[PPT] - RARE MISSPELLINGS OF THE GENOME, DOPAMINE MISHANDLING, AND ADHD PowerPoint Presentation

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RARE MISSPELLINGS OF THE GENOME, DOPAMINE MISHANDLING, AND ADHD

RANDY D. BLAKELY, PH.D. FLORIDA ATLANTIC UNIVERSITY BRAIN INSTITUTE

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CONFLICTS OF INTEREST

Dr. Blakely has consulted on research projects in the

past for Amgen, Forest Research Institute, Lundbeck, Jubilant Innovation, Pfizer, Prexa, and Wyeth

Dr. Blakely declares no conflicts of interest derived

from financial support of the research to be discussed

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ACKNOWLEDGEMENTS

Blakely Lab Collaborators

Aurelio Galli Erica Bowton Jonathan Javitch Steve Couch Heng Dai Doug McMahon Stephanie Gantz Michael Freissmuth Roxanne Vaughan Gregg Stanwood John Williams Mark Stein Richard Shelton Harald Sitte Cristina Fenollar -Ferrer Marc Mergy Adele Stewart Max Rabil Rodenia Pert Keeley Spiess Justin Riele Austin Wheeler Nathan Richtand Jeremy Veenstra-VanderWeele Michelle Mazei-Robison Maureen Hahn Paul Gresch Gwynne Davis Raaj Gowrishankar Rania Katamish Felix Mayer Lorena Areal Michael Gill Peter Hamilton DJ Sakrikar

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ACKNOWLEDGEMENTS

Blakely Lab Collaborators

Aurelio Galli Erica Bowton Jonathan Javitch Steve Couch Heng Dai Doug McMahon Stephanie Gantz Michael Freissmuth Roxanne Vaughan Gregg Stanwood John Williams Mark Stein Richard Shelton Harald Sitte Cristina Fenollar -Ferrer Marc Mergy Adele Stewart Max Rabil Rodenia Pert Keeley Spiess Justin Riele Austin Wheeler Nathan Richtand Jeremy Veenstra-VanderWeele Michelle Mazei-Robison Maureen Hahn Paul Gresch Gwynne Davis Raaj Gowrishankar Rania Katamish Felix Mayer Lorena Areal Michael Gill Peter Hamilton DJ Sakrikar

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OUTLINE

ADHD: Diagnosis, Genetics and Dopamine Involvement Identification of Mutations in the Human Dopamine Transporter (DAT) in Subjects with ADHD The In Vivo Consequences of DAT Val559: From Biochemistry to Behavior and Novel Biomarkers

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BASIC FACTS AND STATISTICS ATTENTION-DEFICIT/HYPERACTIVITY DISORDER

Diagnosed solely through behavioral observation, with presentation of symptoms of inattention, impulsivity and hyperactivity (by age 12, DSM-5). Male bias in diagnosis (~4:1) Higher incidence of substance abuse, conduct disorders, unemployment, incarceration if left untreated Approximately 60% are treated by medication, 25% receive no treatment of any form The most commonly diagnosed neurobehavioral disorder in children (8.4% ages 2-17, 5.4 million) in the U.S. have a formal ADHD diagnosis (National Survey of Children’s Health 2016)

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ADHD: DSM-5 Diagnostic Criteria

Careless Mistakes
Difficulty Sustaining Attention
Not Seeming To Listen
Failing To Finish Tasks
Difficulty Organizing
Avoiding Tasks That Require

Sustained Attention

Losing Things
Easily Distracted
Forgetful

Inattentive Symptoms

Fidgeting
Inability To Stay Seated
Moving Excessively
Difficulty Playing Quietly
On the Go
Talks Excessively
Blurting Out Answers
Difficulty Waiting Turns
Interrupting

Hyperactive/Impulsive Symptoms

I C H

6 or more symptons per category

for 6 months or more

symptoms present in two or more

settings and prior to age 12

Symptoms disrupt normal

function/quality of life

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ADHD GENETICS

Heritability estimated at 60-90%, common and rare gene variation linked to neuronal development and synaptic function Co-morbidity and co-occurrence: Bipolar Disorder and Autism Shared genetic heritability with Bipolar Disorder, Major Depression, and Schizophrenia (Brainstorm Consortium, 2018)

