Centre for Molecular Medicine Medical Genetics Section University of Edinburgh Molecular Medicine Centre Western General Hospital Campus EDINBURGH The.

Centre for Molecular Medicine

Medical Genetics SectionUniversity of Edinburgh Molecular Medicine CentreWestern General Hospital Campus

EDINBURGH

The DISC1 Pathway in Schizophrenia, Learning, Memory

and Mood

THANKS TO..

• Douglas Blackwood• Walter Muir

• Kirsty Millar• Shaun Mackie• Ben Pickard• Rachel James• William Hennah• Pippa Thomson • Sheila Christie• Sebby Cooper• Fumiaki Ogawa • Jennifer Chubb• Nick Bradshaw• Kathy Evans • Pat Malloy

COLLABORATORS at Merck Sharp & Dohme• Nick Brandon, Miguel Camargo, Thomas Rosahl, Paul

Whiting

COLLABORATORS on PDE4B• Miles Houslay, Elaine Huston, Elaine Hill, George

Bailie, Hannah Murdoch, Glasgow

COLLABORATORS on Disc1 Mouse Models• Steve Clapcote & John Roder, Toronto, Canada• Yoishi Gondo, RIKEN, Japan

THE DISC1 CONSORTIUMEdinburgh (Hennah, Thomson, Blackwood, Muir & Porteous)Aberdeen (D St Clair), University College London (H Gurling),Helsinki (L Peltonen)

SPONSORS• Medical Research Council• Wellcome Trust• Merck Sharp & Dohme• Organon Laboratories• Chief Scientists Office of the Scottish Executive Health

Department• Cunningham Trust• Stanley Medical Research Institute• Scottish Hospitals Endowment Research Trust

1. Unipolar depression6. Bipolar Affective Disorder (manic depression)9. Schizophrenia10. Obsessive compulsive disorders

• One in a hundred will develop schizophrenia• One in a hundred will develop bipolar disorder• Both conditions are highly heritable• 10 to 15 fold increased risk in first degree relatives• Higher risk of bipolar if you have a relative with

schizophrenia and vice versa• 60-80% concordance in identical twins

The Leading Causes of Disability Worldwide (Years of Life with Disability)

Lopez & Murray, Nature Medicine, 4, 1241-1243 (1998)

Schizophrenia positive symptoms of

visual & auditory hallucinations,

delusions, incoherent speech

Bipolar Disorder(Manic

Depression)manic phase:

energetic, grandiose, elated, irritable, reduced

sleep

Bipolar Disorder(Manic

Depression)despair, emptiness,

inability to experience joy, lack

of energy

Schizophrenia

negative symptoms of withdrawal & isolation, impaired attention &

blunted emotions

Psychiatric Genetics

• Clear evidence for major genetic component• Familial clustering, but no simple patterns of

inheritance• Predictions:

– Genetic Heterogeneity– Gene-Gene Interaction– Gene-Environment Interaction

• Hypotheses:– Common Disease, Common Variant (CDCV)– Common Disease Rare Variant (CDRV)

• Methods:– Family based linkage– Population based association– Candidate genes– Cytogenetics

Major psychosis co-segregating with a balanced t(1;11) translocation

St Clair et al Lancet (1990), Blackwood et al AmJHumGenet (2001)

major psychiatric illness (SCZ, BPAD, RMD)minor psychiatric illness (ANX, ALC, MIND)unaffected

t(1:11)

1

1q42

11q14

11

The t(1;11) breakpoint & linkage to psychosis t(1;11)

