
NoradrenalineNET
SerotoninSERT
Mammalian Vesicular Transporters
VMAT2 in all aminergic brain cells
Focus on Monoamine Neurotransmitters
NoradrenalinAttention
Blood pressure
HistamineGastric acid release
Immune response
DopamineReward
SerotoninAppetite,Mood
Gastrointestinal motility
Dopamine transporterSerotonin “ “Norepi. “ “
Monoamine Transporters the Major Drug Targets for Stimulants and Antidepressants
Vesicular monoamine Transporter
Dopamine transporterSerotonin “ “Norepi. “ “
Blockade of Plasma Membrane Amine Transporter Increases Extracellular Neurotransmitter
CocaineRitalin ProzacWellbutrinZoloft
Dopamine transporterSerotonin “ “Norepi. “ “
Amphetamines Reverses the Flow(Mechanism is complex)
AmphetaminesMethamphetaminesAdderall- ADHD
Vesicular monoamine Transporter (VMAT2)
Vesicular Transporters are Drug Targets
Vesicular monoamine Transporter (VMAT)
Block VMAT with reserpine: Decreases monoamine storage and thus release-Antihypertensive effects-Depression
Reserpine
What would happen if VMAT worked better? Or there was more of it?
Could that change behavior? Act as a stimulant? Antidepressant?
Increases monoamine release
• Force cells to make more VMAT
• Record amine release• More amines comes out of
each vesicle• (Vesicles get a little bigger)
Sulzer labPothos et al, J. Neurosci. 2000
More VMAT in vitro:
“Normal cells”
Extra VMAT
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
DopamineGrooming
Locomotion
Serotonin Aggression
OctopamineEgg layingLocomotion
How can we tell if there are more extracellular monoamines in flies? -Look at their known functions
DopamineGrooming
Locomotion
Serotonin Aggression
OctopamineEgg layingLocomotion
Stimulants as positive control
DVMAT overexpression in vivomimics the effects of stimulants
Locomotion GroomingChang et al 2006
A New Way to Increase Extracellular Amines
WellbutrinProzacRitalin 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 stimulantsSome mechanisms not exploited
Amine agonist
Activate VMAT Increase exocytotic release
Block reuptake.degradation
CurrentDrugs:
No CurrentDrugs:
VMAT
neuronAminergic
Receptor
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 ScreenTest drugs in dVMAT mutant
1 2 3
4 5 6
1 2 4 5 6Drug: 3 ..1042
Mix into food, Allow larvae to feed, record movement
Example of Primary Screen Data
Number of grids crossed by P/P; UAS-VMAT. Screen on Drugs 105-122 (2A5-2B7)
0
1
2
3
4
5
6
7
2A
5-H
2A
5-L
2B
5-H
2B
5-L
2C
5-H
2C
5-L
2D
5-H
2D
5-L
2E
5-H
2E
5-L
2F
5-H
2F
5-L
2G
5-H
2G
5-L
2H
5-H
2H
5-L
2A
6-H
2A
6-L
VE
H
2B
6-H
2B
6-L
2C
6-H
2C
6-L
2D
6-H
2D
6-L
2E
6-H
2E
6-L
2F
6-H
2F
6-L
2G
6-H
2G
6-L
2H
6-H
2H
6-L
2A
7-H
2A
7-L
2B
7-H
2B
7-L
VE
H
0 hr Ave
2 hr Ave
24 hr Ave
-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
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 bitswith derivatives
Dacarbazine AICA
MethdiazoniumDNA alkylation
AICA: Amino-4-imidazole-carboxamide Removes the toxic bit
AICA also stimulates larval locomotion
Dacarbazine AICA
How does Dacarbazine/AICAIncrease 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?