Translating Genetic Findings Into Biological Mechanisms for ADHD Through Animal Models Klaus-Peter Lesch

Translating genetic findings into biological mechanisms for ADHD through animal models Klaus-Peter Lesch

ECNP, Berlin, October 19h 2014

Division of Molecular Psychiatry Laboratory of Translational Neuroscience Department of Psychiatry, Psychosomatics and Psychotherapy University of Würzburg, Germany Syndromal dimensions of ADHD

Attention deficit

Increased impulsivity Hyperactivity

Emotional dysregulation Oppositional- defiant Risk seeking behaviour behaviour – substance use, behavioural addictions Impaired social behaviour A lifetime of ADHD

Children

100 % Adults > 30 %

Prevalence: 3 - 7% DSM-IV-TR (APA, 2000) 4.4%, 18 - 44 years 8 - 12% world-wide (Kessler et al., 2006, (Faraone et al., 2003) Fayyad et al. 2007) Adult ADHD – Diagnostic challenge !

 Age- and sex-specific symptom modification Females: inattentive subtype

 Symptom modification by axis-I disorders Depression, anxiety disorders, substance use disorders

 Symptom modification by personality disorders Persistent pattern of perception, internal experience processing and individual behavioural responses to adversity: - obsessive-compulsive pd: order, perfection, control - antisocial pd: legal difficulties, delinquency

 Within-family symptom variability

 Psychosocial compensation mechanisms and resources What are the neurobiological mechanisms underlying a complex clinical syndrome, such as adult ADHD ? The behavioural endophenotype concept

ADHD

Gottesman & Gould, 2003 Brain networks implicated in ADHD: fronto-striatal dysfunction

Impulsive and Prefrontal reflective cortex + neurocircuits insula

DAT DAT

Striatum + amygdala

Lesch et al., 2013 Genetics: generating hypotheses for pathogenetic mechanisms and neurobiologic subtyping

. ADHD is an extreme of a common (!) behavioural trait, which accumulates in families, but is genetically complex

. Twin and family studies indicate a remarkable concordance (MT : DT = 70 : 30%) and high heritability (h2, 70 - 80%)

. Genome-wide linkage scans confirmed several susceptibility loci (e.g., 4q13.2, 5p15.33, 9q22, 12p13, 16p13, and 16q24.1)

. Molecular genetic studies identified dopaminergic, noradrenergic, and serotonergic candidate genes with very small effects on disease risk Translating genetic findings into biological mechanisms for ADHD through animal models

 Dopaminergic and serotonergic candidate genes with small effects on disease risk

 Genes identified by fine-mapping of chromosomal regions of confirmed linkage

 Common and rare variants screened by genome-wide association studies (GWAS) and whole exome sequencing (WES) Translating genetic findings into biological mechanisms for ADHD through animal models

 Dopaminergic and serotonergic candidate genes with small effects on disease risk

 Genes identified by fine-mapping of chromosomal regions of confirmed linkage

 Common and rare variants screened by genome-wide association studies (GWAS) and whole exome sequencing (WES) Animal models of human genetic disease

Genetically modified mice (or other species) based on human findings can be used to

. understand the basic pathophysiology of the disease

. detect phenotypes associated with the genetic lesion

. test novel compounds for efficacy against the disease pathology or phenotypes

Wee, sleeket, cowran, tim'rous beastie, O, what panic's in thy breastie! Robert Burns Inactivation of the gene encoding the dopamine transporter in the mouse

Striatum and nucleus accumbens

Sora et al., 1998 Gainetdinov et al., 1999 Association between a functional TPH2 variant with amygdala activation, disorders of emotion regulation and ADHD

Effect of TPH2 genotype on amygdala activity as assessed by ADHD BOLD fMRI percent signal change

(SNP G-703T, TPH2 rs4570625) FEAR SAD HAPPY NEUTRAL

Fear - Neutral

Anxiety- and depression-associated personality dimensions Happy - Neutral

Disorders of emotional regulation Sad - Neutral Walitza et al., 2005 Mössner et al., 2006 depression Gutknecht et al., 2007 bipolar disorder OCD Tph2 deficient mouse

