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

• 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?