YES

N=36 NO

N=35 Schizophrenia 7 0

Bipolar affective disorder 1 0

Recurrent major depression 10 0

Conduct disorder with anxiety in adolescence

2 1

Minor depression 1 3

Alcoholism 0 1

50-fold elevated risk

LOD = 3.4

MLOD = 7.1

Dominant, variable penetrance, broad diagnosis

CHROMOSOME 1 BREAKPOINTMillar et al, Hum. Mol. Gen, 2000

translocation breakpoint

Novel, 13 exons, ~7.5kb mRNA, 854 amino acids,

~100kD protein

Disrupted in Schizophrenia 1

DISC1

DISC1 Association Timeline

1990

Translocation between chromosomes 1 and 11 associates with SCZ & major mental illness

2000 2006

DISC1 Identified at 1q42;Translocation association with SCZ, BPAD, Major depressionSCZ-like P300 deficit in all t(1;11) carriers

Schizoaffective disorder

Bipolar disorder

Major depression

Working memoryAttentionCognitionGray Matter Volume

20072002 2004

Schizophrenia

SCHIZOPHRENIA

SCHIZOAFFECTIVE DISORDER

BIPOLAR AFFECTIVE DISORDER

Linkage & Association between DISC1 & Psychiatric Disorder

translocation

BRITAIN & ICELAND

TAIWAN FINLAND

FINLAND

NORTH-AMERICA

SCOTLAND EUROPECHINA

MAJOR DEPRESSION

JAPAN

Leu607Phe Ser704Cys

Hennah et al, Scz Bulletin, 2006

Hashimoto et al, HumMolGen, 2006

Multiple lines of supportive genetic evidence, but no convergence or validated functional variants

Association between DISC1 & Cognition

VISUAL WORKING MEMORY

(SCHIZOPHRENIA)

NORMAL COGNITIVE

AGING

SPATIAL WORKING MEMORY

VERBAL LEARNING &

MEMORY

HIPPOCAMPAL STRUCTURE &

FUNCTION

GRAY MATTER VOLUME

HIPPOCAMPAL VOLUME

RAPID VISUAL SEARCH VERBAL WORKING MEMORY

(SCHIZOPHRENIA)

Leu607Phe Ser704Cys

Biology of DISC1

ExpressionInteraction

Biochemistry

DISC1 Expression• Widely expressed

– SCZ associated regions of the brain (hippocampus, dentate gyrus)

– peripheral blood lymphocytes / lymphoblastoid cell lines

• Developmentally regulated– Highest during active neurogenesis– Neonatal & adult

• Multiple isoforms• Multiple cell compartments

– Nucleus– Centrosome– Cytoskeleton– Cytoplasm– Mitochondria

10098

80n-75

71

DISC1 Mutiple, Isoform-specific Subcellular Locations

& Interactions

REGIONS OF COILED-COIL FORMING POTENTIAL

NUCLEAR LOCALISATION SIGNALS

S/F-RICH MOTIF

DISC1 Structure & Interacting Proteins

• Yeast 2 Hybrid• Co-localisation• Co-immunoprecipitation• Functional interaction

DISC1 interacts with proteins required for neurodevelopment &

neuronal function

• NUDE / NUDEL: neuronal migration, corticogenesis & axonal outgrowth

• NUDEL: endo oligopeptidase that may regulate neuropeptide action in the CNS

• LIS1: lissencephaly 1 (‘smooth brain’), binds NUDE/NUDEL

• FEZ1: fasciculation and elongation zeta protein 1, neurite outgrowth, neuronal differentiation

• PDE4B: regulates compartmentalised cAMP signalling

Effect of t(1;11) DISC1 mutation Millar et al., Science, 2005

protein levels are reduced

DISC1

GAPDH

T T T T T

50% of normal

100% of normal

Evidence for DISC1 haploinsufficiency

Prediction: reduced interaction with all DISC1 interactors

DISC1 and PDE4 Are Interacting Genetic Factors in Schizophrenia

that Regulate cAMP SignallingScience, 310, 1187-1191 (2005)

J. Kirsty Millar1, Benjamin S. Pickard1, Shaun Mackie1, Rachel James1, Sheila

Christie1, Sebastienne R. Buchanan1, M. Pat Malloy1, Jennifer E. Chubb1, Elaine

Huston2, George S. Baillie2, Elaine V. Hill2, Miles D. Houslay2, Nicholas J. Brandon3,

Jean-Christophe Rain4, L. Miguel Camargo3, Paul J. Whiting3, Douglas H.R.

