Transcript J. Kirsty Millar1, Benjamin S. Pickard1, Shaun Mackie1

The DISC1 Pathway in
Schizophrenia, Learning, Memory
and Mood
Centre for Molecular Medicine
Medical Genetics Section
University of Edinburgh
Molecular Medicine Centre
Western General Hospital Campus
EDINBURGH
THANKS TO..
COLLABORATORS at Merck Sharp & Dohme
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Nick Brandon, Miguel Camargo, Thomas Rosahl, Paul
Whiting
COLLABORATORS on PDE4B
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Miles Houslay, Elaine Huston, Elaine Hill, George
Bailie, Hannah Murdoch, Glasgow
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Douglas Blackwood
Walter Muir
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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 on Disc1 Mouse Models
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Steve Clapcote & John Roder, Toronto, Canada
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Yoishi Gondo, RIKEN, Japan
THE DISC1 CONSORTIUM
Edinburgh (Hennah, Thomson, Blackwood, Muir & Porteous)
Aberdeen (D St Clair), University College London (H Gurling),
Helsinki (L Peltonen)
SPONSORS
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Medical Research Council
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Wellcome Trust
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Merck Sharp & Dohme
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Organon Laboratories
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Chief Scientists Office of the Scottish Executive Health
Department
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Cunningham Trust
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Stanley Medical Research Institute
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Scottish Hospitals Endowment Research Trust
The Leading Causes of Disability Worldwide
(Years of Life with Disability)
Lopez & Murray, Nature Medicine, 4, 1241-1243 (1998)
1. Unipolar depression
6. Bipolar Affective Disorder (manic depression)
9. Schizophrenia
10. Obsessive compulsive disorders
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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
Bipolar Disorder
(Manic Depression)
manic phase: energetic,
grandiose, elated,
irritable, reduced sleep
Schizophrenia
positive symptoms of visual
& auditory hallucinations,
delusions, incoherent speech
Schizophrenia
Bipolar Disorder
(Manic Depression)
despair, emptiness,
inability to experience
joy, lack of energy
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:
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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)
The t(1;11) breakpoint & linkage to psychosis
t(1;11)
LOD = 3.4
Schizophrenia
YES NO
N=36 N=35
7
0
Bipolar affective disorder
1
0
Recurrent major depression
10
0
Conduct disorder with anxiety in
adolescence
Minor depression
2
1
1
3
Alcoholism
0
1
MLOD = 7.1
11q14
11
1q42
1
50-fold elevated risk
Dominant, variable penetrance, broad diagnosis
CHROMOSOME 1 BREAKPOINT
Millar 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
Schizoaffective disorder
Schizophrenia
1990
2000
Translocation between
chromosomes 1 and 11
associates with SCZ &
major mental illness
Major depression
Bipolar disorder
2002
2004
DISC1 Identified at 1q42;
Translocation association
with SCZ, BPAD, Major
depression
SCZ-like P300 deficit in all
t(1;11) carriers
2006
2007
Working memory
Attention
Cognition
Gray Matter Volume
Linkage & Association between
DISC1 & Psychiatric Disorder
translocation
SCOTLAND
BRITAIN & ICELAND
TAIWAN
FINLAND
CHINA
FINLAND
NORTH-AMERICA
Leu607Phe
EUROPE
JAPAN
Ser704Cys
SCHIZOPHRENIA
SCHIZOAFFECTIVE DISORDER
Hennah et al, Scz Bulletin, 2006
BIPOLAR AFFECTIVE DISORDER
MAJOR DEPRESSION
Hashimoto et al, HumMolGen, 2006
Multiple lines of supportive genetic evidence, but no convergence
or validated functional variants
Association between DISC1 &
Cognition
RAPID VISUAL SEARCH
VERBAL WORKING MEMORY
(SCHIZOPHRENIA)
SPATIAL
WORKING
MEMORY
VISUAL WORKING