GENETIC ARCHITECTURE

Common genetic variation of small effect (GWAS) Rare genetic variation of large effect (e.g. Fragile X mutation) Rare genetic variation of small to moderate effect (risk factor)

ATTENTION-DEFICIT/HYPERACTIVITY DISORDER

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DOPAMINE: KEY BRAIN NEUROTRANSMITTER

HUMAN BRAIN 100 BILLION NEURONS

DOPAMINE

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DA CORE FUNCTIONS PERTURBED IN ADHD

DOPAMINE

Cue Saliency Attention Executive Function Learning Reward and Motivation Locomotor Activity

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BRAIN DOPAMINE PATHWAYS

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CONTROL OF DOPAMINE AVAILABILITY BY THE DOPAMINE TRANSPORTER (DAT)

GIROS ET AL 2006

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DOPAMINE TRANSPORTER STRUCTURE

EXTRACELLULAR (SYNAPSE) INTRACELLULAR (CYTOPLASM)

DOPAMINE

DAT Model Courtesy of Cristina Fenollar- Ferrer

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REDUCED DAT LEVELS IN ADHD - TONIC ELEVATION IN DA?

VOLKOW ET AL 2009 DA TRANSPORTER DENSITY VOLKOW ET AL 2007

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MAJOR MEDICATIONS TO TREAT ADHD TARGET DAT

Methylphenidate Amphetamine

Methylphenidate (Ritalin™)- DAT Antagonist Amphetamine (Adderall™)- Competitive Substrate and Induces DA Efflux

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COMMON GENETIC VARIATION IN DAT GENE AND ADHD

HUMAN DAT GENE - SLC6A3

MILLER AND MADRAS, 2012 CHROMOSOME 5

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DAT CODING VARIATION: RARE OPPORTUNITIES FOR PROGRESS?

ADHD is a brain disorder that may possess, and even be enriched for, highly penetrant but overall rare, genetic variants that target DA signaling including DAT. Characterization of such variants in vitro and in vivo is needed to support the hypothesis that a perturbation of DA signaling can contribute to the traits of ADHD Identification of rare, conserved genetic variation in DAT may help build useful animal models that can elucidate molecular, cellular and circuit level mechanisms linked to ADHD.

HYPOTHESIS

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MULTIPLE DAT CODING VARIANTS IDENTIFIED IN ADHD

MAZEI-ROBISON ET AL., 2005 SAKRIKAR ET AL, 2012 MERGY ET AL, 2014

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MULTIPLE DAT CODING VARIANTS IDENTIFIED IN ADHD

A559V R615C L167F V24M

MAZEI-ROBISON ET AL., 2005 SAKRIKAR ET AL, 2012 MERGY ET AL, 2014

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IDENTIFICATION OF DAT VAL559 IN TWO MALE ADHD SIBLINGS

ADHD ASSOCIATED DAT 3’VNTR

BIPOLAR DISORDER: GRUNHAGE ET AL 2000 AUTISM: BOWTON ET AL, 2014

MAZEI-ROBISON ET AL., 2005, 2008

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DAT VAL559: INITIAL IN VITRO STUDIES COME UP DRY

MAZEI-ROBISON ET AL 2008

AND NOW FOR SOMETHING COMPLETELY NORMAL...

Normal DAT total protein levels Normal DAT surface expression Normal dopamine transport activity Normal affinity for dopamine Normal affinity for amphetamine and methylphenidate Normal despondent reaction of graduate student

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DAT VAL559 DISPLAYS ANOMALOUS DA EFFLUX (ADE)

MAZEI-ROBISON ET AL 2008

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DAT VAL559 DISPLAYS ANOMALOUS DA EFFLUX (ADE)

MAZEI-ROBISON ET AL 2008

Outward leak blocked by cocaine or methylphenidate

Block of Dopamine Leak by DAT Antagonist VAL559 ALA559

VAL559 ALA559 ALA559 VAL559

VAL559 ALA559

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PROPOSED SYNAPTIC IMPACT OF DAT VAL559 NORMAL DAT

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PROPOSED SYNAPTIC IMPACT OF DAT VAL559 LEAKY MUTANT DAT = VAL559

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DAT VAL559 MICE: MICE WITH ADHD TRAITS?