Gutknecht et al., 2008 Raphe and Raphe Hippocampus Tph2 5HT 5HTT Tph2 fl/fl

LoxP LoxP Exon 5

Nestin-Cre WT Cre Nestin 5’ region Nestin enh II

Selection for germline transmission Tph2 -/- Tph2 -/-

Constitutive knockout model Neurobiological mechanisms of 5HT deficiency ? Tph2 deficient mice: open field activity

** Popp et al., 2012 #

Genotype effect: F(2,30)=5.92; p=0.007

Sex x genotype interaction: F(2,57)=4.13; p=0.021 Genotype effect: F(2,27)=1.09; p=0.352 Time x genotype interaction: F(14.12,190.44)=2.50; p=0.003 Tph2 deficient mice: homecage activity

Popp et al., 2012

Male: F(2,18)=8.61; p=0.002

Sex x genotype interaction: F(2,45)=5.07; p=0.010 Female: F(2,27)=0.13; p=0.883 Time x genotype interaction: F(28,378)=2.42; p<0.001 Aggressive behaviour Popp et al., 2012

Resident / intruder test

CMS

CMS CMS Fear conditioning CS US

CMS

Freezing Depression-like behaviour Popp et al., 2012

Forced swim test (FST)

Video tracking of movement (5 min)

CMS Behavioural despair

Hyperactivity / impulsivity > Innate anxiety < Fear learning / memory > Aggression > Depression >

Pet-1 Monoamine neurotransmitters

Gutknecht et al., 2008 5-HT lacking neurons retain electrophysiological properties

Araragi et al., 2011 5-HT1A receptor binding

5-HT1A receptor binding and signal transduction

5-HT1A receptor coupling Raphe cells in Tph2-/- mice lack 5-HT but retain properties of serotonergic neurons

5-HTT = 5-HT < 94-99% 5-HT1A > 12-73% Pet-1 =

5-HT1B > 28-64% Firing =

Pet-1 Translating genetic findings into biological mechanisms for ADHD through animal models

 Dopaminergic and serotonergic candidate genes with small effects on disease risk

 Genes identified by fine-mapping of chromosomal regions of confirmed linkage

 Common and rare variants screened by genome-wide association studies (GWAS) and whole exome sequencing (WES) Locus linked to adult ADHD: chromosome 4q13.2

Chr. 4q13.2

Haplotypes at 4q13.2 Paisa: Genetic isolate of Europeans in Antioquia, Columbia

Arcos-Burgos et al., 2004 ADHD risk haplotype in the Latrophilin-3 gene (LPHN3)

Chr. 4q13.2

ADHD: N = 2627 Relatives: N = 2161 Controls: N = 2531

LPHN3: P < 3.01 x 10-8, RR 1.3 Risk allele frequency: 22 % Arcos-Burgos et al., 2010 LPHN3 is an adhesion G protein-couple receptor (GPCR) Glutamatergic synapse Dendrites

Axon Lesch et al. 2013 Latrophilin 3

Black widow Latrodectus mactans -Latrotoxin: Latrodectism

Massive release of neurotransmitters - Lethargia Hyperexcitability - Anxiety - Hallucinations

Treatment ? Tarantella ! Overview of Lphn3 studies in zebrafish Morpholino injection

Exon 1 Exon 2 Exon 3 Exon 4 Exon 17

1 cell embryo Morpholino 1

Neuroanatomy Locomotor behaviour Focus on dopaminergic system Zebrabox set-up

Posterior tuberculum homologous of mammalian SN and VTA (ISH of dat, th)

In collaboration with: M. Lange, W. Norton, L. Bally-Cuif, CNRS, Gif-sur-Yvette, France Lphn3 morphants are hyperactive

Locomotor activity measured as the total distance swum in 5 min (P≤0.001)

Lphn3 knock-down triggers hyperactivity and increased impulsivity in the zebrafish juvenile

Lange et al. 2012 Methylphenidate blocks hyperactivity and impulsivity in Lphn3 morphants

Lphn3-CO 2000

1800 Lphn3-MO

1600 CO+15µM 100 1400 MPH 80 CO 60 +M 1200 MO+10µM 40 PH 1000 MPH 20 800 0 8uM 10uM 12uM 15uM 20uM 600 -20 -40 400 -60 200

