RARE MISSPELLINGS OF THE GENOME, DOPAMINE MISHANDLING, AND ADHD RANDY D. BLAKELY, PH.D. FLORIDA ATLANTIC UNIVERSITY BRAIN INSTITUTE 1
Dr. Blakely declares no conflicts of interest derived from financial support of the research to be discussed Dr. Blakely has consulted on research projects in the past for Amgen, Forest Research Institute, Lundbeck, Jubilant Innovation, Pfizer, Prexa, and Wyeth CONFLICTS OF INTEREST 2
ACKNOWLEDGEMENTS Blakely Lab Collaborators Lorena Areal Marc Mergy Erica Bowton Doug McMahon Rodenia Pert Steve Couch Richard Shelton Gwynne Davis Max Rabil Heng Dai Harald Sitte Raaj Gowrishankar Nathan Richtand Cristina Fenollar -Ferrer Gregg Stanwood Paul Gresch Michael Freissmuth Mark Stein Maureen Hahn Justin Riele Aurelio Galli Roxanne Vaughan Rania Katamish DJ Sakrikar Stephanie Gantz John Williams Michelle Mazei-Robison Keeley Spiess Jonathan Javitch Felix Mayer Adele Stewart Michael Gill Jeremy Veenstra-VanderWeele Peter Hamilton Austin Wheeler 3
ACKNOWLEDGEMENTS Blakely Lab Collaborators Lorena Areal Marc Mergy Erica Bowton Doug McMahon Rodenia Pert Steve Couch Richard Shelton Gwynne Davis Max Rabil Heng Dai Harald Sitte Raaj Gowrishankar Nathan Richtand Cristina Fenollar -Ferrer Gregg Stanwood Paul Gresch Michael Freissmuth Mark Stein Maureen Hahn Justin Riele Aurelio Galli Roxanne Vaughan Rania Katamish DJ Sakrikar Stephanie Gantz John Williams Michelle Mazei-Robison Keeley Spiess Jonathan Javitch Felix Mayer Adele Stewart Michael Gill Jeremy Veenstra-VanderWeele Peter Hamilton Austin Wheeler 4
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 5
ATTENTION-DEFICIT/HYPERACTIVITY DISORDER 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) 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) Approximately 60% are treated by medication, 25% receive no treatment of any form Higher incidence of substance abuse, conduct disorders, unemployment, incarceration if left untreated BASIC FACTS AND STATISTICS 6
ADHD: DSM-5 Diagnostic Criteria Inattentive Hyperactive/Impulsive Symptoms Symptoms • Careless Mistakes • Fidgeting • Di ffi culty Sustaining Attention • Inability To Stay Seated • Not Seeming To Listen • Moving Excessively • Failing To Finish Tasks • Di ffi culty Playing Quietly • Di ffi culty Organizing • On the Go • Avoiding Tasks That Require • Talks Excessively Sustained Attention • Blurting Out Answers • Losing Things • Di ffi culty Waiting Turns • Easily Distracted • Interrupting • Forgetful • 6 or more symptons per category for 6 months or more • symptoms present in two or more settings and prior to age 12 I C H • Symptoms disrupt normal function/quality of life 7
ATTENTION-DEFICIT/HYPERACTIVITY DISORDER 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 e ff ect (GWAS) Rare genetic variation of large e ff ect (e.g. Fragile X mutation) Rare genetic variation of small to moderate e ff ect (risk factor) ADHD GENETICS 8
HUMAN BRAIN 100 BILLION NEURONS DOPAMINE DOPAMINE: KEY BRAIN NEUROTRANSMITTER 9
Cue Saliency DOPAMINE Attention Executive Function Learning Reward and Motivation Locomotor Activity DA CORE FUNCTIONS PERTURBED IN ADHD 10
BRAIN DOPAMINE PATHWAYS 11
GIROS ET AL 2006 CONTROL OF DOPAMINE AVAILABILITY BY THE DOPAMINE TRANSPORTER (DAT) 12
DOPAMINE EXTRACELLULAR (SYNAPSE) DAT Model Courtesy of Cristina Fenollar- Ferrer INTRACELLULAR (CYTOPLASM) DOPAMINE TRANSPORTER STRUCTURE 13
VOLKOW ET AL 2009 DA TRANSPORTER DENSITY VOLKOW ET AL 2007 REDUCED DAT LEVELS IN ADHD - TONIC ELEVATION IN DA? 14
Methylphenidate (Ritalin™)- DAT Antagonist Amphetamine (Adderall™)- Competitive Substrate and Induces DA E ffl ux Methylphenidate Amphetamine MAJOR MEDICATIONS TO TREAT ADHD TARGET DAT 15
CHROMOSOME 5 HUMAN DAT GENE - SLC6A3 MILLER AND MADRAS, 2012 COMMON GENETIC VARIATION IN DAT GENE AND ADHD 16