MERGY ET AL 2014

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DAT VAL559 MICE: NORMAL TRANSPORTER PROTEIN LEVELS AND DOPAMINE UPTAKE INTO NERVE TERMINALS

e

n

WT Het Homo 50 100 150

A559V Genotype DAT Expression (Normalized to Actin)

Normalized A559V DAT Expression

72# 42#

IB:# DAT# IB:# Ac,n#

WT#(A/A)# Het#(A/V)# Homo#(V/V)#

DA UPTAKE DAT PROTEIN

MERGY ET AL 2014

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ELEVATED EXTRACELLULAR DOPAMINE IN DAT VAL559 MICE

MICRODIALYSIS SAMPLING OF EXTRACELLULAR DA LEVELS

MERGY ET AL 2014

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DISTINCT DA PROJECTIONS SUBSERVE DIFFERENT BEHAVIORAL DOMAINS

SNpc/VTA VTA/SNpc DS DS

LOCOMOTION HABIT LEARNING IMPULSE CONTROL

VS VS

REWARD SALIENCE

PFC

EXECUTIVE FUNCTION ATTENTION

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MUTANT DAT IN ALL DA BRAIN REGIONS BUT DAT VAL559 EFFECTS ONLY IN SOME

GOWRISHANKAR ET AL, 2018

A DORSAL STRIATUM B VENTRAL STRIATUM

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DAT VAL559 MOUSE: BEHAVIOR

Spontaneous and Drug- Modulated Locomotor Activation

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FULL LOSS OF DAT IN MICE: PROFOUND HYPERACTIVITY

GIROS ET AL, 1996 KURIAN ET AL, 2009

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DAT VAL559 MICE: A SURPRISE - LACK OF SPONTANEOUS HYPERACTIVITY

MERGY ET AL 2014

10 20 30 500 1000 1500 Time (min) Distance Traveled (cm) WT HET HOM

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DAT VAL559 KI MICE: BLUNTED LOCOMOTOR AND DOPAMINE RESPONSE TO AMPHETAMINE

MERGY ET AL 2014 AMPH 3MG/KG I.P. AMPH- 3 MG/KG I.P.

5 0

10 20 30 40 50 60 500 1000 1500 2000 2500

Time Post AMPH Injection (min) Distance (cm)

