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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
4
24 hr Ave
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?