The DISC1 Pathway in Schizophrenia, Learning, Memory and Mood

1. 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

2. 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 CONSORTIUM Edinburgh (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

3. 1. Unipolar depression 6. Bipolar Affective Disorder (manic depression) 9. Schizophrenia 10. 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)

6. 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

7. Major psychosis co-segregating with a balanced t(1;11) translocation St Clair et al Lancet (1990), Blackwood et al AmJHumGenet (2001)

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

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

10. DISC1 Association Timeline

11. No genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and TaiwanNo genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and Taiwan

12. No genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and TaiwanNo genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and Taiwan

13. Biology of DISC1 Expression Interaction Biochemistry

14. 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

15. DISC1 Mutiple, Isoform-specific Subcellular Locations & Interactions

16. DISC1 Structure & Interacting Proteins Yeast 2 Hybrid Co-localisation Co-immunoprecipitation Functional interaction

17. 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

18. Effect of t(1;11) DISC1 mutation Millar et al., Science, 2005 No genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and TaiwanNo genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and Taiwan

19. 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

20. 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

21. 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.

24. No genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and TaiwanNo genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and Taiwan

25. DISC1multiple functional motifs & interaction domainsmultiple targets for mutation & modulationmutation class specific effects

26. Does modulation / mutation of Mouse Disc1 inform on the human conditions of SCZ & BPAD? Limitations of Human Studies of Brain Disorders

27. Disc1 RNAi in utero inhibits neuronal migration in mouse brainKamiya et al Nature Cell Biol 2005

29. ENU Missense Mutations in Disc1 Exon2

30. Reduced Brain Volume in Disc1 Mutant Mice

31. Mouse Behavioural Tests

32. 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.

33. Pre-Pulse Inhibition is low in 31L and lower still in 100P Disc1 mutants +/+ < 31L/+ < 31L/31L < 100P/+ < 100P/100P = 100P/31L

34. 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)

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

36. The Antipsychotic Clozapine Rescues the Latent Inhibition Deficit in 100P Disc1 Mutants

37. Open Field Activity100P Disc1 mutants are hyperactive 100P/100P >> 100P/+ = 31L/31L = 31L/+ = +/+

38. Choice Delay Test of Working Memory100P Disc1 mutants are slower learners

39. 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

40. 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

41. Learning, Memory & Mood cAMP Signalling & PDE4 Reduced PDE4B activity in 31L, but not 100P

42. No genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and TaiwanNo genes found at chromosome 11 breakpoint Contrast to chromosome 1 breakpoint DISC1 large gene ~500 kb Antisense RNA gene DISC1 encodes large protein predicted to consist of two distinct domains N-terminus consists of helix & sheet C-terminus long helical domain with potential to form numerous coiled-coils Fairly poorly conserved, particularly within N-terminus although aa composition maintained Evidence for alternative splicing and presence of multiple protein isoforms – WB probed with antibody to N-terminus Don’t yet know how these relate to transcripts but v complex Linkage findings – Finland and Taiwan

43. 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)

44. 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

45. 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

46. 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