Blackwood1,5, Walter J. Muir1,5, David J. Porteous1.

Medical Genetics Section, Molecular Medicine Centre, University of Edinburgh, Edinburgh EH4 2XU, UK2Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, IBLS, Wolfson Building,

University of Glasgow, Glasgow G12 8QQ, UK3Merck Sharp and Dohme, The Neuroscience Research Centre, Terlings Park, Harlow, Essex, CM20 2QR, UK43HYBRIGENICS S.A., 3-5 Impasse Reille–75014 Paris, France5Division of Psychiatry, University of Edinburgh, Royal Edinburgh Hospital, Edinburgh EH10 5HF, UK

Genomics & Biology Converge & Corroborate

PDE4B identified as a DISC1 interactor AND Psychosis associated t(1;16) disrupts PDE4B

Millar et al Science 2005

Proband with SCZ & cousin with psychosis

Half normal levels of PDE4B Expression: Haploinsufficiency

PDE4B Association in Schizophrenia

Pickard et al, 2007

Phosphodiesterase 4B (PDE4B) is very interesting because

• PDE’s are sole means of inactivating intracellular cAMP, a key second messenger in the brain.

• PDE4 is involved in learning, memory and mood in fly & mouse.

• PDE4 is target of antidepressant rolipram.

The head domain of DISC1 binds the UCR2 domain of PDE4B

Binding is dynamic, cAMP regulated, phosphorylation dependent and PKA mediated

+cAMP

-cAMP

PDE4D1PDE4D2PDE4D3PDE4D4PDE4D5PDE4D6PDE4D7PDE4D8PDE4D9

PDE4C1PDE4C2PDE4C3

PDE4B1PDE4B2PDE4B3PDE4B4 (rodents)

PDE4A1

PDE4A4BPDE4A5PDE4A6PDE4A7PDE4A8PDE4A10PDE4A11

PDE4 A, B, C & D isoforms

*

UCR2 / DISC1

Domain

***

**

**

**

**

**

**

****

?

?

?

Unique N-termini

Sub-cellular targeting

cAMP compartmentalised signalling

cAMP gradients

An

tid

epre

ssan

t ro

lipra

m s

ensi

tive

N-terminal ‘globular’ head

C-terminal helical tail

B B All B All

UCR2 Catalytic

DISC1-PDE4 Interaction is Isoform Specific

Murdoch et al, 2007 J Neuroscience, under revision

? ? All All

100kD

71kD?

DISC1

multiple functional motifs

& interaction domains

multiple targets for mutation &

modulation

mutation class specific effects

Does modulation / mutation of Mouse Disc1 inform on the human conditions of

SCZ & BPAD?

Limitations of Human Studies of Brain Disorders

Disc1 RNAi in utero inhibits neuronal migration in mouse brain

Kamiya et al Nature Cell Biol 2005

ReducedMigration,Polarity &

Arborisation

Behavioral Phenotypes of Disc1 Missense Mutations in Mice

Clapcote et al, 2007 Neuron, 54, 387-402

ENU Missense Mutations in Disc1 Exon2

The Neurodevelopmental Hypothesis in SCZ

-6% in 31L

-13% in 100P

Cerebellum, thalamus, cortex, entorhinal cortex

Reduced Brain Volume in Disc1 Mutant Mice

Arguello & Gogos, Neuron, 2006

Mouse Behavioural Tests

Pre-Pulse Inhibition (PPI)& Latent Inhibition (LI)

Measures of attention, information processing and working memory

• PPI: does a low level stimulus inhibit a startle response to a strong stimulus?