Lange et al. 2012 Similar effects also observed for the norepinephrine reuptake inhibitor atomoxetine DA neurons in the posterior tuberculum

Dopamine-positive neurons

Distribution of DA neurons in posterior tuberculum groups 1, 2 and 4/5 is altered in morphants

Control lphn3 knockdown

Lange et al. 2012 Neuroanatomical studies of the serotonin system Lange et al. 2012

No difference in 5-HT neurons in MO fish compare to CO Neuroanatomical studies of other system: GABA and glutamate Lange et al. 2012

Glutamic acid decarboxylase (gad67) GABA specific Vesicular glutamate transporter 1 (vglut1) glut specific No modification in the GABA and glutamate neurons in MO compared to CO Translating genetic findings into biological mechanisms for ADHD through animal models

 Dopaminergic and serotonergic candidate genes with small effects on disease risk

 Genes identified by fine-mapping of chromosomal regions of confirmed linkage

 Common and rare variants screened by genome-wide association studies (GWAS) and whole exome sequencing (WES) Next: Whole-genome exome sequencing (WES) in extended pedigrees

Romanos et al., 2008 ADHD subclinical symptoms P1 no ADHD not known 50K Genome-wide parametric linkage in 8 families

Narrow phenotype (blue: MODglobal; red: MODsingle)

Broad phenotype (blue: MODglobal; red: MODsingle)

Romanos et al. 2008 Whole-genome exome sequencing (WES) in an extended pedigree ADHD subclinical symptoms no ADHD P1 not known

Rare variants DGKB DGKB USP20 MDH2 FAM190A HERC4 LRCH3 CCNL1 PHLPP1 WDR17 SEPT8 PON1 MYCBP2 5 deleterious

13 rare SNVs (MAF<1%) shared, but also many common SNPs (>4,000) ! 50K Genome-Wide Parametric Linkage in 8 Families

Narrow phenotype (blue: MODglobal; red: MODsingle) P1 Chr. 9q31.1-33.1

BAAT ABCA1 ZNF462 EPB41L4B AKAP2 PALM2 SVEP1 (2+1) KIAA0368 PTGR1 C9orf84 (5) FKBP15 Broad phenotype (blue: MODglobal; red: MODsingle) HDHD3 35 SNVs RGS3 13 deleterious DFNB31 (2) TNC (3) NIPSNAP3B (2) ABCA1 FSD1L FKTN ZNF462 CTNNAL1 MUSK WDR31 BSPRY C9orf43 (2) Romanos et al. 2008

Complex Aetiology of ADHD Genetic background

Rare Common, low-frequency, ADHD variants Neural and and rare Neuronal De novo or network comorbidity variants Protein cell recurrent failure defect Variation e.g. e.g. Syndromal Epistasis (GxG) in altered neurite altered structure sensitivity of dimensions outgrowth or and / or motivational Gene expression axonal and / or emotional guidance Intermediate Developmental trajectory circuits phenotypes

Environment G x E x T

Epigenetic factors - Prenatal toxins - Early life stress Cascading effects Plasticity - Psychosocial adversity Homeostatic processes Maturation

Compensatory mechanisms Resilience Acknowledgements

ADHD Clinical Research Network

Division of Molecular Psychiatry, U Würzburg

O. Rivero C.P. Jacob J. Waider M. Herrmann M. Heine N. Araragi T. Hahn A. Boreatti-Hümmer S. Merker A. Schmitt S. Gross-Lesch T. Töpner L. Gutknecht S. Kreiker N. Steigerwald L. Hommers H. Weber

Dept. of Translational Neuroscience, U Maastricht T. Strekalova Many thanks to the patients H. Steinbusch Dept. of Genetics, U Nijmegen and their families D. Van den Hove B. Franke KJPP, U Würzburg M. Klein

M. Romanos KPPP, U Frankfurt T. Renner A. Warnke A. Reif CNRS, Paris DPSS, U Tartu MPI MG, Berlin L. Bally-Cuif J. Harro W. Norton R. Ullmann K. Laas M. Lange H.H. Ropers