WT HET HOM

AMPH- 3 MG/KG I.P. AMPH- 0.1µM IN VIVO MICRODIALYSIS DA-LEVELS

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DAT VAL559 MOUSE: BEHAVIOR

Attention, Learning and Impulsivity

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DAVIS ET AL 2017

A TASK TO EVALUATE THE LEARNING CAPACITY AND IMPULSIVITY OF DAT VAL559

START SIGNAL FIXED DELAY TO CHOOSE

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DAVIS ET AL 2017

TRAINING 5-CHOICE

1 3 5 7 9 1 1 1 3 1 5 1 7 1 9 2 1 2 3 2 5 2 7 2 9 3 1 3 3 3 5 3 7 3 9 4 1 4 3

1 2 3 4 5 6 7 8 9

A c q u is itio n C u rv e

D ays

S ta g e P ro g re s s io n

W T H O M

* **** ****** ** *

TASK ACQUISITION

A TASK TO EVALUATE THE LEARNING CAPACITY AND IMPULSIVITY OF DAT VAL559

START SIGNAL FIXED DELAY TO CHOOSE

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DAVIS ET AL 2017

W T H O M 5 1 0 1 5

to ta l p re m a tu re c o m b in e d

P re m a tu re R e s p o n s e s

*

W T H O M 5 1 0 1 5 2 0 2 5

L o n g D e la y P re m a tu re re s p o n s e s c o m b in e d

# o f P re m a tu re R e s p o n s e s

*

MORE PREMATURE RESPONSES AFTER TRAINING ON FIXED DELAY FEWER PREMATURE RESPONSES AFTER TRAINING ON VARIABLE DELAY

* *

A TASK TO EVALUATE THE LEARNING CAPACITY AND IMPULSIVITY OF DAT VAL559

START SIGNAL FIXED OR VARIABLE DELAY TO CHOOSE

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DAVIS ET AL 2018

5 1 0 1 5 2 0 2 5 0 .0 0 .4 0 .8 1 .2 1 .6

A v e ra g e R e s p o n s e R a te

T im e (s ) re s p o n s e /s

W T V a l5 5 9 T ra in e d F ixe d In te rva l

5 1 0 1 5 2 0 0 .0 0 .5 1 .0 1 .5

A v e ra g e R e s p o n s e R a te

T im e (s ) re s p o n s e /s

W T V a l5 5 9 T ra in e d F ixe d In te rva l

DAT VAL559 MICE CAN ACCURATELY CALCULATE ELAPSED TIME BUT EXHIBIT ELEVATED RESPONSE RATES AS DELAYS INCREASE

PEAK INTERVAL TESTING

START SIGNAL CHANGE FROM 5 SEC TO 15 SEC DELAY

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DAT VAL559 MOUSE: BIOMARKERS

The Eyes Have It: Retinal Changes in DAT and Dopamine

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MONITORING DOPAMINE IN THE RETINA: BIOMARKER FOR ADHD?

DAI ET AL IN 2017 RETINAL AMACRINE CELLS MAKE AND RELEASE DOPAMINE DAT VAL559 MICE SHOW ALTERATIONS IN LIGHT-EVOKED ELECTRICAL RESPONSES LIGHT FLASH

2 . 6 5 . 2 7 . 8 1 . 4 1 3 . 1 5 . 6 1 8 . 2 2 . 8 60 70 80 90 100 110 120 130 140 150

Light Adaptation Time (min) Photopic b wave amplitude (µV)

Male Photopic ERG

HET (n=12) HOM (n=9) WT (n=14)

2.6 5.2 7.8 10.4 13.0 15.6 18.2 20.8 60 70 80 90 100 110 120 130 140 150

Light Adaptation Time (min) Photopic b wave amplitude (µV)

Female Photopic ERG

HET (n=9) HOM (n=7) WT (n=3)

A B C

CONTINI AND RAVIOLA, 2003

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DAT VAL559 MOUSE: THE FUTURE

Is DAT All There Is?

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NO DAT IS AN ISLAND - AN ADHD GENE NETWORK?

DAT

PICK1 PKCβ D2R Stx.1A CamKII PKCα Gq. GPCR Rin$ GTPase Rho$ GTPase FloDllin PIP.2 GβƔ Kir$3.2 VMAT2 Syb ERK.1/2

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DAT VAL559: SUMMARY OF FINDINGS

The DAT Val559 mutation exhibits an abnormal outward leak of dopamine that can be suppressed by the two most commonly used ADHD medications. Although the DAT Val559 mutation is expressed throughout the brain, dopamine handling by DAT in the dorsal striatum, a region involved in reward learning, habit formation and goal directed behavior, is particularly impacted Rare variation in the human dopamine transporter (DAT) gene has been identified in individuals with ADHD and ADHD comorbid disorders (BPD, ASD). Adolescent male DAT Val559 mice are not spontaneously hyperactive, but display stress- induced darting behavior and increased struggling when handled, suggesting deficits in aversive learning or impulse control DAT Val559 mice display increased motivation for reward, elevated reward-based learning, and waiting impulsivity when trained on predictable outcomes DAT Val559 mice may be useful in identifing biomarkers that could provide for improved diagnosis as well as treatments tailored to to those with dopamine disturbances

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WHERE DISCOVERY COMES TO MIND

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