• LI: can irrelevant stimuli be disregarded and memory formation inhibited to prevent information overload?

• Both PPI and LI are low in SCZ. • Both PPI and LI can be measured in the

mouse.

Pre-Pulse Inhibition is low in 31L and lower still in 100P Disc1 mutants

+/+ < 31L/+ < 31L/31L < 100P/+ < 100P/100P = 100P/31L

Pharmacological Responses in PPI

• 31L rescued by buproprion, but not rolipram• 100P rescued by rolipram, but not buproprion• 100P, but not 31L, rescued by clozapine (atypical)

and haloperidol (dopamine D2 antagonist)

Latent Inhibition is low in 31L and lower still in 100P Disc1

Mutants

The Antipsychotic Clozapine Rescues the Latent Inhibition Deficit

in 100P Disc1 Mutants

Open Field Activity100P Disc1 mutants are hyperactive

100P/100P >> 100P/+ = 31L/31L = 31L/+ = +/+

Choice Delay Test of Working Memory

100P Disc1 mutants are slower learners

Forced Swim Test31L Disc1 mutants show marked

despair

• Despair is rescued by bupropion AND rolipram in wild type, but ONLY by bupropion in 31L/31L

Summary of Behaviour & Drug Responses in 100P & 31L Disc1 Mutants

• PPI & LI deficits indicate sensory motor gating & attention deficits

• 100P more severe than 31L• PPI deficit in 100P partially alleviated by

typical (haloperidol) & atypical (clozapine) antipsychotics

• LI deficit in 100P rescued by clozapine• 31L homozygotes show depressive like

behaviour in the forced swim test which is reversed by bupoprion, but not rolipram

Learning, Memory & Mood cAMP Signalling & PDE4

Reduced PDE4B activity in 31L, but not 100P

0

25

50

75

100

125

150

L/L L/L L/L L/L P/P P/P P/P P/P

Pd

e4

b a

cti

vit

y (

% +

/+)

L/L P/P

P < 0.00001

N-terminal ‘globular’ head

C-terminal helical tail

B B All B All

Q31L L100P L607F C704S

UCR2 Catalytic

DISC1 Missense Mutations & PDE4 Binding Domains

DISC1 Interactors as Independent & Co-dependent Genetic Risk Factors

• FEZ1– Association (Yamada et al, 2004)

• PDE4B– Cytogenetics (Millar et al, 2005)– Association (Tomppo et al, 2006; Pickard et al, 2007)

• PDE4D– Association (Tomppo et al, 2006)

• NUDEL (NDEL1)– DISC1 ser704cys stratified association & functional brain

imaging (Malhorta et al, 2006)

• NUDE (NDE1)– DISC1 HEP3 stratified linkage & association (Hennah et al,

2007)

DISC1 Genetic Heterogeneity & Genetic Interplay

• Combined analysis of Aberdeen, Edinburgh, London & Helsinki cohorts

• Finnish SNP and London SNP associate with BPAD

• Stratification on Finnish or London SNP identifies a third SNP

• Combined SNPs increase risk of SCZ

1 SNP alone: BPAD >> SCZ

2 SNP’s together: SCZ > BPAD

Conclusions

• The genetic & biological evidence supports a generalised role for DISC1 in major mental illness & cognition

• DISC1 is a scaffold protein that assembles & regulates key neurodevelopmental & neurosignaling proteins

• Disc1 missense mutants model behavioural, pharmacological & brain structural features of schizophrenia & mood disorder

• The DISC1 pathway offers a route towards a mechanistic understanding of these poorly understood & debilitating disorders, prerequisite to development of rational interventions

Schizophrenia & Bipolar Disorder

• One in fifty will develop schizophrenia or bipolar disorder

• Current treatment is unsatisfactory

• Major unmet need• Discovery of DISC1

pathway paves way to– Molecular basis of

disease– Biomarker discovery– Diagnosis, prognosis &

treatment response– Rational drug

development