HYPOTHESIS 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. DAT CODING VARIATION: RARE OPPORTUNITIES FOR PROGRESS? 17
MAZEI-ROBISON ET AL., 2005 SAKRIKAR ET AL, 2012 MERGY ET AL, 2014 MULTIPLE DAT CODING VARIANTS IDENTIFIED IN ADHD 18
A559V L167F V24M R615C MAZEI-ROBISON ET AL., 2005 SAKRIKAR ET AL, 2012 MERGY ET AL, 2014 MULTIPLE DAT CODING VARIANTS IDENTIFIED IN ADHD 19
ADHD ASSOCIATED DAT 3’VNTR BIPOLAR DISORDER: GRUNHAGE ET AL 2000 AUTISM: BOWTON ET AL, 2014 MAZEI-ROBISON ET AL., 2005, 2008 IDENTIFICATION OF DAT VAL559 IN TWO MALE ADHD SIBLINGS 20
AND NOW FOR SOMETHING COMPLETELY NORMAL... Normal DAT total protein levels Normal DAT surface expression Normal dopamine transport activity Normal a ffi nity for dopamine Normal a ffi nity for amphetamine and methylphenidate Normal despondent reaction of graduate student MAZEI-ROBISON ET AL 2008 DAT VAL559: INITIAL IN VITRO STUDIES COME UP DRY 21
MAZEI-ROBISON ET AL 2008 DAT VAL559 DISPLAYS ANOMALOUS DA EFFLUX (ADE) 22
Outward leak blocked by cocaine or methylphenidate Block of Dopamine Leak by DAT Antagonist VAL559 ALA559 ALA559 VAL559 ALA559 VAL559 VAL559 ALA559 MAZEI-ROBISON ET AL 2008 DAT VAL559 DISPLAYS ANOMALOUS DA EFFLUX (ADE) 23
NORMAL DAT PROPOSED SYNAPTIC IMPACT OF DAT VAL559 24
LEAKY MUTANT DAT = VAL559 PROPOSED SYNAPTIC IMPACT OF DAT VAL559 25
MERGY ET AL 2014 DAT VAL559 MICE: MICE WITH ADHD TRAITS? 26
DAT PROTEIN DA UPTAKE Homo#(V/V)# Het#(A/V)# WT#(A/A)# IB:# 72# DAT# IB:# 42# Ac,n# Normalized A559V DAT Expression 150 (Normalized to Actin) DAT Expression 100 50 0 WT Het Homo n A559V Genotype MERGY ET AL 2014 DAT VAL559 MICE: NORMAL TRANSPORTER PROTEIN LEVELS AND DOPAMINE UPTAKE INTO NERVE TERMINALS e 27
MICRODIALYSIS SAMPLING OF EXTRACELLULAR DA LEVELS MERGY ET AL 2014 ELEVATED EXTRACELLULAR DOPAMINE IN DAT VAL559 MICE 28
PFC EXECUTIVE FUNCTION ATTENTION VS VS REWARD SALIENCE DS DS LOCOMOTION HABIT LEARNING IMPULSE CONTROL SNpc/VTA VTA/SNpc DISTINCT DA PROJECTIONS SUBSERVE DIFFERENT BEHAVIORAL DOMAINS 29
DORSAL STRIATUM VENTRAL STRIATUM A B GOWRISHANKAR ET AL, 2018 MUTANT DAT IN ALL DA BRAIN REGIONS BUT DAT VAL559 EFFECTS ONLY IN SOME 30
DAT VAL559 MOUSE: BEHAVIOR Spontaneous and Drug- Modulated Locomotor Activation 31
GIROS ET AL, 1996 KURIAN ET AL, 2009 FULL LOSS OF DAT IN MICE: PROFOUND HYPERACTIVITY 32
1500 Distance Traveled (cm) WT HET HOM 1000 500 0 10 20 30 Time (min) MERGY ET AL 2014 DAT VAL559 MICE: A SURPRISE - LACK OF SPONTANEOUS HYPERACTIVITY 33
AMPH 3MG/KG I.P. AMPH- 3 MG/KG I.P. AMPH- 3 MG/KG I.P. AMPH- 0.1µM IN VIVO 2500 WT HET Distance (cm) 2000 HOM 1500 1000 500 0 -5 0 10 20 30 40 50 60 Time Post AMPH Injection (min) MICRODIALYSIS DA-LEVELS MERGY ET AL 2014 DAT VAL559 KI MICE: BLUNTED LOCOMOTOR AND DOPAMINE RESPONSE TO AMPHETAMINE 34
DAT VAL559 MOUSE: BEHAVIOR Attention, Learning and Impulsivity 35
START SIGNAL FIXED DELAY TO CHOOSE DAVIS ET AL 2017 A TASK TO EVALUATE THE LEARNING CAPACITY AND IMPULSIVITY OF DAT VAL559 36
START SIGNAL FIXED DELAY TO CHOOSE A c q u is itio n C u rv e TASK ACQUISITION H O M 9 * ** W T 8 7 S ta g e P ro g re s s io n 6 5-CHOICE ****** 5 **** * 4 3 TRAINING 2 1 0 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 D ays DAVIS ET AL 2017 A TASK TO EVALUATE THE LEARNING CAPACITY AND IMPULSIVITY OF DAT VAL559 37
START SIGNAL MORE PREMATURE RESPONSES AFTER TRAINING FIXED OR VARIABLE DELAY ON FIXED DELAY 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 * TO CHOOSE * 2 5 # o f P re m a tu re R e s p o n s e s 2 0 1 5 1 0 5 0 T M W O H FEWER PREMATURE RESPONSES AFTER TRAINING ON VARIABLE DELAY to ta l p re m a tu re c o m b in e d 1 5 * * P re m a tu re R e s p o n s e s 1 0 5 0 T M W O H DAVIS ET AL 2017 A TASK TO EVALUATE THE LEARNING CAPACITY AND IMPULSIVITY OF DAT VAL559 38
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