MEMORY
(SCHIZOPHRENIA)
HIPPOCAMPAL
STRUCTURE &
FUNCTION
VERBAL
LEARNING &
MEMORY
NORMAL
COGNITIVE
AGING
HIPPOCAMPAL
VOLUME
GRAY MATTER
VOLUME
Leu607Phe
Ser704Cys
Biology of DISC1
Expression
Interaction
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
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–
–
–
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Nucleus
Centrosome
Cytoskeleton
Cytoplasm
Mitochondria
100
98
80
n-75
71
DISC1
Mutiple, Isoform-specific Subcellular Locations
& Interactions
DISC1 Structure & Interacting
Proteins
REGIONS OF COILED-COIL FORMING POTENTIAL
S/F-RICH
MOTIF
NUCLEAR
LOCALISATION
SIGNALS
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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
100% of normal
50% of normal
DISC1
GAPDH
T
T
T
T
T
Evidence for DISC1 haploinsufficiency
Prediction: reduced interaction with all DISC1 interactors
DISC1 and PDE4 Are Interacting
Genetic Factors in Schizophrenia
that Regulate cAMP Signalling
Science, 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, UK
2Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, IBLS, Wolfson Building,
University of Glasgow, Glasgow G12 8QQ, UK
3Merck Sharp and Dohme, The Neuroscience Research Centre, Terlings Park, Harlow, Essex, CM20 2QR, UK
43HYBRIGENICS S.A., 3-5 Impasse Reille–75014 Paris, France
5Division 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
Unique N-termini
Sub-cellular targeting
cAMP compartmentalised
signalling
cAMP gradients
PDE4A1
PDE4A4B
PDE4A5
PDE4A6
PDE4A7
PDE4A8
PDE4A10
PDE4A11
UCR2 / DISC1
Domain
?
PDE4B1
PDE4B2
PDE4B3
PDE4B4 (rodents)
PDE4C1
PDE4C2
PDE4C3
PDE4D1
PDE4D2
PDE4D3
PDE4D4
PDE4D5
PDE4D6
PDE4D7
PDE4D8
PDE4D9
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*
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*
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*
*?
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*?
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Antidepressant rolipram sensitive
PDE4 A, B, C & D
isoforms
DISC1-PDE4 Interaction is
Isoform Specific
Murdoch et al, 2007 J Neuroscience, under revision
UCR2
Catalytic
B
B
All
B
All
100kD
?
?
All
?
All
71kD
N-terminal ‘globular’ head
C-terminal helical tail
DISC1
multiple
functional motifs
& interaction
domains
multiple targets
for mutation &
modulation
mutation class
specific effects
Limitations of Human
Studies of Brain Disorders
Does modulation / mutation
of Mouse Disc1 inform on
the human conditions of
SCZ & BPAD?
Disc1 RNAi in utero inhibits
neuronal migration in mouse brain
Kamiya et al Nature Cell Biol 2005
Reduced
Migration,
Polarity &
Arborisation
Behavioral Phenotypes of Disc1 Missense
Mutations in Mice
Clapcote et al, 2007 Neuron, 54, 387-402
ENU Missense Mutations in
Disc1 Exon2
Reduced Brain Volume in Disc1 Mutant Mice
-6% in 31L
-13% in 100P
Cerebellum, thalamus,
cortex, entorhinal cortex
The Neurodevelopmental Hypothesis in SCZ
Mouse Behavioural Tests
Arguello & Gogos, Neuron, 2006
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 Activity
100P Disc1 mutants are hyperactive
100P/100P >> 100P/+ = 31L/31L = 31L/+ = +/+
Choice Delay Test of Working Memory
100P Disc1 mutants are slower learners
Forced Swim Test
31L 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
150
Pde4b activity (% +/+)
125
100
P < 0.00001
75
50
25
0
L/L
L/LL/L L/L
L/L
P/P
P/PP/PP/P
P/P
DISC1 Missense Mutations & PDE4
Binding Domains
UCR2
B
B
All
Catalytic
B
Q31L L100P
N-terminal ‘globular’ head
All
L607F C704S
C-terminal helical tail
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
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Combined analysis of Aberdeen, Edinburgh, London & Helsinki cohorts
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Finnish SNP and London SNP associate with BPAD
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Stratification on Finnish or London SNP identifies a third SNP
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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